ORIGINAL ARTICLE

MicroRNA Let-7f: A Novel Regulator of Innate Immune Response in Human Endocervical Cells Ameya Sathe1, Mandar S. Patgaonkar2, Tahir Bashir1, Kudumula Venkata Rami Reddy1 1

Division of Molecular Immunology and Microbiology (MIM), National Institute for Research in Reproductive Health (ICMR), Mumbai, India; Department of Biological Sciences, Tata Institute for Fundamental Research, Mumbai, India

2

Keywords Blimp1, cytokines, let-7f, miRNA, pathogen recognition receptors, tolerance Correspondence K. V. R. Reddy, Division of Molecular Immunology and Microbiology (MIM), National Institute for Research in Reproductive Health (ICMR), J.M. Street, Parel, Mumbai 400012, India. E-mail: [email protected] Submission July 31, 2013; accepted September 8, 2013. Citation Sathe A, Patgaonkar MS, Bashir T, Reddy KVR. MicroRNA let-7f: a novel regulator of innate immune response in human endocervical cells. Am J Reprod Immunol 2014; 71: 137–153

Problem Endocervical epithelial cells express pattern recognition receptors (PRRs) that aid in innate immune responses. Mechanisms regulating signaling of PRRs are poorly understood. Methods of Study Endocervical cells (End1/E6E7) were treated with ligands of TLR9 and RIG-I once or after pre-stimulation with same ligand. Cytokine responses were determined by ELISA. Differential gene expression was analyzed by microarray. Differentially expressed genes were validated by qPCR /Western blot. Role of let-7f was studied by inhibition and over-expression studies using commercial inhibitors and let-7f encoding plasmids, respectively. Results Single stimulation of cells with TLR9 ligand, but not RIG-I ligand, induced tolerance to subsequent challenge to the same ligand. Stimulation with TLR9 decreased let-7f and increased its target Blimp-1. Conversely, RIG-I stimulation increased let-7f and decreased Blimp-1 expression. Inhibition and over-expression revealed let-7f is involved in induction of immune tolerance.

doi:10.1111/aji.12165

Conclusion We identify let-7f as a novel regulator of PRR signaling in endocervical cells.

Introduction Innate immunity is the first line of host defence represented by conserved structural components unique to the pathogens known as pathogen-associated molecular patterns (PAMPs).1 The female reproductive tract (FRT) is one of the primary routes of entry to the body for invading pathogens.2 Epithelial cells of lower FRT play a crucial role in the initiation, regulation, and resolution of both innate and adaptive immune functions in response to microbial infection. PAMPs such as lipopolysaccharide (LPS), unmethylated CpG motifs, double-stranded RNA and peptidoglycan are recognized by pathogen recognition

receptors (PRRs).3 Human cervico-vaginal epithelial cells (CVECs) express several PRRs, which include Toll-like receptors (TLRs), NOD-like receptors (NLRs), retinoic acid–inducible gene-I (RIG-I)-like receptors (RLRs), etc.4 Among TLRs, TLR3, TLR7, and TLR9 are membrane proteins localized to the endosome that recognize viral double-stranded RNA (dsRNA), single-stranded RNA (ssRNA) and unmethylated CpG motifs, respectively, while RLRs, which include RIG-I, Mda5 and LGP2 are cytoplasmic proteins that recognize viral RNA.5 Expression of PRRs enables cervico-vaginal epithelial immune response against viral pathogens. However, these receptors can also recognize commensal

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organisms and other endogenous ligands. For example, TLR3 can sense endogenous mRNA from dying/ necrotic cells6 and can thus amplify inflammation in the absence of viral infection. RIG-I and TLR9 have also been implicated in maintaining immune homoeostasis via recognition of commensal derived nucleic acids.7,8 As commensals are present constitutively in the female genital tract, cervical cells exposed constantly to their nucleic acids. Hence, any perturbation in signaling of these receptors may lead to excessive inflammation. Kobayashi and Flavell9 reported either excessive or prolonged activation of PRR/NF-jB-signaling pathway has devastating effects on the host causing the pathological manifestations of both acute and chronic inflammatory disorders. In contrast, a delayed or insufficient response can cause severe infections. Hence, it is not surprising that a distinct group of TLR/RLR-activated genes are involved in modulation of PRR/NF-jB signal transduction by interfering with upstream-signaling pathways. Several classes of negative regulators of the PRR system have been described in the past: soluble decoy receptors (sTLR2/4), cell-surface transmembrane receptors (ST2, SIGRR, and TRAILR) and several intracellular proteins such as IRAK, suppressor of cytokine signaling 1 (SOCS1), MyD88 and inhibitor of NF-jB (IjB).10 Recently, it has been shown that microRNAs (miRNAs) functionally modulate TLR-mediated immune responses at every step of the innate immune network including inflammation and infection.11–14 MicroRNAs are an evolutionarily conserved class of endogenous regulatory ~22 NT non-coding RNAs involved in post-transcriptional gene repression by targeting 3′ untranslated regions of mRNAs15,16 Let-7 was the first identified human miRNA.17 Its family members are highly conserved across species in sequence and function. Let-7 is linked to inflammation as it degrades Ras mRNA18 and over-expression of let-7 inhibits activation of ERK/MAPK pathways.19 Let-7 is also involved in the regulation of innate immunity. For example, live Salmonella typhimurium and LPS were shown to down-regulate members of let-7 family in HeLa and murine macrophage cell lines.20 The down-regulation of let-7 was inferred to be essential for immune response as let-7 can also target IL-6 and IL-10.20 Let-7 can also modulate expression of TLR4 in biliary epithelial cells,21 thus underlining its vital role in innate immunity. The aim of the present study was to investigate whether stimu-

