Chlamydia muridarum infection associated host MicroRNAs in the murine genital tract and contribution to generation of host immune response.

Chlamydia trachomatis (CT) is the major cause of bacterial sexually transmitted infections (STI) in humans 1 , and is associated with long term reproductive damage 2 , increased chance of acquiring HIV 3 , and development of cervical cancer 4 . In 2013, the Center for Disease Control (USA) reported 19.7 million new STI cases with approximately 63% between 15–24 years of age infected with CT 5 . In order to control increasing incidence rates of genital CT through effective prevention programs, it is imperative to address gaps in our current knowledge of the underlying molecular mechanisms at the initial site of infection that contribute to anti-CT immunity 3 , 6 , 7 . Chlamydia muridarum ( C. mur ), a murine pathogen used in the murine genital chlamydial infection model 8 – 12 , mimics several aspects of infection observed in CT-infected women including bacterial colonization/ascension, tissue histopathology, immune responses, and long term pathological sequelae 8 . Intravaginal ( i. vag .) inoculation of C. mur in mice results in vulvitis and vaginitis in the lower genital tract (LGT, vagina and cervix) with subsequent ascension to and infection of the upper genital tract tissues (UGT, uterine horns and oviducts) 10 . Host immune responses following C. mur infection induce collateral tissue damage and sequelae that are typically non-homogeneously distributed in different segments of the reproductive tract 9 . The host immune response of the genital compartment involves migration of neutrophils and macrophages early on 11 , 13 with subsequent development of humoral and cell mediated immune responses 14 – 16 . However, only limited information on the underlying molecular mechanisms that may modulate the anti-CT immune response after infection is currently available. There is growing evidence that small non-coding species of regulatory RNA, i.e. microRNAs (miRs) contribute to critical processes including immune cell development/function 17 – 19 and reproductive biology 20 – 22 . MicroRNAs modulate gene function post transcriptionally by direct binding to target gene mRNA 23 , 24 . To this end, loss of Drosha and DICER; essential components of the RNA Induced Silencing Complex (RISC) responsible for generation of miRs has been associated with alteration of lymphocyte differentiation and associated immune responses 25 , 26 . Expression of miR-125b in naïve CD4 + T cells has been reported to regulate expression of the genes IFNG , IL2RB , IL10RA involved in T cell differentiation 27 . MicroRNA-29 has been observed to control innate and adaptive immune responses against Listeria monocytogenes and Mycobacterium bovis by modulating IFN-γ mRNA 28 . Conjunctival miR expression in inflammatory trachomatous scarring following CT infection in humans has recently been characterized 29 and of the 754 miRs analyzed, 82 were found to be differentially regulated and reported to control genes involved in inflammation, fibrosis, and scarring 29 . Recently, Igietseme et al . reported the role of host caspases in tissue apoptosis and associated genital chlamydial pathogenesis 30 . Importantly, these investigators provide evidence on inactivation of DICER and involvement of miRs that regulate growth and differentiation in CT infected mice 30 . Given that immunological events 10 occurring relatively early in genital chlamydial infection lead to inflammation and may influence the development of upper genital pathology 15 , additional insight into modulation of host miRs during this period may increase our understanding of this process. To address this knowledge gap, we investigated the regulation of 88 inflammation and immunopathology-associated miRs ( Supp. Fig. 4 ) in the immunologically diverse 9 , 14 , 31 , 32 lower ‘non-sterile’ (vagina and cervix) and upper ‘sterile’ (uterine horns and oviducts) compartments of the murine genital tract at day 6, and 12 post C. mur challenge. Comparative profiling revealed 9 miRs (miRs-125b-5p, −214, −23b, −135a, −182, −183, −30c, −30e, and −146) to be significantly regulated at day 6 post challenge in the LGT and were assessed for probable role(s) in chlamydial ascension and host immune modulation. In vitro knockdown using miR-specific inhibitors was associated with significant increase in C. mur numbers in 3 of these 9 miRs i.e −125b-5p, −30c, and −182. Additionally, following C. mur infection, significant regulation of inflammatory molecules, i.e ., NF-κB (miR-125b-5p, −30c, and −182 knockdown conditions), and IRF-1 levels (miR-125b-5p knockdown conditions) was observed. STAT-1 levels were insignificantly affected upon miR-125b-5p, −30c, and −182 knockdown. Similarly, IRF-1 levels were not affected upon −30c, and −182 knockdown. Comparison of these 9 miRs in WT and CD4 −/− animals’ LGT isolated at day 6 post C. mur challenge showed significant regulation of miR-125b-5p, −135a, −182, and −183. Further, mass spectrometric and in silico analysis of proteins in CD4 −/− and WT LGT tissues suggested putative regulation of Heat Shock Protein B1 (HSPB1), and α2HS-glycoprotein (AHSG) by miR-135a, −182 following C. mur infection.

