Protists protecting food tolerance

Abstract

Celiac disease is an immune disorder characterized by gluten intolerance that can be unleashed by enteric viral infections in mice. However, Sanchez-Medina et al recently identified a murine commensal protist, Tritrichomonas arnold, that protects against reovirus-induced intolerance to dietary protein by counteracting virus-induced epithelial stress and proinflammatory dendritic cell activation.

Celiac disease (CeD) is a complex inflammatory disease of the small intestine characterized by the development of a type 1 immune response to dietary gluten¹. In animal models of the disease, dendritic cells, particularly CD103⁺CD11b⁻ conventional type 1 DCs (cDC1), have been implicated in CeD pathogenesis. In healthy settings, cDC1s promote the induction of peripheral regulatory T (pTreg) cells to food antigens and oral tolerance². However, in CeD, cDC1s adopt a pro-inflammatory profile promoting a CD4⁺ T helper cell 1 (Th1) response to gluten, dampening pTreg cell induction¹. While 30% of the population carries the CeD-predisposing HLA molecules DQ2 and DQ8, only 1% develops CeD¹, indicating that environmental factors play a significant role in CeD development. Enteric viruses, specifically reovirus type 1 Lang (T1L), have recently been shown to precipitate loss of oral tolerance (LOT) to gluten by rendering cDC1s proinflammatory and subsequently promoting a Th1 response to gluten^3,4. While the role of gut commensal bacteria in CeD has been studied⁵, the potential roles of other classes of intestinal microbes remain unknown. In a recent publication in Immunity, Sanchez-Medina et al identify a role for commensal protists in protecting against T1L-mediated LOT in mice⁶.

Sanchez-Medina and colleagues first noticed that C57BL/6 mice raised in their vivarium (“in-house”) did not exhibit T1L-mediated LOT to the dietary antigen ovalbumin (OVA), unlike what was observed in mice directly delivered from Jackson Laboratories (JAX). Gastrointestinal T1L infection led to increased expression of IFNγ in dietary antigen (OVA)-specific CD4⁺ T cells in JAX mice but not in-house mice. Because in-house mice displayed evidence of elevated type 2 immunity in the small intestine relative to JAX mice, the authors suspected a difference in the intestinal content of eukaryotic commensals and performed Internal Transcribed Spacer (ITS) sequencing of cecal contents. They identified a previously uncharacterized species of Tritrichomonas, which the authors referred to as T. arnold, that was only found in the cecum of in-house and not JAX mice. Importantly, in-house mice displayed a higher conversion of dietary antigen-specific CD4⁺ T cells into food tolerance-promoting pTreg cells compared to JAX mice at baseline, a phenomenon attributed to the presence to T. arnold. The mechanism by which this occurs is currently unknown, but it does not appear to involve directly reinforcing common pTreg inducers such as TGFβ and retinoic acid. This also occurred without altering Th2 or Th17 cell responses to food, indicating that T. arnold harbored specific properties that promoted pTreg cell induction at homeostasis. Moreover, the authors proved the sufficiency of T. arnold to protect against T1L-mediated LOT, because JAX mice inoculated with T. arnold prior to T1L infection failed to mount a Th1 response to dietary antigen (OVA). Additionally, depletion of T. arnold within in-house mice with a cellulose-rich diet rendered these mice susceptible to T1L-mediated LOT.

Finally, the authors performed several experiments to determine the relevance of their findings to human CeD. First, they inoculated mice expressing the CeD-associated allele HLA-DQ8 (HLA-DQ8 mice) with T. arnold prior to T1L infection and gluten feeding because human HLA-DQ8 is necessary to present immunogenic gluten peptides. T. arnold prevented LOT to gluten in HLA-DQ8 mice upon T1L infection as measured by decreased serum titers of anti-gliadin IgG2c antibodies and reduced ear swelling upon skin challenge with gluten (delayed type hypersensitivity response) compared to mice lacking T. arnold. In addition, using a human cohort of CeD patients and healthy controls, the authors showed a striking enrichment of protists in the Parabasalia family (including Tritrichomonas) in healthy controls compared to CeD patients; this suggested that a lack of protective protists might represent an environmental factor contributing to CeD manifestation in humans.