lation of End1/E6E7 cells with the agonists of TLR9 (CpG-ODN) and RIG-1[poly (I: C) LL] induce tolerance and to unravel molecules regulating innate immune responses mediated by these receptors. Materials and methods Reagents CpG-ODN2006 (henceforth named as CpG-ODN) [TLR9 ligand], poly(I:C) LMW-Lyovec (henceforth named as poly(I:C)-LL) [RIG-I ligand] and poly(I:C) [TLR3 ligand] were purchased from Invivogen. Let7f inhibitor and Trichostatin A (TSA) were from Ambion, Austin, TX, USA and Upstate, Biotech; EMD Millipore, Billerica, MA, USA, respectively. Antibodies of Blimp-1, phosphoERK 1/2, total ERK1/2 (cellsignaling technology), HDAC2 (Sigma-Aldrich, Saint Louis, MO, USA), mouse monoclonal b-actin (Abcam, Cambridge, UK), horseradish peroxidase (HRP)-tagged secondary antibodies (Sigma-Aldrich), ECL Advance Chemiluminescence Detection Kit (GE Healthcare, Chalfont, UK), Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) were procured from the companies given in the parentheses. Cell Culture Immortalized End1/E6E7 human endocervical epithelial cells (American Type Culture Collection, Manassas, VA, USA; ATCC CRL-2615) were maintained in keratinocyte serum-free medium (KSFM; Gibco BRL Life Technologies, Grand Island, NY, USA) supplemented with 50 lg/mL bovine pituitary extract (BPE), 0.1 ng/mL epidermal growth factor (EGF), and CaCl2 to a final concentration of 0.4 mM, 100 U/mL penicillin, and 100 lg/mL of streptomycin. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2 at 37°C. Knockdown of Let-7f To manipulate intrinsic let-7f expression, we utilized an antisense approach to knockdown let-7f expression in End1/E6E7 cells. Let-7f inhibitor and inhibitor control were reconstituted in nuclease-free water at a concentration of 20 lM. One day prior to transfection, End1/E6E7 cells (5 9 104) were plated in a 24-well plate in complete KSFM medium. Next day, these cells were transfected with 30 nM of let-7f inhibitor or inhibitor control using Lipofectamine 2000 American Journal of Reproductive Immunology 71 (2014) 137–153

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(Invitrogen) following manufacturer’s instructions. Complete growth medium was added to the cells and incubated for 48 hrs. Over-Expression of Let-7f To over-express let-7f in End1/E6E7 cells, we utilized plasmid encoding let-7f (pH1-RNApuro-let-7f) to increase let-7f expression as reported previously.19 The plasmid pH1-RNApuro-let-7f and its control vector (pH1-RNApuro) were obtained as gift from Dr. Edna Kimura (University, Sao Paulo, Brazil). One day prior to transfection, End1/E6E7 cells (5 9 104) were plated in a 24-well plate. Next day, 400 ng of pH1-RNApuro-let-7f or pH1-RNApuro were transfected using Lipofectamine 2000 according to manufacturer’s instructions. All experiments were performed 48 hrs post-transfection. Stimulation with Pathogen Recognition Receptor Ligands End1/E6E7 cells (2 9 104 cells/well) were plated in a 96-well plate 1 day prior to stimulation with selected PRR ligands. Cells were treated either with CpG-ODN or poly (I: C)-LL at a concentration of 10 lg/mL for 24 hrs or till 48 hrs by re-stimulation with same ligand for an additional 24 hrs. In few experiments, initial stimulation was performed with CpG-ODN for 24 hrs followed by poly(I:C)LL for an additional 24 hrs. Poly (I: C; 25 lg/mL) used as positive control for poly (I: C) LL. Same treatment regimen was followed for the stimulation of End1/E6E7 cells in which let-7f expression was either knockdown or over-expressed. Both cell and cell-free supernatants were collected from all the treatment and control groups at 24 and 48 hrs post-treatment and stored in 80°C until used for cytokines analysis. Microarray Analysis To study transcriptome of End1/E6E7 cells stimulated with CpG-ODN and poly (I: C) LL, microarray was performed. Briefly, 2 9 105 End1/E6E7 cells were plated in 12-well culture plates. Next day, cells were stimulated with CpG-ODN (10 lg/mL) or poly (I: C) LL (10 lg/mL) for 1 hr. Unstimulated cells served as control. RNA was extracted; integrity was checked using Agilent 2100 Bioanalyzer. The RNA integrity number (RIN) value for all the samples was found

to be greater than 8. Briefly, biotin-labeled cRNA samples for hybridization were prepared according to Illumina’s recommended sample labeling procedure. Labeled, amplified material (750 ng/array) was hybridized to a ver. 3 of the Illumina Human-Ht-12 BeadChip (48K) according to the manufacturer’s instructions (Illumina, Inc., San Diego, CA, USA). Arrays were scanned with an Illumina Bead array Reader confocal scanner (BeadStation 500GXDW; Illumina, Inc.) according to the manufacturer’s instructions. To export gene expression data based on unique gene, Sample Gene Profile option of Illumina BeadStudio software was used. The whole 48803 probes on the Human-Ht12 beadChip ver. 3 were used. Raw data obtained in .chp and .cel files were normalized using GeneSpring GX v 12.0. Intra-array normalization was performed by Quantile normalization for each chip/samples. Interarray normalization was performed by taking median of all the samples. Quantification of Blimp-1, Let-7f, IL-6, IL-8, and HDAC2 by qPCR To determine IL-6, IL-8, and HDAC2 mRNA expression, we stimulated End1/E6E7 cells with three PRR ligands and performed qPCR analysis using SYBR Green chemistry (Bio-Rad, Hercules, CA, USA). In studies involving validation of microarray data of let7f and Blimp-1 mRNAs, End1/E6E7 cells were treated for 1, 2, and 24 hrs. Total RNA was extracted using Trizol reagent (Invitrogen) following manufacturer’s instructions. One microgram RNA was then reverse transcribed using iScript first-strand cDNA Synthesis Kit (Bio-Rad). Sequences of primers used for analysis are given in Table I. Data were analyzed with untreated values set as calibrator and expression normalized to 18S rRNA. Fold change was calculated by 2 DDCt method. For let-7f qPCR, small RNAs were extracted from End1/E6E7 cells using PureLink miRNA Isolation Kit (Ambion) following manufacturer’s instructions. RNA (10 ng) was then reverse transcribed using stem loop primers specific for let-7f and snRNA RNU6B (Table II). Abundance of mature let-7f was analyzed using TaqMan MicroRNA assays (Applied Biosystems, Foster City, CA, USA). Reactions were performed in triplicates, and expression was normalized to RNU6B. For Blimp1 analysis, TaqMan probes were employed (Invitrogen), and expression was normalized to 18S rRNA.

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Table I Gene-Specific Primers Used in the Study Product size (bp)

Annealing temperature (°C)

Gene

Sequence 5′?3′

Accession no

IL-6

F: GAAAGCAGCAAAGAGGC R: GAAGCATCCATCTTTTTCA F:CACCGGAAGGAACCATCTCACT R:TGCACCTTCACACAGAGCTGCA F:TCATTGGAAAATTGACAGCATAGT R: CATGGTGATGGTGTTGAAGAAG F:GTAACCCGTTGAACCCCATT R: CCATCCAATCGGTAGTAGCG

NM_000600.3

72

62

NM_000584.3

101

62

NM_001527.3

151

60

NR_003286.2

151

60

IL-8 HDAC2 18S rRNA

Table II TaqMan Accession Details of Genes Analyzed in This Study by qPCR Using TaqMan Chemistry Gene

TaqMan accession no.

Blimp-1 18S rRNA Let-7f RNU6B

Hs00153357_m1 Hs99999901_s1 002416 001973

Determination of Cytokines IL-6 and IL-8 concentrations were quantified using cross-reactive kits (human IL-6 and IL-8) by ELISA according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). Concentrations were calculated by quadratic regression analysis based on logarithmically transformed optical densities. Control and treated samples were assayed in duplicate wells, and the experiment was repeated thrice. Sample concentrations were calculated from standard curve. The amount of cytokines in the culture supernatants was expressed as picograms/mL.

