Th17 Biology Research Articles

P130 Transcriptomic analysis of synovitis identifies inhibition of CXCL1 as a mechanism contributing to resolution of inflammatory arthritis under IL-27 control

Abstract Background/Aims Interleukin-27 (IL-27) regulates adaptive immune responses and is critical for the timely resolution of inflammation to restore tissue homeostasis. Studies have highlighted roles for IL-27 in limiting immune cell effector function, stromal cell responses and erosive joint pathology in clinical rheumatoid arthritis and experimental models of the disease. In the murine antigen-induced arthritis (AIA) model, IL-27 receptor-deficient (Il27ra-/-) mice develop severe synovitis associated with increased infiltration of synovial CD4+ T cells, development of synovial ectopic lymphoid-like structures and exacerbated cartilage and bone erosion. Similarly, mice with collagen-induced arthritis (CIA) administered IL-27 show improved joint pathology and reduced serum IFN-γ and IL-17 levels. Identifying mechanisms by which IL-27 regulates arthritis progression is key to understanding its therapeutic potential. Using RNA-sequencing of synovial tissue and joint-infiltrating CD4+ T cells we reveal that IL-27 regulates the magnitude of synovitis and genes in CD4+ T cells central to neutrophil recruitment. Methods Antigen-induced arthritis (AIA) was established in Il27ra-/- and wild-type control mice. RNA-sequencing was performed on whole synovial tissue and joint-infiltrating CD4+ T cells during the peak of joint inflammation and during the resolution of synovitis. Differentially expressed genes were interrogated by molecular pathway analysis and gene set enrichment analysis. CD4+ T cell culture assays, qPCR and ELISA were used to test and validate genes under IL-27 regulation. Results RNA-sequencing of whole synovial tissue revealed that pathways relating to CD4+ T cell and Th17 biology were significantly overrepresented in Il27ra-/- mice with AIA, consistent with a pathogenic role for these cells in inflammatory arthritis. Genes associated with neutrophil effector function (e.g. Elane, Padi4, Prtn3) and recruitment (e.g. Cxcl1, Cxcr2) were also highly expressed in Il27ra-/- synovium and was reflected in the increased number of Ly6G+CD11b+ neutrophils recovered from the joints of Il27ra-/- mice. In contrast to the transcriptomic analysis of inflamed synovial tissue, surprisingly RNA-sequencing of joint-infiltrating CD4+ T cells revealed that the expression of signature Th17 genes (e.g. Il17a, Il17f, Rorc) was comparable between WT and Il27ra-/- T cells. This implies that IL-27 predominantly regulates the magnitude of the joint CD4+ T cell infiltrate more than the effector characteristics of the infiltrating T cells. Differential gene expression analysis revealed heightened Cxcl1 expression in both whole synovial tissue and joint-infiltrating CD4+ T cells recovered from Il27ra-/- mice with AIA. CD4+ T cell differentiation cultures confirmed upregulation of Cxcl1 following T cell receptor activation and under Th1 polarizing conditions. Here, IL-27 suppressed the expression of CXCL1 at the mRNA and protein level. Conclusion Together, our data reveals a role for IL-27 in limiting CXCL1 expression in activated and joint-infiltrating CD4+ T cells, highlighting a novel mechanism by which IL-27 may integrate innate and adaptive arms of the immune response to regulate arthritis progression. Disclosure I. Burridge: None. S. Eastham: None. D.G. Hill: None. R. Andrews: None. B. Szomolay: None. N. Williams: None. S.A. Jones: None. G.W. Jones: None.

Linked Th17 and Calgranulin Responses in Maternal-cord Blood Dyads of Preterm Gestations with Histologic Chorioamnionitis.

