Dendritic Cells Promote the Differentiation of ILCs into NCR-ILC3s in the Lungs of Mice Exposed to Cigarette Smoke.

The involvement of Group 3 innate lymphoid cells (ILC3s) and dendritic cells (DCs) in chronic lung inflammation has been increasingly regarded as the key to understand the inflammatory mechanisms of smoke-related chronic obstructive pulmonary disease (COPD). However, the mechanism underlying the engagement of both remains unclear. Our study aimed to explore NCR-ILC3 differentiation in the lungs of mice exposed to cigarette smoke (CS) and to further investigate whether DCs activated by CS exposure contribute to the differentiation of ILCs into NCR-ILC3s. The study involved both in vivo and in vitro experiments. In the former, the frequencies of lung NCR-ILC3s and NKp46-IL-17A+ ILCs and the expression of DCs, CD40, CD86, IL-23, and IL-1ß quantified by flow cytometry were compared between CS-exposed mice and air-exposed mice. In the latter, NKp46-IL-17A+ ILC frequencies quantified by flow cytometry were compared after two cocultures, one involving lung CD45+Lin-CD127+ ILCs sorted from air-exposed mice and DCs sifted by CD11c magnetic beads from CS-exposed mice and another including identical CD45+Lin-CD127+ ILCs and DCs from air-exposed mice. The results indicated significant increases in the frequencies of NCR-ILC3s and NKp46-IL-17A+ ILCs; in the expression of DCs, CD40, CD86, IL-23, and IL-1ß in CS-exposed mice; and in the frequency of NKp46-IL-17A+ ILCs after the coculture with DCs from CS-exposed mice. In conclusion, CS exposure increases the frequency of lung ILCs and NCR-ILC3s. CS-induced DC activation enhances the differentiation of ILCs into NCR-ILC3s, which likely acts as a mediating step in the involvement of NCR-ILC3s in chronic lung inflammation.

The role of the aryl hydrocarbon receptor (AhR) in modulating intestinal ILC3s to optimise gut pathogen resistance in lupus and benefits of nutritional AhR ligands.

BACKGROUND AND AIMS: Dysbiosis is emerging as a potential trigger of systemic lupus erythematosus (SLE). Group 3 innate lymphoid cells (ILC3s) are recognised as key regulators of intestinal homeostasis. The aryl hydrocarbon receptor (AhR) is critical to intestinal ILC3 development and function. This mechanistic review aimed to investigate whether AhR activation of gut ILC3s facilitates IL-22-mediated antimicrobial peptide (AMP) production to enhance colonisation resistance and ameliorate SLE pathology associated with intestinal dysbiosis. Furthermore, nutritional AhR ligand potential to enhance pathogen resistance was explored. METHODOLOGY: This mechanistic review involved a three-tranche systematic literature search (review, mechanism, intervention) using PubMed with critical appraisal. Data was synthesised into themes and summarised in a narrative analysis. RESULTS: Preclinical mechanistic data indicate that AhR modulation of intestinal ILC3s optimises pathogen resistance via IL-22-derived AMPs. Pre-clinical research is required to validate this mechanism in SLE. Data on systemic immune consequences of AhR modulation in lupus suggest UVB-activated ligands induce aberrant AhR signalling while many dietary ligands exert beneficial effects. Data on xenobiotic-origin ligands is varied, although considerable evidence has demonstrated negative effects on Th17 to Treg balance. Limited human evidence supports the role of nutritional AhR ligands in modulating SLE pathology. Preclinical and clinical data support anti-inflammatory effects of dietary AhR ligands. CONCLUSION: Current evidence is insufficient to fully validate the hypothesis that AhR modulation of intestinal ILC3s can enhance pathogen resistance to ameliorate lupus pathology driven by dysbiosis. However, anti-inflammatory effects of dietary AhR ligands suggest a promising role as a therapeutic intervention for SLE.

GLP-1 receptor agonists alleviate colonic inflammation by modulating intestinal microbiota and the function of group 3 innate lymphoid cells.

