Cytokine 73 (2015) 66–73

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The potential protective role of the combination of IL-22 and TNF-a against genital tract Chlamydia trachomatis infection Xiumin Zhao a,1, Danyang Zhu a,1, Jiangbin Ye b, Xingqun Li b, Zhibin Wang c, Lifang Zhang c, Wen Xu c,⇑ a

Department of Obstetrics and Gynecology, Taizhou First People’s Hospital, Taizhou, Zhejiang 318020, PR China First Affiliated Hospital, Wengzhou Medical University, Wengzhou, Zhejiang 325035, PR China c Department of Microbiology and Immunology, Wengzhou Medical University, Wengzhou, Zhejiang 325035, PR China b

a r t i c l e

i n f o

Article history: Received 13 October 2014 Received in revised form 20 January 2015 Accepted 23 January 2015 Available online 28 February 2015 Keywords: IL-22 Defensin Chemokine Oviduct epithelial cell Th22 cell

a b s t r a c t Th22 cells are a novel class of lymphocytes characterized by the secretion of both IL-22 and TNF-a. In summary, Th22 cells have little or no direct impact on other immune cells, but exert selective effects on epithelia. It is not known, however, whether Th22 cells play a role in genital mucosal immunity. Here, we demonstrate that IL-22 and TNF-a synergistically induce several immunomodulatory molecules, such as the antimicrobial peptide mBD-2 (murine b-defensin 2) and the antimicrobial chemokines CXCL-9, -10, and -11 in primary murine oviduct epithelial cells (MOECs). The induction of innate immunity is relevant in an in vitro infection model, in which MOECs stimulated with Th22 cell supernatants or recombinant IL22 and TNF-a effectively inhibit the growth of Chlamydia trachomatis and maintain the survival of the epithelia compared with IL-22 or TNF-a alone. In summary, we demonstrate that the Th22 cell cytokines IL-22 and TNF-a play important roles in genital tract infection. The potential for Th22 cell cytokines to modulate innate immune mediators may lead to the development of new topical agents to treat and/or prevent immune-mediated sexually transmitted diseases (STDs). In summary, we demonstrate that IL-22 and TNF-a represent a potent, synergistic cytokine combination for inducing genital mucosal immunity. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction The female genital tract epithelium plays an important role in host resistance to pathogens [1]. It serves as a physical barrier to pathogen invasion, and certain cytokines and antimicrobial peptides can also be produced by the genital tract epithelium [2]. Some sexually transmitted pathogens cause damage to the epithelium. For example, in Chlamydia trachomatis (Ct) infection, epithelial cells become infected and are targeted by adaptive and innate immune responses. Ct pathology can be attributed to severe inflammatory processes that lead to scarring and the loss of functional epithelial tissue. In a subset of female patients, ascending genital tract infections have been linked to severe health issues, such as salpingitis,

Abbreviations: STD, sexually transmitted diseases; MOEC, murine oviduct epithelial cell; mBD-2, murine b-defensin 2; SPG, sucrose–phosphate–glutamic acid; SEM, standard error of the mean. ⇑ Corresponding author at: Department of Microbiology and Immunology, Wenzhou Medical University, University-town, Wenzhou, Zhejiang 325035, PR China. Tel.: +86 577 86689910. E-mail address: [email protected] (W. Xu). 1 Equal contribution as co-first author. http://dx.doi.org/10.1016/j.cyto.2015.01.027 1043-4666/Ó 2015 Elsevier Ltd. All rights reserved.

