Gene coexpression networks reveal a broad role for lncRNAs in inflammatory bowel disease.

The role of long noncoding RNAs (lncRNAs) in disease is incompletely understood, but their regulation of inflammation is increasingly appreciated. We addressed the extent of lncRNA involvement in inflammatory bowel disease (IBD) using biopsy-derived RNA-sequencing data from a large cohort of deeply phenotyped patients with IBD. Weighted gene correlation network analysis revealed gene modules of lncRNAs coexpressed with protein-coding genes enriched for biological pathways, correlated with epithelial and immune cell signatures, or correlated with distal colon expression. Correlation of modules with clinical features uncovered a module correlated with disease severity, with an enriched interferon response signature containing the hub lncRNA IRF1-AS1. Connecting genes to IBD-associated single nucleotide polymorphisms (SNPs) revealed an enrichment of SNP-adjacent lncRNAs in biologically relevant modules. Ulcerative colitis-specific SNPs were enriched in distal colon-related modules, suggesting that disease-specific mechanisms may result from altered lncRNA expression. The function of the IBD-associated SNP-adjacent lncRNA IRF1-AS1 was explored in human myeloid cells, and our results suggested IRF1-AS1 promoted optimal production of TNF-α, IL-6, and IL-23. A CRISPR/Cas9-mediated activation screen in THP-1 cells revealed several lncRNAs that modulated LPS-induced TNF-α responses. Overall, this study uncovered the expression patterns of lncRNAs in IBD that identify functional, disease-relevant lncRNAs.

Identification of highly selective SIK1/2 inhibitors that modulate innate immune activation and suppress intestinal inflammation.

The salt-inducible kinases (SIK) 1-3 are key regulators of pro- versus anti-inflammatory cytokine responses during innate immune activation. The lack of highly SIK-family or SIK isoform-selective inhibitors suitable for repeat, oral dosing has limited the study of the optimal SIK isoform selectivity profile for suppressing inflammation in vivo. To overcome this challenge, we devised a structure-based design strategy for developing potent SIK inhibitors that are highly selective against other kinases by engaging two differentiating features of the SIK catalytic site. This effort resulted in SIK1/2-selective probes that inhibit key intracellular proximal signaling events including reducing phosphorylation of the SIK substrate cAMP response element binding protein (CREB) regulated transcription coactivator 3 (CRTC3) as detected with an internally generated phospho-Ser329-CRTC3-specific antibody. These inhibitors also suppress production of pro-inflammatory cytokines while inducing anti-inflammatory interleukin-10 in activated human and murine myeloid cells and in mice following a lipopolysaccharide challenge. Oral dosing of these compounds ameliorates disease in a murine colitis model. These findings define an approach to generate highly selective SIK1/2 inhibitors and establish that targeting these isoforms may be a useful strategy to suppress pathological inflammation.

Identification of small-molecule protein-protein interaction inhibitors for NKG2D.

NKG2D (natural-killer group 2, member D) is a homodimeric transmembrane receptor that plays an important role in NK, γδ+, and CD8+ T cell-mediated immune responses to environmental stressors such as viral or bacterial infections and oxidative stress. However, aberrant NKG2D signaling has also been associated with chronic inflammatory and autoimmune diseases, and as such NKG2D is thought to be an attractive target for immune intervention. Here, we describe a comprehensive small-molecule hit identification strategy and two distinct series of protein-protein interaction inhibitors of NKG2D. Although the hits are chemically distinct, they share a unique allosteric mechanism of disrupting ligand binding by accessing a cryptic pocket and causing the two monomers of the NKG2D dimer to open apart and twist relative to one another. Leveraging a suite of biochemical and cell-based assays coupled with structure-based drug design, we established tractable structure-activity relationships with one of the chemical series and successfully improved both the potency and physicochemical properties. Together, we demonstrate that it is possible, albeit challenging, to disrupt the interaction between NKG2D and multiple protein ligands with a single molecule through allosteric modulation of the NKG2D receptor dimer/ligand interface.

Role of IL-36 cytokines in psoriasis and other inflammatory skin conditions.

