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Emerging evidence suggests that microbiome-host crosstalk regulates intestinal immune activity and predisposition to inflammatory bowel disease (IBD). NF-κB is a master regulator of immune function and a validated target for the treatment of IBD. Here, we identify five Clostridium strains that suppress immune-mediated NF-κB activation in epithelial cell lines, PBMCs, and gut epithelial organoids from healthy human subjects and patients with IBD. Cell-free culture supernatant from Clostridium bolteae AHG0001 strain, but not the reference C. bolteae BAA-613 strain, suppresses inflammatory responses and endoplasmic reticulum stress in gut epithelial organoids derived from Winnie mice. The in vivo responses to Clostridium bolteae AHG0001 and BAA-613 mirror the in vitro activity. Thus, using our in vitro screening of bacteria capable of suppressing NF-κB in the context of IBD and using an ex vivo organoid-based approach, we identify a strain capable of alleviating colitis in a relevant pre-clinical animal model of IBD.

Dendritic cells (DCs) are specialized antigen-presenting cells which link the innate and adaptive immune responses, activating and priming effector CD4+ T cells, cross-presenting antigen to CD8+ T cells, and promoting B-cell antibody production. DCs also play important roles in the maintenance of immune tolerance. DCs and T cells underpin the basis of the autoimmune response in rheumatoid arthritis. In this chapter we describe the function of DCs and the response of T cells in rheumatoid arthritis pathogenesis, introduce the DC and T-cell players and their function in the immune system, then review the evidence for their involvement in the pathogenesis of rheumatoid arthritis (RA), particularly through the presentation of antigen that triggers the differentiation of autoreactive T cells, as well as innate immune effector functions. Finally, the emerging prospects for DC targeting for immunotherapy are covered.

Objectives Adoptive transfer of chimeric antigen receptor (CAR)‐modified T cells is a form of cancer immunotherapy that has achieved remarkable efficacy in patients with some haematological cancers. However, challenges remain in CAR T‐cell treatment of solid tumours because of tumour‐mediated immunosuppression. Methods We have demonstrated that CAR T‐cell stimulation through T‐cell receptors (TCRs) in vivo can generate durable responses against solid tumours in a variety of murine models. Since Clec9A‐targeting tailored nanoemulsion (Clec9A‐TNE) vaccine enhances antitumour immune responses through selective activation of Clec9A+ cross‐presenting dendritic cells (DCs), we hypothesised that Clec9A‐TNE could prime DCs for antigen presentation to CAR T cells through TCRs and thus improve CAR T‐cell responses against solid tumours. To test this hypothesis, we used CAR T cells expressing transgenic TCRs specific for ovalbumin (OVA) peptides SIINFEKL (CAROTI) or OVA323‐339 (CAROTII). Results We demonstrated that the Clec9A‐TNEs encapsulating full‐length recombinant OVA protein (OVA‐Clec9A‐TNE) improved CAROT T‐cell proliferation and inflammatory cytokine secretion in vitro. Combined treatment using the OVA‐Clec9A‐TNE and CAROT cells resulted in durable responses and some rejections of tumours in immunocompetent mice. Tumour regression was accompanied by enhanced CAROT cell proliferation and infiltration into the tumours. Conclusion Our study presents Clec9A‐TNE as a prospective avenue to enhance CAR T‐cell efficacy for solid cancers., Clec9A‐targeting tailored nanoemulsion (Clec9A‐TNE) induced extensive proliferation, persistence and activation of chimeric antigen receptor (CAR) T cells, leading to the eradication of solid tumours in murine models. This novel approach could lead to the development of new CAR T‐cell therapies against some common solid tumour types in patients.

ZAP-70SKG, Il17a-deficient SKG, and BALB/c female mice were infected with C muridarum or C muridarum luciferase in the genitals. C muridarum dissemination was assessed by in vivo imaging or genomic DNA amplification. Macrophages were depleted using clodronate liposomes. Anti-tumor necrosis factor (anti-TNF) and anti-interleukin-23p19 (anti-IL-23p19) were administered after infection or arthritis onset. Gene expression of Hspa5, Tgtp1, Il23a, Il17a, Il12b, and Tnf was compared in SKG mice and BALB/c mice.One week following infection with C muridarum, macrophages and neutrophils were observed to have infiltrated the uteri of mice and were also shown to have carried C muridarum DNA to the spleen. C muridarum load was higher in SKG mice than in BALB/c mice. Macrophage depletion was shown to reduce C muridarum load and prevent development of arthritis. Compared with BALB/c mice, expression of Il23a and Il17a was increased in the uterine and splenic neutrophils of SKG mice. The presence of anti-IL-23p19 during infection or Il17a deficiency suppressed arthritis. Tnf was overexpressed in the joints of SKG mice within 1 week postinfection, and persisted beyond the first week. TNF inhibition during infection or at arthritis onset suppressed the development of arthritis. Levels of endoplasmic reticulum stress were constitutively increased in the joints of SKG mice but were induced, in conjunction with immunity-related GTPase, by C muridarum infection in the uterus.C muridarum load is higher in SKG mice than in BALB/c mice. Whereas proinflammatory IL-23 produced by neutrophils contributes to the initiation of C muridarum-mediated ReA, macrophage depletion reduces C muridarum dissemination to other tissues, tissue burden, and the development of arthritis. TNF inhibition was also shown to suppress arthritis development. Our data suggest that enhanced bacterial dissemination in macrophages of SKG mice drives the TNF production needed for persistent arthritis.

Copyright © 2018 by the American Thoracic Society RelB is a member of the NF-kB family, which is essential for dendritic cell (DC) function and maturation. However, the contribution of RelB to the development of allergic airway inflammation (AAI) is unknown. Here, we identify a pivotal role for RelB in the development of spontaneous AAI that is independent of exogenous allergen exposure. We assessed AAI in two strains of RelB-deficient (RelB2/2) mice: one with a targeted deletion and one expressing a major histocompatibility complex transgene. To determine the importance of RelB in DCs, RelB-sufficient DCs (RelB1/1 or RelB2/2) were adoptively transferred into RelB2/2 mice. Both strains had increased pulmonary inflammation compared with their respective wild-type (RelB1/1) and heterozygous (RelB1/2) controls. RelB2/2 mice also had increased inflammatory cell influx into the airways, levels of chemokines (CCL2/3/4/5/11/17 and CXCL9/10/13) and T-helper cell type 2-associated cytokines (IL-4/5) in lung tissues, serum IgE, and airway remodeling (mucus-secreting cell numbers, collagen deposition, and epithelial thickening). Transfer of RelB1/2 CD11c1 DCs into RelB2/2 mice decreased pulmonary inflammation, with reductions in lung chemokines, T-helper cell type 2-associated cytokines (IL-4/5/13/25/33 and thymic stromal lymphopoietin), serum IgE, type 2 innate lymphoid cells, myeloid DCs, gd T cells, lung Vb131 T cells, mucus-secreting cells, airway collagen deposition, and epithelial thickening. These data indicate that RelB deficiency may be a key pathway underlying AAI, and that DC-encoded RelB is sufficient to restore control of this inflammation.

The HLA-DRB1 locus is strongly associated with rheumatoid arthritis (RA) susceptibility, whereupon citrullinated self-peptides bind to HLA-DR molecules bearing the shared epitope (SE) amino acid motif. However, the differing propensity for citrullinated/non-citrullinated self-peptides to bind given HLA-DR allomorphs remains unclear. Here, we used a fluorescence polarization assay to determine a hierarchy of binding affinities of 34 self-peptides implicated in RA against three HLA-DRB1 allomorphs (HLA-DRB1*04:01/*04:04/*04:05) each possessing the SE motif. For all three HLA-DRB1 allomorphs, we observed a strong correlation between binding affinity and citrullination at P4 of the bound peptide ligand. A differing hierarchy of peptide-binding affinities across the three HLA-DRB1 allomorphs was attributable to the β-chain polymorphisms that resided outside the SE motif and were consistent with sequences of naturally presented peptide ligands. Structural determination of eight HLA–DR4–self-epitope complexes revealed strict conformational convergence of the P4-Cit and surrounding HLA β-chain residues. Polymorphic residues that form part of the P1 and P9 pockets of the HLA-DR molecules provided a structural basis for the preferential binding of the citrullinated self-peptides to the HLA-DR4 allomorphs. Collectively, we provide a molecular basis for the interplay between citrullination of self-antigens and HLA polymorphisms that shape peptide–HLA-DR4 binding affinities in RA.