Glucagon-like peptide-1 receptor agonists (GLP-1RAs), which are drugs used for treating type 2 diabetes, have been reported to exert anti-inflammatory effects on inflammatory bowel disease (IBD), the mechanism of which remains elusive. Here, we report that GLP-1RAs ameliorate dextran sulfate sodium (DSS)-induced colitis in both wild-type and T/B-cell-deficient mice through modulating group 3 innate lymphoid cells (ILC3s), a subset of innate lymphoid cells that regulate intestinal immunity. GLP-1RAs promote IL-22 production by ILC3, and the protective effect of GLP-1RAs on DSS-induced colitis was abrogated in ILC3-deficient RORgtgfp/gfp mice. Furthermore, the treatment effect of GLP-RAs on colitis, as well as the generation of IL-22-producing ILC3s by GLP-RAs, is dependent on the gut microbiota. GLP-1RAs increase the abundance of Firmicutes and Proteobacteria in the gut, particularly beneficial bacteria such as Lactobacillus reuteri, and decrease the abundance of enteropathogenic Staphylococcus bacteria. The untargeted gas chromatography (GC)/liquid chromatography (LC)-mass spectrometry (MS) of faecal metabolites further revealed enrichment of N,N-dimethylsphingosine (DMS), an endogenous metabolite derived from sphingosine, in the GLP-1RA-treated group. Strikingly, DMS ameliorates colitis while promoting intestinal IL-22-producing ILC3s. Taken together, our findings show that GLP-1RAs exert a therapeutic effect on colitis possibly by regulating the microbiota-DMS-IL-22+ILC3 axis, highlighting the potential beneficial role of GLP-RAs in inflammatory intestinal disorders with diabetes complications.

Disease pathogenesis and barrier functions regulated by group 3 innate lymphoid cells.

The mucosal surface is in constant contact with foreign antigens and is regulated by unique mechanisms that are different from immune responses in the peripheral organs. For the last several decades, only adaptive immune cells such as helper T (Th) cells, Th1, Th2, or Th17 were targeted to study a wide variety of immune responses in the mucosal tissues. However, since their discovery, innate lymphoid cells (ILCs) have been attracting attention as a unique subset of immune cells that provide border defense with various functions and tissue specificity. ILCs are classified into different groups based on cell differentiation and functions. Group 3 innate lymphoid cells (ILC3s) are particularly in close proximity to mucosal surfaces and therefore have the opportunity to be exposed to a variety of bacteria including pathogenic bacteria. In recent years, studies have also provided much evidence that ILC3s contribute to disease pathogenesis as well as the defense of mucosal surfaces by rapidly responding to pathogens and coordinating other immune cells. As the counterpart of helper T cells, ILC3s together with other ILC subsets establish the immune balance between adaptive and innate immunity in protecting us from invasion or encounter with non-self-antigens for maintaining a complex homeostasis. In this review, we summarize recent advances in our understanding of ILCs, with a particular focus on the function of ILC3s in their involvement in bacterial infection and disease pathogenesis.

Understanding Type 3 Innate Lymphoid Cells and Crosstalk with the Microbiota: A Skin Connection.

Innate lymphoid cells (ILCs) are a diverse population of lymphocytes classified into natural killer (NK) cells, ILC1s, ILC2s, ILC3s, and ILCregs, broadly following the cytokine secretion and transcription factor profiles of classical T cell subsets. Nonetheless, the ILC lineage does not have rearranged antigen-specific receptors and possesses distinct characteristics. ILCs are found in barrier tissues such as the skin, lungs, and intestines, where they play a role between acquired immune cells and myeloid cells. Within the skin, ILCs are activated by the microbiota and, in turn, may influence the microbiome composition and modulate immune function through cytokine secretion or direct cellular interactions. In particular, ILC3s provide epithelial protection against extracellular bacteria. However, the mechanism by which these cells modulate skin health and homeostasis in response to microbiome changes is unclear. To better understand how ILC3s function against microbiota perturbations in the skin, we propose a role for these cells in response to Cutibacterium acnes, a predominant commensal bacterium linked to the inflammatory skin condition, acne vulgaris. In this article, we review current evidence describing the role of ILC3s in the skin and suggest functional roles by drawing parallels with ILC3s from other organs. We emphasize the limited understanding and knowledge gaps of ILC3s in the skin and discuss the potential impact of ILC3-microbiota crosstalk in select skin diseases. Exploring the dialogue between the microbiota and ILC3s may lead to novel strategies to ameliorate skin immunity.

