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Identification of human macrophage genes differentially expressed by infection with Mycobacterium tuberculosis Tang, Raymond Kwok-Cheung 1997

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IDENTIFICATION OF HUMAN MACROPHAGE GENES DIFFERENTIALLY EXPRESSED BY INFECTION WITH Mycobacterium tuberculosis by • RAYMOND K W O K - C H E U N G TANG B.Sc, University of British Columbia A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF T H E REQUIREMENTS FOR T H E DEGREE OF MASTER OF SCIENCE in THE F A C U L T Y OF GRADUATE STUDIES (Department of Microbiology and Immunology) We accept.this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA AUGUST 1997 © Raymond Kwok-Cheung Tang, 1997 ABSTRACT Mycobacterium tuberculosis infects mononuclear phagocytes and manifests disease by triggering a strong delayed type hypersensitivity response which is detrimental to the host. In most healthy individuals, infection is resolved; however, the bacillus possesses many evasion strategies which may allow it under certain conditions to survive and replicate within the macrophage. M. tuberculosis has been found to alter several host defences to promote its survival so the ability of M. tuberculosis to modulate the expression of genes in human macrophages was investigated, using a novel method which combined subtractive hybridization with differential display. Macrophage genes which appeared to be induced or suppressed by infection were identified and isolated. A total of 25 such cDNAs were isolated. Five of the cDNAs were identified as known genes: N A D H ubiquinone oxidoreductase chain 2; p22-phox; an antioxidant enzyme, A O E 37-2; a possible growth arrest gene, B4B; and a human protein phosphatase g. Seven other cDNAs matched human cDNA sequences, and the remaining 13 were novel sequences. Successful quantitation of three cDNAs revealed that two were induced in macrophages infected with M. tuberculosis, while the other was constitutively expressed. In addition, three other cDNAs were also identified to be induced by infection. The sequence of one of the cDNAs matched an IFN-inducible nuclear phosphoprotein sequence, another matched a cDNA sequence, while the last one was unique. These cDNAs were clearly induced by infection in THP-1 macrophages; however, a pattern of expression of these clones did not emerge in human peripheral blood macrophages. Further research must be performed before conclusions can be made regarding the expression and function of these clones. i i TABLE OF CONTENTS page A B S T R A C T LIST OF T A B L E S V. LIST OF FIGURES V«; LIST OF ABBREVIATIONS viii A C K N O W L E D G E M E N T S I. INTRODUCTION 1 A. Incidence of tuberculosis 1 B. Lifecycle of Mycobacterium tuberculosis 3 1. Infection 3 2. Replication 6 3. Containment 8 C. Immunoprotection and immunopathology of tuberculosis 10 1. Cell types 10 a) Macrophages 10 b) N K cells and y/S T cells 11 c) a /pT cells 12 2. Immunoprotective Thl-type response in resistance to tuberculosis 13 3. Immunosuppressive Th2-type response in susceptibility to tuberculosis 15 4. Immunopathology of tuberculosis 18 D. Focus 20 II. M A T E R I A L S A N D METHODS 23 A. Cell types and growth conditions 23 1. Monocytes 23 2. Bacteria 24 3. Infection of macrophages 24 B. Molecular biology techniques 25 1. R N A extraction from macrophages 25 2. Subtractive hybridization 25 3. Differential display 29 4. Cloning of isolated products 29 5. Sequencing analysis 31 6. Semi-quantitative PCR 31 7. Slot blotting 33 8. Northern blot 34 page III. RESULTS 35 A. Subtractive hybridization and differential display 35 B. Identification of modulated cDNAs 40 C. Slot blotting to measure the expression of the isolated cDNA transcripts 45 D. Quantitation by Northern blotting 48 E. Semi-quantitative PCR 48 1. Induction of H8.3 and H8.5 cDNA by infection; isolation of two new cDNAs 49 2. Semi-quantitative PCR of N A D H ubiquinone oxidoreductase chain 2 55 3. Semi-quantitative PCR of H15.2 57 4. Semi-quantitative PCR of the N l fragment 66 5. Semi-quantitative PCR of the N2 fragment 71 IV. DISCUSSION 77 A . Analysis of subtractive hybridization and differential display 77 B. Analysis of transcripts isolated by subtractive hybridization and differential display 80 C. Analysis of the expression of the IFN-inducible nuclear phosphoprotein 81 D. Analysis of the expression of the N l clone 83 E. Analysis of the expression of the N2 clone 84 F. Problems with human macrophage samples 85 V. CONCLUSIONS 88 REFERENCES 89 LIST OF TABLES page Table 1. Oligonucleotide sequences used for semi-quantitative PCR 32 Table 2. Clones isolated from subtractive hybridization and differential display 45 • LIST OF FIGURES page Figure 1. Lifecycle of Mycobacterium tuberculosis 5 Figure 2. Subtractive hybridization using Oligotex beads to isolate induced genes 28 Figure 3. Differential display on non-subtracted and subtracted macrophage mRNA using oligodT n G and H4 primers 36 Figure 4. Differential display on non-subtracted and subtracted macrophage mRNA using oligodT n G and H8 primers 37 Figure 5. Differential display on non-subtracted and subtracted macrophage mRNA using oligodT n G and HI5 primers 38 Figure 6. Differential display on non-subtracted and subtracted macrophage mRNA using oligodT u G and H26 primers 39 Figure 7. Sequences of 28 isolated cDNA clones 41 Figure 8. Slot blot of putatively modulated clones isolated by subtractive hybridization and differential display 47 Figure 9. Semi-quantitative RT-PCR of THP-1 macrophage R N A using primers specific for H8.3 50 Figure 10. Semi-quantitative RT-PCR of THP-1 macrophage R N A using primers specific for H8.5 51 Figure 11. Identification of putatively induced fragments, N l and N2 53 Figure 12. Semi-quantitative RT-PCR of THP-1 macrophage R N A using primers specific for N l and N2 54 Figure 13 Semi-quantitative RT-PCR of THP-1 macrophage R N A using primers specific for N A D H ubiquinone oxidoreductase chain 2 56 Figure 14. Identification of a putatively induced fragment 58 Figure 15. Semi-quantitative RT-PCR of THP-1 macrophage R N A using primers specific for the IFN-induced nuclear phosphoprotein 61 page Figure 16. Quantitation of the nuclear phosphoprotein in untreated and IFN-y treated THP-1 macrophage R N A 62 Figure 17. PCR Quantitation of the IFN-induced nuclear phosphoprotein in R N A from human macrophages 63 Figure 18. PCR Quantitation of the IFN-induced nuclear phosphoprotein in R N A from human macrophages 64 Figure 19. PCR Quantitation of the IFN-induced nuclear phosphoprotein expression in human macrophages 65 Figure 20. Semi-quantitative RT-PCR of human macrophage R N A using primers specific for N l 67 Figure 21. PCR Quantitation of N l expression in RNA from human macrophages 68 Figure 22. PCR Quantitation of N l expression in human macrophages 69 Figure 23. PCR Quantitation of N l expression in human macrophages 70 Figure 24. Semi-quantitative RT-PCR of human macrophage R N A using primers specific for N2 72 Figure 25. PCR Quantitation of N2 in R N A from human macrophages 74 Figure 26. PCR Quantitation of N2 in RNA from human macrophages 75 Figure 27. PCR Quantitation of N2 expression in human macrophages 76 yii-LIST OF ABBREVIATIONS bp base pairs CMI cell mediated immunity dCTP deoxyribocytidine 5' triphosphate DEPC diethyl pyrocarbonate dNTP deoxyribonucleoside 5' triphosphate DTH delayed type hypersensitivity E D T A ethylenediaminetetra-acetic acid FCS fetal calf serum IFN interferon IL interleukin L A M lipoarabinomannan L B Luria broth MOPS 3 -(N-morpholino)-propanesulfonic acid N A D H nicotinamide adenine dinucleotide (reduced form) N K natural killer P B M C peripheral blood mononuclear cells PCR polymerase chain reaction P M A phenol 12-myristate 13-acetate SDS sodium dodecyl sulphate TGF transforming growth factor TNF tumor necrosis factor tris tris(hydroxymethyl)aminomethane V G H Vancouver General Hospital Vii l ACKNOWLEDGEMENTS I would like to thank everyone in Dr. Robert McMaster's lab for providing an excellent and interesting working and learning environment. Without the helpful discussion and advice from Dr. Phalgun Joshi, Dr. Ben Kelly, and Craig Kreklywich, the novel method of combining subtractive hybridization and differential display would not have been possible. I would also like to thank Raymond Lo for all his help in preparing many of the R N A samples. In addition, I would like to sincerely thank Dr. Nicole Lawrence and Dr. Ben Kelly for their critical reading of this thesis. I have also greatly appreciated the direction and advice provided by my committee, Dr. Pauline Johnson and Dr. Neil Reiner, and I would also like to thank Dr. Pauline Johnson for introducing me to research. I would especially like to thank Dr. Robert McMaster not only for his excellent supervision, but also his advice, encouragement, and support. I. INTRODUCTION A. Incidence of tuberculosis The number of cases of tuberculosis has been increasing since the mid-1980's, despite a steady decline in the last four decades (11). Infection with Mycobacterium tuberculosis remains the leading cause of mortality among infectious diseases worldwide in adults (37, 104) and accounts for approximately three million deaths per year (135). Estimates suggest that a third of the world population is infected with M. tuberculosis and it has been predicted that the burden will increase to 90 million new cases by 1999 if the upward trend continues (104). The number of cases of tuberculosis closely correlate to poverty (132) and its spread is associated socially with decreased healthcare, immigration, and non-compliancy to drug treatments. The incidence of tuberculosis is highest in developing countries, but the disease is reemerging as a problem in developed countries (11). A major contributor to the spread of tuberculosis is the pandemic spread of infection with human immunodeficiency virus (HIV) (39). Approximately ten percent of HIV negative individuals exposed to M. tuberculosis develop disease, mainly as a result of non-compliancy with drug therapy, genetic predisposition to tuberculosis, immune deficiencies caused by malnutrition or alcoholism, and/or differences in the virulence of the mycobacterium (86). However, individuals infected with acquired immune deficiency syndrome (AIDS) are unable to mount an effective defence and become a source for transmission of disease. Tuberculosis is one of the earliest opportunistic infections to develop in AIDS patients (30, 124) and with the progression of AIDS, M. tuberculosis which was previously contained by the host immune system may be reactivated (131). The problem of tuberculosis has been further complicated by the emergence of multidrug resistant (MDR) strains which are nearing epidemic proportions (11). 1 M D R tuberculosis is closely associated with AIDS and HIV whereby its development and spread is also attributed to infection of non-compliant populations such as drug users and the homeless individuals who have a high risk of HIV infection (3, 131). The subsequent spread of different drug resistant strains among HIV individuals then permits the generation of more potent strains with multiple drug resistances (131). The global burden of tuberculosis has increased significantly primarily due to the pandemic spread of HIV and AIDS. With the recent emergence of M D R strains, the development of new drugs and treatments is even more critical for the containment of disease. A n integral part of developing new therapies is gaining a better understanding of host-pathogen interactions. M. tuberculosis modulates many host immune responses to promote its survival; however, the extent of the changes it mediates has not been fully elucidated. In this study, the effects of M. tuberculosis infection were investigated at the molecular level. Because mycobacteria infect macrophages and mediate many effects by manipulating the macrophage response to infection, such as by downregulating antimicrobial mechanisms, it is hoped that the understanding of macrophage- M. tuberculosis interactions may be furthered. Through a novel method of subtractive hybridization and differential display, macrophage genes modulated by infection were identified and characterized. 2 B. Lifecycle of Mycobacterium tuberculosis 1. Infection Mycobacterium tuberculosis is an obligate intracellular pathogen that infects host mononuclear phagocytic cells such as macrophages and usually initiates infection and disease in the lung. The principle lines of defence against the bacillus are the oxygen-dependent and oxygen-independent systems of the macrophages (33); however, M. tuberculosis is able to evade these defence mechanisms and has been shown to suppress macrophage antimicrobial activity (13, 14, 20, 114, 127, 133, 134). Disease in the host is then manifested by the initiation of an inflammatory response to stop the multiplication of the bacteria. For example, caseous lesions form in the lung to contain the site of infection. From this primary manifestation, the progression or regression of disease depends on the ability of the host's cell mediated immunity to clear the mycobacteria (27). Tuberculosis is transmitted primarily through inhalation of aerosolized particles from the lung of an infected host and breathing in fewer than ten bacilli is able to cause disease (Figure 1) (106). The bacilli travel into an alveolus where they are phagocytosed by alveolar macrophages. Depending on the inherent microbicidal activity of the macrophages and the virulence of the mycobacterium, the host may destroy the pathogen; otherwise, disease may develop. Differences between the interaction of macrophages with avirulent and virulent strains of M. tuberculosis have been observed as early as binding. While complement receptors are involved in the phagocytosis of both avirulent and virulent forms (63, 118, 119), mannose receptors are only involved in the uptake of virulent strains (118, 120). This is because virulent strains such as Erdman have mannosyl residues at the terminal end of their cell surface lipoarabinomannan (LAM) as opposed to the arabinose residues of avirulent strains (16). L A M has been implicated 3 as a virulence factor of M. tuberculosis (14, 17, 107) and differences in L A M have also been found to mediate differences in uptake (121). Phagocytosis of Erdman L A M compared to H37Rv or H37Ra L A M was found to be the most efficient, which would better facilitate the transport of the pathogen into a favorable milieu where it may survive and replicate. The ingestion by macrophages of less than three bacilli (limited by the size of the alveolus) does not provide enough antigenic stimulation to mount either a primary or a secondary immune response (27). The survival of the bacilli within the macrophage phagosome determines whether an infection may be established or not. In most healthy individuals, the bacilli are successfully eliminated by the oxygen dependent or independent defence mechanisms; however, M. tuberculosis possesses several counter-defences which provide it with an opportunity to survive and replicate. 4 , B £^ S O XI 83 c g h! - B 0 3 00 CD i c3 oo 00 P oo - B 00 B o 2 43 •4—> oo O oo a CD S-H on c O C oo S <D *S 00 «j a « . B •S — © o U M < £ aw M O P3 <y CD " 3 £ 5 "8 - 2 M O p 00 m g •— . B fa & <D cj § S i -2 1 i • - H s sT £ •2 ^ 3 ° •II " | l o Q oo D"1 (D i—I CD > *J (D J§ O *5 i+H CD • J J 2 - B g .S § < = o * '3 t: CD c3 B -S - ° .2 t n © 80 <S - 5 - B 2 » g cd B q CD B oo O B O O 6 8 Q 00 oo'.S - o I « 1 1 O ™ B ^ Cij G) !fl S ^ M B O g B 00 52 >> cd " S o -a t£. CD O S oo O > 00 -J3 c3 O - B c3 B H o  O s Cj is. 00 B eS D CD CD «3 00 (D g O B — O 3 e a 5 2. Replication Within the phagosome, the bacterium hinders the phagolysosome fusion to prevent the production of radical oxygen intermediates and the accumulation of proton ATPase complexes which would acidify the phagosome (20, 60, 134, 149). However, M. tuberculosis-occupied vacuoles are not transformed into fusion-incompetent vesicles as they have been shown to fuse with other vesicles (114) suggesting that the bacilli are able to prevent fusion with specific endosomal/ lysosomal compartments. This is achieved by arresting the maturation of the endosome at an intermediary stage, which subsequently restricts fusion with lysosomes (114, 133). Because of the lack of acidification in the endosome, proteolytic enzymes acquired by early fusion events, such as cathepsin D, are not activated (133) allowing the pathogen to evade another line of defence. The degree to which the organism is able to reduce the fusigenicity of the phagosome depends on the virulence of the species. (59), the genetics of the host (29), and whether the macrophage is activated or not (128). In addition to avoiding phagolysosome fusion, the bacilli have several other strategies to antagonize host defences. M. tuberculosis releases a large amount (15 mg/g of bacteria) of L A M (65) which has numerous effects on the host. Within the phagolysosome, L A M is able to undermine the oxygen dependent defence mechanism by scavenging reactive oxygen intermediates (ROI) and inhibiting protein kinase C (PKC), an enzyme integral to oxidative burst activation (14). M. tuberculosis also produces ammonia which alkalinizes the phagolysosome and is thought to have inhibitory effects on phagolysosome fusion (52, 60). A n additional product of mycobacteria are the sulfatides which also downregulate ROI production (13, 100) and possibly inhibit phagolysosome fusion (53). M. tuberculosis has also been observed to escape the phagolysosome compartment into the macrophage cytoplasm (85, 93) by releasing 6 hemolytic factors (76). With the successful evasion of host defences, mycobacteria are able to replicate within the host macrophage until the cell bursts, releasing the mycobacteria into the surrounding area. The released bacilli are subsequently phagocytosed by surrounding alveolar macrophages as well as peripheral blood monocytes attracted to the site of infection by the release of bacilli, cellular debris, and chemotactic factors from macrophages (27). Some mycobacteria may also actively invade the surrounding alveolar epithelial cells, which would offer a less hostile environment for replication than macrophages (8). Within 48 hours, the majority of infected cells are peripheral blood monocytes attracted to the site of infection by the secretion of high levels of MCAF/MCP-1 (monocyte chemotactic and activating factor/ monocyte chemoattractant protein 1) (1, 47, 73, 107). M. tuberculosis initially stimulates the production of IL-8, which recruits neutrophils and leukocytes; however, at two hours post-infection, levels of IL-8 drop significantly and MCAF/MCP-1 is produced in large amounts. This leads to chronic inflammation and the formation of lesions to contain the infection (73). In mice, L A M from the H37Ra strain but not the Erdman strain induces the production of the chemoattractants JE, the homologue for human MCAF/MCP-1 , and K C . Failure of Erdman L A M to induce these factors may therefore serve to protect the Erdman strain (107). During this initial period of infection, the host has not developed tuberculin-type sensitivity yet and the monocytes have not been activated, so the replication of mycobacteria and the recruitment of more peripheral blood monocytes continues. The mycobacterial L A M also induces the expression of TNF-a in macrophages (17, 107) which is an essential component of both protection and pathology in tuberculosis. Erdman L A M 7 however, does not stimulate and actually blocks TNF-a expression, even with the addition of IFN-y (17, 107). TNF-a is a proinflammatory cytokine which initiates the formation of granulomas to contain disease; by preventing the release of TNF-a, Erdman M. tuberculosis may maintain its intracellular environment favourable to growth (17, 107). As a consequence of the release of chemoattractants and TNF-a, a caseous centre develops where lysed macrophages and bacilli are surrounded by large numbers of other non-activated macrophages which ingest the pathogens. In this way, the mycobacteria are continually provided with cells in which to replicate and lyse, thereby expanding the caseous centre. 