Perinatal foodborne titanium dioxide exposure-mediated dysbiosis predisposes mice to develop colitis through life.

BACKGROUND: Perinatal exposure to titanium dioxide (TiO2), as a foodborne particle, may influence the intestinal barrier function and the susceptibility to develop inflammatory bowel disease (IBD) later in life. Here, we investigate the impact of perinatal foodborne TiO2 exposure on the intestinal mucosal function and the susceptibility to develop IBD-associated colitis. Pregnant and lactating mother mice were exposed to TiO2 until pups weaning and the gut microbiota and intestinal barrier function of their offspring was assessed at day 30 post-birth (weaning) and at adult age (50 days). Epigenetic marks was studied by DNA methylation profile measuring the level of 5-methyl-2'-deoxycytosine (5-Me-dC) in DNA from colic epithelial cells. The susceptibility to develop IBD has been monitored using dextran-sulfate sodium (DSS)-induced colitis model. Germ-free mice were used to define whether microbial transfer influence the mucosal homeostasis and subsequent exacerbation of DSS-induced colitis. RESULTS: In pregnant and lactating mice, foodborne TiO2 was able to translocate across the host barriers including gut, placenta and mammary gland to reach embryos and pups, respectively. This passage modified the chemical element composition of foetus, and spleen and liver of mothers and their offspring. We showed that perinatal exposure to TiO2 early in life alters the gut microbiota composition, increases the intestinal epithelial permeability and enhances the colonic cytokines and myosin light chain kinase expression. Moreover, perinatal exposure to TiO2 also modifies the abilities of intestinal stem cells to survive, grow and generate a functional epithelium. Maternal TiO2 exposure increases the susceptibility of offspring mice to develop severe DSS-induced colitis later in life. Finally, transfer of TiO2-induced microbiota dysbiosis to pregnant germ-free mice affects the homeostasis of the intestinal mucosal barrier early in life and confers an increased susceptibility to develop colitis in adult offspring. CONCLUSIONS: Our findings indicate that foodborne TiO2 consumption during the perinatal period has negative long-lasting consequences on the development of the intestinal mucosal barrier toward higher colitis susceptibility. This demonstrates to which extent environmental factors influence the microbial-host interplay and impact the long-term mucosal homeostasis.

The parabrachial nucleus elicits a vigorous corticosterone feedback response to the pro-inflammatory cytokine IL-1ß.

The central nervous system regulates systemic immune responses by integrating the physiological and behavioral constraints faced by an individual. Corticosterone (CS), the release of which is controlled in the hypothalamus by the paraventricular nucleus (PVN), is a potent negative regulator of immune responses. Using the mouse model, we report that the parabrachial nucleus (PB), an important hub linking interoceptive afferent information to autonomic and behavioral responses, also integrates the pro-inflammatory cytokine IL-1ß signal to induce the CS response. A subpopulation of PB neurons, directly projecting to the PVN and receiving inputs from the vagal complex (VC), responds to IL-1ß to drive the CS response. Pharmacogenetic reactivation of these IL-1ß-activated PB neurons is sufficient to induce CS-mediated systemic immunosuppression. Our findings demonstrate an efficient brainstem-encoded modality for the central sensing of cytokines and the regulation of systemic immune responses.

ILC3s select microbiota-specific regulatory T cells to establish tolerance in the gut.

Microbial colonization of the mammalian intestine elicits inflammatory or tolerogenic T cell responses, but the mechanisms controlling these distinct outcomes remain poorly understood, and accumulating evidence indicates that aberrant immunity to intestinal microbiota is causally associated with infectious, inflammatory and malignant diseases1-8. Here we define a critical pathway controlling the fate of inflammatory versus tolerogenic T cells that respond to the microbiota and express the transcription factor RORγt. We profiled all RORγt+ immune cells at single-cell resolution from the intestine-draining lymph nodes of mice and reveal a dominant presence of T regulatory (Treg) cells and lymphoid tissue inducer-like group 3 innate lymphoid cells (ILC3s), which co-localize at interfollicular regions. These ILC3s are distinct from extrathymic AIRE-expressing cells, abundantly express major histocompatibility complex class II, and are necessary and sufficient to promote microbiota-specific RORγt+ Treg cells and prevent their expansion as inflammatory T helper 17 cells. This occurs through ILC3-mediated antigen presentation, αV integrin and competition for interleukin-2. Finally, single-cell analyses suggest that interactions between ILC3s and RORγt+ Treg cells are impaired in inflammatory bowel disease. Our results define a paradigm whereby ILC3s select for antigen-specific RORγt+ Treg cells, and against T helper 17 cells, to establish immune tolerance to the microbiota and intestinal health.

