Cytotoxic innate intraepithelial lymphocytes control early stages of Cryptosporidium infection

Abstract

Background: Intraepithelial lymphocytes (IELs) are the first immune cells to contact and fight intestinal pathogens such as Cryptosporidium, a widespread parasite which infects the gut epithelium. IFN-γ producing CD4⁺ T IELs provide an efficient and a long-term protection against cryptosporidiosis while intraepithelial type 1 innate lymphoid cells limits pathogen spreading during early stages of infection in immunodeficient individuals. Yet, the role of T-cell like innate IELs, the most frequent subset of innate lymphocytes in the gut, remains unknown.

Methods: To better define functions of innate IELs in cryptosporidiosis, we developed a co-culture model with innate IELs isolated from Rag2^-/- mice and 3D intestinal organoids infected with C. parvum using microinjection.

Results: Thanks to this original model, we demonstrated that innate IELs control parasite proliferation. We further showed that although innate IELs secrete IFN-γ in response to C. parvum, the cytokine was not sufficient to inhibit parasite proliferation at early stages of the infection. The rapid protective effect of innate IELs was in fact mediated by a cytotoxic, granzyme-dependent mechanism. Moreover, transcriptomic analysis of the Cryptosporidium-infected organoids revealed that epithelial cells down regulated Serpinb9b, a granzyme inhibitor, which may increase their sensitivity to cytolytic attack by innate IELs.

Conclusion: Based on these data we conclude that innate IELs, most likely T-cell-like innate IELs, provide a rapid protection against C. parvum infection through a perforin/granzymes-dependent mechanism. C. parvum infection. The infection may also increase the sensitivity of intestinal epithelial cells to the innate IEL-mediated cytotoxic attack by decreasing the expression of Serpin genes.

Keywords: cryptosporidium; cytotoxicity; gut; innate intraepithelial lymphocytes; organoids.

Figure 1

Very early immune responses induced by C. parvum infection in the ileum of Rag2^-/- mice. Rag2^-/- mice were infected by oral gavage with C. parvum for 24h. Quantitative RT-qPCR analysis was performed to compare expression of genes in the whole small intestine (WI) and in the epithelium (E) (A) between non-infected (NI) (n=14) and infected (I) (n=15) mice and (B) between sites. Results were pooled from 3 independent experiments. Medians and ranges are shown. (C) Frequencies of innate IELs subsets in the small intestine of NI (n=4) and I (n=5) Rag2^-/-mice using flow cytometry; representative dot plots and histograms of means values. (D) Immunohistochemistry, staining of CD3γ and CD8α on ileal sections from NI (n=5) and I (n=5) Rag2^-/- mice. Scatter plots summarizes results and average values. ****p < 0.00005, **p < 0.005 and *p < 0.05.

Figure 2

Innate IELs controls C. parvum infection in murine intestinal organoids. (A) The parasitic load was measured at 0, 24- and 48-hours PI in intestinal organoids using RT-qPCR to quantify C. parvum 18S rRNA. Results were normalized to the β-actin transcript level and median with range are shown in 4 independent experiments. (B) Volcano plot of differentially expressed genes (DEGs) between non-infected (n=4) and infected (n=4) organoids. (blue dot padj ≤ 0.1; red dots FC ≥ 1.5 and padj ≤ 0.1). (C) Immunofluorescence of co-cultures with non-infected (NI) and organoids infected (I) for 24h with C. parvum. Nuclei are stained in blue (DAPI), the actin in red (phalloidin) and innate IELs in yellow (CFSE). The histogram shows the number of innate IELs present in organoids. (D) Expression of innate IELs signature genes (Cd3g, Cd8a and Ncr1) in co-cultures (n=6) with NI and I organoids using RT-qPCR. **p < 0.005 and *p < 0.05).

Figure 3

Innate IELs limit the development of C parvum in intestinal murine organoids. C parvum 18S rRNA expression was measured by RT-qPCR in infected organoids co-cultured or not with innate IELs (n=8). Results were normalized to the condition without lymphocytes and histograms present medians with interquartile range (A). Innate IELs were FACS-sorted and co-cultured with infected organoids (n=2); Results are represented using box and whiskers show individual values (B). *p < 0.05; ***p < 0.0005.

Figure 4

Innate IELs normalize expression of genes deregulated by C parvum in intestinal murine organoids. (A) Medians of log-fold changes of gene expression between infected organoids alone (in blue) or co-cultured with innate IELs (in red) and non-infected organoids. Twenty-seven genes differentially expressed between infected vs non-infected organoids are shown. (B) Heat map representing an unsupervised, hierarchical cluster analysis of all experimental conditions (p-value adjusted ≤ 0.1 and FC ≥ 1.5). *p < 0.05.

Figure 5

The anti-parasitic effect of innate IELs is independent of IFN-γ in co-cultures. Quantification of IFN-γ (Ifng) (A) using RT-qPCR (n=8) and (B) ELISA (n=4) in infected organoids with or without innate IELs. (C) Amounts of C parvum 18S rRNA in infected organoids co-cultured with innate IELs treated with a blocking anti-IFN-γ mAb or an isotype control Ab. Results show individual points from 3 independent experiments.

Figure 6

Innate IELs limit C parvum expansion in intestinal organoids through perforin and a serine-protease dependent mechanism. (A) Quantification of LDH release (DO 490 nm) after 48h of infection in supernatant of organoids non-infected (NI), infected (I) alone or co-cultured with innate IELs. (n=9). (B) Quantification of C parvum 18S rRNA using RT-qPCR in infected organoids co-cultured with innate IELs treated or not with the perforin inhibitor Concanamycin A (CMA) (n=2 independent experiments). (C) Expression of Gzmb in infected organoids cultured with or without innate IELs using RT-qPCR. Quantification of C parvum 18S rRNA using RT-qPCR in infected organoids co-cultured with innate IELs treated or not with (D) the GZMB inhibitor I or (E) with Aprotinin (n=3 independent experiments). Box and whiskers show individual value. (F) Expression of Serpinb9b measure by RT-qPCR in organoids infected or not for 24h with C parvum (n=10). **p < 0.005 and *p < 0.05.