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Attenuating immune pathology using a microbial-based intervention in a mouse model of cigarette smoke-induced… Bazett, Mark; Biala, Agnieszka; Huff, Ryan D; Zeglinksi, Matthew R; Hansbro, Philip M; Bosiljcic, Momir; Gunn, Hal; Kalyan, Shirin; Hirota, Jeremy A May 15, 2017

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RESEARCH Open AccessAttenuating immune pathology using amicrobial-based intervention in a mousemodel of cigarette smoke-induced lunginflammationMark Bazett1, Agnieszka Biala2, Ryan D. Huff2, Matthew R. Zeglinksi3, Philip M. Hansbro4, Momir Bosiljcic1,Hal Gunn1, Shirin Kalyan1,5 and Jeremy A. Hirota2,6*AbstractBackground: Cigarette smoke exposure is the major risk factor for developing COPD. Presently, available COPDtreatments focus on suppressing inflammation and providing bronchodilation. However, these options have varyingefficacy in controlling symptoms and do not reverse or limit the progression of COPD. Treatments strategies usingbacterial-derived products have shown promise in diseases characterized by inflammation and immune dysfunction.This study investigated for the first time whether a novel immunotherapy produced from inactivated Klebsiella(hereafter referred to as KB) containing all the major Klebsiella macromolecules, could attenuate cigarette smokeexposure-induced immune responses. We hypothesized that KB, by re-directing damaging immune responses, wouldattenuate cigarette smoke-induced lung inflammation and bronchoalveolar (BAL) cytokine and chemokine production.Methods: KB was administered via a subcutaneous injection prophylactically before initiating a 3-week acute nose-onlycigarette smoke exposure protocol. Control mice received placebo injection and room air. Total BAL and differential cellnumbers were enumerated. BAL and serum were analysed for 31 cytokines, chemokines, and growth factors. Lung tissueand blood were analysed for Ly6CHI monocytes/macrophages and neutrophils. Body weight and clinical scores wererecorded throughout the experiment.Results: We demonstrate that KB treatment attenuated cigarette smoke-induced lung inflammation as shown byreductions in levels of BAL IFNγ, CXCL9, CXCL10, CCL5, IL-6, G-CSF, and IL-17. KB additionally attenuated the quantity ofBAL lymphocytes and macrophages. In parallel to the attenuation of lung inflammation, KB induced a systemic immuneactivation with increases in Ly6CHI monocytes/macrophages and neutrophils.Conclusions: This is the first demonstration that subcutaneous administration of a microbial-based immunotherapy canattenuate cigarette smoke-induced lung inflammation, and modulate BAL lymphocyte and macrophage levels, whileinducing a systemic immune activation and mobilization. These data provide a foundation for future studies exploringhow KB may be used to either reverse or prevent progression of established emphysema and small airways diseaseassociated with chronic cigarette smoke exposure. The data suggest the intriguing possibility that KB, which stimulatesrather than suppresses systemic immune responses, might be a novel means by which the course of COPD pathogenesismay be altered.Keywords: COPD, Immunomodulators, Klebsiella, mucosal immunology* Correspondence: hirotaja@mcmaster.ca2Department of Medicine, Division of Respiratory Medicine, University ofBritish Columbia, Vancouver, BC, CanadaV6H 3Z66Firestone Institute for Respiratory Health, Division of Respirology,Department of Medicine, McMaster University, Hamilton, ON, CanadaL8N4A6Full list of author information is available at the end of the article© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Bazett et al. Respiratory Research  (2017) 18:92 DOI 10.1186/s12931-017-0577-yBackgroundChronic obstructive pulmonary disease (COPD) is an in-flammatory airway disease that results in progressive irre-versible airflow limitation. The global prevalence ofCOPD, as determined by the World Health