ZBTB46 defines and regulates ILC3s that protect the intestine.

RORγt is a lineage-specifying transcription factor that is expressed by immune cells that are enriched in the gastrointestinal tract and promote immunity, inflammation and tissue homeostasis1-15. However, fundamental questions remain with regard to the cellular heterogeneity among these cell types, the mechanisms that control protective versus inflammatory properties and their functional redundancy. Here we define all RORγt+ immune cells in the intestine at single-cell resolution and identify a subset of group 3 innate lymphoid cells (ILC3s) that expresses ZBTB46, a transcription factor specifying conventional dendritic cells16-20. ZBTB46 is robustly expressed by CCR6+ lymphoid-tissue-inducer-like ILC3s that are developmentally and phenotypically distinct from conventional dendritic cells, and its expression is imprinted by RORγt, fine-tuned by microbiota-derived signals and increased by pro-inflammatory cytokines. ZBTB46 restrains the inflammatory properties of ILC3s, including the OX40L-dependent expansion of T helper 17 cells and the exacerbated intestinal inflammation that occurs after enteric infection. Finally, ZBTB46+ ILC3s are a major source of IL-22, and selective depletion of this population renders mice susceptible to enteric infection and associated intestinal inflammation. These results show that ZBTB46 is a transcription factor that is shared between conventional dendritic cells and ILC3s, and identify a cell-intrinsic function for ZBTB46 in restraining the pro-inflammatory properties of ILC3s and a non-redundant role for ZBTB46+ ILC3s in orchestrating intestinal health.

The neuropeptide VIP potentiates intestinal innate type 2 and type 3 immunity in response to feeding.

The nervous system and the immune system both rely on an extensive set of modalities to perceive and act on perturbations in the internal and external environments. During feeding, the intestine is exposed to nutrients that may contain noxious substances and pathogens. Here we show that Vasoactive Intestinal Peptide (VIP), produced by the nervous system in response to feeding, potentiates the production of effector cytokines by intestinal type 2 and type 3 innate lymphoid cells (ILC2s and ILC3s). Exposure to VIP alone leads to modest activation of ILCs, but strongly potentiates ILCs to concomitant or subsequent activation by the inducer cytokines IL-33 or IL-23, via mobilization of cAMP and energy by glycolysis. Consequently, VIP increases resistance to intestinal infection by the helminth Trichuris muris and the enterobacteria Citrobacter rodentium. These findings uncover a functional neuro-immune crosstalk unfolding during feeding that increases the reactivity of innate immunity necessary to face potential threats associated with food intake.

ILC precursors differentiate into metabolically distinct ILC1-like cells during Mycobacterium tuberculosis infection.

Tissue-resident innate lymphoid cells (ILCs) regulate tissue homeostasis, protect against pathogens at mucosal surfaces, and are key players at the interface of innate and adaptive immunity. How ILCs adapt their phenotype and function to environmental cues within tissues remains to be fully understood. Here, we show that Mycobacterium tuberculosis (Mtb) infection alters the phenotype and function of lung IL-18Rα+ ILC toward a protective interferon-γ-producing ILC1-like population. This differentiation is controlled by type 1 cytokines and is associated with a glycolytic program. Moreover, a BCG-driven type I milieu enhances the early generation of ILC1-like cells during secondary challenge with Mtb. Collectively, our data reveal how tissue-resident ILCs adapt to type 1 inflammation toward a pathogen-tailored immune response.

Bacterial sensing via neuronal Nod2 regulates appetite and body temperature.

