3.1 C. psittaci enter hPMNs in a short time successfully
Our research group has previously demonstrated that C. psittaci can successfully survive and proliferate in hPMNs(He et al., 2022). In this study, we focuses on the molecular mechanism of ROS production by C. psittaci infected with hPMNs in a short period. C. psittaci infected hPMNs at different times (0 min, 30 min, 60 min, 90 min, 120 min). Ultrastructure analysis of C. psittaci in hPMNs was conducted by transmission electron microscopy, and C. psittaci could successfully enter hPMNs (Figure.1A). 30 min after infection, EBs were present in the cells as inclusion bodies. The number of EBs in cells increased with the increase of infection time.
In this study, after infecting hPMNs cells with C. psittaci of different MOI for different periods, the changes of C. psittaci at the transcription level were detected by qRT-PCR. To demonstrate the ability of C. psittaci on infect hPMNs. No infection is a negative control, PMA stimulation is a positive control. C. psittaci of MOI (1, 10, 20, 40) were used to infect hPMNs. As shown in Figure.1B, as the MOI of C. psittaci continues to increase, so does the load of C. psittaci. Subsequently, C. psittaci with MOI of 10 was used to infect hPMNs at different times (0 min, 30 min, 60 min, 90 min, 120 min). the expression of C. psittaci could also be detected with the extension of infection time to 120 min after C. psittaci infection(Figure.1C). However, the load of infected C. psittaci in hPMNs did not increase significantly, which may be related to the short infection time and not reaching the reproductive cycle of C. psittaci. These results indicate that C. psittaci enter hPMNs in a short time.
3.2 The survival rate of hPMNs increases after C. psittaci infection.
Our team has previously demonstrated that C. psittaci can successfully infect hPMNs cells and can be produced in vivo (Lee et al., 2017). The survival rate of hPMNs after ingestion of C. psittaci was studied by two-color fluorescence staining (live/dead staining). After staining with SYTO 9 (green) and ethidium bromide (red), living cells show green staining, while dead cells with damaged membranes show red staining. The results showed that the survival rate of hPMNs infected with C. psittaci was not significantly different from that of hPMNs not infected in the short term. However, compared with the positive control group, the survival rate of hPMNs stimulated by PMA after infection with C. psittaci was higher, indicating that C. psittaci infection did not lead to the direct death of hPMNs and was more conducive to its intracellular parasitism (Fig. 2A,B). In the case of PMA stimulation, the toxic effect of hPMNs can be reduced, and the survival time of hPMNs can be prolonged by inhibiting the production of ROS, which is more conducive to the intracellular parasite and survival of C. psittaci.
3.3 C. psittaci infection with hPMNs did not produce significant ROS but inhibited ROS produced by PMA-induced hPMNs
HPMNs have a short life span and can produce ROS in vitro or in vivo after 0 ~ 2 h(Criss and Seifert, 2008). Current studies have shown that chlamydia is resistant to oxidative killing by the host and contributes to its survival by producing small amounts of ROS. Therefore, chlamydia resistance to oxidative killing by host cells is an essential mechanism for achieving immune escape. In order to demonstrate the oxidative stress response after C. psittaci infection with hPMNs, ROS production in hPMNs infected and uninfected with C. psittaci was measured using DCFH-DA with and without PMA. After the hPMNs cells were infected with C. psittaci of different MOI (1, 10, 20, 40) for 60 min, the ROS were detected by flow cytometry and statistically analyzed. As shown in Figure.3A,B,C,D, unstimulated hPMNs did not produce detectable ROS. In contrast, hPMNs exposure to PMA effectively stimulated the production of ROS within 5 min and was significantly higher than that produced by PMA-added hPMNs. HPMNs infected with C. psittaci of different MOI did not produce significant ROS but inhibited the ROS induced by PMA in hPMNs.