Maternal-fetal immune crosstalk mechanisms are increasingly identified in the pathogenesis of gestational disorders, including histologic chorioamnionitis (HCA). Although an inflammatory Th17 immune phenotype has been described in preterm neonates with HCA, the associated maternal Th17 response is relatively unknown. To refine our understanding of Th17 biology in this context, we examined Th17 responses in maternal-cord blood dyads of preterm gestations. Paired maternal and cord blood (CB) samples were prospectively collected from preterm gestations (23-34 weeks) with HCA or controls. Th17-linked cell frequencies and plasma calgranulin (S100A8, S100A12) levels were determined by flow cytometry and enzyme-linked immunoassay, respectively. Analyses of 47 maternal-cord blood pairs showed striking parallel increases in Th17 cell frequencies as well as plasma calgranulin levels in the presence of fetal inflammation. Cord blood S100A12 levels were directly correlated with Th17 cell frequencies. In CB cultures, rh-S100A12 promoted in vitro propagation of Th17-type CD4+ cells. Maternal and CB Th17-linked responses are dually amplified in gestations with HCA, supporting a biological role for maternal-fetal interactions in this disorder. In addition to advancing current knowledge of neonatal Th17 mechanisms, these data shed new light on their association with maternal inflammation.

P390 Ustekinumab modulates IL-12 and IL-23-related cell type-specific inflammatory transcriptional states in Ulcerative Colitis induction therapy

Abstract Background Ustekinumab (UST) is a monoclonal antibody specific for the p40 subunit of interleukin (IL)-12 and IL-23. In a randomised phase 3 trial (UNIFI; NCT02407236) in patients with moderate-to-severe ulcerative colitis (UC), UST was shown to be more effective for inducing and maintaining clinical remission than placebo. There has yet to be a mechanistic understanding of p40 blockade in inflammatory bowel disease. Here, we investigated the underlying molecular mechanism of action of UST in UC. Methods Paired colon biopsies for transcriptomic analysis were obtained at screening and week 8 in patients who received a single intravenous induction treatment of placebo (n=145 subjects), UST 130 mg (n=155 subjects), or a weight-range-based dose (approximated to UST 6 mg/kg of body weight; n=171 subjects). Tissue transcriptional profiles were determined using Affymetrix microarrays. Transcriptional changes were analysed in the context of cell type-specific co-expression modules derived from single cell UC data. Gene set variation analysis (GSVA) was used to quantitatively assess changes in specific biologic modules in the context of endoscopic and histologic-endoscopic mucosal improvement (HEMI) responder and non-responder analyses. Nichenet, which predicts ligand–target gene links, was used with single cell UC data to identify ligands that are predicted to be associated with our cell type-specific modules. Results By week 8, both UST treatment arms induced a greater magnitude of transcriptional changes associated with Th17, Th1 cytotoxicity, myeloid inflammatory, epithelial inflammatory, and inflammatory fibroblast modules compared with placebo. Ligand-target gene predictions indicate IL-12 and IL-23 are associated with changes in the Th1 cytotoxicity and Th17 module expression, respectively. Population level molecular changes in both UST treatment arms are consistent with endoscopic and HEMI response at week 8. When evaluating sub-groups stratified by endoscopic improvement or HEMI status, significant changes were observed in transcriptional modules in both UST responders and UST non-responders identifying mechanistic processes associated with dual blockade of IL-12 and IL-23 following exposure to UST. Placebo non-responders showed no significant changes. Conclusion Molecular analysis of colon biopsies from UC patients after UST induction revealed IL-12 and IL-23-related transcriptional changes associated with Th1 cytotoxicity and Th17 biology. By leveraging UC single cell derived transcriptional modules, we have identified key disease-relevant processes that significantly changed with UST treatment, regardless of response status and have extended the mechanistic understanding of p40 blockade in UC.

