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Aims To evaluate the safety, pharmacokinetics and pharmacodynamics of single- and multiple-rising doses (MRDs) of BI 705564 and establish proof of mechanism. Methods BI 705564 was studied in 2 placebo-controlled, Phase I clinical trials testing single-rising doses (1-160 mg) and MRDs (1-80 mg) of BI 705564 over 14 days in healthy male volunteers. Blood samples were analysed for BI 705564 plasma concentration, Bruton's tyrosine kinase (BTK) target occupancy (TO) and CD69 expression in B cells stimulated ex vivo. A substudy was conducted in allergic, otherwise healthy, MRD participants. Safety was assessed in both studies. Results All doses of BI 705564 were well tolerated. Geometric mean BI 705564 plasma terminal half-life ranged from 10.1 to 16.9 hours across tested doses, with no relevant accumulation after multiple dosing. Doses ≥20 mg resulted in ≥85% average TO that was maintained for ≥48 hours after single-dose administration. Functional effects of BTK signalling were demonstrated by dose-dependent inhibition of CD69 expression. In allergic participants, BI 705564 treatment showed a trend in wheal size reduction in a skin prick test and complete inhibition of basophil activation. Mild bleeding-related adverse events were observed with BI 705564; bleeding time increased in 1/12 participants (8.3%) who received placebo vs 26/48 (54.2%) treated with BI 705564. Conclusion BI 705564 showed efficient target engagement through durable TO and inhibition of ex vivo B-cell activation, and proof of mechanism through effects on allergic skin responses. Mild bleeding-related adverse events were probably related to inhibition of platelet aggregation by BTK inhibition.

SummaryTissue repair is disturbed in fibrotic diseases like systemic sclerosis (SSc), where the deposition of large amounts of extracellular matrix components such as collagen interferes with organ function. LAIR-1 is an inhibitory collagen receptor highly expressed on tissue immune cells. We questioned whether in SSc, impaired LAIR-1-collagen interaction is contributing to the ongoing inflammation and fibrosis.We found that SSc patients do not have an intrinsic defect in LAIR-1 expression or function. Instead, fibroblasts from SSc patients deposit disorganized collagen products in vitro, which are dysfunctional LAIR-1 ligands. This can be mimicked in healthy fibroblast stimulated by soluble factors that drive inflammation and fibrosis in SSc and is dependent of matrix metalloproteinases and platelet-derived growth factor receptor signaling.In support of a non-redundant role of LAIR-1 in the control of fibrosis, we found that LAIR-1-deficient mice have increased skin fibrosis in the bleomycin mouse model for SSc. Thus, LAIR-1 represents an essential control mechanism for tissue repair. In fibrotic disease, excessive collagen degradation may lead to a disturbed feedback loop. The presence of functional LAIR-1 in patients provides a therapeutic opportunity to reactivate this intrinsic negative feedback mechanism in fibrotic diseases.Abstract Figure

Fibrosis is defined as an excessive deposition of connective tissue components and can affect virtually every organ system, including the skin, lungs, liver and kidney. Fibrotic tissue remodelling often leads to organ malfunction and is commonly associated with high morbidity and mortality. The medical need for effective antifibrotic therapies is thus very high. However, the extraordinarily high costs of drug development and the rare incidence of many fibrotic disorders hinder the development of targeted therapies for individual fibrotic diseases. A potential strategy to overcome this challenge is to target common mechanisms and core pathways that are of central pathophysiological relevance across different fibrotic diseases. The factors influencing susceptibility to and initiation of these diseases are often distinct, with disease-specific and organ-specific risk factors, triggers and sites of first injury. Fibrotic remodelling programmes with shared fibrotic signalling responses such as transforming growth factor-β (TGFβ), platelet-derived growth factor (PDGF), WNT and hedgehog signalling drive disease progression in later stages of fibrotic diseases. The convergence towards shared responses has consequences for drug development as it might enable the development of general antifibrotic compounds that are effective across different disease entities and organs. Technological advances, including new models, single-cell technologies and gene editing, could provide new insights into the pathogenesis of fibrotic diseases and the development of drugs for their treatment. A number of core pathways and mechanisms of fibrosis, outlined in this Review, are shared across different tissues and might therefore present targets for general antifibrotic strategies. Organ-specific and disease-specific differences in fibrotic diseases could also provide insights for drug development efforts.

ObjectiveTo evaluate the safety, efficacy and therapeutic mechanism of BI 655064, an antagonistic anti-CD40 monoclonal antibody, in patients with rheumatoid arthritis (RA) and an inadequate response to methotrexate (MTX-IR).MethodsIn total, 67 patients were randomised to receive weekly subcutaneous doses of 120 mg BI 655064 (n=44) or placebo (n=23) for 12 weeks. The primary endpoint was the proportion of patients who achieved 20% improvement in American College of Rheumatology criteria (ACR20) at week 12. Safety was assessed in patients who received at least one dose of study drug.ResultsAt week 12, the primary endpoint was not met, with 68.2% of patients treated with BI 655064 achieving an ACR20 vs 45.5% with placebo (p=0.064); using Bayesian analysis, the posterior probability of seeing a difference greater than 35% was 42.9%. BI 655064 was associated with greater changes in CD40–CD40L pathway-related markers, including reductions in inflammatory and bone resorption markers (interleukin-6, matrix metalloproteinase-3, receptor activator of nuclear factor-κB ligand), concentration of autoantibodies (immunoglobulin [Ig]G rheumatoid factor [RF], IgM RF, IgA RF) and CD95+ activated B-cell subsets. No serious adverse events (AEs) related to BI 655064 treatment or thromboembolic events occurred; reported AEs were mainly of mild intensity.ConclusionAlthough blockade of the CD40–CD40L pathway with BI 655064 in MTX-IR patients with RA resulted in marked changes in clinical and biological parameters, including reductions in activated B-cells, autoantibody production and inflammatory and bone resorption markers, with a favourable safety profile, clinical efficacy was not demonstrated in this small phase IIa study.Trial registration numberNCT01751776