3. Containment Approximately three weeks after, initial infection, IL-8 generated, by infected monocytes increases to levels capable of recruiting lymphocytes (27). N K cells and y/8 T lymphocytes are the first to arrive at the site of infection (15). N K cells generate IFN-y - which activates macrophages and initiates their antimicrobial functions (15) but the role of y/8 T lymphocytes in host defence remains unclear and is discussed below. The primary effector cell against M. tuberculosis is the CD4 + a/p T cell which is recruited by macrophages in numbers greatly exceeding both N K and y/8 T lymphocytes (15). Upon stimulation by mycobacterial antigens presented on major histocompatibility complex (MHC) class II molecules on macrophages, the a/p T lymphocytes secrete large amounts of IFN-y, which in human macrophages induces production of 1,25-dihydroxy vitamin D 3 (calcitriol). Acting alone or synergistically with IFN-y and TNF-a, calcitriol further activates human macrophages which then kill or inhibit the growth of mycobacteria (11, 23, 109). Conversely, the 8 inhibition of P K C by L A M may counteract this mechanism resulting in reduced transcription of IFN-y induced genes in human macrophages (14, 40). In addition, mycobacteria induce IL-10 production in human macrophages (2, 129), which suppresses the Thl-type cell mediated immune (CMI) response. In most healthy individuals, the immune system overcomes the mycobacteria counter-defences and successfully activates macrophages and expands T cell populations specific for mycobacterial antigens. Activated macrophages contain the infection by surrounding the caseous centre and killing any mycobacteria that escape. The necrotic centre then becomes encapsulated by a fibrotic wall which further prevents dissemination and lowers the partial 0 2 pressure of the necrotic centre to inhibit the growth of M. tuberculosis (15). The infection may be contained within the granuloma indefinitely but the granuloma may rupture i f the CMI response becomes uncontrolled, promoting excessive cell destruction and necrosis (15). The liquefaction of the granuloma releases a rich growth medium, consisting of cellular debris, into an oxygen rich • environment which allows the latent mycobacteria to divide extracellularly. The large bacterial burden triggers the primed immune system causing extensive necrosis of the nearby lung tissue and airways, forming a cavity (27). The accumulation of bacteria along the airways then allows for the infection of other parts of the lung and droplet transmission of tuberculosis to other hosts. Mycobacterium tuberculosis is able to invade and replicate within the hostile environment of macrophage phagosomes by using mechanisms that counter or reverse the host defences. In addition, M. tuberculosis modifies the expression of several macrophage genes such as IL-10 , TNF-a, and IFN-y inducible genes (14, 17, 107) and thus lowers the potency of the 9 host defences. Mycobacteria may also modulate the expression of other macrophage genes and this possibility is examined in the current research project. C. IMMUNOPROTECTIONAND IMMUNOPATHOLOGYOF TUBERCULOSIS 1. Cell types a) Macrophages Macrophages are the first cells M. tuberculosis encounters and their interactions determine whether a CMI response is initiated. Alveolar macrophages are invariably activated because they encounter many stimulants including inhaled particles and thus offer a good first line of defence, able to kill mycobacteria in most individuals (27). However, as mentioned above, M. tuberculosis may escape the activated macrophage through its many evasion mechanisms. If the bacilli are able to multiply and lyse the host cell, macrophages are again responsible for initiating the next line of defence. Macrophages engulf the mycobacteria and secrete chemoattractants and inflammatory cytokines such as IL-8, IL-1, and TNF-a, which are essential to granuloma formation (75). Macrophages also release IL-12 (22, 150) and present mycobacterial antigens on their surface in the context of M H C molecules such that mycobacteria-specific CD4 + T cells may proliferate and initiate a CMI response. T cells then activate macrophages by secreting IFN-y which enhances their bactericidal functions. To prevent excessive inflammation, macrophages also control the immune response by secreting IL-10, TGF-P and calcitriol (which is also responsible for macrophage activation) (110), and this is discusses further below. Therefore, macrophages are key mediators of protection against infection with M. tuberculosis. First, they attempt to clear the bacteria, and i f unsuccessful, they 10 summon the help of other macrophages and T cells by secreting cytokines and presenting antigen. Finally, macrophages limit tissue damage by releasing immunosuppressive cytokines. b) NK cells and y/8 T cells Before a CMI response is initiated, N K cells and y/8 T cells arrive at the site of infection. In response to IL-12 from infected macrophages, N K cells release IFN-y which is involved in the activation of macrophages (147). In addition, N K cells are able to lyse infected macrophages (87) which release the mycobacteria into the extracellular environment which is less favorable to growth, y/8 T lymphocytes preferentially accumulate at the site of mycobacteria infection (4, 67, 71, 142), and like N K cells, they release IFN-y and are involved in the lysis of infected macrophages (68, 90). However, additional roles of y/8 T cells in host defence still remain unclear, y/8 T cells are also able to secrete the cytokines common to Thl-type cells, but because they reside in mucosal tissues, they may offer an initial response to M. tuberculosis infection-prior to the arrival of a/p T cells (46). In human peripheral blood mononuclear cells (PBMC) cultured with M. tuberculosis, the y/8 T cell population expands preferentially (61) and a large fraction of cells appear reactive to the bacteria (71). In mice, a disrupted 8 chain gene allows for the dissemination of M. tuberculosis suggesting a role for y/8 T cells in the resolution of infection (21, 77, 137). However, recent evidence with 8 chain gene disrupted mice suggests y/8 T cells have a protective role only in high dose infections and are not essential in low or moderate dose infections (38). 11 c) a/p T cells a/p T cells consist of CD4 + T cells which recognize antigen in the context of M H C class II, and CD8 + T cells which recognize antigen complexed with M H C class I. CD4 + T cells are the main effector cells that produce a Thl-type response against M. tuberculosis as they accumulate in large numbers at the site of infection (15). CD4 + cells also secrete IL-2, GM-CSF, and IFN-y which in combination with IL-12 from macrophages, promote a protective Thl-type CMI response. In addition to mediating protection by secreting cytokines, CD4 + cells are able to lyse infected macrophage and non-macrophage cells (99). This may serve to lyse heavily laden macrophages, releasing mycobacteria which may be engulfed by greater numbers of activated macrophages with stronger bactericidal activity (7). However, excessive cytolytic activity causes detrimental host tissue damage and must be regulated by immunosuppressive cytokines (27). The importance of CD4 + T cells in the resolution of disease is exemplified in HIV infected individuals. The severity of infection correlates strongly with the depletion of CD4 + T cells since macrophages remain unactivated and a strong Thl-type response is not initiated (70). Further evidence for the importance of a T cell response is that mice with a disruption in the M H C class II gene or the P chain of the T cell receptor become highly susceptible even to the avirulent M. bovisBCG(ll). CD8 + cytotoxic T cells, like CD4 + T cells are able to lyse infected cells. CD8 + T cells recognize M H C class I molecules, suggesting that they may lyse cells with M. tuberculosis that have escaped the phagosome into the cytoplasm (15, 85, 93). CD8 + T cells can transfer immunity to susceptible mice (97, 98) and depletion of these cells renders mice susceptible to infection (44, 101). The role of CD8 + T cells in humans though, is uncertain. M. tuberculosis reactive CD8 + T 12 cells have only been recently isolated (145) which suggests that they may have a role in protection. However, CD8 + T cells do not aggregate at the site of infection (6) and cytotoxic activity at the site of infection was attributed to CD4 + rather than CD8 + T cells (82). In addition, unlike CD4 + T cells, CD8 + T cell numbers do not correlate with disease in HIV + individuals (70), hence the extent to which CD8 + T cells contribute to protection against M. tuberculosis in humans remains to be clarified. It is possible that due to the limited extent of M. tuberculosis escaping the phagosomes, CD8 + T cells do not play a prominent role in humans. However, the greater number of mycobacteria reactive CD8 + T cells in mice suggests that the escape of mycobacteria in mice may be more common than in humans. It would be interesting to investigate whether the frequency of escape is higher in mice and the reasons behind this phenomenon, considering that the entry of bacilli into the cytoplasm has been observed in both humans and murine macrophages in vitro (85). 2. Immunoprotective Thl-type response in resistance to tuberculosis The CD4 + lymphocyte is the primary effector cell which orchestrates the host immune response in tuberculosis(15). CD4 + T cells may be classified into a spectrum of functional subsets based on their cytokine secretion profiles ranging from Thl to Th2 cells (74). These subsets were first identified in mice (88) and later in humans (108), where it was observed that certain antigens led to a polarized Thl-type or Th2-type response. CD4 + Thl-type cells are characterized by secretion of IFN-y and IL-2 which mediate a predominantly CMI response, while Th2-type cells produce IL-4, IL-5, IL-6, IL-10, and IL-13 which are responsible for initiating a humoral immune response (89). 13 The resistance to tuberculosis requires the differentiation of CD4 + T cells towards a T h l -type response. Because the development of one of the subsets inhibits the other, it is important to establish the correct response initially (125). It has been shown that a Thl-type CMI response is effective against M. tuberculosis while a Th2-type humoral immune response is not (18, 51, 116, 136). The inability to mount an appropriate response to mycobacteria is believed to be a factor in disease as the production of cytokines typically seen in a Thl-type response correlates inversely with the severity of tuberculosis. The production of Thl-type cytokines in response to M. tuberculosis or purified protein derivative (PPD) is greatest in healthy tuberculin reactors followed by H I V TB patients, and lowest in HIV + TB patients (95, 139, 144, 151, 152). In addition, mRNA levels and secretion of IL-1, IFN-y, and TNF-a are higher in PPD positive healthy individuals in comparison to PPD positive tuberculosis patients (69, 116). However, IL-12 is tknown to be the most critical cytokine required to initiate a Thl response and this is supported by IFN-y (22, 45, 130). IL-12 is released by infected macrophages and activates N K cells and T cells to produce IFN-y (25, 48, 91, 150). IL-12 also acts synergistically with TNF-a to activate y/8 T cells which increases their expression of TNF-a receptors and IFN-y (142). In vitro evidence suggests that IL-12 alone is able to direct a CD4 + response towards a Thl-type phenotype (96, 123). IFN-y knockout mice treated with IL-12 were able to generate a Thl-type response (148). However, these mice still succumbed to infection with M. tuberculosis, which confirms the additional requirement for IFN-y. IFN-y helps maintain a Thl-type response by forming a positive feedback loop. In response to IL-12, N K and Thl-type cells release IFN-y, which augments IL-12 production (18) and may increase expression of IL-12 receptors (146). HIV + individuals are unable to mount a Thl-type response as their IL-12 levels 14 are low. IL-12 expression may however, be restored with IFN-y priming, giving further evidence of this positive feedback system (58). HIV" tuberculosis patients have normal IL-12 levels (150), but may still show a decreased Thl-type response. This lowered Thl-type response may therefore be attributed to other factors. For example, the expression of cytokine receptors such as for IL-12 may be reduced (51), TB patients may have T cell signal transduction defects that are inherent or caused by M. tuberculosis infection (51), or expression of immunosuppressive cytokines or Th2-type cytokines may be elevated (18, 116, 136). 