The Opposite Functions of CD30 Ligand Isoforms.

TNFSF8/CD30 ligand is a TNF superfamily member expressed on several major immune cell types, including activated monocytes, B, and T cells. The signaling of CD30 ligand through its cognate CD30 receptor has been shown to have effects on cell differentiation, cell death/survival, and cytokine production. The signaling pair has been implicated in hematopoietic malignancies and inflammatory disease, and a chemotherapy-CD30 antibody combination for the treatment of Hodgkin and other lymphomas has been developed. There are two recorded isoforms of CD30 ligand. All hitherto studies of CD30 ligand are of the first, canonical isoform, while the second isoform has never been described. This study aims to elucidate the properties and signaling functions of the second CD30 ligand isoform. We have found mRNA expression of both isoforms in the PBMCs of all six healthy donors tested. Through methods in cell biology and biochemistry, we were able to discover that the second CD30 ligand isoform has no discernable pro-inflammatory function and, in fact, isoform 2 can restrict the capacity of the canonical isoform to signal through the CD30 receptor by preventing their interaction. This discovery has implications for the future development of therapeutics targeting the CD30/CD30 ligand signaling pair in cancer and inflammatory disease.

Select animal models of colitis and their value in predicting clinical efficacy of biological therapies in ulcerative colitis.

Introduction: Advancing new therapies from discovery to development usually requires proof-of-concept in animal models to justify the costs of continuing the program. While animal models are useful for understanding the mechanism of action (MOA) of a target, limitations of many published colitis models restrict their value to predict clinical efficacy.Areas covered: The authors focused their literature search on published studies of chronic animal models used to evaluate the pre-clinical efficacy of therapeutic molecules subsequently evaluated in clinical trials for UC. The UC therapies evaluated were anti-α4ß7, anti-IL13, anti-IL12p40, and anti-IL23p19. The models of chronic colitis evaluating these molecules were: mdra1a-/-, chronic dextran sulfate sodium (DSS), chronic 2,4,6-trinitrobenzene sulfonic acid (TNBS), and the T cell transfer model.Expert opinion: While some models provide insight into target MOA in UC, none is consistently superior in predicting efficacy. Evaluation of multiple models, with varying mechanisms of colitis induction, is needed to understand potential drug efficacy. Additional models of greater complexity, reflecting the disease chronicity/heterogeneity seen in humans, are needed. Although helpful in prioritizing targets, animal models alone will likely not improve outcomes of UC clinical trials. Transformational changes to clinical efficacy will likely only occur when precision medicine approaches are employed.

Performance Characteristics of a Clinical Decision Support Tool for Disease Complications in Crohn's Disease.

Background: Patients with Crohn's disease (CD) are at risk of complications. Performance characteristics of a decision support tool assessing the risk of CD complications were evaluated. Methods: CDPATH (formerly called the Personalized Risk and Outcome Prediction Tool [PROSPECT]) was calibrated and validated in 2 cohorts. Tool prediction of disease characteristics was assessed using Cox regression and Harrell's C-statistic. Results: All associations of CD complications and CDPATH components were significant except perianal location. There was a significant association between individualized risk assessment scores and CD complications in both cohorts. Conclusion: CDPATH is validated as a clinical decision support tool for assessing the risk of CD complications.

Altered Intestinal ACE2 Levels Are Associated With Inflammation, Severe Disease, and Response to Anti-Cytokine Therapy in Inflammatory Bowel Disease.