IgE induced by type 2 immune responses in atopic dermatitis is implicated in the progression of atopic dermatitis to other allergic diseases, including food allergies, allergic rhinitis, and asthma. However, the keratinocyte-derived signals that promote IgE and ensuing allergic diseases remain unclear. Herein, in a mouse model of atopic dermatitis-like skin inflammation induced by epicutaneous Staphylococcus aureus exposure, keratinocyte release of IL‑36α along with IL-4 triggered B cell IgE class-switching, plasma cell differentiation, and increased serum IgE levels-all of which were abrogated in IL-36R-deficient mice or anti-IL‑36R-blocking antibody-treated mice. Moreover, skin allergen sensitization during S. aureus epicutaneous exposure-induced IL-36 responses was required for the development of allergen-specific lung inflammation. In translating these findings, elevated IL‑36 cytokines in human atopic dermatitis skin and in IL‑36 receptor antagonist-deficiency patients coincided with increased serum IgE levels. Collectively, keratinocyte-initiated IL‑36 responses represent a key mechanism and potential therapeutic target against allergic diseases.

IL-36, which belongs to the IL-1 superfamily, is increasingly linked to neutrophilic inflammation. Here, we combined in vivo and in vitro approaches using primary mouse and human cells, as well as, acute and chronic mouse models of lung inflammation to provide mechanistic insight into the intercellular signaling pathways and mechanisms through which IL-36 promotes lung inflammation. IL-36 receptor deficient mice exposed to cigarette smoke or cigarette smoke and H1N1 influenza virus had attenuated lung inflammation compared with wild-type controls. We identified neutrophils as a source of IL-36 and show that IL-36 is a key upstream amplifier of lung inflammation by promoting activation of neutrophils, macrophages and fibroblasts through cooperation with GM-CSF and the viral mimic poly(I:C). Our data implicate IL-36, independent of other IL-1 family members, as a key upstream amplifier of neutrophilic lung inflammation, providing a rationale for targeting IL-36 to improve treatment of a variety of neutrophilic lung diseases., Carolin Koss et al. show that IL-36 produced from neutrophils is a key amplifier of lung inflammation in mice by activating neutrophils, macrophages and fibroblasts in cooperation with GM-CSF and the viral mimic poly(I:C). This study suggests that targeting IL-36 may improve the treatment of neutrophilic lung diseases.

Introduction Crohn’s disease (CD) is a chronic inflammatory condition of the gastrointestinal tract, characterised by an aberrant immune response towards commensal microbiota. Despite the availability of target-specific front-line therapeutics, 30–40% of CD patients still require surgery to manage disease. This project aims to identify different systemic and mucosal CD immunopathotypes and map their associations to distinct treatment responses and behaviours. Methods To study local immune response in CD, colonic mucosal biopsies of inflamed patients (CD, n=4) and non-IBD controls (NC, n=6) were analysed by bulk RNA sequencing. Raw counts were normalised using DESeq and further analysed in R studio with a specific pipeline to select differentially expressed genes associated with the immune system. All findings were validated in a selection of three cohorts comparing gene expression of colonic inflamed CD tissue with non-IBD controls (nCD=36, nNC=24). Differences in the systemic immune response were studied in two separate cohorts by isolating plasma and peripheral mononuclear cells (PBMCs) from fresh whole blood of CD patients with different levels of disease activity (n=30) and NCs (n=42). Subsequently, cytokine levels and leukocyte frequencies were measured using multiplex assays and flow cytometry analysis. Results Gene expression analysis of colonic mucosal tissue biopsies highlighted an immunophenotype driven by macrophage and neutrophil activation and infiltration. After validating this gene cluster in a selection of cohorts, we find that CD patients with colonic active disease can be stratified into three different groups based on their macrophage activation phenotype. In the peripheral blood, we observed that patients have different levels of systemic disease activation, characterised by their leukocyte and cytokine concentrations, independent of their disease activity. Conclusions Our analyses of mucosal tissue and peripheral blood have provided evidence of different immunopathotypes, both mucosal and systemic. Ongoing work will involve the correlation of these phenotypes with clinical information, such as treatment response and disease progression, to better understand whether pathotypes predict disease behaviour in CD.

Lupus nephritis (LN) is a serious end organ complication of systemic lupus erythematosus. Nephrotoxic serum nephritis (NTN) is an inducible model of LN, which utilizes passive transfer of pre-formed nephrotoxic antibodies to initiate disease. In previous studies, we demonstrated that the Bruton's tyrosine kinase inhibitor, BI-BTK-1, prevents the development of nephritis in NTN when treatment was started prior to nephrotoxic serum transfer, and reverses established proteinuria as well. We manipulated the initiation and duration of BI-BTK-1 therapy in NTN to study its delayed therapeutic effects when treatment is given later in the disease course, as well as to further understand what effect BI-BTK-1 is having to prevent initiation of nephritis with early treatment. Early treatment and remission induction each correlated with decreased inflammatory macrophages, CD4+ and CD8+ T cells, and decreased B220+ B cells. Additionally, an increased proportion of resident macrophages within the CD45+ population favored a delay of disease onset and remission induction. We also studied the cellular processes involved in reactivation of nephritis by withdrawing BI-BTK-1 treatment at different time points. Treatment cessation led to either early or later onset of renal flares inversely dependent on the initial duration of BTK inhibition, as assessed by increased proteinuria and BUN levels and worse renal pathology. These flares were associated with an increase in kidney CD45+ infiltrates, including myeloid cell populations. IL-6, CD14, and CCL2 were also increased in mice developing late flares. These analyses point to the role of macrophages as an important contributor to the pathogenesis of immune mediated nephritis, and further support the therapeutic potential of BTK inhibition in this disease and related conditions.