Improved understanding of the molecular mechanisms underlying dysregulated inflammatory responses in severe infection and septic shock is urgently needed to improve patient management and identify new therapeutic opportunities. The WNT signaling pathway has been implicated as a novel constituent of the immune response to infection, but its contribution to the host response in septic shock is unknown. Although individual WNT proteins have been ascribed pro- or anti-inflammatory functions, their concerted contributions to inflammation in vivo remain to be clearly defined. Here we report differential expression of multiple WNT ligands in whole blood of patients with septic shock and reveal significant correlations with inflammatory cytokines. Systemic challenge of mice with lipopolysaccharide (LPS) similarly elicited differential expression of multiple WNT ligands with correlations between WNT and cytokine expression that partially overlap with the findings in human blood. Molecular regulators of WNT expression during microbial encounter in vivo are largely unexplored. Analyses in gene-deficient mice revealed differential contributions of Toll-like receptor signaling adaptors, a positive role for tumor necrosis factor, but a negative regulatory role for interleukin (IL)-12/23p40 in the LPS-induced expression of Wnt5b, Wnt10a, Wnt10b, and Wnt11. Pharmacologic targeting of bottlenecks of the WNT network, WNT acylation and β-catenin activity, diminished IL-6, tumor necrosis factor, and IL-12/23p40 in serum of LPS-challenged mice and cultured splenocytes, whereas IL-10 production remained largely unaffected. Taken together, our data support the conclusion that the concerted action of WNT proteins during severe infection and septic shock promotes inflammation, and that this is, at least in part, mediated by WNT/β-catenin signaling.

Autoimmune diseases resulting from MHC class II–restricted autoantigen-specific T cell immunity include the systemic inflammatory autoimmune conditions rheumatoid arthritis and vasculitis. While currently treated with broad-acting immunosuppressive drugs, a preferable strategy is to regulate antigen-specific effector T cells (Teffs) to restore tolerance by exploiting DC antigen presentation. We targeted draining lymph node (dLN) phagocytic DCs using liposomes encapsulating 1α,25-dihydroxyvitamin D3 (calcitriol) and antigenic peptide to elucidate mechanisms of tolerance used by DCs and responding T cells under resting and immunized conditions. PD-L1 expression was upregulated in dLNs of immunized relative to naive mice. Subcutaneous administration of liposomes encapsulating OVA(323–339) and calcitriol targeted dLN PD-L1(hi) DCs of immunized mice and reduced their MHC class II expression. OVA(323–339)/calcitriol liposomes suppressed expansion, differentiation, and function of Teffs and induced Foxp3(+) and IL-10(+) peripheral Tregs in an antigen-specific manner, which was dependent on PD-L1. Peptide/calcitriol liposomes modulated CD40 expression by human DCs and promoted Treg induction in vitro. Liposomes encapsulating calcitriol and disease-associated peptides suppressed the severity of rheumatoid arthritis and Goodpasture’s vasculitis models with suppression of antigen-specific memory T cell differentiation and function. Accordingly, peptide/calcitriol liposomes leverage DC PD-L1 for antigen-specific T cell regulation and induce antigen-specific tolerance in inflammatory autoimmune diseases.

Type I Interferon (IFN) signaling plays a critical role in dendritic cell (DC) development and functions. Inhibition of hyper type I IFN signaling promotes cDC2 subtype development. Relb is essential to development of cDC2 subtype and here we analyzed its effect on type I IFN signaling in DCs. We show that Relb suppresses the homeostatic type I IFN signaling in cDC2 cultures. TLR stimulation of FL-DCs led to RelB induction coinciding with fall in IFN signatures; conforming with the observation Relb expression reduced TLR stimulated IFN induction along with decrease in ISGs. Towards understanding mechanism, we show that effects of RelB are mediated by increased levels of IκBα. We demonstrate that RelB dampened antiviral responses by lowering ISG levels and the defect in cDC2 development in RelB null mice can be rescued in Ifnar1-/- background. Overall, we propose a novel role of RelB as a negative regulator of the type I IFN signaling pathway; fine tuning development of cDC2 subtype.

Ag-specific tolerizing immunotherapy is considered the optimal strategy to control type 1 diabetes, a childhood disease involving autoimmunity toward multiple islet antigenic peptides. To understand whether tolerizing immunotherapy with a single peptide could control diabetes driven by multiple Ags, we coencapsulated the high-affinity CD4+ mimotope (BDC2.5mim) of islet autoantigen chromogranin A (ChgA) with or without calcitriol (1α,25-dihydroxyvitamin D3) into liposomes. After liposome administration, we followed the endogenous ChgA-specific immune response with specific tetramers. Liposome administration s.c., but not i.v., induced ChgA-specific Foxp3+ and Foxp3− PD1+ CD73+ ICOS+ IL-10+ peripheral regulatory T cells in prediabetic mice, and liposome administration at the onset of hyperglycemia significantly delayed diabetes progression. After BDC2.5mim/calcitriol liposome administration, adoptive transfer of CD4+ T cells suppressed the development of diabetes in NOD severe combined immunodeficiency mice receiving diabetogenic splenocytes. After BDC2.5mim/calcitriol liposome treatment and expansion of ChgA-specific peripheral regulatory T cells. IFN-γ production and expansion of islet-specific glucose-6-phosphatase catalytic subunit–related protein–specific CD8+ T cells were also suppressed in pancreatic draining lymph node, demonstrating bystander tolerance at the site of Ag presentation. Thus, liposomes encapsulating the single CD4+ peptide, BDC2.5mim, and calcitriol induce ChgA-specific CD4+ T cells that regulate CD4+ and CD8+ self-antigen specificities and autoimmune diabetes in NOD mice.

Summary A safe and controlled manipulation of endocytosis in vivo may have disruptive therapeutic potential. Here, we demonstrate that the anti-emetic/anti-psychotic prochlorperazine can be repurposed to reversibly inhibit the in vivo endocytosis of membrane proteins targeted by therapeutic monoclonal antibodies, as directly demonstrated by our human tumor ex vivo assay. Temporary endocytosis inhibition results in enhanced target availability and improved efficiency of natural killer cell-mediated antibody-dependent cellular cytotoxicity (ADCC), a mediator of clinical responses induced by IgG1 antibodies, demonstrated here for cetuximab, trastuzumab, and avelumab. Extensive analysis of downstream signaling pathways ruled out on-target toxicities. By overcoming the heterogeneity of drug target availability that frequently characterizes poorly responsive or resistant tumors, clinical application of reversible endocytosis inhibition may considerably improve the clinical benefit of ADCC-mediating therapeutic antibodies.

Background Patients with rheumatoid arthritis (RA) have higher mortality rates than the general population, primarily from cardiovascular disease (CVD). Diabetes is an acknowledged risk factor for early death, mainly due to CVD. The reported risk for CVD is increased for both patients with RA and diabetes, however the effect of diabetes appears to be greater than that of RA (1). Objectives To assess whether the increase in all-cause mortality rates in patients with RA and patients with diabetes are similar, compared to the general population. Methods Data were obtained from the 2nd and 3rd survey of the population-based Nord Trondelag Health Study (HUNT) and the Norwegian National Cause of Death Registry. The RA diagnoses have previously been validated (2). The definition of diabetes was self-reported diabetes, self-reported use of antidiabetica, or non-fasting blood glucose level >11.1 mmol/L. The participants were followed from their participation in HUNT to death or to 31.12.2014, whichever came first. Participants with other types of inflammatory arthritis or missing data for any of the variables in the model were excluded. Furthermore, the patients with both RA and diabetes were not included in the analysis (n=33). Participants with neither RA nor diabetes were defined as controls. RA patients, diabetes patients and controls were compared using Cox proportional hazard regression modelling, with age as the time scale. The model was adjusted for sex, smoking status, previous CVD, body mass index (BMI), hypertension, total cholesterol and creatinine. To obtain proportional hazards, an interaction term with age ≤75 years vs >75 years was included for diabetes, smoking and previous CVD. The effects of the groups were compared using overlap of confidence intervals. Results There were 123 (32%) deaths in the RA group, 1280 (44%) deaths in the diabetes groups and 11,641 (18%) deaths in the control group. The hazard ratio (HR) for RA was 1.24 (95% confidence interval: CI 1.02-1.46, p-value 0.013). The HR for diabetes was 1.82 (CI 1.60-2.05, p-value 75 years. RA patients had a significantly lower HR compared to diabetes patients for age ≤75 years, but not for age >75 years. Conclusion Patients with RA or diabetes had significantly higher mortality rates than controls, with larger HR for diabetes than for RA. The HR for RA was significantly lower than for diabetes for patients ≤75 years, but not for patients >75 years. This may be due to survival bias in diabetes patients >75 years. References 1 Curtis, J. R. et al., Is Rheumatoid Arthritis a Cardiovascular Risk-Equivalent to Diabetes Mellitus? Arthritis Care Res, 2018. 70(11): p.1694-1699 2 Videm, V, et al., Self-reported Diagnosis of Rheumatoid Arthritis or Ankylosing Spondylitis has Low Accuracy: Data from the Nord-Trondelag Health Study. J Reumatol, 2017. 44(8):p.1134-1141 Disclosure of Interests Ingrid Saether Houge: None declared, Mari Hoff: None declared, Ranjeny Thomas Grant/research support from: Janssen Biotec Inc, Merck and Co, Consultant for: Janssen Biotec Inc, Speakers bureau: Janssen, Merck, Vibeke Videm: None declared

