The intestinal parasite Cryptosporidium is controlled by an enterocyte intrinsic inflammasome that depends on NLRP6 Adam Saterialea,1, Jodi A. Gullicksruda, Julie B. Engilesa, Briana I. McLeoda, Emily M. Kuglera, Jorge Henao-Mejiab,c, Ting Zhoud, Aaron M. Ringd, Igor E. Brodskya, Christopher A. Huntera, and Boris Striepena,2 aDepartment of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104; bDepartment of Pathology and Laboratory Medicine, Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; cDivision of Protective Immunity, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104; and dDepartment of Immunology, Yale School of Medicine, Yale University, New Haven, CT 06519 Edited by Stephen M. Beverley, Washington University School of Medicine, St. Louis, MO, and approved December 1, 2020 (received for review April 24, 2020) The apicomplexan parasite Cryptosporidium infects the intestinal Murine infection with C. tyzzeri resembles human cryptospo- epithelium. While infection is widespread around the world, chil- ridiosis in location, pathology, and resolution and provides an dren in resource-poor settings suffer a disproportionate disease important tool to define the host and parasite factors that burden. Cryptosporidiosis is a leading cause of diarrheal disease, determine the outcome of infection and to identify the im- responsible for mortality and stunted growth in children. CD4 mune mechanisms required for vaccine-mediated protection T cells are required to resolve this infection, but powerful innate to prevent cryptosporidiosis. mechanisms control the parasite prior to the onset of adaptive While the resolution of Cryptosporidium infection in humans immunity. Here, we use the natural mouse pathogen Cryptospo- (7) and mice (6, 8) depends on T cell-mediated adaptive im- ridium tyzzeri to demonstrate that the inflammasome plays a crit- munity, there is a potent innate mechanism of resistance that ical role in initiating this early response. Mice lacking core inflammasome depends on the early production of IFN-γ, which restricts the components, including caspase-1 and apoptosis-associated speck-like pro- initial replication of this pathogen (6, 9, 10). The cytokines IL-12 tein, show increased parasite burden and caspase 1 deletion solely in and IL-18 have been implicated in lymphocyte production of MICROBIOLOGY enterocytes phenocopies whole-body knockout (KO). This response IFN-γ, but the mechanism of innate recognition of Cryptospo- was fully functional in germfree mice and sufficient to control Crypto- ridium that leads to this early cytokine response remains poorly sporidium infection. Inflammasome activation leads to the release of IL- defined. The inflammasome is a multiprotein complex that plays 18, and mice that lack IL-18 are more susceptible to infection. Treatment a key role in innate immune responses to viral, bacterial, and of infected caspase 1 KO mice with recombinant IL-18 is remarkably ef- ficient in rescuing parasite control. Notably, NOD-like receptor family protozoan pathogens (11, 12). Distinct members of the NOD- pyrin domain containing 6 (NLRP6) was the only NLR required for innate like receptor (NLR) family act as cytosolic sensors that enable parasite control. Taken together, these data support a model of innate innate recognition of a wide variety of intracellular molecules recognition of Cryptosporidium infection through an NLRP6-dependent associated with infection. In response to specific signals, some and enterocyte-intrinsic inflammasome that leads to the release of IL-18 NLRs were shown to oligomerize and recruit the adaptor protein required for parasite control. Significance Cryptosporidium | inflammasome | immunity | NLRP6 | parasite The intestinal immune system is able to control pathogens lobally, 9% of deaths in children under the age of 5 are while tolerating and interpreting microbial cues from an Gdirectly attributed to diarrheal disease caused by infection abundant microbiome. The mechanisms of innate recognition (1). Diarrheal diseases are also an important cause of mal- are crucial to differentiating between pathogen and commen- nourishment in children, a condition that enhances susceptibility sal in this tissue and to mounting an appropriate inflammatory to many other infections including malaria, pneumonia, and mea- response. Persistent inflammation can alter the cellular archi- sles, resulting in significantly higher mortality rates (2). Frequent tecture and physiology of the gut and have lasting impact on episodes of enteric infection can also cause growth stunting and the nutritional state of children who face frequent infection persistent developmental delay, a syndrome known as “environ- with certain enteric pathogens. We demonstrate that the Cryptosporidium