The gut mounts secretory immunoglobulin A (SIgA) responses to commensal bacteria through nonredundant T cell-dependent (TD) and T cell-independent (TI) pathways that promote the establishment of mutualistic host-microbiota interactions. SIgAs from the TD pathway target penetrant bacteria, and their induction requires engagement of CD40 on B cells by CD40 ligand on T follicular helper cells. In contrast, SIgAs from the TI pathway bind a larger spectrum of bacteria, but the mechanism underpinning their production remains elusive. Here, we show that the intestinal TI pathway required CD40-independent B cell-activating signals from TACI, a receptor for the innate CD40 ligand-like factors BAFF and APRIL. TACI-induced SIgA responses targeted a fraction of the gut microbiota without shaping its overall composition. Of note, TACI was dispensable for TD induction of IgA in gut-associated lymphoid organs. Thus, BAFF/APRIL signals acting on TACI orchestrate commensal bacteria-specific SIgA responses through an intestinal TI program.

Background Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that affects different end organs, including skin and brain. We and others have previously shown the importance of macrophages in the pathogenesis of cutaneous and neuropsychiatric lupus. Additionally, autoantibodies produced by autoreactive B cells are thought to play a role in both the skin and central nervous system pathologies associated with SLE. Methods We used a novel inhibitor of Bruton’s tyrosine kinase (BTK), BI-BTK-1, to target both macrophage and B cell function in the MRL-lpr/lpr murine model of SLE, and examined the effect of treatment on skin and brain disease. Results We found that treatment with BI-BTK-1 significantly attenuated the lupus associated cutaneous and neuropsychiatric disease phenotypes in MRL/lpr mice. Specifically, BI-BTK-1 treated mice had fewer macroscopic and microscopic skin lesions, reduced cutaneous cellular infiltration, and diminished inflammatory cytokine expression compared to control mice. BTK inhibition also significantly improved cognitive function, and decreased accumulation of T cells, B cells, and macrophages within the central nervous system, specifically the choroid plexus. Conclusions Directed therapies may improve the response rate in lupus-driven target organ involvement, and decrease the dangerous side effects associated with global immunosuppression. Overall, our results suggest that inhibition of BTK may be a promising therapeutic option for cutaneous and neuropsychiatric disease associated with SLE. Electronic supplementary material The online version of this article (doi:10.1186/s13075-017-1500-0) contains supplementary material, which is available to authorized users.

Background The small molecule BI 705564 is a highly selective, covalent and potent inhibitor (B cell CD69 activation IC50=2.2 nM) of the Brutons tyrosine kinase (BTK). BTK plays a critical role in the differentiation and function of B cells and myeloid cell lineages and may play a major role in autoimmune diseases. Blocking the BTK pathways may be a promising new treatment of autoimmune diseases like SLE. Methods BI 705564 has been studied in 43 male healthy volunteers (HV) in a single-blinded, partially randomized, placebo-controlled trial testing single rising doses from 10 to 160 mg. In a double-blinded, randomized, placebo-controlled study BI 705564 was administered to 50 male HV at doses from 10 to 80 mg once daily for 14 days. Blood samples were analyzed for BI 705564 plasma concentrations, BTK target occupancy (TO) and CD69 expression in B cells stimulated ex-vivo with anti-IgD. Results All doses of BI 705564 were well-tolerated in both studies. There were no serious adverse events (AEs) and reported AEs were mainly of mild intensity and not dose-limiting. There was no difference in the total number of subjects with AEs (combined data from both trials) between BI 705564 [28/75 (37.3%)] and placebo [8/18 (44.4%)] and no dose-relationship of AEs. The most frequently reported AE was headache (BI 705564: 8.0% vs placebo: 5.6%, combined data). There was no difference in the occurrence of infections (BI 705564: 6.5% vs placebo: 11.1%) or gastrointestinal disorders (BI 705564: 13.3% vs. placebo 11.1%) in both studies (combined data). There were no relevant drug-related changes in vital signs, ECGs and standard safety laboratory tests. Analysis of BI 705564 plasma exposures showed a terminal half-life between 5.7 and 14.2 hour with no relevant accumulation after multiple dosing. Doses of 20 mg and above resulted in an average TO 85% which is expected to result in clinical efficacy based on preclinical studies. After a single dose administration, TO was maintained for at least 48 hour consistent with the mechanism of a covalent irreversible inhibitor. Functional effects on BTK signalling were demonstrated by dose-dependent inhibition up to 100% of CD69 expression on anti-IgD stimulated B cells. Conclusions BI 705564 was well-tolerated in healthy subjects after single and multiple doses. The favorable clinical safety profile and the high potential based on pre-clinical studies and effects on CD69 support further investigation of BI 705564 in patients with autoimmune diseases like SLE. Funding Source(s): This study was funded by Boehringer Ingelheim.

Pathophysiology of systemic sclerosis (SSc, Scleroderma), an autoimmune rheumatic disease, comprises of mechanisms that drive vasculopathy, inflammation and fibrosis. Understanding of the disease and associated clinical heterogeneity has advanced considerably in the past decade, highlighting the necessity of more specific targeted therapy. While many of the recent trials in SSc failed to meet the primary end points that predominantly relied on changes in modified Rodnan skin scores (MRSS), sub-group analysis, especially those focused on the basal skin transcriptomic data have provided insights into patient subsets that respond to therapies. These findings suggest that deeper understanding of the molecular changes in pathways is very important to define disease drivers in various patient subgroups. In view of these challenges, we performed meta-analysis on 9 public available SSc microarray studies using a novel pathway pivoted approach combining consensus clustering and machine learning assisted feature selection. Selected pathway modules were further explored through cluster specific topological network analysis in search of novel therapeutic concepts. In addition, we went beyond previously described SSc class divisions of 3 clusters (e.g. inflammation, fibro-proliferative, normal-like) and expanded into a much finer stratification in order to profile SSc patients more accurately. Our analysis unveiled an important 80 pathway signatures that differentiated SSc patients into 8 unique subtypes. The 5 pathway modules derived from such signature successfully defined the 8 SSc subsets and were validated by in-silico cellular deconvolution analysis. Myeloid cells and fibroblasts involvement in different clusters were confirmed and linked to corresponding pathway activities. Collectively, our findings revealed more complex disease subtypes in SSc; Key gene mediators such as IL6, FGFR1, TLR7, PLCG2, IRK2 identified by network analysis underscored the scientific rationale for exploring additional targets in treatment of SSc.

