C3a/C3aR Affects the Propagation of Cryptosporidium parvum in the Ileum Tissues of Mice by Regulating the Gut Barrier, Cell Proliferation, and CD4+ T Cell Main Effectors

Simple Summary The complement system plays important roles in both innate and adaptive immunity. The present study explored the function of host C3a/C3aR signaling during Cryptosporidium parvum infection, and found that the C3a/C3aR signaling likely affected the propagation of C. parvum in mouse ileum tissues by regulating the gut barrier, cell proliferation and CD4+ T cell main effectors. Abstract Cryptosporidium parvum is an important zoonotic protozoon that threatens the health of humans and animals, but the interaction mechanisms between C. parvum and hosts are poorly understood. Our previous study indicated that the expression levels of C3a and C3aR were up-regulated in mice during C. parvum infection, but the mechanisms of C3a/C3aR signaling during C. parvum infection have not been elucidated. In the present study, an optimized BALB/c suckling mouse model infected with C. parvum was used to explore the function of C3a/C3aR signaling during C. parvum infection. The expression levels of C3aR in the ileum tissues of mice infected with C. parvum were analyzed using real-time PCR, Western blot and immunohistochemistry. The mRNA levels of the Cryptosporidium 18S rRNA gene, tight junction proteins (zo-1, claudin 3, and occludin), intestinal stem cell marker lgr5, cell proliferation marker ki67, Th1 cell-related cytokine ifn-γ, and Treg cell-related cytokine tgf-β in mouse ileum tissues were analyzed by real-time PCR. The pathological injury of ileal mucosa was examined by histopathology analysis. The mRNA expression levels of Cryptosporidium 18S rRNA gene were significantly up-regulated in the ileum tissues of C3aR-inhibited mice during C. parvum infection. Meanwhile, histopathology analysis of ileal mucosa in mice showed that inhibition of C3aR significantly aggravated the changes in villus length, villus diameter, mucosal thickness and the ratio of villus length to crypt depth during C. parvum infection. Further studies found inhibition of C3aR aggravated the down-regulation of occludin at most time points during C. parvum infection. The mRNA levels of ki67 and lgr5 in the ileum tissues of mice infected with C. parvum were significantly down-regulated. Inhibition of C3aR significantly down-regulated the mRNA expression levels of lgr5 at most time points, but significantly up-regulated the mRNA expression levels of ki67 at most time points. The mRNA expression levels of ifn-γ and tgf-β were significantly up-regulated and down-regulated in the ileum tissues of mice infected with C. parvum, respectively. However, inhibition of C3aR significantly increased the mRNA expression levels of ifn-γ and tgf-β in the ileum tissues of mice infected with C. parvum. Taken together, C3a/C3aR signaling could possibly affect the propagation of C. parvum in mouse ileum tissues by regulating the gut barrier, cell proliferation and CD4+ T cell main effectors, which would contribute to our understanding of the interaction between Cryptosporidium and hosts.


Western Blot Analysis
The protein expression levels of C3aR in mouse ileum tissues at 2 dpi and 6 dpi were analyzed by Western blotting. Briefly, the mouse ileum tissues were homogenized by grinding, and treated with 1 mL RIPA (ComWin, Beijing, China) containing 1% PMSF (Beyotime, Shanghai, China) for the isolation of proteins. Proteins were separated by SDS-PAGE analysis, and transferred to nitrocellulose membranes pre-activated with methanol. Subsequently, the membranes were blocked with TBST containing 5% non-fat milk for 2 h, and incubated with anti-C3aR antibodies (Sanying, Wuhan, China) with 1:1,000 dilution at 4 • C overnight. The membranes were washed in TBST for 5 min thrice. HRP-conjugated goat-anti-rabbit antibodies (Sangon, Shanghai, China) with 1:5,000 dilution were applied as the secondary antibodies to incubate the membranes for 1 h at room temperature. Then, the membranes were washed in TBST for 5 min thrice and treated with Super ECL Plus (Applygen, Beijing, China) for photography under an automatic gel imaging analysis system (Sage, Beijing, China).