Organization,is 11.7% [1], and it is predicted that by 2030 it will be thefourth leading cause of death worldwide [2]. Mainstreamor second-hand cigarette smoke exposure is a risk factorfor developing COPD [3], which can be exacerbated bygenetic factors [4, 5] and environmental exposures [6, 7].Chronic cigarette smokers that develop COPD maypresent with varying degrees of cough, sputum produc-tion, dyspnea, wheezing, and chest tightness [8, 9]. Pres-ently, available treatments primarily focus on suppressinginflammation and providing bronchodilation. However,these options have varying efficacy in controlling symp-toms and do not reverse or limit, completely, the progres-sion of COPD [10, 11].The pathology of COPD includes emphysema and ob-struction of the small airways as a result of chronic bron-chitis, which is associated with inflammation and immunedysfunction [12, 13]. In COPD patients, the inflammatoryimmune response is altered, and often involves increasedcytokines, including IFN-γ, CXCL9 (MIG), CXCL10(IP-10), and CCL5 (RANTES) [14–16]. This lung cytokineand chemokine milieu recruits and activates inflammatorycells including neutrophils, macrophages, B cells, CD4+ Tcells, and CD8+ T cells [17, 18]. In addition to inflamma-tion, abnormal immune function has also been describedin COPD patients, including altered macrophage function[19]. Treatment options that can target the immuneskewing and dysfunction, rather than broad immunerepression, may present a more attractive approach tomanage cigarette smoke-induced COPD.Treatments strategies using bacterial-derived productshave shown promise in diseases characterized by inflam-mation and immune dysfunction [20–23]. This has pri-marily been demonstrated in animal models of allergicairway disease where different treatment strategies usingbacteria or bacterial derived products have been used tomodulate immune responses [20–22, 24]. Towards thatend, intervention strategies encompassing everything fromlive bacteria to specific pattern recognition receptor ago-nists have been used in models of allergic airway disease[21, 22, 25–27]. The apparent mechanism of action worksthrough a reduction in inflammation and an altering ofthe immune response. In studies of smoking-induced lungdisease, Lactobacillus rahmnosus and Bifidobacteriumbreve have been shown to attenuate pro-inflammatorycytokine production in a macrophage cell line treated withcigarette smoke extract [28].The current study investigated for the first time whethera novel immunotherapy produced from inactivatedKlebsiella (hereafter referred to as KB) containing all themajor Klebsiella macromolecules, would attenuate mal-adaptive cigarette smoke exposure-induced immune re-sponses. We hypothesized that KB would re-directdamaging immune responses and attenuate cigarettesmoke-induced lung cellular inflammation and bronchoal-veolar lavage (BAL) cytokine and chemokine production.We demonstrate that subcutaneous administration of KBattenuated cigarette smoke-induced lung inflammationand the quantity of airway BAL lymphocytes and macro-phages while inducing systemic immune activation mim-icking the response to acute infection.MethodsAnimalsFemale mice C57BL/6 age 8–10 weeks old were purchased(Jackson Labs, Farmington, Connecticut, USA), acclima-tized, and housed for one additional week prior to thecommencement of experiments. Female mice were usedas recent studies suggest that they are more susceptible tocigarette smoke induced lung pathology, as are womencompared to men [29]. The experiments included tenmice per group, which were housed as five mice per cagein environmentally-controlled specific pathogen free con-ditions with a 12:12 h light/dark cycle for the duration ofthe study. All protocols were reviewed and approved bythe Animal Care Committee of the University of BritishColumbia (Vancouver, BC, Canada).Cigarette/air smoking protocolAir or cigarette smoke exposure was done for five