E. hellem Ser/Thr protein phosphatase PP1 targets the DC MAPK pathway and impairs immune functions

Abstract

Microsporidia are difficult to be completely eliminated once infected, and the persistence disrupts host cell functions. Here in this study, we aimed to elucidate the impairing effects and consequences of microsporidia on host DCs. Enterocytozoon hellem, one of the most commonly diagnosed zoonotic microsporidia species, was applied. In vivo models demonstrated that E. hellem-infected mice were more susceptible to further pathogenic challenges, and DCs were identified as the most affected groups of cells. In vitro assays revealed that E. hellem infection impaired DCs' immune functions, reflected by down-regulated cytokine expressions, lower extent of maturation, phagocytosis ability, and antigen presentations. E. hellem infection also detained DCs' potencies to prime and stimulate T cells; therefore, host immunities were disrupted. We found that E. hellem Ser/Thr protein phosphatase PP1 directly interacts with host p38α (MAPK14) to manipulate the p38α(MAPK14)/NFAT5 axis of the MAPK pathway. Our study is the first to elucidate the molecular mechanisms of the impairing effects of microsporidia on host DCs' immune functions. The emergence of microsporidiosis may be of great threat to public health.

Figure S1.. Proof of E. hellem persistence and unobvious manifestations.

(A) Spleen size measurement showed no edema after microsporidia infections. (B) Body weights were measured and monitored until 17 dpi. Both uninfected and E. hellem-infected groups showed no body weight loss but slight body weight gain during this period (n = 10 per group; ns, no significance). (C) Persistence of E. hellem in the host. Peripheral blood, stool, and urine samples were taken on the 17 dpi from E. hellem-infected mice; uninfected mice were controls (n = 10 per group). The existence of E. hellem was verified by qRT-PCR using primers targeting the E. hellem SSU-rDNA region.

Figure 1.. E. hellem infection and persistence increase host disease susceptibility and affect DCs.

(A) LPS treatment in E. hellem–pre-infected mice (E. hellem+LPS) caused more body weight loss and slower/less body weight re-gain compared with LPS alone (n = 8 per group; * = P < 0.05, ** = P < 0.01, and *** = P < 0.001). (B) S. aureus infection in E. hellem–pre-infected mice (E. hellem+S. aureus) caused more body weight loss and slower/less body weight re-gain compared with S. aureus alone (n = 8 per group; * = P < 0.05, ** = P < 0.01, and *** = P < 0.001). (C) Hematoxylin–eosin staining of spleen samples after E. hellem infection alone had no obvious effects on tissue pathology compared with control, as white pulps (white arrows) and red pulps (orange arrows) arranged normally. However, E. hellem pre-infection plus LPS treatment caused more damages to tissues compared with LPS alone, as shown by more enlarged/distorted cells/cytosols and vacuolations (golden arrowheads) (scale bar = 20 μm). (D) E. hellem pre-infection plus S. aureus infection caused more damages to tissues compared with S. aureus alone, as shown by more enlarged/distorted cells/cytosols and vacuolations (golden arrowheads) (scale bar = 20 μm). (E) ELISA showed that the E. hellem infection (E. hellem) significantly down-regulated the expression of IL-6, compared with uninfected controls and LPS treatment (n = 8 per group; * = P < 0.05 and ** = P < 0.01). (F) ELISA showed that the E. hellem infection (E. hellem) significantly down-regulated the expression of IL-6, compared with uninfected controls and S. aureus infection (n = 8 per group; * = P < 0.05 and ** = P < 0.01). (G) ELISA showed that the E. hellem infection (E. hellem) significantly down-regulated the expression of IL-12, compared with uninfected controls and LPS treatment (n = 8 per group; * = P < 0.05 and ** = P < 0.01). (H) ELISA showed that the E. hellem infection (E. hellem) significantly down-regulated the expression of IL-12, compared with uninfected controls and S. aureus infection (n = 8 per group; * = P < 0.05 and ** = P < 0.01). All co-infection conditions, either E. hellem plus LPS (EH+LPS) or E. hellem plus S. aureus (EH+SA), did not arose higher but caused a slight lower level of cytokine levels compared with single challenges. (I) Flow cytometry analysis of the immune cell profiles from mouse mesenteric lymph nodes. Mesenteric lymph nodes were isolated from control or E. hellem-infected mice, and were teased into single-cell mixture (n = 3–5 lymph nodes from each mouse, eight mice per group). (J) Flow cytometry analysis showed (J) that DCs (CD11b+CD11c+) were significantly disturbed after E. hellem infection; (K) no significant changes in inflammatory monocytes (CD11b+Ly6c^high); (L) no significant changes in T cells (CD3⁺); and (M) no significant changes in B cells (CD19⁺) (** = P < 0.01 and ns, no significance).