(PVDF) membranes (Bio-Rad). The strips were blocked with 5% BSA in 0.1% TBST for 1 hr at RT and then probed with antiphospho-antibody (rabbit monoclonal, 1:2000 dilution). The membranes were washed with 0.1% TBST buffer for 2 hrs followed by probing with HRP-conjugated goat anti-rabbit secondary antibody (1:100,000 dilution). The membranes were then washed 0.1% TBST for 3 hrs, and bands were visualized using advance chemiluminescence detection system (ECL; GE Healthcare). The same membranes were stripped and reprobed with total ERK antibody (rabbit monoclonal, 1:2000 dilution) following same protocol. For Blimp-1 detection (rabbit monoclonal, 1:1000 dilution) HDAC2 (mouse monoclonal, 1:1000 dilution), same protocol was employed except that 20 lg/lane protein was loaded. The expression of Blimp1 and HDAC2 was normalized to b-actin (mouse monoclonal, 1:1000 dilution). Intensities of the bands were quantified using Quanty-one software by densitometry scanning (Bio-Rad). Relative expression ratios were calculated and normalized to the values obtained with b-actin control. Luciferase Reporter Assay

Western Blotting To assess ERK activation, whole-cell protein extracts were prepared from End1/E6E7 cells with ice-cold RIPA buffer supplemented with a protease and phosphatase inhibitor cocktail (Sigma). Cell lysates were then centrifuged at 9300 g for 20 min at 4°C, and supernatants were collected. Protein concentration was quantified with the BCA protein assay (Pierce, USA). Ten microgram of protein per group was separated by 10% SDS-PAGE electrophoresis, and proteins were transferred to polyvinylidene difluoride

NF-jB-dependant transcriptional activity was monitored by NF-jB driven luciferase reporter assay. Briefly, cells (2 9 104) were seeded in 24-well plates. Next day, these cells were co-transfected either with 30 nM of let-7f inhibitor or 0.2 lg pH1-RNApuro-let7f plasmid along with 0.1 lg of firefly luciferase reporter construct containing 3X-NF-jB response element and 0.1 lg of SV40 Renilla luciferase control plasmid using Lipofectamine 2000. Forty-eight hours post-transfection, cells were treated with ligand CpG-ODN / poly (I: C) LL (10 lg/mL each) for addiAmerican Journal of Reproductive Immunology 71 (2014) 137–153

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tional 24 hrs. Luciferase activity was measured in a luminometer (Synergy 2, BioTek, Winooski, VT, USA) using the dual-Luciferase Reporter Assay Kit (Dual Glow, Promega, Madison, WI, USA) according to the manufacturer’s instructions. Firefly luciferase activities were normalized to Renilla luciferase levels. Statistical Analysis All the statistical analysis was carried out using GraphPad Prism software version 5.0 (GraphPad software, La Jolla, CA, USA). All values are expressed as the mean  standard deviation (S.D.) of at least three independent experiments. The values were evaluated using the two-tailed unpaired Student’s ttest and considered to be significant if the ‘P’ value was less than 0.05 (P < 0.05). Results Stimulation of End1/E6E7 Cells with The Ligands of TLR9 but not TLR3 or RIG1 Induces Immune Tolerance Based on the data obtained from initial standardization, the dose required for all three PRR ligands to induce optimum immune response in End1/E6E7 cells was determined. The cells elicited maximum response to CpG-ODN (10 lg/mL), poly (I: C) LL (10 lg/mL) and poly (I: C; 25 lg/mL) at concentrations indicated in the parentheses. Poly (I: C) was used as a positive control for poly (I: C) LL. Culture supernatants were collected from End1/ E6E7 cells stimulated for 24 hrs with CpG-ODN or poly (I: C)-LL. After adding fresh media, cells were re-stimulated with the same ligands for an additional 24 hrs, and supernatants were collected for the quantification of pro-inflammatory cytokines viz., interleukin-6 (IL-6) and interleukin-8 (IL-8). The results revealed that End1/E6E7 cells responded robustly to CpG-ODN stimulation and secreted higher amounts of IL-6 and IL-8. Upon re-stimulation with the same ligand, however, these cytokine levels were abrogated and returned to the level of unstimulated cells (Fig. 1a). On the other hand, re-stimulation of End1/E6E7 with poly (I: C) LL did not induce the state of tolerance as these cells responded significantly to re-stimulation with the same ligand (Fig. 1b). The levels of IL-6 were elevated upon re-stimulation than initial stimulation. Similarly, stimulation with poly (I: C) did not induce

tolerance to subsequent re-stimulation with this ligand (data not shown). Interestingly, primary stimulation with CpG-ODN also induced tolerance to subsequent stimulation with poly(I:C)LL (Fig. 1c). We also measured abundance of IL-6 and IL-8 transcripts at different time points after stimulation with CpG-ODN, poly (I: C)LL, and poly (I: C). Two hours post-stimulation, transcripts of IL-6 and IL-8 were increased significantly in response to these ligands (Fig. 1d, P < 0.001). However, at 24-hr poststimulation, transcripts of both cytokines returned to unstimulated level in CpG-ODN-treated cells but remained significantly elevated in poly(I:C) and poly (I:C)LL-induced cells, suggesting transcriptional activation of IL-6 and IL-8 lasts longer in cells stimulated with these ligands (Fig. 1e). Microarray Analysis Revealed Differential Gene Regulation in Response to CpG-ODN and Poly (I: C) LL Among all TLR ligands, LPS tolerance is the most widely studied model of TLR tolerance. It has been reported that stimulation of macrophages with TLR4 agonist LPS, early signaling events enhance negative regulators of TLR pathway that induce a state of hypo-responsiveness to subsequent LPS stimulation.3 Therefore, we hypothesized that stimulation of End1/E6E7 cells either with CpG-ODN or poly (I: C) LL might induce different gene profiles, and analysis of such genes would reveal the mechanism by which TLR9 and RIG-I stimulation induce tolerance to subsequent stimulation. To test this, End1/E6E7 cells were treated with CpG-ODN or poly (I: C) LL for 1 hr, gene expression was analyzed by microarray. The results demonstrated that over 3000 genes were differentially expressed. We focused on those genes that are known to be involved in PRR-mediated signaling as well as oppositely modulated in CpG-ODN and poly(I:C)LL-induced cells. Microarray data revealed that treatment of End1/ E6E7 cells with CpG-ODN and poly (I: C) LL led to up-regulation of several pro-inflammatory cytokines/ chemokines and antimicrobial proteins (Table III). However, subtle differences in genes activated between the two treated groups were observed (Fig. 2). For example, treatment with poly (I: C) LL, but not CpG-ODN resulted in significant up-regulation of IFN-b. Among genes known to be modulated in PRR-signaling pathway, we observed CpG-ODN significantly down-regulated miRNA let-7f expres-

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(a)

Control 24 hr

CpG-ODN 24 hr

Control 48 hr

CpG-ODN (R)

(b)

1000

500

**

** *

***

***

**

1500 ***

1000 500 0

IL6 Control 24 hr Control 48 hr

IL8

IL6

CpG-ODN 24 hr CpG-ODN + Poly(I:C) LL **

1000

n.s.

