https://orcid.org/0000-0001-9055-3749
Abstract
Chlamydia genital infection caused by Chlamydia trachomatis is the most common bacterial sexually transmitted disease worldwide. A mouse model has been developed in our laboratory to better understand the effect of cold-induced stress on chlamydia genital infection and immune response. However, the stress mechanism affecting the host response to Chlamydia muridarum genital infection remains unclear. Here, we demonstrate a role for the beta2-adrenergic receptor (β2-AR), which binds noradrenaline and modulates the immune response against chlamydia genital infection in a mouse model. A successful β2-AR homozygous knockout (KO) mouse model was used to study the infection and analyze the immune response. Our data show that stressed mice lacking the β2-AR are less susceptible to C. muridarum genital infection than controls. A correlation was obtained between lower organ load and higher interferon-gamma production by CD4+ and CD8+ cells of the KO mice. Furthermore, exposure of CD4+ T cells to noradrenaline alters the production of cytokines in mice during C. muridarum genital infection. This study suggests that the blockade of β2-AR signaling could be used to increase resistance to chlamydia genital infection. We value the β2-AR KO as a viable model that can provide reproducible results in investigating medical research, including chlamydia genital infection.
Introduction
Chlamydia genital infection caused by Chlamydia trachomatis is the most common bacterial sexually transmitted disease worldwide (Paavonen 1999, Stamm 1999). A complication of chlamydia genital infection leads to pelvic inflammatory disease, fallopian tube scarring, ectopic pregnancy, infertility, and neonatal conjunctivitis (Nikkanen et al. 1980, Weström et al. 1992, Cohen and Brunham 1999).
It is reported that more than 90 million new cases of chlamydia genital infections occur annually worldwide, and 4 million in the USA alone (De Schryver and Meheus 1990, Kularatne et al. 2018). In the USA, chlamydia genital infection disproportionately affects populations of low socioeconomic status (Ellen et al. 1995, Budai 2007). The reasons for the high prevalence of chlamydia in populations of low socioeconomic status are not well known, but stress may have a role in the persistently high rate of the disease associated with low socioeconomic conditions (Campbell et al. 1998). Information on the impact of stress-induced changes on immune response and the risk of infection is emerging (Adler et al. 1994, Sheridan et al. 1994), and studies reported the association between perceived stress and sexually transmitted infections (Ellen et al. 1995, Turpin et al. 2019, Scheidell et al. 2020).
Chlamydia trachomatis leads to the recruitment of protective immune responses to the site of infection or the infiltration of inflammatory products that may also contribute to pathological damage in the host. Several animal model studies have exhibited vital aspects of human chlamydial genital infection, including pathologic consequences or sequelae of infection, tubal inflammation, hydrosalpinx formation, and infertility (Brunham and Peeling 1994, Rank 1994, Darville et al. 1997, Gracey et al. 2015, Lijek et al. 2018, Murthy et al. 2018). This is evidenced during repeated chlamydia genital infection in animal models (Patton and Kuo 1989, Yang 2003) and human subjects (Darville and Hiltke 2010, Lijek et al. 2018) known to have a much greater risk of a tubal obstruction than those with less exposure to C. trachomatis.
Several studies show that psychological or physical stress, resulting from the hardship of life in society has major impacts on public health (Bonneau et al. 1990, Coe and Laudenslager 2007, Zieziulewicz et al. 2013, Avitsur et al. 2015). Moreover, it is well documented that interaction between the sympathetic nervous system and the immune system exists (Padro and Sanders 2014).
Catecholamines [noradrenaline or norepinephrine (NE)], adrenaline [epinephrine (EP)], and glucocorticoids 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 (Madden et al. 1995, Elenkov et al. 1996, Swanson et al. 2001, Zieziulewicz et al. 2013). It is documented that noradrenaline binds and stimulates the beta2-adrenergic receptor (β2-AR), which is predominantly expressed in CD4+ and B cells (Sanders 2012, Lorton and Bellinger 2015). Studies have further shown the direct binding of noradrenaline to β2-AR modulates immune responses, including altering cytokine production, lymphocyte proliferation, and antibody secretion (Elenkov et al. 1996, Gupta et al. 2009, Guereschi et al. 2013).
Although stress is implicated as a risk factor for various infections, the mechanisms of chlamydia genital infection are unknown. Here, we sought to determine the role of β2-AR in stressed mice during C. muridarum genital infection. We hypothesize that noradrenaline produced by cold-induced stress (CIS) stimulates β2-AR to suppress the protective immune response against C. muridarum genital infection. As a result, we anticipate eliminating the noradrenaline receptor in our stress mouse model enhances (i) C. muridarum clearance and (ii) restoration of protective immune cells.
Materials and methods
Chlamydia stock culture and McCoy cell line
Stock culture of C. muridarum (previously known as C. trachomatis), an agent of mouse pneumonitis (MoPn), was provided by Dr. Joseph Igietseme while he was at Morehouse School of Medicine and CDC in Atlanta, GA. McCoy cell line for isolation of C. muridarum and stock culture of C. muridarum were supplied by Dr. Vida A. Dennis’s Lab at Alabama State University, Montgomery, AL.
Animals
Beta2-AR knockout (KO) mice developed from the β1-AR/β2-AR and the wildtype (WT) C57BL/6J were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were housed in a vivarium at Bluefield State University located in the Basic Science Building of the Department of Applied Science and Mathematics. Mice were given food and water ad libitum in an environmentally controlled room with equal daylight and night hours. Animal use was approved by the Institutional Animal Care and Use Committee (IACUC) of Bluefield State University.
Cold-water stressing protocol
Before the start of actual experiments, mice were acclimated for 7 days to avoid the effect of transport stress. To induce chronic stress, the stressing period was designed for 21 days as previously described (Belay and Woart 2013). Five mice were placed in a packet filled with four centimeters of cold water (4°C–5°C) for 5 min daily for 21 days. The water level was deep enough to cover the backs of the mice while swimming in the cold water. At the end of each stressing period, mice were dried with towels to avoid hypothermia. A total of 10 non-stressed mice were kept at room temperature without cold-water treatment.
Body and spleen weight measurements
Body and spleen weight were used as criteria for the impact of CIS on WT and KO mouse physiology. Mice were weighed upon arrival and at 3-day intervals during the stressing period of 21 days. Mice were sacrificed at day 21 by CO2 inhalation followed by cervical dislocation, and the spleens were harvested and weighed as well. Splenic T cells and bone marrow-derived cells (BMDCs) were isolated as previously described in our lab using EasySep mouse T cell enrichment kits (Belay et al. 2020). BMDCs were differentiated with treatment Granulocyte-macrophage colony-stimulating factor (GM-CSF) for 8 days, then matured with lipopolysaccharide (LPS) treatment for 24 h. A t-test statistical method was used to compare the body and spleen weights of stressed and non-stressed mice.
