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The bronchial epithelial cell bacterial microbiome and host response in patients infected with human… Sze, Marc A; Xu, Stella; Leung, Janice M; Vucic, Emily A; Shaipanich, Tawimas; Moghadam, Aida; Harris, Marianne; Guillemi, Silvia; Sinha, Sunita; Nislow, Corey; Murphy, Darra; Hague, Cameron; Leipsic, Jonathon; Lam, Stephen; Lam, Wan; Montaner, Julio S; Sin, Don D; Man, S. F P Nov 9, 2016

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RESEARCH ARTICLE Open AccessThe bronchial epithelial cell bacterialmicrobiome and host response in patientsinfected with human immunodeficiencyvirusMarc A. Sze1*, Stella Xu1, Janice M. Leung1, Emily A. Vucic2, Tawimas Shaipanich3, Aida Moghadam4,Marianne Harris4,5,6, Silvia Guillemi4,5,6, Sunita Sinha7, Corey Nislow7, Darra Murphy8, Cameron Hague8,Jonathon Leipsic8, Stephen Lam2, Wan Lam2, Julio S. Montaner6,9, Don D. Sin1,3 and S. F. Paul Man1,3AbstractBackground: Chronic Obstructive Pulmonary Disease (COPD) is an important comorbidity in patients living withhuman immunodeficiency virus (HIV). Previous bacterial microbiome studies have shown increased abundance ofspecific bacterium, like Tropheryma whipplei, and no overall community differences. However, the host response tothe lung microbiome is unknown in patients infected with HIV.Methods: Two bronchial brush samples were obtained from 21 HIV-infected patients. One brush was used for bacterialmicrobiome analysis using the Illumina MiSeqTM platform, while the other was used to evaluate gene expression patternsof the host using the Affymetrix Human Gene ST 2.0 array. Weighted gene co-expression network analysis was used todetermine the relationship between the bacterial microbiome and host gene expression response.Results: The Shannon Diversity was inversely related to only one gene expression module (p= 0.02); whereas evennesscorrelated with five different modules (p≤ 0.05). After FDR correction only the Firmicutes phylum was significantlycorrelated with any modules (FDR < 0.05). These modules were enriched for cilia, transcription regulation, and immuneresponse. Specific operational taxonomic units (OTUs), such as OTU4 (Pasteurellaceae), were able to distinguish HIVpatients with and without COPD and severe emphysema.Conclusion: These data support the hypothesis that the bacterial microbiome in HIV lungs is associated with specifichost immune responses. Whether or not these responses are also seen in non-HIV infected individuals needs to beaddressed in future studies.Keywords: Bronchial brushing, Bacterial microbiome, HIV, Lungs, Gene expressionBackgroundThe increased susceptibility of patients infected withhuman immunodeficiency virus (HIV) to lung diseases,in particular chronic obstructive pulmonary disease(COPD), has now been recognized in numerousepidemiological studies [1–3]. Because cigarette smokeexposure only partially explains this elevated risk [1], thepathogenesis of comorbid lung disease in HIV is largelya mystery. Investigation of the HIV lung bacterial micro-biome using bronchoalveolar lavage fluid has suggesteda greater abundance of Tropheryma whipplei in the HIVlung [4] and no significant impact of anti-retroviral ther-apy on the bacterial community composition in HIV-infected individuals [5]. However, the impact of the lungmicrobiota on the pathogenesis of chronic lung diseasessuch as COPD in HIV is unclear. Moreover, there is lit-tle information whether the lung microbiome is associ-ated with significant host responses in the lungs.* Correspondence: marcsze@med.umich.edu1Centre for Heart Lung Innovation, St. Paul’s Hospital & Department ofMedicine, University of British Columbia, Rm 166 – 1081 Burrard St.,Vancouver, BC V6Z 1Y6, CanadaFull list of author information is available at the end of the article© The Author(s). 2016 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.Sze et al. BMC Pulmonary Medicine  (2016) 16:142 DOI 10.1186/s12890-016-0303-4Although characterization of bacterial communitycomposition in a disease state is an important first stepin uncovering the possible clinical relevance of the lungmicrobiome [4, 6], the next logical step is to discoverwhether or not changes in the lung microbiome inducea host response that may be important in disease patho-genesis. We have recently shown, using lung tissue sam-ples from non-HIV infected individuals with COPD, thatshifts in the lung microbiome are associated with im-portant changes in inflammatory response in these lungs[7]. One important limitation of that study was that themicrobiome was characterized in a block of lung tissueand as such cell-specificity could not