pelvic inflammatory disease (PID), or tubal infertility, an increased risk of infection by HIV, and increased HIV shedding [3,4]. During infection, different proinflammatory cytokines, such as interleukin (IL)-1, IL-6, and IL-8, are induced and likely affect the disease outcome [5,6]. Th22 cells are a newly identified helper T-cell subset with a specific phenotype and distinct function [7,8]. They have a CCR4+CCR6+CCR10+ phenotype and do not express IFN-c, IL-4, or IL-17, but do secrete TNF-a and IL-22 [9]. Th22 cells have little or no direct impact on other immune cells, but exert selective effects on epithelial cells [10,11]. The characteristic functional profile of Th22 cells is mediated by distinct cytokines. IL-22 is a glycoprotein that belongs to the IL-10 family, and the IL-22 receptor is composed of the IL-22R1 and IL-10R2 subunits. The IL-22R1 is found on nonhematopoietic cells in the lung, skin, kidney, liver and gut, and allows for IL-22-mediated regulation of local epithelial, endothelial, and stromal cell responses after infection or exposure to inflammatory stimuli [12]. In vitro studies showed that IL-22 activates STAT3 and upregulates the expression of proinflammatory and antimicrobial molecules, such as b-defensin 2, S100 proteins, and the chemokines CXCL9 (monokine induced by gamma-interferon [MIG]), CXCL10 (interferon-inducible protein 10 [IP-10]),

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CXCL11 (interferon-inducible T cell chemoattractant [I-TAC]) in keratinocytes, alone or in synergy with TNF-a [9,13,14] . Although it has been suggested that IL-22 might function at all mucosal epithelial surfaces, there have been very few studies to elucidate the role that IL-22 plays in the female genital tract mucosa. Because IL-22 is increased in STDs generally [15,16]. Therefore, we hypothesized that Th22 cells and their associated cytokines maybe play important roles in genital mucosal immunity. In the study, we established Th22 cell lines and observed the synergy effects of Th22 cell cytokines IL-22 and TNF-a on inducing MOEC expression of mBD-2 and the chemokines CXCL-9, -10, and -11 and characterized the functional impact of this synergistic treatment.

2. Materials and methods 2.1. Mice Specific pathogen-free female BALB/c mice that were 6 weeks old were purchased from Shanghai Laboratory Animal Center (Shanghai, China). All animals were housed in the pathogen-free mouse room in the experimental animal center (Experimental Animal Center of Wenzhou Medical University, Zhejiang, China), and all experiments were carried out in accordance with protocols approved by the Wenzhou Medical University Animal Ethics Committee.

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Flow cytometric analysis [9], Th22-type cells were cultured with Golgi-Plug (1 ll/106 cells) during activated for 4 h before intracellular staining. To stain CCR10 molecules, T cells were stained with PElabeled anti-mouse CCR10 Abs (R&D Systems) and FITC-labeled anti-mouse CD4 Abs. PE-conjugated mouse IgG2a was used as the isotype control. After permeabilization, we used PE-labeled antimouse IL-22 Ab, FITC-labeled anti-mouse TNF-a Ab (BioLegend), These Cells (1  106) were stained for 30 min at 4 °C in the dark. 2.4. Isolation of murine oviduct epithelial cells MOEC were isolated from BALB/c mice, as previously described with slight modifications [17]. Oviduct lumens were rinsed with 0.1% protease for 15 min at 37 °C. Released cells were recovered and grown in (1:1) Dulbecco’s modified Eagle medium:F12 K medium supplemented with 10% heat-inactivated FBS, 50 U/mL penicillin, and 50 mg/mL streptomycin. Experiments were performed in cells at passages 3 and 4. An epithelial cell origin was confirmed by the expression of cytokeratin by Immunofluorescence. 2.5. MOEC activation To obtain cell culture supernatants, MOECs were seeded in 24well plates at a density of 3  104 MOECs/mL in the medium described above. One day after plating, cells were stimulated with 25 ng/mL IL-22, 10 ng/mL TNF-a, or Th22 cell supernatant for 24, 48, or 72 h. Supernatants were harvested and stored at 80 °C.

