Structural Activation of Pro-inflammatory Human Cytokine IL-23 by Cognate IL-23 Receptor Enables Recruitment of the Shared Receptor IL-12Rß1.

Interleukin-23 (IL-23), an IL-12 family cytokine, plays pivotal roles in pro-inflammatory T helper 17 cell responses linked to autoimmune and inflammatory diseases. Despite intense therapeutic targeting, structural and mechanistic insights into receptor complexes mediated by IL-23, and by IL-12 family members in general, have remained elusive. We determined a crystal structure of human IL-23 in complex with its cognate receptor, IL-23R, and revealed that IL-23R bound to IL-23 exclusively via its N-terminal immunoglobulin domain. The structural and functional hotspot of this interaction partially restructured the helical IL-23p19 subunit of IL-23 and restrained its IL-12p40 subunit to cooperatively bind the shared receptor IL-12Rß1 with high affinity. Together with structural insights from the interaction of IL-23 with the inhibitory antibody briakinumab and by leveraging additional IL-23:antibody complexes, we propose a mechanistic paradigm for IL-23 and IL-12 whereby cognate receptor binding to the helical cytokine subunits primes recruitment of the shared receptors via the IL-12p40 subunit.

IL-23 induces CLEC5A+ IL-17A+ neutrophils and elicit skin inflammation associated with psoriatic arthritis.

IL-23-activation of IL-17 producing T cells is involved in many rheumatic diseases. Herein, we investigate the role of IL-23 in the activation of myeloid cell subsets that contribute to skin inflammation in mice and man. IL-23 gene transfer in WT, IL-23RGFP reporter mice and subsequent analysis with spectral cytometry show that IL-23 regulates early innate immune events by inducing the expansion of a myeloid MDL1+CD11b+Ly6G+ population that dictates epidermal hyperplasia, acanthosis, and parakeratosis; hallmark pathologic features of psoriasis. Genetic ablation of MDL-1, a major PU.1 transcriptional target during myeloid differentiation exclusively expressed in myeloid cells, completely prevents IL-23-pathology. Moreover, we show that IL-23-induced myeloid subsets are also capable of producing IL-17A and IL-23R+MDL1+ cells are present in the involved skin of psoriasis patients and gene expression correlations between IL-23 and MDL-1 have been validated in multiple patient cohorts. Collectively, our data demonstrate a novel role of IL-23 in MDL-1-myelopoiesis that is responsible for skin inflammation and related pathologies. Our data open a new avenue of investigations regarding the role of IL-23 in the activation of myeloid immunoreceptors and their role in autoimmunity.

IL-27 attenuates IL-23 mediated inflammatory arthritis.

Interleukin 27 has both pro-inflammatory and anti-inflammatory properties in autoimmunity. The anti-inflammatory effects of IL-27 are linked with inhibition of Th17 differentiation but the IL-27 effect on myeloid cells is less studied. Herein we demonstrate that IL-27 inhibits IL-23-induced inflammation associated not only with Th17 cells but also with myeloid cell infiltration in the joints and splenic myeloid populations of CD11b+ GR1+ and CD3-CD11b+CD11c-GR1- cells. The IL-27 anti-inflammatory response was associated with reduced levels of myeloid cells in the spleen and bone marrow. Overall, our data demonstrate that IL-27 has an immunosuppressive role that affects IL-23-dependent myelopoiesis in the bone marrow and its progression to inflammatory arthritis and plays a crucial role in controlling IL-23 driven joint inflammation by negatively regulating the expansion of myeloid cell subsets.

Autophagy pathways in autoimmune diseases.