Immunohistochemistry Analysis
The mouse ileum tissues at the peak of infection were analyzed by immunohistochemistry using SP-0023 Histostain TM -Plus Kits (MBL, Beijing, China). Briefly, paraffin sections were made from the ileum tissues at the peak of infection and subsequently incubated with anti-C3aR antibodies (Sanying, Wuhan, China) with 1:200 dilution at 4 • C overnight and HRP-conjugated goat-anti-rabbit antibodies (Sangon, Shanghai, China) for 15 min at room temperature. These were then photographed under a microscope (Olympus, Hataya, Japan). Image-Pro Plus was used to assess the distribution of C3aR on the surface of mouse ileum tissues by calculating the mean optical density value. Accumulative optical density divided by the area of cell distribution equals the mean optical density value.

Data Analysis
Statistical difference analysis was conducted by GraphPad Prism V.8.0 (https://www. graphpad.com/scientific-software/prism/, accessed on 7 July 2022) using Student's t test and ANOVA [32]. Significant difference was identified if the p value was less than 0.05.

Optimization of a Mouse Model Infected with C. parvum
Optimization of the excystation scheme could significantly increase the excystation rate from 40% to over 85%, as shown in Figure 1A (p < 0.05). Compared with the control group, the relative mRNA expression levels of Cryptosporidium 18S rRNA gene in the mouse ilea in C. parvum-infected group increased gradually and reached a peak at 6 dpi, then gradually decreased. The duration period of C. parvum infection in the mouse model reached 29 days ( Figure 1B). Acid-fast stain analysis of the large intestine contents at 6 dpi recognized rosy oocysts in the infected group ( Figure 1C). Histopathology analyses of mouse ileum tissues at 6 dpi found the existence of C. parvum and the infiltration of the inflammatory cells in the ileum tissues of the infected group ( Figure 1D); subsequently statistical analysis indicated C. parvum infection significantly deceased the length of villi Animals 2023, 13, 837 5 of 14 and the ratio of villus height to crypt depth, and significantly increased the diameter of villi ( Figure 1E). reached 29 days ( Figure 1B). Acid-fast stain analysis of the large intestine contents at 6 dpi recognized rosy oocysts in the infected group ( Figure 1C). Histopathology analyses of mouse ileum tissues at 6 dpi found the existence of C. parvum and the infiltration of the inflammatory cells in the ileum tissues of the infected group ( Figure 1D); subsequently statistical analysis indicated C. parvum infection significantly deceased the length of villi and the ratio of villus height to crypt depth, and significantly increased the diameter of villi ( Figure 1E). Unexcysted and excysted oocysts appeared as light refractile spheres (red arrow) and dark black spheres (blue arrow), respectively. Bars = 20 μm. (B) qRT-PCR amplification results of Cryptosporidium 18S rRNA gene. ** means p < 0.01. (C) Acid-fast staining results for contents in large intestines of mice infected with C. parvum at 6 dpi. Oocysts appeared as rosy spheres (red arrow). Bars = 20 μm. (D) HE staining results in ileum tissues of mice in the control and infected groups at 6 dpi. C. parvum in the brush border of ileum villi in infected group (red arrow) and lymphocytes (green arrow) and neutrophils (blue triangle) infiltration in the lamina propria were indicated. Bars = 20 μm. (E) Analysis of pathological lesions in ileum tissues of mice infected with C. parvum at 6 dpi. Error bars mean standard deviation with each group, ** means p < 0.01, and ns means p > 0.05.

Expression of C3aR in Mouse Ileum Tissues during C. parvum Infection
Compared with control group, the mRNA expression levels of C3aR in mouse ileum tissues of the infected group were significantly down-regulated from 1 to 8 dpi, and then up-regulated until 36 dpi (Figure 2A). Western blot analysis also indicated the down-regulation of protein levels of C3aR in mouse ileum tissues of the infected group at both 2 dpi and 6 dpi (Figures 2B, S1 and S2). Further immunohistochemistry analysis indicated C3aR was majorly distributed on the surface of ileal epithelial cells at 6 dpi ( Figure 2C), and subsequently statistical analysis also showed significantly decreased distribution of C3aR in mouse ileum tissues of the infected group ( Figure 2D).