All experiments involving animals in this study were performed in compliance with the Animal Welfare Act, the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals, the “Guide for the Care and Use of Laboratory Animals” published by the National Research Council, and guidelines set forth by the University of Texas at San Antonio Institutional Animal Care and Use Committee (IACUC) under approved protocol MU012-03/14A1. C. muridarum was grown on confluent Hela cell monolayers and purified as previously described 33 . Briefly, infected Hela cells were lysed using a sonicator (40 V pulses for 10 s on ice; Fisher, Pittsburgh, PA), and chlamydial elementary bodies (EBs) were harvested and purified on renograffin gradients. Titered aliquots of bacteria were stored at −70°C in sucrose-phosphate-glutamine (SPG) buffer until used. Four to six-week old female C57BL6 wild type (WT), CD4 + T-cell deficient (CD4 −/− ), CD8 + T-cell deficient (CD8 −/− ), and Interferon Gamma Receptor deficient (IFN-γR −/− ) mice were purchased from Jackson Laboratory (Bar Harbor, ME). Mice were housed and bred at the University of Texas at San Antonio. C57BL6 WT mice were immunized as described previously 34 . Specifically, groups of mice were anesthetized (3% isofluorane) and immunized intranasally on day 0 with 15µg of rCPAF in 25 µl sterile PBS. On days −1, 0, and +1 10 µg CpG containing 1% normal mouse serum was administered. Mice were boosted intranasally with the same dose on days 14 and 28. The selected rCPAF dose (15 µg/mouse) provided optimal protection against genital C. mur challenge 33 . In order to render mice anestrous and more receptive to genital infection, animals were injected subcutaneous with 2.5 mg Depoprovera (medroxy-progesterone acetate; Pharmacia & Upjohn Co., NY) on day 5 before infection 35 . Mice were inoculated i.vag . with 5 × 10 4 C. mur inclusion forming units (IFU). For determination of miR dose dependence groups of WT mice were infected i.vag with 5 × 10 4 , 5 × 10 5 , or 5 × 10 6 IFU. Bacterial burdens were monitored using vaginal swabs on indicated days post C. mur challenge and subsequent plating on Hela cell monolayers grown on culture coverslips 36 . Chlamydial inclusions were detected using an anti- Chlamydia genus specific murine monoclonal primary antibody and goat anti-mouse IgG secondary antibody conjugated to Cy3 plus Hoescht nuclear stain. The average number of inclusions in 5 random microscopic fields was calculated for each animal for time-points up to day 12 post challenge and entire coverslips for days 15–30 post challenge, and results expressed as average number of inclusions per animal group 34 . At the indicated days following infection, mice were euthanized and the genital tract dissected out, cut into two sections, i.e . LGT and UGT, and snap-frozen until used. Total RNA was extracted from snap-frozen tissue sections using the miRNeasy RNA extraction Kit (Qiagen, Valencia, CA) according to manufacturer’s instructions. Total RNA was assessed using a Nanodrop Spectrophotometer (ThermoScientific, Asheville, NC) and RNA samples (1 µg with A 260/280 and A 260/230 values of ≅ 2.0 and ≅1.8 or higher, respectively) were converted to cDNA using a RT 2 First Strand cDNA Kit according to manufacturer’s instructions (Qiagen). For miRNA screening experiments, real-time quantitative PCR was performed on Murine Inflammation and Immunopathology Focused RT 2 miRNA plates (MAM 104D, Qiagen) using a RT 2 SYBR Green qPCR Mastermix per manufacturer’s instructions (Qiagen) and BioRad CFX96 thermocycler (BioRad, Hercule, CA). Individual miR PCR amplifications were performed using custom designed miScript Primer Assays (Qiagen) per manufacturer’s instructions on a DNA Engine Opticon 2 continuous fluorescence detection system supported with Opticon Monitor Software v2.02 (MJ Research, Waltham, MA). All miR expression analyses were normalized