Modulation of T helper 1 and T helper 2 immune balance in a stress mouse model during Chlamydia muridarum genital infection

A mouse model to study the effect of cold-induced stress on Chlamydia muridarum genital infection and immune response has been developed in our laboratory. Our previous results show that cold-induced stress increases the intensity of chlamydia genital infection, but little is known about the effect of cold-induced stress on differentiations and activities of T cell subpopulations and bone marrow derived dendritic cells (BMDCs). The factors that regulate the production of T helper 1 (Th1) or T helper 2 (Th2) cytokines is not clear. The objective of this study was to examine whether cold-induced stress modulates the expression of transcription factors and hallmark cytokines of Th1 and Th2 or differentiation of BMDCs during C. muridarum genital infection in mice. Our results show that mRNA level of beta2-adrenergic receptor (β2-AR) compared to β1-AR and β3-AR was high in mixed population of CD4+ T cells and BMDCs. Further, decreased expression of T-bet and low level of interferon-gamma (IFN-γ) production and increased expression of GATA-3 and interleukin-4 (IL-4) production in CD4+ T of stressed mice was observed. Exposure of BMDCs to feroterol (β2-AR agonist) or ICI,118551 (β2-AR antagonist), respectively, revealed significant stimulation or inhibition of β2-AR in stressed mice. Moreover, co-culturing of mature BMDC and naïve CD4+ T cells resulted in increased production of IL-4, IL-10, and IL-17 in culture supernatants, suggesting that stimulation of β2-AR leads to the increased production of Th2 cytokines. Overall, our results show for the first time that cold-induced stress is able to modulate the pattern of Th1 and Th2 cytokine environment, suggesting that it promotes the differentiation to Th2 rather than Th1 by the overexpression of GATA-3 correlated with elevated production of IL-4, IL-10, IL-23, and IL-17 in contrast to a low expression of T-bet correlated with less IFN-γ secretion in the mouse model.


INTRODUCTION
Chlamydia genital infection caused by Chlamydia trachomatis, the most common bacterial sexually transmitted disease (STD) worldwide [1] This infection, if left untreated, leads to the development of pelvic inflammatory disease (PID), fallopian tube scarring, ectopic pregnancy, infertility, and neonatal conjunctivitis [2,3]. Epidemiologic data from the Centers for Disease Control and Prevention and World Health Organization indicate that more than 90 and 4 million new cases occur annually worldwide and, in the US, respectively [4]. In the United States, chlamydia genital infection disproportionately affects populations of low socio-economic status, particularly African-Americans [5,6]. The reasons are not well known, but stress may have a major role in the persistent high rate of the disease associated with low socioeconomic conditions [7].
Several studies in animal models have demonstrated that anti-chlamydial T cell responses in the local genital mucosa play a role in the clearance of C. trachomatis from the genital tract [8][9][10][11][12]. It is known that immunity to C. trachomatis is mediated by T-helper (Th), specifically Thelper 1 (Th1), cells through interferon-gamma (IFN-) -dependent and -independent pathways [13][14][15][16].Moreover, other studies in human subjects have identified and characterized factors that may be involved in the development of immunopathogenesis after chlamydia genital infection caused by the human strain C. trachomatis [17,18]. Those murine model studies have exhibited vital aspects of human genital chlamydial infection, including similar course of active infection, pathologic consequences or sequelae of an infection, tubal inflammation, hydrosalpinx formation and infertility [19][20]. It is well recognized that genital infection with C. trachomatis leads both to the recruitment of protective immune responses to the site of infection and infiltration of inflammatory products that may also contribute to pathological damage in the host [2,[21][22][23].
Repeated chlamydia genital infection in animal models [23][24][25] and human subjects [26,27] is known to have a much greater risk of tubal obstruction than those with less exposure to C.