BACKGROUND AND AIMS: The host receptor for severe acute respiratory syndrome coronavirus 2, angiotensin-converting enzyme 2 (ACE2), is highly expressed in small bowel (SB). Our aim was to identify factors influencing intestinal ACE2 expression in Crohn's disease (CD), ulcerative colitis (UC), and non-inflammatory bowel disease (IBD) controls. METHODS: Using bulk RNA sequencing or microarray transcriptomics from tissue samples (4 SB and 2 colonic cohorts; n = 495; n = 387 UC; n = 94 non-IBD), we analyzed the relationship between ACE2 with demographics and disease activity and prognosis. We examined the outcome of anti-tumor necrosis factor and anti-interleukin-12/interleukin-23 treatment on SB and colonic ACE2 expression in 3 clinical trials. Univariate and multivariate regression models were fitted. RESULTS: ACE2 levels were consistently reduced in SB CD and elevated in colonic UC compared with non-IBD controls. Elevated SB ACE2 was also associated with demographic features (age and elevated body mass index) associated with poor coronavirus disease 2019 outcomes. Within CD, SB ACE2 was reduced in patients subsequently developing complicated disease. Within UC, colonic ACE2 was elevated in active disease and in patients subsequently requiring anti-tumor necrosis factor rescue therapy. SB and colonic ACE2 expression in active CD and UC were restored by anti-cytokine therapy, most notably in responders. CONCLUSIONS: Reduced SB but elevated colonic ACE2 levels in IBD are associated with inflammation and severe disease, but normalized after anti-cytokine therapy, suggesting compartmentalization of ACE2-related biology in SB and colonic inflammation. The restoration of ACE2 expression with anti-cytokine therapy might be important in the context of severe acute respiratory syndrome coronavirus 2 infection and potentially explain reports of reduced morbidity from coronavirus disease 2019 in IBD patients treated with anti-cytokines.

Reduced expression of COVID-19 host receptor, ACE2 is associated with small bowel inflammation, more severe disease, and response to anti-TNF therapy in Crohn's disease.

Angiotensin-Converting Enzyme 2 ( ACE2 ) has been identified as the host receptor for SARS-coronavirus 2 (SARS-CoV-2) which has infected millions world-wide and likely caused hundreds of thousands of deaths. Utilizing transcriptomic data from four cohorts taken from Crohn's disease (CD) and non-inflammatory bowel disease (IBD) subjects, we observed evidence of increased ACE2 mRNA in ileum with demographic features that have been associated with poor outcomes in COVID-19 including age and raised BMI. ACE2 was downregulated in CD compared to controls in independent cohorts. Within CD, ACE2 expression was reduced in inflamed ileal tissue and also remarkably, from uninvolved tissue in patients with a worse prognosis in both adult and pediatric cohorts. In active CD, small bowel ACE2 expression was restored by anti-TNF therapy particularly in anti-TNF responders. Collectively our data suggest that ACE2 downregulation is associated with inflammation and worse outcomes in CD.

Ileal Gene Expression Data from Crohn's Disease Small Bowel Resections Indicate Distinct Clinical Subgroups.

BACKGROUND AND AIMS: Heterogeneity in Crohn's disease [CD] provides a challenge for the development of effective therapies. Our goal was to define a unique molecular signature for severe, refractory CD to enable precision therapy approaches to disease treatment and to facilitate earlier intervention in complicated disease. METHODS: We analysed clinical metadata, genetics, and transcriptomics from uninvolved ileal tissue from CD patients who underwent a single small bowel resection. We determined transcriptional risk scores, cellular signatures, and mechanistic pathways that define patient subsets in refractory CD. RESULTS: Within refractory CD, we found three CD patient subgroups [CD1, CD2, and CD3]. Compared with CD1, CD3 was enriched for subjects with increased disease recurrence after first surgery [OR = 6.78, p = 0.04], enhanced occurrence of second surgery [OR = 5.07, p = 0.016], and presence of perianal CD [OR = 3.61, p = 0.036]. The proportion of patients with recurrence-free survival was smaller in CD3 than in CD1 (p = 0.02, median survival time [months] in CD1 = 10 and CD3 = 6). Overlaying differential gene expression between CD1 and CD3 on CD subgroup-associated genetic polymorphisms identified 174 genes representing both genetic and biological differences between the CD subgroups. Pathway analyses using this unique gene signature indicated eukaryotic initiation factor 2 [eIF2] and cyclic adenosine monophosphate [cAMP] signalling to be dominant pathways associated with CD3. Furthermore, the severe, refractory subset, CD3, was associated with a higher transcriptional risk score and enriched with eosinophil and natural killer T [NKT] cell gene signatures. CONCLUSION: We characterized a subset of severe, refractory CD patients who may need more aggressive treatment after first resection and who are likely to benefit from targeted therapy based on their genotype and tissue gene expression signature.