Cang-ai volatile oil ameliorates imiquimod-induced psoriatic skin lesions by suppressing the ILC3s.

ETHNOPHARMACOLOGICAL RELEVANCE: Cang-ai volatile oil (CAVO) is an aromatic Chinese medicine with potent antibacterial and immune regulatory properties. While CAVO has been used to treat upper respiratory tract infections, depression, otomycosis, and bacterial infections in the skin, its effect on psoriasis is unknown. AIM OF THE STUDY: This study explores the effect and mechanism of CAVO in psoriasis intervention. MATERIAL AND METHODS: The effect of CAVO on the expression of IL-6 and IL-1ß was assessed in TNF-α-induced HaCaT cells using enzyme-linked immunosorbent assay (ELISA). Mice were given imiquimod (IMQ) and administered orally with different CAVO doses (0.03 and 0.06 g/kg) for 5 days. The levels of inflammatory cytokines related to group-3 innate lymphoid cells (ILC3s) in the skin were assessed using hematoxylin and eosin (H&E) staining, ELISA, and western blotting (WB). The frequency of ILC3s in mice splenocytes and skin cells was evaluated using flow cytometry. RESULTS: The results demonstrated that CAVO decreased the expression of IL-6 and IL-1ß in TNF-α- induced HaCaT cells. CAVO significantly reduced the severity of psoriatic symptoms in IMQ-induced mice. The expression of inflammatory cytokines in the skin, such as IL-1ß, IL-6, IL-8, IL-22, IL-23, and IL-17 A were decreased, whereas IL-10 levels were increased. The mRNA expressions of TNF-α, IL-23 A, IL-23 R, IL-22, IL-17 A, and RORγt were down-regulated in skin tissues. CAVO also decreased the levels of NF-κB, STAT3, and JAK2 proteins. CONCLUSIONS: CAVO potentially inhibits ILC3s activation to relieve IMQ-induced psoriasis in mice. These effects might be attributed to inhibiting the activation of NF-κB, STAT3, and JAK2 signaling pathways.

Intestinal factors promoting the development of RORγt+ cells and oral tolerance.

The gastrointestinal tract has to harmonize the two seemingly opposite functions of fulfilling nutritional needs and avoiding the entry of pathogens, toxins and agents that can cause physical damage. This balance requires a constant adjustment of absorptive and defending functions by sensing environmental changes or noxious substances and initiating adaptive or protective mechanisms against them through a complex network of receptors integrated with the central nervous system that communicate with cells of the innate and adaptive immune system. Effective homeostatic processes at barrier sites take the responsibility for oral tolerance, which protects from adverse reactions to food that cause allergic diseases. During a very specific time interval in early life, the establishment of a stable microbiota in the large intestine is sufficient to prevent pathological events in adulthood towards a much larger bacterial community and provide tolerance towards diverse food antigens encountered later in life. The beneficial effects of the microbiome are mainly exerted by innate and adaptive cells that express the transcription factor RORγt, in whose generation, mediated by different bacterial metabolites, retinoic acid signalling plays a predominant role. In addition, recent investigations indicate that food antigens also contribute, analogously to microbial-derived signals, to educating innate immune cells and instructing the development and function of RORγt+ cells in the small intestine, complementing and expanding the tolerogenic effect of the microbiome in the colon. This review addresses the mechanisms through which microbiota-produced metabolites and dietary antigens maintain intestinal homeostasis, highlighting the complementarity and redundancy between their functions.

The crosstalk between ILC3s and adaptive immunity in diseases.

RORγt+ group 3 innate lymphoid cells (ILC3s), the innate counterpart of Th17 cells, are enriched in the mucosal area and lymphoid tissues. ILC3s interact with a variety of cells through their effector molecules and play an important role in the host defense against a spectrum of infections. Recent studies suggest that the extensive crosstalk between ILC3s and adaptive immune cells, especially T cells, is essential for maintaining tissue homeostasis. Here we discuss recent advances in the crosstalk between ILC3s and adaptive immune responses in multiple tissues and diseases. Understanding how ILC3s engage with adaptive immune cells will enhance our comprehension of diseases and facilitate the identification of novel therapeutic targets.

IL-23 receptor signaling licenses group 3-like innate lymphoid cells to restrict a live-attenuated oral Chlamydia vaccine in the gut.