500 **

n.s.

0 IL6

IL8

Relative mRNA expression (Fold change)

(e)

6

15

*** ***

10

IL8 Poly(I:C) LL Poly(I:C) *** ***

*** ***

5 0

IL6

IL8

Poly(I:C) LL Poly(I:C)

Control CpG-ODN

8

Control CpG-ODN

(d) Relative mRNA expression (Fold change)

1500

Conc. (pg/mL)

Poly(I:C) LL (R)

2000

0

(c)

Control 48 hr

Poly(I:C) LL 24 hr

2500 ***

Conc. (pg/mL)

Conc. (pg/mL)

1500

Control 24 hr

***

***

***

***

4 2 0

n.s.

n.s.

IL6

IL8

Fig. 1 Quantitative analysis of proinflammatory cytokines (IL-6 and IL-8) in End1/ E6E7 cells. End1/E6E7 cells were stimulated with ligands of TLR9 (CpG-ODN:10 lg/mL) (a, c) and RIG-1{poly(I:C)LL, 10 lg/mL} (b) for 24 hrs. Supernatants were collected, cells were re-stimulated with CpG-ODN (a) or poly (I:C)LL (b,c) or CpG-ODN+poly(I:C)LL(c) for an additional 24 hrs. Supernatants were collected for IL-6 and IL-8 determination by ELISA. Real-time PCR analysis of IL-6 and IL-8 mRNAs in End1/E6E7 cells at 2 hrs (d) and 24 hrs (e) post-stimulation with CpG-ODN, poly(I:C) and poly(I:C)LL. Values represent mean (S.D.) of three experiments performed in duplicates (*P < 0.05, ** P < 0.01,*** P < 0.001, NS, not significant, R, re-stimulated).

Table III List of Genes Up-Regulated in End1/E6E7 Cells After Treatment with CpG-ODN and Poly (I: C)-LL Gene CXCL6 {Chemokine (C-X-C motif) ligand 6} IL-8 (Interleukin -8) TFF3 (Trefoil factor 3) S100A7 (S100 calcium-binding protein A7) ULBP1 (UL16 binding protein 1) HMOX1 {Hemeoxygenase (decycling) 1}

CpG-ODN

Poly(I:C)LL

2.8 1.8 3.1 3.7 2.5 2.4

2.3 2.6 2.1 2.3 2.2 1.8

sion, whereas it was up-regulated in poly(I:C)LL-treated cells. The expression of Blimp-1, a known target of let-7f was also found to be up-regulated in CpG-ODN-stimulated cells, while in poly (I: C)LLtreated group, it was unchanged. The expression of IL-6 was up-regulated in CpG-ODN as well as poly (I: C)LL-induced groups, but the increase was significantly higher in latter group.

Functions Chemotactic for neutrophil granulocytes42 Chemotactic for neutrophil granulocytes and lymphocytes43 Maintain barrier function, mucosal healing44 Calcium binding protein; chemotactic factor45 Ligand for NKG2D Receptor (NK cell receptor); activates NK cells46 Response to oxidative stress47

Reverse Modulation of Let-7f by CpG-ODN and Poly (I: C)LL Microarray data revealed that let-7f expression was oppositely modulated by CpG-ODN, poly (I: C)LL in End1/E6E7 cells. Therefore, we validated changes in Let-7f expression in End1/E6E7 cells upon stimulation with CpG-ODN, poly (I: C)LL and poly (I: C) by American Journal of Reproductive Immunology 71 (2014) 137–153

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4

Blimp-1

Let-7f

IFN-β

IL-8

IL-6

Poly(I:C) LL CpG-ODN

Fold change

2

0

–2

–4

Genes modulated

Fig. 2 Microarray analysis of differential gene regulation in End1/E6E7 cells at 1-hr post-stimulation with CpG-ODN and poly(I:C)LL(10 lg/mL each). RNA was extracted and gene expression profile was evaluated by microarray analysis. Let-7f expression was inversely regulated between two treatment groups. Blimp-1 expression was significantly elevated by CpG-ODN as compared to poly(I:C)LL-induction. The expression of IL-6 and IL-8 was up-regulated by these ligands. Increased expression of Blimp-1 is accompanied by lesser induction of its target gene IFN-b .

fore, we validated changes in Blimp-1 expression in End1/E6E7 cells by qPCR. For this purpose, cells were treated with CpG-ODN, poly (I: C)LL, and poly (I: C) for 1, 2, and 24 hrs. The up-regulation of Blimp-1 transcripts at 1-hr post-stimulation with CpG-ODN was ~2.10-fold as per microarray data, while the elevation was 1.5-fold as per the qPCR results and continued to increase by 3.45- and 2.0-fold at 2 and 24 hrs post-stimulation, respectively (Fig. 3b). In contrast, the levels of Blimp-1 transcript at 1-hr poststimulation with poly (I: C)LL was unchanged as per microarray data, while it was down-regulated 1.8-fold as per the qPCR and continued to decrease to 2.7- and 3.9-fold at 2 and 24 hrs post-stimulation, respectively (Fig. 3b). These results were confirmed at protein level by Western blot analysis. The expression of Blimp-1 remain up-regulated at 24 hrs post-CpGODN stimulation, while stimulation with poly(I:C)LL down-regulated its expression (Fig. 3c). Let-7f Modulates Basal Expression of ProInflammatory Cytokines (IL-6 and IL-8)

qPCR. The down-regulation of let-7f at 1-hr poststimulation with CpG-ODN was ~2.10 fold as per microarray data, while the suppression was 2.80-fold as per the qPCR results and continued to decrease by 3.2- and 4.1-fold at 2- and 24-hr post–stimulation, respectively (Fig. 3a, P < 0.001). The fold change up-regulation of let-7f was found to be 2.72 at 1-hr post-stimulation with poly(I:C)LL as per microarray data, while the increase was 1.3fold as per the qPCR results and expression was remained elevated by 2.81- and 3.0-fold at 2 and 24 hrs post-stimulation, respectively (Fig. 3a). Similarly, stimulation of cells with poly (I: C) also up-regulated let-7f expression at all time points tested (Fig. 3a, P < 0.001). Thus, we infer that CpGODN (induce tolerance) suppress let-7f, while poly (I: C)LL (prevent tolerance) up-regulate let-7f expression.