be ascertained.Moreover, this study did not include any patients withHIV infection. Here, we extend these observations byinvestigating the interactions between the host geneexpression response and the bacterial microbiome inbronchial epithelial cells of small airways collected fromthe same site in patients infected with HIV. The specificaims of this study were to describe the bacterial commu-nity composition of the HIV bronchial epithelium and todetermine whether the bacterial microbiome of the HIVbronchial epithelium is associated with specific geneexpression signatures of the host that may reveal theunderlying pathogenesis of chronic airways disease inHIV-infected individuals.MethodsPatient populationAll subjects provided written informed consent for thecollection of cytologic brushings for research purposesunder the UBC Providence Health Care ethics protocolH14-03267. Subjects were recruited from patients under-going bronchoscopy for pulmonary nodules, masses, orpneumonia (all conditions were diagnosed radiographic-ally by computed tomography (CT) imaging at St. Paul’sHospital, Vancouver, BC). Entry criteria into the study in-cluded documented HIV-1 infection and ≥19 years of age.All subjects performed spirometry according to theAmerican Thoracic Society/European Respiratory Societyguidelines [8] within three months, except for five subjectswho underwent bronchoscopy for acute infection. COPDwas defined by post-bronchodilator forced expiratoryvolume in one second (FEV1)/forced vital capacity (FVC)ratio of less than 70 %.Patients underwent thoracic CT imaging using a 64 de-tector CT scanner (Discovery HD 750 or a VCT, GEHealthcare, Milwaukee, WI). A central imaging core la-boratory (SPH CT Corelab), blinded to spirometry andclinical data, interpreted the CT images for emphysemabased on a modified method of Kazerooni, et al. [9].Emphysema severity was qualitatively scored according toan established algorithm (see Additional file 1). CT scanswere also qualitatively scored for respiratory bronchiolitis(none, trivial, mild, moderate, and severe) and bron-chiectasis (presence or absence). Details on bronchos-copy and specimen collection can be found in theAdditional file 1. Bronchial epithelial cells were ob-tained from sites away from the acute infection,masses or nodules.Bacterial microbiome analysisDNA was extracted using the Qiagen DNeasy Blood andTissue Kit (Qiagen, Toronto, Ontario) from both patientsamples and background negative environmentalcontrols. Total 16S load was quantified using a dropletdigital polymerase chain reaction (ddPCR) assay [10].These background controls were used to assess whetherthe bacterial community of the HIV samples were im-pacted by the instruments and reagents used during theextraction and PCR process. To assess the 16S loadwithin the samples the average 16S load from thenegative controls were subtracted from each HIV 16Ssample. Touchdown PCR [11] of the 16S rRNA gene V4region was used to generate a DNA template for sequen-cing. Cycle conditions for the touchdown PCR can befound in the Additional file 1. Sequencing was per-formed on an Illumina MiSeqTM (Illumina, RedwoodCity, CA, USA) with 2 × 250 paired end-read chemistry.The protocol established by Kozich, et al. was used forthe sequencing and subsequent data cleanup within theprogram mothur (V1.34.4) [12]. After processing,sequence cleanup, and chimera removal, a total of3,559,398 reads remained. Data analysis was performedin R (V3.2.0) and R studio (V0.99.441) employing thevegan (V2.3-0) package [13, 14]. In order to adequatelyperform alpha and beta diversity analysis subsampling tothe lowest total reads (3164) was performed [15]. Alongwith a 97 % similarity threshold, a total of 451 differentOperational Taxonomic Units (OTUs) were identified.Sequence data has been deposited in the NCBI sequenceread archive under the accession number SRP068430.The corresponding metadata can be found at http://www.ncbi.nlm.nih.gov/Traces/study/?acc=SRP068430&go=go.Alternatively, one can use the following link http://www.ncbi.nlm.nih.gov/Traces/study/ and search for SRP068430to bring up the relevant information needed. The relevantsamples used for this study are BIDC1-4, BIDC7-10,BIDC14-26 for the HIV samples and Neg1-4 for the back-ground negative control samples.Microarray analysisRNA was extracted using the Qiagen RNeasy PlusUniversal Kit (Qiagen, Toronto, Ontario). 