Gut bacteria influence brain functions and metabolism. We investigated whether this influence can be mediated by direct sensing of bacterial cell wall components by brain neurons. In mice, we found that bacterial peptidoglycan plays a major role in mediating gut-brain communication via the Nod2 receptor. Peptidoglycan-derived muropeptides reach the brain and alter the activity of a subset of brain neurons that express Nod2. Activation of Nod2 in hypothalamic inhibitory neurons is essential for proper appetite and body temperature control, primarily in females. This study identifies a microbe-sensing mechanism that regulates feeding behavior and host metabolism.

Group 3 innate lymphoid cells produce the growth factor HB-EGF to protect the intestine from TNF-mediated inflammation.

Tumor necrosis factor (TNF) drives chronic inflammation and cell death in the intestine, and blocking TNF is a therapeutic approach in inflammatory bowel disease (IBD). Despite this knowledge, the pathways that protect the intestine from TNF are incompletely understood. Here we demonstrate that group 3 innate lymphoid cells (ILC3s) protect the intestinal epithelium from TNF-induced cell death. This occurs independent of interleukin-22 (IL-22), and we identify that ILC3s are a dominant source of heparin-binding epidermal growth factor-like growth factor (HB-EGF). ILC3s produce HB-EGF in response to prostaglandin E2 (PGE2) and engagement of the EP2 receptor. Mice lacking ILC3-derived HB-EGF exhibit increased susceptibility to TNF-mediated epithelial cell death and experimental intestinal inflammation. Finally, human ILC3s produce HB-EGF and are reduced from the inflamed intestine. These results define an essential role for ILC3-derived HB-EGF in protecting the intestine from TNF and indicate that disruption of this pathway contributes to IBD.

Dietary Lactobacillus-Derived Exopolysaccharide Enhances Immune-Checkpoint Blockade Therapy.

Microbes and their byproducts have been reported to regulate host health and immune functions. Here we demonstrated that microbial exopolysaccharide produced by Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 (EPS-R1) induced CCR6+ CD8+ T cells of mice and humans. In mice, ingestion of EPS-R1 augmented antitumor effects of anti-CTLA-4 or anti-PD-1 monoclonal antibody against CCL20-expressing tumors, in which infiltrating CCR6+ CD8+ T cells were increased and produced IFNγ accompanied by a substantial immune response gene expression signature maintaining T-cell functions. Of note, the antitumor adjuvant effect of EPS-R1 was also observed in germ-free mice. Furthermore, the induction of CCR6 expression was mediated through the phosphorylated structure in EPS-R1 and a lysophosphatidic acid receptor on CD8+ T cells. Overall, we find that dietary EPS-R1 consumption induces CCR6+ CD8+ T cells in Peyer's patches, favoring a tumor microenvironment that augments the therapeutic effect of immune-checkpoint blockade depending on CCL20 production by tumors. SIGNIFICANCE: Gut microbiota- and probiotic-derived metabolites are attractive agents to augment the efficacy of immunotherapies. Here we demonstrated that dietary consumption of Lactobacillus-derived exopolysaccharide induced CCR6+ CD8+ T cells in Peyer's patches and improved the tumor microenvironment to augment the therapeutic effects of immune-checkpoint blockade against CCL20-producing tumors. See related commentary by Di Luccia and Colonna, p. 1189. This article is highlighted in the In This Issue feature, p. 1171.

Antigen-presenting innate lymphoid cells orchestrate neuroinflammation.

Pro-inflammatory T cells in the central nervous system (CNS) are causally associated with multiple demyelinating and neurodegenerative diseases1-6, but the pathways that control these responses remain unclear. Here we define a population of inflammatory group 3 innate lymphoid cells (ILC3s) that infiltrate the CNS in a mouse model of multiple sclerosis. These ILC3s are derived from the circulation, localize in proximity to infiltrating T cells in the CNS, function as antigen-presenting cells that restimulate myelin-specific T cells, and are increased in individuals with multiple sclerosis. Notably, antigen presentation by inflammatory ILC3s is required to promote T cell responses in the CNS and the development of multiple-sclerosis-like disease in mouse models. By contrast, conventional and tissue-resident ILC3s in the periphery do not appear to contribute to disease induction, but instead limit autoimmune T cell responses and prevent multiple-sclerosis-like disease when experimentally targeted to present myelin antigen. Collectively, our data define a population of inflammatory ILC3s that is essential for directly promoting T-cell-dependent neuroinflammation in the CNS and reveal the potential of harnessing peripheral tissue-resident ILC3s for the prevention of autoimmune disease.

