TY - JOUR
AU1 - Johnson, Raymond M.
AU2 - Kerr, Micah S.
AB - Chlamydia trachomatis urogenital serovars are intracellular bacteria that parasitize human reproductive tract epithelium. As the principal cell type supporting bacterial replication, epithelial cells are central to Chlamydia immunobiology initially as sentries and innate defenders, and subsequently as collaborators in adaptive immunity-mediated bacterial clearance. In asymptomatic individuals who do not seek medical care a decisive struggle between C. trachomatis and host defenses occurs at the epithelial interface. For this study, we modeled the immunobiology of epithelial cells and macrophages lining healthy genital mucosa and inflamed/infected mucosa during the transition from innate to adaptive immunity. Upper reproductive tract epithelial cell line responses were compared to bone marrow-derived macrophages utilizing gene expression microarray technology. Those comparisons showed minor differences in the intrinsic innate defenses of macrophages and epithelial cells. Major lineage-specific differences in immunobiology relate to epithelial collaboration with adaptive immunity including an epithelial requirement for inflammatory cytokines to express MHC class II molecules, and a paucity and imbalance between costimulatory and coinhibitory ligands on epithelial cells that potentially limits sterilizing immunity (replication termination) to Chlamydia-specific T cells activated with limited or unconventional second signals. Chlamydia, epithelial cells, macrophages, cytokines, gene expression, micro array INTRODUCTION In the United States, Chlamydia trachomatis causes roughly 3 million new genital tract infections yearly in spite of public health efforts and effective antibiotic therapy thereby demonstrating evasion of both public policy makers and host biological defenses (Centers for Disease Control and Prevention; http://www.cdc.gov/std/stats). Though C. trachomatis is successful in maintaining a presence in the population, it is clear that humans and rodents are capable of self-clearing natural and experimental infections, and that individuals who clear infections are significantly protected from reinfection (Brunham and Rekart 2008; Geisler et al.2013). These demonstrations of sterilizing host immunity are proof-in-principal for development of a Chlamydia vaccine. Because replication of C. trachomatis urogenital serovars is restricted almost exclusively to reproductive tract epithelium, a successful vaccine will have to generate sterilizing immunity at the epithelial interface. In the past, epithelial cells were viewed as passive partners in host defenses generally, and in Chlamydia defenses specifically. Working models of sterilizing immunity had Chlamydia-specific CD4 T cells activated to make IFN-γ in mucosal immune compartments distant from the epithelium travel to the epithelial interface to eliminate the intracellular bacteria by inducing epithelial iNOS and nitric oxide production or by direct killing of infected epithelial cells by CD8 T cells recognizing Chlamydia antigens in the context of MHC class I (Loomis and Starnbach 2002). These models were informed by the fact that epithelial cells have few cell surface MHC class II molecules required for presenting antigens to CD4 T cells and were widely viewed as dampeners of inflammation based on lumenal contact with bacterial flora and a relatively limited array of costimulatory ligands (secondary signals for T cell activation) (Marelli-Berg et al.1997; Marelli-Berg and Lechler 1999). Subsequent studies argued against a passive epithelial model. Chlamydia-Infected epithelial cells were shown to produce chemokines and inflammatory mediators likely responsible for recruiting innate and adaptive effector cells, and possibly contributing directly to immunopathology (Rasmussen et al.1997; Wyrick et al.1999; Dessus-Babus, Knight and Wyrick 2000; Hess et al.2001; Xia et al.2003; Johnson 2004). Our lab demonstrated that Chlamydia-specific CD4 T cells directly recognized infected syngeneic upper reproductive tract epithelial cells in an IFN-γ- and CD4-dependent fashion, and showed a correlation between CD4 T cell clones relative ability to be activated by infected epithelial cells and their ability to terminate Chlamydia replication in them (Jayarapu et al.2009). To further the understanding of epithelial cells in Chlamydia immunobiology, we investigated their response to a cytokine milieu patterned on the genital tract secretions during the transition from innate to adaptive immunity, comparing epithelial responses to those of macrophages, which are professional phagocytes and antigen presenting cells. That analysis revealed many similarities and a few important differences with respect to innate defenses and cooperation with T cell-mediated immunity. We present those interesting results here. MATERIALS AND METHODS Mice C57BL6 female mice were purchased from Harlan Laboratories (Indianapolis, IN). Mice were housed in a specific pathogen-free (SPF) facility with food and water ad libitum. Experimental protocols were approved by the IUPUI Institutional Animal Care and Use Committee. Cell lines and bacteria Bm1.11, Bm12.4 and C57epi.1 upper reproductive tract murine epithelial cell lines were maintained in 1:1 DME:F12 10% FBS supplemented with insulin and recombinant keratinocyte growth factor as previously described (Johnson 2004; Derbigny, Kerr and Johnson 2005; Jayarapu et al.2009). Multifunctional Chlamydia-specific CD4 T cell clone uvmo-2 was maintained weekly on UV-C. muridarum-pulsed irradiated splenocytes in RPMI cytokine-supplemented media with 15% secondary mixed lymphocyte supernatant as previously described (Jayarapu et al.2009). Mycoplasma-free C. muridarum (Nigg) was grown in McCoy cells as previously described (Johnson 2004). Bone marrow-derived macrophages Bone marrow was harvested from tibia and femur of C57BL/6 female mice and plated in RPMI CM supplemented with 20 ηg/ml recombinant murine GM-CSF (Pierce-Endogen, Rockford, IL) in 6-well plates according to the protocol of Inaba et al. (1992) as detailed in Current Protocols in Immunology (Kruisbeek 1997). Bone marrow-derived macrophages (BMDM) monolayers were fed with same media once or twice as needed and used on day 5 or 6. Model inflammatory cytokine milieu Bm1.11 epithelial cell monolayers in 6-well plates pretreated with 10 ηg/ml recombinant IFN-γ for 16 h were infected with 3 IFU/cell C. muridarum (300 g × 30 min) for 4 h, then monolayers were rinsed with RPMI media, then 750 000 uvmo-2 CD4 T cells were added per well (∼1 T cell for every 3 infected epithelial cells) in RPMI FBS media without cytokine supplements. 28 h later culture supernatant was collected, dead and detached cells spun out at 300 × g for 5 min, then supernatant stored aliquoted at –80 C. This supernatant containing epithelial and T cell origin cytokines was analyzed by ELISA to determine IFN-γ content. Supernatants’ IFN-γ content was supplemented to a concentration of 12 ηg/ml at the time of experimentation (recombinant murine IFN-γ; R and D Systems, Minneapolis, MN); supernatants were used at 33% volume-for-volume for a final standardized IFN-γ concentration of 4 ηg/ml. 4 ηg/ml IFN-γ reflect levels measured in genital secretions of mice infected with C. muridarum in the 4–10 days window postinfection as determined by others (Darville et al.2003; Scurlock et al.2011). In addition to measuring IFN-γ, viable C. muridarum in the supernatants was quantified on McCoy monolayers as previously described (Johnson 2004). Two independent supernatant batches were prepared and used in four separate experiments. Microarray experiments and statistics Bm12.4 and C57epi.1 epithelial monolayers and BMDM monolayers in 6-well plates were exposed to RPMI control media or the patterned cytokine milieu detailed above. Two reproductive tract epithelial cell lines, Bm12.4 and C57epi.1, were used to avoid identifying idiosyncratic cell-line specific, rather than lineage-specific, biology. In addition to the epithelial and T cell cytokines, and 4 ηg/ml interferon gamma, the inflammatory supernatants contained one C. muridarum IFU for every ∼500 cells (0.002 IFU/cell). At 24 h total RNA was harvested from all wells using a commercial