con-secutive days for the first 2 weeks and for four consecutivedays in the third week (experimental days: 1–5; 8–12,15–18, Fig. 1). Mice were euthanized 24 h after the last ex-posure (experimental day 19). Briefly, cigarette smokeexposure (University of Kentucky Research GradeCigarettes) was performed by placing mice into plexiglass“nose only” exposure chambers as previously described[29, 30]. Each mouse smoked three cigarettes per day for atotal of 45 min of exposure. Control room air-exposedmice were restrained for a similar duration without expos-ure to smoke. Animals were monitored throughout thesmoke exposure procedure and for an additional 30 minpost-smoke exposure.Microbe-based intervention strategyThe microbe-based intervention, KB, is a proprietary im-munomodulator consisting of all major macromoleculesof an inactivated pathogenic Klebsiella strain was origin-ally isolated from a patient with acute pneumonia. KBwas supplied by Qu Biologics (Vancouver, BC). For thetreatment intervention, KB or a placebo vehicle control(physiological saline containing 0.4% phenol) wasprophylactically administered on the experimental day−7, −5, −3, and the regimen continued throughout theBazett et al. Respiratory Research  (2017) 18:92 Page 2 of 11experiment on days 1, 3, 5, 8, 10, 12, 15, 17 (Fig. 1). Eachadministration involved a subcutaneous injection of30 μL of placebo or KB, which was alternatively deliv-ered into the lower right abdomen, the lower left abdo-men, the upper right chest, and the upper left chest,rotating clockwise for each injection day.Blood collection, BAL, and cytospin analysis of BAL celldifferentialsProcessing and analysis of collected terminal blood andBAL samples was done as described previously [22].Cytospins were performed and cells in BAL evaluatedbased on morphology and Wright-Giemsa staining. BALcell differentials were then counted using the preparedcytospin slide with 100 cells per mouse counted in ablinded fashion.Immune mediator profiling of BAL and serum samplesSoluble mediator analysis in BAL and serum wasperformed using a 31 cytokine, chemokine, growth factormultiplex kit according to the manufacturer’s protocol(Millipore, St. Charles, MO, USA) using the Bio-Plex™ 200system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).The multiplex was performed by Eve Technologies (EveTechnologies Corp, Calgary, AB, Canada). The 31-plexassay included the following mediators: Eotaxin, G-CSF,GM-CSF, IFNγ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15,IL-17, IP-10 (CXCL10), KC (CXCL1), LIF, MCP-1 (CCL2),M-CSF, MIG (CXCL9), MIP-1α (CCL3), MIP-1β (CCL4),MIP-2 (CXCL2), RANTES (CCL5), TNFα, and VEGF.The assay sensitivities of these markers range from 0.1 -33.3 pg/mL.Flow cytometric analysis of Ly6CHI monocytes/macrophages and neutrophilsBlood was collected in EDTA coated tubes (BDMicrotainer) to prevent clotting and stored on ice priorto staining. Anti-coagulant-treated whole blood wasstained with antibodies (CD11b-FITC, Ly6G-PE, CD11c-PerCPCy5.5 and Ly6C-APC) before red blood cell lysis(BD lysis buffer). Flow cytometry was run on a FACSCa-libur (BD Bioscience). Analysis was completed using theFlowJo V10.1 program. Neutrophils were defined asLy6G+CD11b+ cells. Ly6CHI monocytes/macrophagewere defined as Ly6CHILy6G−CD11b+ cells. Lymphocytepopulations were gated on by forward scatter and sidescatter and then defined as B220+, CD3+CD4+, or CD3+CD8+.Data analysisGraphPad Prism 6 Software (GraphPad Software, SanDiego, CA) was used to perform statistical analysis ofthe results. Data are expressed as mean ± SD. One-wayANOVA analysis followed by multiple comparisonsusing a Sidak post-hoc test was performed for groupcomparisons. Four experimental group combinationswere compared; room air-placebo vs. room air-KB, roomair-placebo vs. cigarette smoke-placebo, room air-KB vs.cigarette smoke-KB, cigarette smoke-placebo vs.cigarette smoke-KB. Differences were reported as statis-tically