However, the mechanisms via which T. arnold exerted pTreg cell-promoting and T1L-countering effects remained elusive. T1L viral load and anti-viral immunity were not compromised in T. arnold-harboring mice, and none of the type 2 responses elicited by T. arnold on their own conferred protection against T1L-induced LOT. This suggested that neither direct intestinal niche competition nor immune skewing towards type 2 immunity explained the phenomena. However, T. arnold prevented T1L-mediated upregulation of stress-induced transglutaminase 2 (TG2) in intestinal epithelial cells (IEC) and of IRF1 and NFκB target genes in cDC1s; this suggested that T. arnold was involved in the reprogramming of at least these two cell types (see Figure 1). The authors did in fact report on the transcriptional changes in cDC1s upon T. arnold inoculation in mice in the absence of T1L. Specifically, while they focused on the lack of a striking tolerance-promoting profile in cDC1s, it is noteworthy that cDC1s exhibited an interferon or virus “hypersensitive” signature, with Il27, Irf7, and several Oas gene family members being amongst the most upregulated genes. This implied that T. arnold could lower the threshold of viral sensing and potentially destruction, presumably supporting the conservation or quick restoration of tolerance-promoting processes in cDC1s.

Figure 1. The intestinal protist Tritrichomonas arnold protects from virus-mediated loss of oral tolerance in mice.

The enteric reovirus T1L leads to loss of oral tolerance to dietary antigen by rendering migratory conventional dendritic cells (cDC1s) pro-inflammatory, which then promote Th1 polarization of dietary antigen-specific CD4⁺ T cells (center). Conversely, the intestinal protist Tritrichomonas arnold promotes pTreg cell induction and supports oral tolerance through unknown mechanisms, but which might involve tolerogenic reprogramming of intestinal epithelial cells, or direct action on migratory cDC1s (left). Additionally, Tritrichomonas arnold leads to upregulation of anti-viral genes in cDC1s, suggesting a poised “virus-alert” state. The tolerance-promoting effects of Tritrichomonas arnold dominate during T1L infection and result in protection from T1L-mediated loss of oral tolerance (right)[6]. Figure created with BioRender.com.

Intestinal protists such as Giardia intestinalis and Toxoplasma gondii are largely considered to be pathogenic to humans⁷. However, recent studies have demonstrated that certain commensal protists can be protective in the context of intestinal infections. For example, Tritrichomonas musculis activates the inflammasome in IEC to induce IL-18 release, promoting Th1 and Th17 responses that protect against Salmonella enterica Typhimurium infection⁸. Overall, protists have been described to reshape the bacterial gut microbiome. The findings by Sanchez-Medina et al⁶ therefore stand out in two ways: T. arnold promotes pTreg rather than pro-inflammatory T cell induction, and interkingdom crosstalk between protist-viruses exists here (rather than focusing on bacteria).

Given the central role of cDC1s in the development and maintenance of oral tolerance, the authors focused on how T. arnold affected mesenteric lymph node cDC1s. While their results certainly demonstrated the ability of T. arnold to ultimately dampen a T1L-mediated proinflammatory program of cDC1s presenting dietary antigen, the authors did not consider the possibility that T. arnold might exert its protective effect further upstream of cDC1s, e.g. on IEC. Indeed, because T1L-mediated TG2 activation was blunted by T. arnold, it is possible that T. arnold could somehow reprogram IEC to render them less susceptible to T1L-mediated damage or stress. In this model, antigen presenting cells in the gut might in turn integrate multiple local cues, including signals from microbes, food, and epithelium. Thus, we posit that further research should explore the potential crosstalk between tolerance-promoting commensal protists and IEC, both at homeostasis and in the context of enteric viral infection.

A growing body of literature suggests that type 2 immunity-inducing microbes are protective in intestinal inflammatory diseases including inflammatory bowel diseases (IBD), and recently, intestinal helminths were explored as an alternative putative therapy for IBD⁹. Typically, loss of intestinal helminths in high-sanitation countries is often blamed for the rise in food allergies and IBDs. However, the findings discussed here by Reinhard Hinterleitner’s group⁶ suggest that in addition to losing protection against viral stress and contributing to LOT, loss of commensal protists might be equally responsible for influencing intestinal disease susceptibility. We propose that pTreg cell-inducing protists such as Tritrichomonas sp. (contributing to a tolerogenic response) should be investigated in putative preventive or treatment approaches in intestinal inflammatory diseases (e.g. CeD and IBD) and food allergies.