Lupus nephritis (LN) is a significant complication of systemic lupus erythematosus (SLE), increasing its morbidity and mortality. Although the current standard of care helps suppress disease activity, it is associated with toxicity and ultimately does not cure SLE. At present, there are no therapies specifically indicated for the treatment of LN and there is an unmet need in this disease where treatment remains a challenge. The CD40-CD40L pathway is central to SLE pathogenesis and the generation of autoantibodies and their deposition in the kidneys, resulting in renal injury in patients with LN. CD40 is expressed on immune cells (including B cells, monocytes and dendritic cells) and also non-haematopoietic cells. Interactions between CD40L on T cells and CD40 on B cells in the renal interstitium are critical for the local expansion of naive B cells and autoantibody-producing B cells in LN. CD40L-mediated activation of myeloid cells and resident kidney cells, including endothelial cells, proximal tubular epithelial cells, podocytes and mesangial cells, further amplifies the inflammatory milieu in the interstitium and the glomeruli. Several studies have highlighted the upregulated expression of CD40 in LN kidney biopsies, and preclinical data have demonstrated the importance of the CD40-CD40L pathway in murine SLE and LN. Blocking this pathway is expected to ameliorate inflammation driven by infiltrating immune cells and resident kidney cells. Initial experimental therapeutic interventions targeting the CD40-CD40L pathway, based on CD40L antibodies, were associated with an increased incidence of thrombosis. However, this safety issue has not been observed with second-generation CD40/CD40L antibodies that have been engineered to prevent platelet activation. With these advancements, together with recent preclinical and clinical findings, it is anticipated that selective blockade of the CD40-CD40L pathway may address the unmet treatment needs in SLE, LN and other autoimmune diseases.

Background Crohn’s disease (CD) is a chronic inflammatory gastrointestinal condition, with globally increasing incidence. Patients with CD suffer from a loss of tolerance towards their commensal microbiota causing an aberrant immune response, occurring in a protracted relapse and remission cycle. Although a variety of frontline therapies is currently available, including targeted therapies such as biologic drugs, 30–40% of CD patients still require surgery to manage the disease. At present, the immunobiology of CD is not fully understood. However, differences in immune responses between patients might play an important role in diverse treatment responses. The aim of this study was to identify differences in peripheral and local immune responses of CD to understand differences in disease behaviour and treatment outcome. Methods Peripheral blood mononuclear cells and plasma were isolated from whole blood of a cross-sectional CD patient cohort (nCD = 12) and normal controls (NC, nNC = 28). Flow cytometry analysis and multiplex assays were used to quantify immune cell populations and cytokine levels, respectively. The local immune response was analysed by bulk RNA sequencing of mucosal colonic biopsies either from inflamed CD or normal tissue. Gene signatures were then followed up by validation in publicly deposited gene expression datasets (nCD = 36, nNC = 24), and by measurement of specific proteins using our archived samples. Results Peripheral immunophenotyping of the initial cross-sectional study displayed three different types of CD patients, characterised by either a decrease in leukocyte populations, an increase of cytokines, or a change in both. Analysis of the RNAseq data derived from colonic biopsies revealed four distinct clusters in genes associated with the immune response in CD patients. Further pathway analysis showed one cluster with an enriched B cell signature and another cluster with an elevated macrophage and neutrophil response. We utilised publicly available gene expression datasets to validate these signatures in a larger cohort and identified a selection of patients with an up-regulated pro-inflammatory macrophage response. Using correlation analysis, we suggest an immunopathotype with increased macrophage activation which is potentially associated with a more severe form of the disease. Conclusion We have identified distinct immunopathotypes in both the peripheral and local immune response of CD patients. Further investigation will correlate these distinct immune responses in CD with clinical parameters, to understand associations between diverse treatment responses and disease behaviours.

Lupus nephritis is a common disease manifestation of SLE, in which immune complex deposition and macrophage activation are important contributors to disease pathogenesis. Bruton's tyrosine kinase (BTK) plays an important role in both B cell and FcgammaR mediated myeloid cell activation. In the current study, we examined the efficacy of BI-BTK-1, a recently described irreversible BTK inhibitor, in the classical NZB × NZW F1 (NZB/W) and MRL/lpr spontaneous mouse models of SLE. NZB/W mice were randomly assigned to a treatment (0.3 mg/kg, 1 mg/kg, 3 mg/kg and 10 mg/kg) or control group and began treatment at 22 weeks of age. The experimental setup was similar in MRL/lpr mice, but with a single treated (10 mg/kg, beginning at 8–9 weeks of age) and control group. A separate experiment was performed in the MRL/lpr strain to assess the ability of BI-BTK-1 to reverse established kidney disease. Early treatment with BI-BTK-1 significantly protected NZB/W and MRL/lpr mice from the development of proteinuria, correlating with significant renal histological protection, decreased anti-DNA titers, and increased survival in both strains. BI-BTK-1 treated mice displayed a significant decrease in nephritis-associated inflammatory mediators (e.g. LCN2 and IL-6) in the kidney, combined with a significant inhibition of immune cell infiltration and accumulation. Importantly, BI-BTK-1 treatment resulted in the reversal of established kidney disease. BTK inhibition significantly reduced total B cell numbers and all B cell subsets (immature, transitional, follicular, marginal zone, and class switched) in the spleen of NZB/W mice. Overall, the significant efficacy of BI-BTK-1 in ameliorating multiple pathological endpoints associated with kidney disease in two distinct murine models of spontaneous lupus nephritis provides a strong rationale for BTK inhibition as a promising treatment approach for lupus nephritis.