Centrally Determined Standardization of Flow Cytometry Methods Reduces Interlaboratory Variation in a Prospective Multicenter Study.

OBJECTIVES: Flow cytometry (FC) aids in characterization of cellular and molecular factors involved in pathologic immune responses. Although FC has potential to facilitate early drug development in inflammatory bowel disease, interlaboratory variability limits its use in multicenter trials. Standardization of methods may address this limitation. We compared variability in FC-aided quantitation of T-cell responses across international laboratories using three analytical strategies. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from three healthy donors, stimulated with phorbol 12-myristate 13-acetate and ionomycin at a central laboratory, fixed, frozen, and shipped to seven international laboratories. Permeabilization and staining was performed in triplicate at each laboratory using a common protocol and centrally provided reagents. Gating was performed using local gating with a local strategy (LGLS), local gating with a central strategy (LGCS), and central gating (CG). Median cell percentages were calculated across triplicates and donors, and reported for each condition and strategy. The coefficient of variation (CV) was calculated across laboratories. Between-strategy comparisons were made using a two-way analysis of variance adjusting for donor. RESULTS: Mean interlaboratory CV ranged from 1.8 to 102.1% depending on cell population and gating strategy (LGLS, 4.4-102.1%; LGCS, 10.9-65.6%; CG, 1.8-20.9%). Mean interlaboratory CV differed significantly across strategies and was consistently lower with CG. CONCLUSIONS: Central gating was the only strategy with mean CVs consistently lower than 25%, which is a proposed standard for pharmacodynamic and exploratory biomarker assays.

Association of Ribonuclease T2 Gene Polymorphisms With Decreased Expression and Clinical Characteristics of Severity in Crohn's Disease.

BACKGROUND & AIMS: Variants in the tumor necrosis factor superfamily member 15 gene (TNFSF15, also called TL1A) have been associated with risk for inflammatory bowel disease (IBD). TL1A affects expression of multiple cytokines to promote mucosal inflammation. Little is known about the TL1A-response pathways that regulate cytokine expression. We investigated T-cell gene expression patterns to determine the mechanisms by which TL1A regulates cytokine production, and whether these associate with outcomes of patients with Crohn's disease (CD). METHODS: Peripheral T cells isolated from normal donors were cultured with TL1A. We performed gene expression profile analysis by RNA sequencing of subsets of interferon gamma (IFNG)-producing and non-producing cells purified by flow cytometry. Unsupervised hierarchical clustering analysis was used to identify gene expression differences between these subsets. Ribonuclease T2 gene (RNASET2) expression and methylation were assessed by quantitative trait loci analyses. Clinical characteristics of patients (complications, resistance to therapy, and recurrence time) were associated with single nucleotide polymorphisms in RNASET2. We performed motif screening to identify polymorphisms that disrupt transcription factor binding sites. Levels of RNASET2 were knocked down with small interfering RNA in CD4+ T cells and the effect on protein expression was determined by proteomic analysis and cytokine production. Cell aggregation was measured by flow cytometry. RESULTS: We identified 764 genes with at least a 2-fold difference in TL1A-mediated expression between IFNG-secreting and non-secreting T cells (P < 1 × 10-5). Many of these genes were located near IBD susceptibility variants. RNASET2 was the only IBD risk-associated gene with >5-fold down-regulation in the IFNG-secreting subset. RNASET2 disease risk variants were associated with decreased expression in peripheral and mucosal tissues and DNA hypermethylation in CD patients requiring surgical intervention. RNASET2 disease risk variants were associated in CD patients with more complicated disease or resistance to therapy, defined in part by failed response to treatment, increased length of intestinal resection, shorter time to repeat surgery, and high Rutgeerts score (>2) in postoperative endoscopy. The RNASET2 variant rs2149092 was predicted to disrupt a consensus binding site for the transcription factor ETS within an enhancer region. Expression of RNASET2 correlated with expression of ETS. RNASET2 knockdown in T cells increased expression of IFNG and intercellular adhesion molecule 1 (ICAM1) and induced T-cell aggregation. A blocking antibody against (ILFA1), disrupting the lymphocyte function-associated antigen 1-intercellular adhesion molecule 1 interaction, reduced T-cell production of IFNG. CONCLUSIONS: We identified decreased expression of RNASET2 as a component of TL1A-mediated increase in production of IFNG and as a potential biomarker for patients with severe CD. Further study of the role of RNASET2 in regulating mucosal inflammation may lead to development of novel therapeutic targets.