2.2. Reagents

2.6. TransAMÒ assay

HeLa 229 cells (from ATCC) were cultured with RPMI-1640 supplemented with 10% fetal bovine serum (FBS; Hyclone), 100 U/mL penicillin, 100 U/mL streptomycin, 2 mM L-glutamine, and 5  105 M 2-mercaptoethanol (Gibco). GolgiPlug™ was purchased from BD Biosciences. PE-labeled anti-mouse IL-22 Ab, FITC-labeled anti-mouse TNF-a Ab were purchased from BioLegend. Dulbecco’s modified Eagle medium: F12 K medium were from Gibco; protease were from Sigma. Anti-mouse CD3 Ab, antimouse CD28 Ab, FITC-labeled anti-mouse CD4 Ab, and anti-CXCR3 Ab, PE-labeled anti-mouse CXCR3 Ab were purchased from Pharmingen. Anti-FITC microbeads were purchased from Miltenyi Biotech. Anti-mouse IFN-c Ab and anti-mouse IL-4 Ab; recombinant mouse (rm) TNF-a, IL-6, and IL-2anti-mouse CCR10 Abs, anti-IL-22 and TNF-a mAbs, mouse IL-22, TNF-a and CXCL-9, -10, and -11 ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). The mBD-2 kit was purchased from Phoenix Pharmaceuticals (Burlingame, CA, USA). IL-9, IL-10 IL-21 and TGF-b1 ELISA kits were purchased from Aushon BioSystems. The Nuclear Extract Kit and TransAMÒ ELISA kit were obtained from Active Motif (Carlsbad, CA, USA). The Resorufin CellTiter-Blue Reagent was purchased from Promega (Madison, WI, USA).

For TransAM™ analyses, MOECs were incubated with medium alone, rmIL-22 at a concentration of 12.5, 25, or 50 ng/mL for 48 h. For neutralization experiments, MOECs were preincubated for 1 h with 10 lg/mL anti-IL-R1 mAbs and then were incubated with 25 ng/mL rmIL-22. Nuclear extracts were prepared using the TransAM nuclear extract kit and analyzed for activated transcription factors using TransAm Kits according to the manufacturer’s instructions.

2.3. In vitro differentiation of T cells For Th22 cell polarization [7,9], CD4+ T cells were magnetically isolated from BALB/c mouse splenocytes with a combination of FITC-labeled anti-CD4 antibody and anti-FITC microbeads (Miltenyi Biotech). Purified CD4+ T cells were activated with 1 lg/mL anti-mouse CD3 Ab, 2 lg/mL anti-mouse CD28 Ab, and the following cytokines and blocking antibodies: 5 lg/mL anti-mouse IFN-c Ab, 5 lg/mL anti-mouse IL-4 Ab, and recombinant mouse proteins, including 50 ng/mL TNF-a, 20 ng/mL IL-6, and 100 U/mL IL-2 (Th22 conditions). After 5 days of culture, the harvested T cells were stimulated with 1 lg/mL anti-CD3 Abs and 2 lg/mL anti-CD28 Abs for 48 h, and supernatants were harvested and stored at 80 °C until they were used for analysis.

2.7. ELISA The concentrations of IL-22, TNF-a, IL-17, IFN-c, and IL-4 in Th22 cell supernatants and mBD-2 and CXCL-9, -10, and -11 and in MOEC culture supernatants were measured using ELISA kits according to the manufacturer’s instructions. 2.8. Chemotaxis assay Migration assays were performed using 5-lm pore Transwell inserts (Corning Costar). CD4+ T cells from spleens of naive BALB/ c mice were isolated and activated in vitro with plate-bound anti-CD3 (1 lg/mL), anti-CD28 (2 lg/mL), 25 ng/mL IL-12, and 5 lg/mL anti-IL-4 (Th1 cell conditions) for 48 h [7,9]. Cells were then cultured in fresh media without CD3/CD28 stimulation for 48 h to induce CXCR3 expression. After washing with medium without serum three times, 2  105 activated CD4+ T cells were added to the upper wells. MOEC culture supernatants (as described above) were added to the lower chambers. After incubation for 4 h at 37 °C with 5% CO2 and then at 4 °C for 20 min to loosen leukocytes bound to the undersides of membranes, migrated CD4+ T cells were counted and analyzed for CXCR3 expression by flow cytometry (BD FACS Calibur). A chemotaxis index was calculated by dividing the number of cells in the bottom well in response to a chemokine by the number of migrating cells in wells containing medium alone. Each experiment was performed in triplicate. For neutralization experiments, activated CD4+ T cells were pretreated with 5 lg/mL anti-CXCR3 Abs or control mAb for 1 h at 37 °C.