kit (RNeasy with DNAse step; Qiagen, Valencia, CA), generating 4 RNA samples per experiment: unperturbed epithelial cells, unperturbed BMDM, and cytokine milieu-exposed (and low-grade C. muridarum infected) epithelial cells and BMDM. This experiment was repeated 4 times to minimize false discovery. With assistance from The Indiana University Center for Medical Genomics, gene expression patterns were analyzed using the Affymetrix Mouse ST 1.0 Array that analyzes 28 853 murine genes. Samples were labeled using the standard Affymetrix protocol for the WT Target Labeling and Control Reagents kit according to the Affymetrix user manual: GeneChip® Whole Transcript (WT) Sense Target Labeling Assay GeneChip. Individual labeled samples were hybridized to the Mouse Gene 1.0 ST GeneChips for 17 h then washed, stained and scanned with the standard protocol using Affymetrix GCOS (GeneChip Operating System). GCOS was used to generate data (CEL files). Arrays were visually scanned for abnormalities or defects. CEL files were imported into Partek Genomics Suite (Partek, Inc., St. Louis, Mo). RMA signals were generated for the core probe sets using the RMA background correction, quantile normalization and summarization by Median Polish. Summarized signals for each probe set were log2 transformed. These log transformed signals were used for Principal Components Analysis, hierarchical clustering and signal histograms to determine if there were any outlier arrays. Untransformed RMA signals were used for fold change calculations. Data was analyzed using a one-way Anova (analysis of variance) using log2 transformed signals for all four experimental samples, and contrasts were made comparing BMDM to the combined expression of the epithelial cell lines Bm12.4 and C57epi.1 under control conditions and after exposure to the experimental inflammatory supernatant. Fold changes were calculated using the untransformed RMA signals. Genes up or down regulated 5-fold with P- values <0.001 were considered in the final analysis (Table 1, Supporting Information). The microarray data presented, here, is available in the Gene Expression Omnibus database (www.ncbi.nlm.nih.gov/geo) under accession number (GSE74317). INGENUITY Pathway Analysis was performed for the comparison of ‘resting epithelial cells’ versus ‘resting BMDM’ and ‘activated epithelial cells’ versus ‘activated BMDM’. ANALYSIS OF MICROARRAY DATA INGENUITY Pathways analysis (IPA) was performed importing the p-value, FDR and fold change for the 2 contrasts into Qiagen's Ingenuity Pathway Analysis (IPA) summer release 2015. Genes were selected for analysis if they had an FDR < 0.01 and absolute fold change ≥ 2. This yielded slightly over 4000 genes for each contrast. For pathway analysis a cut-off of FDR < 0.05 was used for significance; for the upstream regulator analysis an absolute z-score ≥ 2.0. The INGENUITY discovery software designed for genome wide association studies on tissues (e.g. blood, solid organ biopsies) is not well-suited for analysis of one cell type versus another under different experimental conditions. Therefore we also did our own pathway analysis looking clusters of genes with specific biological implications. For that purpose the log2 signals from the micro array data were converted to a semi-quantitative + to ++++++ format as follows: mRNA log2 signal 2–3.99 = 0; 3.00–4.99 = +; 5.00–5.99 = ++; 6.00–6.99 = +++; 7.00–7.99 = ++++; 8.0–9.99 = +++++; 10.00+ = ++++++. RESULTS The model Biologically intact upper reproductive epithelial cell lines were previously derived from bm1 (B6.C-H2-Kbm1/ByJ), bm12 (B6(C)-H2-Ab1bm12/KhEgJ), and C57BL/6 female mice. Bone marrow-derived macrophages were generated from female C57BL/6 mice as comparators. An inflammatory cytokine milieu patterned on cytokines in genital secretions days 4–10 of a C. muridarum infection was generated by infecting Bm1.11 epithelial cells with C. muridarum and coculturing them with a syngeneic Chlamydia-specific CD4 T cell clone uvmo-2; the resulting conditioned medium was supplemented with physiologic amounts of recombinant IFN-γ as described in the materials and methods. Replicate monolayers of epithelial cell lines and BMDM were exposed to control RPMI media or RPMI media with 33% inflammatory supernatant containing ∼1 C. muridarum IFU per 500 cells for 24 h, then total RNA isolated for microarray analysis. Macrophages are much less permissive for replication than epithelial cells (Steele, Balsara and Starnbach 2004) but make comparable levels of important regulatory cytokines including IFN-β (Derbigny, Kerr and Johnson 2005; Prantner, Darville and Nagarajan 2010). Under our experimental conditions, secondary infections of adjacent epithelial cells is unlikely or very limited because activated uvmo-2 T cell supernatants markedly limit C. muridarum replication (Johnson, Kerr and Slaven 2012), and C57epi.1 and Bm12.4 epithelial monolayers infected at low multiplicities (in this case 1 IFU for every 500 epithelial cells) are intact with intact inclusions at 24 h postinfection (unpublished observation). We have previously shown that chemokines and IL-6, the earliest innate immune responses of epithelial cells to infection, are detectable in the culture supernatant 6–12 h postinfection (Johnson 2004); therefore, secondarily infected epithelial cells would not likely have an innate response at the 24 h experimental time point sufficient to skew the microarray comparison. The experiment was repeated four times with two independent batches of inflammatory supernatant. Lineage validation To validate the microarray data, we examined the results for lineage specific markers: keratin 19 for epithelial cells, CD11b for macrophages, CD3e for T cells and B220 for B cells. As shown in Table 1, keratin 19 mRNA was abundant in the epithelial cell lines but not the bone marrow-derived macrophages. Conversely CD11b (Itgam; Mac-1) was abundant in the macrophages but not the epithelial cell lines. mRNA signals for T cells (CD3e) and B cells (B220) were weak and did not differ between the epithelial cell lines and BMDM. Table 1. Lineage specific markers. Marker gene symbol  Lineage  Bone marrow derived macs Signal  Fold difference epithelial cells vs BMDM  Reproductive tract epithelial lines Signal (Bm12.4/C57epi.1)  Keratin 19 (Krt19)  Epithelia  4.3  148*  11.70/11.31  CD11b  Macrophage  12.12  –368*  3.44/3.59  CD3e  T cell  5.00  1  4.77/4.71  B220 (Ptprc)  B cell  5.58  1  5.63/5.48  Marker gene symbol  Lineage  Bone marrow derived macs Signal  Fold difference epithelial cells vs BMDM  Reproductive tract epithelial lines Signal (Bm12.4/C57epi.1)  Keratin 19 (Krt19)  Epithelia  4.3  148*  11.70/11.31  CD11b  Macrophage  12.12  –368*  3.44/3.59  CD3e  T cell  5.00  1  4.77/4.71  B220 (Ptprc)  B cell  5.58  1  5.63/5.48  *P- values < 10−15. View Large Table 1. Lineage specific markers. Marker gene symbol  Lineage  Bone marrow derived macs Signal  Fold difference epithelial cells vs BMDM  Reproductive tract epithelial lines Signal (Bm12.4/C57epi.1)  Keratin 19 (Krt19)  Epithelia  4.3  148*  11.70/11.31  CD11b  Macrophage  12.12  –368*  3.44/3.59  CD3e  T cell  5.00  1  4.77/4.71  B220 (Ptprc)  B cell  5.58  1  5.63/5.48  Marker gene symbol  Lineage  Bone marrow derived macs Signal  Fold difference epithelial cells vs BMDM  Reproductive tract epithelial lines Signal (Bm12.4/C57epi.1)  Keratin 19 (Krt19)  Epithelia  4.3  148*  11.70/11.31  CD11b  Macrophage  12.12  –368*  3.44/3.59  CD3e  T cell  5.00  1  4.77/4.71  B220 (Ptprc)  B cell  5.58  1  5.63/5.48  *P- values < 10−15. View Large INGENUITY PATHWAY ANALYSIS Summary INGENUITY analysis documents are included as Supporting Information comparing epithelial cells and BMDM in the resting and activated states. This analysis did not prove enlightening due to the limitations of GWAS analysis applied to comparisons of dissimilar cell types; many pathways are not relevant or have results that reflect known differences in epithelial and macrophages. One meaningful pathway is the comparison for production of reactive nitrogen and oxygen species. Interestingly, there was no significant difference in RNS production pathways while macrophages had a more robust pathway for production of ROS due to low (green fill) epithelial