The IL-36 family of cytokines includes three pro-inflammatory agonists (IL-36α, IL-36ß, and IL-36γ) and a receptor antagonist (IL-36Ra), which bind and signal through a heterodimeric receptor composed of IL-36R and the IL-1R accessory protein (IL-1RAcP). Individuals with inactivating mutations in the gene encoding IL-36Ra develop generalized pustular psoriasis, a severe form of psoriasis, a finding which clearly links dysregulated IL-36 pathway activation to inflammatory skin conditions. The purpose of this review is to highlight the cellular source of IL-36 cytokines, the effects of IL-36 signaling across cell types, and the association of IL-36 to a spectrum of inflammatory skin diseases including various forms of psoriasis as well as hidradenitis suppurativa, atopic dermatitis, and allergic contact dermatitis.

QRICH1 dictates the outcome of ER stress through transcriptional control of proteostasis.

Tissue homeostasis is perturbed in a diversity of inflammatory pathologies. These changes can elicit endoplasmic reticulum (ER) stress, protein misfolding, and cell death. ER stress triggers the unfolded protein response (UPR), which can promote recovery of ER proteostasis and cell survival or trigger programmed cell death. Here, we leveraged single-cell RNA sequencing to define dynamic transcriptional states associated with the adaptive versus terminal UPR in the mouse intestinal epithelium. We integrated these transcriptional programs with genome-scale CRISPR screening to dissect the UPR pathway functionally. We identified QRICH1 as a key effector of the PERK-eIF2α axis of the UPR. QRICH1 controlled a transcriptional program associated with translation and secretory networks that were specifically up-regulated in inflammatory pathologies. Thus, QRICH1 dictates cell fate in response to pathological ER stress.

The CSF-1-receptor inhibitor, JNJ-40346527 (PRV-6527), reduced inflammatory macrophage recruitment to the intestinal mucosa and suppressed murine T cell mediated colitis.

BACKGROUND & AIMS: Originally believed to be primarily a disorder of T-cell signaling, evidence shows that macrophage-lineage cells also contribute to the pathogenesis of Crohn's disease (CD). Colony stimulating factor-1 (CSF-1) is a key regulator of the macrophage lineage, but its role in CD has not been well established. We examined transcriptional data from CD mucosa for evidence of CSF-1 pathway activation and tested JNJ-40346527 (PRV-6527), a small molecule inhibitor of CSF-1 receptor kinase (CSF-1R), for its ability to inhibit disease indices in murine colitis. METHODS: A CSF-1 pathway gene set was created from microarray data of human whole blood cultured ex vivo with CSF-1 and compared to a TNFα-induced gene set generated from epithelial-lineage cells. Gene set variation analysis was performed using existing Crohn's mucosa microarray data comparing patients who either responded or failed to respond to anti-TNFα therapy. Commencing day 14 or day 21, mice with T-cell transfer colitis were treated with vehicle or JNJ-40346527 until study termination (day 42). Endpoints included colon weight/length ratios and histopathology scores, and macrophage and T cells were assessed by immunohistochemistry. Mucosal gene expression was investigated using RNAseq. RESULTS: Both the CSF-1 and the TNFα gene sets were enriched in the colonic mucosal transcriptomes of Crohn's disease and in mouse colitis, and expression of both gene sets was highest in patients who did not respond to anti-TNFα therapy. In these patients neither set was reduced by therapy. In the mouse model, JNJ-40346527 inhibited the increase in colon weight/length ratio by ∼50%, reduced histological disease scores by ∼60%, and reduced F4/80+ mononuclear cell and CD3+ lymphocyte numbers. RNAseq analysis confirmed the CSF-1 gene set was sharply reduced in treated mice, as were gene sets enriched in "M1" inflammatory and "M0" resident macrophages and in activated T cells. CONCLUSIONS: CSF-1 biology is activated in Crohn's disease and in murine T cell transfer colitis. Inhibition of CSF-1R by JNJ-40346527 was associated with attenuated clinical disease scores and reduced inflammatory gene expression in mice. These data provide rationale for testing JNJ-40346527 (PRV-6527) in human inflammatory bowel disease.

A Role for IL-12 in IBD after All?

The interplay between intestinal epithelial barrier function, the intestinal microbiota, and cytokine signaling in the pathogenesis of inflammatory bowel disease is not completely understood. In this issue of Immunity, Eftychi et al. examined the cell types and cytokines that drive colitis in mice with an intestinal epithelial barrier defect and uncovered a surprising role for IL-12 in initiation of disease.