trachomatis.
Chlamydia muridarum, previously known as the mouse pneumonitis strain of C. trachomatis, is commonly used for the study of chlamydia immunity, pathogenesis and vaccine development [28,29]. However, significant differences in the biology and pathogenesis of infections caused C. trachomatis and C. muridarum have been reported [30]. Mouse strain-dependent studies show that C. muridarum produces shorter courses of infection in C57BL/6 and BALB/c mice than in C3H/HeN mice, even though more resistance to oviduct pathology was observed in C57BL/6 mice [31]. A study has demonstrated the successful infectivity and elicitation of pathologies with both human and murine strains of Chlamydia in mice [32]. It is mostly agreed that no model is perfect, extensive literature reports suggest that immunological findings made using C. muridarum infection in mice is not vastly different from those that occur with C. trachomatis infections in humans [33].
Psychological or physical stress resulting from hardship of life in society has major impacts on public health [34][35][36][37][38]. Glucocorticoids and catecholamines are stress hormones that serve as the major mediators of stress responses by modulating signaling events, which result in either immunosuppression or immunostimulation in the host [39][40][41]. Norepinephrine (NE) is one of the catecholamines of the sympathetic nervous system released during stressful conditions including cold-water application [39,42,43]. Studies document the existence of at least eight different alpha or beta subtypes of NE receptors [44]. Norepinephrine binds and stimulates the beta2 adrenergic receptor (2-AR), which is predominantly expressed on CD4 + and B cells [45][46][47]. Studies have further shown that production and direct binding of NE to 2-AR modulates immune responses, including altering cytokine production, lymphocyte proliferation, and antibody secretion [47][48][49][50][51].
More studies have shown that dendritic cells (DCs) have an essential role in the induction of T cell responses to clear C. muridarum during genital infection [29,52]. Furthermore, DCs pulsed with a recombinant chlamydial major outer membrane protein antigen elicit a CD4(+) type T-helper 2 (Th2) rather than type Th1 immune response that is not protective [53]. Other studies have shown that transcription factors T-box expressed in T cells (T-bet) and GATAbinding protein-3 (GATA-3) and play important a regulation role in the differentiation of naive Th cells towards Th1 or Th2 cells in human subjects and animal models [18,54]. Specifically, modulation of cytokines and transcription factors (T-bet and GATA-3 in CD4+ T cells of chlamydia patients was reported [18], but the expression patterns of T-bet and GATA-3 in CD4+ T cells in our stress model during chlamydia genital infection yet is unknown.
Application of cold water as a stressor in animal models, including mice, has resulted in changes in immune responses that correlate with the activity of the neuroendocrine system of corticosteroids and catecholamines [55][56][57][58][59][60]. Although stress is implicated as a risk factor for various infections, its effect on chlamydia genital infection is unknown. We have previously shown that cold-water stress induces the production of catecholamines, which may play a critical role in the modulation of the immune system leading to increased intensity of C. muridarum genital infection [59,60]. Furthermore, we have shown that supplement of NE to in vitro exerts an immunosuppressive effect on splenic T cell production of cytokines, decreased C. muridarum shedding in the genital tract of β1Adr/β2Adr KO mice, and severe pathology with a higher rate of infertility in C. muridarum infected mice [60]. However, our understanding of the expression of and function of Th1 and Th2 during cold-induced stress remains limited.
Here we sought to determine whether cold-stress treatment to mice could lead to imbalanced Th subsets during C. muridarum genital infection. Our overall hypothesis was that cold-stress treatment leads to the skewing CD4+ Th1 cell to Th2 cell function as determined by mRNA levels of T-bet and GATA-3 transcriptions factors and the level of signature cytokines produced during in vitro proliferation. We evaluated the differentiation and cytokine production ability of bone marrow derived dendritic cells (BMDCs) and its influence on the induction of Th cells upon co-culturing in vitro.

Chlamydia stock culture and McCoy cell line
McCoy mouse cell line and stock culture of Chlamydia muridarum (previously known as Chlamydia trachomatis agent of mouse pneumonitis (MoPn) were kindly provided by Dr. Joseph Igietseme, then at Morehouse School of Medicine and currently at CDC, Atlanta, GA.

Animals
Six-to seven-week-old female BALB/c strain mice were purchased from Hilltop Lab Animals, Inc. (Scottsdale, PA). Mice were housed in a vivarium at Bluefield State College (BSC) located in the Basic Science Building of the School of Arts and Sciences. Mice were given food and water ad libitum in an environmentally controlled room with equal daylight and night hours.
all animal experimental protocols. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by Bluefield State College Institutional Animal Care and Use Committee (IACUC) (Assurance # A521-01) prior to beginning of the research.
Chlamydia muridarum inclusion bodies was deposited into genital tract of each mouse under Ketamine-Xylazine induced anesthesia and all efforts were made to minimize animal suffering.

Cold-water stressing protocol
Before the start of actual experiments, mice were acclimated for 7 days to relieve the stress of transport. Mice were then divided into three groups: stressed, handled/non-stressed and not handled/non-stressed. To induce chronic stress, five mice at a time were placed in a packet filled with four cm of cold water (4 to 5° C) for five minutes daily for 24 days, as previously described [56]. The water level was deep enough to cover the backs of the mice and force them to swim in the cold water. At the end of each stressing period, mice were dried with towels to avoid hypothermia. Hereafter this group is referred to as the "test" or "stressed" group. To ensure that the stress was caused by the water treatment, and not simply by handling, the same protocol was applied, with the exception that the water was room temperature (22 O C). A group of non-stressed mice were kept at room temperature without water treatment. Initial data on stress hormone production and C. muridarum course of infection showed no significant difference between the handled/non-stressed and the not-handled/non-stressed groups. For simplicity, the not-handled/non-stressed group was chosen as the control, and hereafter it is this group that is referred to by the terms "non-stressed" and "control "Throughout the study.