to housekeeping RNU6-2_1 or SNORD68 expression values and determined using 2 (−Average ΔΔCT) 37 . Hela 229 cells were seeded at 2.5 × 10 5 cells/well in 24-well culture plates. At the time of seeding, miRNA specific inhibitors (miScript miRNA Inhibitor, Qiagen) were transfected at a final concentration of 20 µM per well using Attractene transfection reagent (Qiagen) and per manufacturer’s specifications, transfection efficiency and knockdown was observed to be >80%. At 16 h post transfection, cells were infected with 0.1 MOI C. mur EBs. Infected cells were fixed 24 h post infection using paraformaldehyde, and stained for chlamydial IFU enumeration as previously described 34 . Non-specific inhibitors (Qiagen) were used as negative control, and infectivity data calculated as the difference in C. mur IFU infected wells and wells with miR-specific inhibitors. To monitor modulation of N uclear F actor i.e NF-κB, and the S ignal T ransducer and A ctivator of T ranscription i.e STAT-1, and I nterferon R egulatory F actor i.e IRF-1 following C. mur infection in miR knockdown conditions, Dual luciferase/renilla activity for NF-κB, STAT-1, and IRF-1 was determined using respective reporter constructs in the CIGNAL Reporter Assay Kit (Qiagen), which encodes the firefly luciferase reporter gene under the control of a minimal (m)CMV promoter. Reporters for all three pathways were transfected either alone or in combimation with miScript miRNA inhibitors for miR-125b-5p, −30c, and −182 in 2.5 × 10 5 Hela cells. At 16 h post transfection, these transiently transfected cells were infected with 0.1 MOI C. muridarum EBs. NF-κB (6 h post infection), STAT-1, and IRF-1 (24 h post infection) activity were determined by monitoring luciferase/renilla levels using the Dual Glo Luciferase assay (Promega, Madison, WI) according to manufacturer’s instructions and a Synergy 2 Muti-Mode Microplate Reader (Biotek, Winooski, VT). For analysis of putative downselected miR- targets, mass spectrometry was performed on genital tract tissue from day 6 naïve, C. mur infected WT, and CD4 −/− mice. Total cell protein extraction was accomplished using RIPA Lysis Buffer Kit (Santa Cruz Biotechnology, Santa Cruz, CA) according to the manufacturer’s protocol. Protein concentration was determined using an Invitrogen EZQ Protein Quantitation Kit (Invitrogen, Grand Island, NY). Quantitative tandem mass spectrometry-based “microwave & magnetic (M 2 )” proteomics was performed on individual specimens and pooled reference materials from two different groups of three specimens as previously described 38 . Tryptic peptides, including reference material were terminally tagged with TMT-6plex isobaric labeling reagents (ThermoScientific, San Jose, CA). Each specimen was derivatized with one TMT reagent i.e 127–130 while pooled reference materials were encoded with TMT reagents 126 or 131 ( C. mur infected CD4 −/− and infected WT, respectively). To normalize across all specimens, TMT-encoded cell lysate were mixed with pooled reference material, and subjected to triplicate analysis with capillary liquid chromatography-Fourier-transform-tandem mass spectrometry (LC/FT-MS/MS). Bioinformatic analysis for putative binding sites in genes encoding proteins observed to be modulated by C. muridarum infection related miRs was performed using a miR target predictive algorithm ( www.microRNA.org , Memorial Sloan-Kettering Cancer Center, NY). All experimental results are calculated on the mean ± SEM of two to five independent experiments. GraphPad Prism 5 (La Jolla, CA) was used to perform all tests of significance. MiR analysis was performed using RT 2 Profiler PCR Array Data Analysis version 3.5 (Qiagen). Student’s t -test was used for comparisons between two groups and ANOVA with Tukey B post hoc test for three or more groups. Differences were considered statistically significant if P values were < 0.05.

