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

Abstract

A murine model to study the effect of cold-induced stress (CIS) on Chlamydia muridarum genital infection and immune response has been developed in our laboratory. Previous results in the lab show that CIS increases the intensity of chlamydia genital infection, but little is known about the effects and mechanisms of CIS on the differentiation and activities of CD4+ 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 are not well defined. In this study, we examined whether CIS modulates the expressions of beta-adrenergic receptor (β-AR), transcription factors, hallmark cytokines of Th1 and Th2, and differentiation of BMDCs during C. muridarum genital infection in the murine model. Our results show that the mRNA level of the beta2-adrenergic receptor (β2-AR) compared to β1-AR and β3-AR was high in the mixed populations of CD4+ T cells and BMDCs. Furthermore, we observed decreased expression of T-bet, low level of Interferon-gamma (IFN-γ) production, increased expression of GATA-3, and Interleukin-4 (IL-4) production in CD4+ T cells of stressed mice. Exposure of BMDCs to Fenoterol, β2-AR agonist, or ICI118,551, β2-AR antagonist, revealed significant β2-AR stimulation or inhibition, respectively, in stressed mice. Moreover, co-culturing of mature BMDCs and naïve CD4+ T cells increased the production of IL-4, IL-10, L-17, and IL-23 cytokines, suggesting that stimulation of β2-AR leads to the increased production of Th2 cytokines. Overall, our results show for the first time that CIS promotes the switching from a Th1 to Th2 cytokine environment. This was evidenced in the murine stress model by the overexpression of GATA-3 concurrent with elevated IL-4 production, reduced T-bet expression, and IFN-γ secretion.

Fig 1. Course of C. muridarum genital infection in stressed and non-stressed mice as measured by quantitation of viable organisms shed from the genital tract.

Each data is mean +/_ SEM of inclusion forming units/milliliter determined from McCoy cell culture representing combined results of two separate experiments (n = 5 mice per group). A statistical difference in counts was observed between stressed and non-stressed mice on day 9 and 15 after infection (p <0.05). Shedding of C. muridarum in non-stress mice or stressed mice after day 28, indicating the resolution of infection.

Fig 2. Gene expression profiles of β-AR) subtypes in CD4+ T cells isolated from the genital tract of stressed and non-stressed mice during C. muridarum genital infection.

Amplification cures and fold-changes of mRNA levels of β2-AR subsets normalized to the housekeeping gene, GADPH, is shown. A) β2-AR, B) β1 –AR, C) β3-AR, D) qPCR products on gel electrophoresis. Abbs: St = stressed, Non-St = none-stressed. Data shown are representative of two or more independent experiments run on different dates.

Fig 3. Gene expression profiles of GATA-3 and T-bet in T cells isolated from the whole or the parts of the genital tract of stressed and non-stressed mice during Chlamydia muridarum genital infection.

Amplification cures and fold-changes of mRNA levels of transcription factors of T cells isolated A) whole genital tract, B) uterus horns, C) oviduct, D) T-bet in T cells of the whole genital tract, E) uterus horns F oviduct), and G) gel electrophoresis of GATA-3 are shown. Abbs: St = stressed, Non-St = none-stressed. Shown data are representatives of two or more independent experiments.

Fig 4. Fold-changes of gene expression of transcription factors and signature cytokines of Th1 or Th2.

A) GATA-3, B) T-bet, C) IL-4) and D) IFN-γ in naïve CD4+ T cells isolated from spleen cells normalized to the internal control gene, GAPDH compared to the non-stressed mice are shown. Data shown 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).

Fig 5. The persistence of the effect of CIS on the modulation of transcription factors and signature cytokines of Th1 and Th2 cells in the genital tract of stressed/non-stressed mice.

Gene expression of A), GATA-3, B) T-bet, IL-4 C), and D) IFN-γ during two or 11-days post-C. muridarum genital infection relative to non-infected mice. Results are mean +/- SEM of two independently performed experiments. *Denotes significant statistical differences between treatment groups at the level of (p < 0.05).

Fig 6

Presence of β2-AR agonist or antagonist on in vitro production of A) IL-4, B) IL-12, and C) IL-23 in naïve CD4 T-cells isolated from stressed non-infected mice. Immature dendritic cell culture was pre-exposed to NE (1 μM), Fenoterol (1 μM) and ICI 118,551 (1 μM) for 1 h before stimulation with LPS (5 μg/mL) for 24 h before RNA isolation. Data shown are a mean +/_ SEM of two or more independent experiments performed on different dates. *Denotes significant statistical differences between treatment groups at the level of (p < 0.05).

Fig 7. Polarization of naïve CD4+ cells in the production of signature cytokines of Th1 and Th2 cells determined by ELISA.

A) IFN-γ, B) IL-4, C) IL-23. For differentiation of Th1 cells, murine IL-12, murine IFN-γ, human IL-2, and murine anti-IL-4 were added to cell cultures in plates precoated with murine anti-CD3 for stimulation. For Th2 cells, murine IL-4, murine anti-IFN-γ and murine and anti-IL-12 were added in plates precoated with murine anti-CD3 for stimulation. The production of cytokines was determined by ELISA. Data shown is a mean +/_ SEM of two or more independent experiments performed on different dates.

Fig 8

Western blot analysis of Protein expression of A) GATA-3, B) β2-AR, GATA-3, STAT 4 & 6, and β-Actin, C) β2-AR intensity, D) GATA-3, STAT4 and STAT6 intensities in CD4+T cells isolated from the genital tract of stressed and non-stressed mice. Amplified Opti-4CN Substrate Kit from Bio-Rad was used to detect protein concentration following the manufacturer’s instructions. Relative protein expression was calculated as described in materials and methods. Data shown are a representative of two or more independent experiments performed on different dates.

Fig 9. Differential cytokine production of immature BMDCs pre-exposed to NE, Fenoterol, or ICI118,551.

A) non-stressed-non-infected (NSNI) or non-stressed-infected mice(NSI); B, C) Stressed-non-infected (SNI) or stressed-infected (SI) mice; to NE, Fenoterol (F), or ICI118,551 results in irregular production IL-12 and IL-10 production. Immature dendritic cell culture was pre-exposed to NE (1 μM), Fenoterol (1 μM) or ICI 118, (1 μM) for 1 h before stimulation with LPS (2.5 μg/mL) for 24 h before RNA isolation. Data shown are a mean +/_ SEM of two or more independent experiments performed on different dates. *Denotes significant statistical differences between treatment groups at the level of (p < 0.05).

Fig 10. Cytokine production kinetics of CD4+T cells and dendritic cells co-cultured in vitro.

CD4+ T cells from stressed and non-stressed mice isolated by negative selection were co-cultured for 48 h. Shown data are mean+/_SEM two or more independent experiments performed on separate dates. *Denotes statistically significant differences between stressed and non-stress mice.

Fig 11. Cystoscope image of functional gene expression and potential regulation during C. muridarum genital infection in mice.

The color of the nodes shows up- or down-regulation of genes in stressed mice during C. muridarum genital infection. Nodes shapes are made to show transcription factors, cell signaling receptors, and cytokines. The edges are colored to show nodes that are affected by up-and down-regulation in stressed mice during C. muridarum genital infection.