Inoculation and isolation of Chlamydia muridarum
To regulate estrous cycles, all mice were injected subcutaneously with 2.5 mg of progesterone in 100 µL of phosphate buffered saline (PBS) on day 17 of the 24-day stressing period. On day 24, stressed and non-stressed mice were inoculated intravaginally with 10 5 IFU of inclusion-forming units per mL (IFU/mL) was determined as previously descried (4).

Purification of T cells from spleen and genital tract of mice
Spleens were removed aseptically then minced and teased with forceps in RPMI 1640 complete medium supplemented with 10% fetal bovine serum 1% penicillin/streptomycin, and 0.1% gentamicin (Sigma, St. Louis, MO). Spleen cell suspensions were pressed through a 70-m cell strainer (Becton Dickinson, Franklin Lakes, NJ) to remove tissue debris. Splenocyte CD4+ T cells were isolated using EasyStep mouse T cell enrichment kits based on immunomagnetic negative selection, (STEMCELL Technologies, Vancouver, Canada) following the manufacturer's instructions. Briefly, suspension of spleen cells was incubated with the EasySep Ab mixtures to non-CD4+ T cells followed by biotin selection mixture and magnetic beads. Labeled non-CD4+ T cells were removed using EasySep magnets. Genital tract lysates were produced by homogenization and lysates were pressed through a 70-m cell strainer to purify CD4+ T cells as above.

Antigen presenting cell preparation
Antigen Presenting Cells (APCs) from splenocytes of non-stressed mice were prepared by treatment with Mytomycin C. Briefly, splenocyte cells were washed by centrifugation at 300 x g for 10 minutes and freshly prepared Mytomycin C at the concentration of 25 g per mL was added to 10 7 splenocyte cells per mL and incubated at 37 °C for 30 minutes. Cells were washed four times as above and a final cell count of 5 x 10 5 Mytomycin C-treated cells was prepared for proliferation assay.

T cell proliferation assay
T cell proliferation in vitro was performed as described previously (7) with some modifications. Briefly, cells were seeded in triplicate at a density of 5 × 10 5 cells mixed with 5 x 10 5 Mytomycin C treated cells per well in a 96-well culture plate (Becton Dickinson). The cells were stimulated with 2.5 μg/mL of concanavalin A (Con A) cultured for 72 h in a water-jacketed incubator at 37°C and 5% CO 2 (NuAire, Plymouth, MN). Culture supernatants were collected after 72 h of proliferation then stored at −86°C in preparation of measuring cytokine production using ELISA.

2-AR antagonists on T cell proliferation
We investigated the influence of synthetic norepinephrine bitartrate salt (NE), 2-AR agonist or antagonist on T cell cytokine production. Spleens were harvested as above from stressed/infected (SI), non-stressed/infected (NSI), and non-stressed/non-infected (NSNI) mice and isolated using Dynabeads® Magnetic Separation following the manufacturer's instructions.
Purified T cells were seeded at a density of 5 x 10 5 per well in 96-well plates along with the same number of antigen presenting cells (APCs were exposed to NE (1 μM), fenoterol (2-AR antagonist) (1 μM) and ICI 118,551 (2-AR antagonist) (1 μM) for 1 h. Cells were proliferated for 72 h at 37° C in the presence/absence of Con A (5 gmL-1), (0.3 gmL-1) irradiated chlamydia antigen. Culture supernatants were collected after 72 h of proliferation and immediately stored at -20°C to be tested for the production of selected cytokines by ELISA.

Isolation and generation of bone marrow-derived dendritic cells (BMDCs) and bone marrow-derived macrophages (BMDMs)
To collect bone marrow cells, the femur and tibia bones were crushed using a mortar and pestle and debris was removed by passing the suspension through a 70 m mesh nylon strainer. in Trypan blue and their viability was over 80%.

Co-culturing of Naïve CD4+ T cells and LPS-stimulated BMDCs in vitro
To examine the effect of BMDCs on T cell proliferation, freshly isolated CD4+ T cells isolated from stressed and non-stressed Chlamydia muridarum-infected mice were co-cultured with matured BMDCs for four days in 96-well plates at BMDC: T cell ratio of 1:10. Culture supernatant was collected at day four of co-culturing and cytokine production was determined by ELISA.

Priming Th1 or Th2 in vitro under polarizing conditions
Naïve CD4 + T cells from stressed and non-stressed groups of mice were isolated using the mouse naïve CD4+ T cell isolation kit for activation with polarizing milieu. Briefly, splenic naïve CD4+ T cells were seeded at 1 x 10 5 cells/well of a plate coated with 1 g/mL anti-CD3 and anti-CD28 and incubated for 72 h in the presence of recombinant murine interferon-gamma (IFN- (10 ng/mL) recombinant murine IL-12 (10 ng/mL) and anti-mouse IL-4 (100 ng/mL) for Th1 polarization or recombinant IL-4 (100 ng/mL) anti-mouse IFN- (10 ng/mL) and anti-mouse IL-12 (10 ng/mL) for Th2 polarization. The CD4+ T cells were stimulated again for another four days in coated plates and fresh medium. Culture supernatants were collected to determine the polarization of IL-4 and IFN- in stressed and non-stressed CD4+ T cell samples as determined by ELISA Cytokine measurement using ELISA.
The level of cytokines in supernatants collected from in vitro proliferating CD4+ T cells and bone-marrow derived dendritic cells (BMDCs) of different treatment groups was measured using an ELISA kit (Invitrogen, Camarillo, CA) following the manufacturer's instructions as previously described (59). Briefly, standard samples and test samples were diluted and added to wells in duplicate and incubated as directed. Optical densities were measured at 450 nm with an ELISA plate reader from Biotech (Winooski, VT). The concentration of the cytokine in each sample was obtained by extrapolation from the standard calibration curve generated in each assay.