We first determined the effects of let-7f knockdown and over-expression in End1/E6E7 cells. To accomplish this task, let-7f was knocked down with antilet7f (miRVana). As let-7f is known to target IL-6, we anticipated knockdown of let-7f may up-regulate IL-6 expression. As expected, we observed a significant increase in IL-6 mRNA and protein expression in cells transfected with the antilet-7f compared with cells transfected with the inhibitor control. Unexpectedly, in let-7f-deficient cells, IL-8 expression was found to be up-regulated (Fig. 4a,b). Next, End1/E6E7 cells were transfected with let-7f plasmid to over-express let-7f and levels of IL-6 and IL-8 were determined at 48 hrs post-transfection. The results indicated decreased expression of IL-6 and IL-8 (Fig. 4c,d) in let-7f over-expressing cells. Although IL-8 is not known to be a target of let-7f, a let-7f-binding site has been predicted in the 3′-UTR region of IL-8 mRNA using online bioinformatic tools (https://cm. jefferson.edu/rna22v1.0/; Fig. 4e), and therefore, we speculate that IL-8 might also be a target of let-7f.

Pathogen Recognition Receptor Ligands Modulates Blimp-1 Expression in End1/E6E7 Cells

Let-7f Modulates Ligand-Stimulated Expression of IL-6 and IL-8

Microarray data revealed that Blimp-1 expression was significantly up-regulated in CpG-ODN-treated cells as compared to poly (I: C)LL-stimulated cells. There-

Our results so far indicated that poly (I: C) LL increases let-7f and prevents tolerance while CpG-ODN decreases let-7f and induces tolerance. Therefore, we

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Control

Poly(I:C) LL

CpG-ODN

Poly(I:C)

4

** **

3 n.s.

2 1

n.s.

*

*

**

*

**

0 1 hr

2 hr

24 hr

(c)

1

Control

(b) Relative expression of Blimp-1

Relative expression of let-7f

(a)

2

Poly(I:C) LL

CpG-ODN

3

Poly(I:C)

**

* *

2

*

** *

*** *** **

1

0 1 hr 3

2 hr

24 hr

4 Blimp-1

Normalized band density

β-actin

(d) 0.8 0.6 0.4 0.2 0.0

1

2

3

Treatment 1: Medium control ; 2: CpG-ODN 3: Poly(I:C)LL ; 4: Negative control

Fig. 3 (a) Determination of let-7f expression in End1/E6E7 cells stimulated with CpG-ODN (10 lg/mL), poly(I:C; 25 lg/mL), and poly(I:C)LL (10 lg/ mL) for indicated time points. RNA was extracted and expression of let-7f was analyzed by qPCR. Relative expression in fold change was calculated after normalization with snRNA RNU6B. Results expressed as the amount of let-7f in ligand-stimulated cells relative to the unstimulated cells. Values represent mean (S.D.) of three experiments performed in duplicates on different days (*P < 0.05, **P < 0.01, ***P < 0.001). (b) Determination of Blimp-1 expression in End1/E6E7 cells stimulated with CpG-ODN (10 lg/mL), poly(I:C) (25 lg/mL) and poly(I:C)LL (10 lg/mL) for indicated time points. RNA was extracted and expression of Blimp-1 was analyzed by qPCR. Relative expression in fold change was calculated after normalization with 18S rRNA. Results expressed as the amount of Blimp-1 in ligand-stimulated cells relative to the unstimulated cells. Values represent mean (S.D.) of three experiments performed in duplicates on different days (*P < 0.05, **P < 0.01, ***P < 0.001). (c) Western blot analysis show Blimp-1 expression in End1/E6E7 cells at 24 hrs post-stimulation with CpG-ODN and poly(I:C)LL. Representative images of Western blot analysis of Blimp-1 (100 kDa) is shown. b-actin (43 kDa) blot confirmed roughly equivalent loading of samples. (d) Quantitative analysis of intensity of Blimp-1 bands after normalization with b-actin as determined by densitometry.

then reasoned that if let-7f is knocked down, stimulation of poly (I: C)LL should induce tolerance. Similarly, over-expression of let-7f should prevent CpG-ODN-induced tolerance. 1. To validate role of let-7f, we knocked down let-7f expression in End1/E6E7 cells and then treated with poly (I: C)LL (10 lg/mL) for 24 hrs and IL-6 and IL-8 levels were measured in culture supernatants. As shown in Fig. 5a, we found that, cells deficient in let-7f produced significantly

lesser amounts of IL-6 and IL-8 proteins compared with inhibitor control treated cells. When cells of both the groups (were re-stimulated for 24 hrs with poly(I:C)LL, IL-6, and IL-8 levels in let-7f knockdown cells were decreased to basal levels, thus mimicking a tolerance-like condition (Fig. 5b), suggesting decreased expression of let-7f induces tolerance. 2. Next, let-7f was over-expressed in End1/E6E7 cells and these cells were stimulated with CpGAmerican Journal of Reproductive Immunology 71 (2014) 137–153

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Fig. 4 Effect of let-7f expression on cytokine changes in End1/E6E7 cells. Effect of let-7f knockdown (a,b) and over-expression (c,d) on changes in cytokines (IL-6 and IL-8) mRNA (a, c) and protein expression (b,d) in End1/E6E7 cells was determined by qPCR and ELISA, respectively. Expression of both cytokine was significantly elevated in let-7f knockdown cells, whereas decreased in let-7f overexpressing cells. Values represent mean (S.D.) of three experiments performed in duplicates on different days (*P < 0.05, **P < 0.01). (e) Target site for let-7f (indicated in black) in 3′UTR region of IL-8 mRNA (indicated in red).

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ODN (10 lg/mL) for 24 hrs, and levels of IL-6 and IL-8 levels were determined by ELISA. The results revealed that although cells responded to CpGODN, they secreted lesser amount of IL-6 and IL-8 as compared to the control plasmid transfected cells (Fig. 6a). This may be attributed to the fact that let-7f by itself targets mRNAs of IL-6 and IL-8. However, when these cells were re-stimulated with CpG-ODN for another 24 hrs, the Ievels of IL-6 and IL-8 were significantly elevated compared with control plasmid (Fig. 6b), suggesting over-expression of let-7f prevents tolerance in End1/E6E7 cells. Our data thus show that over-expression of let-7f prevented CpG-ODN-mediated tolerance. Let-7f did not Affect the Activation of ERK/MAPK Pathway It is documented that PRR ligands induce IL-6 and IL-8 expression via ERK pathway in epithelial cells. Therefore, we next investigated whether let-7f affects activation of ERK pathway. As shown in Fig. (7a,b), let-7f-deficient End1/E6E7 cells were stimulated with agonist of RIG-1, and activation of