Blood collection and plasma levels of stress hormone determination
We hypothesized that the stress hormones adrenaline and noradrenaline, known to modulate the host immune function, may play no role in β2-AR KO mice during C. muridarum genital infection. Mice were euthanized by CO2 inhalation followed by cervical dislocation, and blood collection was performed via cardiac puncture. Briefly, plasma was prepared by centrifugation from blood and stored in the freezer until adrenaline and noradrenaline levels in serum were determined using the 3-cat EIA kit as previously described (Belay and Woart 2013, Belay et al. 2021). Each point represents the means ± standard deviation (SD) of two experiments (n = 5–7 mice for each group of experiments).
RNA isolation, cDNA synthesis, and PCR analysis
Based on our previous findings, we hypothesized that differential gene expression of β-AR subsets might play an essential role in modulating the immune function against chlamydia genital infection, which is altered during stressful conditions. Total RNA extracted from CD4+ T cells of the β2-AR KO and WT was used for gene expression analysis using real-time quantitative polymerase chain reaction (qPCR) as previously described (Belay et al. 2020). Total RNA from cultured splenocyte and lymph node T cells was isolated using the FastPrep-24™ or FastPrep® FP120 Instrument and FastRNA® Pro Green Kit (MP Biomedicals) following the manufacturer’s instructions. Then, the isolated RNA was measured using a Theme Scientific Nanodrop (Thermo Fisher). 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.
The mRNA level was determined and used to make cDNA for gene expression analysis using real-time qPCR. The iScript cDNA synthesis kit from Bio-Rad was used following the manufacturer’s instructions. Briefly, a volume of 4 µl of 5x iScript reaction mix, 1 µl of iScript reverse transcriptase, and 1 µg of total RNA was mixed in clean Eppendorf tubes. Each tube was then brought to 20 µl using RNase/DNase-free water. The tubes were then incubated at 25°C for 5 min, 30 min at 42°C, and 5 min at 85°C. The tubes were placed at −20°C until they were used in QPCR.
Oligo primers of target molecules for beta-adrenergic receptor subsets mRNA determination were purchased from Integrated DNA Technologies (Skokie, IL). Quantitative polymerase chain reaction (PCR) was conducted using the first-strand cDNA by mixing the 2x iTaq SYBR Green Supermix of Bio-Rad following the manufacturer’s instructions (Belay et al. 2020). 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 first-strand cDNA samples were added to PCR strips containing eight 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 three-step cycle.
Sequences of oligo primers used in the study
Mouse β1-AR forward: 5′-AAACTCTGGTAGCGAAAGGGGAC-3′
reverse 5′ TCTGCTCATCGTGGTGGGTAAC-3′
Mouse β2-AR forward: 5′-AGCCGTTCCCATAGGTTTCG-3′
reverse 5′-CGTCCTCGATTGTGTCTTTCTACG-3′
Mouse β3-AR forward: 5′-CGAAGAGCATCACAAGGAGGG-3′
reverse 5′-CGAAACTGGTTGCGGAACTGTGT-3′
QPCR data analysis
Quantitative PCR was performed using Bio-Rad iTaq™ Universal SYBR® Green Supermix following the manufacturer instructions on a CFX96 Real-Time System. Analysis was performed using Bio-Rad CFX Manager Software with GAPDH as a housekeeping gene for reference. Briefly, ITaq™ Universal SYBR® (Belay et al. 2020). To determine the fold change using this method, the following equations were used:
The target genes in the sample refer to beta-adrenergic receptor subsets. 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 the experimental and control samples was determined. 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 non-stressed sample 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. The comparison was at the level of P < .05.
Chlamydia muridarum genital inoculation and isolation
Kinetics of C. muridarum shedding in the genital tract of stressed and non-stressed β2-AR KO and WT mice were assessed for 42 days. 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 21-day stressing period. Then, mice were inoculated intravaginally with 105 IFU of C. muridarum in a volume of 30 µl of PBS while under Ketamine–Xylazine-induced anesthesia (Belay et al. 2017).
The course of infection was monitored by cervicovaginal swabbing at 3-day intervals for the first 42 days of the primary course of infection. Chlamydia muridarum was recovered from swabs by staining infected monolayers of McCoy tissue culture with fluorescein isothiocyanate-labeled, genus-specific anti-chlamydial antibodies purchased from Fisher Scientific. Inclusion bodies in 10 fields/well were visualized, imaged, and counted under a fluorescent microscope of Moetic Inverted Microscope AE31E, and inclusion-forming units per ml (IFU/ml) were determined as previously described (Belay and Woart 2013, Belay et al. 2020).
Splenic and lymph node T cell harvesting and purification
Spleens and lymph nodes 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). Cell suspensions were passed through a 70-µm cell strainer (Becton Dickinson, Franklin Lakes, NJ) to remove tissue debris. Following the manufacturer’s instructions, the splenocyte and lymph node T cells were isolated using EasySep mouse T cell enrichment kits based on immunomagnetic negative selection (STEMCELL Technologies, Vancouver, Canada). Briefly, the spleen and lymph node cell suspensions were incubated with the EasySep Ab mixtures to non-CD4+ T cells, followed by a biotin selection mixture and magnetic beads. Labeled non-CD4+ T cells were removed using EasySep magnets. Culturing of T cells in vitro from stressed and non-stressed mice as previously described (Belay et al. 2021).
Isolation and collection of bone marrow-derived cells
As previously described, the femurs and tibia bones from stressed and non-stressed mice were aseptically collected (Belay et al. 2021). Briefly, bones were cleaned with 70% alcohol and then crushed to obtain monocytes, and debris was removed by passing the cell suspension through a 70-µm nylon cell strainer. Purified bone marrow cells were cultured in a tissue culture flask containing 20 ml of RPMI 1640 supplemented with recombinant GM-CSF (10 ng/ml). On day 3 of culturing, 10 ml of GM-CSF (10 ng/ml) was added to the culture. On day 8, all floating (dendritic cells) and loosely adherent cells (macrophages) were collected as immature BMDCs. All cells were treated with 5 µg/ml of LPS for maturation by incubation for 24 h at 37°C. All cells were counted by trypan blue exclusion method, and their viability was >80%.
Testing the action of synthetic noradrenaline on T cell viability and cytokine production
To investigate the influence of synthetic NE bitartrate salt (Sigma) on cytokine production, we investigated whether NE inhibits immune cell activities as measured by the level of important cytokines produced. Vi-Cell XR Viability Analyzer (Beckman Coulter), an immunofluorescent imaging system using the trypan blue exclusion method, was used to count the viable cells. Briefly, T cells were seeded at a density of 5 × 105 cells/well in 96-well tissue culture plates with the addition of 2.5 µg/ml of Concanavalin A (Con A), 5 µg/ml of LPS, or 2 µl of 10−4 mM NE as previously described (Belay et al. 2017). Plates were incubated in a water-jacketed incubator at 37°C in 5% CO2, viability was measured during 24 h proliferation, and culture supernatants were collected after 72 h of proliferation to detect cytokine production by enzyme-linked immunosorbent assay (ELISA).