RNA isolation, cDNA synthesis and polymerase chain reaction (PCR) analysis
Total RNA was isolated from cultured splenocyte and genital tract T cells, BMDCs using the FastPrep24™ or FastPrep® FP120 Instrument and Fast RNA® Pro Green Kit (MP Biomedicals) following the manufacturer's instructions. Briefly, total RNA was released into the protective RNApro™ solution by homogenizing samples in 2 mL tubes using Lysing Matrix D.
Total RNA Extraction was completed using chloroform and precipitation. Then the isolated RNA was quantified using a Theme Scientific Nanodrop (Thermofisher). RNA sample values ranging from 1.9 to 2.1 at 260/280 and > 2.0 at 260/230 ratio were used for cDNA synthesis.

cDNA synthesis and quantitative real time PCR (qPCR) analysis
Oligo primers of target molecules for mRNA determination were purchased from Integrated DNA Technologies (Skokie, IL). First-strand cDNA for gene expression analysis using real-time qPCR was carried out using the iScript cDNA synthesis kit from Bio-Rad following the manufacturer's instructions. Briefly, a volume of 4 µL of 5x iScript reaction mix and 1 µL of iScript reverse transcriptase and 1 µg of total RNA was mixed in clean Eppendorf tubes. Each tube was then brought to a total of 20 µL using RNase/DNase-free water. The tubes were then incubated at 25 O C for 5 minutes, for 30 minutes at 42 O C, and for 5 minutes 85 O C. The tubes were placed at -20 O C until usage in qPCR, which was conducted using 2x iTaq SYBR Green Supermix of Bio-Rad following the manufacturer's instructions. Briefly, 10 µL of the iTaq Universal SYBR Green Supermix, 4 µL of the forward and reverse primer mix for each gene, 4 µL of RNase/DNase free water and 2 µL of the cDNA samples were added to PCR strips containing 8 wells with two replicates for each sample per plate. The strips were then placed in a Bio-Rad CFX96 PCR machine set to run a 3-step cycle.

Sequences of oligo primers used in the study
Mouse T-bet forward: 5'-TCAACCAGCACCAGACAGAG-3' The target genes in the sample refer to genes of interest, such as cytokines. The reference gene refers to the GAPDH gene used to normalize the CT value of the sample. Using this equation, the fold change of the mRNA of test and control samples were determined. Some of qPCR products were further analyzed by running on gel electrophoresis.
The ∆∆CT method was used to calculate relative changes in gene expression of target genes determined from real-time quantitative PCR experiments. Expression in stressed and nonstressed was normalized to the internal control gene, GAPDH and relative to the non-stressed sample as a control. One representative of two independent experiments is shown. Comparison was at the level of P <0.05.

Western blotting of analysis
Protein from mouse genital tract lysate, CD4+ T cells or dendritic cells were isolated Equivalent protein loading and transfer efficiency were verified by using actin as a control.

Establishing interaction networks of gene and proteins
Cytoscape is a social network of open source software that is used to visualize molecular interaction networks and biological pathways. Data of gene expression and cytokine production in stressed and non-stressed mice with or without Chlamydia muridarum genital infection were collected for interaction with other important genes or proteins extracted from data base. Related protein networks of up-regulated or down-regulated genes or proteins were visualized using the cystoscope software.

Statistical analysis
All data are expressed as a mean+/-standard errors of the mean (SEM) unless otherwise stated. Statistical significance between any two groups were tested using Student's t-test and ANOVA was used to test a statistical difference between more than two groups. Statistical significance was at the level of P < 0.05.

Cold water-induced stress increases the intensity of Chlamydia muridarum genital infection during primary infection
To determine the level of infection in stressed and non-stressed mice along with immune response analysis, we measured the number of viable C. muridarum present on vaginal swabs collected every 3 to 6 days post infection. As shown in Table 1, there are greater numbers of chlamydia inclusion counts in the swabs of stressed mice compared to non-stressed mice, indicating that cold-induced stress increases the intensity of C. muridarum genital infection in mice. Our results are consistent with our previous findings of increased C. muridarum shedding in regions of the genital tract of stressed mice compared to non-stressed mice [59,60].