3. Immunosuppressive Th2-type response in susceptibility to tuberculosis M. tuberculosis infection induces macrophages to produce IL-10 (2, 129), a central mediator of the reduced IFN-y response by T cells (43). Infected cells treated with monoclonal antibodies to IL-10 restore IFN-y production (151) and indirectly enhance IL-12 production (51). It has also been suggested that IL-10 does not have any effects on T cell cytokine secretion but acts directly on macrophages by downregulating macrophage antimicrobial functions activated by IFN-y (92). IL-10 does not promote a Th2-type response as was once believed (92) but suppresses the Thl-type response (43) hence promoting pathogen survival. In addition, IL-10 overcomes the inhibitory effects of IFN-y on the development of a Th2-type response. Another factor which inhibits a protective Thl-type response is the secretion of TGF-P by monocytes. Macrophages and granulomatous lesions of patients contain enhanced amounts of TGF-P, and PPD as well as L A M induce its expression (24, 138, 140). TGF-P reverses the effects of IFN-y and TNF-a by lowering the generation of reactive oxygen intermediates by macrophages and by decreasing T cell proliferation (36, 141). TGF-P has also been implicated in 15 inhibiting the expression of IFN-y, TNF-a, IL-1, and IL-6, and decreasing the cytotoxic activity of CD8 + T cells and N K cells (64, 103). By inducing the expression of TGF-P, M. tuberculosis is able to attenuate macrophage antimicrobial functions, thereby promoting its own proliferation (64). With the inhibitory effects of IL-10 and TGF-P on the Thl-type response, Th2-type cytokines may be secreted, further weakening the protective response. However, there exists many discrepancies in this area of research which have not been resolved. Several groups have observed that a Th2-type response correlates with disease (116, 117, 136), while other groups have failed to observe this correlation (81, 151, 152). These differences may be a result of inherent differences in individuals, such as their responses to infection, as well as inconsistencies between experimental procedures Although the model of tuberculosis being caused by an inappropriate Th2-type response is attractive, it may only be the case in certain individuals. The cytokine profile of tuberculosis patients versus healthy tuberculin reactors are different and this suggests that the development of a Th2-type response may lead to disease. PBMCs isolated from tuberculosis patients and stimulated with PPD respond with decreased T cell proliferation, higher levels of IL-4, and lower levels of IL-2 and IFN-y compared to healthy tuberculin reactors (116, 117, 136). IFN-y secretion was found to be decreased (69, 116), indicating that tuberculosis patients may have deficient IFN-y production and/or enhanced IL-4 production. However, Surcel and coworkers (1994) saw no differences in IFN-y levels between tuberculosis patients and tuberculin reactors, suggesting that IL-4 is overexpressed rather than IFN-y being underexpressed. In either case, the outcome is an augmented humoral response with 16 higher levels of IgE and IgG against M. tuberculosis in patients (110), while a more prominent cellular immune response is observed in healthy controls (116). In contrast, other groups have failed to observe a Th2-type response or elevated levels of IL-4 production. Early studies were performed on PBMCs from only healthy PPD + individuals and the sample sizes were very small (5, 12, 32), which may explain the discrepancy. IL-4 was detected in some T cell clones stimulated with PPD and the clones did not correlate to disease in the PPD + individuals (12); but all the clones were derived from two individuals which questions the statistical significance of the results. More recent studies in which cytokine secretion patterns were analyzed in tuberculosis patients versus PPD + healthy controls also failed to show differences in IL-4 production in PBMCs or in the lymph nodes (81, 151, 152). Even with the selective expansion of Th2-type cells by incubation of PBMCs with IL-4 and M. tuberculosis did not yield any differences between patients and controls (81). The same study also found decreased IFN-y proliferation by PBMCs from TB patients but no differences in IFN-y production. These discrepancies may be due to several different reasons. Preculturing PBMCs with mycobacterial antigens in vitro may be unreliable as N K and Thl-type cells secrete IFN-y which would downregulate Th2-type cytokines (110). Also, different culture conditions may promote or prevent specific cytokine production, and different interactions between individuals and M. tuberculosis may alter the cytokine profile. If a Th2-type profile does develop, the cytokines observed may also depend largely on the stage of infection of the individual when the PBMCs are taken. PBMCs taken early would produce Thl-type cytokines while those taken late in disease would produce Th2-type cytokines. Thus, it is not clear whether tuberculosis is promoted 17 by the mounting of a Th2-type response, an inadequate Thl-type response, or a combination of both. The secretion of Th2-type cytokines may actually be promoted by the activation of macrophages (110). IFN-y released by T cells and N K cells causes macrophages to express the enzyme, 1-a-hydroxylase, which converts 25(OH)-vitamin-D3 to calcitriol (109, 111). Calcitriol, in addition to activating macrophages, also inhibits the synthesis of the Thl-type cytokines IFN-y and IL-2, and promotes secretion of the Th2-type cytokines, IL-4 and IL-5 (28, 105). This in combination with increased glucocorticoid concentrations may possibly favour a switch from a T h l - to a Th2-type response (110). Glucocorticoids are released to control the toxic effects of TNF-a and IL-1 (9) by reducing macrophage activation and Thl-type activity (28). It may be possible that the switch from T h l - to Th2-type is meant to downregulate or prevent an overactive Thl-type response to minimize tissue damage. This suggests that timing may be crucial as a switch occurring too early would allow M. tuberculosis to disseminate, whereas a switch too late would cause excessive necrosis (27). 4. Immunopathology of tuberculosis An appropriate response to infection is a balance between inflammatory cytokines responsible for containing and eliminating the pathogen, and immunosuppressive cytokines that limit the extent of inflammation and necrosis. Tissue damaging responses which are part of the delayed type hypersensitivity (DTH) reaction are necessary to kill infected non-activated macrophages which otherwise permit bacterial multiplication (26). This inflammatory response 18 requires TNF-a, IFN-y, as well as calcitriol to activate macrophages, and these same mediators are also responsible for the pathogenesis of tuberculosis. TNF-a is produced by macrophages in response to infection by M. tuberculosis and is essential for the formation of a granuloma to contain the infection (75). The importance of TNF-a is illustrated in mycobacteria-infected mice treated with anti-TNF-a antibodies, which cause dissemination of bacteria and death of the animal (75). However, TNF-a is also lethal to infected macrophages as infection renders macrophages TNF-a sensitive (41, 42). Only infected tissue is damaged in response to TNF-a because TNF-a synergizes with bacterial products to produce cytotoxic effects (112). In a localized infection, the selective action of TNF-a is beneficial, but in chronic disseminated'infections, the selectivity is lost and TNF-a becomes detrimental to the host. Tuberculosis patients experience fever, weight loss, and tissue damage as a result of TNF-a and IL-1 (10, 34). Evidence of the negative effects are demonstrated by relief of tuberculosis symptoms following treatment with thalidomide, which lowers TNF-a levels (110). In addition, the production of calcitriol as mentioned above promotes a Th2-type response which may further aggravate the effects of TNF-a (110). Therefore, the same cytokine that contains the infection may cause greater damage than the infection itself, if it is not controlled. Though the predominance of a Th2-type response in tuberculosis has still not been resolved, experiments with M. vaccae suggests that TNF-a in the presence of a Th2-type response is pathogenic. A low dose of M. vaccae in mice initiates a Thl-type response and the administration of TNF-a has no deleterious effects in these animals (62). However, with a high dose of M. vaccae, both a Thl and Th2-type responses are detected, and the mice become sensitive to TNF-a (62). Although other changes caused by the increased dose, in parallel to the 19 development of a Th2-type response, may be responsible for the TNF-a sensitivity, this explanation is consistent with other disease models such as schistosomiasis (54). Both diseases require Thl-type and Th2-type responses, and the presence of TNF-a for disease (110). In addition, this fits the model of late stage disease being accompanied by Th2-type cytokines and excessive necrosis. In most individuals infected with M. tuberculosis, a balance is achieved between the necrotic and immunosuppressive effects of TNF-a leading to the resolution of disease. However, in some individuals, there is an imbalance, possibly because of a premature Th2-type response, which not only causes incomplete destruction of the bacteria, but also detrimental tissue damage. D. FOCUS Mycobacterium tuberculosis infects mononuclear phagocytes and manifests disease by triggering a strong delayed type hypersensitivity response which is detrimental to the host. In most healthy individuals, infection is resolved; however, the bacilli possess many evasion strategies which may allow it under certain conditions to survive and replicate within the harsh environment of the macrophage. As the host depends heavily on the macrophage to eliminate mycobacteria, it may be advantageous for the bacteria to infect macrophages and attempt to disarm its arsenal. The L A M coating on M. tuberculosis protects it from the oxidative burst and stimulates the secretion of TNF-a which promotes tissue destruction and provides the bacteria with an opportunity to infect and replicate in more cells. L A M also stimulates the release of IL-10 which may suppress the Thl-type CMI response prematurely, before all the mycobacteria are contained, and may also enable a Th2-type response to develop. 20 The resolution of infection involves the balance between a CMI response, which causes damage, with an immunosuppressive response; M. tuberculosis attempts to disrupt this balance within the macrophage by altering the proper immune response. In this research project, macrophage gene expression, which is altered by mycobacteria infection, is investigated. Changes in macrophage cytokine production and antimicrobial function have already been observed. Thus in order to identify additional changes in the physiology of infected macrophages which compromise the host response, gene expression studies have been initiated. The PCR-based method of differential display was used for comparing non-infected and M. tuberculosis infected macrophage mRNA populations (80). mRNA from non-infected and infected macrophages was reverse transcribed to allow for the subsequent amplification of cDNA. Various 5' random lOmers generated based on the GC content of the mRNA, and a 3' oligodT,,G to restrict the products to one-quarter of the population of products were used to create the final PCR products. Restricting the population in this manner allowed bands to be distinguished from one another following electrophoresis. A low annealing temperature (40°C) was also used during PCR amplification along with a large number of cycles (40 cycles), which allowed for the amplification of a range of products. After electrophoresis of the PCR products through a 6% denaturing poly aery lamide gel, the banding patterns of the non-infected and infected macrophage products were compared and differences were attributed to changes in mRNA levels. The differential display technique alone gave rise to many PCR products, common to both non-infected and infected cells, and these products often co-migrated with the modulated products. To eliminate these common bands, a unique system was developed which incorporated subtractive hybridization using latex beads (56, 57) followed by differential display (80). Prior to f 21 differential display, R N A isolated from infected macrophages was subtracted with a cDNA library derived from non-infected macrophages, covalently attached to latex beads, such that the mRNA population obtained should be enriched for infection-specific induced messages (Figure 2). Likewise, the subtraction of non-infected macrophage R N A with infected macrophage cDNA should yield mRNA suppressed by infection. The isolated cDNAs were then sequenced to ascertain their identity and function. The expression of these genes was quantitated in mRNA isolated from non-infected and infected macrophages. Because avirulent and virulent strains of M. tuberculosis mediate different effects on the host, differences in gene expression were also examined between infection with the two strains. By better understanding changes in macrophages caused by mycobacteria infection, it may be possible to develop treatments and drugs to reverse the harmful effects of M. tuberculosis. 22 II. MATERIALS AND METHODS A. Cell types and growth conditions 1. Monocytes The human monocyte cell line, THP-1 (ATCC TIB 202), was grown in cell culture medium RPMI 1640 (Gibco B R L Life Technologies) supplemented with 10% FCS (Hyclone Laboratories), 100 U/mL penicillin G sodium, 100 iig/mL streptomycin sulphate, and 2x10~5 M 2-mercaptoethanol (BDH). Cultures were maintained at densities between 2x l0 5 to l x l O 6 cells/mL at 37°C , 90% humidity, and 5% C 0 2 . Differentiation of THP-1 monocytes into macrophages was performed by addition of phenol 12-myristate 13-acetate (PMA) (Sigma) at 10 ng/mL to mid-logarithmic stage cells at 5xl0 5 cells/mL. 50 mL aliquots of cells were transferred to 75 cm 2 tissue culture flasks (Falcon) and incubated in the conditions above for 24 hours. Human monocytes were obtained from fresh whole blood (Cell Separator Unit, Vancouver General Hospital [VGH]) except for one sample which was obtained from blood which had been stored at 4°C for over 24 hours. In either case, 40 mL of blood was layered on 10 mL of Ficoll-Paque (Pharmacia) and centrifuged at 2000 g, 15 min. to isolate PBMCs located at the interface. PBMCs were pooled and pelleted at 2000 g, 10 min., and washed twice with Hank's Balanced Salt Solution (StemCell Technologies) at 1000 g, 10 min. Monocytes were then isolated by incubating 3xl0 7 PBMC for 2 hours at 37°C, 90% humidity, and 5% C 0 2 in RPMI prepared as above except that 20% FCS was added. Non-adherent cells were removed with three washes in serum-free RPMI and RPMI with 20% FCS was replaced. Cultures were maintained for 5 days to differentiate the monocytes into macrophages at which point infections were performed. 23 2. Bacteria DH5a F ' E. coli cells (Gibco BPvL) were used for transformations and were grown in Luria Broth media (Gibco BRL). Media was supplemented with 100 jag/mL ampicillin (Sigma) to select for transformants. The strains of M. tuberculosis used for infecting macrophages were the Erdman strain (provided by Dr. Neil Reiner, Division of Infectious Disease, Department of Medicine, UBC), and the H37Rv and H37Ra strains (supplied by Dr. Richard Stokes, Department of Pediatrics, B.C. Childrens' Hospital). 3. Infection of macrophages M. tuberculosis was directly added to flasks containing the macrophage monolayers. The infection ratio was 50:1 of mycobacteria to macrophages, except for the macrophage sample isolated from the stored blood which were infected at a 5:1 ratio. To determine the proportions of cells infected, the infected macrophages were stained with Kinyoun Carbol-Fuchsin stain (PML Microbiologicals) for 20 min., destained 3 times with 3% HC1 in ethanol for 30 s., counterstained with a 4.4% Malachite green (BDH) solution for 3 min., and viewed under a microscope. At a 50:1 ratio, greater than 95% of the cells were infected by 24 hrs., and at a 5:1 ratio, the H37Rv and H37Ra had infection rates of 70% and 30%, respectively. Macrophage samples were also treated with dead H37Rv, zymosan, L A M , and IFN-y (prepared by Raymond Lo, laboratory of Dr. Neil Reiner). H37Rv was killed by heating the bacilli for two hours at 60°C. Dead H37Rv were added to the macrophages at the same infection ratio as above and were found to be phagocytosed as efficiently as live H37Rv. Zymosan was 24 added to the macrophages at 1 mg/mL to induce phagocytosis, and L A M isolated from H37Rv was added to cells at a concentration of 1 |j,g/mL. Following washes with RPMI to remove the P M A from the media, differentiated THP-1 macrophages were treated with 151 U/mL IFN-y. B. Molecular Biology Techniques 1. RNA extraction from macrophages R N A was purified from non-infected and infected cells, or from cells treated with dead H37Rv, zymosan, L A M , or IFN-y at 24 hrs. and 48 hrs. post-infection (IFN-y was isolated only at 24 hours post-treatment) using Trizol (Gibco BRL) according to the manufacturer's instructions. RNeasy Midi Kit (Qiagen) was also used to isolate R N A from non-infected and M. tuberculosis Erdman infected macrophages following the protocol for isolating cytoplasmic RNA. RNase-free DNase I (Ambion) treatment removed D N A that may have been co-purified -with the RNA. 2 uL lOx DNase I buffer (Gibco BRL), 1 uL DNase I (2 U/uL), and 1 uX cloned RNase inhibitor (10 U/uL) (Gibco BRL) was added to 5 ug of R N A in 16 uL of dH 2 0 treated with 0.1% diethyl pyrocarbonate (DEPC) (BDH). The reaction was carried out at 37°C for 30 min. and the DNase I was inactivated by heating the mixture to 65°C for 15 min. 2. Subtractive Hybridization mRNA common to non-infected and infected macrophages were removed by a subtractive hybridization procedure adapted from Hara et. al. (Figure 2) (56, 57). 15 \ig of total R N A from non-infected or infected macrophages was bound to Oligotex latex beads (Qiagen) 25 according to the batch protocol, except that the elution step was omitted. The beads were then pelleted and resuspended in 55 u.L of DEPC treated dH 2 0. The mRNA was then reverse transcribed with 1000 U Superscript II (Gibco BRL) in a total volume of 100 uL as described, except that the incubation time was extended to 90 min. OligodT 3 0 covalently bound to the latex beads served as the primer to create a cDNA library bound to the beads. To remove the mRNA template, the reaction was heated to 90°C for 3 min. and immediately cooled on ice. Following centrifugation at maximum speed in a microcentrifuge for 10 min., the supernatant containing the mRNA was discarded. The beads containing the cDNA from non-infected or infected macrophages were washed a further two times with TE buffer (lOmM Tris-HCl, ImM EDTA, pH 7.5) and were then ready to be hybridized with mRNA from infected or non-infected macrophages, respectively. 15 jag of mRNA from infected and non-infected macrophages was purified with the Oligotex spin column protocol and eluted through the spin columns to a total volume of 40 uL. The beads containing the non-infected and infected macrophage cDNA were then resuspended in 20 uL of hybridization buffer (0.5 M NaCl, 0.5% SDS, 5xl0" 3 M EDTA [pH 8.0], 0.02 M Tris [pH 7.5]), 2 uL oligodT1 8 (100 uM), 38 uL DEPC treated H 2 0 , and 40 uL of infected or non-infected macrophage mRNA, respectively. Hybridization was performed at 55°C for 50 min. to allow for the binding of the common mRNA to the cDNA. Following centrifugation for 10 min., the supernatant was stored in another tube while the common mRNA was removed to regenerate the beads. The beads were resuspended in 400 uL of TE buffer, heated to 90°C for 3 min, chilled on ice for 2 min., and pelleted as before. The supernatant was removed and the beads were washed with another 200 uL of TE buffer. The beads were then rehybridized to the supernatant 26 for another round of subtraction. The supernatant after four rounds of subtraction should be enriched for unique mRNA; residual beads were removed with spin-columns provided with the Oligotex Midi Kit. 0.1 volume of 5 M ammonium acetate and 2.5 volumes of 95% ethanol were added to the eluate and precipitation of the mRNA was carried out at -70°C overnight. After centrifugation at maximum speed in a microcentrifuge for 15 min. at 4°C, the mRNA pellet was washed with 70% ethanol and resuspended in 20 uL of DEPC treated dH 2 0. The mRNA was stored at -70°C until it was used for differential display. 