Polyamines and hypusination are important for Clostridioides difficile toxin B (TcdB)-mediated activation of group 3 innate lymphocytes (ILC3s).

Clostridioides difficile is the most common cause of nosocomial gastrointestinal tract bacterial infections. We lack fully effective reliable treatments for this pathogen, and there is a critical need to better understand how C. difficile interacts with our immune system. Group 3 innate lymphocytes (ILC3s) are rare immune cells localized within mucosal tissues that protect against bacterial infections. Upon activation, ILC3s secrete high levels of the cytokine interleukin-22 (IL-22), which is a critical regulator of tissue responses during infection. C. difficile toxin B (TcdB), the major virulence factor, directly activates ILC3s, resulting in high IL-22 levels. We previously reported that polyamines are important in the activation of ILC3s by the innate cytokine interleukin-23 (IL-23) but did not identify a specific mechanism. In this study, we examine how a pathogen impacts a metabolic pathway important for immune cell function and hypothesized that polyamines are important in TcdB-mediated ILC3 activation. We show that TcdB upregulates the polyamine biosynthesis pathway, and the inhibition of the pathway decreases TcdB-mediated ILC3 activation. Two polyamines, putrescine and spermidine, are involved. Spermidine is the key polyamine in the hypusination of eukaryotic initiation factor 5A (eIF5A), and the inhibition of eIF5A reduced ILC3 activation. Thus, there is potential to leverage polyamines in ILC3s to promote activation of ILC3s during C. difficile infection and other bacterial infections where ILC3s serve a protective role.

Modulation of innate lymphoid cells by enteric bacterial pathogens.

Innate lymphoid cells (ILCs) are key regulators of tissue homeostasis, inflammation, and immunity to infections. ILCs rapidly respond to environmental cues such as cytokines, microbiota and invading pathogens which regulate their function and phenotype. Even though ILCs are rare cells, they are enriched at barrier surfaces such as the gastrointestinal (GI) tract, and they are often critical to the host's immune response to eliminate pathogens. On the other side of host-pathogen interactions, pathogenic bacteria also have the means to modulate these immune responses. Manipulation or evasion of the immune cells is often to the pathogen's benefit and/or to the detriment of competing microbiota. In some instances, specific bacterial virulence factors or toxins have been implicated in how the pathogen modulates immunity. In this review, we discuss the recent progress made towards understanding the role of non-cytotoxic ILCs during enteric bacterial infections, how these pathogens can modulate the immune response, and the implications these have on developing new therapies to combat infection.

Cx3cr1 controls kidney resident macrophage heterogeneity.

Kidney macrophages are comprised of both monocyte-derived and tissue resident populations; however, the heterogeneity of kidney macrophages and factors that regulate their heterogeneity are poorly understood. Herein, we performed single cell RNA sequencing (scRNAseq), fate mapping, and parabiosis to define the cellular heterogeneity of kidney macrophages in healthy mice. Our data indicate that healthy mouse kidneys contain four major subsets of monocytes and two major subsets of kidney resident macrophages (KRM) including a population with enriched Ccr2 expression, suggesting monocyte origin. Surprisingly, fate mapping data using the newly developed Ms4a3Cre Rosa Stopf/f TdT model indicate that less than 50% of Ccr2+ KRM are derived from Ly6chi monocytes. Instead, we find that Ccr2 expression in KRM reflects their spatial distribution as this cell population is almost exclusively found in the kidney cortex. We also identified Cx3cr1 as a gene that governs cortex specific accumulation of Ccr2+ KRM and show that loss of Ccr2+ KRM reduces the severity of cystic kidney disease in a mouse model where cysts are mainly localized to the kidney cortex. Collectively, our data indicate that Cx3cr1 regulates KRM heterogeneity and niche-specific disease progression.

The Polyamine Putrescine Is a Positive Regulator of Group 3 Innate Lymphocyte Activation.