Cytokine measurement using ELISA
The level of cytokines in supernatants collected from peritoneal fluid cells of stressed and non-stressed mice was measured using an ELISA kit (Invitrogen, Camarillo, CA) following the manufacturer’s instructions as previously described (Belay et al. 2017).
Flow cytometry procedures
Single-cell suspensions were prepared from CD4+ T cells enriched T cells of all treatment groups by negative selection following the procedures of STEMCELL Technologies Inc. BMDCs cells harvested from bone marrow were differentiated for 8 days in the presence of GM-CSF and treated overnight with LPS for maturation as previously described (Belay et al. 2021). Isolated cells were washed and stained with fluorochromes-conjugated antibodies. Fc blocker and all antibodies were purchased from BD Biosciences (San Jose, CA). Briefly, cells were incubated with anti-CD16/CD32 FcRII/III blocker at 4°C for 10 min and stained with fluorochrome-conjugated antibodies listed above following the vendor’s instructions. Then, anti-mouse antibodies against CD3 (clone 17A2), CD4 (clone RM4-5), CD44 (clone IM7), CD62L (clone MEL-14), CD11c (clone HL3), CD80 (clone 16–10A1), CD40 (clone 3/23), CD86 (clone GL1), CD11c (clone HL3), and after two times of washing, positive cells for immunostaining were identified using a flow cytometry analyzer (BD FACSDiva 6.1.2). Further analysis and the generation of histograms were performed using FlowJo Software analysis (Belay et al. 2021).
Statistical analysis
All data are expressed as a mean ± standard deviation or error of the mean (SEM). Statistical analyses were performed using GraphPad Prism based on a t-test and analysis of variance (ANOVA), followed by Turkey’s multiple-comparison test to compare the recovered IFU/ml, in stressed and non-stressed mice. Statistical significance was considered at the level of P-values ≤ .05(*), 0.01(**), .001(***), and .0001(****).
Results
Successful generation of β2-AR homozygous KO mice
Previous data in our laboratory show that β1/β2-AR KO (purchased from Jackson Lab) is less susceptible to C. muridarum genital infection than WT C57BL/6J (Belay et al. 2017). Furthermore, elevated noradrenaline release in the spleen and lymph nodes of stressed mice is reported (Bonneau et al. 1998, Cao 2002). Those observations prompted us to use a β2-AR single KO and explore the immunopathogenesis of chlamydia genital infection. A homozygous β2-AR KO mouse model developed by the Jackson Laboratories (Bar Harbor, ME) and a WT C57BL/6J mouse as a control were used in this study. Jackson Laboratories’ data confirm that the C57BL/6J and β2-AR KO have the closest genetic match, and also, β2-AR KO has a higher percentage of C57BL/6J origin DNA in the genome. Following the Jackson Laboratory’s effective colony management protocols and IACUC protocol approval, we have established a breeding protocol in our lab. We value the β2-AR KO as other labs use it (Sanders 2012, Lorton and Bellinger 2015), a viable model that can provide reproducible results in investigating medical research, including chlamydia genital infection.
Stress results in similar plasma levels of stress hormones in β2-AR KO and WT mice
As shown in Fig. 1, β2-AR KO and WT mice showed almost the same catecholamine hormone levels; however, the plasma levels of noradrenaline and adrenaline were significantly higher in the stress β2-AR KO and WT group than in their non-stressed group. The observation justifies that β2-AR was removed from the surface of immune cells, so noradrenaline has less possibility of binding to immune cells to suppress the immune system. Thus, the results show that β2-AR is a key receptor critical in promoting chlamydia genital infection during stressful conditions.
Effect of stress on the production of (A) plasma noradrenaline and (B) adrenaline in stressed and non-stressed mice measured by ELISA. Each point represents the mean ± SD of two experiments (n = 5–7 mice for each experiment). Statistical analysis was performed using GraphPad Prism based on a t-test and analysis of variance (ANOVA) followed by multiple comparisons.
CIS resulted in a comparable body and spleen weight loss of mice in β2-AR KO and WT mice
In previous studies, we have shown that stressed mice have lost more weight than non-stressed mice. Furthermore, the restoration of body or spleen weight loss of Active Hexose Correlated Compound-fed mice compared to Phosphate Buffered Saline-fed stressed mice was observed.
CIS in mice has proven useful in defining the relationships between the central nervous and immune defense systems. In our studies, cold stress was induced by immersing mice in cold water for 5 min daily for 21 days, as previously established protocols (Belay and Woart 2013). Cold-water-treated mice were visually smaller than control mice and weighed ~1.0 g less than non-cold-water-treated control mice (Table 1).
Comparison of body/spleen weight and splenic CD4+ T cell and matured BMDCs counts of stressed and non-stressed beta2-adrenergic receptors and KO and WT C57BL/6J.
| Treatment groups | ||||
|---|---|---|---|---|
| Parameters | Stressed β2-AR KOa | Non-stressed β2-AR KO | Stressed C57BL/6J | Non-stressed C57BL/6J |
| Body weighta | 18.4 ± 0.97 | 20.28 ± 1.4 | 18.1 ± 0.67 | 19.8 ± 0.50 |
| Spleen weightb | 0.097 ± 0.014 | 0.105 ± 001 | 0.086 ± 009 | 0.091 ± 0.004 |
| T cell counts | 6.65 ± 0.72 | 6.63 ± 0.06 | 6.32 ± 0.29 | 6.69 ± 0.14 |
| BMDC counts | 6.12 ± 0.34 | 6.31 ± 0.03 | 6.16 ± 0.71 | 6.41 ± 0.06 |
| Treatment groups | ||||
|---|---|---|---|---|
| Parameters | Stressed β2-AR KOa | Non-stressed β2-AR KO | Stressed C57BL/6J | Non-stressed C57BL/6J |
| Body weighta | 18.4 ± 0.97 | 20.28 ± 1.4 | 18.1 ± 0.67 | 19.8 ± 0.50 |
| Spleen weightb | 0.097 ± 0.014 | 0.105 ± 001 | 0.086 ± 009 | 0.091 ± 0.004 |
| T cell counts | 6.65 ± 0.72 | 6.63 ± 0.06 | 6.32 ± 0.29 | 6.69 ± 0.14 |
| BMDC counts | 6.12 ± 0.34 | 6.31 ± 0.03 | 6.16 ± 0.71 | 6.41 ± 0.06 |
Stressing mouse began at age 5 weeks for 21 days. The mean ± SEM bodyweight of 10 female stressed or non-stressed mice on day 21 is shown.
Five stressed and five non-stressed mice were sacrificed at the end of the stressing period. The mean ± SEM spleen weight of five females is shown. Significant statistical differences between treatment groups were compared at (P < .05, P < .01).