An IFNγ-susceptible mutant of Chlamydia muridarum is attenuated in pathogenicity in the genital tract and was recently licensed as an intracellular Oral vaccine vector or intrOv. Oral delivery of intrOv induces transmucosal protection in the genital tract, but intrOv itself is cleared from the gut (without shedding any infectious particles externally) by IFNγ from group 3-like innate lymphoid cells (ILC3s). We further characterized the intrOv interactions with ILC3s in the current study, since the interactions may impact both the safety and efficacy of intrOv as an oral Chlamydia vaccine. Intracolonic inoculation with intrOv induced IFNγ that in return inhibited intrOv. The intrOv-IFNγ interactions were dependent on RORγt, a signature transcriptional factor of ILC3s. Consistently, the transfer of oral intrOv-induced ILC3s from RORγt-GFP reporter mice to IFNγ-deficient mice rescued the inhibition of intrOv. Thus, IFNγ produced by intrOv-induced ILC3s is likely responsible for inhibiting intrOv, which is further supported by the observation that oral intrOv did induce significant levels of IFNγ-producing LC3s (IFNγ+ILC3s). Interestingly, IL-23 receptor knockout (IL-23R-/-) mice no longer inhibited intrOv, which was accompanied by reduced colonic IFNγ. Transfer of oral intrOv-induced ILC3s rescued the IL-23R-/- mice to inhibit intrOv, validating the dependence of ILC3s on IL-23R signaling for inhibiting intrOv. Clearly, intrOv induces intestinal IFNγ+ILC3s for its own inhibition in the gut, which is facilitated by IL-23R signaling. These findings have provided a mechanism for ensuring the safety of intrOv as an oral Chlamydia vaccine and a platform for investigating how oral intrOv induces transmucosal protection in the genital tract.

A cis-element at the Rorc locus regulates the development of type 3 innate lymphoid cells.

Background: As an important early source of IL-17A and IL-22 in immune responses, type 3 innate lymphoid cells (ILC3s) are critically regulated by the transcription factor retinoic-acid-receptor-related orphan receptor gamma t (RORγt). Previously, we have identified a crucial role of the conserved non-coding sequence 9 (CNS9), located at +5,802 to +7,963 bp of the Rorc gene, in directing T helper 17 differentiation and related autoimmune disease. However, whether cis-acting elements regulate RORγt expression in ILC3s is unknown. Results: Here we show that CNS9 deficiency in mice not only decreases ILC3 signature gene expression and increases ILC1-gene expression features in total ILC3s, but also leads to generation of a distinct CD4+NKp46+ ILC3 population, though the overall numbers and frequencies of RORγt+ ILC3s are not affected. Mechanistically, CNS9 deficiency selectively decreases RORγt expression in ILC3s, which thus alters ILC3 gene expression features and promotes cell-intrinsic generation of CD4+NKp46+ ILC3 subset. Conclusion: Our study thus identifies CNS9 as an essential cis-regulatory element controlling the lineage stability and plasticity of ILC3s through modulating expression levels of RORγt protein.

ILC3s restrict the dissemination of intestinal bacteria to safeguard liver regeneration after surgery.

It is generally believed that environmental or cutaneous bacteria are the main origin of surgical infections. Therefore, measures to prevent postoperative infections focus on optimizing hygiene and improving asepsis and antisepsis. In a large cohort of patients with infections following major surgery, we identified that the causative bacteria are mainly of intestinal origin. Postoperative infections of intestinal origin were also found in mice undergoing partial hepatectomy. CCR6+ group 3 innate lymphoid cells (ILC3s) limited systemic bacterial spread. Such bulwark function against host invasion required the production of interleukin-22 (IL-22), which controlled the expression of antimicrobial peptides in hepatocytes, thereby limiting bacterial spread. Using genetic loss-of-function experiments and punctual depletion of ILCs, we demonstrate that the failure to restrict intestinal commensals by ILC3s results in impaired liver regeneration. Our data emphasize the importance of endogenous intestinal bacteria as a source for postoperative infection and indicate ILC3s as potential new targets.

Regulation of Yujin Powder alcoholic extracts on ILC3s-TD IgA-colonic mucosal flora axis of DSS-induced ulcerative colitis.