Group 3 innate lymphocytes (ILC3s) rapidly respond to invading pathogens or inflammatory signals, which requires shifting cellular metabolic demands. Metabolic adaptations regulating ILC3 function are not completely understood. Polyamines are polycationic metabolites that have diverse roles in cellular functions and in immunity regulate immune cell biology, including Th17 cells. Whether polyamines play a role in ILC3 activation is unknown. In this article, we report that the polyamine synthesis pathway is important for ILC3 activation. IL-23-activated mouse ILC3s upregulate ornithine decarboxylase, the enzyme catalyzing the rate-limiting step of the conversion of ornithine to putrescine in polyamine synthesis, with a subsequent increase in putrescine levels. Inhibition of ornithine decarboxylase via a specific inhibitor, α-difluoromethylornithine, reduced levels of IL-22 produced by steady-state or IL-23-activated ILC3s in a putrescine-dependent manner. Thus, the polyamine putrescine is a positive regulator of ILC3 activation. Our results suggest that polyamines represent a potential target for therapeutic modulation of ILC3 activation during infection or inflammatory disorders.

Clostridioides difficile Toxin B Activates Group 3 Innate Lymphocytes.

Group 3 innate lymphocytes (ILC3s) are rare immune cells localized in mucosal tissues, especially the gastrointestinal (GI) tract. Despite their rarity, they are a major source of the cytokine interleukin-22 (IL-22), which protects the GI epithelium during inflammation and infection. Although ILC3s have been demonstrated to be important for defense against Clostridioides difficile infection, the exact mechanisms through which they sense productive infection and become activated to produce IL-22 remain poorly understood. In this study, we identified a novel mechanism of ILC3 activation after exposure to C. difficile. Toxin B (TcdB) from C. difficile directly induced production of IL-22 in ILC3s, and this induction was dependent on the glucosyltransferase activity of the toxin, which inhibits small GTPases. Pharmacological inhibition of the small GTPase Cdc42 also enhanced IL-22 production in ILC3s, indicating that Cdc42 is a negative regulator of ILC3 activation. Further gene expression analysis revealed that treatment with TcdB modulated the expression of several inflammation-related genes in ILC3s. These findings demonstrate that C. difficile toxin-mediated inhibition of Cdc42 leads to the activation of ILC3s, providing evidence for how these cells are recruited into the immune response against the pathobiont.

Group 3 innate lymphocytes (ILC3s) upregulate IL-22 in response to elevated intracellular cAMP levels.

Group 3 innate lymphocytes (ILC3s) are important immune cells within mucosal tissues and protect against bacterial infections. They can be activated in response to the innate cytokines IL-23 or IL-1ß, which rapidly increases their production of effector molecules that regulate barrier functions. Pathogens can subvert these anti-bacterial effects to evade mucosal defenses to infect the host. Bacillus anthracis, the causative agent of anthrax, produces two major toxins that can modulate the immune response. We have previously shown that lethal toxin downmodulates the function of ILC3s. On the other hand, edema toxin has been shown promote T helper 17 (Th17) cell differentiation, adaptive counterparts of ILC3s, via elevation of cyclic adenosine monophosphate (cAMP). We hypothesized that edema toxin may also modulate ILC3 function. In this study, we show that edema toxin has the opposite effect of lethal toxin; edema toxin directly activates ILC3s independently of innate cytokine stimulation. Treatment of a mouse ILC3-like cell line with edema toxin, a potent adenylate cyclase, upregulated production of the cytokine IL-22, a major effector molecule of ILC3s and a critical factor in maintaining mucosal barriers. Forskolin treatment phenocopied the effect observed with edema toxin and led to an increase in CREB phosphorylation in ILC3s. This observation has potential implications for a role for cAMP signaling in the activation of ILC3s.

IL-22 Binding Protein (IL-22BP) in the Regulation of IL-22 Biology.

