MAP4K4 promotes Chlamydia infectivity
In our previous studies, C. trachomatis regulated host cell antiapoptosis by upregulating MAP4K4(14). Many studies have shown that Chlamydia inhibits the apoptosis of host cells to maintain their survival(17). To further explore the effect of MAP4K4 on the intracellular survival of Chlamydia in persistent infection, HeLa cells were transfected with specific siRNA and then infected with C. trachomatis E (MOI=1) for 24 hpi. By detecting inclusion bodies, we found that the IFU of C. trachomatis in the Ct-siMAP4K4 group was significantly lower than that in the control group (Figure 1A, P<0.05).
It has been reported that macrophages can be used as carriers of Chlamydia transmission in vivo(28, 29). The genital tracts of mice infected with Chlamydia muridarum are a classic model for investigating the pathogenesis of the human pathogen C. trachomatis. Therefore, we constructed lentivirus vectors (LV10N-siMAP4K4 and LV10N-siNC) to explore the effect of MAP4K4 on C. muridarum infectivity in vitro and in vivo. In C. muridarum-infected RAW 264.7 cells, qRT-PCR results showed that MAP4K4 expression was significantly upregulated in the infection group (Figure 1B, P<0.05). We verified the effectiveness of lentivirus LV10N-siMAP4K4 and negative control infection. The results showed that lentivirus-infected RAW 264.7 cells successfully expressed the mCherry red fluorescence gene vector (Figure 1C). The expression of MAP4K4 in the LV10N-siMAP4K4 group was significantly downregulated compared with that in the negative infection group (Figure 1D, P<0.05). The cell density and morphology of RAW 264.7 cells were normal under a white light microscope (Figure 1E). By counting inclusion bodies of C. muridarum in each experimental group, it was found that the LV10N-siMAP4K4 group significantly inhibited the infectivity of C. muridarum compared with the negative control group (Figure 1F, P<0.01).
MAP4K4 is upregulated in Chlamydia-infected BALB/c mice
After 6-week-old mice were infected with C. muridarum through the vagina, some mice were sacrificed 7 days later. The spleen, liver, kidney, and uterus tissues of mice were isolated, and the total RNA of the tissues was extracted. Then, qRT-PCR was used to detect that MAP4K4 was significantly upregulated in the spleen, kidney, and uterus tissues of infected mice compared to the control group (Figure 2A, P<0.05). The spleen cells were isolated and cultured, and IFA was used to detect the expression of mCherry in the vector. The results showed that mCherry was successfully expressed in lentivirus-infected spleen cells (Figure 2B). Meanwhile, compared with the Cm-LV10N-siNC group, the expression of MAP4K4 in the spleen, liver, kidney, and uterus of the Cm-LV10N-siMAP4K4 group was significantly downregulated (Figure 2B, P<0.05).
In addition, the remaining mice were sacrificed after 60 days of infection, and the expression of MAP4K4 in uterine tissues was detected by immunohistochemistry. The results showed that the brown staining of uterus sections in the infection group was deeper than that in the SPG control group, indicating a higher expression of MAP4K4 in the infection group (Figure 2C). Compared with the Cm-LV10N-siNC group, the expression of MAP4K4 in the uterus of the Cm-LV10N-siMAP4K4 group was downregulated (Figure 2D).
MAP4K4 is involved in Chlamydia clearance in the genital tract of BALB/c mice
In the first test (D3) after infection, exfoliated Chlamydia was detected in the vaginal secretions of all infected mice. On Day 6 postinfection, the Chlamydia load in each group reached the highest level and then began to decrease until Chlamydia shedding in each group was zero (Table 1). Then, IFA analysis was performed on Chlamydia load in vaginal secretions of the Cm-LV10N-siNC and Cm-LV10N-siMAP4K4 groups. The results showed that on day 6 postinfection, the bacterial loads of Cm-LV10N-siNC and Cm-LV10N-siMAP4K4 were 40.29±1.53 × 105 and 26.57±1.53 × 105, respectively. MAP4K4 silencing significantly reduced the bacterial load of the vagina (Figure 3). These results suggest that MAP4K4 loss is beneficial to the clearance of Chlamydia in the genital tract of mice.
MAP4K4 inhibits the secretion of proinflammatory cytokines both in vitro and in vivo
It has been shown that some cytokines such as IFN-γ, IL-12, IL-6, and TNF-α secreted by macrophages have phagocytosis, bactericidal, antigen presentation, and promotion of Th1 immunity(30-33). To further explore the effect of MAP4K4 on inducing cytokine expression in Chlamydia-infected RAW 264.7 cells, we examined 32 potential cytokines and chemokines simultaneously using a mouse cytokine array microarray. Compared with the control infection group, the Cm+siMAP4K4 group showed a trend of upregulation of G-CSF, IL-12, and IL-6, and IL-6 was significantly upregulated by four times (Figure 4A-B, P<0.05). In GO analysis, it was found that differentially expressed cytokines and chemokines were enriched in the regulation of the inflammatory response, JAK-STAT pathway, and leukocyte-mediated immunity (Figure 4C). Therefore, we hypothesized that G-CSF, in combination with IL-6 and IL-12, might regulate the intracellular survival of Chlamydia, but the molecular mechanisms remain to be further studied.