Comparison of body/spleen weight and splenic CD4+ T cell and matured BMDCs counts of stressed and non-stressed beta2-adrenergic receptors and KO and WT C57BL/6J.
| Treatment groups | ||||
|---|---|---|---|---|
| Parameters | Stressed β2-AR KOa | Non-stressed β2-AR KO | Stressed C57BL/6J | Non-stressed C57BL/6J |
| Body weighta | 18.4 ± 0.97 | 20.28 ± 1.4 | 18.1 ± 0.67 | 19.8 ± 0.50 |
| Spleen weightb | 0.097 ± 0.014 | 0.105 ± 001 | 0.086 ± 009 | 0.091 ± 0.004 |
| T cell counts | 6.65 ± 0.72 | 6.63 ± 0.06 | 6.32 ± 0.29 | 6.69 ± 0.14 |
| BMDC counts | 6.12 ± 0.34 | 6.31 ± 0.03 | 6.16 ± 0.71 | 6.41 ± 0.06 |
| Treatment groups | ||||
|---|---|---|---|---|
| Parameters | Stressed β2-AR KOa | Non-stressed β2-AR KO | Stressed C57BL/6J | Non-stressed C57BL/6J |
| Body weighta | 18.4 ± 0.97 | 20.28 ± 1.4 | 18.1 ± 0.67 | 19.8 ± 0.50 |
| Spleen weightb | 0.097 ± 0.014 | 0.105 ± 001 | 0.086 ± 009 | 0.091 ± 0.004 |
| T cell counts | 6.65 ± 0.72 | 6.63 ± 0.06 | 6.32 ± 0.29 | 6.69 ± 0.14 |
| BMDC counts | 6.12 ± 0.34 | 6.31 ± 0.03 | 6.16 ± 0.71 | 6.41 ± 0.06 |
Stressing mouse began at age 5 weeks for 21 days. The mean ± SEM bodyweight of 10 female stressed or non-stressed mice on day 21 is shown.
Five stressed and five non-stressed mice were sacrificed at the end of the stressing period. The mean ± SEM spleen weight of five females is shown. Significant statistical differences between treatment groups were compared at (P < .05, P < .01).
The average spleen weight of stressed mice sacrificed on day 21 of stressing was 72.61 mg ± 6.44 mg, compared to 90.55 ± 11.29 mg for non-stressed mice. The spleens of cold-water-treated mice had an average of 17.94 mg less than spleens from non-stressed mice (P < .05). These findings indicate that cold-water treatment induces physiological stress in mice, reflected in a slight loss of body weight and a greater negative loss of spleen weight, indicating stress-related immunosuppression.
Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of beta-adrenergic receptor subsets assures the β2-AR deficient status of the KO model
In this study, CD4+ T cells isolated from the experimental mice after 24 h C. muridarum genital infection tested for altered gene expression patterns of β-AR subsets using qPCR as previously described (Belay et al. 2020). Our previous study demonstrated differential gene expression levels of β-AR subtypes in splenic CD4+ T cells of stressed and non-stressed BALB/c mice during C. muridarum genital infection (Belay et al. 2020). In that study, the gene expression of β2-AR in splenic CD4+ T cells of stressed mice was about five times higher than in non-stressed mice. In contrast, the gene expression of β1-AR and β3-AR of splenic CD4+ T cells showed no difference between stressed and non-stressed mice, indicating β2-AR is stimulated during CIS and C. muridarum genital infection. To confirm that the β2-AR KO colony is not recuperating the receptor gene, we assessed the gene expression profile of β2-AR along with β1- and β3-AR sunsets. Because the parental of the β2-AR KO is a homozygous KO, gene expression of β2-AR was not detected (Table 2). In the absence of β2-AR, a slight upregulation of β1- and β3-AR was observed primarily in CD4+ T cells of β2-AR KO mice. The slight up-regulation of the β1-a and β3-AR may bind noradrenaline to compensate for the loss of β2-AR.
Relative fold-change in mRNA of beta-adrenergic subsets in splenic CD4+ T cells of stressed and non-stressed β2-AR OK and C5BL/6J mice during C. muridarum genital infection.
| Treatment groups | ||||
|---|---|---|---|---|
| β-AR subtypes | Stressed β2-AR KOa | Non-stressed β2-AR KOa | Stressed C57BL/6J | Non-stressed C57BL/6Ja |
| β1-AR | +2.3 ± 0.79 | +0.9 ± 002 | +1.2 ± 0.003 | +0.19 ± 0.005 |
| β2-AR | −0.4 ± 0.001 | −3 ± 0.003 | +7.3 ± 0.75 | +2.9 ± 0.6 |
| β3-AR | +2.5 ± 0.42 | +1.0 ± 0.005 | +3.5 ± 0.42 | +1.0 ± 0.001 |
| Treatment groups | ||||
|---|---|---|---|---|
| β-AR subtypes | Stressed β2-AR KOa | Non-stressed β2-AR KOa | Stressed C57BL/6J | Non-stressed C57BL/6Ja |
| β1-AR | +2.3 ± 0.79 | +0.9 ± 002 | +1.2 ± 0.003 | +0.19 ± 0.005 |
| β2-AR | −0.4 ± 0.001 | −3 ± 0.003 | +7.3 ± 0.75 | +2.9 ± 0.6 |
| β3-AR | +2.5 ± 0.42 | +1.0 ± 0.005 | +3.5 ± 0.42 | +1.0 ± 0.001 |
Fold change values of β-AR subsets are the mean of two independent experiments normalized with GAPDH housekeeping gene. We considered positive fold change values to indicate upregulation, whereas negative values indicate downregulation compared to control. The P-value of the treatment groups was < .05, showing a significant statistical difference compared to the control.
Relative fold-change in mRNA of beta-adrenergic subsets in splenic CD4+ T cells of stressed and non-stressed β2-AR OK and C5BL/6J mice during C. muridarum genital infection.
| Treatment groups | ||||
|---|---|---|---|---|
| β-AR subtypes | Stressed β2-AR KOa | Non-stressed β2-AR KOa | Stressed C57BL/6J | Non-stressed C57BL/6Ja |
| β1-AR | +2.3 ± 0.79 | +0.9 ± 002 | +1.2 ± 0.003 | +0.19 ± 0.005 |
| β2-AR | −0.4 ± 0.001 | −3 ± 0.003 | +7.3 ± 0.75 | +2.9 ± 0.6 |
| β3-AR | +2.5 ± 0.42 | +1.0 ± 0.005 | +3.5 ± 0.42 | +1.0 ± 0.001 |
| Treatment groups | ||||
|---|---|---|---|---|
| β-AR subtypes | Stressed β2-AR KOa | Non-stressed β2-AR KOa | Stressed C57BL/6J | Non-stressed C57BL/6Ja |
| β1-AR | +2.3 ± 0.79 | +0.9 ± 002 | +1.2 ± 0.003 | +0.19 ± 0.005 |
| β2-AR | −0.4 ± 0.001 | −3 ± 0.003 | +7.3 ± 0.75 | +2.9 ± 0.6 |
| β3-AR | +2.5 ± 0.42 | +1.0 ± 0.005 | +3.5 ± 0.42 | +1.0 ± 0.001 |
Fold change values of β-AR subsets are the mean of two independent experiments normalized with GAPDH housekeeping gene. We considered positive fold change values to indicate upregulation, whereas negative values indicate downregulation compared to control. The P-value of the treatment groups was < .05, showing a significant statistical difference compared to the control.