The intestinal flora maintained by the immune system plays an important role in healthy colon. However, the role of ILC3s-TD IgA-colonic mucosal flora axis in ulcerative colitis (UC) and whether it could become an innovative pathway for the treatment of UC is unknown. Yujin Powder is a classic prescription for treatment of dampness-heat type intestine disease in traditional Chinese medicine and has therapeutic effects on UC. Hence, the present study aimed to investigate the regulatory mechanism of Yujin Powder alcoholic extracts (YJP-A) on UC via ILC3s-TD IgA-colonic mucosal flora axis. The UC mouse model was induced by drinking 3.5% dextran sodium sulfate (DSS), meanwhile, YJP-A was given orally for prevention. During the experiment, the clinical symptoms of mice were recorded. Then the intestinal injury and inflammatory response of mice about UC were detected after the experiment. In addition, the relevant indicators of ILC3s-TD IgA-colonic mucosal flora axis were detected. The results showed that YJP-A had good therapy effects on DSS-induced mice UC: improved the symptoms, increased body weight and the length of colon, decreased the disease activity index score, ameliorated the intestinal injury, and reduced the inflammation etc. Also, YJP-A significantly increased the ILC3s proportion and the expression level of MHC II; significantly decreased the proportion of Tfh cells and B cells and the expression levels of Bcl6, IL-4, Aicda in mesenteric lymph nodes of colon in UC mice and IgA in colon. In addition, by 16S rDNA sequencing, YJP-A could restore TD IgA targets colonic mucus flora in UC mice by decreasing the relative abundance of Mucispirillum, Lachnospiraceae and increasing the relative abundance of Allprevotella, Alistipes, and Ruminococcaceae etc. In conclusion, our results demonstrated that the ILC3s-TD IgA-colonic mucosal flora axis was disordered in UC mice. YJP-A could significantly promote the proliferation of ILC3s to inhibit Tfh responses and B cells class switching through MHC II, further to limit TD IgA responses toward colonic mucosal flora. Our findings suggested that this axis may be a novel and promising strategy to prevent UC.

The Transcription Factor ThPOK Regulates ILC3 Lineage Homeostasis and Function During Intestinal Infection.

Innate lymphoid cells (ILCs) have been identified as a heterogeneous population of lymphocytes that mirrors the cytokine and transcriptional profile of adaptive T cells. The dynamic balance between key transcription factors determines the heterogeneity, plasticity, and functions of ILC subsets. The transcription factor ThPOK is highly conserved in biological evolution and exerts pivotal functions in the differentiation of T cells. However, the function of ThPOK in ILC3s has not been identified. Here, we found that ThPOK regulated the homeostasis of ILC3s, as mice lacking ThPOK showed decreased NKp46+ ILC3s and increased CCR6- NKp46- ILC3s. ThPOK-deficient mice were more sensitive to S. typhimurium infection due to the impaired IFN-γ secretion of NKp46+ ILC3s. Furthermore, ThPOK participates in ILC3-mediated control of C. rodentium infection by negatively regulating IL-17A secretion. ThPOK preserves the identity of NKp46+ ILC3s by repressing RORγt, which indirectly releases T-bet expression. On the molecular level, ThPOK directly binds to Rorc and Il23r to restrain their expression which further modulates IL-17A secretion. Collectively, our analysis revealed a critical role of ThPOK in the homeostasis and functions of ILC3 subsets.

Crosstalk between macrophages and innate lymphoid cells (ILCs) in diseases.

Innate lymphoid cells (ILCs) and macrophages are tissue-resident cells that play important roles in tissue-immune homeostasis and immune regulation. ILCs are mainly distributed on the barrier surfaces of mammals to ensure immunity or tissue homeostasis following host, microbial, or environmental stimulation. Their complex relationships with different organs enable them to respond quickly to disturbances in environmental conditions and organ homeostasis, such as during infections and tissue damage. Gradually emerging evidence suggests that ILCs also play complex and diverse roles in macrophage development, homeostasis, polarization, inflammation, and viral infection. In turn, macrophages also determine the fate of ILCs to some extent, which indicates that network crossover between these interactions is a key determinant of the immune response. More work is needed to better define the crosstalk of ILCs with macrophages in different tissues and demonstrate how it is affected during inflammation and other diseases. Here, we summarize current research on the functional interactions between ILCs and macrophages and consider the potential therapeutic utility of these interactions for the benefit of human health.