Cytokines are powerful mediators of inflammation. Consequently, their potency is regulated in many ways to protect the host. Several cytokines, including IL-22, have coordinating binding proteins or soluble receptors that bind to the cytokine, block the interaction with the cellular receptor, and thus prevent cellular signaling. IL-22 is a critical cytokine in the modulation of tissue responses during inflammation and is highly upregulated in many chronic inflammatory disease patients, including those with psoriasis, rheumatoid arthritis, and inflammatory bowel disease (IBD). In healthy individuals, low levels of IL-22 are secreted by immune cells, mainly in the gastrointestinal (GI) tract. However, much of this IL-22 is likely not biologically active due to the high levels of IL-22 binding protein (IL-22BP) produced by intestinal dendritic cells (DCs). IL-22BP is a soluble receptor homolog that binds to IL-22 with greater affinity than the membrane spanning receptor. Much is known regarding the regulation and function of IL-22 in health and disease. However, less is known about IL-22BP. In this review, we will focus on IL-22BP, including its regulation, role in IL-22 biology and inflammation, and promise as a therapeutic. IL-22 can be protective or pathogenic, depending on the context of inflammation. IL-22BP also has divergent roles. Ongoing and forthcoming studies will expand our knowledge of IL-22BP and IL-22 biology, and suggest that IL-22BP holds promise as a way to regulate IL-22 biology in patients with chronic inflammatory disease.

E3 Ubiquitin Ligase Von Hippel-Lindau Protein Promotes Th17 Differentiation.

Von Hippel-Lindau (VHL) is an E3 ubiquitin ligase that targets proteins, including HIF-1α, for proteasomal degradation. VHL and HIF regulate the balance between glycolysis and oxidative phosphorylation, which is critical in highly dynamic T cells. HIF-1α positively regulates Th17 differentiation, a complex process in which quiescent naive CD4 T cells undergo transcriptional changes to effector cells, which are commonly dysregulated in autoimmune diseases. The role of VHL in Th17 cells is not known. In this study, we hypothesized VHL negatively regulates Th17 differentiation and deletion of VHL in CD4 T cells would elevate HIF-1α and increase Th17 differentiation. Unexpectedly, we found that VHL promotes Th17 differentiation. Mice deficient in VHL in their T cells were resistant to an autoimmune disease, experimental autoimmune encephalomyelitis, often mediated by Th17 cells. In vitro Th17 differentiation was impaired in VHL-deficient T cells. In the absence of VHL, Th17 cells had decreased activation of STAT3 and SMAD2, suggesting that VHL indirectly or directly regulates these critical signaling molecules. Gene expression analysis revealed that in Th17 cells, VHL regulates many cellular pathways, including genes encoding proteins involved indirectly or directly in the glycolysis pathway. Compared with wild-type, VHL-deficient Th17 cells had elevated glycolysis and glycolytic capacity. Our finding has implications on the design of therapeutics targeting the distinct metabolic needs of T cells to combat chronic inflammatory diseases.

Glucocorticoids Inhibit Group 3 Innate Lymphocyte IL-22 Production.

Glucocorticoids (GCs) are commonly prescribed to patients with a variety of inflammatory disorders, including inflammatory bowel disease (IBD). GCs mediate their immunomodulatory effects through many different mechanisms and target multiple signaling pathways. The GC dexamethasone downmodulates innate and adaptive immune cell activation. IBD is the manifestation of a dysregulated immune response involving many different immune cells. Group 3 innate lymphocytes (ILC3s) have critical roles in mucosal inflammation. ILC3s secrete high levels of the cytokine IL-22, promoting epithelial proliferation, antimicrobial peptides, and mucins. In this study, we examined the effects of dexamethasone on IL-22 production by ILC3s. We found that dexamethasone suppressed IL-23-mediated IL-22 production in human and mouse ILC3s. This was mediated in part through dexamethasone modulation of the NF-κB pathway. Inhibition of NF-κB signaling with a small molecule inhibitor also downmodulated IL-23- and IL-1ß-mediated IL-22 production in ILC3s. These findings implicate NF-κB as a regulator of IL-22 in ILC3s and likely have repercussions on GC treatment of IBD patients.

IL-22 deficiency increases CD4 T cell responses to mucosal immunization.