β2-AR deficiency in stressed mice results in decreased organ load of C. muridarum in the genital tract
We value the β2-AR KO as other labs use it (Sanders 2012, Lorton and Bellinger 2015), a viable model that can provide reproducible results in investigating medical research, including chlamydia genital infection. To determine the level of infection in β2-AR and C57BL/6J stressed and non-stressed mice, we measured the number of viable C. muridarum present on vaginal swabs collected every 3 days post-infection. We have previously demonstrated that CIS enhances bacterial shedding in BALB/c mice (Belay et al. 2017). In this study, we compared vaginal chlamydial shedding in the stressed β2-AR KO and non-stressed mice. As shown in Fig. 2A, except on Days 3 and 6, no significant statistical differences were observed in chlamydial shedding between stressed and non-stressed β2-AR KO mice. In contrast, WT C57BL/6J, the parental strain of our newly generated β2-AR showed increased susceptibility to infection after exposure to stress, especially for 12 days post-infection (Fig. 2B). We compared CIS WT and stressed β2-AR KO mice and found significantly enhanced chlamydial shedding in non-stressed WT C57BL/6J compared to the non-stressed β2-AR KO mice (Fig. 2C). As shown in Fig. 2D, significantly enhanced chlamydia shedding was observed in stressed WT C57BL/6J compared to stressed β2-AR KO mice. Furthermore, enhanced chlamydial shedding in WT C57BL/6J compared to stressed β2-AR KO mice was observed between days 9 and 15 following intravaginal infection. Overall, the significant reduction in chlamydia shedding in the β2-AR KO suggests the contribution of β2-AR in the enhancement of C. muridarum genital infection that may lead to severe pathology in the genital tract of stressed mice.
Kinetics of C. muridarum shedding in the genital tract of stressed and non-stressed beta2 Adrenergic Receptor (β2-AR) KO and wildtype (WT mice). (A) Non-stressed and stressed β2-AR KO, (B) stressed and non-stressed C57BL/6J, (C) non-stressed β2-AR KO and non-stressed C57BL/6J, (D) stressed β2-AR KO and stressed WT. Inclusion forming unit/milliliter (IFU/ml) was determined. Statistical differences between treatments were evaluated using GraphPad Prism based on a t-test and analysis of variance (ANOVA), followed by Turkey’s multiple comparisons in stressed and non-stressed mice. Each data point represents the mean ± SD of two combined experiments (n = 5–6 mice per experiment). * denotes statistical difference (*P ≤ .05, **.01, ***.001, ****.0001).
Exposure of CD4+ T cells to noradrenaline alters the production of cytokines in mice during C. muridarum infection
This specific experiment was to determine if synthetic norepinephrine (NE) representing noradrenaline impacts the viability and cytokine production ability of CD4+ T cells during proliferation in vitro. Cells stimulated with Con A showed short bars representing dead cells (Fig. 3A). On the other hand, supplementation of synthetic NE to proliferating T cells results in longer red bars (dead cells) suggesting that the synthetic stress hormone is toxic to immune cells to lower the number of viable cells (Fig. 3B). Similarly, supplementation of synthetic NE resulted in a slight decrease in the viability of CD8 + T cells (Fig. 3C) compared to control cells (Fig. 3D).
Viability percentage of immune cells proliferating in 24 h (A) CD4+ T cells + concanavalin A (con A), (B) CD4+ T cells + synthetic NE, (C) CD8 + T cells + NE, (D) cells only. Vi-CELL BLU cell analyzer using the trypan blue dye exclusion method was used. The green and brown bars represent the percentage of live and dead cells.
Another experiment examined whether synthetic NE contributes to decreased cytokine production of proliferating T cells of stressed or non-stressed mice during C. muridarum genital infection. Exposure of T cells stimulated with LPS increased the secretion of IL-1β (Fig. 4A), whereas the addition of NE in the presence or absence of LPS to proliferating T cells resulted in reduced production of IL-1β. As shown in Fig. 4B, the production of TNF-α was substantially increased due to exposure to LPS, whereas exposure to NE resulted in a significantly reduced production of TNF-α (P < .05). Interestingly, the production of IFN-γ in CD4+ T cells with Con A stimulation with/without infection was substantially increased due to exposure to stress and genital infection, but the production was TNF-α slightly reduced by NE supplement (Fig. 4C). The production of IL-10 in CD4+ T cells was substantially increased due to exposure to stress and infection but significantly reduced its production during infection and the presence of NE (Fig. 4D). Moreover, C. muridarum-infected stressed mice showed a slightly increased production of cytokines compared to non-infected mice. The data indicate that synthetic NE affects the production of signature cytokines during C. muridarum genital infection.
Relative production of cytokines in T cells of mice stressed or non-stressed with cold water with/without C. muridarum genital infection. The levels of cytokines (A) Inteleukin-1β, (B) tumor necrosis factor-alpha (TNF-α), (C) interleukin-10, and (D) interferon-gamma (IFN-γ) were measured by using ELISA. Abbreviations: Con A = Concanavalin A, NE = synthetic NE. Statistical differences between treatments were evaluated using GraphPad Prism based on a t-test and analysis of variance (ANOVA), followed by Turkey’s multiple comparisons in stressed and non-stressed mice. Each point value represents the means ± SD of 2 or 3 wells (n = 5 or 6 mice for each group of experiments).
Deficiency of β2-AR restores the secretion of protective cytokines during chlamydia genital infection
Cytokine production in T cells of stressed and non-stressed of both β2-AR KO and WT C57BL/6J was evaluated using ELISA. We aimed to evaluate the secretion of IFN-γ, IL-4, and IL-10 by T cells. As shown in Fig. 5A, the production of IFN-γ by CD4+ T cells in stressed β2-AR KO was significantly increased compared to stressed WT (P < .01). Furthermore, the production of IFN-γ by non-stressed β2-AR KO and non-stressed WT C57BL/6J is almost equal but showed a statistically significant increase over the stressed WT (P < .001). The production of IL-4 by stressed β2-AR KO and non-stressed WT C57BL/6J showed a statistically significant increase over the non-stressed WT (P < .05). However, no significant difference in the production of IL-4 between stressed β2-AR KO and WT was obtained (Fig. 5B). The production of IL-10 by stressed and non-stressed β2-AR KO showed a statistically significant decrease over the stressed and non-stressed WT (P < .01) Furthermore, a significant difference in the production of IL-10 between stressed and non-stressed WT was obtained (P < .001) (Fig. 5C). The production of IFN-γ by CD8 + T cells of β2-AR KO was significantly increased compared to stressed WT, indicating the restoration of IFN-γ production (P < .01) (Fig. 5D). Overall, the data indicate the restoration of IFN-γ production by CD4+ and CD + 8 T cells and decreased production of IL-10 in contrast to a slight change in IL-4 production by CD4+ T cells in the β2-AR deficient mice.
