Metabolic diversity in commensal protists regulates intestinal immunity and trans-kingdom competition.

The microbiota influences intestinal health and physiology, yet the contributions of commensal protists to the gut environment have been largely overlooked. Here, we discover human- and rodent-associated parabasalid protists, revealing substantial diversity and prevalence in nonindustrialized human populations. Genomic and metabolomic analyses of murine parabasalids from the genus Tritrichomonas revealed species-level differences in excretion of the metabolite succinate, which results in distinct small intestinal immune responses. Metabolic differences between Tritrichomonas species also determine their ecological niche within the microbiota. By manipulating dietary fibers and developing in vitro protist culture, we show that different Tritrichomonas species prefer dietary polysaccharides or mucus glycans. These polysaccharide preferences drive trans-kingdom competition with specific commensal bacteria, which affects intestinal immunity in a diet-dependent manner. Our findings reveal unappreciated diversity in commensal parabasalids, elucidate differences in commensal protist metabolism, and suggest how dietary interventions could regulate their impact on gut health.

Tuft cell-derived acetylcholine promotes epithelial chloride secretion and intestinal helminth clearance.

Epithelial cells secrete chloride to regulate water release at mucosal barriers, supporting both homeostatic hydration and the "weep" response that is critical for type 2 immune defense against parasitic worms (helminths). Epithelial tuft cells in the small intestine sense helminths and release cytokines and lipids to activate type 2 immune cells, but whether they regulate epithelial secretion is unknown. Here, we found that tuft cell activation rapidly induced epithelial chloride secretion in the small intestine. This response required tuft cell sensory functions and tuft cell-derived acetylcholine (ACh), which acted directly on neighboring epithelial cells to stimulate chloride secretion, independent of neurons. Maximal tuft cell-induced chloride secretion coincided with immune restriction of helminths, and clearance was delayed in mice lacking tuft cell-derived ACh, despite normal type 2 inflammation. Thus, we have uncovered an epithelium-intrinsic response unit that uses ACh to couple tuft cell sensing to the secretory defenses of neighboring epithelial cells.

Microbiota-derived butyrate restricts tuft cell differentiation via histone deacetylase 3 to modulate intestinal type 2 immunity.

Tuft cells in mucosal tissues are key regulators of type 2 immunity. Here, we examined the impact of the microbiota on tuft cell biology in the intestine. Succinate induction of tuft cells and type 2 innate lymphoid cells was elevated with loss of gut microbiota. Colonization with butyrate-producing bacteria or treatment with butyrate suppressed this effect and reduced intestinal histone deacetylase activity. Epithelial-intrinsic deletion of the epigenetic-modifying enzyme histone deacetylase 3 (HDAC3) inhibited tuft cell expansion in vivo and impaired type 2 immune responses during helminth infection. Butyrate restricted stem cell differentiation into tuft cells, and inhibition of HDAC3 in adult mice and human intestinal organoids blocked tuft cell expansion. Collectively, these data define a HDAC3 mechanism in stem cells for tuft cell differentiation that is dampened by a commensal metabolite, revealing a pathway whereby the microbiota calibrate intestinal type 2 immunity.

Epithelial STAT6 O-GlcNAcylation drives a concerted anti-helminth alarmin response dependent on tuft cell hyperplasia and Gasdermin C.

The epithelium is an integral component of mucosal barrier and host immunity. Following helminth infection, the intestinal epithelial cells secrete "alarmin" cytokines, such as interleukin-25 (IL-25) and IL-33, to initiate the type 2 immune responses for helminth expulsion and tolerance. However, it is unknown how helminth infection and the resulting cytokine milieu drive epithelial remodeling and orchestrate alarmin secretion. Here, we report that epithelial O-linked N-Acetylglucosamine (O-GlcNAc) protein modification was induced upon helminth infections. By modifying and activating the transcription factor STAT6, O-GlcNAc transferase promoted the transcription of lineage-defining Pou2f3 in tuft cell differentiation and IL-25 production. Meanwhile, STAT6 O-GlcNAcylation activated the expression of Gsdmc family genes. The membrane pore formed by GSDMC facilitated the unconventional secretion of IL-33. GSDMC-mediated IL-33 secretion was indispensable for effective anti-helminth immunity and contributed to induced intestinal inflammation. Protein O-GlcNAcylation can be harnessed for future treatment of type 2 inflammation-associated human diseases.

Tuft-Cell-Derived Leukotrienes Drive Rapid Anti-helminth Immunity in the Small Intestine but Are Dispensable for Anti-protist Immunity.

Helminths, allergens, and certain protists induce type 2 immune responses, but the underlying mechanisms of immune activation remain poorly understood. In the small intestine, chemosensing by epithelial tuft cells results in the activation of group 2 innate lymphoid cells (ILC2s), which subsequently drive increased tuft cell frequency. This feedforward circuit is essential for intestinal remodeling and helminth clearance. ILC2 activation requires tuft-cell-derived interleukin-25 (IL-25), but whether additional signals regulate the circuit is unclear. Here, we show that tuft cells secrete cysteinyl leukotrienes (cysLTs) to rapidly activate type 2 immunity following chemosensing of helminth infection. CysLTs cooperate with IL-25 to activate ILC2s, and tuft-cell-specific ablation of leukotriene synthesis attenuates type 2 immunity and delays helminth clearance. Conversely, cysLTs are dispensable for the tuft cell response induced by intestinal protists. Our findings identify an additional tuft cell effector function and suggest context-specific regulation of tuft-ILC2 circuits within the small intestine.

Detection of Succinate by Intestinal Tuft Cells Triggers a Type 2 Innate Immune Circuit.