Expression of C3aR in Mouse Ileum Tissues during C. parvum Infection
Compared with control group, the mRNA expression levels of C3aR in mouse ileum tissues of the infected group were significantly down-regulated from 1 to 8 dpi, and then upregulated until 36 dpi (Figure 2A). Western blot analysis also indicated the down-regulation of protein levels of C3aR in mouse ileum tissues of the infected group at both 2 dpi and 6 dpi (Figures 2B, S1 and S2). Further immunohistochemistry analysis indicated C3aR was majorly distributed on the surface of ileal epithelial cells at 6 dpi ( Figure 2C), and subsequently statistical analysis also showed significantly decreased distribution of C3aR in mouse ileum tissues of the infected group ( Figure 2D).

Effect of C3a/C3aR Signaling on the Propagation of C. parvum in the Ilea of Mice
As shown in Figure 3A, the protein level of C3aR in mouse ileum tissues was significantly down-regulated three hours after intraperitoneal injection with SB290157 ( Figures  3A and S3). Further studies indicated that inhibition of mouse C3aR significantly up-regulated the relative mRNA expression levels of the Cryptosporidium 18S rRNA gene in mouse ileum tissues during C. parvum infection (p < 0.05). Meanwhile, the infection duration period increased from 29 days to 35 days in the C3aR-inhibited infected group ( Figure  3B). Significant differences were found between the relative transcriptional expression of Cryptosporidium 18S rRNA among the three groups (PSB, Oocyst, and Oocyst + SB290157) at 1 dpi (F = 359.612, p < 0.001), 2 dpi (F = 58.443, p < 0.001), 4 dpi (F = 44.112, p < 0.001), 6 dpi (F = 215.622, p < 0.001), 8 dpi (F = 393.826, p < 0.001), 9 dpi (F = 114.706, p < 0.001), 10 dpi (F = 427.847, p < 0.001), 29 dpi (F = 41.950, p < 0.001), 30 dpi (F = 55.628, p < 0.001), 35 dpi (F = 25.905, p < 0.001) and 36 dpi (F = 13.106, p = 0.006). Histopathology analyses of mouse ileum tissues at 6 dpi found the existence of C. parvum in the ileum tissues in the infected group and the C3aR-inhibited group ( Figure 3C). Subsequent statistical analysis indicated that C. parvum infection in C3aR-inhibited group further decreased the length of villi and the ratio of villus height to crypt depth, and increased the diameter of villi and mucosal thickness compared with the normal mouse in the infected group ( Figure 3D), suggesting that C3aR likely alleviated the intestinal injury caused by C. parvum.

Effect of C3a/C3aR Signaling on the Propagation of C. parvum in the Ilea of Mice
As shown in Figure 3A, the protein level of C3aR in mouse ileum tissues was significantly down-regulated three hours after intraperitoneal injection with SB290157 ( Figures 3A and S3). Further studies indicated that inhibition of mouse C3aR significantly up-regulated the relative mRNA expression levels of the Cryptosporidium 18S rRNA gene in mouse ileum tissues during C. parvum infection (p < 0.05). Meanwhile, the infection duration period increased from 29 days to 35 days in the C3aR-inhibited infected group ( Figure 3B). Significant differences were found between the relative transcriptional expression of Cryptosporidium 18S rRNA among the three groups (PSB, Oocyst, and Oocyst + SB290157) at 1 dpi (F = 359.612, p < 0.001), 2 dpi (F = 58.443, p < 0.001), 4 dpi (F = 44.112, p < 0.001), 6 dpi (F = 215.622, p < 0.001), 8 dpi (F = 393.826, p < 0.001), 9 dpi (F = 114.706, p < 0.001), 10 dpi (F = 427.847, p < 0.001), 29 dpi (F = 41.950, p < 0.001), 30 dpi (F = 55.628, p < 0.001), 35 dpi (F = 25.905, p < 0.001) and 36 dpi (F = 13.106, p = 0.006). Histopathology analyses of mouse ileum tissues at 6 dpi found the existence of C. parvum in the ileum tissues in the infected group and the C3aR-inhibited group ( Figure 3C). Subsequent statistical analysis indicated that C. parvum infection in C3aR-inhibited group further decreased the length of villi and the ratio of villus height to crypt depth, and increased the diameter of villi and mucosal thickness compared with the normal mouse in the infected group ( Figure 3D), suggesting that C3aR likely alleviated the intestinal injury caused by C. parvum. The relative expression levels of Cryptosporidium 18S rRNA between two groups at each time point were compared, and significant differences were found between the relative expression levels of Cryptosporidium 18S rRNA between two groups if lowercase letters above the bars were different (p < 0.05); (C) HE staining results in ileum tissues of mice infected with C. parvum at 6 dpi. a/b/c indicate ilea in the control group/infected group/C3aR-inhibited group, respectively; (D) Analysis of pathological lesions in ileum tissues of mice infected with C. parvum at 6 dpi. ** means p < 0.01, ns means p > 0.05.
Significant differences were found between the relative transcriptional expression levels of the ki67 gene among three groups (PSB, Oocyst, and Oocyst + SB290157) at 1 dpi Cryptosporidium parvum infection significantly up-regulated the mRNA expression levels of the ki67 gene at 9 dpi, 30 dpi, 35 dpi and 36 dpi, but down-regulated at 1 dpi, 2 dpi, 4 dpi, 8 dpi and 29 dpi. Inhibition of C3aR led to the significant up-regulation of the ki67 at 1 dpi, 2 dpi, 4 dpi, 6 dpi, 29 dpi and 36 dpi, but down-regulation at 9 dpi and 35 dpi ( Figure  5B).  Cryptosporidium parvum infection significantly up-regulated the mRNA expression levels of the ki67 gene at 9 dpi, 30 dpi, 35 dpi and 36 dpi, but down-regulated at 1 dpi, 2 dpi, 4 dpi, 8 dpi and 29 dpi. Inhibition of C3aR led to the significant up-regulation of the ki67 at 1 dpi, 2 dpi, 4 dpi, 6 dpi, 29 dpi and 36 dpi, but down-regulation at 9 dpi and 35 dpi ( Figure 5B).