A SPECTRUM OF IMMUNOLOGIC EVENTS IS ASSOCIATED WITH ELEVATED DONOR-DERIVED CELL-FREE DNA AFTER LUNG TRANSPLANTATION

TOPIC: Transplantation TYPE: Late Breaking PURPOSE: Chronic lung allograft dysfunction (CLAD)-free survival after lung transplant (LT) according to ISHLT data, remains only 3.16-3.58 years, emphasizing an unmet clinical need for a noninvasive biomarker for surveillance. Here we sought to analyze the use of donor-derived cell-free DNA (dd-cfDNA) for the identification of rejection in LT recipients. METHODS: We performed a single-center, prospective analysis of plasma dd-cfDNA using the Prospera™ test (Natera, Inc., San Carlos) collected with concurrent clinical, fiberoptic bronchoscopy (FOB), transbronchial biopsy (TBBx) and bronchoalveolar lavage (BAL) assessments for diagnosis of acute cellular rejection (ACR, ISHLT Grades A0-A4) and antibody-mediated rejection (AMR). BAL was analyzed with routine microbiologic studies, viral multiplex PCR and cytologic differential cell counts (%) to further define cohorts of CLAD/NRAD (Neutrophilic-responsive Allograft Dysfunction), allograft infection (Infxn) and Stable/Healthy. Nonparametric statistics (Mann-Whitney, p<0.05), cohort median with interquartile range (IQR) and AUROC analyses were performed. RESULTS: A total of 95 dd-cfDNA samples with matched FOB procedures in 72 LT recipients (age: 58±14 years, F:M 50.5:49.5%) were obtained; 6 samples were omitted due to nonspecific histology or confounding conditions. Samples were obtained from patients with native lung diseases: COPD (35%), ILD (52%), CF (2%), PAH (5%), Re-LT (6%) and Other (<1%). Median time post-LT was significantly longer for CLAD/NRAD (852 days, 270-13 515; p=0.005) as compared to Stable (196 days, 90.5-335.5), ACR (212.5 days, 117-3 631), and Infxn (100 days, 81-198) cohorts. BAL Neutrophilia was elevated in individuals with ACR (3.5%, 2-17; p=0.01), CLAD/NRAD (13.5%, 3.5-23.5; p=0.004) and Infxn (8.5%, 0-6; p=0.05) as compared to Stable patients (2%, 1-4). BAL microbiology isolates identified in individuals with allograft infection included: Staphylococcus aureus, Streptococcus viridans, and Pseudomonas aeruginosa. ACR absent from AMR included Grade A1 (15) and A2 (7) in this cohort. Median dd-cfDNA was significantly elevated in the ACR (1.28%, 0.53-3.16; p<0.0001; n=22) and CLAD/NRAD cohorts (1.60%, 0.50-2.53; p=0.008; n=8) as compared to the Stable cohort (0.375%, 0.18-0.70; n=52). No significant difference was seen in the Infxn cohort (0.68%, 0.14-1.24; p=0.46; n=7). An AUC of 0.77 (95%CI: 0.64-0.90) was obtained for distinguishing ACR from Stable samples. Performance of dd-cfDNA for the identification of rejection were determined at cut-offs of 0.5% (77% Sensitivity, 56% Specificity, 27% PPV, 92% NPV) and 1.0% dd-cfDNA (59% Sensitivity, 87% Specificity, 48% PPV, and 91% NPV). PPV and NPV were projected using a prevalence of 17% (OPTN/SRTR, 2021). CONCLUSIONS: Elevated dd-cfDNA levels occur in a spectrum of immunologic events, including ACR and CLAD/NRAD. CLINICAL IMPLICATIONS: The observation of elevated dd-cfDNA in cohorts with BAL neutrophilia may highlight potential value in detection of allograft injury as related to an upregulated IL-8 and Th-17 biology. The elevated dd-cfDNA levels associated with rejection suggest a potential non-invasive molecular tool for surveillance of LT allograft health. DISCLOSURES: Research grant relationship with Natera Please note: 11/18/20-Present Added 06/25/2021 by Brian Keller, source=Web Response, value=Grant/Research Support Advisory Board, Research Grant relationship with CareDx Please note: 3/30/20-Present Added 06/25/2021 by Brian Keller, source=Web Response, value=Grant Support/Honoraria Employee relationship with Natera Please note: October 2020 – Added 06/24/2021 by Michael Olymbios, source=Web Response, value=Salary Research support relationship with Natera Please note: 12/2020-06/2021 Added 06/28/2021 by Justin Rosenheck, source=Web Response, value=Grant/Research Support Employee relationship with Natera, Inc. Please note: 2020- present Added 06/22/2021 by David Ross, source=Web Response, value=Salary Employee relationship with Natera Please note: 2018 - present Added 06/23/2021 by Jonathan Sternberg, source=Web Response, value=Salary

IL-17C/IL-17RE: Emergence of a Unique Axis in TH17 Biology.