ObjectivePatients with rheumatoid arthritis (RA) suffer from more cardiovascular disease (CVD), and develop cardiovascular risk factors at an earlier age than the general population. Cardiorespiratory fitness (CRF) is an important predictor of cardiovascular health. There are few data regarding CRF of RA patients, measured as peak oxygen uptake (VO2peak) by the gold standard method; cardiopulmonary exercise testing. We compared CRF in RA patients to those from a healthy population, and investigated if risk factors for CVD and RA-specific variables including subjective and objective disease activity measures were associated with CRF in RA patients.MethodsVO2peak tests of RA patients (n=93) were compared to those of an age-matched and gender-matched healthy population (n=4631) from the Nord-Trøndelag Health Study. Predictors of VO2peak were found using Lasso (least absolute shrinkage and selection operator) regression, followed by standardised multiple linear regression.ResultsWomen with RA ≥40 years and men with RA aged 40–49 years or 60–69 years had up to 20% lower CRF than the healthy population in the same age groups. By relative importance, body mass index (standardised coefficient=−0.25, p2=0.82, gender-adjusted and age-adjusted).ConclusionCRF in RA patients was lower than in a healthy population. CRF was associated with common risk factors for CVD and the PGA score. Focusing on fitness in RA patients may improve cardiovascular health.

Background: Mucosal-associated invariant T (MAIT) cells are innate-like T cells that recognise bacterial metabolites presented by MHC class I-related protein 1 (MR1). Bacterial dysbiosis has been implicated in auto-inflammatory disease development. We investigated MAIT cells in early, untreated rheumatoid arthritis (RA) and spondyloarthritis (SpA) patients.Methods: Blood and synovial fluid mononuclear cells obtained from patients (SpA/RA) and controls were stimulated with fixed Escherichia coli to provide MAIT ligand. Cells were analysed by flow cytometry and MAIT cells were identified by MR1-5-OP-RU tetramers. Synovial biopsies were studied by confocal microscopy.Results: Peripheral and synovial CD3+ MR1-tet+ MAIT cell frequencies were comparable in all groups. MAIT cells were detected in RA and SpA synovium based on CD3, CD161 and Vα7.2 expression. Peripheral RA MAIT cells were mostly CD4+ (controls 8.3%, SpA 12.3%, RA 52.6%; p p E. coli stimulation (control, CD25 MFI = 177, CD69 MFI = 1307; SpA, CD25 MFI = 95, CD69 MFI = 1257; RA, CD25 MFI = 0, CD69 MFI = 467; p p = 0.01 respectively).Conclusion: In early untreated RA patients, the peripheral MAIT cell composition was altered, with reduced levels of CD161 expression, and cells were hyporesponsive to stimulation. MAIT cell dysfunction may provide a link between the microbiome and development of RA.

Figure S2. No differences in synovial MAIT cells in treated SpA and RA patients. In contrast to our PBMC samples, all SFMC samples were obtained from patients both with longstanding disease and whom received various treatments. (A) SFMC MAIT cells divided based on CD4 and CD8 expression. (B) CD161 expression (MFI) of synovial fluid MAIT cells. There were no statically significant differences, neither in MAIT cell subsets nor CD16 expression. (PDF 808 kb)

Figure S1. Gating strategy. Representative plots showing gating strategy for both PBMCs and SFMCs. Lymphocytes selected based on FSC-A and SSC-A. Doublets removed in both the FSC and SSC, and dead cells excluded based on DAPI for PBMCs and Amcyan (Aqua) for SFMCs. T cells defined within single, live population based on CD3 expression, and MAIT cells gated based on CD3+ and MR1-tetramer+. MAIT cell subsets defined based on CD4 and CD8 expression as indicated in last panel (PDF 205 kb)

Table S1. Patient characteristics (PDF 205 kb) (DOCX 24 kb)

Background:An increased risk of severe COVID-19 outcomes may be seen in patients with autoimmune diseases on moderate to high daily doses of glucocorticoids, as well as in those with comorbidities. However, specific information about COVID-19 outcomes in SLE is scarce.Objectives:To determine the characteristics associated with severe COVID-19 outcomes in a multi-national cross-sectional registry of COVID-19 patients with SLE.Methods:SLE adult patients from a physician-reported registry of the COVID-19 GRA were studied. Variables collected at COVID-19 diagnosis included age, sex, race/ethnicity, region, comorbidities, disease activity, time period of COVID-19 diagnosis, glucocorticoid (GC) dose, and immunomodulatory therapy. Immunomodulatory therapy was categorized as: antimalarials only, no SLE therapy, traditional immunosuppressive (IS) drug monotherapy, biologics/targeted synthetic IS drug monotherapy, and biologic and traditional IS drug combination therapy. We used an ordinal COVID-19 severity outcome defined as: not hospitalized/hospitalized without supplementary oxygen; hospitalized with non-invasive ventilation; hospitalized with mechanical ventilation/extracorporeal membrane oxygenation; and death. An ordinal logistic regression model was constructed to assess the association between demographic characteristics, comorbidities, medications, disease activity and COVID-19 severity. This assumed that the relationship between each pair of outcome groups is of the same direction and magnitude.Results:Of 1069 SLE patients included, 1047 (89.6%) were female, with a mean age of 44.5 (SD: 14.1) years. Patient outcomes included 815 (78.8%) not hospitalized/hospitalized without supplementary oxygen; 116 (11.2) hospitalized with non-invasive ventilation, 25 (2.4%) hospitalized with mechanical ventilation/extracorporeal membrane oxygenation and 78 (7.5%) died. In a multivariate model (n=804), increased age [OR=1.03 (1.01, 1.04)], male sex [OR =1.93 (1.21, 3.08)], COVID-19 diagnosis between June 2020 and January 2021 (OR =1.87 (1.17, 3.00)), no IS drug use [OR =2.29 (1.34, 3.91)], chronic renal disease [OR =2.34 (1.48, 3.70)], cardiovascular disease [OR =1.93 (1.34, 3.91)] and moderate/high disease activity [OR =2.24 (1.46, 3.43)] were associated with more severe COVID-19 outcomes. Compared with no use of GC, patients using GC had a higher odds of poor outcome: 0-5 mg/d, OR =1.98 (1.33, 2.96); 5-10 mg/d, OR =2.88 (1.27, 6.56); >10 mg/d, OR =2.01 (1.26, 3.21) (Table 1).Table 1.Characteristics associated with more severe COVID-19 outcomes in SLE. (N=804)OR (95% CI)Age, years1.03 (1.01, 1.04)Sex, Male1.93 (1.21, 3.08)Race/Ethnicity, Non-White vs White1.47 (0.87, 2.50)RegionEuropeRef.North America0.67 (0.29, 1.54)South America0.67 (0.29, 1.54)Other1.93 (0.85, 4.39)Season, June 16th 2020-January 8th 2021 vs January-June 15th 20201.87 (1.17, 3.00)Glucocorticoids0 mg/dayRef.0-5 mg/day1.98 (1.33, 2.96)5-10 mg/day2.88 (1.27, 6.56)=>10 mg/day2.01 (1.26, 3.21)Medication CategoryAntimalarial onlyRef.No IS drugs2.29 (1.34, 3.91)Traditional IS drugs as monotherapy1.17 (0.77, 1.77)b/ts IS drugs as monotherapy1.00 (0.37, 2.71)Combination of traditional and b/ts IS1.00 (0.55, 1.82)Comorbidity BurdenNumber of Comorbidities (excluding renal and cardiovascular disease)1.39 (0.97, 1.99)Chronic renal disease2.34 (1.48, 3.70)Cardiovascular disease1.93 (1.34, 3.91)Disease Activity, Moderate/ high vs Remission/ low 2.24 (1.46, 3.43)IS: immunosuppressive. b/ts: biologics/targeted syntheticsConclusion:Increased age, male sex, glucocorticoid use, chronic renal disease, cardiovascular disease and moderate/high disease activity at time of COVID-19 diagnosis were associated with more severe COVID-19 outcomes in SLE. Potential limitations include possible selection bias (physician reporting), the cross-sectional nature of the data, and the assumptions underlying the outcomes modelling.Acknowledgements:The views expressed here are those of the authors and participating members of the COVID-19 Global Rheumatology Alliance and do not necessarily represent the views of the ACR, EULAR) the UK National Health Service, the National Institute for Health Research (NIHR), or the UK Department of Health, or any other organization.Disclosure of Interests:Manuel F. Ugarte-Gil Grant/research support from: Pfizer, Janssen, Graciela S Alarcon: None declared, Andrea Seet: None declared, Zara Izadi: None declared, Cristina Reategui Sokolova: None declared, Ann E Clarke Consultant of: AstraZeneca, BristolMyersSquibb, GlaxoSmithKline, Exagen Diagnostics, Leanna Wise: None declared, Guillermo Pons-Estel: None declared, Maria Jose Santos: None declared, Sasha Bernatsky: None declared, Lauren Mathias: None declared, Nathan Lim: None declared, Jeffrey Sparks Consultant of: Bristol-Myers Squibb, Gilead, Inova, Janssen, and Optum unrelated to this work., Grant/research support from: Amgen and Bristol-Myers Squibb, Zachary Wallace Consultant of: Viela Bio and MedPace, Grant/research support from: Bristol-Myers Squibb and Principia/Sanofi, Kimme Hyrich Speakers bureau: Abbvie, Grant/research support from: MS, UCB, and Pfizer, Anja Strangfeld Speakers bureau: AbbVie, MSD, Roche, BMS, Pfizer, Grant/research support from: AbbVie, BMS, Celltrion, Fresenius Kabi, Lilly, Mylan, Hexal, MSD, Pfizer, Roche, Samsung, Sanofi-Aventis, and UCB, Laure Gossec Consultant of: Abbvie, Biogen, Celgene, Janssen, Lilly, Novartis, Pfizer, Sanofi-Aventis, UCB, Grant/research support from: Lilly, Mylan, Pfizer, Loreto Carmona: None declared, Elsa Mateus Grant/research support from: Pfizer, Abbvie, Novartis, Janssen-Cilag, Lilly Portugal, Sanofi, Grünenthal S.A., MSD, Celgene, Medac, Pharmakern, GAfPA, Saskia Lawson-Tovey: None declared, Laura Trupin: None declared, Stephanie Rush: None declared, Gabriela Schmajuk: None declared, Patti Katz: None declared, Lindsay Jacobsohn: None declared, Samar Al Emadi: None declared, Emily Gilbert: None declared, Ali Duarte-Garcia: None declared, Maria Valenzuela-Almada: None declared, Tiffany Hsu: None declared, Kristin D’Silva: None declared, Naomi Serling-Boyd: None declared, Philippe Dieudé Consultant of: Boerhinger Ingelheim, Bristol-Myers Squibb, Lilly, Sanofi, Pfizer, Chugai, Roche, Janssen unrelated to this work, Grant/research support from: Bristol-Myers Squibb, Chugaii, Pfizer, unrelated to this work, Elena Nikiphorou: None declared, Vanessa Kronzer: None declared, Namrata Singh: None declared, Beth Wallace: None declared, Akpabio Akpabio: None declared, Ranjeny Thomas: None declared, Suleman Bhana Consultant of: AbbVie, Horizon, Novartis, and Pfizer (all