Dysregulation of ILC3s unleashes progression and immunotherapy resistance in colon cancer.

Group 3 innate lymphoid cells (ILC3s) regulate immunity and inflammation, yet their role in cancer remains elusive. Here, we identify that colorectal cancer (CRC) manifests with altered ILC3s that are characterized by reduced frequencies, increased plasticity, and an imbalance with T cells. We evaluated the consequences of these changes in mice and determined that a dialog between ILC3s and T cells via major histocompatibility complex class II (MHCII) is necessary to support colonization with microbiota that subsequently induce type-1 immunity in the intestine and tumor microenvironment. As a result, mice lacking ILC3-specific MHCII develop invasive CRC and resistance to anti-PD-1 immunotherapy. Finally, humans with dysregulated intestinal ILC3s harbor microbiota that fail to induce type-1 immunity and immunotherapy responsiveness when transferred to mice. Collectively, these data define a protective role for ILC3s in cancer and indicate that their inherent disruption in CRC drives dysfunctional adaptive immunity, tumor progression, and immunotherapy resistance.

ILC3s control splenic cDC homeostasis via lymphotoxin signaling.

The spleen contains a myriad of conventional dendritic cell (cDC) subsets that protect against systemic pathogen dissemination by bridging antigen detection to the induction of adaptive immunity. How cDC subsets differentiate in the splenic environment is poorly understood. Here, we report that LTα1ß2-expressing Rorgt+ ILC3s, together with B cells, control the splenic cDC niche size and the terminal differentiation of Sirpα+CD4+Esam+ cDC2s, independently of the microbiota and of bone marrow pre-cDC output. Whereas the size of the splenic cDC niche depended on lymphotoxin signaling only during a restricted time frame, the homeostasis of Sirpα+CD4+Esam+ cDC2s required continuous lymphotoxin input. This latter property made Sirpα+CD4+Esam+ cDC2s uniquely susceptible to pharmacological interventions with LTßR agonists and antagonists and to ILC reconstitution strategies. Together, our findings demonstrate that LTα1ß2-expressing Rorgt+ ILC3s drive splenic cDC differentiation and highlight the critical role of ILC3s as perpetual regulators of lymphoid tissue homeostasis.

Intestinal-derived ILCs migrating in lymph increase IFNγ production in response to Salmonella Typhimurium infection.

Innate lymphoid cells (ILCs) are enriched in mucosae and have been described as tissue-resident. Interestingly, ILCs are also present within lymph nodes (LNs), in the interfollicular regions, the destination for lymph-migratory cells. We have previously shown that LN ILCs are supplemented by peripheral tissue-derived ILCs. Using thoracic duct cannulations, we here enumerate the intestinal lymph ILCs that traffic from the intestine to the mesenteric LNs (MLNs). We provide, for the first time, a detailed characterisation of these lymph-migratory ILCs. We show that all ILC subsets migrate in lymph, and while global transcriptional analysis reveals a shared signature with tissue-resident ILCs, lymph ILCs express migration-associated genes including S1PRs, SELL (CD62L) and CCR7. Interestingly, we discovered that while Salmonella Typhimurium infections do not increase the numbers of migrating ILCs, infection changes their composition and cytokine profile. Infection increases the proportions of RORyt+ T-bet+ ILCs, levels of IFNγ, and IFNγ/GM-CSF co-expression. Infection-induced changes in migratory ILCs are reflected in colon-draining MLN ILCs, where RORyt+ T-bet+ ILCs accumulate and display corresponding increased cytokine expression. Thus, we reveal that ILCs respond rapidly to intestinal infection and can migrate to the MLN where they produce cytokines.

Metabolic regulation by PPARγ is required for IL-33-mediated activation of ILC2s in lung and adipose tissue.