Background Systemic lupus erythematosus (SLE) is an autoimmune diseases characterised by dysregulation of immune cell function with numerous clinical manifestations. Along with B and T cells, myeloid cells play an important role in disease pathogenesis. Circulating monocytes are recruited to sites of inflammation where they play an active role in mediating tissue inflammation and injury. Objectives To understand the changes in circulating monocyte phenotypes in SLE patients. Methods Peripheral blood was collected from 25 female SLE patients who were autoantibody positive (dsDNA/Ro/La/SM), with SLEDAIs between 2 and 6 (all patients on hydroxychloroquine, 6 patients on steroids, and 3 on MMF); TR BIO Inc, Hawthorne, NY). Gender matched healthy controls (27 HC) were obtained from BI volunteers. Using multicolor flow cytometry, the percent changes in the expression of HLA-DR/DP/DQ, CD163, CD40, CCR1, CX3CR1 and CD11b on CD14+, CD14+CD16+, and CD16+ monocytes were evaluated. In a smaller subset of these patients (8 SLE and 15 HC), circulating endothelial cell (CECs) phenotyping was performed by evaluating: CD45, CD133/1, CD106, CD105, CD31, CD46, CD34, and CX3CL1. Human 38-plex cytokine/chemokine multiplexes (Luminex, Millipore) were used to evaluate serum analytes. To understand the regulation of cytokines and chemokines from the monocyte population, HC monocytes were sorted to 95% purity, stimulated with disease-relevant TLR ligands, and profiled for their analytes. Statistical analyses were performed using GraphPad Prism, version 7, GraphPad Software Inc. and determined by the Mann-Whitney t-test. Results SLE patients had a significant increase in MFI for CD14+CD11b+ and CD16+CD11b+ and percentages as well as MFI for CD14+CD163+, CD16+CD163+, and CD14+CD16+CD163+ as compared to controls,. An increase in the percentage of CX3CR1+ CD14+ and CD14+CD16+ monocytes were observed, see table 1. Concurrently, an increase in CX3CL1 percentage was observed in immature circulating endothelial cells (iCECs) (p Conclusions Cell surface markers of activation and adhesion increased in SLE monocyte subsets compared to HC. In parallel, increased inflammatory cytokines and chemokines that attract monocytes to tissues were increased in the serum of these patients. Linking a possible source of this increase in serum analytes, HC monocyte subsets were stimulated with disease-relevant ligands and evaluated in culture. Along with increased monocyte expression of CX3CR1, preliminary data demonstrates an increase in CX3CL1 expression on endothelial progenitor cells (EPCs) and immature and mature circulating endothelial cells (iCECs and mCEC respectively) in active disease. Disclosure of Interest None declared

Summary Secretory immunoglobulin A (SIgA) enhances host-microbiota symbiosis, whereas SIgM remains poorly understood. We found that gut IgM+ plasma cells (PCs) were more abundant in humans than mice and clonally related to a large repertoire of memory IgM+ B cells disseminated throughout the intestine but rare in systemic lymphoid organs. In addition to sharing a gut-specific gene signature with memory IgA+ B cells, memory IgM+ B cells were related to some IgA+ clonotypes and switched to IgA in response to T cell-independent or T cell-dependent signals. These signals induced abundant IgM which, together with SIgM from clonally affiliated PCs, recognized mucus-embedded commensals. Bacteria recognized by human SIgM were dually coated by SIgA and showed increased richness and diversity compared to IgA-only-coated or uncoated bacteria. Thus, SIgM may emerge from pre-existing memory rather than newly activated naive IgM+ B cells and could help SIgA to anchor highly diverse commensal communities to mucus., Graphical Abstract, Highlights • IgM+ PCs generating SIgM are relatively abundant in human but not mouse gut • IgM+ PCs clonally relate to a large gut repertoire of memory IgM+ B cells • Gut memory IgM+ B cells express a tissue-specific signature and can switch to IgA • Human but not mouse SIgM binds a highly diverse microbiota dually coated by SIgA, Magri et al. found that the human gut includes a large memory IgM+ B cell repertoire clonally related to plasma cells mounting SIgM responses against mucus-embedded commensals co-targeted by SIgA. Dually coated bacteria are detected in humans but not mice and show increased diversity and richness compared to SIgA-only-coated or uncoated bacteria.

Objective Bruton's tyrosine kinase (BTK) plays a critical role in B cell development and function. We recently described a selective BTK inhibitor, RN486, that blocks B cell receptor (BCR) and Fcγ receptor signaling and is efficacious in animal models of arthritis. The aim of this study was to examine the potential efficacy of BTK in systemic lupus erythematosus (SLE), using an NZB × NZW mouse model of spontaneous SLE. Methods Mice received RN486 or its vehicle (administered in chow) at a final concentration of 30 mg/kg for 8 weeks, starting at 32 weeks of age. Results The administration of RN486 completely stopped disease progression, as determined by histologic and functional analyses of glomerular nephritis. The efficacy was associated with striking inhibition of B cell activation, as demonstrated by a significant reduction in CD69 expression in response to BCR crosslinking. RN486 markedly reduced the secretion of IgG anti–double-stranded DNA (anti-dsDNA) secretion, as determined by enzyme-linked immunosorbent and enzyme-linked immunospot assays. Flow cytometric analysis demonstrated depletion of CD138highB220low plasma cells in the spleen. RN486 inhibited secretion of IgG anti-dsDNA but not IgM anti-dsDNA, suggesting that pharmacologic blockade of BTK resembles the reported transgenic expression of low levels of endogenous BTK in B cells. In addition, RN486 may also impact the effector function of autoantibodies, as evidenced by a significant reduction in immune complex–mediated activation of human monocytes in vitro and down-regulation of the expression of macrophage-related and interferon-inducible genes in both the kidneys and spleens of treated mice. Conclusion Collectively, our data suggest that BTK inhibitors may simultaneously target autoantibody-producing and effector cells in SLE, thus constituting a promising therapeutic alternative for this disease.