27 Figure 2. Subtractive Hybridization using Oligotex (Qiagen) beads to isolate induced genes. A cDNA library is reverse transcribed from the RNA of non-infected THP-1 macrophages using the oligodT3 0 primers covalently attached to the latex beads. The beads containing the cDNA library are used to hybridize to common RNA from infected THP-1 macrophages. By centrifugation, the common transcripts are separated from the unique induced transcripts in the supernatant. The common transcripts are removed from the beads with heat to regenerate the beads such that hybridization may be repeated on the supernatant. After four cycles of subtraction, the supernatant containing the putatively induced genes are used for differential display. Suppressed genes are isolated in the same manner except cDNA from infected macrophages is used to subtract non-infected mRNA. 28 3. Differential display Reverse transcription using Superscript II (Gibco BRL) was performed according to the manufacturer's instructions using 10 uL of the subtracted mRNA. 01igodT uG was used as the primer in order to limit the cDNA library to approximately one-quarter of the set. Differential display was performed as described (80). From the reverse transcription reaction, 2 uL was used per reaction and the primers added were a combination of oligodT,,G and one of four, 5' primers designated as H4 (5' TTT TGG CTC C 3'), H8 (5' TGG T A A A G G G 3'), H15 (5' G A T C C A G T A C 3'), or H26 (5' G A T C T A A G G C 3'). The thermal cycling reaction consisted of 94°C for 5 min. followed by 40 cycles of (94°C, 30 s.; 40°C, 2 min.; 72°C, 30 s.) and then 72°C for 5 min. Each reaction consisted of l xPCR buffer (Perkin Elmer), 1.5 m M MgCl 2 , 2 u M dNTP, 2.5 u M 3' primer, 0.5 u M 5'primer, 1 uCi (a-3 2P) dCTP, 1 U AmpliTaq D N A polymerase (Perkin Elmer). Following electrophoreses on a 6% denaturing polyacrylamide gel, the gel was exposed to X - O M A T RP film (Kodak). By loading the differential display products from subtracted non-infected macrophage mRNA adjacent to subtracted infected macrophage mRNA, differences in banding pattterns could be observed. Bands that were induced or suppressed by infection with M. tuberculosis were then identified. 4. Cloning of isolated products The film was aligned with the dried polyacrylamide gel to allow for the excision of the modulated cDNA PCR products. To isolate the cDNA, each band was soaked in 100 uL of dH 2 0 for 15 min. and boiled for another 15 min. After the removal of the gel and paper fragments, the cDNA was precipitated overnight at -20°C following the addition of 10 uL 3 M ammonium 29 acetate (pH 5.2) and 275 uL 95% ethanol. The cDNA was pelleted by centrifugation at maximum speed in a microcentrifuge for 15 min. at 4°C, and washed with 70% ethanol and resuspended in 20 uL dH 2 0. To amplify the product for cloning, PCR was performed on 2 uL of the isolated cDNA using the same primers and thermal cycling as in the differential display procedure. Amplified products were electrophoresed on 1% agarose gels stained with 0.2 (j.g/mL ethidium bromide and the bands were excised and purified with the Qiaex II Gel Extraction Kit (Qiagen) according to the instructions. The products were quantitated using the saran wrap method of ethidium bromide fluorescent quantitation (115) and ligations were performed as directed with the pGEM-T Vector System (Promega) using a 10:1 ratio of insert to vector. The ligations were transformed into CsCl competent (115) DH5a E. coli cells(Gibco BRL) . Briefly, 5 uL of the ligation reaction was placed with 200 uL of cells on ice for 30 min., heat shocked at 42°C for 30 s., and chilled on ice for 2 min. The cells were allowed to recover at 37°C for 1 hour after the addition of 800 uL of Luria Broth (LB) (Gibco BRL) . The DH5a cells were then plated out on L B plates supplemented with 100 |j.g/mL ampicillin to select for transformants. After an overnight incubation at 37°C, white colonies were selected and tested by PCR for successful ligation. Primers for the T7 transcription site (5' T A A T A C G A C T C A C T A T A G GGC G A 3') and the SP6 transcription site (5' TTT A G G T G A C A C T A T A G A A T A C 3'), on either side of the pGEM-T cloning site were used. The thermal cycling program used was 94°C for 5 min., 30x(94°C, 30 s.; 54°C, 1 min.; 72°C, 30 s.), 72°C for 5 min. Each reaction consisted of l x P C R buffer (Perkin Elmer), 3 m M MgCl 2 , 200 u M dNTP, 1 u M of each primer, 0.3 U AmpliTaq D N A polymerase (Perkin Elmer).The products were run on a 2% agarose gel 30 and products greater than 160 bp (size of the cloning site alone) indicated that the ligation of the differential display products was successful. 5. Sequencing analysis Products successfully cloned into the pGEM-T vector (Promega) were sequenced using the ThermoSequenase Kit (Amersham) or the SequiTherm Excel Cycle Sequencing Kit (Epicentre Technologies), according to the manufacturer's directions. The T7 or the SP6 oligonucleotides (sequence as above) were used to prime the reaction. The sequences obtained were processed through the NCBI-BLAST database (www.ncbi.nlm.nih.gov/cgi-bin/BLAST) to determine i f the isolated cDNAs were from known genes. In addition, the sequences were sent to the M. tuberculosis database (www.sanger.ac.uk/projects/M_tuberculosis/blast_server/shtml) to ensure that the cDNAs were not derived from M. tuberculosis mRNA. 6. Semi-quantitative PCR From the sequences obtained, several sets of primers were constructed for PCR quantitation of the mRNA in macrophages (Table 1). 1-2 \ig of total R N A from non-infected and infected macrophages were reverse transcribed into a cDNA library with Superscript II (Gibco BRL) using oligodT l g as the primer. The RNA template was removed either with the addition of 1 uL of RNase H (1-4 U/mL) (Gibco BRL) , or the addition of RNase I (1 mg/mL) (Sigma) after heating the reaction to 94°C for 5 minutes and placing on ice immediately to denature the RNA. The RNase treatment was carried out at 37°C for 30 minutes and the cDNA was stored at -20° C until it was used. 31 CDNA Name Forward Primer (5'-3') Reverse Primer (5'-3') T anneal (°C) Product Size (bp) H8.3 A C C A C T T A A A T A G A A C A G T G T C A A A T G C C C T A G C A G T A T C 50 162 H8.5 C A T G C A C A G T T T G A T A T T T G G A T C A T C T C A C T G A T T T C A T A C 50 145 N A D H A G G C A C A C T C A T C A C A G C G T A G A T T A G G C G T A G G T A G A A G 50 838 Phox-22 G G T G C C T A C T C C A T T G T G C T T C G C T G C G T T T A T T G C D N W 532 H15.2 T T A T C A C C A C C A C C A C A G T T T A T T A A T G C T C A C C T T G G D N W 113 M l G A T G T A T C T G G T C A A C T C C G G G T C A T T C C T T C A C A A C 45 729 N l C A C C A G T G A A G A T G A C T G C A T C T T C T G A A G T G T G A G C A G G T C T G 54 -550 N2 A G T C A A A G T G G C C A T T A C A T C C A A A C A C T G G A G T A T G C T T C A C C 54 -400 P-actin A T C G T G C G T G A C A T T A A G T G T G T T G G C G T A C A G G T C 54 270 Table 1. Oligonucleotide sequences used for semi-quantitative PCR. Nine pairs of oligonucleotides were designed based on the cDNA sequences obtained. The thermal cycling consisted of 94°C, 5 min., Nx(94°C, 30 s.; T ^ , , 1 min.; 72°C, 30 s.), 72°C, 5 min. where N is the number of amplification cycles and T^^ , is the annealing temperature. For unsuccessful PCR reactions, the annealing temperature is represented by D N W (did not work). The expected product size is also indicated but because N l and N2 were not fully sequenced, approximate sizes of the products are given. PCR was performed on the macrophage cDNA using the different sets of primers (Table 1) with the thermal cycling program and annealing temperature (T a n n e a |) indicated. Each reaction consisted of l x P C R buffer (Perkin Elmer), 3 m M MgCl 2 , 200 u M dNTP, 1 u M of each primer, 0.3 U AmpliTaq D N A polymerase (Perkin Elmer). Because the amplification of products follows a sigmoidal curve and the products are desired at the logarithmic amplification stage for comparative purposes, several identical sets of PCR reactions were prepared and the reactions stopped at various (N) cycles. PCR reactions with the P-actin primers were simultaneously performed using 0.1 diluted macrophage cDNA such that the results could be normalized against the quantity of mRNA in each sample. 32 PCR products were electrophoresed on 2% agarose gels stained with 0.2 fag/mL ethidium bromide. Numerical values were assigned to the bands using the program, ImagePC (Scion Corporation), to assess the level of modulation of the macrophage genes. The technique for analyzing electrophoretic gels was applied as directed in the manual. Briefly, the photographic image of the gel was scanned into a file and the colors inverted, because the program measures the density of black pixels. Identical sized boxes were drawn around each lane and a graph of density versus position was plotted. The background was manually subtracted and the peaks were integrated to obtain a numerical value of the band intensities. 7. Slot Blotting Approximately 100 ng of the isolated differential display products were bound to Hybond N + membrane (Amersham) using the Bio-Dot SF apparatus (Bio-Rad) as instructed. An additional P-actin positive control and a pGEM-T vector negative control were also bound to each membrane. Modifications were made in the buffers used: the binding buffer used to wet the membrane and load the samples was 0.4 M NaOH, 10 mM EDTA (pH 8.0), and the wash buffer was 0.4 M NaOH. Duplicate membranes were made such that one was probed with (a-3 2P) dCTP labeled cDNA from non-infected macrophage R N A and the other from infected macrophage RNA. Labeled cDNA was made by reverse transcription of 1-2 ug of R N A as instructed with Superscript II (Gibco BRL) except that the dCTP in the dNTP mix was replaced with 5 uL of (a-3 2P) dCTP (10 uCi/uL) (Dupont). Free nucleotides were removed by centrifugation of cDNA through BioSpin-6 Chromatography Columns (Bio-Rad) as directed. The labeled cDNA was added to the membranes prehybridized overnight according to the Hybond N + membrane 33 protocol (Amersham). The hybridization of the cDNA to the membrane was performed at 65 °C overnight and the high stringency washes were done as instructed. Following exposure of the membranes to Hyperfilm-MP (Amersham), the intensities of the corresponding bands between the non-infected and infected samples could be compared after normalization with the P-actin control. 8. Northern blot 5 p.g of R N A from non-infected and infected macrophages were electrophoresed on a DEPC treated 1% agarose gel with 0.7 M deionized formaldehyde, 0.05 M MOPS, and lxlO" 3 M EDTA. R N A samples were prepared by the addition of 2 uL lOx MOPS buffer (0.5 M MOPS, 0.01 M EDTA), 3.5 uL 37% deionized formaldehyde, and 10 uL deionized formamide, made up to 20 (J.L with DEPC treated water. The gel was run at 60 V in 1 x MOPS buffer and transferred to Hybond N + membrane (Amersham) (115). Probes were produced by random-priming labeling the cDNA clones amplified by PCR. 25 ng of cDNA in 28uL was combined with 10 u.L of N9 primer (27 O.D./uL) and boiled for 5 min. To it was added 5 uL of lOx Klenow (exo') Random Priming Buffer (USB); 1 u.L of 1 m M dGTP, dATP, dTTP; 5 uL (a-3 2P) dCTP (10 uCi/uL); and 1 uL of Klenow enzyme (5000 U/mL) (USB). The reaction was incubated at 37°C for 10 min. and stopped by the addition of 2 uL 0.5 M EDTA (pH 8.0). The probes were then purified by BioSpin-6 Chromatography Columns (Bio-Rad) as directed, and added to the prehybridized membrane to allow for hybridization overnight at 65°C. The membrane was washed with high stringency according to the Hybond N + protocol and exposed to Hyperfilm-MP (Amersham). 34 III. RESULTS A. Subtractive hybridization and differential display Subtractive hybridizations were performed on four different samples: control macrophage and infected macrophage mRNA obtained at 24 and 48 hours post-infection. The mRNA population obtained was then reverse transcribed and amplified for differential display. Four different 5' lOmer primers were used (H4, H8, H15, H26) with oligodT,,G as the 3' primer for the PCR. For comparison, non-subtracted samples were also amplified. The results for non-subtracted versus subtracted and non-infected versus infected macrophage mRNA populations, using H4, H8, HI 5, and H26 lOmers, are shown on Figures 3-6, respectively. Between the non-subtracted and subtracted samples, the most significant difference was the clarity of the bands, especially for larger PCR fragments. Subtractive hybridization appeared to remove many of the bands common to all four samples, thus allowing detection of other similar sized bands. Differentially expressed bands were identified more easily and, in addition,-new bands, not seen in the non-subtracted lanes, were observed. Subtractive hybridization reduced the number of products so that there would be fewer similar sized products and the increased resolution between larger bands allowed for the isolation of larger sized products. In addition, the elimination of common products permitted the identification of low copy-number genes. 35 H4.1(+) H4.4(+) H4.5(+) Figure 3. Differential display on non-subtracted and subtracted macrophage mRNA using oligodT,,G and H4 primers. Differential display was performed on mRNA from non-subtracted (Lanes 1-4) and subtracted (Lanes 5-8) macrophage mRNA using oligodT n G and H4 (5' TTT TGG CTC C 3') primers. Lanes 1 and 2 are products from non-infected and infected macrophage mRNA, respectively, at 24 hours post-infection. Lanes 3 and 4 are products from non-infected and infected macrophage mRNA, respectively, at 48 hours post-infection. Lanes 5 and 6 are products from non-infected macrophage mRNA subtracted with infected macrophage cDNA, and infected macrophage mRNA subtracted with non-infected macrophage cDNA, respectively, at 24 hours. Lanes 7 and 8 are products from similarly subtracted non-infected and infected macrophage mRNA, respectively, at 48 hours. Bands that were excised are indicated by the small boxes. Beside each box is the name of the isolated clone and whether it is putatively induced (+), or suppressed (-). 36 1 2 3 4 5 6 7 8 H8.1 (+) H8.2 (+) H8.3 (+) H8.4 (+) H8.5 (+) H8.6 (+) H8.7 (+) H8.8 (-) Figure 4. Differential display on non-subtracted and subtracted macrophage mRNA using oligodT,,G and H8 primers. Differential display was performed on mRNA from non-subtracted (Lanes 1-4) and subtracted (Lanes 5-8) macrophage mRNA using oligodT n G and H4 (5' TGG T A A A G G G 3') primers. Lanes 1 and 2 are products from non-infected and infected macrophage mRNA, respectively, at 24 hours post-infection. Lanes 3 and 4 are products from non-infected and infected macrophage mRNA, respectively, at 48 hours post-infection. Lanes 5 and 6 are products from non-infected macrophage mRNA subtracted with infected macrophage cDNA, and infected macrophage mRNA subtracted with non-infected macrophage cDNA, respectively, at 24 hours. Lanes 7 and 8 are products from similarly subtracted non-infected and infected macrophage mRNA, respectively, at 48 hours. Bands that were excised are indicated by the small boxes. Beside each box is the name of the isolated clone and whether it is putatively induced (+), or suppressed (-). H8.9, H8.10, and H 8 . l l were excised below the area of the gel shown and are not shown on the figure. 37 1 2 3 4 5 6 7 8 H15.1 (+) H15.2(+) I H15.3 (+) H15.4(+) H15.5 (-) H15.6 (+) H15.7(+) Figure 5. Differential display on non-subtracted and subtracted macrophage mRNA using oligodTnG and H15 primers. Differential display was performed on mRNA from non-subtracted (Lanes 1-4) and subtracted (Lanes 5-8) macrophage mRNA using oligodT,,G and HI5 (5' GAT C C A G T A C 3') primers. Lanes 1 and 2 are products from non-infected and infected macrophage mRNA, respectively, at 24 hours post-infection. Lanes 3 and 4 are products from non-infected and infected macrophage mRNA, respectively, at 48 hours post-infection. Lanes 5 and 6 are products from non-infected macrophage mRNA subtracted with infected macrophage cDNA, and infected macrophage mRNA subtracted with non-infected macrophage cDNA, respectively, at 24 hours. Lanes 7 and 8 are products from similarly subtracted non-infected and infected macrophage mRNA, respectively, at 48 hours. Bands that were excised are indicated by the small boxes. Beside each box is the name of the isolated clone and whether it is putatively induced (+), or suppressed (-). 38 1 2 3 4 5 6 7 8 H26.1 (+) H26.2 (+) H26.3 (+) H26.4 (+) H26.5 (+) H26.6 (-) H26.7 (-) Figure 6. Differential display on non-subtracted and subtracted macrophage mRNA using oligodTuG and H26 primers. Differential display was performed on mRNA from non-subtracted (Lanes 1-4) and subtracted (Lanes 5-8) macrophage mRNA using oligodT n G and H26 (5' G A T C T A A G G C 3') primers. Lanes 1 and 2 are products from non-infected and infected macrophage mRNA, respectively, at 24 hours post-infection. Lanes 3 and 4 are products from non-infected and infected macrophage mRNA, respectively, at 48 hours post-infection. Lanes 5 and 6 are products from non-infected macrophage mRNA subtracted with infected macrophage cDNA, and infected macrophage mRNA subtracted with non-infected macrophage cDNA, respectively, at 24 hours. Lanes 7 and 8 are products from similarly subtracted non-infected and infected macrophage mRNA, respectively, at 48 hours. Bands that were excised are indicated by the small boxes. Beside each box is the name of the isolated clone and whether it is putatively induced (+), or suppressed (-). 