IL-31 expression by inflammatory cells is preferentially elevated in atopic dermatitis.

Interleukin-31 (IL-31) is a recently discovered cytokine expressed in many human tissues, and predominantly by activated CD4(+) T cells. IL-31 signals through a heterodimeric receptor consisting of IL-31 receptor alpha (IL-31RA) and oncostatin M receptor beta (OSMR). Earlier studies have shown involvement of IL-31 and its receptor components IL-31RA and OSMR in atopic dermatitis, pruritus and Th2-weighted inflammation at the mRNA level. The aim of this study was to investigate IL-31 protein expression in skin of such conditions. Immunohistochemical staining for IL-31, IL-31RA and OSMR was performed in formalin-fixed paraffin-embedded biopsy specimens. IL-31 expression was increased in the inflammatory infiltrates from skin biopsies taken from subjects with atopic dermatitis, compared with controls (p ≤ 0.05). IL-31, IL-31RA and OSMR protein immunoreactivity was not increased in biopsies from subjects with other Th2-weighted and pruritic skin diseases. Our results confirm, at the protein level, the relationship between IL-31 expression and atopic dermatitis. Our results do not support a general relationship between expression of IL-31/IL-31R and pruritic or Th2-mediated diseases.

Vstm3 is a member of the CD28 family and an important modulator of T-cell function.

Members of the CD28 family play important roles in regulating T-cell functions and share a common gene structure profile. We have identified VSTM3 as a protein whose gene structure matches that of the other CD28 family members. This protein (also known as TIGIT and WUCAM) has been previously shown to affect immune responses and is expressed on NK cells, activated and memory T cells, and Tregs. The nectin-family proteins CD155 and CD112 serve as counter-structures for VSTM3, and CD155 and CD112 also bind to the activating receptor CD226 on T cells and NK cells. Hence, this group of interacting proteins forms a network of molecules similar to the well-characterized CD28-CTLA-4-CD80-CD86 network. In the same way that soluble CTLA-4 can be used to block T-cell responses, we show that soluble Vstm3 attenuates T-cell responses in vitro and in vivo. Moreover, animals deficient in Vstm3 are more sensitive to autoimmune challenges indicating that this new member of the CD28 family is an important regulator of T-cell responses.

IL-31 receptor (IL-31RA) knockout mice exhibit elevated responsiveness to oncostatin M.

IL-31 signals through the heterodimeric receptor IL-31RA and oncostatin M receptor (OSMR), and has been linked with the development of atopic dermatitis, a Th2 cytokine-associated disease in humans. However, recent studies of IL-31RA knockout (KO) mice have suggested that IL-31 signaling may be required to negatively regulate Th2 type responses rather than exacerbate them. Because those studies were performed on genetically modified mice, we examined whether neutralizing IL-31 with a specific mAb would give similar results to IL-31RA KO mice in two Th2 cytokine-associated immune models. We report no difference in lymphocyte Th2-type cytokine production after Ag immunization between IL-31RA KO mice, mice treated with the IL-31 mAb, or control animals. Second, we tested whether the absence of the IL-31RA subunit in IL-31RA KO mice may allow for increased pairing of the OSMR subunit with another cytokine receptor, gp130, resulting in overrepresentation of the heterodimeric receptor for OSM and increased responsiveness to OSM protein. We found that intranasal OSM challenge of IL-31RA KO mice resulted in increased IL-6 and vascular endothelial growth factor production in the lung compared with wild-type littermate control animals. Moreover, PBS-challenged IL-31RA KO mice already had increased levels of vascular endothelial growth factor, which were further increased by OSM challenge. These data imply that IL-31RA-deficient mice produce increased levels of OSM-inducible cytokines during airway sensitization and challenge, which may be the driving force behind the apparent exacerbation of Th2-type inflammatory responses previously observed in these mice.

