The intestinal parasite Cryptosporidium is controlled by an enterocyte intrinsic inflammasome that depends on NLRP6

Abstract

The apicomplexan parasite Cryptosporidium infects the intestinal epithelium. While infection is widespread around the world, children in resource-poor settings suffer a disproportionate disease burden. Cryptosporidiosis is a leading cause of diarrheal disease, responsible for mortality and stunted growth in children. CD4 T cells are required to resolve this infection, but powerful innate mechanisms control the parasite prior to the onset of adaptive immunity. Here, we use the natural mouse pathogen Cryptosporidium tyzzeri to demonstrate that the inflammasome plays a critical role in initiating this early response. Mice lacking core inflammasome components, including caspase-1 and apoptosis-associated speck-like protein, show increased parasite burden and caspase 1 deletion solely in enterocytes phenocopies whole-body knockout (KO). This response was fully functional in germfree mice and sufficient to control Cryptosporidium infection. Inflammasome activation leads to the release of IL-18, and mice that lack IL-18 are more susceptible to infection. Treatment of infected caspase 1 KO mice with recombinant IL-18 is remarkably efficient in rescuing parasite control. Notably, NOD-like receptor family pyrin domain containing 6 (NLRP6) was the only NLR required for innate parasite control. Taken together, these data support a model of innate recognition of Cryptosporidium infection through an NLRP6-dependent and enterocyte-intrinsic inflammasome that leads to the release of IL-18 required for parasite control.

Keywords: Cryptosporidium; NLRP6; immunity; inflammasome; parasite.

Fig. 1.

Mice deficient in core components of the inflammasome have a higher parasite burden. (A) Schematic map of the strategy used to derive the Ct-NluC strain. (B) Graph of parasite burden measured by qPCR (x axis) and luminescence (y axis). Each measurement represents the average of three technical replicates from the same mouse fecal sample, and the linear relationship has an R² value of 0.94. (C–E) C57BL/6 (WT) and Casp1/11^−/− mice were infected with 50,000 Ct-FluC oocysts each, and parasite burden was measured by whole-animal imaging (C and D) and qPCR (E). Quantitative PCR was performed on pooled collected samples with two technical replicates; mean ± SD and n = 6 mice in total. (F–H) Parasite shedding in C57BL/6 (WT) versus Casp1/11^−/− (F), Casp11^−/− (G), and Asc^−/− (H) mice infected with 50,000 Ct-NluC oocysts per mouse. Fecal luminescence measurements are from pooled cage samples shown as mean ± SD from two technical replicates. (I) Comparative infection of Casp1^fl/fl mice and Casp1^fl/fl -vilCRE mice that are deficient in caspase 1 specifically in their intestinal epithelial cells. Mice were infected with 50,000 Ct-NluC parasites, and fecal luminescence is from pooled collections shown as mean ± SD from two technical replicates per time point. Data are from two separate experiments, each with n = 8 mice. For F and G, n = 8 mice. For H, n = 7 mice in total. Samples in F–I are reported for every 2 d, but the x axis is abbreviated to 4-d increments for clarity.

Fig. 2.

Loss of the inflammasome leads to more severe intestinal pathology. C57BL/6 (WT) mice and Casp1/11^−/- mice were infected with 50,000 Ct oocysts each, followed by histological examination of the small intestine on day 5 post infection. (A) Representative images of infected and uninfected mice with parasites highlighted by arrowheads. (B) Histological scoring from a veterinary pathologist; n = 30, with the scoring from each mouse represented as a single measurement. Mean ± SD with significance determined using one-way ANOVA and Tukey’s multiple comparisons test (*P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.00005).

Fig. 3.