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2.9. In vitro Ct infection model

3. Results

C. trachomatis serovar E (from ATCC), a common urogenital isolate, was used for in vitro antimicrobial studies. Chlamydiae were cultured in HeLa 229 cells, and were purified and stored in sucrose–phosphate–glutamic acid (SPG) buffer (0.22 M sucrose, 0.02 M sodium phosphate, 5 mM glutamic acid; 0.2 mm filtered, pH 7.4) at 80 °C until later use [18,19] . The ability of MOEC culture supernatants to prevent Ct infection was determined by preincubating the MOEC culture supernatants with purified elementary bodies (EB) and titrating out the infectious activity on MOECs in triplicate (3  104 cells/well) in 96-well tissue culture plates, with some modifications of established protocols [20,21]. In brief, for each triplicate, 1.2  103 viable EB mixed with 100 lL various MOEC culture supernatants (described above) were incubated at 37 °C for 45 min. Immediately prior to Chlamydia inoculation, MOECs were washed with PBS and then 33 lL EB mixture was added. Plates were centrifuged for 40 min at 250g at room temperature, and then the supernatants were removed and replaced with 200 lL cell culture medium. Thereafter, MOECs were incubated for an additional 40 h at 37 °C with 5% CO2, fixed in 10% ice-cold methanol for 10 min, and stained using an antibody specific for Ct lipopolysaccharide. Inclusion bodies, a measure of Ct infectious activity, were enumerated microscopically using a 40 objective (Olympus culture microscope). Expression of regenerating protein 3b (Reg3b) mRNA was performed 24 and 48 h after infection by quantitative RT-PCR using 50 -CGCATTAGTTGCCCCAAGG-30 and 50 -TCCAGGCCTCTTTTGGCAG30 primers. Total RNA from MOECs treated with IL-22 was extracted, and cDNA was synthesized using classical procedures. Cell viability was evaluated 24 and 48 h after infection using Resorufin (CellTiter-Blue Reagent, Promega), according to the manufacturer’s protocol. Viable cells with intact metabolism were identified by their ability to reduce cell-permeable resazurin to fluorescent resorufin. After 1 h incubation at 37 °C, fluorescence was measured using a multilabel counter (VICTORTM3V 1420, PerkinElmer).

3.1. IL-22 up-regulates STAT3 expression on MOECs via IL-22R

2.10. Statistical analysis Results are expressed as means ± SEM. Statistical analyses of data were performed with one-way ANOVA and Bonferroni Multiple Comparison Test as post-tests. All data were analyzed using SPSS 17.0 (Chicago, IL, USA). P values less than 0.05 were considered to represent statistically significant differences.

The IL-22 receptor is a dimeric complex composed of IL-10R2 and IL-22R1. IL-10R2 is ubiquitously expressed, whereas IL-22R1 is only expressed by non-hematopoietic cells. Initially, we established primary MOEC cultures and determined the expression levels of IL-22R1 on MOECs by flow cytometry. We found that IL-22R1 was expressed on MOECs (Fig. 1A). TransAM experiments demonstrated that IL-22 could activate the transcription factor STAT3 in MOECs via IL-22R1. 3.2. Induction of polarized Th22-type cells Th22 cells are IL-22-producing CD4+ T cells, which act as a unique subset of Th cells that develop along a pathway distinct from the Th1, Th2, and Th17 cell differentiation pathways [22,23]. In the study, we established polarized Th22-type cells by culturing purified CCR10+CD4+ T cells in the presence of antiCD3+CD28 Abs, anti-IFN-c Ab, anti-IL-4 Ab, IL-6, and TNF-a (Th22 conditions). Intracellular staining of T cells with three color staining with Abs to IL-22, TNF-a, and CCR10 confirmed that these CD4+ T cells highly expressed the Th22 cell marker CCR10 (Fig. 2A). Our data showed that the CCR10+ cells also expressed IL-22 and TNF-a (Fig. 2B–D). We also examined whether these cells could express Th22 cytokines. We found that these cells produced large amounts of IL-22 and TNF-a, but not IFN-c, IL-4, or IL-17, IL-9, IL-10, IL-21 and TGF-b1 which produced by Th1, Th2, Th17, Th9 and Treg (Table 1). 3.3. Th1 cell-associated chemokines and antimicrobial peptides are produced after stimulation with Th22 cell cytokines Although MOECs can express IL-22R1, and IL-22 alone can induce transcription factor STAT-3 activation, studies have shown that Th22 cells activate target cells via the functional interplay of IL-22 and TNF-a [13]. Fig. 3A confirms the synergistic effect of IL22 and TNF-a. First, we investigated the production of the Th1 cellassociated chemokines CXCL-9, -10, and -11 under non-stimulatory and IL-22 and TNF-a stimulatory conditions in a time-dependent manner for 24, 48, or 72 h. IL-22 or TNF-a only slightly induced CXCL-9, -10, and -11 and mBD-2. Combined stimulation with IL-22+TNF-a or Th22 cell supernatants resulted in consistently and significantly enhanced