significant when p < 0.05.ResultsKB attenuated cigarette smoke exposure-induced influxof lymphocytes and macrophages, but not neutrophilsinto the airwaysA three-week acute model of cigarette smoke exposurein mice (Fig. 1) was used to investigate how KB exposurecan modulate lung inflammation. The total BAL cellcounts and cellular differentials for each experimentalgroup were assessed (Fig. 2). In placebo treated animals,cigarette smoke exposure increased the total number ofBAL cells (Fig. 2a, p < 0.0001). The observed increase incellularity resulted from increases in the numbers oflymphocytes, macrophages, and neutrophils (Fig. 2b, d,p < 0.0001), but not eosinophils (p = 0.35, data notshown). KB intervention did not significantly decreaseFig. 1 Cigarette smoke exposure protocol with Klebsiella (KB) intervention. Four groups of mice were exposed to either placebo + room air, KB + roomair, placebo + cigarette smoke, or KB + cigarette smoke. Grey arrows, room air or cigarette smoke; white arrows, subcutaneous injection of placebo orKB. See methods for detailsBazett et al. Respiratory Research  (2017) 18:92 Page 3 of 11the total number of BAL cells, however it did attenuatethe increase in lymphocytes and macrophages in thecigarette smoke-exposed group (p < 0.005, Fig. 2b-c)while having no detectable impact on neutrophils(Fig. 2d, p = 0.59).KB intervention attenuated cigarette smoke exposure-induced lung inflammatory responsesPrevious reports have demonstrated that cigarette smokeexposure models are characterized by a cytokine profilethat includes IFN-γ, CXCL9 (MIG), CXCL10 (IP-10), andCCL5 (RANTES) [14, 15]. A multiplex analysis of 31 cyto-kines, chemokines, and growth factors (Additional file 1:Table S1) was used to investigate cytokine and chemokineproduction induced by cigarette smoke exposure in thisexperimental system. KB intervention had no impact onair-exposed animals for any mediator measured in theBAL fluid. Cigarette smoke exposure induced 15 of the 31measured mediators in the BAL fluid, which were IFNγ,CXCL9, CXCL10, CCL5, IL-6, IL-17, G-CSF, CXCL1, LIF,CCL2, CCL3, CCL4, TNFα, eotaxin, and VEGF (p < 0.05).Although IL-17 was elevated with cigarette smokeexposure, this was only observed in 4 of 10 samples andthe values were close to the level of detection for thiscytokine (0.64 pg/ml). KB intervention attenuatedcigarette smoke-induced increases in IFNγ, CXCL9,CXCL10, CCL5, IL-6, G-CSF, and IL-17 (Fig. 3, p < 0.05)in the BAL fluid. KB also decreased TNFα levels (cigarettesmoke + placebo 7.50 ± 5.98 pg/ml vs cigarette smoke +KB 3.52 ± 3.34 pg/ml), but this was not statisticallysignificant (P = 0.057, Additional file 1: Table S1).Systemic immune cytokine, chemokine and growth factorprofile were not significantly altered with 3-weekcigarette smoke exposure, but were augmented by KBexposureWhen assessing changes in the serum levels of cytokine,chemokines and growth factors in the experimental treat-ment groups, it was found that cigarette smoke exposureinduced an increase in only VEGF, and this was notFig. 2 KB treatment attenuated cigarette smoke exposure induced increases in airway macrophages and lymphocytes but not total cells or neutrophils.BAL cell counts and differentials following placebo and KB treatment in room air or cigarette smoke-exposed groups. a BAL total cells, b lymphocytes, c,macrophages d, and neutrophils. * p< 0.05 comparing to the groups relative control; # p< 0.05 comparing KB group to relative placebo control. Data aremeans ± SD of 9–10 mice per groupBazett et al. Respiratory Research  (2017) 18:92 Page 4 of 11changed with the KB intervention (Fig. 4a, p < 0.05;Additional file 2: Table S2). KB treatment in air-exposedanimals decreased only one mediator, IL12p40, while in-creasing serum levels of IL-1β, CCL2, CXCL9, andCXCL10 (Fig. 4b-d, p < 0.05). In the cigarette smoke-exposed mice, treatment increased the levels of CXCL9,CXCL10, and CCL5 relative to cigarette smoke + placebotreated groups (p < 0.05). Collectively these data suggestthat KB intervention induced systemic immune responsesthat are independent of cigarette smoke exposure, whichmay play a role in the observed local down-regulation ofcigarette smoke exposure-induced lung inflammation.KB induced both a systemic and local lung tissue increasein the proportion of Ly6CHI monocytes/macrophages andneutrophils, with no change in lymphocyte populationsTo investigate if the KB intervention altered systemic orlocal cellular immune profiles, blood and lung cells wereassessed by flow cytometry, with particular focus on thelevels of Ly6CHI monocytes (an inflammatory subgroupof monocytes, characterized as Ly6G−CD11b+ cells) andneutrophils (characterized as Ly6G+CD11b+ cells).Cigarette smoke exposure had no effects on the numbersof blood Ly6CHI monocytes or neutrophils (Fig. 5a-b,p > 0.6). KB intervention increased the blood Ly6CHImonocytes and neutrophils in the cigarette smokeexposure groups (p < 0.005), and the neutrophils in theair-exposed animals (p = 0.05). The increases in systemicLy6CHI monocytes and neutrophils were associated withlocal increases in the lung tissue (Fig. 5c-d), where KBinduced increases in these cell types, which was furtherenhanced by cigarette smoke exposure (p < 0.05).The levels of B and T lymphocytes were also assessedin the lungs. The percentage of B cells in the lungs aftersmoke exposure was elevated (Fig. 6a, p < 0.05), whichwas not attenuated by KB administration. No statisticallysignificant change in the percentage of CD3+CD4+ orCD3+CD8+ cells was observed following cigarette smokeexposure or KB administration (Fig. 6b-c).Intervention with KB had no impact on clinical score andbody weight following cigarette smoke exposureBody weight and clinical score was used to monitor theoverall health of mice exposed to cigarette smoke in thepresence or absence of KB. Body weight was normalizedto the starting weight of each animal and all animals wereobserved throughout the experiment and their healthassessed based on a clinical score (e.g. hunched posture,interaction with other animals, activity levels). KB admin-istration in the air-exposed group did not significantlyalter the body weight (Fig. 7) nor impact the clinical score(data not shown). No adverse effects of repeated KB ad-ministration were observed. Cigarette smoke-exposedmice had a prominent loss in body weight (p < 0.05),which KB intervention did not attenuate (p > 0.05).Fig. 3 KB treatment attenuated cigarette smoke exposure induced increases Th1-skewed lung inflammatory responses. BAL supernatant fluid analysisfollowing placebo and KB treatment in room air or cigarette smoke-exposed groups. a IFNγ, b CXCL9, c CXCL10, d CCL5, e IL-6, f G-CSF, g CXCL1, hIL-17. * p < 0.05 comparing to the groups relative control; # p < 0.05 comparing KB group to relative placebo control. Data are means ± SD of 10 miceper groupBazett et al. Respiratory Research  (2017) 18:92 Page 5 of 11Cigarette smoke exposure did not significantly changedthe clinical score for either placebo or KB-treated mice.DiscussionThere is growing awareness that exposure to microbialproducts can alter the course of inflammatory diseases. Inasthma, several studies have demonstrated promising re-sults for resolution of symptoms with microbial productsin both animal models and clinical studies [21, 22, 24–27];however, there is a paucity of data looking into the use ofthese approaches aimed at modulating the course of theimmune dysfunction in COPD [31]. In this study, wetested the hypothesis that KB, which was produced from aclinical Klebsiella isolate containing all the majorKlebsiella macromolecules, could modulate airway inflam-mation and immune responses in a mouse model of acutecigarette smoke exposure. These results demonstrate thatprophylactic KB treatment attenuated both cigarettesmoke-induced lung inflammation and BAL macrophageand lymphocyte cellularity. In