Inflammatory bowel disease (IBD) is an inflammatory condition with mucosal ulceration, edema and hemorrhage of gastrointestinal tract. Curcumin has been shown to mitigate colitis in animal models. However, its usefulness is reduced due to poor pharmacokinetic behavior and low oral bioavailability. To address this, novel pH-sensitive hydrolyzed polyacrylamide-grafted-xanthan gum (PAAm-g-XG) nanoparticles (NPs) loaded with curcumin were prepared for colonic delivery. Optimized nanoparticles (CN20) were spherical, with an average size of 425 nm. A negligible amount of curcumin (≈8%) was released from CN20 NPs in pH 1.2 and 4.5 solutions. When the pH was increased to 7.2, curcumin release was comparatively faster than that observed with pH 1.2 and 4.5 collectively. In pH 6.8 solution, excellent release of curcumin was observed. Highest curcumin release was observed when rat caecal contents were incorporated in pH 6.8 solution, indicating microflora-dependent drug release property of NPs. In acetic acid-induced IBD in rats, curcumin NPs reduced myeloperoxidase and nitrite levels, prevented weight loss and attenuated colonic inflammation. Curcumin was better absorbed systemically in nanoparticulate form with increased Cmax (∼3 fold) and AUC (∼2.5 fold) than when delivered as free curcumin. We demonstrate successful development of grafted co-polymeric NPs containing drug suitable for colon targeting.

BackgroundWhile efficacy of combination treatment with methotrexate (MTX), sulfasalazine (SSZ) and hydroxychloroquine (HCQ) (triple therapy') has been shown in clinical trials, few studies have examined its longevity in a real-life setting.

Invariant natural killer T (iNKT) cells are a subset of lymphocytes with immune regulatory activity. Their ability to bridge the innate and adaptive immune systems has been studied using the glycolipid ligand α-galactosylceramide (αGC). To better harness the immune adjuvant properties of iNKT cells to enhance priming of antigen-specific CD8+ T cells, we encapsulated both αGC and antigen in a Clec9a-targeted nanoemulsion (TNE) to deliver these molecules to cross-presenting CD8+ dendritic cells (DC). We demonstrate that, even in the absence of exogenous glycolipid, iNKT cells supported the maturation of CD8α+ DCs to drive efficient cross-priming of antigen-specific CD8+ T cells upon delivery of Clec9a/OVA-TNE. The addition of αGC to the TNE (Clec9a/OVA/αGC) further enhanced activation of iNKT cells, NK cells, CD8α+ DCs, and polyfunctional CD8+ T cells. When tested therapeutically against HPVE7-expressing TC-1 tumors, long-term tumor suppression was achieved with a single administration of Clec9a/E7 peptide/αGC TNE. Antitumor activity was correlated with the recruitment of mature DCs, NK cells, and tumor-specific effector CD8+ T cells to the tumor-draining lymph node and tumor tissue. Thus, Clec9a-TNE codelivery of CD8+ T-cell epitopes with αGC induces alternative helper signals from activated iNKT cells, elicits innate (iNKT, NK) immunity, and enhances antitumor CD8+ T-cell responses for control of solid tumors.

Rheumatoid Arthritis (RA) is a severe, chronic autoimmune disease that affects 1% of the world's population. Familial risk contributes 50% of the risk of seropositive RA, with strongest risks seen in first-degree relatives. Smoking increases the risk of developing anti-citrullinated peptide antibody (ACPA)+ RA, particularly in individuals with high-risk RA-susceptibility alleles. Other contributory environmental risks including particulate exposure, periodontal disease, bronchiectasis, diet, obesity and the oral contraceptive impact respiratory, oral, intestinal and genital tract mucosal sites. Furthermore, the first signs of autoimmunity may appear at mucosal sites e.g. sputum ACPA-IgA and IgG. While oral and faecal dysbiosis are well described, there is no consistent single bacterial species that appears to drive RA. Animal and human data suggest a model in which multiple environmental influences impact mucosal immune function through the host genetics through enhanced mucosal permeability and the traffic of pro-inflammatory PAMPs and the amplification of autoimmune responses. In some cases, autoimmunity may be driven by cross-reactivity, or mimicry, to pathogen-specific antigens, particularly where the host immune system fails to support their rapid control and elimination.

Animal models of human diseases are used to study a broad array of autoimmune disorders including spondyloarthritis (SpA). Various animal models, either genetically engineered, spontaneous or induced, have been described to address the fundamental pathological features of human SpA. With the advent of novel techniques for manipulations in animal models, it has become possible to delineate critical pathways involved in SpA disease mechanisms and to develop novel therapeutics. In this chapter, we discuss animal models to study the fundamental characteristics of human SpA. None of the animal models developed exactly mimics human diseases, but some of the models closely resemble the group of human SpA diseases. The SKG mouse, in particular, harbours a point mutation in the ZAP-70 gene yielding reduced T-cell receptor (TCR) signalling, develops multi-organ inflammation under microbial influence, mimics human SpA disease pathogenesis and is a promising tool for designing new therapeutics against SpA. We describe the contribution of animal models to the earliest known disease mechanisms, including how attenuated T cell signalling contributes to disease pathogenesis as a result of genetic or environmental predisposition. We also discuss recent advances in the understanding of key cytokines that drive SpA pathophysiology and the crosstalk of gut microbiota with host genetic determinants of immune function and finally the potential future prospects to study these animal models to translate our knowledge of pathogenesis into clinical practice.