Fig. 1. IL-22 can activate STAT-3 in MOECs via IL-22R1. (A) MOECs from BALB/c mice were stained with FITC-conjugated mAb against IL-22R1 or control mouse IgG2a mAb. Expression of IL-22R1 was analyzed by flow cytometry. (B) MOECs were cultured with medium alone or various concentrations of rm-IL-22. For neutralization experiments, MOECs were preincubated for 1 h at 37 °C with 20 lg/mL anti-IL-22R1. Nuclear extracts were collected and tested for STAT-3 using TransAM kits. Data represent means ± SEM of at least three independent experiments that were performed in triplicate. ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001, compared with the control group; ##p < 0.01, compared with the Ab blocking group.

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Non-polarized A

3.4. CD4+CXCR3+ Th1 cells are attracted by IL-22 and TNF-a stimulated MOECs

Polarized B 4

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3.5. Combination of TNF-a and IL-22 protects MOECs from death after in vitro Ct infection

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TNF-α

We next tested whether activated MOECs could induce the migration of CD4+ T cells. In a transwell chemotaxis assay, Th1 cells (stimulated by Th1 conditions) were added to the upper chamber of 5-lm pore membrane inserts. Unstimulated, IL-22+TNF-a, or Th22 cell supernatant stimulated MOECs were tested for their capacity to attract CD4+CXCR3+ Th1 cells. After 4 h incubation, cells in the lower chamber were harvested and CXCR3 expression on CD4+ T cells was determined (Fig. 4A). Although unstimulated MOECs could CD4+CXCR3+ T cells to some extent, IL-22+TNF-a or Th22 cell supernatant activated MOECs induced a significant migration of CD4+ CXCR3+ T cells (P < 0.05; Fig. 4B). These data indicate that after stimulation by Th22 cell cytokines the MOECs produce Th1 cell associated chemokines that induce the chemotaxis of CD4+ T cells. Pretreatment of CD4+ T cells with anti-CXCR3+ mAbs also can inhibit T cell migration (P < 0.01).

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IL-22 Fig. 2. Polarization of CD4+ T Cells into IL-22- and TNF-a-producing cells. By flow cytometry, freshly isolated CCR10+CD4+ T cells from the spleens of normal mice (left panel) and polarized Th22-type cells (right panel) were stained with anti-CCR10, anti-CD4, anti-IL-22, and anti-TNF-a Abs after permeabilization. The percentage of cells that were double-positive for CCR10 and CD4 (A and B) or IL-22 and TNF-a are shown (C and D).

Table 1 Cytokine production by the polarized Th22-type cells.