Mucosal vaccines are a promising platform for combatting infectious diseases for which we still lack effective preventative measures. Optimizing these vaccines to generate the best protective immune responses with the least complicated immunization regimen is imperative. Mucosal barriers are the first line of defense against many pathogens and, as such, we looked to their biology for strategies to improve vaccine delivery. Interleukin-22 (IL-22) is a key cytokine in both healthy and inflamed mucosal tissues. IL-22 promotes epithelial cell proliferation and inhibits apoptosis, upregulates mucin and antimicrobial peptides, all of which promote mucosal barrier integrity. In this study, we find that IL-22 impairs the development of a T cell response during mucosal immunization. Compared to wild-type control mice, IL-22 deficient mice had increased antigen-specific CD4 T cell responses to intrarectal immunization using a protein and cholera toxin adjuvant vaccine. When immunized systemically with the same protein antigen adsorbed to alum, no differences in the CD4 T cell response between wild-type and IL-22 deficient mice were detected. This suggests that transiently inhibiting IL-22 during mucosal vaccination could enhance T cell responses. The broad-applicability of this proposed approach would allow for improvement of many existing mucosal vaccine regimens and have positive implications in the development of more efficacious mucosal vaccines.

IL-22: There Is a Gap in Our Knowledge.

IL-22 is a critical cytokine in modulating tissue responses during inflammation. IL-22 is upregulated in many chronic inflammatory diseases, making IL-22 biology a potentially rewarding therapeutic target. However, this is complicated by the dual-natured role of IL-22 in inflammation, as the cytokine can be protective or inflammatory depending on the disease model. Although scientific interest in IL-22 has increased considerably in the past 10 y, there is still much we do not know about the environmental, cellular, and molecular factors that regulate the production and function of this cytokine. A better understanding of IL-22 biology will allow us to develop new or improved therapeutics for treating chronic inflammatory diseases. In this article, I will highlight some of the outstanding questions in IL-22 biology.

Bacillus anthracis lethal toxin negatively modulates ILC3 function through perturbation of IL-23-mediated MAPK signaling.

Bacillus anthracis, the causative agent of anthrax, secretes lethal toxin that down-regulates immune functions. Translocation of B. anthracis across mucosal epithelia is key for its dissemination and pathogenesis. Group 3 innate lymphocytes (ILC3s) are important in mucosal barrier maintenance due to their expression of the cytokine IL-22, a critical regulator of tissue responses and repair during homeostasis and inflammation. We found that B. anthracis lethal toxin perturbed ILC3 function in vitro and in vivo, revealing an unknown IL-23-mediated MAPK signaling pathway. Lethal toxin had no effects on the canonical STAT3-mediated IL-23 signaling pathway. Rather lethal toxin triggered the loss of several MAP2K kinases, which correlated with reduced activation of downstream ERK1/2 and p38, respectively. Inhibition studies showed the importance of MAPK signaling in IL-23-mediated production of IL-22. Our finding that MAPK signaling is required for optimal IL-22 production in ILC3s may lead to new approaches for targeting IL-22 biology.

Hypoxic modulation of hepatocyte responses to the cytokine interleukin-22.

The cytokine interleukin-22 (IL-22) is a potent regulator of tissue responses during inflammation. Depending on the context of inflammation, IL-22 can have protective or inflammatory effects on epithelial cells. This dual nature of IL-22 leads us to hypothesize that its activity must be exquisitely regulated to prevent host tissue damage. Environmental factors may act as a cellular cue as to how cells respond to IL-22. Inflammatory environments are characterized by low oxygen and thus we examined whether cells respond differently to IL-22 hypoxia compared with normoxia. In this study, we show that hepatocyte responses to IL-22 stimulation are reduced in hypoxic environments. IL-22 stimulation of hepatocytes incubated in low oxygen led to reduced levels of activated signal transducer and activator of transcription 3 and further downstream effects such as reduced induction of the anti-microbial protein, lipocalin-2. This modulation appears to be independent of the hypoxia-inducible factor-1α signaling pathway. Thus, hypoxia that accompanies chronic inflammation may be a mechanism to regulate the bioactivity of the dual-natured IL-22 cytokine.