In this experiment, with MOI as 10 of C. psittaci infected hPMNs at different times (0 min, 30 min, 60 min, 90 min, 120 min), C. psittaci did not produce obvious ROS after infection with hPMNs (Figure.3E). ROS produced by hPMNs induced by PMA could be inhibited after 120 min, and the inhibition was most apparent at 60 min (Figure.3F). At the same time, fluorescence microscopy was used to detect them in this experiment. HPMNs without C. psittaci infection was the negative control, hPMNs stimulated by PMA were a positive control. When C. psittaci with an MOI of 10 infected hPMNs for 60 min, fluorescence microscopy results with or without PMA showed that C. psittaci infected hPMNs did not produce significant ROS (Figure.3H), showing that the ROS produced by hPMNs caused by inhibition of PMA was consistent with flow cytometry results.
3.4 C. psittaci inhibited ROS production from hPMNs induced by PMA
The ROS release site in hPMN depends on the nature of the stimulus. In general, soluble stimuli release NADPH oxidase targeting ROS into extracellular mediators, while granular stimuli such as OpZ, Neisseria meningitides serogroup B, and other bacteria target enzymes to form phagosomes, where ROS accumulates in the phagocoelum (Naids and Rest, 1991; Steed et al., 1992; Zuo et al., 2013). In this study, NBT was analyzed by a microscope, forming a deep purple precipitate in the presence of superoxide. Compared with control cells (Figure.4A), the positive control cells stimulated by PMA (Figure.4C) had a large amount of dark purple precipitation, while C. psittaci-infected hPMNs had low NBT, and occasional cells with dark purple precipitation were observed (Figure.4B). HPMNs infection by C. psittaci with PMA produced many dark purple precipitates (Fig. 4D). However, ROS production was significantly lower than that of PMA-induced hPMNs, which suggest that C. psittaci inhibits PMA-induced ROS production in hPMNs .
Microscopic NBT analysis is a qualitative analysis usually used in clinical diagnosis. An NBT spectrophotometer was used in this study to obtain quantitative measurements of these results, which determined intracellular and extracellular ROS production(Siemsen et al., 2009). Previous studies have shown that exposure of hPMNs to phagocytic formiformes mobilizes NADPH oxidase reserves in hPMNs, thereby reducing subsequent activation of soluble agonists(JW and Mueller, 2004). Using a similar analysis, this study used PMA as a positive control and found that intracellular phagocytosis of C. psittaci produced few ROS in hPMNs (Figure.4E, F), whereas PMA induced significant levels of ROS. Preinfection of C. psittaci with hPMNs and stimulation with PMA also produced significant ROS (Figure.4G, H), but ROS induced by PMA was slightly reduced compared to 30 min, which seemed to alter the subsequent response of soluble agonist PMA. At the same time, minimal extracellular ROS production was observed in these cells (Figure.4E, G), there was no significant difference between groups, and most ROS were produced intracellularly (Figure.4F, H).
3.5 Inhibiting hPMNs superoxide production is part of inhibiting hPMNs ROS production
Previous studies have suggested that respiratory burst activation coincides with NADPH oxidase assembly, rapidly producing superoxides and other toxic ROS. Therefore, this study detected the change of superoxide in C. psittaci infected with hPMNs with an MOI of 10. The superoxide produced by hPMNs in each group was detected with or without adding PMA. HPMNs without C. psittaci and PMA produced no significant superoxide, while PMA without C. psittaci produced significant superoxide. Similarly, hPMNs produced no significant superoxide in the presence of C. psittaci. In contrast, hPMNs produced superoxide in the presence of C. psittaci and PMA, although in lower amounts than in control without C. psittaci(and lower than in the PMA group). Then, in this experiment, hPMNs and C. psittaci were pre-cultured at different time points to detect the influence of superoxide. HPMNs without C. psittaci and PMA produced no superoxide, while bacteria without PMA produced obvious superoxide (Figure.5). In all experimental groups, the PMA-added samples showed significant superoxide production compared with those without PMA. The superoxide was low but increased and then decreased after the peak at 60 min. After C. psittaci was pre-infected with hPMNs simultaneously, hPMNs produced obvious superoxide when PMA was added. After hPMNs were preincubated with C. psittaci for 60 min, the superoxide produced by PMA was significantly reduced compared with that produced by PMA only (Figure.5). The results showed that inhibiting the production of hPMNs superoxide was a part of inhibiting the production of ROS by hPMNs.