1 ug of RNAwas processed and hybridized onto the AffymetrixHuman Gene ST 2.0 array (Affymetrix Inc, Santa Clara,USA) according to the manufacturer’s protocol at theHospital for Sick Children, Centre for Applied GenomicsSze et al. BMC Pulmonary Medicine  (2016) 16:142 Page 2 of 10(Toronto, Ontario). Raw CEL files were processed andRMA normalized in R (V3.2.0) and R studio (V0.99.441)using a standard protocol from the oligo package(V1.32.0) [16]. Gene symbols and names were obtainedfrom the hugene20sttranscriptcluster.db from biocon-ductor [17].Data analysis and integrationFor phyla level comparisons a t-test with Bonferroni cor-rection was applied. A random forest algorithm withBoruta feature selection [18, 19] was used to identify anyOTUs that could be discriminative of specific clinicaltraits in the patient population (e.g. smoking status,COPD, etc.). Traits were chosen for Boruta featureselection analysis based on whether or not theirPERMANOVA value was less than or equal to 0.1. Wedetermined differences in the bacterial communitycomposition between groups by a Bonferroni correctedPERMANOVA [20] of ≤ 0.0125. Robustly co-expressedsets of genes (i.e. modules) were identified in airwayexpression data using a weighted gene co-expressionnetwork analysis (WGCNA) [21, 22]. Modules eigengenevector values were then compared to alpha diversitymeasures (Shannon Diversity, OTU richness, andevenness) and phyla measures using the WGCNA(V1.46) R package [21, 22]. For this analysis no groupingwas performed by smoking, CD4 cell count, or viral loadstatus as these variables were not significantly associatedwith microbiome measures. The Database for Annota-tion, Visualization, and Integrated Discovery (DAVID)[23] was used to identify the most relevant pathway clus-ters for each module that were significantly correlated tothe specific bacterial microbiome measurements. Thetop 10 enrichment clusters were used as a guide to dis-covering pathways that were most strongly associatedwith each module. A false discovery rate (FDR) of lessthan 0.05 was considered significant. In addition to thismodule comparison versus microbiome metrics for ournetwork used p-values of less than 0.05 as well. TheOTU data was reported to the lowest taxonomic identi-fication, either within the text or in the respective figure.ResultsOverview of the bacterial microbiome in the HIV cohortAn overview of the clinical characteristics of the studysubjects showed that all individuals were between 40and 75 years of age with a majority on highly active anti-retroviral therapy (HAART) at the time of assessment[Table 1]. The total 16S concentration in each subjectfollowing background negative control subtraction was0.42 ± 1.39 16S/ng of DNA (mean ± standard deviation).The Shannon Diversity was 2.13 ± 0.54, OTU richnesswas 37.52 ± 11.83, and evenness was 0.59 ± 0.13 for thispatient population (mean ± standard deviation). Thedistribution of Shannon Diversity, OTU richness, andevenness can be found in the Additional file 1: Table S1.On a cursory overview, the phyla distribution seems tobe quite different than the experimental backgroundnegative controls. However, there was only a significantdifference in the relative abundance of the Acti-nobacteria phylum, following Bonferroni correction, be-tween the HIV group and background negative controls(p = 0.003) [Fig. 1a]. This would suggest that apart fromthe Actinobacteria phylum the other phyla distributionsare similar to the background negative controls. A totalof 23.8 % of HIV subjects contained OTUs that alignedto Tropheryma.Airway microbiome comparisons between those with andwithout COPD by spirometryThere was no difference in Shannon Diversity, evenness,and OTU richness between HIV patients with andwithout COPD (p > 0.05). The diagnosis of COPD hadno influence on the phyla observed (p > 0.05) [Fig. 1b].However, in COPD, there was a trend towards greaterabundance of the Actinobacteria and Proteobacteriaphyla. Using a Bray-Curtis dissimilarity matrix andNon-Metric Multidimensional Scaling (NMDS) withPERMANOVA, we found no significant difference in thebacterial community composition between those withand without COPD (PERMANOVA = 0.10) [Fig. 2a].However, analysis of specific OTUs in relation to COPDstatus revealed 3 OTUs that were able to discriminateHIV patients with and without COPD [Fig. 2b]: OTU4(Pasteurellaceae), OTU15 (Brachybacterium), andOTU38 (Yersinia). In COPD samples, there was a pau-city of OTU4 and OTU15, and a slight enrichment ofOTU38 [Fig. 2b]. Ribosomal database classifier [24, 25]revealed that OTU4 contained sequences of bacteria inthe genus for HaemophilusAirway microbiome comparisons based on CT presence ofemphysema or bronchiectasisWe did not detect a significant difference in the bacterialcommunity composition based on severe emphysemastatus that was detected on CT scans (PERMANOVA =0.06) [Fig. 2c]. However, there were two OTUs