Autophagy comprises a growing range of cellular pathways, which occupy central roles in response to energy deprivation, organelle turnover and proteostasis. Over the years, autophagy has been increasingly linked to governing several aspects of immunity, including host defence against various pathogens, unconventional secretion of cytokines and antigen presentation. While canonical autophagy-mediated antigen processing in thymic epithelial cells supports the generation of a self-tolerant CD4+ T cell repertoire, mounting evidence suggests that deregulated autophagy pathways contribute to or sustain autoimmune responses. In animal models of multiple sclerosis (MS), non-canonical autophagy pathways such as microtubule-associated protein 1 A/1 B-light chain 3 (LC3)-associated phagocytosis can contribute to major histocompatibility complex (MHC) class II presentation of autoantigen, thereby amplifying autoreactive CD4+ T cell responses. In systemic lupus erythematosus (SLE), increased type 1 interferon production is linked to excessive autophagy in plasmacytoid dendritic cells (DCs). In rheumatoid arthritis (RA), autophagy proteins contribute to pathological citrullination of autoantigen. Immunotherapies effective in autoimmune diseases modulate autophagy functions, and strategies harnessing autophagy pathways to restrain autoimmune responses have been developed. This review illustrates recent insights in how autophagy, distinct autophagy pathways and autophagy protein functions intersect with the evolution and progression of autoimmune diseases, focusing on MS, SLE and RA.

Serine and arginine rich splicing factor 1 deficiency alters pathways involved in IL-17A expression and is implicated in human psoriasis.

Serine and Arginine Rich Splicing Factor 1 (SRSF1) is a splicing factor that binds to exonic enhancers and stimulates splicing and is previously implicated with autoimmunity. Herein, we investigate the role of SRSF1 in regulating innate immune functions that are pertinent in the pathogenesis of auto-inflammatory diseases. Specifically, we show that conditional deletion of SRSF1 in mature lymphocytes resulted in higher expression of il-17a and il-17 f and an expansion of IL17A+ CD8 T cells. Mechanistically, the aberrant expression of IL-17A in SRSF1 cKO mice could not be attributed to alternative splicing of il-17a or il-17 f genes but possibly to defective CD11B+LY6C+ myeloid derived suppressor function in the spleen. Finally, meta-analysis of RNA-Seq collected from psoriasis patients demonstrate a clear correlation between SRSF1 and psoriasis that suggests a putative role of SRSF1 in IL-17A-induced psoriasis.

Psoriatic arthritis under the influence of IFNγ.

Psoriasis is a common multifactorial autoimmune disease of the skin, and in a large percentage of patients, immune responses involve nail and joint pathology, which develop psoriatic arthritis (PsA). Historically, T helper 1 (Th1)-derived-IFN-γ was abundantly detected in psoriatic skin and its correlation with development and severity of PsO, led to an early classification of psoriasis as a Th1-mediated disease. Investigations of the cellular and molecular mechanisms of PsO pathogenesis in recent years, together with impressive results of biologics against interleukin 17A (IL-17) have shifted focus on IL-17A. However, the contributions of IFN-γ in IL-17 induced pathology and its involvement in the development of PsA have been largely overshadowed. This review summarizes the current knowledge on IFN-γ and provides new insights on the contribution of IFN-γ to PsO and PsA disease pathogenesis and development.

Ca2+-Dependent Regulation of NFATc1 via KCa3.1 in Inflammatory Osteoclastogenesis.

In inflammatory arthritis, the dysregulation of osteoclast activity by proinflammatory cytokines, including TNF, interferes with bone remodeling during inflammation through Ca2+-dependent mechanisms causing pathological bone loss. Ca2+-dependent CREB/c-fos activation via Ca2+-calmodulin kinase IV (CaMKIV) induces transcriptional regulation of osteoclast-specific genes via NFATc1, which facilitate bone resorption. In leukocytes, Ca2+ regulation of NFAT-dependent gene expression oftentimes involves the activity of the Ca2+-activated K+ channel KCa3.1. In this study, we evaluate KCa3.1 as a modulator of Ca2+-induced NFAT-dependent osteoclast differentiation in inflammatory bone loss. Microarray analysis of receptor activator of NF-κB ligand (RANKL)-activated murine bone marrow macrophage (BMM) cultures revealed unique upregulation of KCa3.1 during osteoclastogenesis. The expression of KCa3.1 in vivo was confirmed by immunofluorescence staining on multinucleated cells at the bone surface of inflamed mouse joints. Experiments on in vitro BMM cultures revealed that KCa3.1-/- and TRAM-34 treatment significantly reduced the expression of osteoclast-specific genes (p < 0.05) alongside decreased osteoclast formation (p < 0.0001) in inflammatory (RANKL+TNF) and noninflammatory (RANKL) conditions. In particular, live cell Ca2+ imaging and Western blot analysis showed that TRAM-34 pretreatment decreased transient RANKL-induced Ca2+ amplitudes in BMMs by ∼50% (p < 0.0001) and prevented phosphorylation of CaMKIV. KCa3.1-/- reduced RANKL+/-TNF-stimulated phosphorylation of CREB and expression of c-fos in BMMs (p < 0.01), culminating in decreased NFATc1 protein expression and transcriptional activity (p < 0.01). These data indicate that KCa3.1 regulates Ca2+-dependent NFATc1 expression via CaMKIV/CREB during inflammatory osteoclastogenesis in the presence of TNF, corroborating its role as a target candidate for the treatment of bone erosion in inflammatory arthritis.