3.6 C. psittaci affects ROS production by affecting the expression of gp91phox and p40phox, which are components of NADPH oxidase
Since the ROS in hPMNs induced by PMA is mainly produced by NADPH oxidase, in order to study the inhibitory mechanism of C. psittaci in producing ROS in hPMNs. This study further explored the molecular mechanism of C. psittaci inhibiting the oxidation of hPMNs by NADPH oxidase. gp91phox and p40phox are essential components of NADPH oxidase. In this study, gp91phox and p40phox expression were detected in separate hPMNs with or without PMA when C. psittaci infected hPMNs with different infection plurals. The results showed no significant changes in gp91phox and p40phox expression of hPMNs infected with different complex C. psittaci for 60 min (Figure.6A,C). After adding PMA, the gp91phox produced by hPMNs was significantly inhibited when the MOI was 10 and 20 compared with that of hPMNs with only PMA and no C. psittaci. There was no significant effect on the expression of p40phox (Fig. 6B,D).
Subsequently, C. psittaci infected hPMNs with MOI 10 at different times (0 min, 30 min, 60 min, 90 min, 120 min) without PMA. The expression of gp91phox decreased after 30 min and 60 min after infection compared with 0 min. while p40phox increased (Figure.6E,G). gp91phox and p40phox produced by hPMNs in the presence of C. psittaci and PMA were significantly reduced compared with those produced by hPMNs only in the presence of PMA, the most obvious at 30 min (Figure.6F,H).
This study used indirect immunofluorescence further to verify changes in NADPH oxidase assembly in hPMNs. HPMNs without C. psittaci infection was the negative control, and hPMNs stimulated by PMA were a positive control. When PMA was added or not, C. psittac infected hPMN at different times (0 min, 30 min, 60 min, 90 min, 120 min) with an MOI of 10. The results showed that gp91phox and p40phox expression were not affected by C. psittaci at different times of C. psittaci infection with hPMNs (Figure.6I,K). However, under PMA stimulation, gp91phox and p40phox expression decreased in hPMNs infected with C. psittaci for 30 and 60 min (Figure.6J,L). Gp91phox is a nuclear membrane protein. Indirect immunofluorescence results showed that PMA can activate NADPH oxidase to increase gp91phox in the nuclear membrane (Figure.6L). These results indicated that C. psittaci could not significantly activate NADPH oxidase in hPMNs, but C. psittaci could inhibit the expression of gp91phox and p40phox proteins, thereby affecting the activation of NADPH oxidase in hPMNs by PMA, leading to ROS production.
3.7 MAPKs pathway is involved in the activation of hPMNs by C. psittaci and the inhibition of ROS production by C. psittaci on PMA-induced hPMNs
Previous studies have shown that the MAPKs signaling pathway plays a vital role in ROS production in other intracellular bacteria. Therefore, to determine whether C. psittaci inhibits PMA-induced hPMNs by generating ROS through MAPKs signaling pathway, this project detected the activation of MAPKs p38 kinase and ERK by Western blotting. C. psittaci with an MOI of 10 infected hPMNs for 0 min, 30 min, 60 min, 90 min, and 120 min. The results showed that C. psittaci could activate p38 and ERK molecules simultaneously and promote their phosphorylation. p38 significantly stimulated hPMNs phosphorylation of p38 60 min after C. psittaci infection, while ERK increased significantly 30 min and 60 min after C. psittaci infection (Figure.7A,B,C).
To explore whether C. psittaci infection of hPMNs affects the expression of gp91phox and p40phox through MAPKs signaling pathway, thus inhibiting the production of ROS. Enhanced phosphorylation of p38 and ERK in C. psittaci-infected hPMNs suggests that the activation of this pathway may be involved in C. psittaci inhibition of hPMNs oxidation. Since other methods, such as siRNA, do not apply to hPMNs, the inhibitor method was used in this study to demonstrate whether the MAKPs signaling pathway plays a role in C. psittaci inhibition of hPMNs production of ROS. hPMNs was pretreated with selective p38 inhibitors (SB203580) and ERK inhibitors (PD98059) to block the phosphorylation of p38 and ERK.