expression of phox genes (Fig. 1). In light of GWAS analysis limitations, we did a separate analysis of mRNA levels of gene clusters with biological implications. Figure 1. View largeDownload slide Comparison of the reactive nitrogen and oxygen species (RNS and ROS) pathways in activated epithelial cells and macrophages using INGENUITY pathway analysis. Green fill = comparatively low epithelial mRNA; red fill = comparatively high epithelial mRNA for the indicated genes. Figure 1. View largeDownload slide Comparison of the reactive nitrogen and oxygen species (RNS and ROS) pathways in activated epithelial cells and macrophages using INGENUITY pathway analysis. Green fill = comparatively low epithelial mRNA; red fill = comparatively high epithelial mRNA for the indicated genes. Sentry duty Epithelial cells lining mucosal surfaces are a frequent point of entry for bacterial and viral pathogens. Accordingly, they are equipped with intracellular and extracellular pattern recognition receptors to detect invading microbes, and recruit innate and adaptive immune responses. In the experimental inflammatory microenvironment, epithelial cells and macrophages similarly upregulated microbial pattern recognition receptors (PRR) RIG-I (Ddx58) and its cofactor Ddx60, and the cytosolic DNA sensor Zbp1; also upregulated was Irf7, a transcription factor downstream of many PPR that positively regulates type I interferon transcription (Table 2). The cytosolic DS-RNA recognizing RIG-I is not involved in Chlamydia recognition, but epithelial cells and macrophages had high basal levels of STING mRNA that were unaffected by inflammatory supernatant (Tmem173; data not shown). STING is an important cytoplasmic PRR for recognition of Chlamydia infection and production of IFN-β (Prantner, Darville and Nagarajan 2010). Zbp1 is a cytosolic DNA sensor not known to be involved in sensing Chlamydia infections. Epithelial cells and macrophages had similar non-inducible levels of mRNA for the cytosolic DNA sensor cGAS, a critical Chlamydia PPR for IFN-β production (Zhang et al.2014) (data not shown). Epithelial and macrophage detection of a Chlamydia invasion event does not appear to be influenced by the inflammatory microenvironment, though the inflammatory microenvironment may amplify epithelial and macrophage type 1 interferon production via upregulated transcription of Irf7. Table 2. Sentinel components.   Bone marrow derived macs  Reproductive tract epithelial lines  Mediator (receptor/effect)  Common alt name  Signal log2  Fold induction  Fold induction  Signal log2 (Bm12.4/C57epi.1)  Ddx58  RIG-I  10.85  7  6  11.18/10.96  Ddx60     9.37  11  74  10.64/10       Irf7    12.55  42  26  12.34/11.43  Zbp1    10.80  17  43  10.65/11.31    Bone marrow derived macs  Reproductive tract epithelial lines  Mediator (receptor/effect)  Common alt name  Signal log2  Fold induction  Fold induction  Signal log2 (Bm12.4/C57epi.1)  Ddx58  RIG-I  10.85  7  6  11.18/10.96  Ddx60     9.37  11  74  10.64/10       Irf7    12.55  42  26  12.34/11.43  Zbp1    10.80  17  43  10.65/11.31  P- values for Fold for the entire table are <0.000001. All induced genes were common to macrophages and epithelial cells. View Large Table 2. Sentinel components.   Bone marrow derived macs  Reproductive tract epithelial lines  Mediator (receptor/effect)  Common alt name  Signal log2  Fold induction  Fold induction  Signal log2 (Bm12.4/C57epi.1)  Ddx58  RIG-I  10.85  7  6  11.18/10.96  Ddx60     9.37  11  74  10.64/10       Irf7    12.55  42  26  12.34/11.43  Zbp1    10.80  17  43  10.65/11.31    Bone marrow derived macs  Reproductive tract epithelial lines  Mediator (receptor/effect)  Common alt name  Signal log2  Fold induction  Fold induction  Signal log2 (Bm12.4/C57epi.1)  Ddx58  RIG-I  10.85  7  6  11.18/10.96  Ddx60     9.37  11  74  10.64/10       Irf7    12.55  42  26  12.34/11.43  Zbp1    10.80  17  43  10.65/11.31  P- values for Fold for the entire table are <0.000001. All induced genes were common to macrophages and epithelial cells. View Large Innate defenses Surprisingly, inflammatory milieu-induced innate defenses for epithelial cells and macrophages against intracellular pathogens look remarkably similar (Table 3). Both cell types upregulated effector molecules that participate in termination of C. muridarum replication including Nos2 and Plac8 (Johnson, Kerr and Slaven 2012). Macrophages had a high basal mRNA level for a third effector molecule Mpeg1 (perforin 2) shown to control C. muridarum replication in macrophages (Fields et al.2013). Mpeg1 mRNA was markedly upregulated in epithelial cells in the experimental inflammatory microenvironment. Interestingly Fields et al. have shown that Chlamydia infection prevents interferon-induced transcription of Mpeg1 (perforin 2) in epithelial cells, but absent infection high levels of Mpeg1 mRNA reflected high levels of protein (Fields et al.2013). In this microarray experiment both cell types upregulated p47 GTPases and guanylate-binding proteins (Gbps) that interact with inclusions to the detriment of Chlamydia replication (Bernstein-Hanley et al.2006; Coers et al.2008; Tietzel, El-Haibi and Carabeo 2009). Irg1, an innate defense mechanism against susceptible intracellular bacteria including Mycobacteria and Salmonella, was induced in both cell types. Irg1 synthesizes itaconic acid that blocks bacterial replication by inhibiting bacterial isocitrate lyase (Michelucci et al.2013); however, C. trachomatis and C. muridarum genomes do not encode an isocitrate lyase. Interestingly, macrophages in their basal state and epithelial cells in their activated state upregulated Samhd1 that depletes intracellular dNTP pools. This may be a relevant defense mechanism as early Chlamydia replication may be dependent on host ATP (Gerard et al.2002). The inflammatory milieu also induced a broad spectrum of interferon-regulated antiviral proteins (e.g. Mx1/Mx2) in epithelial cells and macrophages not likely to interfere with Chlamydia replication. Table 3. Innate defenses.   Bone marrow derived macs  Reproductive tract epithelial lines  Mediator  Common  Signal  Fold  Fold  Signal log2  Mediator  (effect)  alt name  log2  induction  induction  (Bm12.4/C57epi.1)  (effect)  Gbp1     8.51  20  –  4.47/4.39    Dnase1l3 (necrotic cell death)     6.45  7  –  3.89/4.79    Klra2 (nonself killing)  Ly49b  7.82  13  –  2.99/2.93    Klrk1 (nonself killing)  NKG2-D  6.28  6  –  3.08/3.12    Nos2 (nitric oxide)  iNOS  10.51  49  13  6.93/9.47    Plac8 (possible microbicide)  Onzin  6.88  8  6  11.29/6.54    Gbp2    13.49  90  11  13.17/12.71    Gbp3    11.95  8  99  12.07/11.49    Gbp4    11.20  86  125  10.91/10.54    Gbp5    11.10  72  60  10.33/10.50    Gbp6    11.77  11  67  11.88/11.16    Gbp9    9.95  19  63  10.27/9.97    Gbp11    9.62  20  61  10.42/9.41    Igtp  Irgm3  10.87  14  75  12.53/12.14    Iigp1    10.95  192  125  11.81/11.05    Irgm1  LRG-47  12.16  17  18  12.10/12.11    irgb6  Tgtp  11.65  133  117  12.52/12.41    Irgb10  OTTMUSG00000005723  11.29  24  150  12.29/12.06    Irg1 (itaconic acid)    12.67  28  16  9.90/7.30    Cfb (opsonization)    13.20  7  42  10.55/10.41    Trim30d (antiviral protease)    9.96  8  52  8.97/8.93    Apol9b (antiviral)  apolipoprotein L 9b  8.14  11  20  12.30/11.84    Mx1 (antiviral)    9.94  23  54  8.81/10.20    Mx2 (antiviral)    7.00  11  63  8.40/10.19    Usp18 (antiviral)    10.91  11  57  10.67/10.82    Herc5 (antiviral)    10.77  9  32  10.90/10.23                    Perforin 2  13.00  –  85  10.74/10.28  Mpeg1 (possible microbicide)      4.67  –  12  9.90/9.08  C4b (opsonization)      13.04  –  5  12.23/11.43  Samhd1 (depletes dNTP pool)    C1inh  5.24  –  8  8.77/6.55  Serping1 (opsonization)    Bone marrow derived macs  Reproductive tract epithelial lines  Mediator  Common  Signal  Fold  Fold  Signal log2  Mediator  (effect)  alt name  log2  induction  induction  (Bm12.4/C57epi.1)  (effect)  Gbp1     8.51  20  –  4.47/4.39    Dnase1l3 (necrotic cell death)     6.45  7  –  3.89/4.79    Klra2 (nonself killing)  Ly49b  7.82  13  –  2.99/2.93    Klrk1 (nonself killing)  NKG2-D  6.28  6  –  3.08/3.12    Nos2 (nitric oxide)  iNOS  10.51  49  13  6.93/9.47    Plac8 (possible microbicide)  Onzin  6.88  8  6  11.29/6.54    Gbp2    13.49  