thatdiscriminated samples from those with and withoutemphysema: OTU4 (Pasteurellaceae-Haemophilus) andOTU30 (Pedobacter). There was no difference in bacter-ial community composition in relation to emphysemadistribution (whether centrilobular or paraseptal) oracross respiratory bronchiolitis severity (both PERMA-NOVA > 0.10) (data not shown).In those with bronchiectasis on CT, the bacterial commu-nity composition was also not significantly different fromthose without any bronchiectasis (PERMANOVA= 0.04)Sze et al. BMC Pulmonary Medicine  (2016) 16:142 Page 3 of 10[Fig. 2e]. Two OTUs were important for this discrimin-ation: OTU1 (Prevotella), and OTU38 (Yersinia).The impact of acute lung infection on airway microbiomeand host responsesWe found no significant difference in the bacterial com-munity composition between those with and withoutpneumonia (PERMANOVA = 0.30) probably because thebronchoscopic samples were obtained from the lungcontralateral to the site of active infection. We alsofound no significant differences in bacterial communitycomposition between those with CD4 counts above orbelow 200 (PERMANOVA = 0.84), across smoking status(current, past, or never smokers; PERMANOVA = 0.37),or whether Tropheryma was detected or not (PERMA-NOVA = 0.16) [Additional file 1: Figure S1–S5].Significant pathways in WGCNA modules that correlatedwith the bacterial microbiomeThe power measurement of 6 was used to create thegene co-expression network and a single sample wasexcluded since it was an extreme outlier [21, 22][Additional file 1: Figures S6 and S7]. This sample wasconsidered an outlier since on the hierarchical clusteringof the gene expression data it formed its own uniquebranch on the tree versus all other samples [Additionalfile 1: Figure S7]. DAVID was used to assess the mostrelevant pathways involved in the WGCNA modulesthat correlated with the bacterial microbiome. In totalthere were 14/23 (60.8 %) gene expression modules thatcorrelated with at least one measure of the bacterialmicrobiome [Table 2]. Most interesting were theimmune pathways identified by the Tan, Red, Pink, andGreen Yellow modules and the cilia pathways repre-sented by the Green module [Table 2].WGCNA of alpha diversity and phyla with geneexpressionOur analysis revealed a number of gene expression mod-ules that correlated with the Firmicutes phylum. It wasthe only group that had significant correlations with anymodules after FDR correction (two negative and twopositive correlations). The negatively correlated moduleswere Green (FDR = 0.037, p = 4 × 10-4), Midnight Blue(FDR = 0.037, p = 8 × 10-4). The positively correlatedmodules were, Brown (FDR = 0.037, p = 8 × 10-4), andBlue (FDR = 0.037, p = 5 × 10-4) [Fig. 3 and Additionalfile 1: Figure S9].When looking at those that had a p value under 0.05but not an FDR under 0.05 there were additionalTable 1 An overview of clinical traits of HIV infected patients sampled in this studyAge VitalStatusCurrent VL Current CD4 BronchoscopyIndicationSmokingStatusPack-YearsCurrentHAARTCTEmphysemaCTBronchiectasisFEV1 (L) FEV1/FVC(%)60-69 Alive <40 400–499 Cancer Current 30 Yes Yes Yes 1.53 33.0450–59 Alive 1000–9999 100–199 Pneumonia Current 39 No Yes No 1.11 90.0070–79 Alive <40 500–599 Cancer Current 130 Yes Yes No 2.99 70.5550–59 Alive <40 600–699 Cancer Current 12.5 Yes Yes No 3.35 56.6350–59 Alive 40–1000 500–599 Cancer Current 37.5 Yes Yes No 2.71 64.1870–79 Alive 10000–99999 200–299 Cancer Current 30 No No No N/A N/A50–59 Alive <40 900–999 Cancer Current 15 Yes No No 3.33 70.9550–59 Alive 10000–99999 200–299 Pneumonia Current 19.5 No Yes No N/A N/A60–69 Alive <40 700–799 Cancer Past 20 Yes Yes No 2.54 59.8660–69 Alive <40 800–899 Cancer Past 3 Yes Yes No 2.87 71.4460–69 Alive 40–999 200–299 Cancer Past 45 Yes No No 2.41 76.8060–69 Alive <40 ≥1000 Bronchiectasis Past 12 Yes No Yes 3.06 70.5760–69 Alive <40 300–399 Pneumonia Past 75 Yes No Yes 2.47 8540–49 Alive <40 100–199 Cancer Current 30 Yes Yes No 2.41 51.5640–49 Alive 10000–99999 100–199 Pneumonia Current 115 Yes Yes Yes 2 78.2850–59 Alive <40 400–499 Cancer Past 90 Yes Yes No 3.33 75.7670–79 Deceased <40 400–499 Cancer Past 20 Yes Yes Yes 2.75 69.0960–69 Deceased <40 100–199 Cancer Past 4 Yes No Yes 2.45 72.8660–69 Deceased <40 100–199 Cancer None 0 Yes Yes No N/A N/A50–59 Deceased <40 100–199 Pneumonia Current N/A Yes Yes No N/A N/A40–49 Deceased ≥100000 <100 Cancer None 0 No Yes No N/A N/ASze et al. BMC Pulmonary Medicine  (2016) 16:142 Page 4 of 10correlations that have been summarized in Fig. 3. Briefly,the Tan module may be negatively correlated with bothShannon Diversity and evenness (FDR = 0.283, p = 0.02and FDR = 0.368, p = 