HPMNs cells were pretreated with SB203580 (30 µM) and PD98059 (30 µM) for 60 min and then infected with C. psittaci for 60 min with or without PMA. Western blotting techniques were used to detect the expression of NADPH oxygenase-related proteins at the translational level and the phosphorylation levels of ERK and p38 kinases. The results showed that C. psittaci-infected hPMNs inhibited the phosphorylation of p38 and ERK caused by C. psittaci after 60 min, and the pretreatment of SB203580 and PD98059 inhibited the phosphorylation of p38(Figure.7D,E) and ERK caused by C. psittaci (Figure.7G,H). C. psittaci-infected hPMNs were stimulated by PMA after 60 min, and the pretreatment of SB203580 and PD98059 also inhibited the phosphorylation of p38 and ERK (Figure.7D,E,G,H). At the same time, the pretreatment of SB203580 and PD98059 promoted the expression of gp91phox and p40phox at the protein level after 60 min of C. psittaci-infected hPMNs. C. psittaci-infected hPMNs were stimulated with PMA 60 min later. SB203580 could promote the expression of p40phox protein but had no significant change in gp91phox protein expression. Pretreatment of PD98059 promoted the expression of gp91phox protein but did not significantly change the expression of p40phox protein (Figure.7F,I).
The ROS production rate of hPMNs pretreated with p38 and ERK inhibitors was significantly higher than that of the control group. Inhibitor treatment of hPMNs reversed the inhibitory effect of C. psittaci on ROS induced by PMA stimulation of hPMNs (Figure.7J,K). These results suggest that C. psittaci inhibits ROS generated by PMA stimulation of hPMNs, and the MAPKs signaling pathway plays a vital role in this process.
3.8 C. psittaci-induced IL-8 secretion from hPMNs
In this study, the changes of inflammatory cytokines (IL-6, IL-8, and IL-12) were detected by ELISA after C. psittaci of different MOI were applied to hPMNs cells for different periods. As shown in Figure.7.1, compared with negative control, C. psittaci of different MOI could induce IL-8 production in hPMNs at 30 min of infection. However, there was no significant change in IL-8 production with the increasing MOI of C. psittaci and no significant IL-8 secretion in hPMNs stimulated by PMA (Figure.8A). When C. psittaci with an MOI of 10 was infected with hPMNs for different times (0 min, 30 min, 60 min, 90 min, 120 min), the changes of IL-6, IL-8, and IL-12 were detected in the absence or presence of PMA. It was found that C. psittaci induced IL-8 secretion from hPMNs in a time-dependent manner (Figure.8B). PMA stimulated hPMNs without secretion of significant IL-8. However, when PMA and C. psittaci were present together, it was more likely to induce IL-8 secretion from hPMNs than when PMA or C. psittaci were present alone (Figure.8C). C. psittaci significantly induced the secretion of IL-8 by hPMNs cells in a time-dependent manner and produced more significant IL-8 when stimulated with PMA (Figure.8C). The secretion of IL-6 and IL-12 did not change significantly (data not shown).
Chemokines produced by PMNs are thought to influence the inflammatory process by recruiting various activated white blood cell populations. Because the primary target cells of IL-8 are PMNs, inflammatory hPMNs produce this chemokine that acts as an amplifying ring, attracting more hPMNs to the inflammatory site. In order to verify the role of MAPKs pathway in C. psittaci-induced expression of IL-8 in hPMNs cells, specific inhibitors of the MAPKs signaling pathway were applied in this study. After treatment with 30 µM SB203580 and PD98059 and hPMNs cells for 60 min, hPMNs were infected with C. psittaci with MOI of 10 for 60 min. The culture supernatant was collected with or without PMA, and ELISA detected the secretion level of IL-8. The results showed that the expression level of IL-8 in C. psittaci-induced hPMNs was significantly decreased after SB203580 treatment. PMA-stimulated IL-8 expression levels were slightly reduced after C. psittaci infection with hPMNs (Figure.8D). The IL-8 secreted by PMA after C. psittaci pretreatment with PD98059 infected hPMNs was higher than that without PD98059 pretreatment (Figure.8E), indicating that PD98059 inhibited the increase of IL-8 caused by ROS induced by C. psittaci inhibition of PMA. These results indicated that C. psittaci-infected hPMNs affect IL-8 secretion through the p38 signaling pathway. After C. psittaci was infected with hPMNs, adding PMA affected IL-8 production mainly through the ERK signaling pathway.