39 B. Identification of modulated cDNAs Several sequences were identified as being modulated by infection (Figures 3-6) and were therefore excised from the gel for cloning and sequencing. The cDNA population represented by approximately 30 individual bands was eluted and each was specifically amplified using the differential display protocol with the same primers used for generating the original bands. Twenty-five products were successfully cloned into the pGEM-T vector (Promega) and sequenced (Figure 7). The sequences were cross-referenced through NCBI -BLAST and M. tuberculosis databases to determine whether any of the identified cDNAs were from known human genes and to ensure that none of the isolated cDNAs were from M. tuberculosis genes. Five of the 25 cDNAs were found to be from known human genes (Table 2) and none matched any of the M. tuberculosis genes sequenced thus far. Seven cDNAs matched human cDNA sequences which had no assigned function, and the remaining 13 isolated cDNAs were novel sequences. Of the known human genes identified, p22-phox (35) and N A D H ubiquinone oxidoreductase chain 2 (19), which encode products involved in the macrophage oxidative burst. The other genes encoded an anti-oxidant enzyme AOE 37-2, human protein phosphatase y (94), and B4B, which is a possible growth arrest gene (113). 40 H4.1 (T7) 1 c c t g a a t a t c g t a a t g a g c t c a t a g a t t a t t g t t t g c a t t g a g c c a t g t a 51 g t c a c t a t t a a t t g t t t a g a t g c t c a t a t t g t c t c a g g t t a a t a a g t a t c 101 t c t t c a a g t t g a c t c c c a t g g c c t t t t g a c g t g a t c c t g t t g g a c t t g g a 151 t g g c t t c c t t g c t t t c t g g c aaaaaaaaaa a H4.3(T7) 1 t t t t t g g a t a t g g a g a t g c a gcagcacaat g c a a t g c t g t t t c a t g t g t t 51 t g a g g a t g c t t g a a a t t c a c c a t t t c c a g a g a g a g a t g t t t t t t g a t t c g 101 a g t a a c a t c a g c t t c t c g t g c a g c t t g c a g caacgagtgg c c t t t a a a t t 151 c a t a t g c t a a t c t t t c t t t t a a c t g t g g t g tgggagccaa aaaa H4.4 (T7) 1 g g c t c c c a g a a t a a g a c t t t g a a a a t a a t a t a a t a c t c t a c g c t a g g c a t 51 g g t g g c c c a t g c t t g t a a t c c c a g c a c t t t aggaggctgt ggcagaaaga 101 t c a c t g g a g g c c a g g a a t t t gagatcagcc agagcaacat a g t g a g a c c c 151 c c a t c t c t a c aaaaaa H4.5(T7) 1 c a t a t g g t c g actgaggcgg c g a c t a g t g a t t t t t t g g c t c ccagcagac 51 a c a c t g t a g g tgaagaacag a t a t t t a c t g aatgaaaaca g a c t g a g t g c 101 c t t a a a t a c g t c t t c c c a c t - t c a t c a t g a c , actacaaaaa- aaaaa H8.1.1 (T7) 1 t c t c g t g g c a t t t g g c t t c t t t t t c t c t g c a g g c t t t a a a a t c t g a a a a g 51 a a c a c a t t a g t g t t t c a t c c a c a c t c a t c a t g g c a c c t g c a t t g t c a a a c 101 t c t c c g c a a t a a t t g g g c g c agaaaacaga g t g a c c a a c t g c c t c t t t g c 151 aaaaaaaaaa a H8.1.2(T7) 1 ggagaaatga g t t g a t g c t g t a t c g t g t g t g t g t g t g t g t g t g t g t g t g t 51 g t g t g t g t g t g t g t t t t t a a t g g a g t g t t g c c t t g a a t g a a t c a c t g g g a 10.1 a g c c a g c c a t ggtaagggct ggtaagggct g g t g a g g t t g gggagaaagg 151 a a g a g c t t t a t g t t t c t c t g t t g t t t g g a c c c t a c t t g g c aaaaa H8.2(T7) 1 g a g a t t c a g a t g a c t a t c t a t g a c g t g a t t a g a a g a t g t a g t a t c a c a c a 51 t c t c a a c a t g t a t t g t a c t c t t t t g t t g c t a a g g t a g c t c c t g t a g t t a t 101 g c a t g t t g c a a a a a t t t a c a g a t g t t t t a a a a g t g g t t a a c a t c t g t t t t 151 t t t t a a a c g t t a g c c a a a a a aaaaaa 41 H8.3/4(T7) 1 t t a c a c a t g c a t a c a a a c c a g t g t t a a g a a a g t a t t c a c c a t c a t t t a a a 51 c a a a t a a c c a c t t a a a t a g a a c a g t g t c t g c a a t t t t a t c t g t a t a a a a a 101 t a a g a t a c a t t t t t a c a g a a t t c a c g c t c c a g t t c t t a t a g c a a t a a a c a 151 a t a c a c a a c t a t a a t a a a g t a c a a t t g a a c c t g a c c a t g g t t t t t a a t t a 200 g a t a c t g c t a g g g c a t t t t a a t g t g c a a a a aaaa H8.5 (T7) 1 t t a g a a g t t g c t c c a g t g a t a t c a g a t c a t c t c a c t g a t t t c a t a c t c t t 51 g a c a g t a t g t a c a c a t t g a t t t t a c a c a t a t c t g g c t c t a a a c a a g t a t t 101 t a c t g a a t t g a a t a a t t g a a c t t t t a t g a t t g a a g a g c a t c a t t c a a a t c 151 a a a t a t c a a a c t g t g c a t g a a t a t g c t g c a g c t t t c c c c a aaaaaaaaaa H8.6 (T7) 1 ggtaaagggc a c a a a c a t a g g g t t a g a t t g aaggagtaaa t t c a a t g t t t 51 aataacagag t g g g g t g a c t a t a g t c a a c a g a a g t g t a t t g t a c t c a g g t 101 gatggacaca c c a a a t a c c c t g a c t t g a c c a a t a t g c a t t a t a t a c a t g t 151 a a c a a a a t t t c a c a t g t a c c c c a t a a a t t t c t a c a a a a a a H8.7 (T7) 1 t t t c c g t t g ' c c t t g a g a g t t c c c t g t t a c t c c c a c t t c t c c c t a t g g t g a 51 c c t t t t a t a g a a gaccataa a t a a a c c a c t acagcccaaa aaaaaaaa H8.8 (T7) 1 c g c a c t a g t g a t t g g t a a g g c c a g a t a g t a a a t a t t t c a g g c t t g c a g a c 51 c a a a t g a t c t g t c a c a a c t a c t c a a c t c t g c c g t t g t a a t accaaagcag 101 c g a t g g a t a a t a c t g a c t g t g t t c c a g t a a a a c t t t a t t t acaaaaaaaa 151 aaa H8.9 (T7) 1 a t t t g g t a a a g g g c t a a t a t c c a g a a t c t a c a a t g a a c t c a a a c a a a t c t 51 acaaaaaaaa aaa H15.1 (T7) 1 c c t g c c c c c g c t a a c c g c t t t t t g c c a a a t g g g c c a t t a t c g a a g a a t t c 51 acaaaaaaca a t a g c c t c a t c a t c c c c a c c a t c a t a g c c a c c a t c a c c c t 101 c c t t a a c c t c t a c t t c t a c c t a c g c c t a a t c t a c t c c a c c t c a a t c a c a c 151 t a c t c c c c a a aaaaaaa H15.2(T7) 1 c c c c g t t t c g a t a g c a t c a t g t t a t c a c c a c c a c c a c a g c c t a t t t c a c a 51 g a c a c a a a t a g t t t t t c t g t t t c c c a a t a a t t c t t t a a a t c c t t g t a t t g 101 t a a t t g c a t g gccaccaagg t g a g c a t t a a t a a a a t c c a g c t c c t t c a t g 151 t t c a a a a a a a 42 H15.3 (T7) 1 g a t c c a g t a c t a g a g a t t a g g g c a c t t c a a a g c a t t g a a a a a a a t c t a g t 51 g a t a c t t a c t t t c t t a g a c a a g t a g t t c t t . a g t t a a c c a c c a a t g g a a c t 101 g g g t t c a t t c tgaat-cctga g a g g a g c t t c c t c g t g c c a c c a g t g t c t g g 151 cc H15.4 (T7) 1 t t c t a g t t t c t t a c g t g t a c acaggcacat a t a a a t c t g g g t t t a c t t g t 51 t t t a t t t c t t c a c a g a agtc g g c a t a t t a t a t t t a c t g g t t t a t a c a t t g 101 c t t t c t c a t t a a g a a c a t a t g t t g a c c c t t ccaaaaaaaa aaa H15.5(T7) 1 g a t c c a g t a c a c t g a c a a a c acggagaagt c t g c c c t g c t ggctggaaac 51 c t g g t a g t g a a a c a a t a a t c ccagatggag c t g g a a a g c t g a a g t a t t t c 101 g a t a a a c t g a a t t g a g a a a t a c t t c t t c a a g t t a t g a t g c t t g a a a g t t c 151 t c a a t a a a g t t c a c g g t t t c a t t a c a a a a a aaaaa H15.6/7 (T7) 1 t g a t c c a g t a caccagcaga g g a a a c t t a t a a c c t c g g g a g g c a g g t c c t 51 t c c c c t c a g t g c g g t c a c a t a c t t c c a g a a gagcggacca g g g c t g c t g c 101 c a g c a c c t g c c a c t c a g a g c g c c t c t g t c g c t g g g a c c c t t c a g a a c t c t 151 c t t t g c t c a c a a g t t a c c a a aaaaaaaaa H26.1 (T7) 1 c a t c a g t g c t t c c c a c t t a c c g t a t t t g t c t g t c g g t g g c c c c a t a t g g a 51 a a c c c t g c g t g t c t g t t g g c a t a a t a g t t t a c a a a t g g t t t t t t c a g t c c 101 t a t c c a a a t t t a t t g a a c c a acaaaaaaaa aaa H26.2 (T7) 1 t t t g g g a g t g agcagtaaaa aacaaacaaa. c c c a t g c a g g g c t g t t g t g c 51 t g t g g g a a a t c a g a t g t g t t c a c t g c c a t a a g t c t t c a g t gcggccaaac 101 t t a a a a a c c a g c c c t c c a a a aaagatgaag g c a t t c t g g a a a a a t a c a g c 151 a a t t c c a g g g t a t a c t a t g t c t t t t t c t t t a g c a a c a t a a c c t c c a a a a a 201 aaaaaa H26.3 (T7) 1 aaaacaaaca aacaaacccc c c a a a c c c a a a c c t g t a t c a g t g a t t g t t a 51 gaaagccaat c c t c t c t t t c caagacagct a a t c a a t a t a c t c a t t a c t t 101 t t c a a c t a c t c c c t g c t a t a aaaaaaatac a t a a a a c c t g t t t g g a a c a t 151 c a a t a t g c a a a t a c a t t c c a t g a t t t c c a a aaaaaaaaa H26.4 (T7) 1 t t a g a t t t t g c t t a t t a t t a t t a t t t t t t t g a a c t g t g g t c t t g t t c a c a 51 t g a a a g c c t t a g t t t c t c a a c t c a a t a t c a ggggactgat a t t t g a t t t t 101 g c c t a c t c c c t a t a a t a c t t c c t c c t a g c c a a t t g c c a t t ccaaaaaaaa 151 aaa 43 H26.5 (T7) 1 g a a a a t a a c t t t t a a t a a c a a t a t g a g c a a a a t g a t t a a a t a t t g a a t c t 51 gtagaaaagt c c a g a a a t a c a t t g a t g t g g c a t t a c a a c a t g a t a g t g g a 101 c t t t c c a a g c a t t t a t t a c c g t a a a a t a t t t t t g a a a t a c t a a t c t g c a a 151 aaaaaaaaa H26.6 (T7) 1 g a t c t a a g g c c a a t c t t a g t t a a a a g t t g t a a a t g c a a c c a t a g g c t a c a 51 t g t t a c a t t g t a t t t a a t t a a a a a c c t t a a gaggaaaggc a a t a g a g c t t 101 g t a a c a g c t a t t a c c c t t g g t t g t a a a g g a gaaggttgga g a t t a t t t g t 151 a t a c c a a c t a c t t a a g t t t a caaaaa M l (T7) 1 t a a g g t g a c t caaaggaaag a c g a c t c a a c c t g g a a c t c a g a g g t c a t g a t 51 gagggtc Nl (T7 & SP6) 1 a c c a c t t a a a t a g a a c a g t g t c c t t a c a c c a g t g a a g a t g a g t g c a t c a a 51 a g a c t t t g a t gaaaaggagt a t c a g g a g t t g a a t g a g c t g cagaagaagt 101 t a a a t a t t a a c a t t t c c t g g a ccataagag a c c t t t g a t t a a g g t t t g g g 151 a a t t a g c a g a g a t g t g a t g c agctagagat g a a t t g a g g c g a t g a t c cacagacaca aggccacagt t t a t c t g t t c a g c g c c t c a c 201 gaa a t c c a a a g t g a c a t c c c t g c a c a c t g g a g t g a t a t g a agcagcagaa 151 t t t c t g t g t g g t g g a g c t g c t g c c t a g t g a t c c t g a g t a c a a c a c g g t g g 101 ca a g c a a g t t t a a t c a g a c c t g c t c a c a c t t c a g a a t a g a g a a g a t t g a g 51 aggatccaga a t c c a g a t c t c t g g a a t a g c t a c c a g g c a a aaaaaaaaaa 1 N2 (T7 & SP6) 1 c a t g c a c a g t t t g a t a t t t g c a c a a a a g t c a t t t a a a a a a a t c t g a g t a a 51 t t g t c a a a t a t t a a a a g a a a g a t a t t c t t c c t g t a a g a a t a c a g t t t t t a 101 g t c a a a g t g g c c a t t a c a t c c t c t t t t t a a t t t a c a t a a t a c a g a t a c t t 151 g a g a a a g t t g t t g t g g t g t t g t a t g c c t t g a a a t t c t t t t t a t t g g t g c t • 201 a t a t t g t a a c a a t t a t t t t t a t g c a t g t a t t t a t c a c t g t a g a g t c t g a g a t a t t t g c a c t c a t g t t a t g g 201 t t a a t g a a t a t t t t g t a a a a gtaaaagcaa c a a a t t t a t a a a t t g a t t a t 151 t t g a a a c t t t a c a a c a c a a t t g c a t c c c a a a t a c a a a t t g t a t t g c t t a t 101 t c a t t a t a g c t a t t c g t c c t g t a a t c t g t t t c t a g g t g a a g c a t a c t c c a 51 g t g t t t t a g g g g t t t t g a a a a t a a a t a t t t a a a t t t c a c a gtcaaaaaaa 1 Figure 7. Sequences of isolated cDNA clones. 25 sequences isolated by subtractive hybridization and differential display were sequenced and M l , N l , and N2 isolated by other means were sequenced using Thermo Sequenase Kit (Amersham) or the SequiTherm Excel Cycle Sequencing Kit (Epicentre Technologies), as directed. The primers used (T7 or SP6) are shown in brackets beside the name of the sequence. 44 cDNA Identity ^-X^ • Modulation • I-I4.4 p22-phox Increased H15.1 NADH-ubiquinone oxidoreductase chain 2 Increased H15.5 Antioxidant enzyme A O E 37-2 Decreased H8.1.2 Human protein phosphatase y Increased H15.6/7 B4B (Possible growth arrest gene) Increased CL-10 (Squamous cell differentiation gene) H8.1.1 cDNA match Increased H8.3 cDNA match Increased H8.7 cDNA match Increased H8.9 5' flanking region of several genes Increased H15.3 cDNA match Increased H26.1 cDNA match Increased H26.2 cDNA match Increased 13 others No known significant matches Table 2. Macrophage cDNAs isolated by subtractive hybridization and differential display. cDNAs isolated by subtractive hybridization and differential display were cross-referenced through NCBI-BLAST and M. tuberculosis databases. No cDNA matched any known M. tuberculosis sequences, and cDNAs matching human sequences are indicated. The apparent modulation of the cDNA is also shown. C. Slot blotting to measure the expression of the isolated cDNA transcripts Differential display is non-quantitative as the band intensities do not necessarily correlate to the amount of mRNA target (78). In order to avoid problems with PCR artifacts, most bands that were selected for excision were present in the infected or non-infected lanes and absent in the respective non-infected or infected lanes. To confirm whether these cDNAs were actually modulated by infection with M. tuberculosis, it was necessary to quantitate and compare the specific products from R N A of non-infected and infected macrophages. A reverse Northern blot was performed on each cDNA isolated. The 25 cloned fragments were re-amplified and bound to duplicate membranes using a slot blotter (Bio-Rad). Then (32P-ot) dCTP labeled cDNAs of non-infected and infected macrophages were generated by reverse 45 transcription from R N A and hybridized to the membranes. Since the fragments bound to the membranes were in excess, the intensity of the bands represent the quantity of the specified mRNA in either non-infected or infected cells. By normalizing the bands against a p-actin control, to adjust for differences in total cDNA produced, the corresponding bands from the two membranes could be compared qualitatively (Figure 8). Several bands appeared to be modulated, including H8.5 and H8.6; however, these results obtained were not reproducible. The intensities seemed to depend more on the quantity of the cloned fragments bound to the membrane than the level of expression of the genes in macrophages. In addition, when high stringency washes were performed, many of the signals were eliminated, suggesting that the signals observed were a result of non-specific binding. The transcripts were possibly expressed at low levels such that the specific activities of the probes were too low to detect. 46 H4.1 H8.3 H15.1 H26.1 B H4.1 H8.3 H15.1 H26.1 H4.3 H8.4 H15.2 H26.2 H4.4 H4.5 H8.1.1 H8.1.2 H8.5 H8.6 H8.7 H8.9 H15.3 H15.4 H15.5 H15.6 H26.3 H26.4 B-actin p G E M - T H4.3 H4.4 H4.5 H8.1.1 H8.1.2 H8.4 H8.5 H8.6 H8.7 H8.9 H15.2 H15.3 H15.4 H15.5 H15.6 H26.2 H26.3 H26.4 B-actin p G E M - T Figure 8. Slot blot of putatively modulated cDNAs isolated by subtractive hybridization and differential display. cDNA clones were amplified by PCR and the fragments were bound to a Hybond N + membrane (Amersham) using a slot blotter (Bio-Rad). (32P-oc) dCTP labeled cDNA was reverse transcribed from R N A from non-infected (A) and infected (B) THP-1 macrophages, and hybridized to one of the two duplicate membranes. Membranes were then exposed to film for two days and developed. Bands were normalized against the p-actin standard; a negative control of pGEM-T vector (Promega) was also included. 47 D. Quantitation by Northern blotting Northern blots were also performed where macrophage R N A was electrophoresed and transferred to membranes. Probes with high specific activity were generated with random priming labeling of the isolated fragments and then hybridized to the membranes. No signal was obtained after high stringency washes (data not shown). The membrane was reprobed with a (3-actin probe produced in the same manner. Bands were present in each lane (data not shown) indicating that R N A was present and that the genes isolated by differential display were expressed below the level of detection using hybridization techniques. E. Semi-quantitative PCR Since the quantity of the cDNA isolated by differential display was undetectable by the hybridization techniques, attempts were made to quantify by PCR, a more sensitive and specific assay. Specific primers were constructed based on the sequence of five clones (H8.3, H8.5, N A D H ubiquinone oxidoreductase chain 2, p22 phox, and HI5.2) and PCR was performed on cDNA created from non-infected and infected macrophages at 24 and 48 hours post-infection. The number of cycles in the PCR reactions was titrated to obtain a set of reactions in the logarithmic range of amplification for comparative purposes. Multiple reaction sets containing non-infected and infected macrophage cDNAs were amplified and the PCR was stopped at different cycle numbers for each set. To normalize the data based on the total amount of cDNA in each sample, PCR with p-actin primers was simultaneously performed on the samples and the reactions stopped at identical time-points. The products were electrophoresed on 2% agarose gels stained with ethidium bromide so that the bands could be quantified based on intensity. 