Therapeutic targeting of IL-17A and its receptor IL-17RA with antibodies has turned out to be a tremendous success in the treatment of several autoimmune conditions. As the IL-17 cytokine family consists of six members (IL-17A to F), it is intriguing to elucidate the biological function of these five other molecules to identify more potential targets. In the past decade, IL-17C has emerged as quite a unique member of this pro-inflammatory cytokine group. In contrast to the well-described IL-17A and IL-17F, IL-17C is upregulated at very early timepoints of several disease settings. Also, the cellular source of the homodimeric cytokine differs from the other members of the family: Epithelial rather than hematopoietic cells were identified as the producers of IL-17C, while its receptor IL-17RE is expressed on TH17 cells as well as the epithelial cells themselves. Numerous investigations led to the current understanding that IL-17C (a) maintains an autocrine loop in the epithelium reinforcing innate immune barriers and (b) stimulates highly inflammatory TH17 cells. Functionally, the IL-17C/RE axis has been described to be involved in the pathogenesis of several diseases ranging from infectious and autoimmune conditions to cancer development and progression. This body of evidence has paved the way for the first clinical trials attempting to neutralize IL-17C in patients. Here, we review the latest knowledge about identification, regulation, and function of the IL-17C/IL-17receptor E pathway in inflammation and immunity, with a focus on the mechanisms underlying tissue injury. We also discuss the rationale for the translation of these findings into new therapeutic approaches in patients with immune-mediated disease.

Role of nuclear receptors in differentiation of MOG-specific Th17 cells and MOG-induced experimental autoimmune encephalomyelitis

Background: Th17 cells play a key role in several autoimmune disorders including multiple sclerosis (MS) and inflammatory bowel disease (IBD). Their differentiation is mainly regulated by a concerted action of several nuclear receptors such as RORgammaT, RORalpha and, as suggested recently, NFIL3 and NR1D1 as part of the peripheral clock. However, little is known about possibly disease-specific contributions of these elements of Th17 biology.

OP0282 Mavrilimumab Reduces IL-17A and IL-17F Production in Biomap Human Primary Cell Systems Modeling T Cell-Dependent B Cell Activation

BackgroundMavrilimumab, an antibody to the GMCSFR alpha chain has demonstrated significant improvements in disease activity in a Phase IIa study in subjects with moderate to severe rheumatoid arthritis (Burmester et...

Emerging role of IL-17 and Th17 cells in systemic lupus erythematosus.

Despite the success of targeted therapies in managing immune-mediated inflammatory diseases (IMIDs) such as RA, IBDs, MS and psoriasis, unmet needs for such approach in SLE are widely recognized. In the past 2years, exciting insights supporting previous lines of evidence on the role of the IL-23/IL-17 axis in SLE have emerged. This is of particular importance as IL-17 blockers have now moved successfully into the clinical space, as illustrated in psoriasis and ankylosing spondylitis. However, recent fundamental studies also highlighted unexpected aspects of IL-17/Th17 biology whose comprehension may prevent disappointing results of IL-17 targeting such as those obtained in Crohn's disease. Therefore, establishing a current picture of the IL-17 pre-clinical situation in SLE is timely in order to plan future proof-of-concept studies in human.

BET bromodomain inhibition suppresses TH17-mediated pathology

Interleukin (IL) 17-producing T helper (T(H)17) cells have been selected through evolution for their ability to control fungal and bacterial infections. It is also firmly established that their aberrant generation and activation results in autoimmune conditions. Using a characterized potent and selective small molecule inhibitor, we show that the bromodomain and extra-terminal domain (BET) family of chromatin adaptors plays fundamental and selective roles in human and murine T(H)17 differentiation from naive CD4(+) T cells, as well as in the activation of previously differentiated T(H)17 cells. We provide evidence that BET controls T(H)17 differentiation in a bromodomain-dependent manner through a mechanism that includes the direct regulation of multiple effector T(H)17-associated cytokines, including IL17, IL21, and GMCSF. We also demonstrate that BET family members Brd2 and Brd4 associate with the Il17 locus in T(H)17 cells, and that this association requires bromodomains. We recapitulate the critical role of BET bromodomains in T(H)17 differentiation in vivo and show that therapeutic dosing of the BET inhibitor is efficacious in mouse models of autoimmunity. Our results identify the BET family of proteins as a fundamental link between chromatin signaling and T(H)17 biology, and support the notion of BET inhibition as a point of therapeutic intervention in autoimmune conditions.