Publisher Correction: Get the IL-17F outta here!

In the version of this article initially published, a word ("neutraling") in sentence 2 of paragraph 5 is incorrect. The correct phrase is "...neutralizing properties...". The error has been corrected in the HTML and PDF version of the article.

Mutation in IL36RN impairs the processing and regulatory function of the interleukin-36-receptor antagonist and is associated with DITRA syndrome.

The identification of loss-of-function mutations of the IL36RN gene encoding the interleukin-36 receptor antagonist (IL-36Ra) in generalized pustular psoriasis (GPP) emphasized the key role of this pathway in skin innate immunity and systemic inflammation. It has been previously shown in vitro that removal of the N-terminal amino acid IL36Ra (M1) is critical to its biological activity, but the in vivo contribution of this processing remains unknown. We report herein a new homozygous (c4G>T, pV2F) missense IL36RN mutation segregating in a family with three GPP-affected patients. The V2F mutation does not alter IL-36Ra protein expression but was devoid of any antagonist activity. Mass spectrometry showed that the V2F IL-36Ra mutant retains its first N-terminal methionine. These results provide the first in vivo demonstration that removal of N-terminal methionine of native IL-36Ra is a mandatory step to reach optimal antagonist activity and to prevent sustained skin and systemic inflammation in humans.

Abnormal Small Intestinal Epithelial Microvilli in Patients With Crohn's Disease.

BACKGROUND & AIMS: Crohn disease (CD) presents as chronic and often progressive intestinal inflammation, but the contributing pathogenic mechanisms are unclear. We aimed to identify alterations in intestinal cells that could contribute to the chronic and progressive course of CD. METHODS: We took an unbiased system-wide approach by performing sequence analysis of RNA extracted from formalin-fixed paraffin-embedded ileal tissue sections from patients with CD (n = 36) and without CD (controls; n = 32). We selected relatively uninflamed samples, based on histology, before gene expression profiling; validation studies were performed using adjacent serial tissue sections. A separate set of samples (3 control and 4 CD samples) was analyzed by transmission electron microscopy. We developed methods to visualize an overlapping modular network of genes dysregulated in the CD samples. We validated our findings using biopsy samples (110 CD samples for gene expression analysis and 54 for histologic analysis) from the UNITI-2 phase 3 trial of ustekinumab for patients with CD and healthy individuals (26 samples used in gene expression analysis). RESULTS: We identified gene clusters that were altered in nearly all CD samples. One cluster encoded genes associated with the enterocyte brush border, leading us to investigate microvilli. In ileal tissues from patients with CD, the microvilli were of decreased length and had ultrastructural defects compared with tissues from controls. Microvilli length correlated with expression of genes that regulate microvilli structure and function. Network analysis linked the microvilli cluster to several other down-regulated clusters associated with altered intracellular trafficking and cellular metabolism. Enrichment of a core microvilli gene set also was lower in the UNITI-2 trial CD samples compared with controls; expression of microvilli genes was correlated with microvilli length and endoscopy score and was associated with response to treatment. CONCLUSIONS: In a transcriptome analysis of formalin-fixed and paraffin-embedded ileal tissues from patients with CD and controls, we associated transcriptional alterations with histologic alterations, such as differences in microvilli length. Decreased microvilli length and decreased expression of the microvilli gene set might contribute to epithelial malfunction and the chronic and progressive disease course in patients with CD.

Regulation and function of interleukin-36 cytokines.

The interleukin (IL)-36 cytokines include 3 agonists, IL-36α, IL-36ß, and IL-36γ that bind to a common receptor composed of IL-36R and IL-1RAcP to stimulate inflammatory responses. IL-36Ra is a natural antagonist that binds to IL-36R, but does not recruit the co-receptor IL-1RAcP and does not stimulate any intracellular responses. The IL-36 cytokines are expressed predominantly by epithelial cells and act on a number of cells including immune cells, epithelial cells, and fibroblasts. Processing of the N-terminus is required for full agonist or antagonist activity for all IL-36 members. The role of IL-36 has been extensively demonstrated in the skin where it can act on keratinocytes and immune cells to induce a robust inflammatory response that has been implicated in psoriatic disorders. Emerging data also suggest a role for this cytokine family in pulmonary and intestinal physiology and pathology.