control room air-exposedand experimental cigarette smoke-exposed animals, KBinduced systemic immune responses, resulting inmobilization of monocytes and neutrophils. This systemicimmune modulation was mirrored locally in lung tissue,reflected by an increase in Ly6CHI monocytes/macro-phages and neutrophils. These data therefore suggest thatinterventions with microbial components that enhancerather than suppress immune responses may provide anovel strategy to alter the course of cigarette smoke ex-posure related COPD pathogenesis. Future therapeuticintervention-dosing strategies will be aimed at determin-ing how late in the course of smoke-induced lung damagesuch a microbe-based intervention strategy can be admin-istered to reverse pathology.Chronic cigarette smoke exposure in humans is associ-ated with emphysema and chronic bronchitis in COPDpatients. Acute cigarette smoke exposure can lead to in-flammatory responses that may be important precedingevents in the chronic changes to lung physiology [32]. Themouse model of acute cigarette smoke exposure used inthis study was designed to determine the impact of KB onmodulating these earlier alterations in the inflammatoryresponse and not the chronic bronchitic or emphysema-tous phenotype observed in chronic mouse cigarettesmoke exposure models [29, 33–38]. This study thereforefocused on outcome measurements that are impacted byFig. 4 KB treatment differentially modulates cigarette smoke exposure induced changes in serum immune mediators. Serum analysis following placeboand KB treatment in room air or cigarette smoke-exposed groups. a VEGF, b IL-1β, c CCL2, d CXCL9, e CXCL10 and f CCL5. * p< 0.05 comparing to thegroups relative control; # p< 0.05 comparing KB group to relative placebo control. Data are means ± SD of 9–10 mice per groupBazett et al. Respiratory Research  (2017) 18:92 Page 6 of 11Fig. 5 KB treatment increased blood and lung Ly6CHI monocytes and neutrophils. Flow cytometric analysis of blood a-b and lung c-d Ly6CHImonocytes and neutrophils following placebo and KB treatment in room air or cigarette smoke-exposed groups. * p < 0.05 comparing to thegroups relative control. # p < 0.05 comparing KB group to relative placebo control. Data are means ± SD of 10 mice per groupFig. 6 KB treatment has no impact on lung B220+ cells, CD3 + CD4+, or CD3 + CD8+ T cells. Flow cytometric analysis of lung a B220+ B cells, b CD3+ CD4+ T cells, or c CD3 + CD8+ T cells following placebo and KB treatment in room air or cigarette smoke-exposed groups. * p < 0.05 comparing tothe groups relative control. Data are means ± SD of 10 mice per groupBazett et al. Respiratory Research  (2017) 18:92 Page 7 of 11acute cigarette smoke exposure, including lung in-flammation resulting from smoke-induced tissue dam-age, systemic inflammation, immune cell activation,and body weight.Cigarette smoke exposure-induced inflammation hasbeen described as TH1 skewed, although this may be anover simplification [14, 15]. Changes in inflammatory me-diators are accompanied by elevations in macrophages,lymphocytes, and neutrophil populations [17, 18, 32].IFN-γ has been implicated as an important participant inthe development of emphysematous lesions followingcigarette smoke exposure in mice [16] and has been asso-ciated with COPD in humans [14, 15]. IFN-γ induces theCXCR3 ligands, CXCL9 (MIG) and CXCL10 (IP-10) [39]and the CCR5 ligand, CCL-5 (RANTES) [16] whichrecruit lymphocytes and macrophages to sites of inflam-mation. Importantly, blocking this response protects micefrom the pathological impacts of cigarette smoke exposure[16, 40]. KB intervention was found to specifically attenu-ate cigarette smoke-induced elevations in IFN-γ, CXCL9,CXCL10, CCL-5, and IL-6 in the BAL, which wasuncoupled from systemic immune activation. This reduc-tion in BAL inflammatory mediators was associated with aconcomitant reduction in macrophage and lymphocyte re-cruitment to the airways. Future