IL-22 TNF-a IFN-c IL-17 IL-4 IL-9 IL-10 TGF-b1 IL-22

Activated T cells (pg/ml)

Isotype control (pg/ml)

18,543*** 7655*** 56* 9# 21* 5# 3# 3# 3#

15 11 5 4 8 5 3 4 3

We set up Th22-type cells from spleens of BALB/c mice. Supernatants were measured 48 h after anti-CD3/CD28 stimulation. * P < 0.05. *** P < 0.001. # P > 0.05, compared with isotype Ab control.

chemokines or cytokine secretion over the level of an additive effect. Th22 cell supernatants also contain a small amount of IL17, IFN-c, and other cytokines, and combinations of IL-22/IL-17, IL-17/IFN-c, and IFN-c/TNF-a can synergistically induce epithelial cells to produce an abundance of cytokines. These cytokines can be induced by these cytokine combinations, to exclude this possibility we pretreated Th22 cell supernatants with anti-IL-22/TNF-a mAbs prior to the stimulation of MOECs. Anti-IL-22/TNF-a mAbs could inhibit the stimulatory effects of Th22 cell supernatants (Fig. 3B), and we found that IL-22/TNF-a are major factors that induce MOECs to produce chemokines and mBD-2.

We also analyzed effects of TNF-a and IL-22 in an in vitro Ct infection model. Ct growth was inhibited by supernatants of MOECs stimulated with IL-22 or TNF-a, IL-22+TNF-a, or Th22 cell supernatant. Both TNF-a+IL-22 and Th22 cell supernatant (Fig. 5A) exhibited significant antibacterial activities against Ct. By contrast, IL-22 or TNF-a alone had only a weak inhibitory effect on Ct growth. These findings indicate that a Th22 cell-like combination of cytokines synergistically induces an effective innate immune response of MOECs. During infection, Ct initially targets the genital epithelium to replicate, a process that leads to epithelial cell death and pathology [24]. Although IL-22 and/or TNF-a have been described to protect epithelial cells from a variety of environmental insults [7,13], its potential role in the context of Ct genital tract infection has not been evaluated to date. Before addressing this question, we first investigated the effects of IL-22 on primary MOECs. As seen in Fig. 5B, IL-22+TNF-a synergistically induced increased transcript levels of REG3b, which is known to play a role in epithelial fitness and survival [25]. Next, we studied its potential role in the control of cell mortality triggered by Ct infection. As shown in Fig. 5C, IL22+TNF-a strongly prevented Ct-triggered cell death. Thus, IL22+TNF-a may exert a protective function on epithelial cells during Ct infection. 4. Discussion Th22. cells are a unique subset of CD4+ effector T cells that display a specific cytokine profile that includes IL-22 and TNF-a, which are both frequently reported to play important roles in chronic inflammation. Unlike other cytokines, IL-22 receptors are absent on immune cells, being instead restricted to tissues and thereby providing signal directionality from the immune system to tissues [26,27]. IL-22 regulates cell activity via the JAK–STAT3 signaling pathway [28,29]. Several studies have demonstrated that IL-22 is involved in host defense against infections caused by various bacteria, fungi, viruses, and parasites [30]. Previously, IL-22 was shown to be increased in acute Ct and Tv infection [15,16]. This finding raises the possibility that Th22 cells might play an independent role in immune responses at the site of a genital tract infection. Evidence suggests that the effects of T cells on epithelial cells are determined by cytokine combinations rather than by single cytokines [13]. In this study, we investigated the functional synergistic

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A

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IL-22

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Th22 supertant

E

Fig. 3. MOECs produce Th1 cell-associated chemokines and mBD-2 after IL-22+TNF-a stimulation. MOECs were stimulated with medium alone, IL-22, TNF-a, IL-22+TNF-a, or Th22 cell supernatant for 24, 48, or 72 h. For neutralization experiments, Th22 cell supernatants were pretreated with anti-IL-22+TNF-a mAbs and then were used to stimulate MOECs for 48 h. The levels of (A) CXCL-9, (B) CXCL-10, (C) CXCL-11, and (D) mBD-2 over a time-course in cell-free supernatants were quantified using ELISA kits. The inhibition of anti-IL-22+TNF-a mAbs is shown in (E). Bars show the means and SEM of three independent experiments. ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001, compared with the groups treated by TNF-a or IL-22 alone; compared with control; #P < 0.05, ###P < 0.001, compared with corresponding Th22 supernatant untreated by anti-IL-22/TNF-a mAbs.