Compendium of synovial signatures identifies pathologic characteristics for predicting treatment response in rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is therapeutically challenging due to patient heterogeneity and variability. Herein we describe a novel integration of RA synovial genome-scale transcriptomic profiling of different patient cohorts that can be used to provide predictive insights on drug responses. A normalized compendium consisting of 256 RA synovial samples that cover an intersection of 11,769 genes from 11 datasets was build and compared with similar datasets derived from OA patients and healthy controls. Differentially expression genes (DEGs) that were identified in three independent methods were fed into functional network analysis, with subsequent grouping of the samples based on a non-negative matrix factorization method. RA-relevant pathway activation scores and four machine learning classification techniques supported the generation of a predictive model of patient treatment response. We identified 876 up-regulated DEGs including 24 known genetic risk factors and 8 drug targets. DEG-based subgrouping revealed 3 distinct RA patient clusters with distinct activity signatures for RA-relevant pathways. In the case of infliximab, we constructed a classifier of drug response that was highly accurate with an AUC/AUPR of 0.92/0.86. The most informative pathways in achieving this performance were the NFκB-, FcεRI- TCR-, and TNF signaling pathways. Similarly, the expression of the HMMR, PRPF4B, EVI2A, RAB27A, MALT1, SNX6, and IFIH1 genes contributed in predicting the patient outcome. Construction and analysis of normalized synovial transcriptomic compendia can provide useful insights for understanding RA-related pathway involvement and drug responses for individual patients.

Pathophysiology and inhibition of IL-23 signaling in psoriatic arthritis: A molecular insight.

Psoriatic arthritis (PsA) is a chronic inflammatory arthritis of unknown etiology, and currently the cellular and molecular interactions that dictate its pathogenesis remain elusive. A role of the interleukin-23 (IL-23)/IL-23R (IL-23 receptor) interaction in the development of psoriasis and PsA is well established. As IL-23 regulates the differentiation and activation of innate and adaptive immunity, it pertains to a very complex pathophysiology involving a plethora of effectors and transducers. In this review, we will discuss recent advances on the cellular and molecular pathophysiological mechanisms that regulate the initiation and progression of PsA as well as new therapeutic approaches for IL-23/IL-23R targeted therapeutics.

Critical Role of LTB4/BLT1 in IL-23-Induced Synovial Inflammation and Osteoclastogenesis via NF-κB.

IL-23 activates the synthesis and production of leukotriene B4 (LTB4) in myeloid cells, which modulate inflammatory arthritis. In this study we investigated the role of LTB4 and its receptor LTB4R1 (BLT1) in synovial inflammation and osteoclast differentiation. Specifically, we used IL-23 in vivo gene transfer to induce arthritis in mice and showed that elevated serum LTB4 and synovial expression of 5-lipoxygenase correlated with increased disease severity by histological evaluation and paw swelling compared with GFP gene transfer controls. To further investigate the effect of the LTB4 pathway in bone loss, we performed osteoclast differentiation assays by stimulating with M-CSF and receptor activator of NF-κB ligand bone marrow cells derived from BLT1+/+ and/or BLT1-/- mice and used quantitative PCR for gene expression analysis in terminally differentiated osteoclasts. Deficiency in BLT1 resulted in the upregulation of osteoclast-related genes and an increase in the formation of giant, multinucleated TRAP+ cells capable of F-actin ring formation. Additionally, BLT1 deficiency showed an increase of phosphorylated NF-κB and phosphorylated IκB levels in osteoclasts. We also performed real-time calcium imaging to study the effect of BLT1 deficiency in receptor activator of NF-κ-B ligand-induced activation of intracellular calcium flux in vitro. Our data show that LTB4 and its receptor BLT1 exacerbate synovial inflammation in vivo and bone resorption in vitro, suggesting that LTB4 and BLT1 could be effectively targeted for the treatment of musculoskeletal diseases.