In the small intestine, type 2 responses are regulated by a signaling circuit that involves tuft cells and group 2 innate lymphoid cells (ILC2s). Here, we identified the microbial metabolite succinate as an activating ligand for small intestinal (SI) tuft cells. Sequencing analyses of tuft cells isolated from the small intestine, gall bladder, colon, thymus, and trachea revealed that expression of tuft cell chemosensory receptors is tissue specific. SI tuft cells expressed the succinate receptor (SUCNR1), and providing succinate in drinking water was sufficient to induce a multifaceted type 2 immune response via the tuft-ILC2 circuit. The helminth Nippostrongylus brasiliensis and a tritrichomonad protist both secreted succinate as a metabolite. In vivo sensing of the tritrichomonad required SUCNR1, whereas N. brasiliensis was SUCNR1 independent. These findings define a paradigm wherein tuft cells monitor microbial metabolites to initiate type 2 immunity and suggest the existence of other sensing pathways triggering the response to helminths.

Tuft cells mediate commensal remodeling of the small intestinal antimicrobial landscape.

Succinate produced by the commensal protist Tritrichomonas musculis (T. mu) stimulates chemosensory tuft cells, resulting in intestinal type 2 immunity. Tuft cells express the succinate receptor SUCNR1, yet this receptor does not mediate antihelminth immunity nor alter protist colonization. Here, we report that microbial-derived succinate increases Paneth cell numbers and profoundly alters the antimicrobial peptide (AMP) landscape in the small intestine. Succinate was sufficient to drive this epithelial remodeling, but not in mice lacking tuft cell chemosensory components required to detect this metabolite. Tuft cells respond to succinate by stimulating type 2 immunity, leading to interleukin-13-mediated epithelial and AMP expression changes. Moreover, type 2 immunity decreases the total number of mucosa-associated bacteria and alters the small intestinal microbiota composition. Finally, tuft cells can detect short-term bacterial dysbiosis that leads to a spike in luminal succinate levels and modulate AMP production in response. These findings demonstrate that a single metabolite produced by commensals can markedly shift the intestinal AMP profile and suggest that tuft cells utilize SUCNR1 and succinate sensing to modulate bacterial homeostasis.

Identifying a missing lineage driver in a subset of lung neuroendocrine tumors.

Tumor heterogeneity of a primary histologic cancer type has major implications for cancer research and therapeutics. An important and understudied aspect of this heterogeneity is the role of transcription factors that serve as "lineage oncogenes" in a tumor type. A demonstration that different subgroups have distinct dependencies on lineage-specific transcription factors is highlighted in a relatively homogenous cancer type: the pulmonary neuroendocrine cancer small cell lung carcinoma (SCLC). Identification of these factors is providing new insights into the origin of the heterogeneity and subtype-specific vulnerabilities in SCLC and provides a template for studying heterogeneity in other cancer types.

POU2F3 is a master regulator of a tuft cell-like variant of small cell lung cancer.

Small cell lung cancer (SCLC) is widely considered to be a tumor of pulmonary neuroendocrine cells; however, a variant form of this disease has been described that lacks neuroendocrine features. Here, we applied domain-focused CRISPR screening to human cancer cell lines to identify the transcription factor (TF) POU2F3 (POU class 2 homeobox 3; also known as SKN-1a/OCT-11) as a powerful dependency in a subset of SCLC lines. An analysis of human SCLC specimens revealed that POU2F3 is expressed exclusively in variant SCLC tumors that lack expression of neuroendocrine markers and instead express markers of a chemosensory lineage known as tuft cells. Using chromatin- and RNA-profiling experiments, we provide evidence that POU2F3 is a master regulator of tuft cell identity in a variant form of SCLC. Moreover, we show that most SCLC tumors can be classified into one of three lineages based on the expression of POU2F3, ASCL1, or NEUROD1. Our CRISPR screens exposed other unique dependencies in POU2F3-expressing SCLC lines, including the lineage TFs SOX9 and ASCL2 and the receptor tyrosine kinase IGF1R (insulin-like growth factor 1 receptor). These data reveal POU2F3 as a cell identity determinant and a dependency in a tuft cell-like variant of SCLC, which may reflect a previously unrecognized cell of origin or a trans-differentiation event in this disease.

Folate supports IL-25-induced tuft cell expansion following enteroviral infections.

Intestinal tuft cells, a kind of epithelial immune cells, rapidly expand in response to pathogenic infections, which is associated with infection-induced interleukin 25 (IL-25) upregulation. However, the metabolic mechanism of IL-25-induced tuft cell expansion is largely unknown. Folate metabolism provides essential purine and methyl substrates for cell proliferation and differentiation. Thus, we aim to investigate the roles of folate metabolism playing in IL-25-induced tuft cell expansion by enteroviral infection and recombinant murine IL-25 (rmIL-25) protein-stimulated mouse models. At present, enteroviruses, such as EV71, CVA16, CVB3, and CVB4, upregulated IL-25 expression and induced tuft cell expansion in the intestinal tissues of mice. However, EV71 did not induce intestinal tuft cell expansion in IL-25-/- mice. Interestingly, compared to the mock group, folate was enriched in the intestinal tissues of both the EV71-infected group and the rmIL-25 protein-stimulated group. Moreover, folate metabolism supported IL-25-induced tuft cell expansion since both folate-depletion and anti-folate MTX-treated mice had a disrupted tuft cell expansion in response to rmIL-25 protein stimulation. In summary, our data suggested that folate metabolism supported intestinal tuft cell expansion in response to enterovirus-induced IL-25 expression, which provided a new insight into the mechanisms of tuft cell expansion from the perspective of folate metabolism.