48 1. Induction of H8.3 and H8.5 cDNA by infection; isolation of two new cDNAs The H8.3 cDNA did not match any known genes (Table 2) and when PCR was performed on macrophage cDNA, it was found that H8.3 was expressed both in non-infected and infected macrophages (Figure 9). However, at 25 cycles, levels of the H8.3 PCR product were higher from the infected sample. The ratio of intensities of H8.3 to (5-actin for control samples was 1.92 at 24 hours and 1.51 at 48 hours, and increased to 2.55 and 2.06 in infected samples, respectively. The difference between non-infected and infected sample became less evident with increased cycles because the amount of product began to plateau towards the same level despite differences in the quantity of starting template. Similarly, H8.5 was expressed in both non-infected and infected cells and the ratio of H8.5 to P-actin was significantly higher in the infected samples compared to the controls, at both 25 and 26 cycles of amplification (Figure 10). The results obtained for H8.3 and H8.5 were repeated twice on different sets of THP-1 macrophage RNA, yielding the same result (data not shown). 49 25 cycles 27 cycles 28 cycles CO 60 l_ ir, co u. CO \— to CO i_ in ( H -C s— _C -C Jg s— -C i— -C EH DC -t OO n oc oc Tfr t C N oo ^_ _ , c U o u o u c 0 u *c <-i v. — +^ *3 U a u O u -J C c c r"-> c c .*> c 0 0 c '— o 0 '— c U B U c U c u c (J c U '— 43 00 T3 i> *J — C 300 bp^ 100 bp^ 25 cycles 27 cycles 28 c: C24 124 C48 148 C24 124 C48 148 C24 124 C48 148 p-actin 462 708 418 1335 907 834 197 893 1194 828 484 825 H8.3 885 1802 674 2751 1902 2546 773 3513 2342 2685 1518 2498 H8.3/ P-actin 1.91 2.55 1.51 2.06 2.10 3.05 3.92 3.93 1.96 3.24 3.14 3.03 Figure 9. Semi-quantitative R T - P C R of THP-1 macrophage R N A using primers specific for H8.3. PCR was performed on cDNA reverse transcribed from non-infected and infected macrophage RNA, 24 and 48 hours post-infection. The PCR reactions consisted of 95°C, 5 min. and the specified number of cycles of (95°C, 30 s,; 45°C, 1 min.; and 72°C, 30 s.) The products were electrophoresed in a 2% agarose gel to quantitate the 162 bp H8.3 products normalized against the 270 bp p-actin standards. Numerical values were assigned to each band based on their intensity using the program, ImagePC (Scion Corporation). The ratios of the intensities of the H8.3 bands to the P-actin bands are also given. 50 25 cycles 26 cycles H8.5I 25 c: ^ cles 26 cycles C24 124 C48 148 C24 124 C48 148 p-actin 1566 541 575 622 3416 770 743 1482 118.5 367 746 61 674 270 1475 73 1462 H8.5/p-actin 0.23 1.38 0.11 1.08 0.08 1.92 0.10 0.99 Figure 10. Semi-quantitative RT-PCR of THP-1 macrophage RNA using primers specific for H8.5. PCR was performed on cDNA reverse transcribed from non-infected and infected macrophage RNA, 24 and 48 hours post-infection. The PCR reactions consisted of 95°C, 5 min. and the specified number of cycles of (95°C, 30 s.; 45°C, 1 min.; and 72°C, 30 s.) The products were electrophoresed in a 2% agarose gel to quantitate the 145 bp products normalized against the 270 bp p-actin standards. Numerical values were assigned to each band based on their intensity using the program, ImagePC (Scion Corporation). The ratios of the intensities of the H8.5 bands to the P-actin bands are also given. 51 The second time the reactions were repeated, the cDNA was reverse transcribed from macrophage R N A samples with an oligodT1 8 primer rather than an ol igodT n G primer which theoretically increased the number of cDNAs by four times. The H8.3 product was again increased, although not as significantly as before as an apparent plateau of product was obtained at 30 cycles of amplification. The H8.5 product was very faint and could not be photographed, but the band intensities appeared similar between the control and infected samples at 30 cycles. The increased cDNA levels obtained using the oligodT18 primers also resulted in other products being produced during PCR including a 600 bp band from the H8.3 reaction (Nl fragment) and a 500 bp band from the H8.5 reaction (N2 fragment). Both of these products appeared to be induced by infection (Figure 11). This experiment was repeated in order to rule out the possibility that the bands were artifactual. The N l and the N2 cDNAs were then cloned into the pGEM-T vector and sequenced from the 5' and 3' ends using SP6 and T7 primers, respectively, Sequence was obtained for 5' and 3' regions of the cDNAs and specific primers were designed-from these (Table 1). Analysis of the sequence (Figure 7) indicated that residual oligodT 1 8 from the reverse transcriptase reaction served as a 3' primer and that the H8.3 and H8.5 forward primers (Table 1) bound to 5' sites to generate the 2 bands. In order to confirm that the N l and N2 cDNAs were induced by infection of THP-1 macrophages, PCR was performed on reverse-transcribed macrophage R N A using N l and N2 primers. The levels of N l and N2 products were significantly higher in infected THP-1 macrophages than in controls (Figure 12), suggesting that N l and N2 expression was induced by infection with M. tuberculosis. 52 600 bp| 300 bp| 200 bp| B 500 bp| 300 bp| B C24 124 C48 148 C24 124 C48 148 N l 0 453 0 327 N2 0 1031 0 953 P-actin 515 1127 1391 2160 P-actin 528 773 851 873 H8.3 763 1764 693 1870 H8.5 0 0 0 0 Nl/p-actin 0 0.40 0 0.15 N2/p-actin 0 1.33 0 1.09 H8.3/p-actin 1.48 1.57 0.50 0.87 H8.5/p-actin 0 0 0 0 Figure 11. Identification of putatively induced fragments, Nl and N2. cDNA was reverse transcribed from non-infected and infected THP-1 macrophage RNA using an oligodT ] 8 primer. PCR was performed on the cDNA using H8.3 (A) and H8.5 (B) primers with the same cycling protocol as above using 30 cycles. A 600 bp band in A, and a 500 bp band in B, were identified as being induced after normalizing the intensity of the bands against the 270 bp P-actin standard. Numerical values were assigned to each band based on their intensity using the program, ImagePC (Scion Corporation). The ratios of the intensities of the N l , N2, H8.3, and H8.5 bands to the p-actin bands are also given. 53 25 cycles 27 cycles 29 cycles + N l , N2i B-actin1 0 0 - i . T f qo 0 0 _ | . ^ <N <N S? ^ CN ^ t 3 ,— i d , — t 3 ,—, t 3 ,—, t d , "73 — t 3 g < £ g < £ g c £ g £ g £ g < £ 25 cycles 27 c; ^ cles 29 cycles C24 124 C48 148 C24 124 C48 148 C24 124 C48 148 N l 14 239 0 293 0 233 0 264 0 218 0 373 N2 9 139 0 182 0 204 0 130 0 16 0 317 P-actin 346 267 48 156 781 406 189 382 725 659 243 374 N l / P-actin 0.04 0.90 0 1.88 0 0.57 0 0.69 0 0.33 0 1.00 N2/ P-actin 0.03 0.52 0 1.17 0 0.50 0 0.34 0 0.02 0 0.85 Figure 12. Semi-quantitative R T - P C R of THP-1 macrophage R N A using primers specific for N l and N2. PCR was performed on cDNA reverse transcribed non-infected and infected THP-1 macrophage RNA, 24 and 48 hours post-infection. The PCR reaction consisted of 95°C, 5 min. and the indicated number of cycles of (95°C, 30 s.; 54°C, 1 min.; and 72°C, 30 s.). The products were electrophoresed in a 2% agarose gel to quantitate the approximately 550 bp N l and 400 bp N2 products normalized against the 270 bp P-actin standard. Positive controls for N l , N2 and P-actin are also shown. N l and N2 appeared to be induced by infection. Numerical values were assigned to each band based on their intensity using the program, ImagePC (Scion Corporation). The ratios of the intensities of the N l and N2 bands to the p-actin bands are also given. 54 2. Semi-quantitative PCR of NADH ubiquinone oxidoreductase chain 2 Primers for amplification of N A D H ubiquinone oxidoreductase chain 2 and p22-phox were generated to investigate the possibility of increased transcription of oxygen-dependent defence genes in response to infection. The expression of N A D H ubiquinone oxidoreductase chain 2 appeared to be constitutively transcribed and no pattern emerged in the ratios of N A D H ubiquinone oxidoreductase chain 2 to P-actin (Figure 13). The expression of p22-phox could not be measured as the PCR reactions were unsuccessful (data not shown). The primers created for p22-phox did not appear to be specific enough as several products were amplified and none were the expected size of 532 bp, nor were any induced by infection. Increasing the stringency of the PCR annealing step still did not generate the correct size product. 55 25 cycles 27 cycles 29 cycles 800 bp^ 300 b p ^ 25 c; ^ cles 27 cycles 29 cycles C24 124 C48 148 C24 124 C48 148 C24 124 C48 148 N A D H 257 228 540 336 348 150 440 274 722 418 927 512 P-actin 59 32 161 83 265 162 292 220 1571 874 916 682 N A D H / P-actin 4.36 7.13 3.35 4.05 1.31 0.93 1.51 1.25 0.46 0.48 1.01 0.75 Figure 13. Semi-quantitative R T - P C R of THP-1 macrophage R N A using primers specific for N A D H ubiquinone oxidoreductase chain 2. PCR was performed on cDNA reverse transcribed from non-infected and infected THP-1 macrophage RNA, 24 and 48 hours post-infection. The PCR reaction consisted of 95°C, 5 min. and the specified number of cycles of (95°C, 30 s.; 45°C, 1 min.; and 72°C, 30 s.) The products were electrophoresed in a 2% agarose gel to quantitate the 838 bp product normalized against the 270 bp P-actin standards. N A D H appears to be constitutively expressed and not induced by infection. Numerical values were assigned to each band based on their intensity using the program, ImagePC (Scion Corporation). The ratios of the intensities of the N A D H ubiquinone oxidoreductase chain 2 bands to the P-actin bands are also given. 56 3. Semi-quantitative PCR of H15.2 PCR amplification of macrophage cDNA with the HI5.2 primers also did not yield any detectable product. However, when the stringency of the reactions was lowered by decreasing the annealing temperature from 54°C to 45°C, several bands appeared (Figure 14). One of the bands, at 300 bp, was present only in the infected macrophage samples suggesting that it may have been induced by infection. After the result was confirmed by a repeat experiment on another set of cDNA samples, the M l cDNA was cloned and sequenced. The M l sequence matched the sequences of two highly related, IFN-inducible nuclear phosphoproteins (72) which have not been fully characterized yet. To confirm that the transcription of the IFN-induced nuclear phosphoprotein was increased following infection, PCR was carried out on two other cDNA sets, using primers specific for sequences within the phosphoproteins (Table 1). The primers were designed such that they would hybridize to the cDNA of both phosphoproteins to generate a 729 bp product for the larger phosphoprotein and a 580 bp product for the smaller phosphoprotein. However, only the 729 bp product was produced (Figure 15), indicating that only the larger phosphoprotein was expressed, or that the primers selectively hybridized to the cDNA of the larger phosphoprotein. 57 U x: ' o u c o U X ! (N Td CD •*—i Q CD C C M U x: oo O C o U S-H X 0 0 T3 C D +-* o D c 300 bp C24 124 C48 148 300 bp band 81 1464 43 817 P-actin 336 45 1048 52 300 bp band/ p-actin 0.24 32.53 0.41 15.71 Figure 14. Identification of a putatively induced fragment. PCR was performed on cDNA from non-infected and infected THP-1 macrophages using primers specific for the HI5.2 fragment. The annealing temperature was lowered from 54°C to 45 °C such that the reactions consisted of 95°C, 5min and 30 cycles of (95°C, 30 s.; 45°C, 1 min.; 72°C, 30 s.) Following electrophoresis, there was the appearance of a putatively induced 300 bp product. Cloning and sequencing of the band identified it as an IFN-induced nuclear phosphoprotein cDNA. Numerical values were assigned to each band based on their intensity using the program, ImagePC (Scion Corporation). The ratios of the intensities of the 300 bp bands to the p-actin bands are also given. 58 The abundance of the phosphoprotein transcript was greater in the samples from infected THP-1 macrophages than in non-infected macrophages (Figure 15). In order to characterize further the expression of the phosphoprotein, semi-quantitative PCR was performed on cDNA from cells treated with different stimuli. PCR amplification of cDNA from THP-1 cells treated with IFN-y confirmed that the phosphoprotein was IFN inducible (Figure 16). The phosphoprotein expression was induced in human macrophages by infection with live but not dead H37Rv, and was uninducible by treatment with zymosan- indicating that it was not a phagocytosis-related gene (Figure 17). PCR using the M l primers was also done on cDNA from human macrophages infected with H37Rv or H37Ra, or exposed to H37Rv L A M (Figure 18). The results from this experiment were not as clear, as the results from 48 hours post-treatment were not consistent with either those at 24 hours,or the previous results with THP-1 macrophages. In the 24 hours samples, the transcription of the phosphoprotein was induced significantly with H37Rv infection, but not in response to treatment with either H37Ra, or L A M . However, at 48 hours, the phosphoprotein transcript was expressed in all samples, although it was slightly higher in the H37Rv sample. It is also possible that induction of the phosphoprotein expression occurred more rapidly in response to H37Rv than it did with either H37Ra and L A M . The quantitation was repeated with new RNA samples from human macrophages infected with H37Rv or H37Ra (Figure 19). Unlike the previous samples, the expression of the phosphoprotein mRNA in these samples appeared to be constitutive. At 20 cycles of amplification, the phosphoprotein PCR product was increased in 24 hour macrophages infected with H37Ra and 48 hour control macrophages. The ambiguity of the results from these samples did not allow a definitive conclusion to be made on the expression pattern of the phosphoprotein. One difference between these RNA samples and all the previous ones was that the macrophages 59 in these samples were isolated from blood which had been stored at 4°C for over 24 hours instead of fresh blood. i 60 C24 124 C48 148 Phosphoprotein 103 882 324 1235 P-actin 2548 1644 4033 611 Phosphoprotein/ P-actin 0.04 0.54 0.08 2.02 Figure 15. Semi-quantitative R T - P C R of THP-1 macrophage R N A using primers specific for the IFN-induced nuclear phosphoprotein. PCR was performed on cDNA reverse transcribed from non-infected and infected THP-1 macrophage RNA, 24 and 48 hours post-infection. The PCR reactions consisted of 95°C, 5 min. and 30 cycles of (95°C, 30 s.; 45°C, 1 min.; and 72°C, 30 s.). The products were electrophoresed in a 2% agarose gel to quantitate the 729 bp product normalized against the 270 bp P-actin standards. The phosphoprotein transcript appears to be induced by infection. Numerical values were assigned to each band based on their intensity using the program, ImagePC (Scion Corporation). The ratios of the intensities of the phosphoprotein bands to the P-actin bands are also given. 61 C/3 700 b p ^ 300 b p ^ Control IFN-y Phosphoprotein 0 2522 P-actin 5557 7131 Phosphoprotein/ p-actin 0 0.354 Figure 16. Quantitation of the nuclear phosphoprotein in untreated and IFN-y treated THP-1 macrophage R N A . Semi-quantitative PCR was performed as previously described on RNA from macrophages, 24 hours post-treatment, for 30 cycles. Normalizing against the intensity of the p-actin, the phosphoprotein transcription appears to be induced by treatment with IFN-y. Numerical values were assigned to each band based on their intensity using the program, ImagePC (Scion Corporation). The ratios of the intensities of the phosphoprotein bands to the p-actin bands are also given. 62 24 hrs. 48 hrs. Phosphoprotein • B-actin • C24 L.Rv24 D.Rv24 Z24 C48 L.Rv48 D.Rv48 Z48 P-protein 63 550 73 18 83 292 61 42 P-actin 420 203 118 268 367 50 1138 570 P-protein/ P-actin 0.15 2.71 0.62 0.07 0.23 5.84 0.05 0.07 Figure 17. P C R Quantitation of the IFN-induced nuclear phosphoprotein in R N A from human macrophages. Human peripheral macrophages were treated with live (L.Rv) and dead H37Rv (D.Rv) and zymosan (Z). RNA was extracted 24 and 48 hours post-treatment. Following reverse transcription with oligodT I 8 primers, semi-quantitative PCR was performed on the samples as previously described for 30 cycles. The PCR products were run on a 2% agarose gel with positive controls for the phosphoprotein product (P-protein) and p-actin. The phosphoprotein transcript appears to be induced only by infection with H37Rv. Numerical values were assigned to each band based on their intensity using the program, ImagePC (Scion Corporation). The ratios of the intensities of the phosphoprotein bands to the p-actin bands are also given. 63 24 hrs. 48 hrs. Phosphoprotein • B-actin • C24 Rv24 Ra24 LAM24 C48 Rv48 Ra48 LAM48 P-protein 18 267 0 27 134 229 133 131 p-actin 437 365 380 228 284 339 383 456 P-protein/ P-actin 0.04 0.73 0 0.12 0.47 0.68 0.35 0.29 Figure 18. P C R Quantitation of the IFN-induced nuclear phosphoprotein in R N A from human macrophages. Human peripheral macrophages were treated with H37Rv (Rv) and H37Ra (Ra) and L A M , and their RNA was extracted 24 and 48 hours post-treatment. Following reverse transcription with oligodT,8 primers, semi-quantitative PCR was performed on the samples as previously described for 30 cycles. The PCR products were run on a 2% agarose gel with positive controls for the phosphoprotein product (P-protein) and P-actin. The phosphoprotein transcript appears to be induced only by infection with H37Rv at 24 hours but induced in all samples at 48 hours. Numerical values were assigned to each band based on their intensity using the program, ImagePC (Scion Corporation). The ratios of the intensities of the phosphoprotein bands to the P-actin bands are also given. 