effects of two key Th22 cell cytokines, IL-22 and TNF-a, on MOECs. We questioned whether MOECs stimulated by IL-22 and TNF-a, which represent an essential combinatorial key for cutaneous immunity that have the potential to attract Th1 subsets [7,13], could inhibit the growth of sexually transmitted pathogen and protect genital epithelial cells. Our data indicated that IL-22+TNF-a activated MOECs to induce the production of the Th1 cell-associated chemokines CXCL-9, -10, and -11 and the antimicrobial peptide mBD-2. Functionally, CD4+CXCR3+ T cells were attracted by activated MOECs, and splenocytes migrated towards activated and resting MOECs that we tested using chemotaxis assays. Additionally, supernatants from MOECs pre-incubated with the combination of cytokines or Th22 cell supernatants most effectively reduced Ct growth and induced increased transcript levels of REG3b, which encodes a protein known to play a role in epithelial fitness and survival [31]. Adaptive immunity, especially Th1 cell immune responses, plays a critical role in preventing Ct and other sexually transmitted pathogens [32]. Furthermore, the regulation of Th1 cell responses

in the genital tract is a crucial factor for controlling the duration of infection and the development of subsequent tubal pathology [33,34]. While systemic effector T cells can migrate freely through organs such as the spleen and liver, other tissues, such as the intestines, lung airways, central nervous system, skin, and vagina, are restrictive for memory T cell entry [35]. In the latter tissues, inflammation or infection is often required to permit the entry of circulating activated T cells to establish a tissue-resident memory T cell pool that represents a separate compartment from the circulating pool [35]. Essential elements involved in this process are chemokines that are secreted in the tissue and the corresponding chemokine receptors that are present on T cells. CXCR3 is expressed by both effector Th1 cells and CD8+ T cells, as well as other cell types [36]. CXCL-9, -10, and -11 mediate the recruitment of effector CD8+ and CD4+ T cells to the infected tissue via CXCR3 [37]. Thus, we hypothesized that Th22 cell cytokines regulate genital tract mucosal immunity by inducing genital epithelial cells to produce CXCL-9, -10, and -11. To test this hypothesis, we simulated MOECs with Th22 cell cytokines. Both IL-22 and TNF-a or Th22 cell

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Fig. 4. Migration of Th1 cells induced by supernatants of MOECs treated with IL-22, TNF-a, IL-22+TNF-a, or Th22 cell supernatant. (A) Migrated cells were collected and analyzed by flow cytometry to evaluate the CXCR3 expression. (B) MOECs were stimulated by IL-22, TNF-a, IL-22+TNF-a, or Th22 cell supernatant for 48 h. Then, supernatants were collected and analyzed by chemotaxis assay to assess the chemotactic activity of supernatants for Th1 cells. ⁄P < 0.05, ⁄⁄P < 0.01, compared with control; # P < 0.05, ##P < 0.01, compared with corresponding T cells that were not treated with CXCR3 antibody.

Fig. 5. IL-22 and TNF-a co-operate to protect MOECs against Ct. (A) MOEC supernatants from IL-22+TNF-a and Th22 cell supernatant stimulation significantly inhibited Ct growth. Data are expressed relative values compared with the IFU/well resulting from infection with untreated Ct; (B) REG3b mRNA copy numbers were measured by quantitative RT-PCR. Data are normalized to the expression of b-actin, and are expressed as the fold-increase over the average gene expression in untreated MOECs. (C) 24 and 48 h later, cell viability was quantified using Resorufin. Data represent means ± SEM of a representative experiment of three independent experiments. One-way ANOVA was used to analyze variance followed by a Bonferroni multiple comparison test to compare all groups. ⁄P < 0.05, ⁄⁄P < 0.01, compared with the control group; #P < 0.05, ##P < 0.01, compared with the corresponding Th22 cell supernatant untreated by anti-IL-22 or TNF-a mAbs group.

supernatant stimulation lead to an upregulation of CXCL-9, -10, and -11. We also determined the total number of migrated CD4+CXCR3+ T cells in our migration assay experiments. MOECs were able to attract CD4+CXCR3+ T cells. Activation of MOECs with IL22 and TNF-a or Th22 cell supernatant increased the migration

of CD4+CXCR3+ T cells. Our data confirm that activated MOECs are capable of attracting Th1 cells. Although Th1 cell responses are indispensable, innate immunity represents the first line of defense and also plays an important role in the mucosal immune response in the female genital tract [38].