Lupus nephritis (LN) is a potentially dangerous end organ pathology that affects upwards of 60% of SLE patients. Bruton’s tyrosine kinase (BTK) is important for B cell development, Fc receptor signaling, and macrophage polarization. In this study, we investigated the effects of a novel, highly selective and potent BTK inhibitor, BI-BTK-1, in an inducible model of LN in which mice receive nephrotoxic serum (NTS) containing anti-glomerular antibodies. Mice were treated once daily with vehicle alone or BI-BTK-1 (0.3–10 mg/kg, n=16/group), either prophylactically or therapeutically. When compared with control treated mice, NTS-challenged mice treated prophylactically with BI-BTK-1 exhibited significantly attenuated disease which was dose dependent, as measured by proteinuria, serum creatinine, and serum BUN. Histological assessment confirmed marked renal protection in the BI-BTK-1 treatment groups. BI-BTK-1 treatment resulted in decreased recruitment of inflammatory monocytes from the splenic reservoir, and a decrease in infiltrating IBA-1+ cells, as well as C3 deposition, within the kidney. RT-PCR on whole kidney RNA and serum profiling indicated that BTK inhibition significantly decreased levels of LN-relevant inflammatory cytokines and chemokines. Renal RNA expression profiling by RNA-seq revealed that BI-BTK-1 dramatically modulated pathways related to inflammation and glomerular injury. Importantly, when administered therapeutically, BI-BTK-1 reversed established proteinuria and improved renal histopathology. Our results highlight the important role for BTK in the pathogenesis of immune complex-mediated nephritis, and BTK inhibition as a promising therapeutic target for LN.

Lupus nephritis (LN) is a serious manifestation of systemic lupus erythematosus. Therapeutic studies in mouse LN models do not always predict outcomes of human therapeutic trials, raising concerns about the human relevance of these preclinical models. In this study, we used an unbiased transcriptional network approach to define, in molecular terms, similarities and differences among three lupus models and human LN. Genome-wide gene-expression networks were generated using natural language processing and automated promoter analysis and compared across species via suboptimal graph matching. The three murine models and human LN share both common and unique features. The 20 commonly shared network nodes reflect the key pathologic processes of immune cell infiltration/activation, endothelial cell activation/injury, and tissue remodeling/fibrosis, with macrophage/dendritic cell activation as a dominant cross-species shared transcriptional pathway. The unique nodes reflect differences in numbers and types of infiltrating cells and degree of remodeling among the three mouse strains. To define mononuclear phagocyte-derived pathways in human LN, gene sets activated in isolated NZB/W renal mononuclear cells were compared with human LN kidney profiles. A tissue compartment-specific macrophage-activation pattern was seen, with NF-κB1 and PPARγ as major regulatory nodes in the tubulointerstitial and glomerular networks, respectively. Our study defines which pathologic processes in murine models of LN recapitulate the key transcriptional processes active in human LN and suggests that there are functional differences between mononuclear phagocytes infiltrating different renal microenvironments.

The critical role of IFN-α in the pathogenesis of human systemic lupus erythematosus has been highlighted in recent years. Exposure of young lupus-prone NZB/W F1 mice to IFN-α in vivo leads to an accelerated lupus phenotype that is dependent on T cells and is associated with elevated serum levels of BAFF, IL-6, and TNF-α, increased splenic expression of IL-6 and IL-21, formation of large germinal centers, and the generation of large numbers of short-lived plasma cells that produce IgG2a and IgG3 autoantibodies. In this study, we show that both IgG2a and IgG3 autoantibodies are pathogenic in IFN-α–accelerated lupus, and their production can be dissociated by using low-dose CTLA4-Ig. Only high-dose CTLA4-Ig attenuates both IgG2a and IgG3 autoantibody production and significantly delays death from lupus nephritis. In contrast, BAFF/APRIL blockade has no effect on germinal centers or the production of IgG anti-dsDNA Abs but, if given at the time of IFN-α challenge, delays the progression of lupus by attenuating systemic and renal inflammation. Temporary remission of nephritis induced by combination therapy with cyclophosphamide, anti-CD40L Ab, and CTLA4-Ig is associated with the abrogation of germinal centers and depletion of short-lived plasma cells, but relapse occurs more rapidly than in conventional NZB/W F1 mice. This study demonstrates that IFN-α renders NZB/W F1 relatively resistant to therapeutic intervention and suggests that the IFN signature should be considered when randomizing patients into groups and analyzing the results of human clinical trials in systemic lupus erythematosus.