Susceptibility-associated genetic variation at IL12B enhances Th1 polarization in psoriasis

The IL12B gene encodes the common p40 subunit of IL-12 and IL-23, cytokines with key roles in Th1 and Th17 biology, respectively, and genetic variation in this region significantly influences risk of psoriasis. Here, we demonstrate that a psoriasis-associated risk haplotype at the IL12B locus leads to increased expression of IL12B by monocytes and correlated with increased serum levels of IL-12, IFN-γ and the IFN-γ induced chemokine, CXCL10. In contrast, serum IL-23 levels were decreased in risk carriers when compared with non-carriers. We further demonstrate that IL-12 is increased in psoriatic skin and that risk carriers manifest a skewing of the inflammatory network toward stronger IFN-γ responses. Taken together, our data demonstrate that the risk variant in IL12B associates with its increased expression and predisposes to stronger Th1 polarization through deviation of the local inflammatory environment toward increased IL-12/IFN-γ at the expense of IL-23/IL-17 responses.

Targeting Interleukin-17 and Th17 in Immune Inflammatory Diseases

Th17 cells (Th17) are a distinct lineage of CD4 T cells that secrete high amounts of IL-17 under orphan nuclear receptor retinoic acid receptor-related orphan receptor γt (RORγt) which is a lineage-specific transcription factor. TGF-β and inflammatory cytokines, such as IL-6, IL-21, IL-1β, and IL-23, play central roles in the generation of Th17 cells. Th17 cells and their effector molecules, such as IL-17A, IL-17F, IL-21, IL-22, and CCL20, contribute to the progression and pathogenesis of several autoimmune and inflammatory diseases, such as rheumatoid arthritis, psoriasis, multiple sclerosis, inflammatory bowel disease and systemic lupus erythematosus. Studies of Th17 development and the effects of IL-17 signaling in autoimmune responses suggest a high potential for targeting this pathway in immune pathologies. In this review, we discuss Th17 biology in relation to autoinflammatory disorders and the various therapeutic strategies under investigation which target the IL17–Th17 cell pathway in chronic inflammatory autoimmune disorders.

Are Th17 Cells an Appropriate New Target in the Treatment of Rheumatoid Arthritis?

Th17 cells play crucial roles not only in host defense but also in many human autoimmune diseases and corresponding animal models. Although many of the fundamental principles regarding Th17 biology have been rapidly elucidated in mice, there remain numerous controversies regarding the differentiation, plasticity, and pathogenicity of human Th17 cells. In this review, we consider these open questions in comparison to what has already been clarified in mice, and discuss the potential impact of discoveries related to the Th17 pathway on the development of new therapeutic strategies in Th17 driven autoimmune diseases, specifically rheumatoid arthritis.

Embracing novel cytokines in RA – complexity grows as does opportunity!

Current therapeutics for the treatment of rheumatoid arthritis (RA) offer limited efficacy in a restricted number of patients. There is, therefore, an unmet clinical need for the development of more efficacious therapeutics for the treatment of disease. Anti-TNFalpha therapy has provided proof of principle that cytokine blockade is an appropriate strategy by which to inhibit disease progression. In this review, we describe the basic biology of potential novel cytokine targets and the results of recent clinical trials, with particular focus on the cytokines related to Th17 biology, namely interleukin (IL)-12, IL-23 and IL-17, in addition to the TNF superfamily and the adipocytokines.

Aryl Hydrocarbon Receptor independent production of IL-17A in a murine model of Hypersensitivity Pneumonitis (87.12)

Abstract AhR (Aryl hydrocarbon Receptor) is a cytosolic transcription factor functioning as a sensor for environmental toxins. Recently, AhR has been suggested to have a cardinal role in Th17 biology. Although AhR is not a direct regulatory factor for Th17 differentiation its activation is required for the functional differentiation of Th17 cells, and production IL-22. To study the role of AhR in T cell differentiation we have used selective ligands of AhR in an in vitro assay and found that AhR agonist beta-naphthoflavone enhances IL-17 production whereas an AhR inhibitor suppresses it, both at the level of transcription and translation. The Th2 cytokine IL-4 was suppressed by beta-naphthoflavone and induced by the inhibitor and the Th1 cytokine IFNγ was inhibited by both the compounds. To further investigate the role of AhR in Th17 biology we used a well established Th17 dominated murine model of hypersensitivity pneumonitis mediated by a thermophilic actinomycete Staphylopolyspora rectivirgula (SR). Analysis of the cytokine transcripts from the SR exposed lungs revealed that IL-17 was not only expressed in WT mice but was more highly expressed in Ahr-null mice. These data suggest that the AhR may regulate cytokine production by other T cell subsets, and that the requirement for the AhR in IL-17 expression may be restricted to T cells. Further work is required to determine the role of the AhR in the innate immune cells, which are potential sources of IL-17.