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ERK pathway was analyzed by Western blot. Similar level of ERK activation was observed in control and let-7f-deficient cells. Next, we checked whether let7f over-expressing cells can activate ERK pathway after stimulation with CpG-ODN. We observed that activation of ERK pathway was not affected by overexpression of let-7f in End1/E6E7 cells (Fig. 7c,d). Let-7f did not Affect the NF-jB Signaling We then checked whether knockdown or overexpression of let-7f in End1/E6E7 cells has any effect on activation of NF-jB signaling. It is known that nuclear factor-jB (NF-jB) regulates a battery of genes that are critical to innate and adaptive immunity and inflammation.22 NF-jB-dependent transcriptional activity was monitored using NF-jBp65-driven luciferase reporter construct having 3-NF-jB-binding sites followed by luciferase gene. The readings of luciferase were normalized to Renilla luciferase to nullify the differences due to transfection efficiencies. End1/ E6E7 cells were transfected with antilet-7f (knockdown; Fig. 8a) or pH1-let-7f plasmid (over-expression; Fig. 8b) and stimulated either with poly (I: C) LL or

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Fig. 5 Effects of antilet-7f on ligand-induced up-regulation of IL-6 and IL-8 proteins in End1/E6E7 cells as determined by ELISA. To knockdown endogenous let-7f, End1/E6E7 cells were treated with antilet-7f (30 nM). These cells were stimulated for 24 hrs with poly(I:C)LL (10 lg/mL) (a). Supernatants were collected, cells were re-stimulated for an additional 24 hrs with same ligand (10 lg/mL) (b), supernatants were analyzed for IL6 and IL-8 by ELISA. Cells deficient in let-7f synthesized significantly lesser amounts of IL-6 and IL-8. Values represent mean (S.D.) of three experiments performed in duplicates on different days Level of significance (*P < 0.05, **P < 0.01, ***P < 0.001) were calculated by Student’s ttest (NS, Not significant; R, re-stimulated). pH1

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Fig. 6 Effects of let-7f plasmid on ligand-induced up-regulation of IL-6 and IL-8 in End1/E6E7 cells. These cells transfected with let-7f plasmid or control plasmid (400 ng/mL each) and then stimulated with CpG-ODN (10 lg/mL) for 24 hrs (a). Supernatants were collected, cells were restimulated for an additional 24 hrs with same ligand (b), supernatants were analyzed for IL-6 and IL-8 by ELISA. Cells over-expressing let-7f synthesized increased amounts of IL-6 and IL-8 upon re-stimulation with CpG-ODN. Values represent mean (S.D.) of three experiments performed in duplicates on different days. Level of significance (*P < 0.05, **P < 0.01) was calculated using Student’s t-test (NS, Not significant; R, restimulated).

CpG-ODN, respectively. As shown in Fig. (8a,b), stimulation of cells with poly(I:C)LL resulted in activation of NF-jB, and no significant difference was observed the control and let-7f knockdown cells. Similarly, over-expression of let-7f did not affect NF-jB activation post-stimulation with ODN. Let-7f Targets Blimp-1 mRNA We next asked whether let-7f targets Blimp-1 expression in End1/E6E7 cells. The qPCR results revealed that Blimp-1 mRNA in let-7f deficient cells at 48 hrs post-stimulation were up-regulated by ~3-fold (Fig. 9a). In contrast, over-expression of let-7f in End1/E6E7 cells show reduction of Blimp-1 mRNA expression by 3.0-fold (Fig. 9b). Similar modulation of Blimp1 expression upon inhibition and over-expression of let-7f was seen at protein level (Fig. 9c,d).

TLR9-Induced Tolerance is Reversible by HDAC Inhibitor It is known that Blimp-1 represses transcription of its target genes by recruiting histone deacetylases (HDACs) at their promoters.23 Therefore, we studied whether CpG-ODN-induced tolerance is reversed by HDAC inhibitor, trichostatin A (TSA). To perform this task, IL-6 and IL-8 levels were measured by ELISA in End1/E6E7 cells at 24 hrs post-CpG-ODN (10 lg/mL) treatment for 24 hrs. After replacing the medium, fresh medium contain same ligand or TSA or CpGODN+TSA was added and incubated for an additional 24 hrs. Release of cytokine into the medium was determined by ELISA and the results indicated that upon re-stimulation with CpG-ODN, cells displayed tolerance as IL-6 and IL-8 levels were comparable with untreated cells (Fig. 10). Treatment with TSA American Journal of Reproductive Immunology 71 (2014) 137–153

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Fig. 7 Effect of Let-7f knockdown or over-expression on phospho-ERK/MAPK in End1/E6E7 cells. These transfected with antilet-7f or inhibitor control (30 nM each) and then stimulated with poly(I:C)LL (10 lg/mL) for 30 min (a). End1/E6E7 cells were transfected with pH1-let-7f plasmid or pH1 vector control (400 ng/mL each) were induced with CpG-ODN (10 lg/mL) for 30 min (c). Levels of pERK/MAPK and total ERK were determined by Western blot. Representative image of Western blot analysis of pERK and total ERK expression in antilet-7f (a,b) and pH1-let-7f plasmid is shown (c,d). Total ERK blot confirmed equivalent loading of samples. A quantitative assessment of the intensity of the each band after normalization with total ERK was determined by densitometry (b,d). Inhibitor

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Fig. 8 Effect of Let-7f knockdown or over-expression on NF-jBp65 activity in End1/E6E7 cells. (a) End1/E6E7 cells co-transfected with antilet-7f or inhibitor control (30 nM each) and NF-kB-driven luciferase reporter construct were stimulated with poly(I:C)LL (10 lg/mL) for 24 hrs. The activity of p65-NF-jB in nuclear extracts was determined by firefly luciferase reporter assay. Values were normalized to the Renilla control. Luciferase values represent (mean  S.D.) of three independent experiments performed in duplicates (NS, Not significant). (b) End1/E6E7 cells co-transfected with pH1-let-7f plasmid or pH1 vector (400 ng/mL each) and NF-kB-driven luciferase reporter construct were stimulated with CpG-ODN (10 lg/mL) for 24 hrs. The activity of p65-NF-jB in nuclear extracts was determined by firefly luciferase reporter assay. Values were normalized to the Renilla control. Luciferase values represent (mean  S.D.) of three independent experiments performed in duplicates (NS, Not significant).

alone showed marginal increase in IL-6 and IL-8 levels. In contrast, cells treated with CpG-ODN +TSA complex resulted in significant up-regulation of these cytokines as compared to the cells treated either with CpG-ODN or TSA. These results indicate that tolerance induced by CpG-ODN is reversed by HDACs inhibition, attributing the involvement of histone modifications in cellular immune tolerance.