mental enteropathy” (3). Changes in the structure and physiology of widespread parasite acts as a potent trigger the gut have been associated with environmental enteropathy, but for an enterocyte-intrinsic inflammasome that depends on the the underlying immunological mechanisms are poorly understood. NOD-like receptor pyrin domain-6 and results in the local re- The apicomplexan parasite Cryptosporidium is an enteric pathogen lease of the proinflammatory cytokine IL-18. that contributes to this syndrome and is a leading global cause of Author contributions: A.S., J.A.G., C.A.H., and B.S. designed research; A.S., J.A.G., J.B.E., illness and death in young children (4, 5). The molecular mecha- B.I.M., and E.M.K. performed research; J.H.-M., T.Z., A.M.R., and I.E.B. contributed new nisms by which this parasite causes disease and long-term sequelae reagents/analytic tools; A.S., J.A.G., J.B.E., B.I.M., E.M.K., C.A.H., and B.S. analyzed data; are largely unknown. and A.S., J.A.G., C.A.H., and B.S. wrote the paper. Until recently, in vivo studies of Cryptosporidium infection The authors declare no competing interest. were limited to large animal models including calves and piglets This article is a PNAS Direct Submission. or to mice with diminished immune function (through chemical This open access article is distributed under Creative Commons Attribution License 4.0 suppression, genetic insufficiency, or use of neonates). To (CC BY). overcome some of the challenges associated with these studies, 1Present address: The Francis Crick Institute, London NW1 1AT, United Kingdom. we recently developed a mouse model for cryptosporidiosis using 2To whom correspondence may be addressed. Email: [email protected]. Cryptosporidium tyzzeri, a natural pathogen of house mice with a This article contains supporting information online at https://www.pnas.org/lookup/suppl/ genome >95% identical to that of the human-specific parasites doi:10.1073/pnas.2007807118/-/DCSupplemental. Cryptosporidium parvum and Cryptosporidium hominis (6). Published December 28, 2020. PNAS 2021 Vol. 118 No. 2 e2007807118 https://doi.org/10.1073/pnas.2007807118 | 1of8 Downloaded by guest on September 24, 2021 apoptosis-associated speck-like protein (ASC), which in turn re- particularly in macrophages. However, Cryptosporidium invades and cruits and activates the proprotease caspase-1 (11). Active caspase-1 replicates within intestinal epithelial cells, which raises the question cleaves and activates its targets, which include procytokines of the of which inflammasome components contribute to cell-intrinsic IL-1 family, as well as pore-forming molecules such as gasdermin D. sensing of this infection. Using a natural host-adapted Cryptospo- Together, these activities lead to the release of active IL-1β and IL- ridium, we demonstrate that control of this organism relies on an 18 and an inflammatory form of cell death known as pyroptosis. For epithelial cell-intrinsic caspase-1. This response was exclusively de- C. parvum, recent studies by McNair and colleagues showed that pendent on NOD-like receptor 6 (NLRP6) and required gasdermin neonatal mice that lacked caspase-1 or ASC were more susceptible D and IL-18. Moreover, exogenous IL-18 rescued the ability of to infection, providing evidence for the involvement of the inflam- caspase-1–deficient mice to control Cryptosporidium, indicating masome in cryptosporidiosis (13). that the role of inflammasome activation is to promote the release The role of the inflammasome in innate recognition and resis- of IL-18. Collectively, these findings implicate epithelial cell- tance to infection has been studied primarily in innate immune cells, intrinsic NLRP6/caspase-1–mediated secretion of IL-18 as a key A Targeting construct B rib TK mCherry Nanoluciferase NeoR act eno 3 104 WT r2=0.94 CTYZ_00003487 TK CTYZ_00003489 2 104 gRNA + Cas9 4 Ct-NluC 1 10 TK mCherry Nanoluciferase NeoR Relative luminescence units 0 0.0 5.0 105 1.0 106 1.5 106 Oocyts per g feces (18s qPCR) C 36912 DE -/- -/- 1 107 C57BL/6Casp1/1 C57BL/6 Casp1/11 ) 7 3 108 5 10 7 C57BL/6 4 10 2 108 3 107 -/- 2 107 1 108 5 flux (p/s) Total 10 1 107 Casp1/11 0 Oocysts per g feces (qPCR 0 3 6 9 3 6 9 12 12 Days post infection Days post infection F G H I -/- fl/fl -/- 1 fl/fl -/- Casp1 Asc C57BL/6 Casp1/11 C57BL/6 Casp1 C57BL/6 Casp1 vilCre s s 8 105 8 105 1.2 106 1.25 106 1.00 106 6 105 6 105 9.0 105 7.50 105 4 105 4 105 6.0 105 5.00 105 2 105 2 105 3.0 105 2.50 105 Relative luminescence units Relative luminescence unit Relative luminescence unit 0 0 0.0 Relative luminescence units 0.00 4 8 4 8 4 8 4 8 4 8 4 8 4 8 4 8 12 12 12 12 12 12 12 12 Days post infection Days post infection Days post infection Days post infection 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).