Emerging from the shadows: Follicular helper T cells in autoimmunity

Emerging from the shadows: Follicular helper T cells in autoimmunity

Cutting Edge: An IL-17F-CreEYFP Reporter Mouse Allows Fate Mapping of Th17 Cells

The need for reporter lines able to faithfully track Th17 cells in vivo has become an issue of exceptional importance. To address this, we generated a mouse strain in which Cre recombinase is expressed from the IL-17F promoter. Crossing the IL-17F-Cre allele to a conditional enhanced yellow fluorescent protein (EYFP) reporter mouse yielded the IL-17F-Cre(EYFP) strain, in which IL-17F expression is twinned with EYFP in live IL-17F-expressing cells. Although we demonstrate that IL-17F expression is restricted to CD4(+) T cells during experimental autoimmune encephalomyelitis, IL-17F-Cre(EYFP) CD8 T cells robustly expressed IL-17F in response to TGF-beta, IL-6, and IL-23. Fate mapping of IL-17F-expressing reporter T cells revealed a significant down-regulation of Th17 cytokines after homeostatic expansion in RAG1-deficient animals. Despite this loss of effector phenotype, committed Th17 cells were resistant to Foxp3 expression in vitro or in vivo. Thus, the IL-17F-Cre strain furthers our understanding of Th17 biology.

Sequential evolution of IL-17 responses in the early period of allograft rejection

In addition to CD4+CD25+Foxp3+ regulatory T (T(reg)) cells which protect against autoimmune tissue injury, IL-17-producing CD4+ T (T(h)17) cells have been recently described and shown to play a crucial role in autoimmune injury. It appears that there is a reciprocal developmental pathway between T(h)17 and T(reg) cells. Although IL-17 is known to be associated with allograft rejection, the cellular source of IL-17 and the nature of T(h)17 in the context of allograft rejection remain unknown. In the current study, the dynamics of T(reg) and IL-17-producing cells after syngeneic and allogeneic transplantation were examined using a wild-type murine cardiac transplantation model. Ly6G+ cells were found to produce IL-17 during the early postoperative period and CD8+ as well as CD4+ T cells were also found to produce IL-17 during alloimmune response. Graft-infiltrating Ly6G+, CD4+, and even CD8+ cells were found to express IL-17 highly compared to those in spleen. Although the frequencies of T(h)17 and T(reg) were found to gradually increase in both syngeneic and allogeneic recipients, T(h)17/T(reg) ratios were significantly higher in recipients with allograft rejection than in syngeneic recipients. In conclusion, IL-17 is produced by neutrophils during the early postoperative period and subsequently by T(h)17 and CD8+ T cells during allograft rejection. T(h)17/T(reg) imbalance is associated with the development of allograft rejection. This study would provide basic information on T(h)17 biology for future investigation in the field of transplantation.

Th17 cells in human disease

Our understanding of the role of T cells in human disease is undergoing revision as a result of the discovery of T-helper 17 (Th17) cells, a unique CD4(+) T-cell subset characterized by production of interleukin-17 (IL-17). IL-17 is a highly inflammatory cytokine with robust effects on stromal cells in many tissues. Recent data in humans and mice suggest that Th17 cells play an important role in the pathogenesis of a diverse group of immune-mediated diseases, including psoriasis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, and asthma. Initial reports also propose a role for Th17 cells in tumorigenesis and transplant rejection. Important differences, as well as many similarities, are emerging when the biology of Th17 cells in the mouse is compared with corresponding phenomena in humans. As our understanding of human Th17 biology grows, the mechanisms underlying many diseases are becoming more apparent, resulting in a new appreciation for both previously known and more recently discovered cytokines, chemokines, and feedback mechanisms. Given the strong association between excessive Th17 activity and human disease, new therapeutic approaches targeting Th17 cells are highly promising, but the potential safety of such treatments may be limited by the role of these cells in normal host defenses against infection.