Follicular helper T cells recruit eosinophils into host liver by producing CXCL12 during Schistosoma japonicum infection

Abstract Schistosomiasis affects at least 200 million people in tropical and subtropical areas. The major pathology of schistosomiasis is egg‐induced liver granuloma characterized by an eosinophil‐rich inflammatory infiltration around the eggs, which subsequently leads to hepatic fibrosis and circulatory impairment in host. However, the mechanisms how eosinophils are recruited into the liver, which are crucial for the better understanding of the mechanisms underlying granuloma formation and control of schistosomiasis, remain unclear. In this study, we showed that follicular helper T (Tfh) cells participate in recruitment of eosinophils into liver partially by producing CXCL12 during schistosome infection. Our findings uncovered a previously unappreciated role of Tfh cells in promotion of the development of liver granuloma in schistosomiasis, making Tfh‐CXCL12‐eosinophil axis a potential target for intervention of schistosomiasis.

In our previous study, we showed that follicular helper T (Tfh) cells play an obvious central role in hepatic granuloma formation during Schistosoma japonicum (S japonicum) infection. 12 However, the precise pathogenic mechanism by which Tfh cells promote the granuloma formation remains to be determined. Here, we further demonstrated that Tfh cells recruit eosinophils into liver partially through producing CXCL12 during S japonicum infection. Our findings uncovered a novel mechanism of Tfh cells in the recruitment of eosinophils in schistosomiasis, which contributes to a deeper understanding and may help to design better treatment of the schistosomiasis.

| Adoptive transfer experiment
Fresh total cells from spleens of WT mice 8 weeks after infection with S japonicum were pre-sorted by CD4 + T cell negative-isolation kit (Miltenyi Biotec), and then stained with CD3e-Percp-cy5.5

| RNA isolation and reverse transcriptase polymerase chain reaction
Total cellular RNA was extracted using TRIzol (Invitrogen) according to manufacturer's instructions and quantified using a

| Immunohistochemical analysis
Livers were harvested from infected or normal mice and fixed in 10% neutral buffered formalin. Paraffin-embedded sections were incubated with anti-mouse CXCL12 (Cat 14-7992-81, Thermo Fisher Scientific) or isotype antibodies (Thermo Fisher Scientific), followed by an HPR-conjugated secondary antibody. DAB was used as the substrate. All images were captured using an Axiovert 200M microscope (Carl Zeiss).

| Statistical analysis
Data were expressed as the mean ± standard deviation. Student's t test was applied for comparisons between two groups, and one-way ANOVA followed by LSD t test was used to compare differences between more than two different groups (GraphPad Prism software, version 5.01). Pvalues of less than .05 were considered as statistically significant.

| Tfh cells promote hepatic granuloma formation and control liver eosinophil number in S japonicuminfected mice
Although our previously published data documents that Tfh cells represent a key pathogenic player in schistosomiasis japonica, 12

| Tfh cells recruit the eosinophils in vitro
To investigate whether Tfh cells have the potential to recruit eosinophils, we performed the in vitro transwell migration assay. Tfh cells were sorted with a FACSAria and placed in the bottom of tissue culture wells and transwell inserts with MACS-purified eosinophils (>95% pure) were placed on top (Figure 2A). Compared with naïve CD4 + T cells, Tfh cells were able to stimulate eosinophil migration ( Figure 2B). Treatment with PMA and ionomycin markedly enhanced the capacity of Tfh cells to recruit eosinophils in vitro ( Figure 2B).
Taken together, these results suggest that the soluble proteins secreted by Tfh cells may be involved in this recruitment process.

| Eosinophils express CXCR4 in S japonicuminfected mice
Given that CXCR4-mediated signalling has important roles in both eosinophil recruitment in the context of allergic disease 16 and granuloma formation in schistosomiasis, 17  More importantly, the surface expression of CXCR4 protein on eosinophils in the liver of infected mice was also clearly detected by flow cytometry ( Figure 3B).

| Tfh cells are an important in vivo source of CXCL12 in livers of mice with schistosomiasis japonica
We next determine whether Tfh cells produced CXCL12, the principal ligand for CXCR4. 16 Interestingly, 30%-50% of Tfh cells constitutively expressed CXCL12 in spleen, lymph nodes and liver from normal mice or infected mice ( Figure 4A,B). Although no difference in the percentages of CXCL12 + Tfh cells in total Tfh cells was observed in spleen, lymph nodes or liver between normal mice and infected mice ( Figure 4B), the absolute number of CXCL12 + Tfh cells was markedly increased in mice after schistosome infection ( Figure 4C). In addition, the expression of CXCL12 in the liver was considerably decreased in Tfh cell-deficient-infected mice compared with WT-infected mice ( Figure 4D), suggesting Tfh cells as an important in vivo source of CXCL12 in liver in schistosomeinfected mice.

| Tfh cells recruit the eosinophils partially dependent on CXCL12-CXCR4 axis
Finally, we investigated whether Tfh cells recruited eosinophils via producing CXCL12. We found that CXCL12 blockade by neutralizing antibody impaired the capacity of Tfh cells to recruit eosinophils in the in vitro transwell migration assay ( Figure 5). Taken together, these results indicate that CXCL12 produced by Tfh cells contributes to the recruitment of eosinophils in the liver in S japonicum-infected mice.

| D ISCUSS I ON
The eosinophil-rich granulomatous lesions in the liver are the major contributors to the pathogenesis of schistosomiasis. Thus, clarification of the mechanisms responsible for the recruitment of eosinophils Tfh-induced eosinophil migration may be through release of IL-5.
Indeed, these may explain why the blockade of CXCL12-CXCR4 axis has only partially compromised Tfh cell-mediated eosinophil recruitment in our study. Therefore, future studies will be crucial for identifying the other pathways of Tfh cell-mediated eosinophil recruitment.
In summary, our study reveals a novel role of Tfh cells in controlling eosinophil migrating and provides additional insights into granuloma formation during S japonicum infection, making Tfh-CXCL12eosinophil axis a potential target for treating schistosomiasis.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

AUTH O R S ' CO NTR I B UTI O N S
XC and CS conceived and designed the experiments. XC and CS analysed the data. XC, ZX, CW, XY, LX, SZ and JZ performed the experiments. CS and X.C wrote the paper. All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this published article.