Disease modifying strategies are available for treatment of rheumatoid arthritis (RA), and good response rates are achieved. However, limitations include toxicity, a response rate ceiling, cost and rationing of biologic therapies, inability to cure or permanently reverse RA pathology, and inability to prevent disease. Immunotherapies targeting checkpoint molecules are markedly changing the landscape of clinical oncology. In autoimmune diseases such as RA, dendritic cells represent an important target for antigen-specific immunotherapy for T cell tolerance. Antigen-specific strategies promise greater specificity and safety, without general immune suppression, and thus the potential for intervention in at-risk subjects before disease onset. In a proof-of-concept trial, delivery of autoantigenic peptides and autologous tolerogenic dendritic cells was safe and had immunomodulatory effects on T cells including reduction of effector T cells and a relative increase in regulatory T cells. We have developed and are trialling in RA patients, antigen-specific immunotherapy targeting dendritic cells in situ with liposomes encapsulating autoantigenic peptide and calcitriol with the aim of antigen-specific T cell tolerance. I will discuss the development of antigen-specific tolerance strategies and the parallel development of immune monitoring assays to determine T cell outcomes in clinical trials in RA. Disclosure of Interest R. Thomas Grant/research support from: Janssen Biotech Inc, Consultant for: Janssen Biotech Inc

Background Mortality rates are higher in RA patients compared to the general population, mostly due to cardiovascular disease (CVD). Cardiorespiratory fitness (CRF) is inversely associated with CVD, but little is known about CRF levels in RA patients. Objectives To study the association between CRF in RA patients (measured as VO2peak), and CVD risk factors and RA-specific clinical variables. Methods 93 patients were recruited from a hospital rheumatology outpatient clinic. VO2peak was measured by ergospirometry during a treadmill test. Tender and swollen joint count, height, weight, waist circumference, resting heart rate, blood pressure and hsCRP were measured. RA characteristics and information on comorbidity were recorded from an interview and from medical records. Based on a previously published index weighting frequency, intensity and duration of self-reported physical activity (PA) (1), patients were categorized into four different PA categories (table 1). VO2peak was analyzed using multivariate linear regression, adjusting for age and gender. All participants signed an informed consent and the regional ethics committee approved the study. Results Conclusions We observed a strong association between common CVD risk factors (inactivity, high BMI, high resting heart rate) and lower CRF in RA patients, similar to published findings from the general population. Patients in the “Active” and “Very active” PA categories showed significantly higher VO2peak, whereas RA-specific variables such as seropositivity, disease activity, disease duration and medications had no impact on VO2peak. We cannot exclude a selection bias because more fit patients could be more likely to participate in the study. Reference [1]Nes BM, Janszky I, Vatten LJ, Nilsen TI, Aspenes ST, Wisloff U. Estimating VO2peak from a nonexercise prediction model: the HUNT Study, Norway. Med Sci Sports Exerc2011;43(11):2024–30. Acknowledgements: The VO2peak testing was performed at the core facility NeXt Move, NTNU – Norwegian University of Science and Technology. Disclosure of Interest: None declared

Polymorphisms impacting thymic function may decrease peripheral tolerance and hasten autoimmune disease. The NF-κB transcription factor subunit, RelB, is essential for the development and differentiation of medullary thymic epithelial cells (mTECs): RelB-deficient mice have reduced thymic cellularity and markedly fewer mTECs, lacking AIRE. The precise mechanism of this mTEC reduction in the absence of RelB is unclear. To address this, we studied mTECs and dendritic cells (DCs), which critically regulate negative selection, and thymic regulatory T-cells (tTreg) in RelB−/− mice, which have spontaneous multiorgan autoimmune disease. RelB−/− thymi were organized, with medullary structures containing AIRE− mTECs, DCs, and CD4+ thymocytes, but fewer tTreg. Granulocytes infiltrated the RelB−/− thymic cortex, capsule, and medulla, producing inflammatory thymic medullary atrophy, which could be treated by granulocyte depletion or RelB+ DC immunotherapy, with concomitant recovery of mTEC and tTreg numbers. These data indicate that central tolerance defects may be accelerated by autoimmune thymic inflammation where impaired RelB signaling impairs the medullary niche, and may be reversible by therapies enhancing peripheral Treg or suppressing inflammation.

Autoimmune-mediated destruction of pancreatic islet β cells results in type 1 diabetes (T1D). Serum islet autoantibodies usually develop in genetically susceptible individuals in early childhood before T1D onset, with multiple islet autoantibodies predicting diabetes development. However, most at-risk children remain islet-antibody negative, and no test currently identifies those likely to seroconvert. We sought a genomic signature predicting seroconversion risk by integrating longitudinal peripheral blood gene expression profiles collected in high-risk children included in the BABYDIET and DIPP cohorts, of whom 50 seroconverted. Subjects were followed for 10 years to determine time of seroconversion. Any cohort effect and the time of seroconversion were corrected to uncover genes differentially expressed (DE) in seroconverting children. Gene expression signatures associated with seroconversion were evident during the first year of life, with 67 DE genes identified in seroconverting children relative to those remaining antibody negative. These genes contribute to T cell-, DC-, and B cell-related immune responses. Near-birth expression of ADCY9, PTCH1, MEX3B, IL15RA, ZNF714, TENM1, and PLEKHA5, along with HLA risk score predicted seroconversion (AUC 0.85). The ubiquitin-proteasome pathway linked DE genes and T1D susceptibility genes. Therefore, a gene expression signature in infancy predicts risk of seroconversion. Ubiquitination may play a mechanistic role in diabetes progression.

Early treatment ensures optimal outcomes in rheumatoid arthritis (RA) yet there are limited data in Australia quantifying treatment delays in clinical practice.To quantify treatment delays in new RA patients and to explore factors influencing delay and resultant patient outcomes.Data were obtained for 88 patients presenting with a new diagnosis of RA to an early arthritis clinic (EAC) in Australia between 2008 and 2015. Date and details of symptom onset, initial general practitioner (GP) presentation, GP referral and review at EAC were collected. Patient demographics and clinical features were analysed for outcomes and features predictive of delay.Median overall delay from symptom onset to rheumatology review was 26.4 weeks. Patient delay (8.7 weeks) was the longest delay and predicted overall delay. Delays in GP referral and time to EAC review were 4 and 8.4 weeks respectively. Increased overall delay was predicted by lower fatigue and disease activity scores (DAS28) and increased tender joint counts (TJC). Patient delay was increased by socioeconomic disadvantage. Increased GP delay was associated with lower DAS28 and higher TJC and ESR. Patients seen within 16 weeks had greater improvement in DAS28 and probability of remission at 6 months.In this Australian EAC setting, patient delay was the greatest contributor to RA treatment delay. Delays in treatment were associated with lower disease severity and socioeconomic disadvantage. Remission was more likely after prompt initiation of treatment.

Background Clinician-scientists are in decline worldwide. They represent a unique niche in medicine by bridging the gap between scientific discovery and patient care. A national, integrated approach to training clinician-scientists, typically programs that comprise a comprehensive MD-PhD pathway, are customary. Such a pathway is lacking in Australia. The objective was to gather perceptions from Australian medical students on factors they perceive would influence their decision to pursue clinician-scientist training. Methods A cross-sectional mixed methods design used quantitative and qualitative questions in an online self-report survey with medical students from a four-year MD program. Quantitative measures comprised scaled response questions regarding prior experience and current involvement in research, and short- and long-term opinions about factors that influence their decisions to undertake a research higher degree (RHD) during medical school. Qualitative questions gathered broader perceptions of what a career pathway as a clinician-scientist would include and what factors are most conducive to a medical student’s commitment to MD-PhD training. Results Respondents (N = 418; 51% female) indicated Time, Funding and Pathway as the major themes arising from the qualitative data, highlighting negative perceptions rather than possible benefits to RHD training. The lack of an evident Pathway was inter-related to Time and Funding. Themes were supported by the quantitative data. Sixty percent of students have previous research experience of varying forms, and 90% report a current interest, mainly to improve their career prospects. Conclusions The data emphasise the need for an MD-PhD pathway in Australia. A model that provides an early, integrated, and exclusive approach to research training pathways across all stages of medical education is suggested as the best way to rejuvenate the clinician-scientist. A national pathway that addresses factors influencing career decision making throughout the medical education continuum should include an appropriate funding structure, and provide early and continuing advice and mentoring. It should be flexible, gender equitable, and include post-graduate training. The implications of implementing MD-PhD programs represent a substantial investment. However this should not be a deterrent to Australia’s commitment to an MD-PhD pathway, but rather a challenge to help ensure our future healthcare is guided by highly trained and competent clinician-scientists.