In vivo, IFN-γ produced by T cells is considered protective against Chlamydia infection(34). However, Chlamydia-induced proinflammatory cytokines are responsible for tissue damage(35). In particular, TNF-α was reported to be involved in the pathogenesis of upper genital tract pathology post-infection(36). Therefore, the levels of TNF-α and IFN-γ cytokines in genital tract secretions of mice were detected by ELISA. The results showed that the TNF-α and IFN-γ levels in the infection groups were significantly higher than those in the SPG control group on Day 3, Day 6, Day 9, Day 12, and Day 15. The levels of TNF-α and IFN-γ in the infection groups peaked on day 6. By comparing the cytokine secretion levels of the Cm+siNC and Cm+siMAP4K4 groups, we found that the levels of TNF-α and IFN-γ in the Cm+siMAP4K4 group were significantly higher than those in the Cm+siNC group (Figure 4D-E, P<0.05). These results suggest that silencing MAP4K4 may be beneficial to Chlamydia clearance in the genital tract of mice by promoting the secretion of the proinflammatory cytokines IL-6, IL-12, TNF-α, and IFN-γ in the early stage of infection.
MAP4K4 regulates T cell immunity early and reduces IFN-γ and TNF-α secretion
Studies have shown that the clearance of Chlamydia in the genital tract of mice is closely related to T cell immunity(37, 38). On Day 7, Day 15, and Day 21 postinfection, six mice in each group were sacrificed, and the spleens of mice in each group were removed anatomically and placed into a sterile EP tube for precise weighing. The results showed that on day 15, the spleen weight of mice in the Cm+siMAP4K4 group increased compared with that in the Cm+siNC group (Figure 5A, P<0.05). Then, spleen cells were prepared and cultured in each group. After inactivated C. muridarum (106 IFU) was used to stimulate spleen cells, CCK-8 analysis showed that the stimulation indexes (SI) of spleen cells in the SPG, Cm, Cm+siNC, Cm+siMAP4K4, and ConA groups were 0.971±0.01, 1.83±0.08, 1.73±0.08, 2.78±0.16, and 3.16±0.09, respectively. The SI of the infection group was higher than that of the SPG control group, and that of the Cm+siMAP4K4 group was higher than that of the Cm+siNC group (Figure 5B, P<0.01).
Next, we analyzed the T cell response in infected BALB/c mice, and flow cytometry was used to analyze the proportion of CD4+ and CD8+ T cells in each group. On day 7 postinfection, the proportion of CD4+ and CD8+ T cells in the infection group was increased compared with that in the Cm+siNC group, and the proportion of CD8+ T cells was increased in the Cm+siMAP4K4 group (Figure 5C, P<0.05). On Day 15 postinfection, the ratio of CD4+/CD8+ cells in the SPG, Cm, Cm+siNC, and Cm+siMAP4K4 groups was 1.64±0.20, 1.84±0.04, 1.80±0.07, and 2.26±0.01, respectively. The ratio of the Cm+siMAP4K4 group was significantly higher than that of the Cm+siNC group (Figure 5D-E, P<0.05). However, on day 21 postinfection, there was no difference in the proportion of CD4+ and CD8+ T cells among all groups. The results showed that genital tract-infected mice could induce T cell immunity, and MAP4K4 silencing could activate T cell immunity early in the anti-Chlamydia reaction.
The immune response mediated by CD4+ Th1 cells and IFN-γ secretion is the primary mechanism of clearing Chlamydia infection(39, 40). Flow cytometry showed that CD8+/IFN-γ, CD8+/TNF-α, CD4+/IFN-γ, and CD4+/TNF-α levels were significantly increased in the infected group. Compared with those in the Cm+siNC group, the levels of CD8+/IFN-γ, CD8+/TNF-α, and CD4+/TNF-α in the Cm+siMAP4K4 group were significantly increased, while the levels of CD8+/IL-4 and CD4+/IL-4 were not significantly changed (Figure 5F-K, P<0.05). In the early stage of the anti-Chlamydia response, the results showed that IFN-γ instead of IL-4 participates in the immune response, and the silencing of MAP4K4 could further promote CD4+ and CD8+ T cells to produce more IFN-γ and TNF-α to participate in the immune response dominated by Th1 cells.
MAP4K4 correlates with Chlamydia-mediated upper genital tract pathology in mice
Uterine tissue samples of mice from each group (SPG, Cm, Cm+siNC, and Cm+siMAP4K4) were collected, and the pathological changes in the uterus were evaluated by H&E staining. On Day 15 postinfection, total uterine horn enlargement was observed in the infected group. H&E staining sections showed uterine cavity enlargement and infiltration of inflammatory cells dominated by neutrophils in the cavity, which indicated that acute inflammation occurred in the genital tract. Among them, the Cm+siMAP4K4 group showed more inflammatory cell infiltration than the Cm+siNC group (Figure 6A).
On day 60 postinfection, pathological changes in the upper genital tract of each group were observed by the naked eye. The incidence of hydrosalpinx in mice was 75% (n=4) in the Cm+siNC and Cm+siMAP4K4 groups and 100% in the infection group. However, the severity of hydrosalpinx was different, and the scoring analysis was conducted according to the gross hydrosalpinx scoring rules. The results were 0.00±0.00 (SPG), 3.50±0.58 (Cm), 3.00±2.16 (Cm+siNC), and 1.50±1.29 (Cm+siMAP4K4) (Figure 6B-C, P<0.001).
Although silencing MAP4K4 tended to reduce hydrosalpinx scores, there was no significant difference. From another perspective, tubal lumen dilation and inflammatory cell infiltration scores were determined to analyze the pathological conditions of each group. The results showed that tubal lumen dilation occurred in all infection groups, and inflammatory cells mainly infiltrated mononuclear cells (Figure 6D). The score of the oviduct dilation degree of mice in the Cm+siMAP4K4 group was significantly lower than that in the Cm+siNC group (Figure 6E-F, P<0.05). Together, these data suggest that MAP4K4 is beneficial to the development of chronic oviduct lesions.