A Japanese Herbal Formula, Daikenchuto, Alleviates Experimental Colitis by Reshaping Microbial Profiles and Enhancing Group 3 Innate Lymphoid Cells.

Daikenchuto (DKT) is one of the most widely used Japanese herbal formulae for various gastrointestinal disorders. It consists of Zanthoxylum Fructus (Japanese pepper), Zingiberis Siccatum Rhizoma (processed ginger), Ginseng radix, and maltose powder. However, the use of DKT in clinical settings is still controversial due to the limited molecular evidence and largely unknown therapeutic effects. Here, we investigated the anti-inflammatory actions of DKT in the dextran sodium sulfate (DSS)-induced colitis model in mice. We observed that DKT remarkably attenuated the severity of experimental colitis while maintaining the members of the symbiotic microbiota such as family Lactobacillaceae and increasing levels of propionate, an immunomodulatory microbial metabolite, in the colon. DKT also protected colonic epithelial integrity by upregulating the fucosyltransferase gene Fut2 and the antimicrobial peptide gene Reg3g. More remarkably, DKT restored the reduced colonic group 3 innate lymphoid cells (ILC3s), mainly RORγthigh-ILC3s, in DSS-induced colitis. We further demonstrated that ILC3-deficient mice showed increased mortality during experimental colitis, suggesting that ILC3s play a protective function on colonic inflammation. These findings demonstrate that DKT possesses anti-inflammatory activity, partly via ILC3 function, to maintain the colonic microenvironment. Our study also provides insights into the molecular basis of herbal medicine effects, promotes more profound mechanistic studies towards herbal formulae and contributes to future drug development.

Interleukin-1ß secretion induced by mucosa-associated gut commensal bacteria promotes intestinal barrier repair.

The gut microbiota is essential for maintenance and repair of the intestinal epithelial barrier. As shifts in both intestinal epithelial barrier function and microbiota composition are found in inflammatory bowel disease patients, it is critical to understand the role of distinct bacteria in regulating barrier repair. We identified a mouse commensal E. coli isolate, GDAR2-2, that protects mice from Citrobacter rodentium infection and dextran sulfate sodium-induced colitis. Colonization with GDAR2-2 in mice resulted in expansion of CX3CR1+ mononuclear phagocytes, including CX3CR1+ macrophages/dendritic cells and monocytes, along with IL-22-secreting type 3 innate lymphoid cells and improved epithelial barrier function. In vitro co-culture of macrophages with GDAR2-2 resulted in IL-1ß production. In vivo, protection after GDAR2-2 colonization was lost after depletion of CX3CR1+ MNPs, or blockade of IL-1ß or IL-22. We further identified human commensal E. coli isolates that similarly protect mice from C. rodentium infection through CX3CR1+ MNP and IL-1ß production. Together, these findings demonstrate an unexpected role for commensal bacteria in promoting IL-1ß secretion to support intestinal barrier repair.

ILC3s control airway inflammation by limiting T cell responses to allergens and microbes.

Group 3 innate lymphoid cells (ILC3s) critically regulate host-microbe interactions in the gastrointestinal tract, but their role in the airway remains poorly understood. Here, we demonstrate that lymphoid-tissue-inducer (LTi)-like ILC3s are enriched in the lung-draining lymph nodes of healthy mice and humans. These ILC3s abundantly express major histocompatibility complex class II (MHC class II) and functionally restrict the expansion of allergen-specific CD4+ T cells upon experimental airway challenge. In a mouse model of house-dust-mite-induced allergic airway inflammation, MHC class II+ ILC3s limit T helper type 2 (Th2) cell responses, eosinophilia, and airway hyperresponsiveness. Furthermore, MHC class II+ ILC3s limit a concomitant Th17 cell response and airway neutrophilia. This exacerbated Th17 cell response requires exposure of the lung to microbial stimuli, which can be found associated with house dust mites. These findings demonstrate a critical role for antigen-presenting ILC3s in orchestrating immune tolerance in the airway by restricting pro-inflammatory T cell responses to both allergens and microbes.

Type 3 Innate Lymphoid Cells as Regulators of the Host-Pathogen Interaction.