Discussion
Cryptosporidiosis is an important zoonotic disease caused by Cryptosporidium spp., which greatly threatens the health of humans and animals, especially young and immunocompromised hosts [1,2]. However, there are still no effective drugs and vaccines for the prevention and control of cryptosporidiosis. One of the main reasons for this is a lack of knowledge on the interaction between Cryptosporidium and hosts. Previous studies indicated C3a/C3aR signaling played some important roles in the recovery of intestinal injury [22]. Our previous study showed that C. parvum infection could lead to the up-regulation of C3a and C3aR in mouse ileum tissues [24], but the role of C3a/C3aR signaling during C. parvum infection is still unknown. The present study applied an optimized mouse model with C. parvum infection to explore the function of C3a/C3aR signaling during C. parvum infection, enriching our knowledge on the interaction between Cryptosporidium and hosts.
The present study optimized an established suckling mouse model infected with C. parvum in our group by increasing the concentration of sodium taurocholate and the excystation time, and the results indicated that the optimized model significantly increased the excystation of C. parvum. Similar to the previous model, the optimized model also found pathological damage and an inflammatory reaction in the intestines in mice during C. parvum infection [31]. Compared with other infection models, the optimized mouse model in the present study simulated the natural infection process of animals and provided a candidate model for studies on the interaction between Cryptosporidium and hosts without the use of dexamethasone.
In the present study, the optimized mouse model found that the up-regulation of C3aR in the ileum of mice during C. parvum infection and inhibition of C3aR significantly aggravated intestinal damage caused by C. parvum infection. Although opposite results were found for the C3aR mRNA expression at 1 dpi and 2 dpi between the present study and our previous work, the mRNA levels of this gene were increased at later stages of infection for both studies [24]. The differences at the early stage of infection may be due to the distinct immunity of animal models. The present study used suckling mice for parasite infection, while older mice aged 3 weeks were used in the previous study.
Tight junctions are an important structural basis of the intestinal mucosal mechanical barrier, which plays an important role in maintaining the integrity of the intestinal morphology and resisting the invasion of intestinal pathogens [33]. To explore the mechanisms of C3a/C3aR signaling, the mRNA expression levels of three tight junction proteins, namely zo-1, claudin 3 and occludin, were detected by using qPCR. The results found that C. parvum infection down-regulated the mRNA expression levels of the zo-1, claudin 3 and occludin genes at most time points, indicating that C. parvum likely destroyed the integrity of intestinal barrier by down-regulating tight junctions, which was similar to the C57BL/6 mouse model infected with C. parvum [34]. Meanwhile, Cryptosporidium andersoni infection could also destroy the expression of the ZO-1 in the epithelial cells of humans and cattle [35]. Interestingly, inhibition of C3aR significantly down-regulated the mRNA expression of the occludin gene at half of the time points during C. parvum infection, suggesting that C3a/C3aR signaling likely maintained the integrity of the intestinal barrier by upregulating the expression of the tight junction-related gene occludin, thus reducing the infection and propagation of C. parvum in mouse ilea.