Inflammation in bone physiology and pathology.

PURPOSE OF REVIEW: Bone is constantly being remodeled throughout adult life through constant anabolic and catabolic actions that maintain tissue homeostasis. A number of hormones, cytokines growth factors, and the proximity of various cells to bone surfaces influence this process. Inflammatory changes at the bone microenvironment result in alterations leading to both excessive bone loss and bone formation. Detailed understanding of the physiological and pathological mechanisms that dictate these changes will allow us to harness inflammatory signals in bone regeneration. RECENT FINDINGS: Recent reports have suggested that inflammatory signals are able to stimulate transcription factors that regulate osteoblast differentiation from their precursors. SUMMARY: In this review, we summarized current understanding of the roles of inflammation in bone resorption and bone formation, which give rise to different disorders and discuss the huge potential of harnessing these inflammatory signals to achieve bone regeneration.

Concise Review: Stem Cells in Osteoimmunology.

Bone remodeling is a lifelong process in which mature bone tissue is removed from the skeleton by bone resorption and is replenished by new during ossification or bone formation. The remodeling cycle requires both the differentiation and activation of two cell types with opposing functions; the osteoclast, which orchestrates bone resorption, and the osteoblast, which orchestrates bone formation. The differentiation of these cells from their respective precursors is a process which has been overshadowed by enigma, particularly because the precise osteoclast precursor has not been identified and because the identification of skeletal stem cells, which give rise to osteoblasts, is very recent. Latest advances in the area of stem cell biology have enabled us to gain a better understanding of how these differentiation processes occur in physiological and pathological conditions. In this review we postulate that modulation of stem cells during inflammatory conditions is a necessary prerequisite of bone remodeling and therefore an essential new component to the field of osteoimmunology. In this context, we highlight the role of transcription factor nuclear factor of activated T cells cytoplasmic 1 (NFATc1), because it directly links inflammation with differentiation of osteoclasts and osteoblasts. Stem Cells 2017;35:1461-1467.

T Cell-Independent Mechanisms Associated with Neutrophil Extracellular Trap Formation and Selective Autophagy in IL-17A-Mediated Epidermal Hyperplasia.

IL-17A has been strongly associated with epidermal hyperplasia in many cutaneous disorders. However, because IL-17A is mainly produced by αß and γδT cells in response to IL-23, the role of T cells and IL-23 has overshadowed any IL-17A-independent actions. In this article, we report that IL-17A gene transfer induces epidermal hyperplasia in Il23r-/-Rag1-/-- and Tcrδ-deficient mice, which can be prevented by neutrophil depletion. Moreover, adoptive transfer of CD11b+Gr-1hi cells, after IL-17A gene transfer, was sufficient to phenocopy the disease. We further show that the IL-17A-induced pathology was prevented in transgenic mice with impaired neutrophil extracellular trap formation and/or neutrophils with conditional deletion of the master regulator of selective autophagy, Wdfy3. Our data demonstrate a novel T cell-independent mechanism that is associated with neutrophil extracellular trap formation and selective autophagy in IL-17A-mediated epidermal hyperplasia.

Autophagy and autoimmunity.