Production of cytokines by T cells of stressed and non-stressed beta2-adrenergic receptor knockout (β-AR) and WT mice during C. muridarum genital infection. (A) Interferongamma (IFN-γ), (B) IL-4, (C) IL-10 by CD4+ T cells, and (D) IFN-γ by CD8 + T cells. Each point value represents the means ± SD of 2 or 3 experiments (n = 5 or 6 mice for each group of experiments). Statistical differences between treatments were evaluated using GraphPad Prism based on a t-test and analysis of variance (ANOVA), followed by Turkey’s multiple comparisons in stressed and nonstressed mice. * Denotes significant statistical differences between the experimental groups at the level of (P ≤ .05, .01).
Differential proinflammatory cytokine productions in bone marrow-derived macrophages and dendritic cells during C. muridarum genital infection
BMDCs were harvested from the femur and tibia of β2-AR KO and WT stressed and non-stressed mice during C. muridarum genital infection. Monocytes 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 LPS for 24 h.
As shown in Fig. 6A, no statistically significant difference in the production of IL-6 in matured macrophages was observed between treatment groups. A similar IL-6 production pattern was observed in mature dendritic cells (DCs) as well. The results suggest that the presence or absence of β2-AR resulted in little or no difference in IL-6 production in macrophages and dendritic cells. The production of TNF-α in macrophages as shown in Fig. 6B was substantially increased in non-stressed β2-AR KO compared to the stressed β2-AR and WT (P < .05). Furthermore, the absence of β2-AR resulted in no statistically significant production of TNF-α (P < .01) compared to non-stressed WT indicating that β2-AR deficiency showed no impact on TNF-α production. As shown in Fig. 6C, the production of TNF-α in DCs was considerably similar to the production pattern observed in macrophages (P < .01). The production of IL-12 in bone marrow-derived DCs as shown in Fig. 6D was substantially increased in stressed β2-AR KO compared to the other (P < .0001). The data indicate that β2-AR may play an important role in modulating DC function as an essential regulator of the immune system during chlamydia infection.
![Production of cytokines by bone marrow-derived macrophages and dendritic cells of stressed and non-stressed beta2-adrenergic receptor knockout (β2-AR) and WT mice during C. muridarum genital infection. (A) Interleukine-6, (B) tumor necrosis factor-a [TNF-in macrophages and (C) TNF-α], and (D) interleukin-12 by dendritic cells. Each point value represents the means ± SD of 2 or 3 experiments (n = 5 or 6 mice for each group of experiments). Statistical differences between treatments were evaluated using GraphPad Prism based on a t-test and analysis of variance (ANOVA), followed by Turkey’s multiple comparisons in stressed and non-stressed mice. * denotes significant statistical differences between the experimental groups at the level of (P ≤ .05, .01).](https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/femspd/82/10.1093_femspd_ftae029/1/m_ftae029fig6.jpeg?Expires=1747892669&Signature=NaRVxwctb3pf9T-BLvx8fRHoYg3KJZaSJxk2ozSU9cZT4ImoAGjFlLHfqWiasR~fsu5zZ5m0zpfGcm9krvwO3TCCHX26B7MnwlMLZvrc1ge9nVCbNHAdvWSiFscbEPxifh-9nftHP4SXs2IvZ3YdDiJMWqnOjvMamhvubHzpA26Y0awUzQjdW0yEeGMlGpUR6iR5hz94W9FJr9Ocq94dixKrWw23Z6i-~2qsuvvBfV0vYs9xNi~z9uN4Myvb1QpLpXUkQFWsgEk6OXJ0RMhwTEI0ItwWhXVZVf-nWA4K-uMT7bUGxN9SUyYhw-JFpMg3rTipLB5p0KiHkYb83gNDeQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Production of cytokines by bone marrow-derived macrophages and dendritic cells of stressed and non-stressed beta2-adrenergic receptor knockout (β2-AR) and WT mice during C. muridarum genital infection. (A) Interleukine-6, (B) tumor necrosis factor-a [TNF-in macrophages and (C) TNF-α], and (D) interleukin-12 by dendritic cells. Each point value represents the means ± SD of 2 or 3 experiments (n = 5 or 6 mice for each group of experiments). Statistical differences between treatments were evaluated using GraphPad Prism based on a t-test and analysis of variance (ANOVA), followed by Turkey’s multiple comparisons in stressed and non-stressed mice. * denotes significant statistical differences between the experimental groups at the level of (P ≤ .05, .01).
The deficiency of β2-AR increases the expression of surface markers of CD4+ T cells in stressed mice during chlamydia genital infection
We tested whether the absence of β2-AR in CD4+ T cells and DCs of stressed modulates the surface marker expression during chlamydia genital infection. We hypothesized that the absence of stressed mice modulates the expression of surface markers of splenic CD4+ T cells during chlamydia genital infection. Cells were gated with CD3 markers (Fig. 7A; a, d, g, i) and plotted for CD4 positive cells (Fig. 7A; b, e, h, k) and then analyzed for memory and effectors T cells (Fig. 7A; c, f, i, l). Data in Fig. 7A show that the CD4 (16.1 vs. 17.3%) T cells were not changed in number, but an increase in memory CD44+ CD62L+ (21.5 vs. 11.3%) (Fig. 7A; b, e) T cells was observed. Data also show that the stressed WT memory CD44 + CD62L + T cells decreased (2.71 vs. 15.6%) (Fig. 7A; i, l), which correlates with lower CD4 (9.69 vs. 19.2%) (Fig. 7A; h, k) in stressed WT compared to non-stressed WT mice. Data also indicate an increase in memory CD44 + CD62L+ (21.5 vs. 11.3%) (Fig. 7A; c, f), while stressed WT decreased (2.71 vs. 15.6%) (Fig. 7A; i, l). The data indicate that CD4+ T cell memory phenotypes (CD44 + and CD62L+) increased ~2-fold in stressed β2-AR KO compared to stressed WT. In contrast, stressed WT mice showed a 3-fold increase in expression of effector T cells (CD44 + and CD62L−) phenotype and a 7-fold and 2-fold increase in memory and naïve T cell phenotypes. The differential expression of T cell phenotypes in stressed and non-stressed β2-AR KO and WT suggests that it can enhance or suppress the CD4+ T cells’ function during chlamydia genital infection. Thus, further mechanistic studies are needed to define the specific activities of β2-AR on CD4+ T cells in our murine stress model.