Type 3 Innate lymphoid cells (ILC3s) have been described as tissue-resident cells and characterized throughout the body, especially in mucosal sites and classical first barrier organs such as skin, gut and lungs, among others. A significant part of the research has focused on their role in combating pathogens, mainly extracellular pathogens, with the gut as the principal organ. However, some recent discoveries in the field have unveiled their activity in other organs, combating intracellular pathogens and as part of the response to viruses. In this review we have compiled the latest studies on the role of ILC3s and the molecular mechanisms involved in defending against different microbes at the mucosal surface, most of these studies have made use of conditional transgenic mice. The present review therefore attempts to provide an overview of the function of ILC3s in infections throughout the body, focusing on their specific activity in different organs.

Adoptive Transfer of Group 3-Like Innate Lymphoid Cells Restores Mouse Colon Resistance to Colonization of a Gamma Interferon-Susceptible Chlamydia muridarum Mutant.

The obligate intracellular bacterium Chlamydia muridarum can colonize the mouse colon for a long period, but a gamma interferon (IFN-γ)-susceptible mutant clone fails to do so. Nevertheless, the mutant's colonization is rescued in mice deficient in interleukin-7 receptor (IL-7R) (lacking both lymphocytes and innate lymphoid cells [ILCs]) or IFN-γ but not in mice lacking recombination-activated gene 1 (Rag1-/- mice) (lacking adaptive immunity lymphocytes), indicating a critical role of ILC-derived IFN-γ in regulating chlamydial colonization. In the current study, we have used an adoptive transfer approach for further characterizing the responsible ILCs. First, intestinal ILCs isolated from Rag1-/- mice were able to rescue IL-7R-deficient mice to restrict the colonization of the IFN-γ-susceptible Chlamydia muridarum mutant. Second, the responsible ILCs were localized to the intestinal lamina propria since ILCs from the lamina propria but not the intraepithelial compartment conferred the restriction. Third, lamina propria ILCs enriched for RORγt expression but not those negative for RORγt rescued the IL-7R-deficient mice to restrict mutant colonization, indicating a critical role of group 3-like ILCs (ILC3s) since RORγt is a signature transcriptional factor of ILC3s. Fourth, a portion of the ILC3s expressed IFN-γ, thus defined as ex-ILC3s, and the transfer of the ex-ILC3s conferred colon resistance to mutant Chlamydia muridarum colonization in IFN-γ-deficient mice. Finally, genetically labeled RORγt-positive (RORγt+) ILCs were able to inhibit mutant colonization. Thus, we have demonstrated that ILC3s are sufficient for regulating chlamydial colonization, laying a foundation for further revealing the mechanisms by which an obligate intracellular bacterium activates colonic ILC3s.

DOCK8 controls survival of group 3 innate lymphoid cells in the gut through Cdc42 activation.

Innate lymphoid cells (ILCs) are a family of developmentally related leukocytes that rapidly secrete polarized sets of cytokines to combat infection and promote tissue repair at mucosal barriers. Among them, group 3 ILCs (ILC3s) play an important role in maintenance of the gut homeostasis by producing IL-22, and their development and function critically depend on the transcription factor RORγt. Although recent evidence indicates that RORγt+ ILC3s are reduced in the gut in the absence of the Cdc42 activator DOCK8 (dedicator of cytokinesis 8), the underlying mechanism remains unclear. We found that genetic deletion of Dock8 in RORγt+-lineage cells markedly reduced ILC3s in the lamina propria of the small intestine. By analyzing BrdU incorporation, it was revealed that DOCK8 deficiency did not affect the cell proliferation. Furthermore, when lineage marker-negative (Lin-) α4ß7+ CD127+ RORγt- fetal liver cells were cultured with OP9 stromal cells in the presence of stem cell factor (SCF) and IL-7 in vitro, RORγt+ ILC3s normally developed irrespective of DOCK8 expression. However, DOCK8-deficient ILC3s exhibited a severe defect in survival of ILC3s under the condition with or without IL-7. Similar defects were observed when we analyzed Dock8VAGR mice having mutations in the catalytic center of DOCK8, thereby failing to activate Cdc42. Thus, DOCK8 acts in cell-autonomous manner to control survival of ILC3s in the gut through Cdc42 activation.