64 20 cycles 22 cycles s-CN "o 4—> c o u M i l B-actin i ] . C/3 r. E/3 u- •— • £h X X X X H SX oo oo 00 CN CN CN CN > ' o > > H 4-" r-- c r - r - c m CO c co co o ro CO 3C U X + U £ ^ v> r 5 b ^ X X 22 oo oo — ^ ^ O > 3^ ^ O ro ro 20 cycles 22 cycles C Rv Ra C Rv Ra C Rv Ra C Rv Ra 24 24 24 48 48 48 24 24 24 48 48 48 P-protein 2240 1812 2370 3615 2774 1492 1506 2130 2289 3482 2901 2471 P-actin 2638 1891 1575 1458 1564 1082 271 382 461 149 590 524 P-protein/ 0.85 0.96 1.51 2.48 1.68 1.38 5.56 5.58 4.97 4.65 4.92 4.72 P-actin Figure 19. PCR Quantitation of the IFN-induced nuclear phosphoprotein expression in human macrophages. Human peripheral macrophages were treated with H37Rv (Rv) and H37Ra (Ra), and their RNA was extracted 24 and 48 hours post-treatment. Following reverse transcription with oligodT1 8 primers, semi-quantitative PCR was performed on the samples as previously described for 20 and 22 cycles. The PCR products were run on a 2% agarose gel with positive and negative controls for the phosphoprotein product (P-protein) and P-actin. Numerical values were assigned to each band based on their intensity using the program, ImagePC (Scion Corporation). The ratios of the intensities of the phosphoprotein bands to the P-actin bands are also given. 65 4. Semi-quantitative PCR of the Nl fragment The N l fragment, which was identified indirectly from the PCR of H8.3, did not match any known genes when the sequence was cross-referenced through the N C B I - B L A S T database. In THP-1 macrophages, infection with Erdman M. tuberculosis induced the transcription of N l (Figure 12). However, the induction was not as prominent in human peripheral macrophages infected with M. tuberculosis (Figure 20-23). N l expression was induced by live, but not dead H37Rv which suggested that induction of N l required the active stimulation of macrophages or that the inducing factor was destroyed when the bacteria was heat killed. The inability of zymosan to stimulate N l transcription indicated that N l was not a phagocytosis gene. In addition, the transcription of the N l at 24 hours post-infection with H37Rv was induced when compared to uninfected macrophages and macrophages infected with H37Ra or treated with L A M (Figure 22). However, at 48 hours post-infection, N l was transcribed equally in macrophages infected with H37Rv and H37Ra or treated with L A M . This suggested that H37Rv may have the ability to induce N l expression before H37Ra and the initial induction by H37Rv was not mediated by L A M . Contrary to the previous findings, no differences in N l expression were observed in the human macrophage samples derived from stored blood infected with either H37Rv or H37Ra. Similar to the phosphoprotein transcript expression results obtained from this R N A sample (Figure 19), the expression of N l was constitutive (Figure 23). Again, the discrepancy in expression patterns may be due to individual sample differences or macrophages being derived from stored, rather than fresh blood. The findings of the N l expression were ambiguous. Although the expression of N l appeared to be induced by infection in THP-1 macrophages, its expression was variable in the 66 experiments with human macrophages. However, the preliminary data obtained suggests that the N l expression may have been induced by infection with H37Rv. N i l B-actinl C24 124 C48 148 N l 589 1492 1313 2404 P-actin 3039 3412 4338 5056 N l / P-actin 0.19 0.44 0.30 0.48 Figure 20. Semi-quantitative R T - P C R of human macrophage R N A using primers specific for N l . PCR was performed on cDNA reverse transcribed from non-infected and infected human macrophage RNA, 24 and 48 hours post-infection. The PCR reaction consisted of 95°C, 5 min. and 27 cycles of (95°C, 30 s.; 54°C, 1 min.; and 72°C, 30 s.) The products were electrophoresed along with positive and negative controls in a 2% agarose gel to quantitate the approximately 550 bp product normalized against the 270 bp P-actin standard. Numerical values were assigned to each band based on their intensity using the program, ImagePC (Scion Corporation). The ratios of the intensities of N l bands to the P-actin bands are also given. 67 24 hrs. 48 hrs. N l ^ B-actin^ C24 L.Rv24 D.Rv24 Z24 C48 L.Rv48 D.Rv48 Z48 N l 0 1528 10 17 210 300 354 227 P-actin 330 223 257 178 1837 156 2059 929 N l / P-actin 0 6.85 0.04 0.10 0.11 1.92 0.17 0.24 Figure 21. P C R Quantitation of N l expression in R N A from human macrophages. Human peripheral macrophages were treated with live (L.Rv) and dead H37Rv (D.Rv) and zymosan (Z), and their R N A was extracted 24 and 48 hours post-treatment. Following reverse transcription with oligodT 1 8 primers, semi-quantitative PCR was performed on the samples as previously described for 29 cycles. The PCR products were run on a 2% agarose gel with positive and negative controls for N l and p-actin. N l appears to be induced only by infection with H37Rv at 24 hours. Numerical values were assigned to each band based on their intensity using the program, ImagePC (Scion Corporation). The ratios of the intensities of N l to the p-actin bands are also given. 68 28 cycles 30 cycles N i l B-actinl SZ "o i— > c o C/5 V5 sz sz xz t / 3 22 00 00 — ^ T l " o > <tf 5 ; iz C/3 00 r 5 b r/ £ ^ *-j3 £ , C j3 ^ ^ Tt -c 00 oo (N M r t ^ ^j-O > cd <N -q > cS 0 < ^ < ^ " < O r<^ <r UXX^UXX^l s. 1-00 28 cycles C24 Rv24 Ra24 LAM24 C48 Rv48 Ra48 LAM48 N l 1194 2213 651 480 1071 3145 2350 3418 P-actin 1204 1157 960 779 1057 1375 1141 954 N l / P-actin 0.99 1.91 0.68 0.62 1.01 2.29 2.08 3.58 30 cycles C24 Rv24 Ra24 LAM24 C48 Rv48 Ra48 LAM48 N l 812 1553 313 802 883 2437 1823 2495 P-actin 628 898 932 819 1243 957 943 717 N l / P-actin 1.29 1.73 0.34 0.98 0.71 2.55 1.93 3.48 Figure 22. PCR Quantitation of Nl expression in human macrophages. Human peripheral macrophages were treated with H37Rv (Rv) and H37Ra (Ra) and L A M , and their R N A was extracted 24 and 48 hours post-treatment. Following reverse transcription with oligodT1 8 primers, semi-quantitative PCR was performed on the samples as previously described for 28 and 30 cycles. The PCR products were run on a 2% agarose gel with positive and negative controls for N l and P-actin. Numerical values were assigned to each band based on their intensity using the program, ImagePC (Scion Corporation). The ratios of the intensities of the N l bands to the P-actin bands are also given. 69 20 cycles 22 cycles N i l B-actinl hrs hrs. hrs. hrs hrs. hrs. CN -i- oo rr oo 00 1- -f *o > *o > 4—1 e r- c r- r-o m m o m m u X U X X — ! I — CM c j^ > co X o U CM C3 X 00 c o U s-- C 00 > X C/3 oo TI-CS m 20 c: ^ cles 22 cycles c 24 Rv 24 Ra 24 C 48 Rv 48 Ra 48 C 24 Rv 24 Ra 24 C 48 Rv 48 Ra 48 N l 240 212 301 451 450 335 215 437 781 1037 847 676 P-actin 2264 2513 3105 4004 3072 3049 253 684 887 1091 910 1129 N l / P-actin 0.11 0.08 0.10 0.11 0.15 0.11 0.85 0.64 0.88 0.95 0.93 0.60 Figure 23. P C R Quantitation of N l expression in human macrophages. Human peripheral macrophages were treated with H37Rv (Rv) and H37Ra (Ra), and their RNA was extracted 24 and 48 hours post-treatment. Following reverse transcription with oligodT1 8 primers, semi-quantitative PCR was performed on the samples as previously described for 20 and 22 cycles. The PCR products were run on a 2% agarose gel with positive and negative controls for N l and P-actin. Numerical values were assigned to each band based on their intensity using the program, ImagePC (Scion Corporation). The ratios of the intensities of the N l bands to the P-actin bands are also given. 70 5. Semi-quantitative PCR of N2 fragment The N2 cDNA product was obtained by PCR using the H8.5 forward and oligodT1 8 primers. By cross-referencing the sequence through the NCBI-BLAST database, N2 was found to match a human cDNA EST clone with no assigned function. PCR with non-infected and Erdman M. tuberculosis infected human macrophages indicated that N2 was transcribed in significantly higher amounts in infected macrophages than in controls (Figure 24). PCR of N2 in macrophages exposed to live or dead M. tuberculosis, or zymosan revealed that it was upregulated in response to all three stimuli, suggesting that N2 may be a gene involved in phagocytosis (Figure 25). The expression of N2 relative to P-actin also appeared to be lower at 48 hours versus 24 hours in macrophages exposed to dead H37Rv. However, because the relationship of band intensity versus the quantity of nucleic acid follows a sigmoidal curve, it is difficult to accurately assess the amount of D N A when the intensities are high, as.in this case. Therefore, it can only be concluded that the treatment of human macrophages with live or dead H37Rv, or zymosan induced the transcription of N2. 71 C24 124 C48 148 N2 1262 6207 3286 5144 P-actin 493 861 719 691 N2/ p-actin 2.56 7.21 4.57 7.44 Figure 24. Semi-quantitative R T - P C R of human macrophage R N A using primers specific for N2. PCR was performed on cDNA reverse transcribed from non-infected and infected human macrophage RNA, 24 and 48 hours post-infection. The PCR reaction consisted of 95°C, 5 min. and 27 cycles of (95°C, 30 s.; 54°C, 1 min.; and 72°C, 30 s.) The products were electrophoresed in a 2% agarose gel along with positive and negative controls to quantitate the approximately 400 bp product normalized against the 270 bp P-actin standard. Numerical values were assigned to 72 PCR of cDNA from macrophages infected with H37Rv or H37Ra, or treated with L A M indicated that N2 was induced by all three stimuli (Figure 26). After 27 cycles of amplification, the only prominent band produced was from amplification of the 24 hour sample of H37Rv infected macrophages. Very faint bands were also observed in the 48 hour sample of H37Rv infected macrophages and the 24 hour sample of H37Ra infected macrophages. After 30 cycles of amplification, N2 appeared to be expressed in all samples. At 24 hours post infection, both H37Rv and H37Ra significantly induced N2 transcription and at 48 hours, all the treatments induced N2 transcription. With the R N A samples from the macrophages isolated from stored blood, the expression of N2 was slightly induced by H37Rv (Figure 27). However, the results from H37Ra infection were not clear as discrepancies existed between 24 and 48 hour samples as well as between different cycles of PCR. These differences could not be resolved by repeating the PCR as the results remained variable. Similar to the results obtained from the analysis of N l expression, a consistent pattern did not emerge for the expression of N2. In THP-1 macrophages, N l was clearly induced by infection, but in human peripheral macrophages, the results were variable between samples. To confirm the expression pattern of N2, these experiments must be repeated on more macrophage samples. 73 24 hrs 48 hrs N 2 ^ B-actin^ C24 L.Rv24 D.Rv24 Z24 C48 L.Rv48 D.Rv48 Z48 N2 1451 5542 4739 5062 1709 6016 4659 7645 p-actin 630 837 609 631 1244 1305 1801 1374 N2/ P-actin 2.30 6.62 7.78 8.02 1.37 4.61 2.58 5.56 Figure 25. P C R Quantitation of N2 in R N A from human macrophages. Human peripheral macrophages were treated with live (L.Rv) and dead H37Rv (D.Rv) and zymosan (Z), and their RNA was extracted 24 and 48 hours post-treatment. Following reverse transcription with oligodT, s primers, semi-quantitative PCR was performed on the samples as previously described for 28 cycles. The PCR products were run on a 2% agarose gel with positive and negative controls for N2 and P-actin. The N2 appears to be induced by all stimuli. Numerical values were assigned to each band based on their intensity using the program, ImagePC (Scion Corporation). The ratios of the intensities of N2 bands to the p-actin bands are also given. 74 N2i B-actin' 27 cycles + 30 cycles § g on x: 2 <N CN fN O > <N J3 ^ c r- ^ t / 3 c on 3$ oo 00 ^ T t on 00 > a Pi Ol\ ^ r -m m <J U PC X i—l 27 cycles C24 Rv24 Ra24 LAM24 C48 Rv48 Ra48 LAM48 N2 0 656 0 0 0 0 0 0 P-actin 787 756 650 777 841 1035 728 729 N2/ P-actin 0 0.87 0 0 0 0 0 0 30 c: pcles C24 Rv24 Ra24 LAM24 C48 Rv48 Ra48 LAM48 N2 742 1750 1029 496 532 1093 1504 882 p-actin 368 201 142 177 200 123 144 108 N2/ P-actin 2.02 8.71 7.25 2.80 2.66 8.90 10.44 8.17 Figure 26. P C R Quantitation of N2 in R N A from human macrophages. Human peripheral macrophages were treated with H37Rv (Rv) and H37Ra (Ra) and L A M , and their R N A was extracted 24 and 48 hours post-treatment. Following reverse transcription with oligodT1 8 primers, semi-quantitative PCR was performed on the samples as previously described for 27 and 30 cycles. The PCR products were run on a 2% agarose gel with positive and negative controls for N2 and P-actin. Numerical values were assigned to each band based on their intensity using the program, ImagePC (Scion Corporation). The ratios of the intensities of the N2 bands to the p-actin bands are also given. 75 20 cycles 22 cycles N2i B-actin' + O 4—> a o U X X (N ^= 00 o U J/5 C/3 4 3 J 3 00 00 m m SG OC 20 cycles 22 c: ^ cles C 24 Rv 24 Ra 24 C 48 Rv 48 Ra 48 C 24 Rv 24 Ra 24 C 48 Rv 48 Ra 48 N2 93 484 315 422 684 147 1630 2877 3082 1995 3132 1038 P-actin 4692 3297 3897 3998 2727 1832 2431 2845 3006 2067 1292 789 N2/ P-actin 0.07 0.15 0.08 0.11 0.25 0.08 0.67 1.01 1.03 0.97 2.42 1.32 Figure 27. P C R Quantitation of N2 expression in human macrophages. Human peripheral macrophages were treated with H37Rv (Rv) and H37Ra (Ra), and their R N A was extracted 24 and 48 hours post-treatment. Following reverse transcription with oligodT l 8 primers, semi-quantitative PCR was performed on the samples as previously described for 20 and 22 cycles. The PCR products were run on a 2% agarose gel with positive and negative controls for N2 and P-actin. Numerical values were assigned to each band based on their intensity using the program, ImagePC (Scion Corporation). The ratios of the intensities of the N2 bands to the P-actin bands are also given. 76 IV. DISCUSSION A. Analysis of subtractive hybridization and differential display A novel method of isolating genes whose expression is modulated was developed by combining subtractive hybridization (56, 57) with differential display (80). The addition of a subtractive hybridization step solved many of the problems encountered with differential display alone, including co-migrating products, short products, and the inability to isolate low copy-number transcripts (31, 126). The resolution of the bands was increased compared to the non-subtracted samples, especially for larger products (Figure 3-6). In addition, the bands which were isolated had a greater chance of being differentially expressed because the subtractive step should have decreased or eliminated the transcripts common to both non-infected and infected macrophage mRNA. With fewer common transcripts, there would have also been less competition for primers, thus facilitating the amplification and identification of low abundance gene products. This was evident in the appearance of new bands, present only in the subtracted samples, during differential display. In addition, many of the cDNAs isolated by this method were only detectable by PCR and not by hybridization techniques. The advantage of using differential display was that it allowed for the identification of novel transcripts since the PCR conditions had low stringency conditions. By applying this method of subtractive hybridization with differential display to other systems such as signaling pathways, new genes and/or novel downstream signaling components could be identified. An improvement to this procedure could be the use of (3 3P-a) dCTP instead of (3 2P-a) dCTP because 3 3 P would provide sharper bands (79). This would allow for easier identification of large modulated transcripts towards the top of the gel such that longer regions of sequence may be 77 obtained. In addition, the sharper bands would make the extraction of bands more accurate and decrease the chance that an adjacent band may be extracted. Using the four different pairs of primers, 25 different cDNAs, that were putatively modulated by infection were isolated. The expression patterns of five different genes in response to infection with M. tuberculosis infection were investigated by semi-quantitative PCR. Two of the clones, H8.3 and H8.5, were found to be induced by infection. In contrast, another clone, N A D H ubiquinone oxidoreductase chain 2 was found to be constitutively expressed, suggesting that the subtractive hybridization process was not completely effective at eliminating common transcripts. Possible ways that could improve the subtractive hybridization could be to increase the number of subtractive hybridization steps; however, this would compromise the amount of mRNA obtained from the process, as excessive heating steps would promote the degradation of the mRNA. Alternatively, the amount of cDNA covalently attached to the beads could be increased relative to the mRNA to be subtracted, by increasing the amount of R N A used in the reverse transcription reaction. Another possible method for increasing the library size bound to the beads could be to perform PCR on the RNA using the oligodT3 0 on the beads and the same lOmer later to be used for differential display as primers. In this way, the majority of the beads would contain cDNA, which would increase their subtractive power; however, this method could be limited to the thermal stability of the beads as the continuous heating could destroy them. PCR amplification from two clones, phox-22 and HI5.2, was unsuccessful as no products were produced of the expected size. It was possible that primer design was the main reason for this failure. Differential display provides short cDNA sequences and the cDNA was sequenced only from one end. By using (33P-oc) dCTP, larger fragments might be obtained, and by 78 sequencing from both ends of the isolated fragments, more specific primers might be designed from the longer sequences. The short sequences isolated from differential display may have also resulted in limitations during semi-quantitative PCR. Quantitation of the PCR products relies on ethidium bromide staining which has a sigmoidal relationship between fluorescent intensity and quantity of DNA. Also, the intensity depends on the number of base pairs rather than the number of products. Therefore, it requires a greater number of short PCR products than long PCR products for a band to be visualized. Since PCR also has a sigmoidal relation between the number of products and the number of cycles, short products would only be visible as the reaction approaches the end of the exponential amplification stage. In this respect, there would be a limited number of cycles where products and differences in expression would be both seen for short products. In contrast, large products would be visible earlier in the exponential phase of amplification so differences in expression would be more evident. Because the amplification of products during PCR follows a sigmoidal curve, it is not possible to assess differences in the expression levels quantitatively. If endpoint products are considered, a five-fold increase in intensity does not equate to a five-fold increase in the original template; it can only be concluded that an increase in expression was observed. To determine the amount of starting template, competitive PCR may be performed (50, 102, 153). Using this approach, a modified template, which would yield a different size PCR product than the original, is added at different known concentrations to a PCR reaction such that when the amount of both templates are equal, the intensity of both products would also be equal. In this way, the amount of final product may be extrapolated back to the amount of starting template. 