Days post infection
Stressed mice Non-stressed b A statistical difference in counts was observed between stressed and non-stressed mice on day 9 and 15 after infection (P <0.05).
C No Chlamydia muridarum count in non-stress mice and stressed mice thereafter indicating the resolution of infection.

Cold-induced stress results in differential gene expression of beta-adrenergic receptor (-AR) subtypes in mouse genital tract T cells during Chlamydia muridarum infection
This experiment was to determine the gene expression levels of -adrenergic receptor (-AR) subtypes in splenic CD4+ T cells of stressed and non-stressed mice during C. muridarum genital infection. We hypothesized that differential gene expression of β-AR subsets may play an   Data shown are a representative of two or more independent experiments ran on different dates.

Cold-induced stress leads to increased gene expression of transcription factor of GATA-3 in CD4+ T cells of genital tract during Chlamydia muridarum infection
Our laboratory showed previously that cold-induced stress treatment to mice results in differential gene expression and secretion of cytokines during C. muridarum genital infection.
However, little is known about the effect cold-induced stress on the differentiation of the two major T helper (Th) Th1 and Th2 subsets. This study was to determine the gene expression Furthermore, a significant increase in gene expression of GATA-3 in CD4+ T cells isolated from the uterus and cervix of stressed mice is shown compared to that of non-stressed mice ( Figure   3C). In contrast, T-bet mRNA expression was not elevated in either the whole genital tract or specific regions of the genital tract of both stressed and non-stressed mice ( Figure 3D, 3E).
Furthermore, T bet gene expression was down-regulated in oviduct CD4+ T cells of stressed mice compared to that of non-stressed mice ( Figure 3F). DNA intensity of GATA-3 PCR endproduct was significantly higher in stressed mice than non-stressed mice ( Figure 3G). These results suggest that transcription factor GATA-3 may play a big role in the switching of Th1 to Th2, arming the immune system during stressful conditions of chlamydia genital infection.

Cold-induced stress leads to increased gene expression of transcription factor GATA-3 in lysate of whole and regions of the genital tract of stress mice during Chlamydia muridarum infection
The purpose of this study was to determine if there is a differential gene expression in the

Cold-induced stress alters gene expression patterns of transcription factors and signature cytokines in mouse splenic naïve CD4+ T cells during Chlamydia muridarum genital infection
Our aim was to further evaluate the gene expression of transcription factors (GATA3 and T-bet) and IL-4 and -IFN in splenic naïve CD4+ T cells isolated from stressed and non-stressed mice with/without C. muridarum genital infection. As shown Figure 5A, the lowest Ct value and highest fold-change (p < 0.05) of GATA-3 gene expression was observed in stressed/infected mice compared to the other treatment groups. In contrast, the highest Ct value and negative foldchange of T-bet gene expression (p <0.05) was obtained in stressed/infected mice ( Figure 5B).
Similarly, the Ct value and the highest fold-changes of IL-4 was observed in stressed/infected mice ( Figure 5C), whereas the highest Ct value and negative value in fold-changes of IFN-was observed in the stressed group ( Figure 5D).  dates. *Denotes significant statistical differences between treatment groups at the level of (p < 0.05).

Persistence of stress effect on modulation of gene expression of transcription factors and secretion of Cytokines
To determine how long the effect of stress persists, we compared the gene expression of T cell transcription factors or secretion of signature cytokines by CD4+ T cells at two-or 11days post infection. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control to normalize target gene expression levels, which were determined using the 2- CT method. As shown in Figure 6A, a marked increase in expression of mRNA encoding GATA-3 was demonstrated at day two but expression was significantly decreased at day 11 in stressed and infected mice compared to non-stressed and infected mice (P<0.05). As shown in  independently performed experiments. *Denotes significant statistical differences between treatment groups at the level of (p < 0.05).

Pre-exposure of splenic mouse naïve CD4+ T cells to 2-AR agonist or antagonist alters gene expression patterns of transcription factors and signature cytokines
Whether the gene expression of signature cytokines of splenic naïve CD4+ T cells or transcription factors and are influenced by exposure to 2-AR agonist or antagonist were tested.
As shown in Figure 7A,

Preexposure of splenic naïve CD4+ T cells to 2-AR agonist or antagonist affects the production of signature cytokines
In this study, production of IL-4 and IFN- after exposure of CD4+ T cells to a 2-AR agonist was evaluated. As shown in Figure 8A, the production of IL-4 in naïve CD4+ T cells was substantially increased with exposure to the 2-AR agonist Feroterol, whereas pre-exposure to 2-AR antagonist ICI118,551 resulted in a significantly reduced production of IL-4 (P<0.05).
As shown in Figure 8B, the production of interleukin-12 (IL-12) in naïve CD+ T cells was considerably decreased due to pre-exposure to the 2-AR agonist Feroterol, whereas preexposure with ICI118,551 resulted in a significantly increased production of IL-12 (p <0.05).