0.03 respectively) [Fig. 3a]. Even-ness may also be correlated with the Midnight Bluemodule (FDR = 0.184, p = 0.009) [Fig. 3a]. The Proteo-bacteria phylum may also be positively correlated withthe Magenta module (FDR = 0.283, p = 0.02) and theTurquoise module (FDR = 0.368, p = 0.03) [Fig. 3b].WGCNA of the important OTUs and gene expressionNo modules and OTUs found to be predictive byrandom forest for COPD, severe emphysema, orbronchiectasis were found above an FDR of 0.05. are de-scribed in the Additional file 1: Figure S10. However,some correlations between modules and these OTUs oc-curred with a p-value under 0.05. This included OTU4[Fig. 3c] which was negatively correlated with the Grey(FDR = 0.941, p = 0.04), Green (FDR = 0.941, p = 0.04),and Green Yellow (FDR = p = 0.03) modules. It was alsopositively correlated with the Blue (p = 0.004) and DarkGreen (p = 0.04) modules.DiscussionThe interplay between the microbiome and host geneexpression is increasingly recognized as a key element ofhealth and disease. Our study extensively examined therelationship between the bacterial microbiome and hostgene expression from bronchial epithelial cells takenfrom the same small airways of patients infected withHIV. We found that the small airway microbiome ofHIV-infected patients demonstrated only modest differ-ences in the global bacterial community compositioncompared with background negative controls [Fig. 1].However, we did not find any significant differences inthe global airway bacterial composition between thoseFig. 1 Breakdown of major phyla. a Comparison between HIV patient samples (n = 21) and background negative controls (n = 4). There was asignificant difference in the Actinobacteria phylum between HIV and background negative controls (p = 0.003). There were also slightly moreProteobacteria in the HIV group than in the background negative controls (p > 0.05). b Comparison between HIV patients with (n = 6) andwithout COPD (n = 10). No difference between the different phyla was observed (p > 0.05)Sze et al. BMC Pulmonary Medicine  (2016) 16:142 Page 5 of 10with and without COPD, between those with elevated orreduced CD4 counts, between those with bronchiectasis,or between those with and without emphysema on CTscans [Fig. 2]. However, when we investigated individualOTUs using an unocrrected PERMANOVA threshold of0.10 or below, we discovered OTU signatures that weredistinct for those with COPD (measured by spirometry),severe emphysema (detected on CT), and bronchiectasis.Spirometry-based COPD was associated with OTU4,OTU15, and OTU38, severe emphysema was associatedwith OTU4 and OTU30, while bronchiectasis wasassociated with OTU1 and OTU38. More importantly,we found that measures of the airway microbiome in-cluding alpha diversity measures, phyla, and OTUs, weresignificantly related to distinct host response in the sameairway as captured by gene expression modules. Many ofthese modules involved immune and inflammatoryresponses, cell signaling, and cilia pathways suggestingimmunomodulatory role of the airway microbiota in thehost’s ability to process and remove irritants and aero-pathogens. Additional work will be needed to validatethis hypothesis.Fig. 2 Bacterial community composition and COPD status, severe emphysema, and bronchiectasis. a Non-metric multidimensional scaling analysis ofindividuals with and without COPD, PERMANOVA = 0.10. b Boxplot of the relative abundance of each of the discriminative OTUs for COPD status.c Non-metric multidimensional scaling analysis of individuals with and without severe emphysema, PERMANOVA = 0.06. d Boxplot of the relativeabundance of each of the discriminative OTUs for severe emphysema. e Non-metric multidimensional scaling analysis of individuals with and withoutbronchiectasis, PERMANOVA = 0.04. f Boxplot of the relative abundance of each of the discriminative OTUs for bronchiectasisTable 2 An overview of significant gene expression module pathwaysGene Expression Module Number of Genes Pathway Identified FDRTan 201 Lysosome, Immune Response, Plasma Membrane <5.0 × 10-4Red 554 Immune Response, Defense Response, InflammatoryResponse, Response to Wounding<1.0 × 10-12Midnight Blue 82 Magnesium Ion Binding <0.05Green 791 Cilia <2 × 10-4Turquoise 6050 Intracellular Organelle, Membrane-Enclosed Lumen <2 × 10-7Dark Green 43 None Identified N/ABlack 452 Cell to Cell Signaling, Cell Membrane <1.0 × 10-5Magenta 365 Oxidation/Reduction, Microsomes <2.0 × 10-2Pink 427 Immune Response, Immunoglobulion, Antigen Presentation <1.0 × 10-3Brown 1274 Glycoprotein, Plasma Membrane, Immune Response <1.0 × 10-6Blue 5675 Nucleus, Transcription Regulation, Nuclear Lumen <2 × 