Free to read Objective Chlamydia trachomatis is a sexually transmitted obligate intracellular pathogen that causes inflammatory reactive arthritis, spondylitis, psoriasiform dermatitis, and conjunctivitis in some individuals after genital infection. The immunologic basis for this inflammatory response in susceptible hosts is poorly understood. As ZAP-70W163C–mutant BALB/c (SKG) mice are susceptible to spondylo-arthritis after systemic exposure to microbial β-glucan, we undertook the present study to compare responses to infection with Chlamydia muridarum in SKG mice and BALB/c mice. Methods After genital or respiratory infection with C muridarum, conjunctivitis and arthritis were assessed clinically, and eye, skin, and joint specimens were analyzed histologically. Chlamydial major outer membrane protein antigen–specific responses were assessed in splenocytes. Treg cells were depleted from FoxP3-DTR BALB/c or SKG mice, and chlamydial DNA was quantified by polymerase chain reaction. Results Five weeks after vaginal infection with live C muridarum, arthritis, spondylitis, and psoriasiform dermatitis developed in female SKG mice, but not in BALB/c mice. Inflammatory bowel disease did not occur in mice of either strain. The severity of inflammatory disease was correlated with C muridarum inoculum size and vaginal burden postinoculation. Treatment with combination antibiotics starting 1 day postinoculation prevented disease. Chlamydial antigen was present in macrophages and spread from the infection site to lymphoid organs and peripheral tissue. In response to chlamydial antigen, production of interferon-γ and interleukin-17 was impaired in T cells from SKG mice but tumor necrosis factor (TNF) responses were exaggerated, compared to findings in T cells from BALB/c mice. Unlike previous observations in arthritis triggered by β-glucan, no autoantibodies developed. Accelerated disease triggered by depletion of Treg cells was TNF dependent. Conclusion In the susceptible SKG strain, Chlamydia-induced reactive arthritis develops as a result of deficient intracellular pathogen control, with antigen-specific TNF production upon dissemination of antigen, and TNF-dependent inflammatory disease.

Genetic discoveries in arthritis and their associated biological pathways spanning the innate and adaptive immune system demonstrate the strong association between susceptibility to arthritis and control of exogenous organisms. The canonical theory of the aetiology of immune-mediated arthritis and other immune-mediated diseases is that the introduction of exogenous antigenic stimuli to a genetically susceptible host sets up the environment for an abnormal immune response manifesting as disease. A disruption in host-microbe homeostasis driven by disease-associated genetic variants could ultimately provide the source of exogenous antigen triggering disease development. We discuss genetic variants impacting the innate and adaptive arms of the immune system and their relationship to microbial control and arthritic disease. We go on to consider the evidence for a relationship between HLA-B27, infection and arthritis, and then emerging evidence for an interaction between microbiota and rheumatoid arthritis.

ObjectiveThe pathogenetic mechanisms by which HLA-DRB1 alleles are associated with anticitrullinated peptide antibody (ACPA)-positive rheumatoid arthritis (RA) are incompletely understood. RA high-risk HLA-DRB1 alleles are known to share a common motif, the ‘shared susceptibility epitope (SE)’. Here, the electropositive P4 pocket of HLA-DRB1 accommodates self-peptide residues containing citrulline but not arginine. HLA-DRB1 His/Phe13β stratifies with ACPA-positive RA, while His13βSer polymorphisms stratify with ACPA-negative RA and RA protection. Indigenous North American (INA) populations have high risk of early-onset ACPA-positive RA, whereby HLA-DRB1*04:04 and HLA-DRB1*14:02 are implicated as risk factors for RA in INA. However, HLA-DRB1*14:02 has a His13βSer polymorphism. Therefore, we aimed to verify this association and determine its molecular mechanism.MethodsHLA genotype was compared in 344 INA patients with RA and 352 controls. Structures of HLA-DRB1*1402-class II loaded with vimentin-64Arg59-71, vimentin-64Cit59-71 and fibrinogen β−74Cit69-81 were solved using X-ray crystallography. Vimentin-64Cit59-71-specific and vimentin59-71-specific CD4+ T cells were characterised by flow cytometry using peptide-histocompatibility leukocyte antigen (pHLA) tetramers. After sorting of antigen-specific T cells, TCRα and β-chains were analysed using multiplex, nested PCR and sequencing.ResultsACPA+ RA in INA was independently associated with HLA-DRB1*14:02. Consequent to the His13βSer polymorphism and altered P4 pocket of HLA-DRB1*14:02, both citrulline and arginine were accommodated in opposite orientations. Oligoclonal autoreactive CD4+ effector T cells reactive with both citrulline and arginine forms of vimentin59-71 were observed in patients with HLA-DRB1*14:02+ RA and at-risk ACPA- first-degree relatives. HLA-DRB1*14:02-vimentin59-71-specific and HLA-DRB1*14:02-vimentin-64Cit59-71-specific CD4+ memory T cells were phenotypically distinct populations.ConclusionHLA-DRB1*14:02 broadens the capacity for citrullinated and native self-peptide presentation and T cell expansion, increasing risk of ACPA+ RA.

Non-antigen-specific stimulatory cancer immunotherapies are commonly complicated by off-target effects. Antigen-specific immunotherapy, combining viral tumor antigen or personalized neoepitopes with immune targeting, offers a solution. However, the lack of flexible systems targeting tumor antigens to cross-presenting dendritic cells (DCs) limits clinical development. Although antigen-anti-Clec9A mAb conjugates target cross-presenting DCs, adjuvant must be codelivered for cytotoxic T lymphocyte (CTL) induction. We functionalized tailored nanoemulsions encapsulating tumor antigens to target Clec9A (Clec9A-TNE). Clec9A-TNE encapsulating OVA antigen targeted and activated cross-presenting DCs without additional adjuvant, promoting antigen-specific CD4+ and CD8+ T cell proliferation and CTL and antibody responses. OVA-Clec9A-TNE-induced DC activation required CD4 and CD8 epitopes, CD40, and IFN-α. Clec9A-TNE encapsulating HPV E6/E7 significantly suppressed HPV-associated tumor growth, while E6/E7-CpG did not. Clec9A-TNE loaded with pooled B16-F10 melanoma neoepitopes induced epitope-specific CD4+ and CD8+ T cell responses, permitting selection of immunogenic neoepitopes. Clec9A-TNE encapsulating 6 neoepitopes significantly suppressed B16-F10 melanoma growth in a CD4+ T cell-dependent manner. Thus, cross-presenting DCs targeted with antigen-Clec9A-TNE stimulate therapeutically effective tumor-specific immunity, dependent on T cell help.

Objective Non-alcoholic fatty liver disease (NAFLD) is characterized by hepatic macrophage inflammation, steatosis and fibrosis. Liposomes injected intravenously passively target hepatic myeloid cells and have potential to deliver immunomodulatory compounds and treat disease. We investigated targeting, delivery, immunomodulation and efficacy of liposomes in mice with diet-induced NASH. Methods Liposome-encapsulated lipophilic curcumin or 1,25-dihydroxy-vitamin D3 (calcitriol) were injected intravenously into mice with diet-induced NASH. Liver and cell liposome uptake was assessed by in vivo imaging and flow cytometry. Immunomodulation of targeted cells were assessed by RNA transcriptome sequencing. NASH was assessed by histological scoring, serum liver enzymes and fasting glucose/insulin and liver RNA transcriptome sequencing. Results Liposomes targeted lipid containing MHC class-II+ hepatic dendritic cells in mice and humans. Delivery of liposomal curcumin to hepatic dendritic cells shifted their inflammatory profile towards a regulatory phenotype. Delivery of liposomal curcumin or calcitriol to mice with diet-induced NASH led to reduced liver inflammation, fibrosis and fat accumulation, and reduced insulin resistance. RNA transcriptome sequencing of liver from treated mice identified suppression of pathways of immune activation, cell cycle and collagen deposition. Conclusions Liposomes are a new strategy to target lipid rich inflammatory dendritic cells and have potential to deliver immunomodulatory compounds to treat NASH.