There is accumulating evidence on the role of miRs in modulating inflammatory processes, cell signaling, differentiation, homeostasis 18 , 19 and controlling intracellular bacterial and viral infections 28 , 39 but there is little information on genital chlamydial infection. A PCR array ( Suppl. Fig. 4 ) containing 88 miRs reported to contribute to immuno-pathological processes in several disease models 40 and regulated in the female reproductive tract 22 was used to identify putative miRs associated with C. mur infection. MiR profiling in the lower and upper regions 9 , 14 , 31 , 32 of the murine genital tract were evaluated ( Fig. 1 ) at 6 and 12 days post i. vag C. mur infection (5 × 10 4 IFU) in C57BL6 mice. Bacterial burden at day 6 following C. mur challenge (307300±121700 IFU) were reduced by day 12 (12700±8900 IFU) post challenge with resolution of vaginal infection by day 30 post challenge ( Fig. 2 ). As shown in Fig. 1 , 77 of the 88 miRs (> 80%) associated with inflammation and immunopathology were downregulated (green rectangles and dots in Fig. 1A ) at day 6 in the LGT of C. mur challenged mice compared to mock infected control mice. In contrast, a greater proportion (≅45%) of UGT associated miRs remained unchanged ( Fig. 1B , black rectangles and dots). At day 12 post infection, more than 40% of the miRs were upregulated in the LGT ( Fig. 1C , red rectangles and dots) while less than 15% and approximately 35% of the miRs were upregulated or unaffected respectively in the UGT ( Fig. 1D ). Overall, regulation of immunopathology related miRs was greater in LGT than UGT during first 12 days of infection with initial downregulation (day 6) followed by upregulation at day 12. Eleven of the 88 miRs ( P < 0.05) exhibiting greater than ±2 fold change following C. mur challenge compared to controls were selected for further study ( Table 1 ). Importantly, 4 of these 11 miRs (miR-125b, −16, −23b, 182) are abundant in the female reproductive tract 22 . These 11 miRs are referred to as ‘infection associated’ and modulated in a spatial (lower vs. upper) and temporal (day 6 vs. day 12) fashion following C. mur challenge ( Table 1 ). These 11 infection associated miRs were not significantly regulated in a spatio-temporal fashion in rCPAF vaccinated mice compared to mock vaccinated ( i.e C. mur challenge) mice at day 6 and 12 post challenge ( Supp Fig. 1 and Supp Table 1 ). Furthermore, miR expression analysis using miR-specific PCR primers confirmed 9 of the 11 miRs changed greater than ±2 fold at 6 days post-infection with 5 × 10 4 IFU C. mur in the LGT ( Fig. 3 ) with the exception of miR-16 and −451 ( Table 1 ). Increased C. mur challenge dose 5 × 10 5 and 5 × 10 6 previously reported to elevate immune response 41 , 42 , did not significantly alter expression of 6 (miR- 135a, 183, 182, 30e, 23 and 30c) of the 9 infection specific miRs. Although significant differences in the fold change of miR-146 and −125b were observed following increased challenge dose, the trend of regulation (upregulated for miR-146a and downregulated for 125b compared to mock challenged controls respectively) was not consistent with increasing challenge doses ( Fig. 3 ). Because no overall difference in miR modulation was observed with increasing infection dose previously shown to elevate immune response 41 , 42 , an infection dose of 5 × 10 4 IFU was used for all subsequent analyses. These 9 miRs have been previously reported to contribute to critical functions including initiation of innate immune signaling and controlling inflammatory cytokines 17 , 18 , 27 , 43 , 44 their role in genital CT infection remains to be elucidated. To this end, knockdown of miR-125b, −30c, and −182 in Hela cells with miR specific inhibitors (with >80% transfection knockdown efficiency), resulted in significant increase in chlamydial IFU compared to control ( Fig. 4A ), and modulation of NF-κβ activation and IRF-1 levels ( Fig. 4B ) suggests these miRs may affect chlamydial infectivity and inflammatory gene function. Significant reduction in NF-κβ activation was detected upon knockdown of miR-125b, −30c, and −182, respectively whereas, IRF-1 levels were significantly increased only upon miR-125b knockdown ( Fig. 4B ). STAT-1 levels were not affected by knockdown of miR-125b, −30c, and −182 ( Fig. 4B ). Our laboratory 33 , 34 , 45 and others 8 , 9 , 15 have shown that antigen specific CD4 + T cells and IFN-γ provide robust protection in primary infections and are