Renal infiltration with mononuclear cells is associated with poor prognosis in systemic lupus erythematosus. A renal macrophage/dendritic cell signature is associated with the onset of nephritis in NZB/W mice, and immune-modulating therapies can reverse this signature and the associated renal damage despite ongoing immune complex deposition. In nephritic NZB/W mice, renal F4/80hi/CD11cint macrophages are located throughout the interstitium, whereas F4/80lo/CD11chi dendritic cells accumulate in perivascular lymphoid aggregates. We show here that F4/80hi/CD11cint renal macrophages have a Gr1lo/Ly6Clo/VLA4lo/MHCIIhi/CD43lo/CD62Llo phenotype different from that described for inflammatory macrophages. At nephritis onset, F4/80hi/CD11cint cells upregulate cell surface CD11b, acquire cathepsin and matrix metalloproteinase activity, and accumulate large numbers of autophagocytic vacuoles; these changes reverse after the induction of remission. Latex bead labeling of peripheral blood Gr1lo monocytes indicates that these are the source of F4/80hi/CD11cint macrophages. CD11chi/MHCIIlo dendritic cells are found in the kidneys only after proteinuria onset, turnover rapidly, and disappear rapidly after remission induction. Gene expression profiling of the F4/80hi/CD11cint population displays increased expression of proinflammatory, regulatory, and tissue repair/degradation-associated genes at nephritis onset that reverses with remission induction. Our findings suggest that mononuclear phagocytes with an aberrant activation profile contribute to tissue damage in lupus nephritis by mediating both local inflammation and excessive tissue remodeling.

Systemic lupus erythematosus (SLE) can affect multiple different organ systems, including the kidneys (lupus nephritis, LN), brain (neuropsychiatric SLE, NPSLE), and skin (cutaneous lupus, CLE). B cells and macrophages are implicated in the pathogenesis of these disease manifestations. We have previously shown the importance of Bruton’s tyrosine kinase (BTK), an enzyme important in B cell and macrophage signaling, in reversing disease in a model of immune nephritis induced by the passive transfer of nephrotoxic antibodies. To extend our findings to a more severe type of nephritis more similar to human disease and examine the effects of BTK inhibition on extra-renal lupus manifestations, we treated a spontaneous model of lupus, MRL/lpr mice, with the novel BTK inhibitor, BI-BTK-1. Early treatment with BI-BTK-1 normalized proteinuria (p MRL/lpr mice develop brain and skin disease that model the pathology of NPSLE and CLE, respectively. Early BI-BTK-1 treatment significantly improved cognitive function and decreased inflammatory cell infiltration into the brain choroid plexus. Moreover, skin disease was also significantly improved as determined by attenuated macroscopic and histologic skin scores. Our results further highlight the therapeutic potential of BI-BTK-1 in SLE, not only in treatment of LN, but potentially also for skin and brain disease as well.

Systemic lupus erythematosus (SLE) is an autoimmune disorder in which loss of tolerance to nucleic acids is associated with the development of pathogenic autoantibodies that damage target organs. Lupus nephritis develops in up to 60% of adult SLE patients and is even more common in children. Induction of remission of lupus nephritis requires the use of potent immunosuppressive treatment with significant adverse effects, and frequent relapses (1). B cells are therapeutic targets in SLE because they produce pathogenic autoantibodies and because they have multiple effector functions including antigen presentation to T cells, cytokine production and migration to sites of inflammation (2). One way to modulate B cell function is by inhibiting the B cell survival molecule BAFF (BLyS). Therapeutic antagonism of BAFF and its homolog APRIL (a proliferation ligand) is based on the discoveries that BAFF provides a crucial homeostatic signal for B cell survival and selection (3–6) and that soluble BAFF and APRIL are highly expressed in the serum of SLE patients (7) and in the target organs of SLE prone mice (8, 9). BAFF binds to three receptors, BAFF-R, TACI and BCMA that are differentially expressed during B cell ontogeny (10), whereas APRIL binds only to TACI and BCMA. Selective blockade of BAFF can be achieved with a soluble BAFF-R-Ig fusion protein or an antibody to BAFF whereas blockade of both BAFF and APRIL is achieved with soluble TACI-Ig. Initial phase 2 and 3 studies of a selective antibody to soluble BAFF (belimumab) were recently completed (11) and studies of TACI-Ig (atacicept) are currently in progress. Questions remain about the mechanism of action of these reagents and about whether blocking both BAFF and APRIL will be more efficacious than blocking BAFF alone. The NZM2410 mouse is an inbred strain derived from NZB/W. NZM2410 mice manifest antibodies to nucleosomes and dsDNA and they develop rapidly progressive glomerulosclerosis with little lymphocytic infiltrate in the kidneys. These mice express high levels of IL-4 and they secrete large amounts of IgG1 antibodies (12). NZM2410 mice have a defect in migration of plasma cells to the bone marrow and retain large numbers of plasma cells in their spleens (13). We therefore hypothesized that disease in these mice might be more responsive to TACI-Ig, that depletes splenic plasma cells (14), than to BAFF-R-Ig. Our study shows that BAFF-R-Ig and TACI-Ig are equally effective at preventing disease and that a short course of either agent induces sustained remission when used as a single therapeutic. This appears to be due to prolonged B cell depletion and a decrease in the inflammatory response to renal immune complex deposition.

Glomerulonephritis is a major cause of morbidity in patients with systemic lupus erythematosus. Although substantial progress has been made in the identification of pathogenic triggers that result in autoantibody production, little is known about the pathogenesis of aggressive proliferative processes that lead directly to irreversible glomerular damage and compromise of renal function. In this study, we describe a model of polyinosinic: polycytidylic acid-accelerated lupus nephritis in NZB/W mice that is characterized by severe glomerular proliferative lesions with de novo crescent formation, findings that are linked with decreased survival and adverse outcomes in lupus. Proliferative glomerulonephritis was associated with infiltrating kidney macrophages and renal expression of IFN-inducible genes, matrix metalloproteinases (MMPs), and growth factors. Crescent formation and renal MMP and growth factor expression were dependent on renal macrophages that expressed Il10 , MMPs, osteopontin , and growth factors, including Pdgfc and Hbegf . Infiltrating macrophages and renal MMP expression were induced by type I IFN. These findings reveal a role for type I IFNs and alternatively activated macrophages in aggressive proliferative lesions of lupus nephritis.