Anti-interleukin-31-antibodies ameliorate scratching behaviour in NC/Nga mice: a model of atopic dermatitis.

BACKGROUND: Interleukin-31 (IL-31), a novel cytokine, is upregulated in atopic dermatitis skin lesions in humans and skin lesions in the NC/Nga mice, a murine model for atopic dermatitis. OBJECTIVE: Here, we investigated the effect of a monoclonal IL-31 antibody on scratching behaviour, weight gain and dermatitis in NC/Nga mice. METHODS: Mice were divided into three groups, n = 10 in each group. Mice were given monoclonal IL-31 rat-anti-mouse antibody 10 mg/kg or albumin intraperitoneally every fifth day for seven weeks. In addition, the mice in one group were not exposed to any form of intervention. The dermatitis score was clinically assessed twice a week. The scratching behaviour was automatically detected and objectively evaluated. RESULTS: Intervention with IL-31 antibody 10 mg/kg intraperitoneally every fifth day in NC/Nga mice from age 7 weeks reduced the scratching behaviour, but did not have any impact on weight gain or dermatitis. CONCLUSION: IL-31 antibody reduces scratching behaviour in an atopic dermatitis-like murine model during the onset of clinical skin manifestations. Our findings suggest IL-31 antibody as a new potential therapeutic approach for pruritus in atopic dermatitis and other pruritic diseases.

IL-31 is associated with cutaneous lymphocyte antigen-positive skin homing T cells in patients with atopic dermatitis.

BACKGROUND: IL-31 is a newly discovered T-cell cytokine that, when overexpressed in mice, results in pruritus and skin dermatitis resembling human atopic dermatitis (AD). OBJECTIVE: We sought to investigate the expression of IL-31 and IL-31 receptor A (IL-31RA) in skin biopsy specimens and peripheral blood cells from patients with AD and healthy individuals. METHODS: Expression of IL-31 and IL-31RA was evaluated in skin biopsy specimens from patients with AD and healthy individuals by means of immunohistochemistry and RT-PCR. IL-31 protein production by skin-homing cutaneous lymphocyte antigen (CLA)-positive T cells was also assessed. RESULTS: IL-31RA protein was expressed by keratinocytes and infiltrating macrophages in skin biopsy specimens from patients with AD. Comparisons between skin from patients with AD and healthy skin showed IL-31RA expression at higher levels on epidermal keratinocytes in AD samples. Infiltrating cells, more numerous in skin from patients with AD compared with that of healthy individuals, expressed IL31 mRNA. Histomorphometric analysis of these cells indicated they were of the lymphocytic lineage, with the majority of cells staining positive for CLA and CD3. IL31 mRNA and protein expression is largely restricted to CD45RO(+) (memory) CLA(+) T cells in peripheral blood of patients with AD and healthy volunteers. Moreover, circulating CLA(+) T cells from patients with AD, but not from patients with psoriasis, are capable of producing higher levels of IL-31 compared with CLA(+) T cells from healthy individuals. However, the average levels of IL-31 were not significantly different between patients with AD and healthy individuals. CONCLUSION: We provide evidence that IL-31 expression is associated with CLA(+) T cells and might contribute to the development of AD-induced skin inflammation and pruritus.

From model to mechanism: lessons of mice and men in the discovery of protein biologicals for the treatment of inflammatory bowel disease.