essential for vaccination generated protection against chlamydial infection. Antigen specific CD8 + T cells have been reported to contribute to anti-chlamydial immune responses 46 , 47 as well as development of pathology 12 . To investigate the contribution of CD4 + and CD8 + T cells as well as interferon-γ in modulating these 9 miRs at the site of infection, we compared miR expression in WT mice to immuno-deficient mice 6 days post-challenge. As shown in Fig. 5 , significant differences in expression of mir-125b-5p, −135a, −183, and −182 in CD4 −/− mice (white bars) compared to WT mice challenged with C. mur was observed suggesting that CD4 + T cells may contribute to modulation of infection associated miR response at the infection site. In contrast, there were no significant difference in expression of these miRs in C. mur infected IFN-γR −/− ( Supp. Fig. 2A ) and CD8 −/− ( Supp. Fig 2B ) mice compared to WT LGT at the same time (day 6). Importantly, no statistically significant differences in expression of these miRs in the LGT of mock infected WT, CD4, CD8 and IFN-γ knockout mice (data not shown). MicroRNAs have been reported to modulate cellular functions and immune responses by regulation of gene products 17 , 18 , 27 , 28 , 48 . In order to correlate host miR regulation with host protein expression following C. mur challenge, we conducted proteomic analyses to identify differentially expressed proteins at day 6 in the LGT of mock and C. mur challenged mice. We observed significant regulation of acute phase response signaling, structural reorganization, and stress/inflammation related proteins in C. mur challenged mice ( Supp. Fig. 3 ). Among the differentially expressed proteins, we observed two proteins, HSPB1 (Heat Shock Protein B1) and AHSG (Alpha-2HS-Glycoprotein, or Fetuin-1 contain predicted binding sites for miR-135a, and −182 ( Fig. 6A , in silico prediction using www.microRNA.org ). Interestingly, miR-135a was predicted to have binding sites in both, HSPB1 and AHSG suggesting the robustness and redundancy of miR-protein modulation ( Fig. 6A ). These two proteins were significantly upregulated in C. mur infected WT compared to mock infected WT mice, and correlated with miR-135a, and −182 expression which was downregulated following C. mur challenge ( Fig. 1A and Table 1 ). Additionally, these two proteins were significantly downregulated in CD4 −/− mice compared to WT following infection ( Fig. 6B, C ) and these changes correlated to significant upregulated expression of miR-135a and −182 ( Fig. 5 ). We next mapped i.e identified the “interactome” network for known protein-protein interaction of C. mur infection modulated proteins by mass spectrometry. These IPA analyses revealed HSPB1 and Fibronectin 1 (FN1) ( P = 2.3 × 10 −2 ), and AHSG and Decorin (DCN) ( P = 8.59 × 10 −8 ) to interact significantly with each other (data not shown). Comparative protein expression assays indicated that both FN1 and DCN were downregulated upon chlamydial infection in WT but not CD4 −/− mice ( Fig. 7 ) consistent with IPA predicted protein networking with HSPB1 and AHSG, respectively. As shown in Fig. 7A , FN1 levels were significantly reduced in WT mice compared to mock infected WT or C. mur infected CD4 −/− mice. Fibronectin binding proteins have been reported to interact with HSP family proteins 49 . FN-Integrin signaling is associated with HSP induction and reduced apoptosis in gut injury 50 . Fibronectin has been reported to interact with bacterial proteins and be involved in bacterial pathogenesis 51 – 53 . Fibronectin is known to be recruited by C. trachomatis serovar L2 and D EBs to facilitate tissue colonization 54 , but its role in bacterial pathogenesis is not known. We also observed DCN ( Fig 7B ), to be regulated in a manner opposite to its interacting member AHSG ( Fig 6C ) consistent with a previous report on DCN-AHSG interaction 55 . Decorin has been indicated previously in microbial adhesion 56 , and contribute to bacterial pathogenesis 57 . Chlamydia pneumoniae EB adhesion and infectivity is affected by interactions between Invasin Protein Pmp21, and human epidermal growth factor receptor 58 known to have decorin binding sites 59 . Collectively, these results suggest that C. muridarum infection induced host response (protein expression) may be functionally related to C. muridarum infection associated miRs. However, their specific contribution to bacterial pathogenesis warrants further investigation.