Comparison of CD4 T cell phenotypes in cold-stressed β-AR KO and WT mice during C. muridarum genital mice. The expression of effector (CD44 + CD62L−), memory (CD44 + CD62L+), and naïve (CD44− CD62L+) CD4+ T cell surface isolated from different treatment groups is shown in percentiles on the right end of the panels. Stressed β2-AR KO, (a–c) non-stressed β2-AR KO, (d–f) stressed WT (g–i) and non-stressed WT (j–l). Cells were gated with CD3 markers and plotted for CD4 positive cells, which were then analyzed for memory and effectors T cells.
To clarify how the absence of β2-AR affects the function of BMDCs in our murine stress model, we assessed the expression of surface markers and co-stimulatory molecules in matured BMDCs during C. muridarum genital infection. Cells were gated with CD11c (not shown) and plotted for co-stimulatory molecules (CD40, CD80, and CD86) as histograms. Our results in Fig. 7B show enhanced surface expression of CD40 (23.9%), CD80 (8.47%), and CD86 (9.93%) in the DCs population (Fig. 7B; a, e, i) of β2-AR KO compared to CD40 (6.83%), CD80 (3.51%), and CD86 (4.54%) of non-stressed β2-AR KO mice (Fig. 7B; b, f, j). The increase in expression was 2-fold or higher in the stressed β2-AR KO compared to non-stressed β2-AR KO mice (Fig. 7B; c, g, k). In contrast, the expressions of CD40 and CD80 in stressed and non-stressed WT showed no major difference (Fig. 7B; d, h, l). The CD86 expression in the DCs population was lower in stressed and non-stressed β2-AR KO and WT-stressed compared to WT non-stressed mice. The results suggest that stress enhances or brings the expression of the co-stimulators in DCs to the non-stress level of expression except CD86. This chain activation may result in the upregulation of antigen presentation and thus effectively activating CD4+ T cells, strengthening the DC and CD4+ T cell pathway to enhance IFN-γ secreted by CD4+ T cells so that the immune responses become intensified in the clearance of C. muridarum from the genital tract.
Comparison of the expression of co-stimulatory molecules on dendritic cells of cold-stressed βAR KO and WT mice during C. muridarum genital mice. The expression of co-stimulatory CD40 (a–d), CD80 (e–h), and CD86 (i–l) molecules. Bone marrow-derived dendritic cells were isolated from different treatment groups, as shown in percentiles on the right end of the panels. Stressed β2-AR KO (a, e, i), non-stressed β2-AR KO (b, f, j), stressed WT (c, g, k), and non-stressed (d, h, l).
Discussion and conclusion
Previous studies in our lab show that β2-AR KO subsets play important roles in modulating the immune response when triggered through stress hormones or catecholamines (Goyarts et al. 2008, Mohammadpour et al. 2019). Although stress is implicated as a risk factor for various infections, the mechanisms of stress concerning chlamydia genital infection are unknown. We have previously shown that exposure of mice to CIS results in (i) increased susceptibility to C. muridarum genital infection and (ii) increased plasma noradrenaline or adrenaline level as previously determined by ELISA (Guereschi et al. 2013), but the mechanism(s) is not explored.
The significance of the present study is expanding the mounting evidence that stress increases susceptibility to infections by modulating the immune system in animal models, which may be applicable in humans. β2-AR is commonly expressed on the surface of immune cells, including Th1, and impairs Th1 differentiation and function, leading to inhibition of cell proliferation and decreased production of IL-12 and IFN-γ (Guereschi et al. 2013). Because we were determined to use the β2-AR KO and WT C57BL/6J mice for infection and immune response analysis, we first tested whether the β2-AR and the WT C57BL/6J have similar physiological conditions to compare pathogenesis, immunity, histopathology, and infertility. Weight loss is an important indicator of the difference between treated and untreated groups of animals. Thus, this study demonstrated that the β2-AR KO and WT C57BL/J6 mice have almost the same body and spleen weights during stress. Moreover, gene expression of β2-AR was not detected in β2-AR KO T cells. Furthermore, our KO results are consistent with β2-AR KO model organisms previously reported (Sanders 2012, Lorton and Bellinger 2015).
Our mouse model has unveiled the course of infection as Fig. 2 illustrates that the intensity of C. muridarum infection was notably lower in the β2-AR KO compared to the WT, implying the involvement of β2-AR in the pathogenesis of C. muridarum genital infection during stressful conditions. These findings align with the principle that the sympathetic nervous system communicates with the immune system through NE binding to β2-AR, a receptor abundantly expressed in immune cells, including T cells (Gupta et al. 2009, Murthy et al. 2011). This insight is a significant contribution to our understanding of immune response modulation.
We predict that producing catecholamines, which may play a critical role in modulating the immune system, leads to increased intensity of C. muridarum genital infection (Aviles et al. 2004, Belay and Woart 2013). The interaction of stress and β2-AR is relevant to an investigation in our model. We have shown that a supplement of synthetic NE in vitro proliferation of T cells in the presence of Con A exerts an immunosuppressive effect on splenic T cell cytokine production. Further, our results show that the viability of CD4+ and CD8 + T cells treated with NE was decreased, indicating NE has a suppressive effect on immune cells.
The production of key cytokines in differentiated BMDCs stimulated by LPS was tested. A marked increase of TNF-α production was observed in bone marrow-derived and splenic macrophages of stressed infected mice compared to non-stressed mice, demonstrating that CIS influences the gene expression and production of cytokines in macrophages during C. muridarum infection.
The present data suggest that β2-AR signaling may play an important role in modulating DC function as an essential regulator of the immune system during chlamydia infection. In previous studies, enhanced IL-1β, TNF-α, and IL-6 productions in stressed mice were observed (Belay and Woart 2013) with the possibility that catecholamines may enhance IL-1β, TNF-α, to promote detrimental effects of proinflammatory cytokines. Previous studies showed that altered macrophages and dysregulated macrophage function may well contribute to the generalized suppression of the immune response in the host as described (Chen et al. 2003). Although produced by macrophages, DCs, and CD8+ T cells, it has been demonstrated that TNF-α contributes significantly to upper genital tract pathology in mice (Murthy et al. 2011).