10-4Grey 5861 Olfactory Transduction <4.9 × 10-42Green Yellow 245 Immunoglobulin, Antigen Processing and Presentation <1 × 10-4Light Green 63 None Identified N/ASze et al. BMC Pulmonary Medicine  (2016) 16:142 Page 6 of 10Fig. 3 (See legend on next page.)Sze et al. BMC Pulmonary Medicine  (2016) 16:142 Page 7 of 10Our findings may be consistent with previous studies onthe lung microbiome in HIV, which found Tropherymawhipplei as a discriminative bacterium in bronchoalveolarlavage fluid (BALF), occurring in 13.4 % of HIV subjectsversus only 1.3 % of HIV-uninfected subjects [4]. In ourstudy, which used bronchial brushes rather than BALF, wedemonstrated the presence of Tropheryma in 23.4 % ofthe samples. Although these data are in line with theprevious literature, additional molecular studies such as aqPCR assay targeting a gene specific for the species wouldbe needed to confirm that the Tropheryma we identifiedwas indeed T.whipplei. We extend the previous findingsby characterizing the host gene expression response to thebacterial microbiome. For instance, we found that Shan-non Diversity and evenness were negatively correlatedwith genes involved with lysosome formation and immuneresponse. This finding is consistent with the evolving con-cept that reduction in bacterial diversity is associated withan elevated risk of clinical infection and increased inflam-matory response by the host [26, 27]. It should be notedthat certain organisms independent of their numbers, aremore likely to elicit an inflammatory response comparedwith others that are less “pathogenic”. For instance, al-though in our study we found that bacteria in the Actino-bacteria phylum were significantly more abundant in HIVlungs than in the background negative environmentalcontrols, these bacteria were not significantly associatedwith gene expression modules. In contrast, bacteria in theFirmicutes phyla (though less abundant compared withActinobacteria) were significantly associated with severaldifferent gene expression modules. Firmicutes were nega-tively related to pathways governing cilium and positivelyassociated with gene expression modules associated withimmune response and transcription regulation. When weexplored all correlations that had a p-value under 0.05[Fig. 3] the Proteobacteria phylum was positively associ-ated with gene expression pathways related to oxidation/reduction and intracellular orgnaelles, whereas the Firmi-cutes phylum was negatively associated with these path-ways. This data is consistent with a previous study whichreported a natural antagonism between the Firmicutesand Proteobacteria phyla in the oropharynx [28]. Thesedata are also consistent with the evolving concept that thelung microbiome is propagated by upper airway seeding[29, 30]. We speculate that the host immune response isregulated in the HIV lung by the seeding of certain organ-isms from the upper airways into the lower airway tract.We posit that the predominance of Firmicutes phylumleads to a heightened inflammatory state. Additional stud-ies into the host interactions with the bacterial micro-biome within the lung will need to be completed toconfirm this hypothesis.Most intriguingly, we found that OTU4 (Pasteurella-ceae-Haemophilus) was predictive of both COPD (byspirometry) and severe emphysema (by CT). Previousstudies suggest that Haemophilus influenzae is an im-portant pathogen in COPD [31] and a recent study usingwhole lung tissue has shown that this organism is foundin control subjects but not in patients with GOLD 4 se-verity [7]. Consistent with this observation, in our studyHaemophilus spp was found in airways of patients with-out COPD by spirometry and without significant emphy-sema on CT scan [Figs. 2 and 3], although there were nocorrelations under and FDR < 0.05 when exploring OTUcorrelations with gene expression modules. Intriguingly,those correlations that were under a p value of 0.05showed that OTU4 negatively correlated with both path-ways involved with cilia and antigen processing andpresentation [Fig. 3]. This raises the tantalizing hypoth-esis that up-regulation of immune genes which activatethe adaptive immune processes may enable processingand removal of Haemophilus spp in the airways. How-ever, this result would need to be validated in a studywith more power to asses this relationship. Up-regulation of genes involved in cilia may have a similareffect. We speculate that COPD airways have altered im-mune and/or cilia function that may prevent effectiveclearance of Haemophilus spp. Additional work will beneeded to validate this hypothesis.There are several limitations to this study. First, thefindings pertain exclusively