Figure 2.. E. hellem interferes with DCs’ immune functions and maturation.

(A) DCs were isolated from normal mice or E. hellem-infected mice, and fluorescent (GFP)-labeled S. aureus were then added to DC culture (MOI = 20:1). The phagocytic ability was assessed by fluorescent microscopy. Most of the added-in S. aureus (green) were engulfed by control DCs, whereas most added-in S. aureus (green) were floating outside of DCs isolated from E. hellem-infected mice (scale bar = 5 μm). Zoomed-in images showed that DCs from the E. hellem-infected group engulfed significant less S. aureus (green; arrows) compared with DCs from uninfected controls. Persistence of E. hellem spores was manifested by calcofluor white stain (blue) (scale bar = 2 μm). (B) Flow cytometry analysis showed that E. hellem infection retained the splenic DCs (33D1+) to the comparable level as the uninfected controls. Yet, LPS treatment significantly up-regulated the DCs (n = 8/group; ns, no significance, ** = P < 0.01, and *** = P < 0.001). (C) E. hellem infection, but not LPS treatment, inhibited the expressions of CD40 on DCs. The expression of CD86 was at a comparable level between E. hellem infection and LPS treatment, both significantly higher than uninfected control (n = 8/group; ns, no significance and * = P < 0.05). (E) Cytokine gene expression profiles from splenic DCs were assessed by qRT-PCR; results showed that the (E) IL-12p40 expression level of DCs from E. hellem-infected mice was significantly down-regulated compared with uninfected controls, and was significantly lower than the LPS-treated ones. (F) IL-6 expression level of DCs from E. hellem-infected mice was retained from uninfected controls, and was significantly lower than the LPS-treated ones. (G) INF-γ expression level of DCs from E. hellem-infected mice was significantly down-regulated compared with uninfected controls, and was significantly lower than the LPS-treated ones. (H) IFN-α expression level of DCs from E. hellem-infected mice was retained from uninfected controls, and was significantly lower than the LPS-treated ones (n = 8/group; ns, no significance, * = P < 0.05, ** = P < 0.01, and *** = P < 0.001).

Figure 3.. E. hellem detained the antigen presentation abilities and T-cell priming potencies of DCs.

The expressions of DC antigen presentation–related surface markers were assessed by qRT-PCR assay. (A) Results showed that (A) E. hellem infection caused the up-regulation of H2Ab; (B) E. hellem infection detained the up-regulation of H2Aa; and (C) E. hellem infection detained the up-regulation of DC-SIGN (ns, no significance and * = P < 0.05). (D, E) Flow cytometry analysis of cell population changes after E. hellem infection showed that there were no significant stimulations or changes in populations of both (D) CD4⁺ T cells and (E) CD8⁺ T cells (n = 8/group; ns, no significance). (F, G) qRT-PCR analysis of the expressions of T-cell markers showed that neither (F) Ctla4 nor (G) Tigit showed any changes after E. hellem infection (ns, no significance). (H) DC2.4 cells, either infected by E. hellem or S. aureus, or uninfected controls, were co-cultured with Jurkat T cells. The DC2.4 cells were isolated later for surface marker analysis. qRT-PCR results showed the representative antigen presentation markers were detained by E. hellem infection, compared with S. aureus infections. (I) Jurkat T cells isolated from co-culture with DC2.4 were also analyzed by qRT-PCR. Results confirmed that T cells co-cultured with E. hellem-infected DCs reluctant to up-regulate the Ctla4, and significantly down-regulated the expressions of T-cell activation surface markers CD4 and PD-1 (n = 10/group; ns, no significance and * = P < 0.05).

Figure 4.. p38α/MAPK signaling pathway is key for E. hellem–DC interactions and modulations.