Type 2 innate lymphoid cells (ILC2s) play a critical role early in the response to infection by helminths and in the development of allergic reactions. ILC2s are also involved in the physiologic regulation of adipose tissue and its metabolic response to cold shock. We find that the metabolic sensor peroxisome proliferator-activated receptor gamma (PPARγ) is highly expressed in ILC2s of the lung and adipose tissue and increases responsiveness to IL-33. In turn, activation of ILC2 by IL-33 leads to increased expression of PPARγ, a prerequisite for proliferation and expression of the effector cytokines IL-5 and IL-13. In contrast, pharmacological inhibition of PPARγ leads to decreased expression of CD36 and fatty acid uptake, a necessary source of energy for ILC2s and of potential ligands for PPARγ. As a consequence, treatment of mice with a PPARγ antagonist reduces the severity of an ILC2-dependent acute airway inflammation. Together, our results demonstrate the critical role of the metabolic sensor PPARγ for the functions of ILC2s.

Effect of gut microbiota on depressive-like behaviors in mice is mediated by the endocannabinoid system.

Depression is the leading cause of disability worldwide. Recent observations have revealed an association between mood disorders and alterations of the intestinal microbiota. Here, using unpredictable chronic mild stress (UCMS) as a mouse model of depression, we show that UCMS mice display phenotypic alterations, which could be transferred from UCMS donors to naïve recipient mice by fecal microbiota transplantation. The cellular and behavioral alterations observed in recipient mice were accompanied by a decrease in the endocannabinoid (eCB) signaling due to lower peripheral levels of fatty acid precursors of eCB ligands. The adverse effects of UCMS-transferred microbiota were alleviated by selectively enhancing the central eCB or by complementation with a strain of the Lactobacilli genus. Our findings provide a mechanistic scenario for how chronic stress, diet and gut microbiota generate a pathological feed-forward loop that contributes to despair behavior via the central eCB system.

Beware of whom you live with: Your intestinal IgA may depend on it.

In the past 15 years, it became clear that the symbiotic microbiota has an important impact on the development and regulation of the immune system. Consequently, it is incorrect to interpret a phenotype solely as a direct result of the genotype, without considering the impact of the microbiota. In fact, ignorance of the effects exerted by the microbiota may account for a large part of the "replication issues" found in many studies. In this issue of the European Journal of Immunology, Beller et al. [Eur. J. Immunol. 2020. 50: 783-794] provide data suggesting that eosinophils are not required to maintain IgA-producing plasma cells in the intestine, contrary to earlier reports. This paper shows that mice lacking eosinophils develop an altered intestinal microbiota, which poorly induces IgA. Normal levels of IgA were obtained in mice lacking eosinophils when these were colonized by microbiota from the WT mice. Therefore, the use of littermate controls carrying the same microbiota, in experiments comparing WT and mutant mice, is necessary to control the potential role of the microbiota. Nevertheless, caution should always be exercised in the interpretation of the results: changes in the microbiota may result from mutations in the host, and thereby, indirectly convey the effect of genotypes on phenotypes.

Changes in Gut Microbiota by Chronic Stress Impair the Efficacy of Fluoxetine.

Major depressive disorders (MDDs) constitute a leading cause of disability worldwide and current pharmacological treatments are partially effective. The gut microbiota (GM) has recently emerged as a target of therapeutic interest for MDDs. In this study, we transfer GM from mice that sustained unpredictable chronic mild stress (UCMS) to healthy recipient mice. The fecal transfer induces despair-like behavior, decreases neurogenesis in the hippocampus (HpC), and impairs the antidepressant and neurogenic effects of a standard selective serotonin (5-HT) reuptake inhibitor, fluoxetine (FLX). These effects are paralleled by deficits in 5-HT bioavailability, biosynthesis, and reuptake in the HpC. Treatment with 5-hydroxytryptophan restores the levels of 5-HT and its precursors in the HpC, improves HpC neurogenesis, and alleviates despair-like symptoms. Our results reveal that stress-induced changes in GM are involved in the pathogenesis of depressive disorders and minimize FLX efficacy via alterations in the serotonergic pathway of Trp metabolism.