90  11  13.17/12.71    Gbp3    11.95  8  99  12.07/11.49    Gbp4    11.20  86  125  10.91/10.54    Gbp5    11.10  72  60  10.33/10.50    Gbp6    11.77  11  67  11.88/11.16    Gbp9    9.95  19  63  10.27/9.97    Gbp11    9.62  20  61  10.42/9.41    Igtp  Irgm3  10.87  14  75  12.53/12.14    Iigp1    10.95  192  125  11.81/11.05    Irgm1  LRG-47  12.16  17  18  12.10/12.11    irgb6  Tgtp  11.65  133  117  12.52/12.41    Irgb10  OTTMUSG00000005723  11.29  24  150  12.29/12.06    Irg1 (itaconic acid)    12.67  28  16  9.90/7.30    Cfb (opsonization)    13.20  7  42  10.55/10.41    Trim30d (antiviral protease)    9.96  8  52  8.97/8.93    Apol9b (antiviral)  apolipoprotein L 9b  8.14  11  20  12.30/11.84    Mx1 (antiviral)    9.94  23  54  8.81/10.20    Mx2 (antiviral)    7.00  11  63  8.40/10.19    Usp18 (antiviral)    10.91  11  57  10.67/10.82    Herc5 (antiviral)    10.77  9  32  10.90/10.23                    Perforin 2  13.00  –  85  10.74/10.28  Mpeg1 (possible microbicide)      4.67  –  12  9.90/9.08  C4b (opsonization)      13.04  –  5  12.23/11.43  Samhd1 (depletes dNTP pool)    C1inh  5.24  –  8  8.77/6.55  Serping1 (opsonization)  P-values for fold Induction for the entire table are <0.000001. Genes uniquely induced in macrophages by inflammatory supernatant in white boxes, both macrophages and epithelial cells in light gray boxes, and uniquely in epithelial cells in dark gray boxes. View Large Table 3. Innate defenses.   Bone marrow derived macs  Reproductive tract epithelial lines  Mediator  Common  Signal  Fold  Fold  Signal log2  Mediator  (effect)  alt name  log2  induction  induction  (Bm12.4/C57epi.1)  (effect)  Gbp1     8.51  20  –  4.47/4.39    Dnase1l3 (necrotic cell death)     6.45  7  –  3.89/4.79    Klra2 (nonself killing)  Ly49b  7.82  13  –  2.99/2.93    Klrk1 (nonself killing)  NKG2-D  6.28  6  –  3.08/3.12    Nos2 (nitric oxide)  iNOS  10.51  49  13  6.93/9.47    Plac8 (possible microbicide)  Onzin  6.88  8  6  11.29/6.54    Gbp2    13.49  90  11  13.17/12.71    Gbp3    11.95  8  99  12.07/11.49    Gbp4    11.20  86  125  10.91/10.54    Gbp5    11.10  72  60  10.33/10.50    Gbp6    11.77  11  67  11.88/11.16    Gbp9    9.95  19  63  10.27/9.97    Gbp11    9.62  20  61  10.42/9.41    Igtp  Irgm3  10.87  14  75  12.53/12.14    Iigp1    10.95  192  125  11.81/11.05    Irgm1  LRG-47  12.16  17  18  12.10/12.11    irgb6  Tgtp  11.65  133  117  12.52/12.41    Irgb10  OTTMUSG00000005723  11.29  24  150  12.29/12.06    Irg1 (itaconic acid)    12.67  28  16  9.90/7.30    Cfb (opsonization)    13.20  7  42  10.55/10.41    Trim30d (antiviral protease)    9.96  8  52  8.97/8.93    Apol9b (antiviral)  apolipoprotein L 9b  8.14  11  20  12.30/11.84    Mx1 (antiviral)    9.94  23  54  8.81/10.20    Mx2 (antiviral)    7.00  11  63  8.40/10.19    Usp18 (antiviral)    10.91  11  57  10.67/10.82    Herc5 (antiviral)    10.77  9  32  10.90/10.23                    Perforin 2  13.00  –  85  10.74/10.28  Mpeg1 (possible microbicide)      4.67  –  12  9.90/9.08  C4b (opsonization)      13.04  –  5  12.23/11.43  Samhd1 (depletes dNTP pool)    C1inh  5.24  –  8  8.77/6.55  Serping1 (opsonization)    Bone marrow derived macs  Reproductive tract epithelial lines  Mediator  Common  Signal  Fold  Fold  Signal log2  Mediator  (effect)  alt name  log2  induction  induction  (Bm12.4/C57epi.1)  (effect)  Gbp1     8.51  20  –  4.47/4.39    Dnase1l3 (necrotic cell death)     6.45  7  –  3.89/4.79    Klra2 (nonself killing)  Ly49b  7.82  13  –  2.99/2.93    Klrk1 (nonself killing)  NKG2-D  6.28  6  –  3.08/3.12    Nos2 (nitric oxide)  iNOS  10.51  49  13  6.93/9.47    Plac8 (possible microbicide)  Onzin  6.88  8  6  11.29/6.54    Gbp2    13.49  90  11  13.17/12.71    Gbp3    11.95  8  99  12.07/11.49    Gbp4    11.20  86  125  10.91/10.54    Gbp5    11.10  72  60  10.33/10.50    Gbp6    11.77  11  67  11.88/11.16    Gbp9    9.95  19  63  10.27/9.97    Gbp11    9.62  20  61  10.42/9.41    Igtp  Irgm3  10.87  14  75  12.53/12.14    Iigp1    10.95  192  125  11.81/11.05    Irgm1  LRG-47  12.16  17  18  12.10/12.11    irgb6  Tgtp  11.65  133  117  12.52/12.41    Irgb10  OTTMUSG00000005723  11.29  24  150  12.29/12.06    Irg1 (itaconic acid)    12.67  28  16  9.90/7.30    Cfb (opsonization)    13.20  7  42  10.55/10.41    Trim30d (antiviral protease)    9.96  8  52  8.97/8.93    Apol9b (antiviral)  apolipoprotein L 9b  8.14  11  20  12.30/11.84    Mx1 (antiviral)    9.94  23  54  8.81/10.20    Mx2 (antiviral)    7.00  11  63  8.40/10.19    Usp18 (antiviral)    10.91  11  57  10.67/10.82    Herc5 (antiviral)    10.77  9  32  10.90/10.23                    Perforin 2  13.00  –  85  10.74/10.28  Mpeg1 (possible microbicide)      4.67  –  12  9.90/9.08  C4b (opsonization)      13.04  –  5  12.23/11.43  Samhd1 (depletes dNTP pool)    C1inh  5.24  –  8  8.77/6.55  Serping1 (opsonization)  P-values for fold Induction for the entire table are <0.000001. Genes uniquely induced in macrophages by inflammatory supernatant in white boxes, both macrophages and epithelial cells in light gray boxes, and uniquely in epithelial cells in dark gray boxes. View Large There was one induced macrophage-specific innate defense. Macrophages in the inflammatory milieu upregulated non-self-recognition killer receptors (Ly49b and NKG2-D) and the necrotic/inflammatory killing molecule Dnase1l3. Macrophages have been shown to utilize non-self-recognition in vivo dependent on prior CD4 T cell priming and CD40L-CD40 interaction (Liu et al.2012). Epithelial cells do not possess cytolytic capabilities and did not transcribe these genes. Macrophages in their basal state appear to be a hostile environment for Chlamydia replication with preformed Mpeg1 and limited ATP (Samhd1). In the experimental inflammatory microenvironment, epithelial cells upregulated Mpeg1 and Samhd1, achieving mRNA levels similar to macrophages. In an inflammatory microenvironment, both cell types markedly upregulated additional hostility including innate effector molecules likely to damage bacteria (Nos2 and Plac8) and disrupt inclusion biology (p47 GTPases and Gbps). Inflammatory mediators During infections, the local microenvironment is shaped by inflammatory mediators that control blood flow, recruit effector cells and influence the nascent adaptive immune response (Table 4). In the experimental inflammatory microenvironment, macrophages and epithelial cells amplified chemokine recruitment through shared production of CCL2 (Th1 inflammation), CCL5 (mixed inflammation), CCL7 (mixed inflammation), Cxcl9 (Th1 inflammation) and Cxcl10 (Th1 inflammation). Epithelial cells uniquely increased transcription of Cxcl11 (Th1 inflammation), IL-15 (IEL and NK expansion/activation), and the enzyme for histamine synthesis (Hdc), potentially influencing local blood flow. In epithelial cells, the inflammatory supernatant induced the non-canonical inflammasome (Casp4) that posttranslationally activates IL-1 and IL-18 precursors. Macrophages in the inflammatory microenvironment uniquely upregulated transcription of genes that would augment prostaglandin synthesis regulating local blood flow and platelet function, promote type 2 innate lymphocyte cells to produce IL-5 and 13 (IL33), influence Th17 development (IL36γ), counterbalance IEL and NK expansion/activation (IL15ra), and promote apoptosis (Fgl2). While it is not possible to predict the net effect of this panoply of inflammatory mediators based on mRNA levels beyond vasodilatation and recruitment of a broad spectrum of inflammatory cells, perhaps there was a slight bias toward recruiting Th1 effectors based on Ccl2, Cxcl9, Cxcl10 and Cxcl11. Table 4. Soluble mediators (Cytokines/Chemokines/Prostaglandins).   