mechanistic studies arerequired to determine how modulation of systemic im-mune function alters the progression of lung immunityimportant in chronic models of cigarette smoke exposure.COPD pathology has many pathologic pathways incommon with other inflammatory diseases, includingasthma and inflammatory bowel disease (IBD). In theseindications, microbial products are actively under investi-gation as treatment options [23, 24, 31, 41]. IBD andCOPD share common pathology relating to mucosal bar-rier disruption including an altered microbiome, immunedysfunction, altered epithelial cell function, and chronicinflammation [31, 41, 42]. Live microbial products are cur-rently being tested for efficacy in IBD [43, 44]. Further-more, a product that is prepared in a similar manner toKB, but produced from Escherichia coli, has shownevidence of efficacy in IBD patients [45] and is currentlybeing investigated in clinical trials as a treatment for IBD.There is also significant overlap between asthma andCOPD including altered respiratory microbiome andimmune dysfunction [31, 42, 46, 47]. In animalmodels of allergic asthma, live bacteria and theircomponents [24, 27, 48, 49], Toll-like receptor (TLR)agonists [50], and the KB product [22], have allreduced lung inflammation. Collectively, the primaryresearch presented in this report and the studiesoutlined above demonstrate some of the similaritiesbetween COPD and other inflammatory diseases thatbenefit from microbial intervention strategies. Takentogether, these findings suggest that enhancing orresetting the immune response with bacterial productscould be a novel therapeutic approach to managingCOPD.Systemically, these data showed that KB administration,which contains all the major macromodules from theKlebsiella, increased certain cytokines conventionallyconsidered as being pro-inflammatory cytokines, such asIL-1β, as well as the proportion of blood Ly6CHImonocytes and neutrophils, similar to the response seenwith an acute infection [51–54]. This immune activationand mobilization was also detected in the lung tissue byflow cytometry where an increase in the proportion ofmonocytes and neutrophils was observed. Conversely, theairways of KB treated animals showed a reduction in themacrophage and lymphocyte levels. This duality of the in-creased inflammation in the lung tissue and decreased in-flammation in the airways highlights the importance ofwhere the immune response occurs for resolution ofFig. 7 Cigarette smoke exposure impaired body weight gain independent of KB treatment. Daily measurements of mice were normalized tostarting weight for each of the four groups. * p < 0.05 comparing to the group’s relative control. Data are means ± SD of 10 mice per groupBazett et al. Respiratory Research  (2017) 18:92 Page 8 of 11symptoms. Although the precise mechanism(s) of thisphenomenon are not yet clear, the prevailing evidencesuggests that inflammatory monocytes can differentiateinto multiple different cells types in inflamed/damagedtissue [19, 55, 56] and that enhancement of immune func-tion in the correct tissue-microenvironment may paradox-ically contribute to an attenuation in overall inflammation,potentially by clearing necrotic/damaged tissue and re-building the loss of barrier function. Indeed, the observedincrease in the number of lung Ly6CHI/CD11b+inflammatory monocytes may have the ability to suppressinflammation [56–58].ConclusionsOur study shows that KB, produced from a clinicalKlebsiella isolate, can suppress the progression of localairway immune responses and lymphocyte and macro-phage influx, while inducing a systemic inflammatoryresponse, in a mouse model of acute cigarette smoke-induced lung inflammation. This is the first demonstra-tion that subcutaneous administration of a microbialderived intervention, KB, can attenuate cigarette smoke-induced inflammation. These data provide a foundationfor future studies exploring how KB may be used toeither