Cryptosporidium inflammasome activation is independent of the microbiome and drives IL-18 secretion. (A) Parasite burden from C57BL/6 (WT), Il1α ^−/−, Il1β ^−/−, and Il18^−/− mice and (C) C57BL/6 and Gsdmd^−/− mice infected with 50,000 Ct-NluC oocysts per mouse. Four mice per group; n = 16 or n = 8 mice total, respectively. Fecal luminescence was measured from pooled cage samples, and mean ± SD is shown from two technical measurements per time point. (B) Parasite burden from rIL-18–treated and untreated C57BL/6 (WT) and Casp1/11^−/− mice. Mice were infected with 50,000 Ct-NluC parasites each, and 2 μg of rIL-18 was administered by intraperitoneal injection every second day starting on day 1 post infection. Fecal luminescence was measured from pooled cage samples, and mean ± SD is shown from two technical measurements per time point. Four mice per group with n = 20 mice total. One group received a single rIL-18 injection which alone decreased parasite burden by 64.4%. (D) C57BL/6 and Casp1/11^−/− mice were cohoused for 2 wk prior to infection with 50,000 Ct-NluC oocytes per mouse. Fecal luminescence shown as mean ± SD from two technical replicates per time point; n = 12 from two separate experiments. (E) C57BL/6 and Casp1/11^−/− mice were treated for 1 wk with an antibiotic mixture (Methods) ad libitum in their cage water and then infected with 50,000 Ct-NluC oocysts per mouse. Fecal luminescence shown as mean ± SD from two technical replicates per time point; n = 16 from one experiment. (F) C57BL/6 germfree mice and matched controls were infected with 50,000 Ct-Nluc parasites by gavage, and fecal luminescence was measured from pooled cage fecal collections. Fecal luminescence shown as mean ± SD from two technical replicates per time point; n = 8 from one experiment. (G and H) ELISA measurements of IL-1β and IL-18 and from intestinal segments of infected and uninfected mice. Mice from G and H were a part of a cohort; therefore, significance was determined using one-way ANOVA with Tukey’s multiple comparisons test. Four mice per condition with n = 16 mice total and six intestinal segments harvested per mouse on day 5 post infection. Each mouse was infected with 50,000 Ct-NluC oocysts. Samples in A and C–F are reported for every 2 d, but the x axis is abbreviated to 4-d increments for clarity.

Fig. 4.

NLRP6 is responsible for resistance to Cryptosporidium infection. (A) Parasite shedding from a comparative infection of C57BL/6, Aim2^−/-, Nlrp3^−/−, Nlrp1b^−/−, Nlrc4^−/−, and Nlrp6^−/− mice infected with 50,000 Ct-Nluc oocysts. Four mice per group with n = 24 mice total. Fecal luminescence was measured from pooled cage collections with mean ± SD shown from two technical replicates. Samples in A are reported for every 2 d, but x axis is abbreviated to 4-d increments for clarity. (B–D) C57BL/6 (WT) and Nlrp6^−/− mice were infected with 50,000 Ct-FluC oocysts, and parasite burden was measured by whole-animal imaging (B and C) and qPCR (D). qPCR was performed on pooled collected samples with two technical replicates; mean ± SD and n = 6 mice in total. (E) ELISA measurements of IL-18 from intestinal segments of infected and uninfected C57BL/6 and Nlrp6^−/− mice. Note that lack of NLRP6 ablates the increase in IL-18 production from Cryptosporidium infection. Significance was determined using one-way ANOVA with Tukey’s multiple comparisons test. Four mice per condition with n = 16 mice total and four intestinal segments harvested per mouse on day 5 post infection (*P < 0.05).

Fig. 5.

Model of inflammasome activation by Cryptosporidium. Cryptosporidium actively invades enterocytes and transforms into an intracellular replicative stage right at the brush border. In this study we demonstrate that parasite infection leads to the activation of an enterocyte intrinsic inflammasome that depends on caspase-1, ASC, and NLRP6. This activation leads to the processing and release of IL-18 (red) in a GSDMD (blue)-dependent fashion. We hypothesize that, once released, IL-18 acts on tissue resident lymphocytes to induce the production of IFNγ (gray), which restricts parasite growth by a yet-to-be-defined mechanism.