Bone marrow derived macs  Reproductive tract epithelial lines  Mediator  Common alt  Signal  Fold  Fold  Signal log2  Mediator  (receptor/ effect)  name  log2      (Bm12.4/C57epi.1)  (receptor/ effect)  Il33 (St2/production IL-5 and IL-13 by type 2 ILC)    7.06  10  –  3.64/3.76    Tnfsf15 (DR3/effector T cell activation)  TL1A  8.98  9  –  5.55/5.82    Il1f9 (IL-1RL2/enhances inflammatory cytokine, esp. Th17-type)  IL36γ  9.52  12  –  3.81/3.59    Fgl2 (Fcgr2b,Fcgtr3/ tolerance via apoptosis)  Fibroleukin  8.77  10  –  3.56/3.65    IL15ra (antagonizes IL-15)    8.08  5  –  6.47/6.49    Acsl1 (inflammatory PG synthesis)    12.42  9  –  7.05/8.15    Ptgs2 (prostaglandin synthesis)  Cox2  8.18  9  –  8.76/11.50    Ccl2 (Th1 recruitment)  MCP1  8.84  20  9  11.77/11.23    Ccl5 (mixed recruitment)  Rantes  13.27  14  6  9.16/9.49    Ccl7 (mixed recruitment)  MCP3  7.61  6  9  12.02/11.15    Cxcl9 (Th1 recruitment)  Mig  10.05  23  76  8.55/10.96    Cxcl10 (Th1 recruitment)  IP-10  9.62  84  62  13.53/13.68    IL18bp (antagonizes IL-18)    11.15  8  7  8.99/8.55      Casp11  9.57  –  6  9.77/9.87  Casp4 (pyroptosis, IL-1 and IL-18 maturation)    I-TAC  5.01  –  11  6.31/9.16  Cxcl11 (Th1 recruitment)      8.02  –  12  7.01/6.29  IL15      5.21  –  5  7.67/7.14  Hdc (histamine synthesis)    Bone marrow derived macs  Reproductive tract epithelial lines  Mediator  Common alt  Signal  Fold  Fold  Signal log2  Mediator  (receptor/ effect)  name  log2      (Bm12.4/C57epi.1)  (receptor/ effect)  Il33 (St2/production IL-5 and IL-13 by type 2 ILC)    7.06  10  –  3.64/3.76    Tnfsf15 (DR3/effector T cell activation)  TL1A  8.98  9  –  5.55/5.82    Il1f9 (IL-1RL2/enhances inflammatory cytokine, esp. Th17-type)  IL36γ  9.52  12  –  3.81/3.59    Fgl2 (Fcgr2b,Fcgtr3/ tolerance via apoptosis)  Fibroleukin  8.77  10  –  3.56/3.65    IL15ra (antagonizes IL-15)    8.08  5  –  6.47/6.49    Acsl1 (inflammatory PG synthesis)    12.42  9  –  7.05/8.15    Ptgs2 (prostaglandin synthesis)  Cox2  8.18  9  –  8.76/11.50    Ccl2 (Th1 recruitment)  MCP1  8.84  20  9  11.77/11.23    Ccl5 (mixed recruitment)  Rantes  13.27  14  6  9.16/9.49    Ccl7 (mixed recruitment)  MCP3  7.61  6  9  12.02/11.15    Cxcl9 (Th1 recruitment)  Mig  10.05  23  76  8.55/10.96    Cxcl10 (Th1 recruitment)  IP-10  9.62  84  62  13.53/13.68    IL18bp (antagonizes IL-18)    11.15  8  7  8.99/8.55      Casp11  9.57  –  6  9.77/9.87  Casp4 (pyroptosis, IL-1 and IL-18 maturation)    I-TAC  5.01  –  11  6.31/9.16  Cxcl11 (Th1 recruitment)      8.02  –  12  7.01/6.29  IL15      5.21  –  5  7.67/7.14  Hdc (histamine synthesis)  P-values for fold for the entire table are <0.000001. Genes uniquely induced in macrophages by inflammatory supernatant in white boxes, both macrophages and epithelial cells in light gray boxes, and uniquely in epithelial cells in dark gray boxes. View Large Table 4. Soluble mediators (Cytokines/Chemokines/Prostaglandins).   Bone marrow derived macs  Reproductive tract epithelial lines  Mediator  Common alt  Signal  Fold  Fold  Signal log2  Mediator  (receptor/ effect)  name  log2      (Bm12.4/C57epi.1)  (receptor/ effect)  Il33 (St2/production IL-5 and IL-13 by type 2 ILC)    7.06  10  –  3.64/3.76    Tnfsf15 (DR3/effector T cell activation)  TL1A  8.98  9  –  5.55/5.82    Il1f9 (IL-1RL2/enhances inflammatory cytokine, esp. Th17-type)  IL36γ  9.52  12  –  3.81/3.59    Fgl2 (Fcgr2b,Fcgtr3/ tolerance via apoptosis)  Fibroleukin  8.77  10  –  3.56/3.65    IL15ra (antagonizes IL-15)    8.08  5  –  6.47/6.49    Acsl1 (inflammatory PG synthesis)    12.42  9  –  7.05/8.15    Ptgs2 (prostaglandin synthesis)  Cox2  8.18  9  –  8.76/11.50    Ccl2 (Th1 recruitment)  MCP1  8.84  20  9  11.77/11.23    Ccl5 (mixed recruitment)  Rantes  13.27  14  6  9.16/9.49    Ccl7 (mixed recruitment)  MCP3  7.61  6  9  12.02/11.15    Cxcl9 (Th1 recruitment)  Mig  10.05  23  76  8.55/10.96    Cxcl10 (Th1 recruitment)  IP-10  9.62  84  62  13.53/13.68    IL18bp (antagonizes IL-18)    11.15  8  7  8.99/8.55      Casp11  9.57  –  6  9.77/9.87  Casp4 (pyroptosis, IL-1 and IL-18 maturation)    I-TAC  5.01  –  11  6.31/9.16  Cxcl11 (Th1 recruitment)      8.02  –  12  7.01/6.29  IL15      5.21  –  5  7.67/7.14  Hdc (histamine synthesis)    Bone marrow derived macs  Reproductive tract epithelial lines  Mediator  Common alt  Signal  Fold  Fold  Signal log2  Mediator  (receptor/ effect)  name  log2      (Bm12.4/C57epi.1)  (receptor/ effect)  Il33 (St2/production IL-5 and IL-13 by type 2 ILC)    7.06  10  –  3.64/3.76    Tnfsf15 (DR3/effector T cell activation)  TL1A  8.98  9  –  5.55/5.82    Il1f9 (IL-1RL2/enhances inflammatory cytokine, esp. Th17-type)  IL36γ  9.52  12  –  3.81/3.59    Fgl2 (Fcgr2b,Fcgtr3/ tolerance via apoptosis)  Fibroleukin  8.77  10  –  3.56/3.65    IL15ra (antagonizes IL-15)    8.08  5  –  6.47/6.49    Acsl1 (inflammatory PG synthesis)    12.42  9  –  7.05/8.15    Ptgs2 (prostaglandin synthesis)  Cox2  8.18  9  –  8.76/11.50    Ccl2 (Th1 recruitment)  MCP1  8.84  20  9  11.77/11.23    Ccl5 (mixed recruitment)  Rantes  13.27  14  6  9.16/9.49    Ccl7 (mixed recruitment)  MCP3  7.61  6  9  12.02/11.15    Cxcl9 (Th1 recruitment)  Mig  10.05  23  76  8.55/10.96    Cxcl10 (Th1 recruitment)  IP-10  9.62  84  62  13.53/13.68    IL18bp (antagonizes IL-18)    11.15  8  7  8.99/8.55      Casp11  9.57  –  6  9.77/9.87  Casp4 (pyroptosis, IL-1 and IL-18 maturation)    I-TAC  5.01  –  11  6.31/9.16  Cxcl11 (Th1 recruitment)      8.02  –  12  7.01/6.29  IL15      5.21  –  5  7.67/7.14  Hdc (histamine synthesis)  P-values for fold for the entire table are <0.000001. Genes uniquely induced in macrophages by inflammatory supernatant in white boxes, both macrophages and epithelial cells in light gray boxes, and uniquely in epithelial cells in dark gray boxes. View Large Macrophage polarization An important bridge between innate and adaptive immunity is the influence of macrophage biology on T cell polarization. Macrophages somewhat analogous to T cells polarize toward fostering inflammatory adaptive responses (M1) or attenuating inflammatory responses to promote healing (M2). Gene expression analyses have identified M1 and M2 polarization-associated genes (Martinez and Gordon 2014). We reviewed our microarray data to see whether the experimental inflammatory condition influenced the polarity of macrophages (Table 5). Baseline polarization of GM-CSF derived macrophages was neither M1 nor M2, though high levels of Arg1 mRNA (polyamine synthesis from L-arginine) showed the untreated BMDM population to be skewed toward M2. In the presence of the inflammatory supernatant containing IFN-γ, there was a marked increase in Nos2 transcription (converts L-arginine to nitric oxide) without a change in Arg1 mRNA levels suggesting either that as a population the BMDM shifted toward M1, or that a significant subpopulation within BMDM shifted toward M1. Nos2 transcription is regulated by interferons, and therefore this crudely defined shift toward M1 based on Nos2 (49-fold induction) was predictable. Table 5. Macrophage polarization. M1 lineage marker  Common alt name  Induction y/n (fold)  Signal log2  Score  M2 lineage marker  Csf2rb    N  12.24  ++++++    IL2ra    N  4.07  +    IL6r    N  9.13  +++++    CD36    N  12.64  ++++++    CD97    N  10.20  ++++++    Icam1    N  9.99  +++++    Itgal    N  8.98  +++++    Itga4    N  8.90  +++++    Itgb7    N  9.90  +++++    Muc1    N  4.17  +    St6gal1  Siat1  N  7.75  ++++    Ptpro    N  5.89  ++    Nmi    N  9.24  +++++    IL15ra    Y (5)  8.08  +++++    CD69    Y (11)  8.86  +++++    Nos2    Y (49)  10.51  ++++++        N (1)  11.71  ++++++  Arg1      Y (7)  9.30  +++++  Ch25h      N  7.03  ++++  Mrc1      N  12.47  ++++++  Tgm2      N  8.94  +++++  C1qa    Dip1  N  7.46  ++++  Tsc22d3      N  12.50  ++++++  Thbs1      N  6.10  +++  IL1r2    Fizz1  N  7.40  ++++  Retnla    Ym1  N  11.51  ++++++  Chi3l3    Ym1  N  3.65  +  Chi3l4      N  3.14  +  CD163      N  9.22  +++++  Ptgs1      N  7.93  ++++  IRF4      N  7.98  ++++  KLF4    CD64  N  11.15  ++++++  Fcgr1    CD32  N  10.53  ++++++  Fcgr2b      N  11.51  ++++++  Fcgr3      N  10.66  ++++++  Fgr4  M1 lineage marker  Common alt name  Induction y/n (fold)  Signal log2  Score  M2 lineage marker  Csf2rb    N  12.24  ++++++    IL2ra    N  4.07  +    IL6r    N  9.13  +++++    CD36    N  12.64  ++++++    CD97    N  10.20  ++++++    Icam1    N  9.99  +++++    Itgal    N  8.98  +++++    Itga4    N  8.90  +++++    Itgb7    N  9.90  +++++    Muc1    N  4.17  +    St6gal1  Siat1  N  7.75  ++++    Ptpro    N  5.89  ++    Nmi    N  9.24  +++++    IL15ra    Y (5)  8.08  +++++    CD69    Y (11)  8.86  +++++    Nos2    Y (49)  10.51  ++++++        N (1)  11.71  ++++++  Arg1      Y (7)  9.30  +++++  Ch25h      N  7.03  ++++  Mrc1      N  12.47  ++++++  Tgm2      N  8.94  +++++  C1qa    Dip1  N  7.46  ++++  Tsc22d3      N  12.50  ++++++  