reverse or prevent progression of establishedemphysema and small airways disease associated withchronic cigarette smoke exposure. Lastly, the datasuggest the intriguing possibility that KB, whichstimulates rather than suppresses systemic immuneresponses, might be a novel means by which the courseof COPD pathogenesis may be altered, highlighting thecomplex interaction between inflammation and COPDpathogenesis.Additional filesAdditional file 1: Table S1. Soluble mediator analysis in BAL fluidfollowing placebo and KB treatment in room air or cigarette smoke-exposed groups. * p < 0.05 comparing to the group’s relative control;# p < 0.05 comparing treated group to untreated relative control. ns= no significant difference. Data are means ± SD of 9–10 mice pergroup. (XLSX 39 kb)Additional file 2: Table S2. Soluble mediator analysis in serum followingplacebo and KB treatment in room air or cigarette smoke-exposed groups. *p < 0.05 comparing to the group’s relative control; # p < 0.05 comparingtreated group to untreated relative control. ns= no significant difference.Data are means ± SD of 9–10 mice per group. (XLSX 36 kb)AbbreviationsANOVA: Analysis of variance; BAL: Bronchoalveolar lavage; COPD: Chronicobstructive pulmonary disease; EDTA: Ethylenediaminetetraacetic acid;G-CSF: Granulocyte-colony stimulating factor; GM-CSF: Granulocytemacrophage colony-stimulating factor; IFNγ: Interferon-gamma; LIF: Leukemiainhibitor factor; M-CSF: Macrophage colony-stimulating factor; TNFα: Tumornecrosis factor-alpha; VEGF: Vascular endothelial growth factor;IBD: Inflammatory bowel disease; TLR: Toll-like receptorAcknowledgementsNot Applicable.FundingThe work was funded by Mitacs and Qu Biologics.Availability of data and materialThe datasets used and/or analysed during the current study available fromthe corresponding author on reasonable request.Authors’ contributionsMark B. helped design the experiments, completed the flow cytometry andthe multiplex analysis, drafted and edited the manuscript. A.B. completed theanimal experiments and drafted the manuscript. R.D.H., M.R.Z., and P.M.H.contributed intellectually to the experiment design and manuscript drafting.Momir B. helped design the experiments, analyse the data and edited themanuscript. H.G. helped design the experiments and edited the manuscript.S.K. helped design the experiments, analyse the data, and edited themanuscript. J.A.H. designed the experiments, completed the experiments,drafted and edited the manuscript, and oversaw the study completion. Allauthors read and approved the final manuscript.Competing interestsMark B. is an employee of Qu Biologics. A.B. was funded by a Mitacs Industrypartnered fellowship with Qu Biologics. R.D.H., M.R.Z., and P.M.H. have nocompeting interests. Momir B. is an employee of Qu Biologics. H.G. is anemployee and co-founder of Qu Biologics. S.K. is an employee of QuBiologics. J.A.H. received consulting fees from Qu Biologics.Consent for publicationNot Applicable.Ethics approval and consent to participateAll studies were approved by the UBC Animal Care Committee.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in publishedmaps and institutional affiliations.Author details1Qu Biologics Inc., Vancouver, BC, CanadaV5T 4T5. 2Department of Medicine,Division of Respiratory Medicine, University of British Columbia, Vancouver,BC, CanadaV6H 3Z6. 3iCORD Research Centre, University of British Columbia,Vancouver, BC, CanadaV5Z 1M5. 4Priority Research Centre for Healthy Lungs,Hunter Medical Research Institute, The University of Newcastle, Newcastle,NSW, Australia. 5Department of Medicine, Division of Endocrinology,CeMCOR, University of British Columbia, Vancouver, BC, CanadaV5Z 1M9.6Firestone Institute for Respiratory Health, Division of Respirology,Department of Medicine, McMaster University, Hamilton, ON, CanadaL8N4A6.Received: 2 January 2017 Accepted: 8 May 2017References1. 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