Transcription Factor HIF-1α Controls Expression of the Cytokine IL-22 in CD4 T Cells.

IL-22 is expressed by activated lymphocytes and is important in modulation of tissue responses during inflammation. The cytokine induces proliferative and antiapoptotic pathways in epithelial cells allowing enhanced cell survival. This can have positive effects, such as in the maintenance of epithelial barriers in the gastrointestinal tract, but also negative effects, such as contributing to colorectal tumorigenesis. Because IL-22 can be dual-natured, we hypothesized that its biological activity should be tightly regulated to limit IL-22 expression to the sites of inflammation. One such environmental cue could be low oxygen, which often accompanies inflammation. We show that in CD4 T cells IL-22 expression is upregulated in hypoxia. The Il22 promoter contains a putative conserved hypoxic response element suggesting that the transcription factor HIF-1α may influence IL-22 expression. Differentiation in the presence of dimethyloxallyl glycine, a stabilizer of HIF-1α at normoxia, increased IL-22 expression. Using HIF-1α-deficient CD4 T cells, we show that hypoxic IL-22 upregulation is dependent on HIF-1α. These findings have implications on the regulation of Il22 gene expression and the presence of the cytokine in different inflammatory environments.

Defective Intestinal Mucin-Type O-Glycosylation Causes Spontaneous Colitis-Associated Cancer in Mice.

BACKGROUND & AIMS: Core 1- and core 3-derived mucin-type O-linked oligosaccharides (O-glycans) are major components of the colonic mucus layer. Defective forms of colonic O-glycans, such as the Thomsen-nouveau (Tn) antigen, frequently are observed in patients with ulcerative colitis and colorectal cancer, but it is not clear if they contribute to their pathogenesis. We investigated whether and how impaired O-glycosylation contributes to the development of colitis-associated colorectal cancer using mice lacking intestinal core 1- and core 3-derived O-glycans. METHODS: We generated mice that lack core 1- and core 3-derived intestinal O-glycans (DKO mice) and analyzed them, along with mice that singly lack intestinal epithelial core 1 O-glycans (IEC C1galt1(-/-) mice) or core 3 O-glycans (C3Gnt(-/-) mice). Intestinal tissues were collected at different time points and analyzed for levels of mucin and Tn antigen, development of colitis, and tumor formation using imaging, quantitative polymerase chain reaction, immunoblot, and enzyme-linked immunosorbent assay techniques. We also used cellular and genetic approaches, as well as intestinal microbiota depletion, to identify inflammatory mediators and pathways that contribute to disease in DKO and wild-type littermates (controls). RESULTS: Intestinal tissues from DKO mice contained higher levels of Tn antigen and had more severe spontaneous chronic colitis than tissues from IEC C1galt1(-/-) mice, whereas spontaneous colitis was absent in C3GnT(-/-) and control mice. IEC C1galt1(-/-) mice and DKO mice developed spontaneous colorectal tumors, although the onset of tumors in the DKO mice occurred earlier (age, 8-9 months) than that in IEC C1galt1(-/-) mice (15 months old). Antibiotic depletion of the microbiota did not cause loss of Tn antigen but did reduce the development of colitis and cancer formation in DKO mice. Colon tissues from DKO mice, but not control mice, contained active forms of caspase 1 and increased caspase 11, which were reduced after antibiotic administration. Supernatants from colon tissues of DKO mice contained increased levels of interleukin-1ß and interleukin-18, compared with those from control mice. Disruption of the caspase 1 and caspase 11 genes in DKO mice (DKO/Casp1/11(-/-) mice) decreased the development of colitis and cancer, characterized by reduced colonic thickening, hyperplasia, inflammatory infiltrate, and tumors compared with DKO mice. CONCLUSIONS: Impaired expression of O-glycans causes colonic mucus barrier breach and subsequent microbiota-mediated activation of caspase 1-dependent inflammasomes in colonic epithelial cells of mice. These processes could contribute to colitis-associated colon cancer in humans.