Thbs1      N  6.10  +++  IL1r2    Fizz1  N  7.40  ++++  Retnla    Ym1  N  11.51  ++++++  Chi3l3    Ym1  N  3.65  +  Chi3l4      N  3.14  +  CD163      N  9.22  +++++  Ptgs1      N  7.93  ++++  IRF4      N  7.98  ++++  KLF4    CD64  N  11.15  ++++++  Fcgr1    CD32  N  10.53  ++++++  Fcgr2b      N  11.51  ++++++  Fcgr3      N  10.66  ++++++  Fgr4  P- values for ‘(fold)’ for induced genes indicated by a ‘Y’ in the table are <0.000001. View Large Table 5. Macrophage polarization. M1 lineage marker  Common alt name  Induction y/n (fold)  Signal log2  Score  M2 lineage marker  Csf2rb    N  12.24  ++++++    IL2ra    N  4.07  +    IL6r    N  9.13  +++++    CD36    N  12.64  ++++++    CD97    N  10.20  ++++++    Icam1    N  9.99  +++++    Itgal    N  8.98  +++++    Itga4    N  8.90  +++++    Itgb7    N  9.90  +++++    Muc1    N  4.17  +    St6gal1  Siat1  N  7.75  ++++    Ptpro    N  5.89  ++    Nmi    N  9.24  +++++    IL15ra    Y (5)  8.08  +++++    CD69    Y (11)  8.86  +++++    Nos2    Y (49)  10.51  ++++++        N (1)  11.71  ++++++  Arg1      Y (7)  9.30  +++++  Ch25h      N  7.03  ++++  Mrc1      N  12.47  ++++++  Tgm2      N  8.94  +++++  C1qa    Dip1  N  7.46  ++++  Tsc22d3      N  12.50  ++++++  Thbs1      N  6.10  +++  IL1r2    Fizz1  N  7.40  ++++  Retnla    Ym1  N  11.51  ++++++  Chi3l3    Ym1  N  3.65  +  Chi3l4      N  3.14  +  CD163      N  9.22  +++++  Ptgs1      N  7.93  ++++  IRF4      N  7.98  ++++  KLF4    CD64  N  11.15  ++++++  Fcgr1    CD32  N  10.53  ++++++  Fcgr2b      N  11.51  ++++++  Fcgr3      N  10.66  ++++++  Fgr4  M1 lineage marker  Common alt name  Induction y/n (fold)  Signal log2  Score  M2 lineage marker  Csf2rb    N  12.24  ++++++    IL2ra    N  4.07  +    IL6r    N  9.13  +++++    CD36    N  12.64  ++++++    CD97    N  10.20  ++++++    Icam1    N  9.99  +++++    Itgal    N  8.98  +++++    Itga4    N  8.90  +++++    Itgb7    N  9.90  +++++    Muc1    N  4.17  +    St6gal1  Siat1  N  7.75  ++++    Ptpro    N  5.89  ++    Nmi    N  9.24  +++++    IL15ra    Y (5)  8.08  +++++    CD69    Y (11)  8.86  +++++    Nos2    Y (49)  10.51  ++++++        N (1)  11.71  ++++++  Arg1      Y (7)  9.30  +++++  Ch25h      N  7.03  ++++  Mrc1      N  12.47  ++++++  Tgm2      N  8.94  +++++  C1qa    Dip1  N  7.46  ++++  Tsc22d3      N  12.50  ++++++  Thbs1      N  6.10  +++  IL1r2    Fizz1  N  7.40  ++++  Retnla    Ym1  N  11.51  ++++++  Chi3l3    Ym1  N  3.65  +  Chi3l4      N  3.14  +  CD163      N  9.22  +++++  Ptgs1      N  7.93  ++++  IRF4      N  7.98  ++++  KLF4    CD64  N  11.15  ++++++  Fcgr1    CD32  N  10.53  ++++++  Fcgr2b      N  11.51  ++++++  Fcgr3      N  10.66  ++++++  Fgr4  P- values for ‘(fold)’ for induced genes indicated by a ‘Y’ in the table are <0.000001. View Large Antigen presentation Antigen presentation (this section) and T cell activation (next section) are two areas of immunobiology that clearly differ between macrophages and epithelial cells. Interestingly, the experimental inflammatory microenvironment did very little to mRNA levels of antigen processing and presentation genes in macrophages; upregulating transcription of MHC class Ib molecules, and Serpinb9 and Siglec1 genes associated with antigen capture for processing (Table 6). In stark contrast, exposure of epithelial cells to the inflammatory supernatant upregulated transcription of MHC class Ia (antigen presentation to CD8 T cells), and MHC class II with its peptide loading partner H2-DM (antigen presentation to CD4 T cells). An oft repeated statement that epithelial cells do not express class II molecules should be modified to epithelial cells express few MHC class II molecules under non-inflammatory conditions. Table 6. Antigen presentation components.   Bone marrow derived macs  Reproductive tract epithelial lines  Component gene  Common    Fold  Fold  Signal log2  Component gene  symbol (effect)  alt name  Signal log2  induction  induction  (Bm12.4/C57epi.1)  symbol (effect)  H2-Q6 (MHC class Ib)  Qa-6  12.07  12  –  6.38/6.10    H2-Q8 (MHC class Ib)  Qa-8  10.41  6  –  7.10/6.26    Serpinb9 (cross presentation)    6.78  11  –  7.86/7.85    Siglec1 (exosome receptor)    9.15  11    4.23/4.00    H2-Q2 (MHC class Ib)  Qa-2  11.65  8  14  9.81/6.54    H2-T22 (MHC class Ib ligand for γ/ζ IEL)    11.64  5  8  11.10/10.2    H2-T24 (MHC class Ib)    10.59  8  11  8.22/8.32    Tap1 (MHC class I loading)    11.51  7  7  11.2/6.28      H-2K  13.41  –  8  12.68/10.64  H2-K1 (MHC class Ia)    I-A alpha chain  12.47  –  41  8.81/8.72  H2-Aa (MHC class II)    I-A beta chain  13.09  –  15  8.98/7.63  H2-Ab1 (MHC class II)    DM  10.15  –  7  7.07/7.18  H2-DMa (class II loading)    I-E beta chain*  12.28  –  8  9.11/8.30  H2-Eb1 (MHC class II)    Bone marrow derived macs  Reproductive tract epithelial lines  Component gene  Common    Fold  Fold  Signal log2  Component gene  symbol (effect)  alt name  Signal log2  induction  induction  (Bm12.4/C57epi.1)  symbol (effect)  H2-Q6 (MHC class Ib)  Qa-6  12.07  12  –  6.38/6.10    H2-Q8 (MHC class Ib)  Qa-8  10.41  6  –  7.10/6.26    Serpinb9 (cross presentation)    6.78  11  –  7.86/7.85    Siglec1 (exosome receptor)    9.15  11    4.23/4.00    H2-Q2 (MHC class Ib)  Qa-2  11.65  8  14  9.81/6.54    H2-T22 (MHC class Ib ligand for γ/ζ IEL)    11.64  5  8  11.10/10.2    H2-T24 (MHC class Ib)    10.59  8  11  8.22/8.32    Tap1 (MHC class I loading)    11.51  7  7  11.2/6.28      H-2K  13.41  –  8  12.68/10.64  H2-K1 (MHC class Ia)    I-A alpha chain  12.47  –  41  8.81/8.72  H2-Aa (MHC class II)    I-A beta chain  13.09  –  15  8.98/7.63  H2-Ab1 (MHC class II)    DM  10.15  –  7  7.07/7.18  H2-DMa (class II loading)    I-E beta chain*  12.28  –  8  9.11/8.30  H2-Eb1 (MHC class II)  *no I-E MHC class II expressed on cell surface in C57BL/6 mice due to absence of an I-E alpha chain. P-values for Fold Induction for the entire table are < 0.000001. Genes uniquely induced in macrophages by inflammatory supernatant in white boxes, both macrophages and epithelial cells in light gray boxes, and uniquely in epithelial cells in dark gray boxes. View Large Table 6. Antigen presentation components.   Bone marrow derived macs  Reproductive tract epithelial lines  Component gene  Common    Fold  Fold  Signal log2  Component gene  symbol (effect)  alt name  Signal log2  induction  induction  (Bm12.4/C57epi.1)  symbol (effect)  H2-Q6 (MHC class Ib)  Qa-6  12.07  12  –  6.38/6.10    H2-Q8 (MHC class Ib)  Qa-8  10.41  6  –  7.10/6.26    Serpinb9 (cross presentation)    6.78  11  –  7.86/7.85    Siglec1 (exosome receptor)    9.15  11    4.23/4.00    H2-Q2 (MHC class Ib)  Qa-2  11.65  8  14  9.81/6.54    H2-T22 (MHC class Ib ligand for γ/ζ IEL)    11.64  5  8  11.10/10.2    H2-T24 (MHC class Ib)    10.59  8  11  8.22/8.32    Tap1 (MHC class I loading)    11.51  7  7  11.2/6.28      H-2K  13.41  –  8  12.68/10.64  H2-K1 (MHC class Ia)    I-A alpha chain  12.47  –  41  8.81/8.72  H2-Aa (MHC class II)    I-A beta chain  13.09  –  15  8.98/7.63  H2-Ab1 (MHC class II)    DM  10.15  –  7  7.07/7.18  H2-DMa (class II loading)    I-E beta chain*  12.28  –  8  9.11/8.30  H2-Eb1 (MHC class II)    Bone marrow derived macs  Reproductive tract epithelial lines  Component gene  Common    Fold  Fold  Signal log2  Component gene  symbol (effect)  alt name  Signal log2  induction  induction  (Bm12.4/C57epi.1)  symbol (effect)  H2-Q6 (MHC class Ib)  Qa-6  12.07  12  –  6.38/6.10    H2-Q8 (MHC class Ib)  Qa-8  10.41  6  –  7.10/6.26    Serpinb9 (cross presentation)    6.78  11  –  7.86/7.85    Siglec1 (exosome receptor)    9.15  11    4.23/4.00    H2-Q2 (MHC class Ib)  Qa-2  11.65  8  14  9.81/6.54    H2-T22 (MHC class Ib ligand for γ/ζ IEL)    11.64  5  8  11.10/10.2    H2-T24 (MHC class Ib)    10.59  8  11  8.22/8.32    Tap1 (MHC class I loading)    11.51  7  7  11.2/6.28      H-2K  13.41  –  8  12.68/10.64  H2-K1 (MHC class Ia)    I-A alpha chain  12.47  –  41  8.81/8.72  H2-Aa (MHC class II)    I-A beta chain  13.09  –  15  8.98/7.63  H2-Ab1 (MHC class II)    DM  10.15  –  7  7.07/7.18  H2-DMa (class II loading)    I-E beta chain*  12.28  –  8  9.11/8.30  H2-Eb1 (MHC class II)  *no I-E MHC class II expressed on cell surface in C57BL/6 mice due to absence of an I-E alpha chain. P-values for Fold Induction for the entire table are < 0.000001. Genes uniquely induced