to HIV-infected patients. Thusit is possible that these OTUs may not help to distinguishCOPD, severe emphysema, or bronchiectasis in HIV-uninfected patients. However, a recent study suggests thatthe bacterial microbiome between HIV-uninfectedpatients and HIV patients on successful antiretroviraltherapy may be similar [5]. Second, no oral wash wasperformed prior to the bronchoscopy. This could have ledto minor contaminations of the bronchial brush samples.However, it was reassuring that the findings of the presentstudy were consistent with others that used mouth rinsingprocedures [6, 29, 32]. Thirdly, all patients enrolled in thisstudy had a clinical indication for bronchoscopy. Whilegreat care was taken to sample epithelial cells from un-infected regions of the lung, and far away from nodules ormasses, our results could be confounded by these(See figure on previous page.)Fig. 3 Network of the module correlations with bacterial microbiome measures. a Gene expression modules and alpha diversity measures. b Geneexpression modules and bacterial phyla. c Gene expression modules and important OTUs for COPD, severe emphysema, and bronchiectasis. Inbrackets under each module is a brief description of pathways identified by DAVID for genes in the module. Red represents significant positivecorrelations while black represents significant negative correlationsSze et al. BMC Pulmonary Medicine  (2016) 16:142 Page 8 of 10underlying conditions. However, we did not find the bac-terial species identified by routine clinical culture in thosepatients with a diagnosis of pneumonia in the analysis of16S, which would support that sampling was indeed fromthe unaffected portions of the lung. This would explainthe fact that there was no difference in the bacterial com-munity composition between those with and withoutpneumonia. Another limitation is that we were unable tovalidate the microarray expression results with RT-PCRdue to the large size of many of the modules. Thus it ispossible that some of the genes within the modules arenot accurate. However, a module is based on more thanone gene and in order for a module to be wrong the ma-jority of gene expression values within it would have to beincorrect. Finally, it is possible that some of the gene ex-pression could be accounted for by infiltrating immunecells that were taken along with the epithelial cells duringsampling. We cannot conclusively rule this possibility outbut samples in this study were obtained away from loca-tions with signs of clear inflammation. Future studies inwhich the bronchial epithelial cell microbiome is assessedin asymptomatic HIV-infected individuals would help toclarify the relationships between the microbiome and hostresponse, and in certain pulmonary phenotypes.Overall, this study provides a preliminary investigationinto the host gene expression interaction with the bac-terial microbiome in the small airways of HIV infectedindividuals. It supports the hypothesis that diversity andevenness of the community are important in modulatinginflammatory responses of the host. This study alsoshows how bacteria in some phyla and OTUs may beimportant in disease pathogenesis by modifying eitherthe host response and/or ecological niche areas. Ourwork supports the possibility that specific interactionsbetween the bacterial microbiome and host cells withinthe airways of the lung occur and may be associatedwith distinct disease phenotypes; these findings wouldrequire additional studies for validation.ConclusionsIn summary this study demonstrates that the bacterialmicrobiome and host gene expression may interact withone another in individuals with HIV infection. It identifiespathways, such as the mucocillary transport system, asimportant in the interaction between host and bacterialmicrobiome. However, more research into this specificarea needs to be accomplished to confirm these resultsand observations.Additional fileAdditional file 1: Table S1. Alpha diversity measures used in theanalysis. Figure S1. NMDS of HIV patients based on pneumonia status.Figure S2. NMDS of HIV patients based on CD4 counts below 200.Figure S3. NMDS of HIV patients based on smoking status. Figure S4.NMDS of HIV patients based on whether Emphysema was present on CTscan. Figure S5. NMDS of HIV patients based on whether Tropherymawas present. Figure S6. Power threshold calculation for WGCNA. Figure S7.Outlier detection for WGCNA. A single sample was removed (BIDC25) since itclustered quite differently than the rest of the data set. Figure S8. Heatmapshowing that BIDC25 is also very different than other samples versus traits ofinterest. Figure S9. Module trait relationship with alpha diversity measuresand phyla. Green squares represent negative correlations while red squaresrepresent positive correlations. The tow numbers displayed in each square isthe R-value correlation and P-value (number in brackets) respectively. FigureS10. Module trait relationship with the important OTUs identified. Greensquares represent negative correlations while red squares represent positivecorrelations. The two numbers displayed in each square is theR-value correlations and P-value (number in brackets respectively).