Let-7f Targets Histone Deacetylase-2 (HDAC2) Blimp1 requires HDAC1 and HDAC2 proteins to repress transcription of its target genes.24 Therefore, we asked whether let-7f targets HDAC proteins. Using ‘RNA22-HAS’ algorithm online software (https://cm.jefferson.edu/rna22v1.0/)25 for miRNA target prediction, it was observed that HDAC2 mRNA

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Treatment 1: Negative control ; 2: Inhibitor control 3:Anti-let-7f ; 4: Plasmid (pH1) control and 5: pH1-let-7f Fig. 9 Effect of Let-7f knockdown or over-expression on Blimp-1 mRNA and protein expression in End1/E6E7 cells. (a) End1/E6E7 cells were stimulated with antilet-7f to knockdown endogenous let-7f. Blimp-1 mRNA expression was determined by qPCR at 48 hrs post-treatment. Fold change was calculated after normalization with 18S rRNA. Blimp-1 mRNA expression was significantly up-regulated in let-7f-deficient cells. Values are mean  S.D. of three independent experiments performed in duplicates (***P < 0.001). (b) End1/E6E7 cells were stimulated with pH1-let-7f to over-express let-7f. Blimp-1 mRNA expression was determined by qPCR at 48 hrs post-treatment. Fold change was calculated after normalization with 18S rRNA. Blimp-1 mRNA expression was significantly decreased in pH1-let-7f plasmid-treated cells. Values are mean  S.D. of three independent experiments performed in duplicates (***P < 0.001). (c) Expression of Blimp-1 was checked at protein level by Western blot at 48 hrs post-treatment with antilet-7f or pH1-let-7f plasmid or their respective controls. b-actin (43 kDa) blot confirmed roughly equivalent loading of samples. Representative image of Western blot of Blimp-1 (100 kDa) expression is shown. (d) Quantitative analysis of intensity of the Blimp-1 bands after normalization with b-actin as determined by densitometry. The results revealed up-regulation of Blimp-1 in let-7f-deficient cells and down-regulation in let-7f over-expressing cells.

has multiple binding sites complementary to let-7f in its 3′UTR region (Fig. 11a). In let-7f over-expressing cells, expression of HDAC2 mRNA and protein was reduced, whereas knockdown of let-7f increased HDAC2 expression (Fig. 11b,c). We also examined HDAC2 expression at 24 hrs post-stimulation with CpG-ODN and poly (I: C) LL in End1/E6E7 cells. These cells treated with CpG-ODN show significantly higher expression of HDAC2 as compared to poly (I: C) LL treated cells (Fig. 11f,g). These results indicated that HDAC2 is a target of let-7f.

Discussion A number of infections that cause damage to FRT are caused by inflammation.26 Recognition of pathogenic microorganisms by cervico-vaginal epithelial cells (CVECs) is required for host defence and to trigger the adaptive immune system to eliminate pathogens.27 Lack and/or poor expression of PRRs by CVECs detrimentally reduce the immune responses to pathogens.28 We used immortalized epithelial cells of endocervical origin (End1/E6E7 cells), American Journal of Reproductive Immunology 71 (2014) 137–153

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Fig. 10 Effect of HDAC inhibitor, trichostatin A (TSA) on CpG-ODNinduced tolerance in End1/E6E7 cells. End1/E6E7 cells were treated with CpG-ODN (10 lg/mL) for 24 hrs. Cells were washed, re-stimulated for an additional 24 hrs either with CpG-ODN (10 lg/mL) or TSA (10 lg/mL) or CpG-ODN+TSA. At 24 hrs post-re-stimulation, supernatants were collected; IL-6 and IL-8 levels were determined by ELISA. Values are the mean (S.D.) of three independent experiments performed on different days. Level of significance (**P < 0.01, *P < 0.05) was calculated by Students t-test (NS, Not significant; R, restimulated).

as the procurement of primary cervical cells is problematic due to several ethical reasons. The morphological and immunocytochemical properties of this cell line have been shown similar to primary endocervical cells.29 Studies carried out with End1/E6E7 cells revealed that induction of pro-inflammatory cytokines (IL-6 and IL-8) in CpG-ODN-stimulated End1/E6E7 cells was abolished after initial stimulation with the same ligand, thus mimicking ‘PRR tolerance’. In contrast, stimulation with ligands of TLR3 and RIG-1 did not induce tolerance as these cells secreted abundant levels of IL-6 and IL-8 upon re-stimulation with same ligands. For such an observation, we reasoned that comparison of differentially regulated genes after ligand treatment by microarray would offer an insight into the mechanisms influencing tolerance. Accordingly, we excluded those genes that were upregulated or down-regulated in treated groups and focused only on those genes which were oppositely regulated. From the microarray data, we observed a significant up-regulation of let-7f with a downregulation of its target gene Blimp-1 in cells stimulated with RIG-I ligands. Real-time PCR results confirmed the microarray data which revealed a

time-dependent increase of let-7f with a decrease in expression of its target protein Blimp-1. Conversely, stimulation with TLR9 ligand significantly reduced the levels of let-7f at all time points checked, with an increase in Blimp-1 transcript and protein expression. It has been reported that stimulation of epithelial cells with TLR3 ligand [poly(I:C)] did not induce tolerance.30 To the best of our knowledge, till date no mechanism has been shown to be responsible for such a phenomenon, although few indirect clues existed. For example, infection of lung epithelial cells with respiratory syncytial virus (a known inducer of TLR3 pathway) has been shown to prevent tolerance to stimulation with poly (I: C).31 Furthermore, let-7f was recently identified as one of the miRNA induced by respiratory synctial virus in lung epithelial cells,32 which may explain inability of this virus to induce TLR tolerance. Therefore, we hypothesized that let-7f may play a crucial role in epithelial inflammatory response. Our hypothesis is further supported by the fact that Blimp1, a target of let-7f was differentially modulated upon CpG-ODN and poly (I: C)LL stimulation.15 Blimp-1 is a nuclear zinc finger containing transcriptional repressor that was initially shown to be a master regulator of B and T lymphocytes33; its expression is repressed in diffuse large B-cell malignancies34 due to members of let-7 family. Blimp-1 represses transcription of IFN-b,35 IL-6 genes36 by recruiting HDAC proteins to their respective gene promoters.37 Interestingly, LPS-induced tolerance has been shown to be mediated by HDAC proteins.38 Subsequent studies also indicated that Blimp-1 also plays an important role in regulation of TLR-mediated cytokine release as Blimp-1 knockout dendritic cells secrete significantly higher amounts of IL-6 upon LPS stimulation.39 Therefore, we reasoned that Blimp-1 may play a similar role in CpG-ODN induced tolerance in other cell types such as cervicovaginal epithelial cells. Our results demonstrate that down-regulation of let-7f significantly reduces cytokine response to subsequent ligand stimulation. As knockdown of let-7f did not impair activation of NF-jBp65 and ERK/ MAPK pathways, it may be suggested that reduced levels of let-7f induce tolerance without compromising pro-inflammatory pathways. On the other hand, Blimp1 can bind to promoters of IL-6 and IL-8 and recruit HDACs to inhibit binding of NF-jB and other transcription factors. Our findings also show that let7f targets HDAC2, which is required by Blimp-1