in macrophages by inflammatory supernatant in white boxes, both macrophages and epithelial cells in light gray boxes, and uniquely in epithelial cells in dark gray boxes. View Large T cell activation T cell activation is the result of engagement of the T cell receptor by MHC molecules bearing cognate antigenic peptides, plus the coreceptor (CD4 or CD8), and the balance between additional positive (costimulatory) and negative (coinhibitory) signals delivered by the target cell presenting antigen (antigen presenting cell). We analyzed the relative mRNA levels of the major costimulatory and coinhibitory ligands in macrophages and epithelial cells (Tables 7 and 8). The major finding was that epithelial cells have a paucity of costimulatory ligands compared to macrophages. Icam1 was the only costimulatory ligand in epithelial cells with a strong mRNA signal, and exposure to inflammatory supernatant increased its mRNA level less than 5-fold. Conversely, macrophages had seven different costimulatory ligands with log2 signals >6.00 (arbitrary scored as +++ out of ++++++). Of these transcription of CD40 (9-fold) and CD86 (6-fold) were significantly upregulated by inflammatory supernatant. In balanced fashion, macrophages had significant mRNA signals for seven different coinhibitory ligands; of them only PDL2 was significantly induced by inflammatory supernatant (9-fold). Table 7. Costimulatory ligands*.   Bone marrow derived macs  Reproductive tract epithelial lines  APC ligand (T cell partner)  Common alternative name  Signal log2  Score  Score  Signal log2 (Bm12.4/C57epi.1)  CD40 (CD154)    8.86  +++++  0/+  2.99/3.01  CD48 (CD2)    8.68  +++++  +  3.12/3.23  CD70 (CD27)    3.81  +  +  3.61/3.70  CD80 (CD28)  B7-1  6.76  +++  ++  5.29/5.00  CD86 (CD28)  B7-2  10.33  ++++++  +  3.13/3.18  Icam1 (LFA-1)    9.99  +++++  +++++  8.32/8.63  Icosl (Icos)  B7-H2  5.49  ++  +  4.37/4.32  Tnfsf4 (Tnfrsf4)  Ox40L  6.40  +++  +  3.68/3.68  Tnfsf8 (CD30)  CD153  7.86  ++++  +  3.24/3.17  Tnfsf9 (Tnfrsf9)  4-1BBL  5.65  ++  ++  5.54/5.97  Tnfsf18 (Tnfrsf18)  GITRL  4.03  +  +++++/+  9.33/4.88    Bone marrow derived macs  Reproductive tract epithelial lines  APC ligand (T cell partner)  Common alternative name  Signal log2  Score  Score  Signal log2 (Bm12.4/C57epi.1)  CD40 (CD154)    8.86  +++++  0/+  2.99/3.01  CD48 (CD2)    8.68  +++++  +  3.12/3.23  CD70 (CD27)    3.81  +  +  3.61/3.70  CD80 (CD28)  B7-1  6.76  +++  ++  5.29/5.00  CD86 (CD28)  B7-2  10.33  ++++++  +  3.13/3.18  Icam1 (LFA-1)    9.99  +++++  +++++  8.32/8.63  Icosl (Icos)  B7-H2  5.49  ++  +  4.37/4.32  Tnfsf4 (Tnfrsf4)  Ox40L  6.40  +++  +  3.68/3.68  Tnfsf8 (CD30)  CD153  7.86  ++++  +  3.24/3.17  Tnfsf9 (Tnfrsf9)  4-1BBL  5.65  ++  ++  5.54/5.97  Tnfsf18 (Tnfrsf18)  GITRL  4.03  +  +++++/+  9.33/4.88  *mRNA signals in the experimental inflammatory state. View Large Table 7. Costimulatory ligands*.   Bone marrow derived macs  Reproductive tract epithelial lines  APC ligand (T cell partner)  Common alternative name  Signal log2  Score  Score  Signal log2 (Bm12.4/C57epi.1)  CD40 (CD154)    8.86  +++++  0/+  2.99/3.01  CD48 (CD2)    8.68  +++++  +  3.12/3.23  CD70 (CD27)    3.81  +  +  3.61/3.70  CD80 (CD28)  B7-1  6.76  +++  ++  5.29/5.00  CD86 (CD28)  B7-2  10.33  ++++++  +  3.13/3.18  Icam1 (LFA-1)    9.99  +++++  +++++  8.32/8.63  Icosl (Icos)  B7-H2  5.49  ++  +  4.37/4.32  Tnfsf4 (Tnfrsf4)  Ox40L  6.40  +++  +  3.68/3.68  Tnfsf8 (CD30)  CD153  7.86  ++++  +  3.24/3.17  Tnfsf9 (Tnfrsf9)  4-1BBL  5.65  ++  ++  5.54/5.97  Tnfsf18 (Tnfrsf18)  GITRL  4.03  +  +++++/+  9.33/4.88    Bone marrow derived macs  Reproductive tract epithelial lines  APC ligand (T cell partner)  Common alternative name  Signal log2  Score  Score  Signal log2 (Bm12.4/C57epi.1)  CD40 (CD154)    8.86  +++++  0/+  2.99/3.01  CD48 (CD2)    8.68  +++++  +  3.12/3.23  CD70 (CD27)    3.81  +  +  3.61/3.70  CD80 (CD28)  B7-1  6.76  +++  ++  5.29/5.00  CD86 (CD28)  B7-2  10.33  ++++++  +  3.13/3.18  Icam1 (LFA-1)    9.99  +++++  +++++  8.32/8.63  Icosl (Icos)  B7-H2  5.49  ++  +  4.37/4.32  Tnfsf4 (Tnfrsf4)  Ox40L  6.40  +++  +  3.68/3.68  Tnfsf8 (CD30)  CD153  7.86  ++++  +  3.24/3.17  Tnfsf9 (Tnfrsf9)  4-1BBL  5.65  ++  ++  5.54/5.97  Tnfsf18 (Tnfrsf18)  GITRL  4.03  +  +++++/+  9.33/4.88  *mRNA signals in the experimental inflammatory state. View Large Table 8. Coinhibitory ligands*.   Bone marrow derived macs  Reproductive tract epithelial lines  APC ligand (T cell partner)  Common alternative name  Signal log2  Score  Score  Signal log2 (Bm12.4/C57epi.1)  CD80 (CTLA-4)  B7-1  6.76  +++  ++  5.29/5.00  CD86 (CTLA-4)  B7-2  10.33  ++++++  +  3.13/3.18  CD276 (unknown)  B7-H3  6.59  +++  ++++  7.99/7.71  Vtcn1 (unknown)  B7-H4  4.15  +  +  4.12/4.00  Tnfrsf14 (BTLA)  HVEM  9.51  +++++  ++++  7.10/7.65  Lilrb4 (Bst2)  gp49B  12.45  ++++++  +  3.72/3.52  CD274 (PD-1)  PDL1  12.61  ++++++  +++++  8.95/8.99  Pdcd1lg2 (PD-1)  PDL2  9.97  +++++  +  3.47/3.32    Bone marrow derived macs  Reproductive tract epithelial lines  APC ligand (T cell partner)  Common alternative name  Signal log2  Score  Score  Signal log2 (Bm12.4/C57epi.1)  CD80 (CTLA-4)  B7-1  6.76  +++  ++  5.29/5.00  CD86 (CTLA-4)  B7-2  10.33  ++++++  +  3.13/3.18  CD276 (unknown)  B7-H3  6.59  +++  ++++  7.99/7.71  Vtcn1 (unknown)  B7-H4  4.15  +  +  4.12/4.00  Tnfrsf14 (BTLA)  HVEM  9.51  +++++  ++++  7.10/7.65  Lilrb4 (Bst2)  gp49B  12.45  ++++++  +  3.72/3.52  CD274 (PD-1)  PDL1  12.61  ++++++  +++++  8.95/8.99  Pdcd1lg2 (PD-1)  PDL2  9.97  +++++  +  3.47/3.32  *mRNA signals in the experimental inflammatory state. View Large Table 8. Coinhibitory ligands*.   Bone marrow derived macs  Reproductive tract epithelial lines  APC ligand (T cell partner)  Common alternative name  Signal log2  Score  Score  Signal log2 (Bm12.4/C57epi.1)  CD80 (CTLA-4)  B7-1  6.76  +++  ++  5.29/5.00  CD86 (CTLA-4)  B7-2  10.33  ++++++  +  3.13/3.18  CD276 (unknown)  B7-H3  6.59  +++  ++++  7.99/7.71  Vtcn1 (unknown)  B7-H4  4.15  +  +  4.12/4.00  Tnfrsf14 (BTLA)  HVEM  9.51  +++++  ++++  7.10/7.65  Lilrb4 (Bst2)  gp49B  12.45  ++++++  +  3.72/3.52  CD274 (PD-1)  PDL1  12.61  ++++++  +++++  8.95/8.99  Pdcd1lg2 (PD-1)  PDL2  9.97  +++++  +  3.47/3.32    Bone marrow derived macs  Reproductive tract epithelial lines  APC ligand (T cell partner)  Common alternative name  Signal log2  Score  Score  Signal log2 (Bm12.4/C57epi.1)  CD80 (CTLA-4)  B7-1  6.76  +++  ++  5.29/5.00  CD86 (CTLA-4)  B7-2  10.33  ++++++  +  3.13/3.18  CD276 (unknown)  B7-H3  6.59  +++  ++++  7.99/7.71  Vtcn1 (unknown)  B7-H4  4.15  +  +  4.12/4.00  Tnfrsf14 (BTLA)  HVEM  9.51  +++++  ++++  7.10/7.65  Lilrb4 (Bst2)  gp49B  12.45  ++++++  +  3.72/3.52  CD274 (PD-1)  PDL1  12.61  ++++++  +++++  8.95/8.99  Pdcd1lg2 (PD-1)  PDL2  9.97  +++++  +  3.47/3.32  *mRNA signals in the experimental inflammatory state. View Large Epithelial cells were richer in coinhibitory ligands, including B7-H3, HVEM and PDL1 with mRNA levels scored as at least +++. Of these only PDL1 was induced by inflammatory supernatant (24-fold). PDL1 is a notorious coinhibitory molecule associated with ‘T lymphocyte exhaustion’ in chronic viral infections like HIV (Day et al.2006). However, in the C. muridarum mouse model Peng et al. have shown that interrupting the PDL1 pathway with monoclonal antibody had no effect on bacterial clearance. If PDL1 coinhibition significantly impeded T cell activation in the murine genital tract one would predict that PDL1 antibody would have accelerated Chlamydia clearance. Instead blocking the PDL1 pathway with antibody led to increased pathology in the upper genital tract without affecting clearance (Peng et al.2011). DISCUSSION We modeled in vitro the immunobiology of epithelial cells and macrophages in healthy genital tract versus inflamed genital tract. The focus of the investigation was reproductive tract epithelial cells using macrophages as a comparator. This experimental model is contrived by its nature, but we believe useful for initial investigations. All the cytokines in the experimental inflammatory milieu, excepting supplemental recombinant IFN-γ, originated from infected epithelial cells and a Chlamydia-specific CD4 T cell clone uvmo-2. Natural killer cells are likely the major source of IFN-γ in the genital tract during the