To determine whether synthetic NE inhibits T cells, we evaluated the proliferation of T cells supplemented with synthetic NE in vitro and obtained different cytokine production profiles in vitro and in vivo. We hypothesized that noradrenaline, through binding to β2-AR expressed on immune cells, leads to decreased resistance to chlamydia genital infection. We questioned whether mice deficient in adrenergic receptors would develop a reduced infection. Our results appear to be substantiated by documented studies that sympathetic nerves in the lymphoid organs release the stress hormone noradrenaline, which stimulates the β2-AR expressed on CD4+, B cells, macrophages, and dendritic cells, leading to modulation of the immune system (Shimizu et al. 1996). Interestingly, our results also showed that NE in vitro significantly alters the production of different cytokines, possibly exerting an immunosuppressive effect on the proliferation of T cells. We believe that the increased release of cytokines from T cells of stressed mice compared to naïve T cells is a result of combined influences such as other stress hormones compared to the action of noradrenaline.
It is known that CD40, CD80, and CD86 in DCs regulate the Th1/Th2 cytokine production. When CD80 and CD86 are expressed on DCs, they are known to bind CD28 on the T cell for activation. β2-AR may suppress DC functions by inhibiting co-stimulatory molecules like CD80, CD86, and CD40 and molecules and accompanying higher production of IL-12. The elevated expression of DC40 in stressed β2-AR KO was associated with increased production of IL-12 in DCs (Fig. 5D), indicating that IL-12 may promote IFN-γ production by T cells for clearance of C. muridarum. The results suggest that stress enhances or brings the expression of the co-stimulators in DCs to the non-stress level except CD86. β2-AR may suppress DC functions by inhibiting co-stimulatory molecules like CD80, CD86, and CD40 and molecules and accompanying higher production of IL-12. Overall, this chain activation may result in the upregulation of antigen presentation and thus effectively activating CD4+ T cells, strengthening the DC and CD4+ T cell pathway to enhance IFN-γ secreted by CD4+ T cells so that the immune responses become intensified in the clearance of C. muridarum from the genital tract.
Several studies of the mouse immune system have led to significant advances in our knowledge of the human immune system. While we agree that no model is perfect, extensive literature presents little evidence suggesting that immunological findings made using C. muridarum genital infection in mice are vastly different from those with C. trachomatis infections in humans. The closeness of the mouse and human serovars has been described. Thus, the results of our studies in mice might extend to what happens in humans who are stressed and exposed to C. trachomatis, which could explain the increased incidence of chlamydial genital infection in some populations.
This study may have some limitations. Our lack of a flow cytometry machine is a major impediment to confirming the purity of immune cells such as naïve, memory, or effector cells. Significant effort was taken to obtain larger cells (5 × 106/ml) to proliferate in vitro. The starting number of cells was sufficient to isolate RNA and collect culture supernatants for ELISA.
This study has been innovative and will continue developing new approaches that will allow us to explore more underlying mechanistic questions using a novel β2-AR KO mouse model compared to WT for the first time to address our understanding of the effect of stress on chlamydia infection. It is innovative in developing and utilizing a CIS model for studying C. muridarum genital infection immune response, combined with pathological analysis in mice to provide insight into the underlying mechanisms of human chlamydia infection. The utilization of the β2-AR KO study may provide good evidence that the interaction of noradrenaline and β2-AR could be restricted in maintaining a normal level of adaptive immunity against C. muridarum in the animal stress model. Thus, understanding and defining mechanisms linking neuroendocrine and immune function during chlamydial genital infection is a novel approach potentially significant to developing new strategies for preventing and treating human chlamydial genital disease. Future mechanistic studies will focus on β2-AR signing pathways involved in regulating the immune response of chlamydia genital infection.
Although the findings in vivo and in vivo are difficult to compare, it is important to note that the effects observed in vivo could result from a combined influence from other hormones such as corticosteroids. Compared to the in vitro action of synthetic NE, several cellular and molecular events and mechanisms, such as coordination with different signaling pathways and/or transcription factors that occur in vivo may influence the level of cytokine production.
Even though C. trachomatis is the causative agent of chlamydia in humans, we used the mouse agent, C. muridarum, in the present study for the following reasons. Studies show that inoculation of C. trachomatis in mice does not cause productive infection, nor does it cause reproductive pathologies as observed following C. muridarum infections in mice (Darville and Hiltke 2010). Furthermore, C. trachomatis in mice may not directly mimic C. trachomatis infections in humans because C. trachomatis and C. muridarum have evolved host-specific mechanisms for IFN-γ evasion (tryptophan synthesizes and GTPase, respectively) (Nelson et al. 2005).
In summary, a reproducible mouse model is developed to study chlamydia genital infection. The viability of immune cells treated with synthetic NE is significantly reduced compared to cells treated with LPS or Con A. Secretion of proinflammatory cytokines was higher in stressed β2-AR KO than WT. Stressed β2-AR KO mice displayed increased production of protective cytokines compared to stressed WT mice, indicating the absence of β2-AR leads to the functional restoration of protective immune cells. Higher surface markers of CD40 and CD80 in CD11c of stressed mice compared to the non-stressed treatment groups; however, no difference in the expression of CD86 was observed. Higher surface markers of CD4 T cells of stressed mice were observed compared to the non-stressed treatment groups. Our results indicate that CIS may influence the role of dendritic and T cells due to the action of noradrenaline production. Overall, the loss of β2-AR restores the protective immune system over the suppressive immune system of CIS.
In conclusion, our findings indicate a role of β2-AR in enhancing chlamydial infection during stressful conditions; however, additional studies are necessary to determine a mechanism(s) by which β2-AR stimulation enhances chlamydial infection and the anti-inflammatory response in the stress mouse model. The present finding, taken together with previous observations (Belay et al. 2017, 2020) indicates that cold-water-induced stress increases the intensity of chlamydia genital infection in mice. In the long term, our study will employ the cold-water-induced stress in a β2-AR KO mouse model, and the hypothesis to be tested is cold-water-induced stress leads to noradrenaline modulation of the immune response against C. muridarum genital infection by enhancing the production of immunopathogenic cytokines that result in disease sequelae.
Acknowledgment
We are grateful to Dr. Joseph Igiesteme, who worked at Morehouse School of Medicine and the Centers for Disease Control and Prevention (CDC), for his expertise and consultation in the Chlamydia project and for providing us with Chlamydia stock cultures throughout several years.
Author contributions
Tesfaye Belay (Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing), Rajnish Sahu (Conceptualization, Data curation, Investigation, Methodology), Vida Dennis (Conceptualization, Formal analysis, Investigation, Validation), Kaitlyn Cook (Data curation, Investigation, Methodology, Software), Alexis Ray (Data curation, Investigation, Methodology, Validation, Writing – original draft), Danielle Baker (Data curation, Investigation, Methodology, Validation, Visualization), Ashlei Kelly (Investigation, Methodology, Software, Supervision, Validation), and Nathasha Woart (Data curation, Investigation, Methodology, Software, Validation, Visualization)
Conflict of interest
None declared.
Funding
This work was supported by the National Institution of Health (NIH) grant NIH grant number 1R15AI174169-01A1 awarded to Bluefield State University and the grant # P20GM103434 to the West Virginia IDeA Network Biomedical Research Excellence (WV-INBRE).
