Eosinophils are dispensable for the regulation of IgA and Th17 responses in Giardia muris infection

IgA and Th17 responses are pivotal for the control of Giardia infections. Eosinophils support IgA class switching, the survival of intestinal IgA+ plasma cells at steady state and can control Th17 activity in the small intestine. To see whether eosinophils regulate adaptive immune responses during giardiasis, we investigated Giardia muris infections in wild‐type BALB/c and eosinophil‐deficient ∆dblGATA‐1 mice.

nematodes. 9,11,12 However, the healthy small intestine harbours high numbers of eosinophils, which support Peyer's patches (PP) development and mucus production 13

and promote homeostatic
IgA antibody class switching (CS). Eosinophils also support IgAproducing plasma cells in the small intestinal lamina propria (siLP) at steady state, and during intestinal nematode and Toxoplasma gondii infection. 14,15 Importantly, eosinophils were also shown to restrict baseline and LPS-induced activity of intestinal Th17 cells 16 and to limit Th1/Th17-induced immunopathology during infections with Helicobacter pylori and Citrobacter rodentium. 17 Whether eosinophils exert regulatory functions of mucosal immune responses during Giardia infection has not been addressed experimentally before. Investigating G muris infection in BALB/c and eosinophil-deficient ∆dblGATA-1 mice, we hence assessed whether eosinophil deficiency was associated with impaired IgA responses during Giardia infection and whether eosinophils promoted susceptibility in BALB/c mice by controlling Th17 activity.

| Mice and G muris infection
Eight-week old female BALB/c mice were purchased from Janvier Labs (Saint-Berthevin, France). Age-matched ∆dblGATA-1 mice (Jackson Laboratory, CA, USA) were bred in house. 10-to 12-week old mice were infected and sacrificed at the indicated time points as previously described. 8 All animal experiments were performed in accordance with the National Animal Protection Guidelines and approved by the Berlin Ethics Committee for the Protection of Animals (G0113/15).

| Cell isolation
Cells from lymphatic organs and siLP were isolated as described previously. 18 Cell numbers were determined using an automated CASY cell counter (Roche-Innovatis).

| Flow cytometric analysis
Surface and intracellular stainings were performed as previously described, 8 applying the antibodies listed in Table S1. Samples were analysed on a Canto II flow cytometer (BD Biosciences), and data were analysed using FlowJo software Version 10 (Tree star Inc.).

| Quantitative real-time PCR analysis
Tissue sampling, RNA isolation, cDNA preparation and quantitative real-time PCR (qRT-PCR) using 10 ng of cDNA and FastStart Universal SYBR Green Master Mix (Roche) were performed as described previously. 8 Efficiencies for each primer pair were determined by generating a standard curve, and mRNA expression was normalized to the housekeeping gene β-glucuronidase (GUSB).
Primers pairs are listed in Table S2.

| Faecal IgA ELISA
Snap-frozen faecal pellets were weighed and homogenized in 1 mL sterile PBS per 100 mg faecal material. After vigorous vortexing and centrifugation at 20 000 ×g (10 minutes, 4°C), supernatants were used for quantification of total faecal IgA via ELISA.

| Statistical analysis
Statistical analysis was performed using GraphPad Prism software (La Jolla, CA, USA). Results are displayed as mean ± SD, and significance is displayed as *P < .05, **P < .01, ***P < .001. Results were tested for normal distribution using the Shapiro-Wilk normality tests, followed by ANOVA or Kruskal-Wallis combined with Tukey's or Dunn's multiple comparison testing.

| Control of G muris infection is not impaired in the absence of eosinophils
Monitoring cyst shedding of wild-type (WT) BALB/c and eosinophildeficient ∆dblGATA-1 mice over 6 weeks after primary G muris infection revealed similar dynamics in parasite control between the two strains ( Figure 1). Nevertheless, on average, ∆dblGATA-1 mice shed mildly elevated cyst numbers over the 6-week infection period, with cumulative cyst counts confirming the trend of overall higher cyst shedding in ∆dblGATA-1 mice ( Figure 1B). Independent of eosinophil deficiency, histopathological scoring of the small intestine confirmed the lack of overt intestinal pathology caused by G muris 8,19,20 ( Figure 1C). In accordance with stable eosinophil frequencies in siLP,

| Defective IgA production recovers in G murisinfected ∆dblGATA-1 mice
Confirming previous reports on poor IgA class switching in the absence of eosinophils, 13,18 naive ∆dblGATA-1 mice harboured lower frequencies of total and IgA + B220 + PNA + germinal centre (GC) B cells in PP than naïve BALB/c mice (

| Eosinophil-deficient mice display transient Th17 responses to G muris
Previous work has demonstrated that efficient control of Giardia infection relies on Th17 cells and IL-17A production. 5,6 As eosinophils were reported to regulate Th17 cells and IL-17A production, 16,17 we assessed whether eosinophil deficiency was associated with deregulated IL-17A production in ∆dblGATA-1 mice at steady state or upon G muris infection. We could show that naïve and infected WT mice previously been suggested as a potential alternative source of IL-17A during giardiasis. 5 Here, ∆dblGATA-1 mice harboured lower frequencies of ILC3 at steady state and poorly sustained ILC3 during G muris infection ( Figure 3D). While ILC3 expanded slightly in G murisinfected WT mice, IL-17A production was barely detectable for ILC3 from either mouse strain ( Figure 3E). The modest differences in mucosal Th17 cells and ILC3 responses between Giardia-infected WT and eosinophil-deficient mice did not translate to changes in small intestinal IL-17A gene expression ( Figure 3F). Hence, eosinophils appear dispensable for the regulation of Th17 and ILC3-derived IL-17A responses during G muris infection.

| D ISCUSS I ON
Several studies have previously demonstrated that small intestinal eosinophils support IgA class switching and the maintenance of intestinal IgA secreting plasma cells in the small intestine. [13][14][15]23 As IgA is important for the control of both the human-pathogenic G lamblia and the rodent G muris infection, 3,4 we investigated whether As small intestinal eosinophil numbers were reported to inversely correlate with IL-17A production, 16 we asked whether eosinophils were responsible for the low Th17 activity previously determined for G muris-infected BALB/c mice by our group. 8 14 and T gondii 15 infections, but restrain IgA responses in the large intestine during infections with the murine whipworm Trichuris muris. 23 In summary, our study therefore demonstrates that eosinophils are dispensable for intestinal IgA responses and the regulation of Th17 responses during G muris infection. Together with previous reports, these findings highlight the contextual nature of eosinophils as regulators of mucosal immune responses during parasitic infections.

ACK N OWLED G EM ENTS
The authors would like to thank Yvonne Weber, Marion Müller, Bettina Sonnenburg, Christiane Palissa and Beate Anders for providing excellent technical support. Open access funding enabled and organized by ProjektDEAL.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

AUTH O R CO NTR I B UTI O N S
IAY and ML carried out the experiments, and IAY and SR analysed the data. AAK performed the histological analysis of tissue samples. SH and SR provided valuable input into the study design, data analysis, critically reviewed and edited the manuscript. SH and SR originally designed the study, and IAY and SR wrote the manuscript.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1111/pim.12791.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data presented here are available from the corresponding author upon reasonable request.