Differing roles for short chain fatty acids and GPR43 agonism in the regulation of intestinal barrier function and immune responses.

Inflammatory bowel disease (IBD) is associated with a loss of intestinal barrier function and dysregulated immune responses. It has been shown that short chain fatty acids (SCFAs) are protective in IBD and that GPR43 mediates the protective effects of SCFAs. In this study, we investigated the effects of SCFAs in comparison to highly specific GPR43 agonists on human intestinal epithelial and immune cells. Our results confirm that SCFAs are enhancers of barrier function in intestinal epithelial cells. Additionally, SCFAs also displayed potent immunoregulatory properties based upon the ability to inhibit LPS-induced cytokine production in PBMC, and human T cell proliferation and cytokine production. Unexpectedly, and in contrast to the current belief, specific GPR43 agonists failed to exhibit similar barrier enhancing and anti-inflammatory properties. These findings demonstrate that SCFA possess broad protective functions in IBD and agonizing GPR43 alone is unlikely to be beneficial in patients.

Effect of IL-17 receptor A blockade with brodalumab in inflammatory diseases.

IL-17 cytokines are expressed by a variety of cells and mediate host defence against extracellular pathogens. IL-17 is upregulated at sites of inflammation and can synergize with other cytokines, such as TNF-α, to amplify the inflammatory response. Activation of these signalling pathways has been hypothesized to contribute to the underlying pathogenesis of several inflammatory diseases, including psoriasis, RA, PsA and asthma. Thus the IL-17 signalling pathway is an attractive target for the development of therapeutic agents to modulate aberrant inflammatory responses. This review of the clinical development of therapeutic agents that target IL-17 signalling pathways in inflammatory diseases focuses on brodalumab, a human anti-IL-17 receptor A mAb. The cumulative findings of early clinical studies with anti-IL-17 agents, including brodalumab, secukinumab and ixekizumab, provide strong evidence for the role of IL-17 signalling in the pathophysiology of certain inflammatory diseases and support the potential use of these agents in treating these diseases.

Cutting Edge: IL-36 Receptor Promotes Resolution of Intestinal Damage.

IL-1 family members are central mediators of host defense. In this article, we show that the novel IL-1 family member IL-36γ was expressed during experimental colitis and human inflammatory bowel disease. Germ-free mice failed to induce IL-36γ in response to dextran sodium sulfate (DSS)-induced damage, suggesting that gut microbiota are involved in its induction. Surprisingly, IL-36R-deficient (Il1rl2(-/-)) mice exhibited defective recovery following DSS-induced damage and impaired closure of colonic mucosal biopsy wounds, which coincided with impaired neutrophil accumulation in the wound bed. Failure of Il1rl2(-/-) mice to recover from DSS-induced damage was associated with a profound reduction in IL-22 expression, particularly by colonic neutrophils. Defective recovery of Il1rl2(-/-) mice could be rescued by an aryl hydrocarbon receptor agonist, which was sufficient to restore IL-22 expression and promote full recovery from DSS-induced damage. These findings implicate the IL-36/IL-36R axis in the resolution of intestinal mucosal wounds.

Differential Roles for Interleukin-23 and Interleukin-17 in Intestinal Immunoregulation.

Interleukin-23 (IL-23) and IL-17 are cytokines currently being targeted in clinical trials. Although inhibition of both of these cytokines is effective for treating psoriasis, IL-12 and IL-23 p40 inhibition attenuates Crohn's disease, whereas IL-17A or IL-17 receptor A (IL-17RA) inhibition exacerbates Crohn's disease. This dichotomy between IL-23 and IL-17 was effectively modeled in the multidrug resistance-1a-ablated (Abcb1a(-/-)) mouse model of colitis. IL-23 inhibition attenuated disease by decreasing colonic inflammation while enhancing regulatory T (Treg) cell accumulation. Exacerbation of colitis by IL-17A or IL-17RA inhibition was associated with severe weakening of the intestinal epithelial barrier, culminating in increased colonic inflammation and accelerated mortality. These data show that IL-17A acts on intestinal epithelium to promote barrier function and provide insight into mechanisms underlying exacerbation of Crohn's disease when IL-17A or IL-17RA is inhibited.