(A) Pie chat of subcellular localization of top differentially expressed DC proteins after E. hellem infection. The top localization is cytoplasm and nucleus. (B) GO enrichment analysis of top differentially expressed DC proteins after E. hellem infection. Many signaling pathways including the MAPK pathway were among the most affected cellular events. (C) Protein–protein interaction network showed that top differentially expressed DC proteins were associated with the MAPK signaling pathway.

Figure 5.. NFAT5/MAPK signal axis is essential during E. hellem infection and cellular modulation.

(A) Western blot analysis of NFAT5 protein levels in DCs showed that NFAT5 expression was suppressed by E. hellem infection, compared with uninfected or S. aureus infection controls. (B) Immunofluorescent microscopy of NFAT5 localization in DCs. NFAT5 (Alexa Fluor 594) is constitutively expressed in the cytoplasm (orange arrows) of uninfected control DCs. The localization of NFAT5 was retained in the cytoplasm (orange arrows) after E. hellem infection. The E. hellem was able to proliferate in DCs and form parasitophorous vacuole (blue arrows). S. aureus infection and osmotic stimulation control (NaCl) were applied to DCs, and both can lead to up-regulation of NFAT5 expressions and re-localization into nucleus (orange arrows) (scale bar = 2 μm). (C) Western blot analysis showed that NFAT5 protein levels are significantly down-regulated after RNAi. (D) qRT-PCR analysis of E. hellem proliferation within host DC2.4 cells, as reflected by the copy numbers of E. hellem-specific PTP4 (n = 8/group; ** = P < 0.01).

Figure 6.. Direct binding of E. hellem PP1 with DC p38α(MAPK14).

(A) Protein sequence alignments of serine/threonine protein phosphatase (PP1), derived from E. hellem (XP_003888309.1), human (P62136.1), and mouse (P62137.1). Alignments confirmed that PP1s, especially at the catalytic regions, were highly conserved among different species. (B) Yeast two-hybrid assay. DC MAPK14 was cloned into pGBKT7 plasmid (BD-MAPK14), and E. hellem–serine/threonine protein phosphatase PP1 was cloned into pGADT7 plasmid (AD-EhPP1). The plasmids were transformed into competent yeast cells, and the binding was validated in synthetic dropout-Leu-Trp-Ade-His medium supplemented with Xα-gal. The fusion strain of pGBKT7-53 with pGADT7-T was used as a positive control, and the fusion strain of pGBKT7-lam with pGADT7-T was used as a negative control. The EhPP1 and MAPK14 fused clones were subjected to PCR and gel electrophoresis to confirm the existence of target sequences.

Figure 7.. Direct interaction of E. hellem PP1 with DC p38α (MAPK14) and the down-regulated MAPK pathway gene expressions.

(A) Expressions of E. hellem PP1 in DCs. Fluorescent microscopy confirmed the expression of E. hellem-derived PP1 (pCMV-mCherry-PP1) in the cytoplasm of DCs (red color) (scale bar = 20 μm). (B) Immunofluorescent microscopy confirmed the co-localization of E. hellem PP1 with DC p38α (MAPK14). Heterologously expressed E. hellem–PP1 (mCherry, red color) is expressed in the cytoplasm of DCs. The DC p38a (MAPK14) is labeled as Alexa Fluor 488 (green color) and is constitutively expressed in the cytoplasm of DCs. E. hellem PP1 co-localized with DC p38α (MAPK14) (arrows) (scale bar = 5 μm). (C) qRT-PCR analysis of DC NFAT5, IL-6, H2Aa, and H2Ab of MHC-II. All these genes were significantly down-regulated when DCs have heterologously expressed E. hellem PP1 (n = 12/group; * = P < 0.05, ** = P < 0.01, and *** = P < 0.001).

Figure 8.. Image illustration and summary of E. hellem expressed the serine/threonine protein phosphatase PP1, directly interacted with DC MAPK14.

The direct interaction would interfere with normal functions of MAPK14 and the subsequent transduction of the MAPK pathway; therefore, the downstream transcription factor NFAT5 expression and localization were disrupted. Eventually, various immune-related genes of DCs were down-regulated and the immune functions of DCs were severely impaired.