Objective. Self-reported diagnoses of inflammatory arthritis are not accurate. The primary study aim was to ascertain self-reported diagnoses of rheumatoid arthritis (RA) and ankylosing spondylitis (AS) in the Norwegian population-based Nord-Trøndelag Health Study (HUNT) using hospital case files. The secondary aim was to provide updated estimates of the prevalence and incidence of RA and AS. Methods. All inhabitants ≥ 20 years old from the county of Nord-Trøndelag were invited. Data from 70,805 unique participants from HUNT2 (1995–1997) and HUNT3 (2006–2008) were included. For participants who self-reported RA or AS, case files from all 3 hospitals in the catchment area were evaluated using standardized diagnostic criteria. Results. Of 2703 self-reported cases of RA, 19.1% were verified in hospital files. Of 1064 self-reported cases of AS, 15.8% were verified. Of 259 cases self-reporting both RA and AS, 8.1% had RA and 5.4% had AS. Overall, a self-report of 1 or both diagnoses could not be verified in 82.1%, including 22.8% with insufficient information or no case file. The prevalence of RA was 768 (95% CI 705–835) per 100,000. The incidence of RA from HUNT2 to HUNT3 was 0.48 (0.41–0.56) per 1000 per year. The prevalence of AS was 264 (228–305) per 100,000. The incidence of AS from HUNT2 to HUNT3 was 0.19 (0.15–0.24) per 1000 per year. Conclusion. Self-reported diagnoses of RA and AS are often false-positive. The prevalence and incidence of RA were comparable to reports from similar populations. The incidence of AS was higher than previously reported in a mixed population from Norway. © 2017. This is a pre-copy-editing, author-produced PDF of an article accepted for publication in The Journal of Rheumatology following peer review. LOCKED until 1.8.2018 due to copyright restrictions. The definitive publisher-authenticated version [Self-reported diagnosis of rheumatoid arthritis or ankylosing spondylitis has low accuracy: Data from the Nord-Trøndelag Health Study] is available online at: http://www.jrheum.org/content/44/8/1134

ObjectiveThe spondyloarthritides share genetic susceptibility, interleukin-23 (IL-23) dependence, and the involvement of microbiota. The aim of the current study was to elucidate how host genetics influence gut microbiota and the relationship between microbiota and organ inflammation in spondyloarthritides.

Objective. Spondyloarthritides (SpA) occur in 1% of the population and include ankylosing spondylitis (AS) and arthropathy of inflammatory bowel disease (IBD), with characteristic spondylitis, arthritis, enthesitis, and IBD. Genetic studies implicate interleukin-23 (IL-23) receptor signaling in the development of SpA and IBD, and IL-23 overexpression in mice is sufficient for enthesitis, driven by entheseal-resident T cells. However, in genetically prone individuals, it is not clear where IL-23 is produced and how it drives the SpA syndrome, including IBD or subclinical gut inflammation of AS. Moreover, it is unclear why specific tissue involvement varies between patients with SpA. We undertook this study to determine the location of IL-23 production and its role in SpA pathogenesis in BALB/c ZAP-70(W163C)-mutant (SKG) mice injected intraperitoneally with beta-1,3-glucan (curdlan).

Graft-versus-host disease (GVHD) in the gastrointestinal (GI) tract is the principal determinant of lethality following allogeneic bone marrow transplantation (BMT). Here, we examined the mechanisms that initiate GVHD, including the relevant antigen-presenting cells. MHC class II was expressed on intestinal epithelial cells (IECs) within the ileum at steady state but was absent from the IECs of germ-free mice. IEC-specific deletion of MHC class II prevented the initiation of lethal GVHD in the GI tract. MHC class II expression on IECs was absent from mice deficient in the TLR adaptors MyD88 and TRIF and required IFNγ secretion by lamina propria lymphocytes. IFNγ responses are characteristically driven by IL-12 secretion from myeloid cells. Antibiotic-mediated depletion of the microbiota inhibited IL-12/23p40 production by ileal macrophages. IL-12/23p40 neutralization prevented MHC class II upregulation on IECs and initiation of lethal GVHD in the GI tract. Thus, MHC class II expression by IECs in the ileum initiates lethal GVHD, and blockade of IL-12/23p40 may represent a readily translatable therapeutic strategy.

The factors initiating pathology in the gastrointestinal (GI) tract are largely unknown. Graft-versus-host disease (GVHD) of the gastrointestinal (GI) tract is the principal determinant of lethality following allogeneic bone marrow transplantation (BMT). MHC class II-dependent GVHD is initiated by recipient alloantigens being presented to donor T cells that in turn undergo Th1 and Th17 differentiation to mediate pathology in the GI tract. Critically, the mechanisms that initiate disease, especially the relevant antigen presenting cells, remain unclear. Here, we identify the response of IL-12-secreting macrophages to the microbiome as the key driver of IFNγ secretion by mucosal type-1 innate lymphoid cells (ILC1). The latter controls MHC class II expression and antigen presentation by intestinal epithelial cells (IEC) specifically within the ileum. When these tightly regulated responses between microbiome and lamina propria lymphocytes are perturbed by inflammatory signals driven by pre-transplant conditioning, dramatic increases in antigen presentation by IEC occur in the ileum and lethal GVHD ensues. Conditional and Villin-specific deletion of MHC class II in IEC, or inhibition of IL-12 pre-transplant, completely prevent the initiation of lethal GVHD, identifying a readily translatable therapeutic strategy.

Bone marrow (BM) or hematopoietic stem cell (HSC) transplantation is used as curative therapy for hematologic malignancies. Incorporation of gene therapy to drive tolerogenic expression of antigens is a promising strategy to overcome the limited long-term efficacy of autologous HSC transplantation for autoimmune diseases. HSC engraftment and tolerance induction is readily achieved after myeloablative or immune-depleting conditioning regardless of the cellular compartment in which antigen is expressed. It is unclear whether the efficiency of engraftment and tolerance induction is influenced by targeting antigen to specific cellular compartments. This is particularly important when using clinically feasible low-intensity conditioning aimed at preserving infectious immunity in individuals where immunologic memory exists to the autoantigen to be expressed. Here we demonstrate that, under immune-preserving conditions, confining expression of a transgenically expressed antigen to dendritic cells permits stable, long-term engraftment of genetically modified BM even when recipients are immune to the expressed antigen. In contrast, broader expression within the hematopoietic compartment leads to graft rejection and therapeutic failure because of antigen expression in HSCs. These findings are relevant to the clinical application of genetically engineered HSCs and provide evidence that careful selection of promoters for HSC-mediated gene therapy is important, particularly where tolerance is sought under immune-preserving conditions.

A comprehensive structural portrait of the association between citrullination, the HLA-DRB1 locus, and T cell autoreactivity in rheumatoid arthritis., Rheumatoid arthritis (RA) is strongly associated with the human leukocyte antigen (HLA)-DRB1 locus that possesses the shared susceptibility epitope (SE) and the citrullination of self-antigens. We show how citrullinated aggrecan and vimentin epitopes bind to HLA-DRB1*04:01/04. Citrulline was accommodated within the electropositive P4 pocket of HLA-DRB1*04:01/04, whereas the electronegative P4 pocket of the RA-resistant HLA-DRB1*04:02 allomorph interacted with arginine or citrulline-containing epitopes. Peptide elution studies revealed P4 arginine–containing peptides from HLA-DRB1*04:02, but not from HLA-DRB1*04:01/04. Citrullination altered protease susceptibility of vimentin, thereby generating self-epitopes that are presented to T cells in HLA-DRB1*04:01+ individuals. Using HLA-II tetramers, we observed citrullinated vimentin- and aggrecan-specific CD4+ T cells in the peripheral blood of HLA-DRB1*04:01+ RA-affected and healthy individuals. In RA patients, autoreactive T cell numbers correlated with disease activity and were deficient in regulatory T cells relative to healthy individuals. These findings reshape our understanding of the association between citrullination, the HLA-DRB1 locus, and T cell autoreactivity in RA.

Type-1 regulatory T (TR1) cells are Foxp3-negative IL-10-producing CD4+ T cells with potent immune suppressive properties but their requirements for lineage development have remained elusive. Here we show that TR1 cells constitute the most abundant regulatory population after allogeneic bone marrow transplantation (BMT), express the transcription factor Eomesodermin (Eomes) and are critical for the prevention of graft-versus-host disease (GVHD). We demonstrate that Eomes is required for TR1 cell differentiation during which it acts in concert with the transcription factor B-lymphocyte-induced maturation protein-1 (Blimp-1) by transcriptionally activating IL-10 expression and repressing differentiation into other Th lineages. We further show that Eomes induction in TR1 cells requires T-bet and donor macrophage-derived IL-27. We thus define the cellular and transcriptional control of TR1 cell differentiation during bone marrow transplantation, opening new avenues to therapeutic manipulation.