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Antimicrobial peptides are a well-recognized component of the innate immune repertoire at mucosal surfaces [16,39]. The defensins are a major family of antimicrobial peptides that include two subfamilies, the alpha- and beta-defensins. In addition to wide spectrum of antimicrobial activities [40,41], recent reports indicate that defensins can exert multiple additional activities, including chemoattraction for adaptive immune system cells and triggering cytokine release [42]. The beta-defensins (BD-1, -2, and -3) are predominantly produced by epithelial cells [43]. To verify the functional impact of Th22 cell cytokines on MOECs, we analyzed the effects of IL-22 and TNF-a or Th22 cell supernatant in an in vitro Ct infection model. Ct growth was inhibited by the supernatant of epithelial cells stimulated with IL-22 and TNF-a or Th22 cell supernatant. By contrast, IL-22 alone showed no effect and TNF-a only showed a weak inhibitory effect on Ct growth. Moreover, our findings indicate that IL-22 and TNF-a play a positive role in the control of epithelial damage early after Ct infection. It is possible that the cytoprotective effect of IL-22 is mediated through the induction of REG3b, which is known to play a role in epithelial fitness and survival. Thus, although this finding remains to be established in vivo, Th22 cell-derived IL-22 and TNF-a cytokines may be important for sustaining the genital epithelium and preserving the epithelial barrier during Ct infection. Additionally, IL-22 may promote the synthesis of epithelial chemokines and antimicrobial peptides, some of which are known to recruit effector T cells and/or neutralize Ct. Through these mechanisms, Th22 cells along with IL-22 and TNF-a may be of particular significance during CT genital infection, and this hypothesis is currently the subject of active investigation. The discoveries presented here may provide information that could contribute to the development of new intervention strategies and vaccines for preventing sexually transmitted infections. Funding This research was supported by Zhejiang Provincial Natural Science Foundation of China under Grant No. Y271067 and Science and Technology Planning Project of Wenzhou Municipal under Grant No. Y20110126. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cyto.2015.01.027. References [1] Ferreira VH, Kafka JK, Kaushic C. Influence of common mucosal co-factors on HIV infection in the female genital tract. Am J Reprod Immunol 2014;71:543–54. [2] Doerflinger SY, Throop AL, Herbst-Kralovetz MM. Bacteria in the vaginal microbiome alter the innate immune response and barrier properties of the human vaginal epithelia in a species-specific manner. J Infect Dis 2014;209:1989–99. [3] Cabeza J, Garcia PJ, Segura E, Garcia P, Escudero F, La Rosa S, et al. Feasibility of Chlamydia trachomatis screening and treatment in pregnant women in Lima, Peru: a prospective study in two large urban hospitals. Sex Transm Infect 2014. [4] Peters RP, Dubbink JH, van der Eem L, Verweij SP, Bos ML, Ouburg S, et al. Cross-sectional study of genital, rectal, and pharyngeal Chlamydia and gonorrhea in women in rural South Africa. Sex Transm Dis 2014;41:564–9. [5] Nikolaitchouk N, Andersch B, Falsen E, Strombeck L, Mattsby-Baltzer I. The lower genital tract microbiota in relation to cytokine-, SLPI- and endotoxin levels: application of checkerboard DNA–DNA hybridization (CDH). APMIS 2008;116:263–77. [6] Mitchell C, Balkus JE, McKernan-Mullin J, Cohn SE, Luque AE, Mwachari C, et al. Associations between genital tract infections, genital tract inflammation, and cervical cytobrush HIV-1 DNA in US versus Kenyan women. J Acquir Immune Defic Syndr 2013;62:143–8. [7] Eyerich S, Eyerich K, Pennino D, Carbone T, Nasorri F, Pallotta S, et al. Th22 cells represent a distinct human T cell subset involved in epidermal immunity and remodeling. J Clin Invest 2009;119:3573–85.

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