Meanwhile, the proliferation and differentiation of intestinal stem cells and the normal renewal of intestinal epithelial cells also play some important roles in maintaining the structure and function of intestinal mucosa [22,36]. Furthermore, the mRNA expression levels of intestinal stem cell marker LGR5 and cell proliferation marker KI67 of ileum tissues during C. parvum infection were detected by qPCR. The results indicated that C. parvum infection down-regulated the mRNA expression levels of the lgr5 and ki67 genes in mouse ileum tissues at half of the time points, reflecting that C. parvum infection possibly retarded the proliferation and renewal of intestinal epithelial cells to prolong the survival time of the parasites in intestinal cells, and then aggravated Cryptosporidium infection in intestine, in accordance with previous reports [37,38]. Inhibition of C3aR led to the down-regulation of the lgr5 gene at most time points, while leading to up-regulation of ki67 at most time points, suggesting that C3a/C3aR signaling likely promoted the regeneration of intestinal epithelial cells and inhibited the proliferation of intestinal epithelial cells to inhibit the propagation of C. parvum in the ilea of mice.
Early moderate IFN-γ can not only initiate intestinal immunity, but can also promote intestinal cell proliferation and mucosal damage repair. However, abnormal over-activation of the immune system will release a large amount of IFN-γ, and excessive IFN-γ can not only kill pathogenic microorganisms, but also activate the JAK/STAT1 pathway of intestinal epithelial cells, leading to programmed necrosis of intestinal epithelial cells and impairment of intestinal barrier function [36]. Unlike IFN-γ, TGF-β showed a protective function on the intestinal barrier [39]. The aforementioned functions of IFN-γ and TGF-β have also been confirmed in intestinal epithelial cells infected with Cryptosporidium [40][41][42]. Meanwhile, C3a/C3aR signaling can function in resisting pathogen invasion by promoting/inhibiting the expression of IFN-γ and TGF-β [43,44]. To explore the link between C3a/C3aR signaling and the expression of IFN-γ and TGF-β, we used qPCR to detect the mRNA expression levels of the ifn-γ and tgf-β genes in mouse ilea during Cryptosporidium infection. The results revealed that C. parvum infection significantly up-regulated and down-regulated the mRNA levels of the ifn-γ and tgf-β genes, respectively, indicating that C. parvum infection may induce a Th1-type immune response, and inhibit the differentiation and function of Treg cells. Therefore, mice infected with C. parvum were likely in a state of continuous inflammation. However, inhibition of C3aR led to the significant up-regulation of mRNA levels of both ifn-γ and tgf-β in the ileum tissues of infected mice, which was possibly related to the dual anti-inflammatory and pro-inflammatory actions of C3a/C3aR signaling [45], suggesting that C3a/C3aR signaling likely participated in the anti-Cryptosporidium effect by down-regulating the expression of Th1 and Treg cells' main effect factors; however, these mechanisms need to be explored in future studies.

Conclusions
The present study firstly explored the preliminary function of host C3a/C3aR signaling during C. parvum infection, and revealed C3a/C3aR signaling likely inhibited the propagation of C. parvum in the ilea of mice by regulating the gut barrier, cell proliferation, and CD4 + T cell main effectors of hosts, which could contribute to our knowledge on the interaction between Cryptosporidium and hosts.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/ani13050837/s1, Figure S1: Original Western blot picture for the protein expression level of C3aR in the mouse ileum tissues of PBS and Oocyst groups at 2 dpi; Figure S2: Original Western blot picture for the protein expression level of C3aR in the mouse ileum tissues of PBS and Oocyst groups at 6 dpi; Figure S3: Original Western blot picture for the protein expression level of C3aR in the mouse ileum tissues of PBS and SB290157 groups at three hours after intraperitoneal injection with SB290157.
Funding: This research was funded by grants from the National Natural Science Foundation of China (32072890; 31572509; 32202838).

Institutional Review Board Statement:
This study was conducted under the approval and instructions of the ethics committee of Northwest A&F University (DY2019042).