(DOC 430 kb)Abbreviations16S: 16 Svedberg; BC: British Columbia; BIDC: Internal naming system forpatient samples; COPD: Chronic Obstructive Pulmonary Disease;CT: Computed Tomography; DAVID: Database for Annotation Visualizationand Integrated Discovery; ddPCR: Droplet digital Polymerase Chain Reaction;FDR: False discovery rate; FEV1: Forced expiratory volume in 1 second;FVC: Forced vital capacity; HAART: Highly active antiretroviral therapy;HIV: Human immunodeficiency virus; NCBI: Nationa Center for BiotechnologyInformation; Neg: Negative; NMDS: Non-metric multidimensional scaling;OTU: Operational taxonomic unit; PCR: Polymerase chain reaction;PERMANOVA: Permutation analysis of variance; RMA: Robust multi-arrayaverage; rRNA: Ribosomal ribonucleic acid; SPH: Saint Paul’s Hospital;SRP: Sequence read project; UBC: University of British Columbia;WGCNA: Weighted gene co-expression network analysisAcknowledgementsNone.FundingFunding was provided by the Canadian Institutes of Health Research grantnumber 342422 and the BC Lung Association.Availability of data and materialsData for this study have been provided at http://www.ncbi.nlm.nih.gov/Traces/study/ (search for SRP068430).Authors’ contributionsStudy conception and design: MAS, JML, EAV, SL, WL, DDS, SFPM. Dataacquisition: SX, JML, EAV, TS, AM, MH, SG, SS, CN, DM, CH, JL, SL, WL, JSM,DDS, SFPM. Data analysis: MAS, JML, EAV. Manuscript writing: MAS, JML.Manuscript editing: MAS, SX, JML, EAC, MH, SG, DM, CH, JL, SL, WL, JSM, DDS,SFPM. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.Consent for publicationConsent to publish was not obtained, therefore key baseline demographicinformation was summarized to prevent identification of subjects.Ethics approval and consent to participateAll patients provided written informed consent to participate. The study wasapproved by the University of British Columbia Providence Health Careethics board (Protocol H14-03267).Author details1Centre for Heart Lung Innovation, St. Paul’s Hospital & Department ofMedicine, University of British Columbia, Rm 166 – 1081 Burrard St.,Vancouver, BC V6Z 1Y6, Canada. 2Department of Integrative Oncology, BCCancer Research Centre, Vancouver, BC, Canada. 3Division of RespiratoryMedicine, St. Paul’s Hospital, University of British Columbia, Vancouver, BC,Canada. 4AIDS Research Program, St. Paul’s Hospital, Vancouver, BC, Canada.5Department of Family Medicine, Faculty of Medicine, University of BritishSze et al. BMC Pulmonary Medicine  (2016) 16:142 Page 9 of 10Columbia, Vancouver, BC, Canada. 6Division of HIV/AIDS, Department ofMedicine, University of British Columbia, Vancouver, BC, Canada. 7Faculty ofPharmaceutical Sciences, Pharmaceutical Sciences Building, University ofBritish Columbia, Vancouver, BC, Canada. 8Department of Radiology andDiagnostic Imaging, St. Paul’s Hospital, Vancouver, BC, Canada. 9BritishColumbia Centre for Excellence in HIV/AIDS, St. Paul’s Hospital, Vancouver,BC, Canada.Received: 20 April 2016 Accepted: 27 October 2016References1. Crothers K, Butt AA, Gibert CL, Rodriguez-Barradas MC, Crystal S, Justice AC,et al. Increased COPD among HIV-positive compared to HIV-negativeveterans. Chest. 2006;130(5):1326–33.2. Crothers K, Huang L, Goulet JL, Goetz MB, Brown ST, Rodriguez-Barradas MC,et al. HIV infection and risk for incident pulmonary diseases in the combinationantiretroviral therapy era. Am J Respir Crit Care Med. 2011;183(3):388–95.3. 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The lung tissue microbiome in chronic obstructive pulmonary disease.Am J Respir Crit Care Med. 2012;185(10):1073–80.•  We accept pre-submission inquiries •  Our selector tool helps you to find the most relevant journal•  We provide round the clock customer support •  Convenient online submission•  Thorough peer review•  Inclusion in PubMed and all major indexing services •  Maximum visibility for your researchSubmit your manuscript atwww.biomedcentral.com/submitSubmit your next manuscript to BioMed Central and we will help you at every step:Sze et al. BMC Pulmonary Medicine  (2016) 16:142 Page 10 of 10


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