Successful therapeutics for inflammatory bowel disease (IBD) must be able to reverse effectively the complex processes involved in the manifestation of inflammatory pathology in intact tissues. Although studies of human tissue samples are important to confirm the biological rationale for developing a particular therapeutic, in vivo rodent models of IBD provide a biological 'flask' in which therapeutics can be tested in a more representative setting. Moreover, gene targeting and transgenic technologies in rodents have exponentially increased the repertoire of available IBD models and provided insight into possible contributions that certain genes may have in the pathogenesis of disease. These models have been key in generating the current arsenal of biological therapeutics that are available, or are presently under investigation, for the treatment of IBD patients.

The mdr1a-/- mouse model of spontaneous colitis: a relevant and appropriate animal model to study inflammatory bowel disease.

There are many types of colitis models in animals that researchers use to elucidate the mechanism of action of human inflammatory bowel disease (IBD). These models are also used to test novel therapeutics and therapeutic treatment regimens. Here, we will review the characteristics of the mdr1a -/- model of spontaneous colitis that we believe make this model an important part of the IBD researcher's toolbox. We will also share new data that will reinforce the fact that this model is relevant in the study of IBD. Mdr1a -/- mice lack the murine multiple drug resistance gene for P-glycoprotein 170 that is normally expressed in multiple tissues including intestinal epithelial cells. These mice spontaneously develop a form of colitis at around 12 wk of age. The fact that the complexity of this model mirrors the complexity of disease in humans, as well as recent literature that links MDR1 polymorphisms in humans to Crohn's Disease and Ulcerative Colitis, makes this an appropriate animal model to study.

Gastrointestinal dendritic cells play a role in immunity, tolerance, and disease.

Discrimination between beneficial commensal organisms and potentially harmful pathogens is a central component of the essential role that gut immune cells play in maintaining the balance between immune activation and tolerance. Antigen presenting cells (APC) are the key to this process, and the type of APC, including epithelial cells, B cells, macrophages, and dendritic cells (DC), in the gut is varied. The purpose of this review is to focus on the vast amount of data that has recently been generated on gastrointestinal dendritic cells in the context of their potential function and contribution to mucosal immunity, tolerance, and disease.

Interleukin 31, a cytokine produced by activated T cells, induces dermatitis in mice.

T cell-derived cytokines are important in the development of an effective immune response, but when dysregulated they can promote disease. Here we identify a four-helix bundle cytokine we have called interleukin 31 (IL-31), which is preferentially produced by T helper type 2 cells. IL-31 signals through a receptor composed of IL-31 receptor A and oncostatin M receptor. Expression of IL-31 receptor A and oncostatin M receptor mRNA was induced in activated monocytes, whereas epithelial cells expressed both mRNAs constitutively. Transgenic mice overexpressing IL-31 developed severe pruritus, alopecia and skin lesions. Furthermore, IL-31 receptor expression was increased in diseased tissues derived from an animal model of airway hypersensitivity. These data indicate that IL-31 may be involved in promoting the dermatitis and epithelial responses that characterize allergic and non-allergic diseases.

In vivo enhancement of dendritic cell function.

Despite the apparent positive recognition of antigen by mucosal T cells after ingesting food, the default functional response in the gut is tolerance. Although dendritic cells (DCs) are classically defined as potent stimulatory antigen-presenting cells, we have previously shown that tolerance is enhanced in vivo in the presence of elevated numbers of DCs. In order to more closely investigate the mechanistic basis of tolerance induction, we have focused our subsequent studies on identifying features peculiar to mucosal dendritic cells and the functional involvement of mucosal DCs in driving the early T cell response to fed antigen. These studies have revealed a population of DCs in the mucosae that exhibit the plasmacytoid phenotype and secrete IFN-alpha following stimulation with CpG, and can drive differentiation of naive T cells into cells that exhibit regulatory properties. The activity of these DCs also failed to sustain robust T cell proliferation and, rather, functioned to enhance the suppressive efficacy of CD4(+)CD25(+) T regulatory cells. Given their significant presence in mucosal tissue, these DCs likely provide a mechanistic basis for the homeostatic regulation prominent in the gut, presumably by eliciting regulatory cell suppressor function and poorly supporting T helper cell proliferation. These studies further underscore the critical role for intestinal DCs in promoting tolerogenic antigen presentation at a site of high antigenic stimulation.