We provide evidence suggesting (1) modulation of host microRNAs following in vivo C. muridarum genital infection, and (2) their possible contribution to infectivity, and associated anti-chlamydial immunity. Specifically, expression of host miRs ( Fig 1 ) were downregulated in the genital tract of infected mice compared to mock-controls at day 6 post bacterial challenge. We also observed that these miRs were significantly regulated in a time dependent fashion (day 6 compared to day 12) in the LGT ( Table 1 ). In a similar fashion, we ascertained ( Supp Fig, 1 ) if these infection associated miRs were regulated following intranasal vaccination using rCPAF (recombinant chlamydial protease-like activity factor, serovar L2, a protective chlamydial antigen 35 reported by our laboratory to provide protection against genital C. mur challenge 36 . We observed no significant regulation of infection associated miRs ( Supp Table 1 ) following rCPAF vaccination at days 6 and 12 post challenge in the LGT and UGT suggesting these 11 infection associated miRs are not modulated in rCPAF vaccination ( Suppl Fig. 1 , Suppl Table 1 , and Fig. 2 ) and maybe associated only with primary infection and not with rCPAF vaccine. The spatio-temporal modulation of infection associated miRs ( Table 1 , Fig. 1 ) suggesting these miRs may be associated with bacterial infectivity at day 6 was corroborated by knockdown of miR 125b-5p, −30c, and −182 in host cells which resulted in an increase of C. mur IFU. These in vitro observations partially support the in vivo findings and suggest a possible involvement of these miRs in modulation of chlamydial infection and colonization. Furthermore, under miR −125b-5p, −30c, and −182 knockdown conditions, we investigated modulation of inflammatory signaling molecules i.e NF-κB, IRF-1, and STAT-1 previously reported to be involved in chlamydial infection and control 60–65 . Using a luciferase reporter assay ( Fig. 4A ), we observed C. mur infection under miR-125b-5p, −30c, and −182 knockdown conditions resulted in significant decrease of NF-κB( Fig. 4B ). MiR-125b and miR-125a knockdown has been reported to downregulate NF-κB activity following tumor necrosis factor, alpha-induced protein 3 targeting 66 . Additionally, comparative profiling of infection associated miRs revealed miR-125b and −182 along with −183 and −135a to be significantly upregulated in C. mur infected CD4 −/− mice compared to WT ( Fig. 5 ). Importantly, mir-125b has been reported to inhibit T cell differentiation by controlling IFNG, IL2RB , and PRDM1 genes 27 . MiR-125b has also been shown to increase macrophage activation, responsiveness to IFN-γ 43 , promote cell survival via NF-κB 67 , and regulate MyD88 68 (a critical molecule involved in signaling cascades leading to NF-κB mediated transcription of proinflammatory genes 69 ). Moreover, mir-182 which belongs to the miR-183, −96, −182 cluster 70 has been reported to upregulate NF-κB in malignant gliomas 71 , and is induced by IL-2 and shown to clonally expand helper T cells 72 . Similar to NF-κB, IRF and STAT family have been reported to be involved in downstream signaling involving cytokine production 73 , 74 . Significantly increased IRF-1 expression was observed following miR-125b inhibition ( Fig. 4B ). Although increased IRF-1 levels lead to production of interferon which controls chlamydial infection 63 , 64 , there is to date no evidence for miR-125b influence on IRF-1 production. Additionally, miR-125 has been reported to be a non-responsive regulator of interferon-β 75 , and may not directly affect IRF-1 mediated chlamydial reduction involving interferon-β. In contrast, knockdown of miR-30c and −182 did not affect IRF-1 ( Fig. 4B ). We observed no significant change in relative levels of STAT-1 expression under miR-125b-5p, −30c, or −182 knockdown conditions compared to C. mur infected Hela cells suggesting STAT-1 may not be associated with these miRs and C. mur infectivity. A recent report 76 , suggests antigen specific CD4 + T cells and expression of MyD88 is required for resolution of genital C. mur infection. Furthermore, interferon regulation 95, 96 and