Autophagy is a highly conserved protein degradation pathway from yeasts to humans that is essential for removing protein aggregates and misfolded proteins in healthy cells. Recently, autophagy-related genes polymorphisms have been implicated in several autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, psoriasis, and multiple sclerosis. Numerous studies reveal autophagy and autophagy-related proteins also participate in immune regulation. Conditional deletions of autophagy-related proteins in mice have rendered protection from experimental autoimmune encephalomyelitis, and TNF-mediated joint destruction in animal models of multiple sclerosis and experimental arthritis respectively. As autophagy is strongly implicated in immune functions such as removal of intracellular bacteria, inflammatory cytokine secretion, antigen presentation, and lymphocyte development, in this review we summarized current understanding of the roles of autophagy and autophagy proteins in autoimmune diseases.

Loss of WDFY3 ameliorates severity of serum transfer-induced arthritis independently of autophagy.

WDFY3 is a master regulator of selective autophagy that we recently showed to interact with TRAF6 and augment RANKL-induced osteoclastogenesis in vitro and in vivo via the NF-κB pathway. Since the NF-κB pathway plays a major role in inflammation herein, we investigate the role of WDFY3 in an arthritis animal model. Our data show that WDFY3 conditional knockout mice (Wdfy3loxP/loxP-LysM-Cre+) were protected in the K/BxN serum transfer-induced arthritis animal model. These effects were independent of alterations in starvation-induced autophagy as evidenced by Western blot analysis of the autophagy marker LC3, autophagosome formation in osteoclast precursors and lysosome formation in osteoclasts derived from WDFY3-cKO mice compared to controls. Moreover, we demonstrate by immunofluorescence and co-immunoprecipitation that WDFY3 interacts with SQSTM1 in macrophages and osteoclasts. Collectively, our data suggest that loss of WDFY3 in myeloid cells leads to reduced severity of inflammatory arthritis independently of WDFY3 function in starvation-induced autophagy.

CD4+ virtual memory: Antigen-inexperienced T cells reside in the naïve, regulatory, and memory T cell compartments at similar frequencies, implications for autoimmunity.

It is widely accepted that central and effector memory CD4+ T cells originate from naïve T cells after they have encountered their cognate antigen in the setting of appropriate co-stimulation. However, if this were true the diversity of T cell receptor (TCR) sequences within the naïve T cell compartment should be far greater than that of the memory T cell compartment, which is not supported by TCR sequencing data. Here we demonstrate that aged mice with far fewer naïve T cells, respond to the model antigen, hen eggwhite lysozyme (HEL), by utilizing the same TCR sequence as their younger counterparts. CD4+ T cell repertoire analysis of highly purified T cell populations from naive animals revealed that the HEL-specific clones displayed effector and central "memory" cell surface phenotypes even prior to having encountered their cognate antigen. Furthermore, HEL-inexperienced CD4+ T cells were found to reside within the naïve, regulatory, central memory, and effector memory T cell populations at similar frequencies and the majority of the CD4+ T cells within the regulatory and memory populations were unexpanded. These findings support a new paradigm for CD4+ T cell maturation in which a specific clone can undergo a differentiation process to exhibit a "memory" or regulatory phenotype without having undergone a clonal expansion event. It also demonstrates that a foreign-specific T cell is just as likely to reside within the regulatory T cell compartment as it would the naïve compartment, arguing against the specificity of the regulatory T cell compartment being skewed towards self-reactive T cell clones. Finally, we demonstrate that the same set of foreign and autoreactive CD4+ T cell clones are repetitively generated throughout adulthood. The latter observation argues against T cell-depleting strategies or autologous stem cell transplantation as therapies for autoimmunity-as the immune system has the ability to regenerate pathogenic clones.

Crosstalk among IL-23 and DNAX activating protein of 12 kDa-dependent pathways promotes osteoclastogenesis.