Islet-specific memory T cells arise early in type 1 diabetes (T1D), persist for long periods, perpetuate disease, and are rapidly reactivated by islet transplantation. As memory T cells are poorly controlled by “conventional” therapies, memory T cell–mediated attack is a substantial challenge in islet transplantation, and this will extend to application of personalized approaches using stem cell–derived replacement β-cells. New approaches are required to limit memory autoimmune attack of transplanted islets or replacement β-cells. Here, we show that transfer of bone marrow encoding cognate antigen directed to dendritic cells, under mild, immune-preserving conditions, inactivates established memory CD8+ T-cell populations and generates a long-lived, antigen-specific tolerogenic environment. Consequently, CD8+ memory T cell–mediated targeting of islet-expressed antigens is prevented and islet graft rejection alleviated. The immunological mechanisms of protection are mediated through deletion and induction of unresponsiveness in targeted memory T-cell populations. The data demonstrate that hematopoietic stem cell–mediated gene therapy effectively terminates antigen-specific memory T-cell responses, and this can alleviate destruction of antigen-expressing islets. This addresses a key challenge facing islet transplantation and, importantly, the clinical application of personalized β-cell replacement therapies using patient-derived stem cells.

Type 1 diabetes is caused by immune-mediated loss of pancreatic beta cells. It has been proposed that inflammatory cytokines play a role in killing beta cells. Expression of interleukin (IL)-1 and tumor necrosis factor (TNF-α) has been detected in islets from patients with type 1 diabetes, and these cytokines can induce beta cell death in vitro. We produced nonobese diabetic (NOD) mice lacking receptors for these cytokines. Islets from mice lacking IL-1RI or TNFR1 were killed when transplanted into wild-type NOD mice, suggesting that cytokine action on beta cells is not required for killing. Mice lacking TNFR1 did not develop diabetes, and mice lacking IL-1R had delayed onset of diabetes, indicating a role for these cytokines in disease development. TNFR1-deficient mice had an increased number of CD4(+) CD25(+) FoxP3(+) regulatory T cells with enhanced suppressive capacity. IL-1 was produced at higher levels in NOD mice and resulted in dilution of suppressor function of CD4(+) CD25(+) FoxP3(+) regulatory T cells. Our data suggest that blocking inflammatory cytokines may increase the capacity of the immune system to suppress type 1 diabetes through regulatory T cells.

Objective The spondylarthritides (SpA), including ankylosing spondylitis (AS), psoriatic arthritis (PsA), reactive arthritis, and arthritis associated with inflammatory bowel disease, cause chronic inflammation of the large peripheral and axial joints, eyes, skin, ileum, and colon. Genetic studies reveal common candidate genes for AS, PsA, and Crohn's disease, including IL23R, IL12B, STAT3, and CARD9, all of which are associated with interleukin-23 (IL-23) signaling downstream of the dectin 1 β-glucan receptor. In autoimmune-prone SKG mice with mutated ZAP-70, which attenuates T cell receptor signaling and increases the autoreactivity of T cells in the peripheral repertoire, IL-17–dependent inflammatory arthritis developed after dectin 1–mediated fungal infection. This study was undertaken to determine whether SKG mice injected with 1,3-β-glucan (curdlan) develop evidence of SpA, and the relationship of innate and adaptive autoimmunity to this process. Methods SKG mice and control BALB/c mice were injected once with curdlan or mannan. Arthritis was scored weekly, and organs were assessed for pathologic features. Anti–IL-23 monoclonal antibodies were injected into curdlan-treated SKG mice. CD4+ T cells were transferred from curdlan-treated mice to SCID mice, and sera were analyzed for autoantibodies. Results After systemic injection of curdlan, SKG mice developed enthesitis, wrist, ankle, and sacroiliac joint arthritis, dactylitis, plantar fasciitis, vertebral inflammation, ileitis resembling Crohn's disease, and unilateral uveitis. Mannan triggered spondylitis and arthritis. Arthritis and spondylitis were T cell– and IL-23–dependent and were transferable to SCID recipients with CD4+ T cells. SpA was associated with collagen- and proteoglycan-specific autoantibodies. Conclusion Our findings indicate that the SKG ZAP-70W163C mutation predisposes BALB/c mice to SpA, resulting from innate and adaptive autoimmunity, after systemic β-glucan or mannan exposure.

Objective Ankylosing spondylitis (AS) is a common inflammatory arthritis affecting primarily the axial skeleton. IL23R is genetically associated with AS. This study was undertaken to investigate and characterize the role of interleukin-23 (IL-23) signaling in AS pathogenesis. Methods The study population consisted of patients with active AS (n = 17), patients with psoriatic arthritis (n = 8), patients with rheumatoid arthritis, (n = 9), and healthy subjects (n = 20). IL-23 receptor (IL-23R) expression in T cells was determined in each subject group, and expression levels were compared. Results The proportion of IL-23R–expressing T cells in the periphery was 2-fold higher in AS patients than in healthy controls, specifically driven by a 3-fold increase in IL-23R–positive γ/δ T cells in AS patients. The proportions of CD4+ and CD8+ cells that were positive for IL-17 were unchanged. This increased IL-23R expression on γ/δ T cells was also associated with enhanced IL-17 secretion, with no observable IL-17 production from IL-23R–negative γ/δ T cells in AS patients. Furthermore, γ/δ T cells from AS patients were heavily skewed toward IL-17 production in response to stimulation with IL-23 and/or anti-CD3/CD28. Conclusion Recently, mouse models have shown IL-17–secreting γ/δ T cells to be pathogenic in infection and autoimmunity. Our data provide the first description of a potentially pathogenic role of these cells in a human autoimmune disease. Since IL-23 is a maturation and growth factor for IL-17–producing cells, increased IL-23R expression may regulate the function of this putative pathogenic γ/δ T cell population.

The immune system has evolved to survey and respond appropriately to the universe of foreign pathogens, while at the same time deploying an intricate repertoire of mechanisms that keep responses to host tissues in check. For the adaptive immune system, specificity and sensitivity are provided by a large repertoire of antigen T-cell receptors (TCRs) constructed in their extracellular domain to recognize antigenic peptide fragments restricted and presented by histocompatibility complex molecules, and coupled through intracellular domains to signal transduction modules that serve to transmit environmental cues inside the cell. In this review we consider recent evidence that has provided insight into how altered TCR signaling thresholds could contribute to human autoimmune arthritis, including rheumatoid arthritis (RA), and the spondyloarthropathies (SpA). We also discuss mechanistic studies that demonstrate how perturbations of T-cell antigen receptor signaling in the SKG mouse model can promote systemic autoimmunity and the intersection with essential innate immune pathways that lead to the development of chronic inflammatory phenotypes.

OBJECTIVE To determine whether and by what mechanism systemic delivery of curcumin-containing liposomes improves insulin resistance in the leptin deficient (ob/ob) mouse model of insulin resistance. RESEARCH DESIGN AND METHODS Insulin resistant ob/ob mice with steatosis were injected intraperitoneally with liposome nanoparticles, entrapping the nuclear factor-κB (NF-κB) inhibitor curcumin (curcusomes), and uptake in liver and adipose tissue was determined by flow cytometry. The effects of curcusomes on macrophage NF-κB activation and cytokine production were assessed. Transfer experiments determined the role of hepatic tumor necrosis factor (TNF)/inducible nitric oxide synthase-producing dendritic cells (Tip-DCs) and adipose tissue macrophages (ATMs) in inflammation-induced insulin resistance, determined by homeostatic assessment of insulin resistance. RESULTS Phagocytic myeloid cells infiltrating the liver in ob/ob mice had the phenotypic characteristics of Tip-DCs that arise from monocyte precursors in the liver and spleen after infection. Targeting Tip-DCs and ATMs with curcusomes in ob/ob mice reduced NF-κB/RelA DNA binding activity, reduced TNF, and enhanced interleukin-4 production. Curcusomes improved peripheral insulin resistance. CONCLUSIONS Both hepatic Tip-DCs and ATMs contribute to insulin resistance in ob/ob mice. Curcusome nanoparticles inhibit proinflammatory pathways in hepatic Tip-DCs and ATMs and reverse insulin resistance. Targeting inflammatory DCs is a novel approach for type 2 diabetes treatment.

Antigen-dependent interactions between T lymphocytes and dendritic cells (DCs) can produce two distinct outcomes: tolerance and immunity. It is generally considered that all DC subsets are capable of supporting both tolerogenic and immunogenic responses, depending on their exposure to activating signals. Here, we tested whether epidermal Langerhans cells (LCs) can support immunogenic responses in vivo in the absence of antigen presentation by other DC subsets. CD4 T cells responding to antigen presentation by activated LCs initially proliferated but then failed to differentiate into effector/memory cells or to survive long term. The tolerogenic function of LCs was maintained after exposure to potent adjuvants and occurred despite up-regulation of the costimulatory molecules CD80, CD86, and IL-12, but was consistent with their failure to translocate the NF-κB family member RelB from the cytoplasm to the nucleus. Commitment of LCs to tolerogenic function may explain why commensal microorganisms expressing Toll-like receptor (TLR) ligands but confined to the skin epithelium are tolerated, whereas invading pathogens that breach the epithelial basement membrane and activate dermal DCs stimulate a strong immune response.