Regulation and function of interleukin-36 cytokines in homeostasis and pathological conditions.

IL-36α, IL-36ß, and IL-36γ are members of the IL-1 family of cytokines that signal through a common receptor composed of IL-36R and IL-1R/AcP to activate NF-κB and MAPKs, such as p38 and JNK, and promote inflammatory responses. IL-36Ra is a natural antagonist of the 3 IL-36 agonists that binds to IL-36R and inhibits binding of the agonistic ligands. These cytokines are expressed predominantly by epithelial cells and act on a number of cells, including immune cells, epithelial cells, and fibroblasts. Processing of the N terminus is required for full agonist or antagonist activity for all IL-36 members. The role of IL-36 has been demonstrated extensively in the skin, where it can act on keratinocytes and immune cells to induce a robust inflammatory response and is implicated strongly through functional and genetic evidence in the pathology of psoriatic disorders. Emerging data also suggest a role for this cytokine family in pulmonary physiology and pathology. Although much has been learned about the biochemistry of IL-36 and its role in various tissues, it is clear that we are at an early stage in our understanding of the full biology of these cytokines.

Combined anti-CD40 and anti-IL-23 monoclonal antibody therapy effectively suppresses tumor growth and metastases.

Tumor-induced immunosuppression remains one of the major obstacles to many potentially effective cancer therapies and vaccines. Host interleukin (IL)-23 suppresses the immune response during tumor initiation, growth, and metastases, and neutralization of IL-23 causes IL-12-dependent antitumor effects. Here, we report that combining agonistic anti-CD40 monoclonal antibodies (mAb) to drive IL-12 production and anti-IL-23 mAbs to counter the tumor promoting effects of IL-23 has greater antitumor activity than either agent alone. This increased antitumor efficacy was observed in several experimental and spontaneous lung metastases models as well as in models of de novo carcinogenesis. The combination effects were dependent on host IL-12, perforin, IFN-γ, natural killer, and/or T cells and independent of host B cells and IFN-αß sensitivity. Interestingly, in the experimental lung metastases tumor models, we observed that intracellular IL-23 production was specifically restricted to MHC-II(hi)CD11c(+)CD11b(+) cells. Furthermore, an increase in proportion of these IL-23-producing cells was detected only in tumor models where IL-23 neutralization was therapeutic. Overall, these data suggest the clinical potential of using anti-CD40 (push) and anti-IL-23 mAbs (pull) to tip the IL-12/23 balance in established tumors.

The severity of experimental arthritis is independent of IL-36 receptor signaling.

INTRODUCTION: Interleukin (IL)-36 refers to three related IL-1 family cytokines, IL-36α, IL-36ß, and IL-36γ, that bind to the IL-36 receptor (IL-36R). IL-36 exerts proinflammatory effects in skin and lung and stimulates T cell responses. In the present study, we examined the expression and function of IL-36R and its ligands in experimental arthritis. METHODS: Collagen-induced arthritis (CIA), antigen-induced arthritis (AIA), and K/BxN serum transfer-induced arthritis were induced according to standard protocols. Messenger RNA levels for IL-36R and its ligands in the joints of mice with CIA were determined by RT-qPCR. Mice with CIA were injected with a blocking monoclonal anti-IL-36R, a blocking anti-IL-1RI, or their isotype-matched control antibodies at the time of arthritis onset. Anti-IL-36R or control antibodies were also injected at the time of AIA induction. Finally, IL-36R-deficient mice were examined in AIA and serum transfer-induced arthritis. The development and severity of arthritis were assessed by clinical and histological scoring. RESULTS: IL-36R, IL-36Ra and IL-36γ mRNA were detected in the joints of mice with CIA, but their levels did not correlate with arthritis severity. As opposed to anti-IL-1RI antibody treatment, the injection of an anti-IL-36R antibody was devoid of effect on the development and severity of CIA. The severity of joint inflammation and structural damage in AIA was also unaltered by anti-IL-36R antibody treatment. Finally, the severity of AIA and K/BxN serum transfer-induced arthritis was similar in IL-36R-deficient and wild-type mice. CONCLUSIONS: The development and severity of experimental arthritis are independent of IL-36R signaling.