Shift work influences the outcomes of Chlamydia infection and pathogenesis

Abstract

Shift work, performed by approximately 21 million Americans, is irregular or unusual work schedule hours occurring after 6:00 pm. Shift work has been shown to disrupt circadian rhythms and is associated with several adverse health outcomes and chronic diseases such as cancer, gastrointestinal and psychiatric diseases and disorders. It is unclear if shift work influences the complications associated with certain infectious agents, such as pelvic inflammatory disease, ectopic pregnancy and tubal factor infertility resulting from genital chlamydial infection. We used an Environmental circadian disruption (ECD) model mimicking circadian disruption occurring during shift work, where mice had a 6-h advance in the normal light/dark cycle (LD) every week for a month. Control group mice were housed under normal 12/12 LD cycle. Our hypothesis was that compared to controls, mice that had their circadian rhythms disrupted in this ECD model will have a higher Chlamydia load, more pathology and decreased fertility rate following Chlamydia infection. Results showed that, compared to controls, mice that had their circadian rhythms disrupted (ECD) had higher Chlamydia loads, more tissue alterations or lesions, and lower fertility rate associated with chlamydial infection. Also, infected ECD mice elicited higher proinflammatory cytokines compared to mice under normal 12/12 LD cycle. These results imply that there might be an association between shift work and the increased likelihood of developing more severe disease from Chlamydia infection.

Figure 1

Light cycle conditions. (A) Normal light: dark cycle (Control)—mice were housed in cages under normal light: dark cycle (LD) conditions of 12 h light on and 12 h lights out. (B) Environmental circadian disruption (ECD) model—mice were housed in cages and received 6-h advance in light cycle every week for 4 weeks.

Figure 2

Effect of ECD on Chlamydia infectivity. ECD and control mice (n = 12 per group) were infected with C. muridarum at ZT3. Data was analyzed using two-way repeat measure ANOVA and Tukey post hoc test (**p < 0.01).

Figure 3

Effect of ECD on gross pathology and histopathology after Chlamydia infection (n = 6). Gross pathology. (A) Infected ECD mice had more periovarian cysts (white arrow) than control mice. (B) There was moderate periovarian and oviductal inflammation (long arrow), hydrosalpinx (asterisks), and cystic endometrial hyperplasia of the uterus (arrowheads). Hematoxylin and eosin (HE) stain. Bar = 500 µm. (C) Mouse uterus in infected ECD mice, with cystic endometrial hyperplasia (long arrow). Asterisk in uterus lumen. HE stain. Bar = 200 µm. (D) Mouse uterus in infected control mice, with cystic endometrial hyperplasia. Asterisk in uterus lumen containing large numbers of neutrophils. HE stain. Bar = 200 µm. (E) Mouse oviduct in infected ECD mice, with hydrosalpinx and lymphocytic (CD4 positive) inflammation (long arrow). Asterisk in dilated oviduct lumen (ampulla), lined by ciliated cells (arrowheads). Hematoxylin counterstain. Bar = 20 µm.

Figure 4

Severity and distribution of histopathology scores in ECD and control mice after Chlamydia infection. (A) Ovarian inflammation. (B) Ovarian inflammation cell infiltrate. (C) Oviduct inflammation. (D) Oviduct ectasia. (E) Uterine inflammation. (F) Uterine necrosis. Samples were collected 34 days post infection (n = 6).

Figure 5

Effect of ECD on cytokine and chemokine secretion after Chlamydia infection. Cytokine and chemokine concentrations in vaginal lavages collected from ECD and control mice infected at ZT3 were determined (n = 12). (A) TNF-α. (B) IL-1β. (C) IFN-γ. (D) IL-4. (E) IL-10. (F) CXCL1. (G) CCL3. Uninfected and infected ECD and control mice were compared with each other. The data was analyzed using a one-way ANOVA and Tukey post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 6

Effect of ECD on anti-chlamydial antibody secretion after Chlamydia infection. Anti-Chlamydia antibody concentrations were determined in vaginal lavages collected weekly from ECD and control mice (n = 12) infected with C. muridarum at ZT3. (A) IgG. (B) IgG_2C. The data was analysed using a one-way ANOVA and Tukey post hoc test. **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 7

Effect of ECD on fertility after Chlamydia infection. Fertility of ECD and control mice were measured using a fertility assay after infection with C. muridarum at ZT3 (n = 6 per group). The fertility rate was determined by analyzing the number of pups per mouse. The data was analysed using a one-way ANOVA and Tukey post hoc test. *p < 0.05.

Figure 8

Effect of ECD on chlamydial pathogenesis after Chlamydia infection in the early active period. (A) Chlamydia infectivity in ECD and control mice (n = 12 per group) infected at ZT15 was determined. Data was analyzed using a two-way repeat measure ANOVA and Tukey post hoc test. (B) ECD mice had periovarian cysts (next to the ovary, as indicated by the white arrow), while infected control mice did not have periovarian cysts (n = 6 per group). (C) Fertility of ECD and control mice infected with C. muridarum at ZT15 was determined by analyzing the number of pups per mouse (n = 6 per group). The data was analysed using a one-way ANOVA and Tukey post hoc test.