Production of IL-23 in naïve CD4+ T cells was substantially increased in the presence of
Feroterol, but decreased in the presence of ICI118,551 ( Figure 8C). This data suggests that 2-AR signaling may play an important role in modulating DC function as an essential regulator of the immune system during chlamydia genital infection. independent experiments ran on different dates. *Denotes significant statistical differences between treatment groups at the level of (p < 0.05).

Norepinephrine, 2-AR agonist, or 2-AR antagonist modulates the differentiation of bone marrow derived dendritic cells (BMDCs) cultured in vitro
Culturing of immature BMDC in the presence or absence of norepinephrine, 2-AR and 2-AR in vitro prior to stimulation with LPS was performed for 24 h and culture supernatants were collected for the detection of cytokine production using ELISA. As shown in Table2, Unexpectedly, preexposure to feroterol, β2-AR agonist resulted in a marked increase of IL-12 in non-stressed-non-infected mice compared to that of non-stressed-infected mice. However, β2-AR antagonist, ICI 118,551, treatment resulted in increasing production of IL-12 by BMDCs of stressed mice compared to that of non-stressed mice. Pretreatment with NE, feroterol, showed a significant decrease in IL-12 production between non-stressed-infected and non-stressed-noninfected (Table 3). However, ICI 118,551 resulted in a significant increase of IL-12 production particularly in stressed-infected mice ( Table 3). Pretreating of BMDCs obtained from stressed mice with NE, Feroterol, or ICI118,551 resulted in significantly decreased production of IL-10 compared to that of BMDCs obtained from non-stressed-non-infected mice ( Table 4). Non-stressed-infected only 450.5 +/-127.3 a Immature BMDCs of non-stressed mice were pretreated with norepinephrine, β2-AR agonist, or β2-AR antagonist for 15 minutes prior proliferation for 24 hours. Culture supernatants were tested for cytokine production using ELISA.
b Pretreatment with feroterol resulted in a significant increase in production of IL-12 in noninfected mice (P < 0.05).
c Pretreatment with ICI 118, 551 resulted in a significant increase in production of IL-12 compared to non-stressed and infected.
Immature dendritic cell (iDC) culture was pre-exposed to NE (1 μM), fenoterol (2-AR antagonist) (1 μM) and ICI 118,551 (2-AR antagonist) (1 μM) for 1 h before stimulation with LPS (5 μg/mL) for 24 h before RNA isolation. Data shown are a representative or a mean +/_ SEM of two or more independent experiments ran on different dates. *Denotes significant statistical differences between treatment groups at the level of (p < 0.05). Stressed-infected only 300.3+/-12.5 a Immature BMDCs of stressed mice were pretreated with norepinephrine, β2-AR agonist, or β2-AR antagonist for 15 minutes prior proliferation for 24 hours. Culture supernatants were tested for cytokine production using ELISA.
b Pretreatment with ICI, 118 551 resulted in a significant increase in production of IL-12 in stressed and infected mice (P < 0.05).
c Pretreatment with exogenous chemicals norepinephrine or forterol resulted in a significant decrease in production of IL-12 compared to the stressed and infected only.
b Pretreatment with feroterol resulted in a significant increase in production of IL-10 in non-infected mice compared to stressed mice (P < 0.05).
c Pretreatment with ICI 118, 551 resulted in a significant decrease in production of IL-10 compared to non-stressed and infected (p <0.05).

Cold-induced stress results in differential gene expression of -AR subtypes in splenic dendritic cells
BMDCs harvested from the femurs and tibias of stressed and non-stressed mice were allowed to differentiate in complete RMPI for 8 days in the presence of 20 ng/mL GM-CSF.
Immature BMDCs were pre-exposed to norepinephrine, Feroterol or ICI118,551 for 20 minutes then stimulated with 1 g/mL LPS for 24 h to obtain mature BMDCs. We hypothesized cold induced-stress results in increased gene expression of β2-AR and decreased gene expression of β1-AR and β3-ARs in splenic DCs. Purified splenic DCs from stressed and non-stressed mice were proliferated with/without LPS for RNA isolation and real-time PCR analysis. As shown in   Following maturation of BMDC using LPS, they were pre-primed for 1 h by 2-AR agonist or antagonist. CD4+ T cells from stressed and non-stressed mice isolated by negative selection were co-cultured for 48 h. Shown data are representatives of two or more independent experiments ran on separate dates. *Denotes statistically significant differences between stressed and non-stress mice.
Whether CD4+ T activation is T cell receptor (TCR)-independent or dependent, BMDC-CD4+ T cell co-culturing was in plates coated with anti-CD3 and anti-CD28 antibodies with/without feroterol or ICI 188,551( Table 5). Culture supernatants were collected after 48 h of proliferation for ELISA analysis. The 2-AR agonist resulted in complete blockage of IL-12 production and a significant blockage of IL-4 production, regardless of stress treatment and plate coating with anti-CD3 coating (p<0.5). 2-AR agonist feroterol resulted in a significant increase of IL-4 production in stressed mice but in plates without anti-CD3 coating (p<0.5). The production of IL-10 in stressed mice was the maximum in plates coated with antiCD3 (p<0.5).
Production of IL-23 was high regardless of stress/no stress conditions and plate coating with ani-CD3 antibodies. The 2-AR agonist, forterol, resulted in a significant increase of IL-4 production in stressed mice in plates without anti-CD3 antibodies (p<0.5). Samples from non-stressed mice showed no significant stimulation or inhibition (data not shown).
c ND indicates non-detectable level of cytokines by ELISA d The production of IL-10 in stressed mice was the maximum in plates coated with anti-CD3 (p<0.5) Immature dendritic cell (iDC) culture was pre-exposed to NE (1 μM), fenoterol (2-AR antagonist) (1 μM) and ICI 118,551 (2-AR antagonist) (1 μM) for 1 h before stimulation with LPS (5 μg/mL) for 24 h before RNA isolation. Data shown are a representative or a mean +/_ SEM of two or more independent experiments ran on different dates. *Denotes significant statistical differences between treatment groups at the level of (p < 0.05).