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Fig. 11 Effect of let-7f knockdown or over-expression on HDAC2 mRNA and protein expression in End1/E6E7 cells. (a) miRNA target sites for let-7f (indicated in black) in 3′-UTR region of HDAC2 mRNA (indicated in red) predicted by RNA22-HSA algorithm online software. (b) End1/E6E7 cells were transfected either with antilet-7f or pH1-let-7f or their respective controls. HDAC2 mRNA at 48 hrs post-treatment by qPCR. Fold change was calculated after normalization with 18S rRNA. In antilet-7f-deficient cells, HDAC2 mRNA was up-regulated, whereas in pH1-let-7f over-expressing cells, it was down-regulated. Values are mean (S.D.) of three independent experiments performed in duplicates (**P < 0.01). (c) End1/E6E7 cells were treated with CpG-ODN (10 lg/mL) or poly(I:C)LL (10 lg/mL) for 24 hrs. HDAC2 expression was evaluated by qPCR. Fold change was calculated after normalization with 18S rRNA. Values are mean (S.D.) of three independent experiments performed in duplicates (**P < 0.01). (d) End1/E6E7 cells were transfected with antilet-7f inhibitor or pH1-let-7f or their controls. HDAC2 (50 kDa) expression was determined by Western blot (d). Representative image of Western blot analysis of HDAC2 expression is shown. b-actin (43 kDa) blot confirmed roughly equivalent loading of samples. A quantitative assessment of the intensity of each band after normalization with b-actin was determined by densitometry (e). Densitometric data show up-regulation of HDAC2 by antilet-7f and down-regulation by pH1-let-7f. (f).End1/E6E7 cells were treated with CpG-ODN (10 lg/mL) or poly(I:C)LL (10 lg/mL) for 24 hrs. HDAC2 expression was evaluated by Western blot. Representative image of Western blot analysis of HDAC2 expression is shown. b-actin (43 kDa) blot confirmed roughly equivalent loading of samples. (g) Quantitative assessment of intensity of the each band after normalization with b-actin was determined by densitometry. Densitometric data show up-regulation of HDAC2 by CpG-ODN and down-regulation by poly(I:C)LL. American Journal of Reproductive Immunology 71 (2014) 137–153

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Fig. 12 Proposed hypothesis for role of let-7f in immune tolerance in End1/E6E7 cells. Stimulation of these cells via TLR3/RIG-I up-regulates let-7f expression. Elevated levels of let-7f in turn targets mRNAs of Blimp-1 and HDAC2, thereby reducing their expression. Upon restimulation with poly(I:C)LL, transcriptional up-regulation occurs at IL-6 and IL-8 promoters and hence, no tolerance is observed. In contrast, stimulation of cells via TLR9 decreases let-7f expression. As a result, expression of Blimp-1 and HDAC2 increases which binds to promoters of IL-6 and IL-8 and repress their transcription via chromatin remodeling. Upon re-stimulation via TLR9, transcription does not occur at these loci, which leads to tolerance.

in turn suppress Blimp-1 expression, thereby preventing the tolerance. In contrast, signaling through TLR9 causes down-regulation of let-7f and increased Blimp-1 expression. Blimp-1 then binds to promoters of IL-6 and IL-8 and turn- off transcriptional activity by recruiting HDACs to these sites leading to tolerance (Fig. 12). Though the present data suggest let-7f plays an integral role in ligand tolerance in End1/E6E7 cells, a more complete understanding of the role of let-7f in vaginal mucosal immunity may aid in design strategies for controlling inflammation and/or infection. Therefore, it will be of interest to extend these studies to analyze the involvement of let-7f in cellular immune responses in vivo using mouse as animal model. In summary, the aforementioned studies reveal that (i) let-7f is expressed in End1/E6E7 cells and regulate ligand-induced expression of cytokines, (ii) let-7f does not impair the activation of NF-jB p65 or pERK in End1/E6E7 cells and (iii) knockdown or over-expression of let-7f causes reciprocal alterations in immune response to PRR ligands in vitro. Based on the present data, we can now add let-7f to the list of potential regulator of PRR signaling in epithelial cells. Acknowledgments

dependent and independent negative regulation of TLR signaling as reported previously.23 The fact that CpG-ODN-induced tolerance was reversible by trichostatin A, further supports our hypothesis that tolerance in End1/E6E7 cells could be due to increased expression of Blimp1. It has been shown that let-7f expression does not vary between primary and malignant human cervical cells,40 hence, results obtained in our study are not artifacts arising during immortalization. However, a few questions remain to be addressed with respect to its expression and function in human endocervical cells. For example, in breast cancer cells, estrogen receptor (ER) signaling was shown to be down-regulated by let-7 family members.41 It is not known whether in primary cervical cells, let-7f expression is regulated by estrogen (E2) and whether ER and let-7f counter-regulate each other. Based on our data, we propose a hypothesis wherein let-7f mediates tolerance to PRR signaling in End1/E6E7 cells. Binding of TLR3 or RIG-I to their ligands induces up-regulation of let-7f, which

The authors are grateful to the Director for giving encouragement in carrying out this study. This study was funded by Indian Council of Medical Research (ICMR, New Delhi) (NIRRH/MS/91/2013). We thank ICMR for providing Senior Research fellowship to AS.

References 1 Kawai T, Akira S: The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol 2010; 11:373–384. 2 Hu G, Zhou R, Liu J, Gong AY, Chen XM: MicroRNA-98 and let-7 regulate expression of suppressor of cytokine signaling-4 in biliary epithelial cells in response to Cryptosporidium parvum infection. J Infect Dis 2010; 202:125–135. 3 Akira S, Takeda K: Toll-like receptor signaling. Nat Rev Immunol 2004; 4:499–511. 4 Hart KM, Murphy AJ, Barrett KT, Wira CR, Guyre PM, Pioli PA: Functional expression of pattern recognition receptors in tissues of the human female reproductive tract. J Reprod Immunol 2009; 80:33–40. 5 Li Y, Shi X: MicroRNAs in the regulation of TLR and RIG-I pathways. Cell Mol Immunol 2013; 10:65–71.

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American Journal of Reproductive Immunology 71 (2014) 137–153 ª 2013 John Wiley & Sons Ltd

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MicroRNA let-7f: a novel regulator of innate immune response in human endocervical cells.

Endocervical epithelial cells express pattern recognition receptors (PRRs) that aid in innate immune responses. Mechanisms regulating signaling of PRR...
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