first 4+ days of infection in C. muridarum infections (Tseng and Rank 1998). It was not practical to add NK cells to epithelial cells and T cells to make conditioned supernatant. In the C. muridarum model IFN-γ in the genital tract secretions peaks on day 4 at 1–5 ηg/ml and then fades to near zero over the following week (Darville et al.2003; Scurlock et al.2011). Humans with Chlamydia genital tract infections similarly have detectable IFN-γ in genital tract secretions (Arno et al.1990). Supplementing IFN-γ to a final experimental concentration of 4 ηg/ml was within physiologic range based on available data. The T cell density used to generate the conditioned media was relatively low (0.33 T cells per epithelial cell), hopefully reflecting T cell to epithelial ratios in foci of infection during the clearance phase of C. muridarum infections. We chose to use a conditioned media rather than recombinant IFN-γ because in our hands 10 ηg/ml of recombinant IFN-γ reduces C. muridarum replication in murine epithelial cells by ∼2.5-fold (Jayarapu et al.2009), while conditioned media from antiCD3-activated CD4 T cell clone uvmo-2 blocks 99.9% of C. muridarum replication in the same epithelial cells (Jayarapu et al.2010); conditioned media is more than its IFN-γ content. Censoring the microarray data for 5-fold differences with a P-value <0.001 comparing epithelial cells and macrophages at rest and exposed to inflammatory supernatant identified over 2000 genes (Table 1, Supporting Information). Many of these genes likely reflect lineage-specific differences between macrophages and epithelial cells. Data not presented includes a large set of genes known to be regulated by interferon, and other genes affected by the inflammatory supernatant that differed between epithelial cells and macrophages without obvious relationship to Chlamydia immunobiology. Genes annotated, here, were limited to those likely reflecting epithelial- and macrophage-specific biology relevant to Chlamydia infections. Analysis of the data yielded several surprising results. In the experimental inflammatory microenvironment, there was little difference in intrinsic (internal) innate defenses of epithelial cells and macrophages with respect to Chlamydia. The basic program for both cell types included defenses directed at the inclusion (p47 GTPases and Gbps) depletion of ATP (Samhd1) and up regulation of antimicrobial effector molecules (Nos2, Plac8 and Mpeg1) that likely directly damage the bacteria and/or the inclusion. These innate defenses were induced by inflammatory supernatant in both cell types except for constitutive high levels of Mpeg1 mRNA in macrophages that was unaffected by supernatant exposure. Uninfected epithelial cells and macrophages within the inflammatory milieu of the genital tract during transition from innate to adaptive immunity are not likely to be very permissive for C. muridarum replication. That result is consistent with the observation that the innate immunity/cytokine wave during the first week of infection clears 1.5 logs (95%) of C. muridarum, before significant adaptive immunity can be demonstrated (Su et al.1999). There were notable differences in extrinsic innate defenses between macrophages and epithelial cells. Macrophages impose innate immunity on their surroundings while epithelial cells keep to themselves. Reflecting that basic difference, macrophages exposed to the inflammatory milieu appeared to take on additional innate defenses including killer receptors typically seen on NK cells and the possibly the ability to cause necrotic/inflammatory cell death in target cells (Dnase1l3). Induced soluble mediators for macrophages and epithelial cells in the experimental inflammatory environment included Th1-biased chemokines CCL2, Cxcl9 and Cxcl10. Macrophages appeared to uniquely up regulate transcription of genes related to prostaglandin synthesis, release IL33 potentially promoting type 2 innate lymphoid cells to produce IL-13, and facilitate IL-17 biology via Il1f9 (IL-36γ). Epithelial cells were uniquely induced to increase transcription of Cxcl11 (Th1 chemokine), and IL-15 important for expansion and activation of intraepithelial lymphocytes and NK cells. C. muridarum is cleared from the genital tract of mice by T cells interacting with infected epithelial cells. Mouse model data support CD4 T cells as the critical subset during primary genital tract infections (Morrison, Feilzer and Tumas 1995; Morrison and Caldwell 2002). Understanding how infected epithelial cells present Chlamydia antigens and activate CD4 T cells is important for understanding sterilizing immunity and has implications for vaccine development. We have previously shown in vitro that epithelial cells have IFN-γ inducible expression of MHC class II molecules, that Chlamydia-specific CD4 T cell clones recognize infected epithelial cells in an IFN-γ and CD4-dependent fashion, and that termination of Chlamydia replicating within epithelial cells correlates with how much IFN-γ produced when activated by infected epithelial cells, which was unrelated to their capacity for IFN-γ production when activated by antigen-pulsed irradiated splenocytes (Jayarapu et al.2009). Data presented here reinforce a sterilizing immunity paradigm based on CD4 recognition of infected epithelial cells presenting antigen in the context of MHC class II molecules. In this study, macrophages had strong mRNA signals for all components of conventional antigen presentation via either MHC class Ia or MHC class II, and are known to be capable of presenting antigen to CD4 or CD8 in their basal state. Epithelial cells did not have strong mRNA signals for MHC class Ia or MHC class II molecules until exposure to an inflammatory milieu containing IFN-γ. Epithelial cells are not competent antigen presenting cells in their basal state; they become competent antigen presenting cells within an inflammatory microenvironment such as that seen during Chlamydia genital tract infections, and this is likely critical during the effector phase of the adaptive immune response. Epithelial cells have very few conventional costimulatory ligands and an imbalance of coinhibitory ligands compared to macrophages (Fig. 2). It is an open question whether Chlamydia-specific T cells effectively activated by infected epithelial cells to terminate Chlamydia replication are able to do so because they recognize relatively abundant epitopes (engagement of multiple T cell receptors), recognize high-affinity antigens (less dependent on secondary signals), or utilize available accessory molecule ligands in a different fashion (are specialized to interact with epithelial cells). Answering that question has important implications for Chlamydia pathogenesis and the assessment of Chlamydia vaccine candidates. Figure 2. View largeDownload slide Model for T cell interaction with macrophages versus epithelial cells in inflamed mucosa highlighting the difference in costimulatory (+) and coinhibitory (–) signals likely delivered by the two cell types. Font size for each receptor–ligand pair is proportional to the mRNA level determined in the microarray analysis. Figure 2. View largeDownload slide Model for T cell interaction with macrophages versus epithelial cells in inflamed mucosa highlighting the difference in costimulatory (+) and coinhibitory (–) signals likely delivered by the two cell types. Font size for each receptor–ligand pair is proportional to the mRNA level determined in the microarray analysis. SUPPLEMENTARY DATA Supplementary Data. Critical assistance with data analysis including the INGENUITY pathway analysis was provided by Jeanette McClintick in the Indiana University Center for Medical Genomics. FUNDING This research was supported by NIH/NIAID grant R01AI070514. Conflict of interest. None declared. 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TI - Modeling the transcriptome of genital tract epithelial cells and macrophages in healthy mucosa versus mucosa inflamed by Chlamydia muridarum infection
JF - Pathogens and Disease
DO - 10.1093/femspd/ftv100
DA - 2015-12-01
UR - https://www.deepdyve.com/lp/oxford-university-press/modeling-the-transcriptome-of-genital-tract-epithelial-cells-and-hvtxhFuW4O
SP - ftv100
VL - 73
IS - 9
DP - DeepDyve
ER -