Western blot analysis on secretion of transcription factors and signal transducer and activator of transcriptions (STATs)
Western blot analysis confirmed that cold-induced stress treatment results in increased secretion of GATA-3 in the genital tract, suggesting that cold induced stress is associated with dysregulation of transcription factor of T-bet while favoring increased signaling of GATA-3 ( Figure 11). Elevated expression of GATA3 transcription factor was detected in CD4+ T cells obtained from stressed mice compared to that of non-stressed mice suggesting that GATA3 is crucial for inducing key attributes of Th2 cells essential for Th2 cytokine production including IL-4. In addition to GATA-3 and T-bet expression patterns, the production of Th1 and Th2

Interconnection of genes and their products that may be essential during stress and genital infection
Up-regulated or down-regulated gene expression profiles from stressed and non-stressed mice during Chlamydia muridarum genital infection were used to generate an interaction network to identify any other gene that could also be affected by their expression. As shown in Figure 13, gene nodes that are colored blue were down-regulated and up-regulated during stress and non-stressed conditions, respectively. Gene nodes in green were up regulated during stress and down regulated during non-stress conditions. The gene node that is colored red was downregulated during both in stressed and non-stressed mice. The node shape shows the type of function that the genes play during stressful conditions. Nodes that are square are transcription factors, nodes that are circles are cytokines and nodes that are triangles are cells signaling receptors. Line color and shape indicates the regulation that their expression could play on other genes during stressful conditions. Green dashed lines indicate the genes could be down regulated and up regulated during stress and non-stressed respectively. Red arrowed lines show genes that could be up regulated and down regulated during stress and non-stressed respectfully.