generation of antigen specific CD4 + Th1 responses which control bacterial infection is well-documented 8, 14, 16 . Taken together, these results suggest that ‘infection associated’ miRs may contribute to chlamydial invasion and modulate inflammatory pathways. Data from our in vitro experiments reported here ( Fig. 4 ) provide evidence for differential production/ expression of inflammatory molecules (NF-κB, and IRF-1) following C. mur infection under miR knockdown conditions (miR-125b-5p, −30c, and −182). However, further investigation using stimulators/agonists of NF-κB, IRF-1, and STAT-1 (not significantly regulated under miR-125b-5p, −30c, and −182 knockdown conditions, Fig. 4B ) in miR-125b-5p, −30c, and −182 deficient Hela cell lines will provide needed insight into downstream pathways that contribute to C. mur infectivity. Importantly, it must be appreciated that (1) in vitro experiments using miR specific inhibitors were were carried out in Hela cells (a human cell line), and (2) murine and human chlamydial genital infection models are dissimilar in certain immunological aspects 3 . Therefore, further investigation of miR regulation of inflammatory molecules and development of adaptive immunity “network” will be greatly strengthened by the availability of specific miR-deficient mice. We next determined cellular targets for infection associated miRs. Mass spectrometry analyses ( Supp Fig 1 ) revealed regulation of structural and stress proteins involved in signaling, cellular actin reorganization, and anti-apoptotic mechanisms 77 – 80 . Amongst these C. mur infection modulated proteins, we observed probable regulation of two proteins, i.e HSPB1 and AHSG by infection associated miRs ( Fig. 6B, C ) corroborating our in silico prediction ( Fig. 6A ). HSPB1 or HSP27 ( Fig. 6B ) belong to a family of proteins reported to play a crucial role in maintaining integrity and function in several cell types 77 . Additionally, this protein family has been reported to initiate adaptive immune responses, and exert immuno-regulatory functions 78 , 79 . The phosphorylation of HSP27 and associated inhibition of chlamydial growth by rottlerin involving p38-regulated/activated protein kinase (PRAK) has recently been reported 81 . AHSG is reported to be associated with inflammatory processes 80 , and has been shown to be produced in elevated levels in women diagnosed with endometriosis 82 , 83 . Additionally, miR-135a and −182 which have binding sites for these two proteins ( Fig. 6A ) have been reported to be functionally active in women with endometriosis 84 or endometrial adenocarcinoma 85 . MiR-135a and −182 were found to be significantly regulated in C. mur infected CD4 −/− mice compared to WT animals ( Fig. 5 ) suggesting a probable link between these miRs, their respective targets, and the contribution of CD4 + T cells in the C. mur infected genital tract. Data described in this report are consistent with the recent findings of Igietseme et al . 30 , indicating miR induced response in UGT pathology following chlamydial infection. Results presented here focus at day 6 post challenge and miRs that may influence bacterial colonization. Recently, the contribution of miRs in ocular CT infection has been reported 29 indicating miR-147b and −1285 to be upregulated in patients with inflammatory trachomatous scarring. Interestingly, miR-23b and −30c were also found to be modulated in patients with conjunctival scarring with or without inflammation compared to healthy individuals suggesting the ubiquitous nature of these two miRs in chlamydial infections. Additionally, the contribution of miRs in the lungs of neonatal mice infected with CT also has recently been described 86 indicating antagomiRs of selected miRs regulated upon CT infection in neonates prevented development of serious respiratory sequelae similar to reports from our laboratory 87 . In summary, this study 29 , 30 , 86 , 88 , 89 30 , 31 , 87 , 89 , 90 corroborates further the importance of host miR regulation in chlamydial infections 29 , 30 , 86 , 88 , 89 , and underscores the need for future studies on the underlying molecular mechanism(s) of pathogenesis.