IL-23 has been well studied in the context of T cell differentiation; however, its role in the differentiation of myeloid progenitors is less clear. In this paper, we describe a novel role of IL-23 in myeloid cell differentiation. Specifically, we have identified that in human PBMCs, IL-23 induces the expression of MDL-1, a PU.1 transcriptional target during myeloid differentiation, which orchestrates osteoclast differentiation through activation of DNAX activating protein of 12 kDa and its ITAMs. The molecular events that lead to the differentiation of human macrophages to terminally differentiated osteoclasts are dependent on spleen tyrosine kinase and phospholipase Cγ2 phosphorylation for the induction of intracellular calcium flux and the subsequent activation of master regulator osteoclast transcription factor NFATc1. IL-23-elicited osteoclastogenesis is independent of the receptor activator of NF-κB ligand pathway and uses a unique myeloid DNAX activating protein of 12 kDa-associated lectin-1(+)/DNAX activating protein of 12 kDa(+) cell subset. Our data define a novel pathway that is used by IL-23 in myeloid cells and identify a major mechanism for the stimulation of osteoclastogenesis in inflammatory arthritis.

Autophagy-linked FYVE containing protein WDFY3 interacts with TRAF6 and modulates RANKL-induced osteoclastogenesis.

Recently, autophagy-related proteins were shown to regulate osteoclast mediated bone resorption, a critical process in autoimmune diseases such as rheumatoid arthritis. However, the role of autophagy-linked FYVE containing protein, WDFY3, in osteoclast biology remains elusive. WDFY3 is a master regulator in selective autophagy for clearing ubiquitinated protein aggregates and has been linked with rheumatoid arthritis. Herein, we used a series of WDFY3 transgenic mice (Wdfy3(lacZ) and Wdfy3(loxP)) to investigate the function of WDFY3 in osteoclast development and function. Our data demonstrate that WDFY3 is highly expressed at the growth plate of neonatal mice and is expressed in osteoclasts in vitro cultures. Osteoclasts derived from WDFY3 conditional knockout mice (Wdfy3(loxP/loxP)-LysM-Cre(+)) demonstrated increased osteoclast differentiation as evidenced by higher number and enlarged size of TRAP(+) multinucleated cells. Western blot analysis also revealed up-regulation of TRAF6 and an increase in RANKL-induced NF-κB signaling in WDFY3-deficient bone marrow-derived macrophages compared to wild type cultures. Consistent with these observations WDFY3-deficient cells also demonstrated an increase in osteoclast-related genes Ctsk, Acp5, Mmp9 and an increase of dentine resorption in in vitro assays. Importantly, in vivo RANKL gene transfer exacerbated bone loss in WDFY3 conditional knockout mice, as evidenced by elevated serum TRAP, CTX-I and micro-CT analysis of distal femurs compared to wild type littermates. Taken together, our data highlight a novel role for WDFY3 in osteoclast development and function, which can be exploited for the treatment of musculoskeletal diseases.

IL-17A gene transfer induces bone loss and epidermal hyperplasia associated with psoriatic arthritis.

BACKGROUND: Psoriatic arthritis (PsA) is a chronic inflammatory disease characterised by clinical features that include bone loss and epidermal hyperplasia. Aberrant cytokine expression has been linked to joint and skin pathology; however, it is unclear which cytokines are critical for disease initiation. Interleukin 17A (IL-17A) participates in many pathological immune responses; however, its role in PsA has not been fully elucidated. OBJECTIVE: To determine the role of IL-17A in epidermal hyperplasia and bone destruction associated with psoriatic arthritis. DESIGN: An in vivo gene transfer approach was used to investigate the role of IL-17A in animal models of inflammatory (collagen-induced arthritis) and non-inflammatory (receptor activator of NF-κB ligand (RANKL)-gene transfer) bone loss. RESULTS: IL-17A gene transfer induced the expansion of IL-17RA(+)CD11b(+)Gr1(low) osteoclast precursors and a concomitant elevation of biomarkers indicative of bone resorption. This occurred at a time preceding noticeable joint inflammation, suggesting that IL-17A is critical for the induction of pathological bone resorption through direct activation of osteoclast precursors. Moreover, IL-17A induced a second myeloid population CD11b(+)Gr1(high) neutrophil-like cells, which was associated with cutaneous pathology including epidermal hyperplasia, parakeratosis and Munro's microabscesses formation. CONCLUSIONS: Collectively, these data support that IL-17A can play a key role in the pathogenesis of inflammation-associated arthritis and/or skin disease, as observed in PsA.