DISCUSSION
We showed previously that repeated exposure of mice to cold water leads to increased norepinephrine and epinephrine production in plasma, spleen, and genital tract lysates that are speculated in increasing the intensity of C. muridarum genital infection [59,60]. Real-time quantitative PCR was used in gene expression profiling based on real-time fluorescence detection and QPCR target concentration measurements using threshold cycle (Ct).
In most cases of our findings, the Ct is inversely proportional to the initial copy number and a direct relationship of a higher initial copy number to a lower threshold cycle was obtained.
However, as shown in our amplification curves, reaction endpoint is not uniform even though the same amounts of cDNA from mRNA were being amplified. This observation is consistent with the fact PCR reaction efficiency can decrease during later amplification cycles as reagents are consumed and accumulation of inhibitors to the reaction do increase. We feel that these differences in final fluorescence values of samples are not related to the starting template concentrations.
Several studies indicate that the stress hormone norepinephrine (NE) suppresses the immune system through the stimulation of 2-AR expressed on various cell surfaces [39,61,62]. In this study, we determined the mRNA levels of -anadrenergic receptor (-AR) subsets in Because 2-AR is the most commonly expressed receptor, we tested whether a 2-AR agonist or 2-AR antagonist affects cytokine production. Our results show that 2-AR agonist decreases Th1 cytokine production probably by blocking T cell receptor (TCR)-mediated signaling, whereas 2-AR antagonist restores Th1 cytokine production. However, controversy exists on the effect of 2-AR agonist or 2-AR antagonist on Th2 cells, but studies have shown that 2-AR agonist modulates T cell functions via direct actions on both Th1 and Th2 cells (63).
Overall, the results of 2-AR and NE interaction suggest that the differential receptor expression may alter the functional responsiveness of CD4+ T cells during cold-stress treatment and C. genital infection [39,62].
Chronic stress generation adopted in our lab has demonstrated to exert a significant suppressive effect on Th1 cell cytokine production. We tested how long cold-induced stress can persist in our mouse model and results show that cold-induced stress has lasting effect on suppression of the immune system after the cessation of stressing process. This observation suggests to consider how the timing of stress is critical in promoting immune suppression in the implicated host. This slow immune response recovery shown in our study is in line with several studies demonstrating stress has long-lasting effects on the immune system that subsequently leads to infection [27,28,36,40,58].
The functional differences in Th1 and Th2 responses as protective and immunopathology, respectively during chlamydia infection was reported [18,30]. In line with those reports, our data implicate that cold induced stress application changes the trends of Th1 and Th2 function that may play important role in reducing the host defense and promoting pathology during chlamydia genital infection. Differences found in Th1 and Th2 subset cytokine secretion in response to cold-stress treatment may provide insight onto the potential intrinsic immune equilibrium established between normal and stressful conditions. This study suggests the balance of Th1 and Th2 cytokines doubtless is important in resolving the immunopathogenesis of chlamydia genital infection during stressful conditions. The revealed changes in expression of transcription factors may indicate the imbalance between T-helper cells of the Th1 and Th2 type cells, which can be one of the causes for the development of pathology and infertility associated with chlamydia genital infection that was previous reported [60]. In contrast, recent studies revealed that C.
trachomatous genital infection is mostly controlled by Th1 immunity, recent report claim that Th2 immunity has a major in controlling chlamydia genital infection in females [40]. Studies in our and other labs have demonstrated that cold-stress treatment to mice brings changes that impact the activity of the immune system. However, it is not yet known how cold stress exposure suppresses or enhances the immune system activities. It is suggested that the sympathetic nervous system activation leads to the release of norepinephrine and epinephrine that interact with the immune system cells through binding to adrenergic receptors on their surfaces. Although there are multiple mechanisms that sympathetic nervous induced system of regulation, -AR signaling may contribute to increased immunosuppression during stress chlamydia genital infection. We feel that the major stress response is norepinephrine production which plays a key role in regulation CD4+ T cells and DCs function. To clarify the role of -AR signaling on the modulation of DC function, we compared the effect of NE, forterol and IC 118,551 on maturation, cytokine production assess the ability of these BMDCs to induce/derive T helper differentiations. We observed cold stress treatment results high secretion of IL-10 while reducing IL-12. This phenomenon corresponded with a Th2 skewing potential of BMDCs preexposed to -AR agonist. We were able to demonstrate the dichotomy between IL-12 and IL-4 in the presence of -AR agonist and antagonist. Although C. trachomatis serovar D infection in mice was used, it was reported the Th1 subset with T-bet and IFN- production dominated the upper genital tract compartment female mice whereas the lower genital tract has IL-10 and GATA-3 suggesting the dominance of Th2 [40,63]. Based on our present findings of th1 and Th2 cytokine production profiles in our stress model, we feel that cold-induced stress leads to Th2 dominated conditions which favor enhanced development of immunopathogenesis during C.
muridarum. Although previous studies showed that b2-AR is absent in Th2 cells (47), other studies demonstrated that 2-AR agonists modulate T cell functions via direct actions on Th1 and Th2 cells, where CD3 + CD28-stimulatd IL-13 (Th2 cytokine) and IFN-g and IL-2 (Th1 cytokines) production were inhibited by 2-AR agonist (40,63). Our study on mixed population of Th1 and Th2 resulted in the inhibition of Th1 cytokines but not Th2 cytokines thus our findings underscore the importance of further study on the action of 2-AR agonists during stressful conditions.
Nowadays, datasets are available in public archives such as the Gene Expression Omnibus (GEO) and cystoscope [69]. Although the scope of this study was not to undertake detailed analysis of biological processes and signaling pathways of genes or their products, we inserted our findings into global datasets and constructed a network and identified the genes shown in Figure 13. We identified a group of proteins that have interconnection during chlamydia infection of stressed model identified several candidate targets for further fundamental experimental research.
In summary, we undertook a detailed assessment of how cold-induced stress modulates a This observation suggests that while it is known that CD4+ Th1 are essential in the clearance of chlamydia, the increased production of IL-10 and IL-17 in our stress model may lead to the establishment of chronic chlamydia infection (66)(67)(68). We believe that this study may offer important direction for further investigation of the mechanisms of stress on chlamydia genital infection and immunity in humans. We feel that this study is important because psychological or physical stressors may lead to increased stress hormone production and have profound effects on health, including prevalence of chlamydia genital infection. Evidence shows that these stressors generally are greater in populations of lower socioeconomic status, in which there are increased health concerns. The present finding, taken together within previous observations [59,60] indicate that cold-water induced stress increases the intensity of chlamydia genital infection in mice. The long-term goal of our study is to employ the cold-water induced stress in a 2-AR KO mouse model, and the hypothesis to be tested is: cold water-induced stress leads to NE modulation of the immune response against C. muridarum genital infection by enhancing the production of immunopathogenic cytokines that result in disease sequelae.

FUNDING
This research work was funded by the WV-INBRE of NIH Grant P20GM103434 and NIH grant # 1R15AI124156-01.