79 B. Analysis of transcripts isolated by subtractive hybridization and differential display Twelve of the 25 cDNAs isolated matched human cDNA sequences and five of these matched known human genes (Table 2). P22-phox, N A D H ubiquinone oxidoreductase chain 2, and possibly the antioxidant enzyme, A O E 37-2, are related to the oxygen-dependent defence mechanism and thus, it may have been expected that these would be modulated in response to M. tuberculosis infection. However, analysis of N A D H ubiquinone oxidoreductase chain 2 revealed that it was constitutively expressed in both control and infected macrophages. Interestingly, the release of reactive oxygen intermediates has previously been found to be upregulated in response to P M A (55), hence the p22-phox and N A D H ubiquinone oxidoreductase chain 2 detected by differential display might have been due to induction and the A O E 37-2 due to suppression by P M A rather than infection. However, the subtractive hybridization step should have removed these transcripts i f the induction was due to P M A , even-if M. tuberculosis further modulated their expression, since the non-infected macrophages were also treated with equal amounts of P M A . Another isolated cDNA matched a growth arrest gene, B4B (113), and a gene associated with squamous cell differentiation, CL-10 (84). Interestingly, Ruegg et. al. also isolated the B4B gene through differential display. It was found by differential display and PCR that B4B was exclusively expressed in the intermediate density cells (T, B , N K , and dendritic cells) and not in low- or high-density cells (monocytes or lymphocytes) (113). However, in the current study, B4B was clearly found in low-density cells as it was identified from differential display of THP-1 macrophages. This suggests either that the results of Ruegg et. al, with monocytes were falsely negative or that B4B is expressed in macrophages, but not monocytes. Consistent with its proposed role as a B cell growth arrest gene (113), B4B may also play a role in the growth arrest of macrophages. If this is true, it is possible that the expression of B4B in macrophages might 80 have been induced by P M A rather than by infection with M. tuberculosis, and that similar to N A D H ubiquinone oxidoreductase chain 2, B4B was not successfully subtracted. It would be interesting to investigate the expression of B4B in monocytes versus macrophages at the mRNA and protein level to see i f its expression correlates with growth arrest. Alternatively, the isolated cDNA may be a novel growth arrest gene specific to macrophages with sequence similarity to both B4B and CL-10, which would also be of interest. The other known cDNA isolated was a human protein phosphatase y (94), which has no known function other than being a serine phosphatase. It is possible that this serine phosphatase is part of a signal transduction pathway initiated by M. tuberculosis infection or simply by differentiation of monocytes to macrophages. Seven of the isolated cDNAs matched human cDNA with no assigned function and the remaining 13-had no significant matches. Although the 13 cDNAs did not match any known M. tuberculosis sequences and were amplified based on their poly A tails, there is still the possibility that they may be from M. tuberculosis transcripts as the M. tuberculosis database is not been yet completed. A possible experiment to determine the source of the cDNAs would be to do a Southern blot on human and M. tuberculosis D N A using the isolated cDNAs as probes. 81 C. Analysis of the expression of the IF~N-inducible nuclear phosphoprotein The cDNA, M l , was discovered while performing PCR when the stringency conditions were lowered while amplifying another isolated clone, H15.2. The 300 bp band corresponding to M l appeared only in the infected macrophage samples (Figure 14). Upon sequencing, it was revealed that the isolated cDNA matched the sequence of a known IFN-inducible human nuclear phosphoprotein (72). The phosphoprotein transcript is known to be inducible by IFN-a and very strongly inducible by IFN-y in Daudi and HeLa cells, and the amino acid sequence suggested that it may have serine and threonine phosphorylation sites (72). In addition, the phosphoprotein is localized to the nucleus and could possibly have a role in gene transcription (72). In THP-1 macrophages, the potential release of IFN-a in response to infection may have induced the expression of the phosphoprotein transcript since macrophages do not secrete IFN-y. IFN-a is an essential cytokine for antiviral responses as IFN-a receptor knockout mice are susceptible to viral infections (66). IFN-a acts synergistically with IL-12 to enhance the priming for IFN-y production to initiate a Thl-type response (83). It may also play a role in the development of a protective response to M. tuberculosis infection. IFN-a has a secondary role to IFN-y in M. tuberculosis infections, as IFN-a is unable to rescue IFN-y knockout mice (143). In any case, it may be postulated that the phosphoprotein may have a role in regulating IFN-inducible genes. Possibly, the initial release of IFN-a in response to infection initiates the production of the phosphoprotein such that it prepares the macrophage for new gene transcription when IFN-y is released later in the course of infection. In the RNA samples from THP-1 macrophages, the phosphoprotein transcript was distinctly upregulated from an off state in response to infection with M. tuberculosis (Figure 15). 82 However, in human peripheral macrophages, expression of the phosphoprotein was observed even in control cells. This observation may be explained by the release of IFNs from other cells, or even by other macrophages if the donor was mounting a Thl-type response to infection at the time that the P B M C samples were taken. In addition, two human sample sets (Figures 17 and 18), showed the phosphoprotein transcription to be induced more strongly by live H37Rv than by dead H37Rv or H37Ra. The percentage of macrophages containing mycobacteria were identical in each sample, which suggests that live H37Rv may have interacted differently with macrophages compared to dead H37Rv or H37Ra. Physiological differences as well as differences in their interactions with macrophages, such as in binding (118, 120), exist between H37Rv and H37Ra, hence it is plausible that they may also induce genes differently. However, H37Rv L A M cannot be the only factor responsible for this difference since induction of the phosphoprotein transcription by L A M or dead H37Rv did not account for the difference. Also, because dead H37Rv did not appear to induce expression of the phosphoprotein mRNA, it also suggested that live H37Rv may have actively induced the phosphoprotein transcription or that the inducing factor was destroyed during the process of heat killing the mycobacteria. The data from the third sample set with macrophages from stored blood infected with H37Rv or H37Ra indicated that the phosphoprotein mRNA was constitutively expressed in all samples, which was inconsistent with all the previous data (Figure 19). The results of the last sample will be discussed below. This discrepancy indicates that more samples must be analyzed to determine i f a pattern exists in the expression of the phosphoprotein. 83 D. Analysis of the expression of the Nl clone The N l clone did not match any known genes but its presence in non-infected human macrophage samples (Figure 20) indicated that it was not an M. tuberculosis transcript. In THP-1 macrophages, the N l transcript was expressed distinctly in M. tuberculosis infected macrophages and not in non-infected cells (Figure 12). However, in non-infected human peripheral macrophage samples, the N l transcript was also detected, although at levels slightly lower than infected cells (Figure 20). The preliminary data suggests that N l may be induced in both THP-1 and peripheral macrophages by infection with H37Rv, H37Ra, and L A M , but not by zymosan or dead H37Rv. This indicated that N l is induced neither by phagocytosis per se nor by inactive M. tuberculosis. Because N l expression was induced by H37Rv L A M and not dead H37Rv, it suggested either that the L A M was destroyed during the heat killing, the concentrations of L A M differed, or that there were different responses to L A M from one human macrophage sample to the next. Similar to the phosphoprotein mRNA expression, the macrophage samples from the stored blood indicated that N l was constitutively expressed. Again, more samples must be tested in order to draw firm conclusions. If N l was induced by infection with H37Rv, H37Ra, and L A M , it would be interesting to determine whether it is also induced by other intracellular pathogens. In addition, because N l does not have any sequence similarity to other known genes, further experiments may determine what role, in any, it has in infection. 84 E. Analysis of the expression of the N2 clone The N2 clone matched a human cDNA EST clone with no known function. Preliminary evidence suggests that N2 may be a phagocytosis related gene as it was induced by all treatments, including zymosan (Figures 24-27). Similar to the phosphoprotein and N l , in THP-1 macrophages, N2 was induced from a virtually off state (Figure 12) while it appeared to be expressed constitutively in infected and non-infected samples obtained from human peripheral macrophages. Again, this may be explained by human peripheral macrophages having been in an environment where they would have encountered other cells as well as antigens which could have induced gene expression, whereas THP-1 cells grown in culture were not exposed to such factors. Differences existed in the N2 transcription seen in macrophages infected with H37Ra between one human macrophage sample set (Figure 26) and another (Figure 27). It is possible that, i f the N2 gene is involved in phagocytosis, this may reflect differences in either phagocytic efficiency of the macrophages or differences in responses between macrophages. To test whether N2 is involved in phagocytosis, phagocytosis inhibitors could be added. F. Problems with human macrophage samples Unlike THP-1 cell line macrophages, the results obtained from human peripheral macrophages were not as consistent and differences in expression levels were not as distinct. Because these macrophage samples are taken from different donors, there are inherent differences between one sample to the next. The histories of the donors are unknown, thus prior exposures to M. tuberculosis or other pathogens could have resulted in different responses to infection. Additionally, differences between non-infected and infected human macrophages were not as striking as the differences observed in THP-1 macrophages, perhaps because cell culture 85 macrophages were not exposed to other cell types, cytokines, or antigens which could have a role in the activation and suppression of the isolated genes. There was also one macrophage sample set which was derived from blood that had been stored at 4°C for over 24 hours. In this sample set, the phosphoprotein and N l mRNAs appeared to be induced in non-infected cells to the same level as infected macrophages. Other researchers have also observed similar results with macrophages from stored blood, where the signaling molecules appeared to be expressed prior to stimulation (Raymond Lo, personal communication). The phosphoprotein has a putative role as a downstream regulator of transcription which would be consistent with this hypothesis. Although the role of N l has not been elucidated yet, its expression may also correlate with macrophage activation. Other changes have also been observed based on the age of the macrophages. It was found that there was an enhanced antiviral response to LPS stimulation, which correlated with the age of the macrophages (49). Thus, it was possible that the macrophages from the stored blood were more activated than those from fresh blood such that the induction of several genes including the phosphoprotein and N l occurred prior to infection with M. tuberculosis. The use of cloned cell lines in all the experiments might have yielded more consistent results. Using this approach, the induction of genes caused solely by M. tuberculosis infection and not by other factors might have been determined. Although more experiments must be done before solid conclusions may be made, many potentially interesting cDNAs have been isolated. For example, the HI5.6/7 cDNA, although it may not have a role in infection with M. tuberculosis, may offer insight into macrophage differentiation. The cDNA has sequence similarity to a potential growth arrest gene, B4B (113), and a squamous cell differentiation gene, CL-10 (84). The expression pattern of H15.6/7 may be 86 studied by measuring its transcription in monocytes and macrophages, to see i f it is upregulated in response to macrophage differentiation. In addition, expression of the IFN-inducible phosphoprotein, N l , and N2 observed in THP-1 cells and fresh human peripheral macrophages, indicated that their expression may correlate to infection with M. tuberculosis. By obtaining more samples of non-infected and infected peripheral macrophages from fresh blood, a pattern of expression of these cDNAs may be found. Specifically, experiments should be done to confirm whether the transcription of IFN-induced phosphoprotein and N l are induced preferentially by virulent and not avirulent forms of M. tuberculosis. Experiments should also be performed to establish if N2 is a transcript related to phagocytosis, and what role it may have. This may offer insight not only into intracellular bacterial infections, but cell physiology, as well. In addition, a process for identifying more cDNAs by subtractive hybridization and differential display has been established and this may be applied to other systems to identify other differentially expressed genes. 87 V. CONCLUSIONS A novel method for isolating differentially expressed genes was developed by combining subtractive hybridization (56, 57) with differential display (80). The addition of the subtractive hybridization step enhanced the method of differential display by theoretically reducing the number of gene products common to both infected and non-infected macrophages, thus improving the clarity of the differential display bands, and increasing the probability of isolating modulated products. Twenty-five cDNAs were isolated using this procedure, of which five were from known human genes and seven from known human cDNA. The remaining 13 cDNAs were novel sequences. The five known genes are: N A D H ubiquinone oxidoreductase chain 2 (19); p22-phox (35); an antioxidant enzyme, A O E 37-2; a possible growth arrest gene, B4B (113); and a human protein phosphatase y (94). PCR analysis confirmed that two novel sequences were moderately induced by infection with M. tuberculosis. However, N A D H ubiquinone oxidoreductase chain 2, which was also predicted to be induced by infection, was actually constitutively expressed in THP-1 macrophages. Three additional cDNAs were identified which were induced by infection of macrophages with M. tuberculosis. One matched an IFN-inducible nuclear phosphoprotein sequence while the other two had no known function. Although these three cDNA were confirmed to be induced in THP-1 macrophages, their expression pattern in human macrophages was unclear. Further work is required to establish i f these cDNAs are inducible by infection with M. tuberculosis, and also to determine their roles in macrophages. 88 Antony, V . 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