Excessive Th1 responses due to the absence of TGF-ß signaling cause autoimmune diabetes and dysregulated Treg cell homeostasis.

TGF-ß signaling in T cells is critical for peripheral T-cell tolerance by regulating effector CD4(+) T helper (Th) cell differentiation. However, it is still controversial to what extent TGF-ß signaling in Foxp3(+) regulatory T (Treg) cells contributes to immune homeostasis. Here we showed that abrogation of TGF-ß signaling in thymic T cells led to rapid type 1 diabetes (T1D) development in NOD mice transgenic for the BDC2.5 T-cell receptor. Disease development in these mice was associated with increased peripheral Th1 cells, whereas Th17 cells and Foxp3(+) Treg cells were reduced. Blocking of IFN-γ signaling alone completely suppressed diabetes development in these mice, indicating a critical role of Th1 cells in this model. Furthermore, deletion of TGF-ß signaling in peripheral effector CD4(+) T cells, but not Treg cells, also resulted in rapid T1D development, suggesting that conventional CD4(+) T cells are the main targets of TGF-ß to suppress T1D. TGF-ß signaling was dispensable for Treg cell function, development, and maintenance, but excessive IFN-γ production due to the absence of TGF-ß signaling in naive CD4(+) T cells indirectly caused dysregulated Treg cell homeostasis. We further showed that T cell-derived TGF-ß1 was critical for suppression of Th1 cell differentiation and T1D development. These results indicate that autocrine/paracrine TGF-ß signaling in diabetogenic CD4(+) T cells, but not Treg cells, is essential for controlling T1D development.

IL-22 deficiency alters colonic microbiota to be transmissible and colitogenic.

IL-22 is a good candidate to play a critical role in regulating gut microbiota because it is an important inducer of antimicrobial peptides and mucins in the gut. However, whether IL-22 participates in immune homeostasis by way of modulating gut microbiota remains to be elucidated. In this study, we find, through 16S rRNA gene-pyrosequencing analysis, that healthy IL-22-deficient mice had altered colonic microbiota, notably with decreased abundance of some genera, including Lactobacillus, and increased levels of others. Mice harboring this altered microbiota exhibited more severe disease during experimentally induced colitis. Interestingly, this altered gut microbiota can be transmitted to cohoused wild-type animals along with the increased susceptibility to this colitis, indicating an important role for IL-22 in shaping the homeostatic balance between immunity and colonic microbiota for host health.

IL-22BP is regulated by the inflammasome and modulates tumorigenesis in the intestine.

Chronic mucosal inflammation and tissue damage predisposes patients to the development of colorectal cancer. This association could be explained by the hypothesis that the same factors and pathways important for wound healing also promote tumorigenesis. A sensor of tissue damage should induce these factors to promote tissue repair and regulate their action to prevent development of cancer. Interleukin 22 (IL-22), a cytokine of the IL-10 superfamily, has an important role in colonic epithelial cell repair, and its levels are increased in the blood and intestine of inflammatory bowel disease patients. This cytokine can be neutralized by the soluble IL-22 receptor, known as the IL-22 binding protein (IL-22BP, also known as IL22RA2); however, the significance of endogenous IL-22BP in vivo and the pathways that regulate this receptor are unknown. Here we describe that IL-22BP has a crucial role in controlling tumorigenesis and epithelial cell proliferation in the colon. IL-22BP is highly expressed by dendritic cells in the colon in steady-state conditions. Sensing of intestinal tissue damage via the NLRP3 or NLRP6 inflammasomes led to an IL-18-dependent downregulation of IL-22BP, thereby increasing the ratio of IL-22/IL-22BP. IL-22, which is induced during intestinal tissue damage, exerted protective properties during the peak of damage, but promoted tumour development if uncontrolled during the recovery phase. Thus, the IL-22-IL-22BP axis critically regulates intestinal tissue repair and tumorigenesis in the colon.