Objective Male (NZW × BXSB)F1 mice develop antiphospholipid syndrome (APS) and proliferative glomerulonephritis that is markedly accelerated by the Yaa locus encoding an extra copy of Tlr7. Female (NZW × BXSB)F1 mice with only 1 active copy of Tlr7 develop late-onset glomerulonephritis but not APS. Because a major function of Toll-like receptor 7 is to induce type I interferons (IFNs), our goal was to determine whether IFNα can induce or accelerate the manifestations of systemic lupus erythematosus (SLE) in female (NZW × BXSB)F1 mice. Methods Eight-week-old female (NZW × BXSB)F1 mice were injected with a single dose of adenovirus expressing IFNα. Mice were monitored for the development of thrombocytopenia and proteinuria. Sera were tested for anticardiolipin and anti-Sm/RNP antibodies. Mice were killed at 17 or 22 weeks of age, and their kidneys and hearts were examined histologically and by immunohistochemistry. Spleen cells were phenotyped, and enzyme-linked immunospot assays for autoantibody-producing B cells were performed. Results IFNα markedly accelerated nephritis and death in female (NZW × BXSB)F1 mice. A significant increase in spleen cell numbers associated with a striking increase in the number of activated B and T cells was observed. Marginal-zone B cells were retained. IFNα-induced increased titers of autoantibodies were observed, but thrombocytopenia was not observed. Cardiac damage was milder than that in male mice. Conclusion IFNα accelerates the development of renal inflammatory disease in female (NZW × BXSB)F1 mice but induces only mild APS and does not induce thrombocytopenia. The effect of IFNα on SLE disease manifestations is strain dependent. These findings are relevant to our understanding of the physiologic significance of the IFN signature.

Systemic lupus erythematosus (SLE) is an autoimmune disease which results in multiple different end organ pathologies, including the kidney. Lupus nephritis (LN) is one of the most serious complications of SLE, and a leading cause of morbidity and mortality. Current treatment options are suboptimal, involving non-specific immunosuppression which exposes patients to potentially serious side effects with no guarantee of remission. More targeted therapeutic approaches may improve treatment results. Many studies have implicated macrophages as actively contributing to LN pathogenesis in both human and murine disease. Indeed, various studies have shown that depletion of macrophage populations, inhibition of macrophage recruitment, and disruption of inflammatory macrophage activation and polarization have significantly ameliorated nephritis in several different murine LN models. The current literature explores targeting macrophages by several different means, including the CSF-1/CSF-1R signaling axis, the CX3CL1/CX3CR1 signaling axis, the CCL2/CCR2 signaling axis, and Bruton's Tyrosine Kinase (BTK), all of which hold promise as targets for future LN treatments. These studies highlight the potential benefit of targeting macrophages in LN, and emphasize the need for future investigations to discern the ideal mean(s) for targeting macrophages in LN.

B cell-activating factor belonging to the TNF family (BAFF) blockade prevents the onset of disease in systemic lupus erythematosus (SLE)-prone NZB/NZW F1 mice. To determine the mechanism of this effect, we administered a short course of TACI-Ig with and without six doses of CTLA4-Ig to 18- to 20-wk-old NZB/NZW F1 mice and evaluated the effect on B and T cell subsets and on anti-dsDNA Ab-producing B cells. Even a brief exposure to TACI-Ig had a beneficial effect on murine SLE; CTLA4-Ig potentiated this effect. The combination of TACI-Ig and CTLA4-Ig resulted in a temporary decrease in serum IgG levels. However, after cessation of treatment, high titers of IgG anti-dsDNA Abs appeared in the serum and IgG Abs deposited in the kidneys. Despite the appearance of pathogenic autoantibodies, the onset of proteinuria was markedly delayed; this was associated with prolonged depletion of B cells past the T1 stage, a decrease in the size of the spleen and lymph nodes, and a decrease in the absolute number of activated and memory CD4+ T cells. TACI-Ig treatment normalized serum levels of IgM that are markedly elevated in NZB/W F1 mice; this appeared to be due to a prolonged effect on the ability of the splenic microenvironment to support short-lived IgM plasma cells. Finally, a short course of combination TACI-Ig and CTLA4-Ig prolonged life and even reversed proteinuria in aged NZB/W F1 mice, suggesting that BAFF blockade may be an effective therapeutic strategy for active SLE.

Lupus nephritis (LN) patients currently lack highly effective and safe treatment options. Bruton’s tyrosine kinase (BTK) is a tyrosine kinase which is important for B cell and macrophage function. Based on preliminary studies in an inducible model of nephritis that highlighted the importance of BTK dependent macrophage effector function in the pathogenesis of disease, we extended our studies to explore the effect of BTK inhibition on remission induction of established, proliferative nephritis. Nephritis was induced in female 129 sv/J mice (10 weeks of age) via the passive transfer of nephrotoxic serum (NTS). Once a mouse developed severe nephritis (>300 mg/dl proteinuria for two consecutive days, or a single measurement of >2000 mg/dl), treatment begun via daily oral gavage with 3 mg/kg of BI-BTK-1, a novel, highly selective and potent BTK inhibitor. Each treated mouse was matched to a control mouse that received daily gavage of the vehicle alone. Within two days of beginning treatment, mice treated with BI-BTK-1 had a significant reversal of the proteinuria compared to vehicle control treated mice (p Therapeutic treatment with BI-BTK-1 reversed severe, established nephritis induced by pathogenic antibodies. These exciting results indicate the novel therapeutic potential of BTK inhibition for treating LN patients with established disease.

The role of three N-linked glycosylation sites in rubella virus (RV) E1 protein on virion release was analyzed by transfecting Vero 76 cells with infectious RV RNA (Robo302WT) containing isoleucine substitutions at N76, N177, and N209 (individually and in combinations). RV RNAs were detected and found to retain substitutions in the transfected cells, but RV capsid indicative of infection was undetectable, except for in Robo302WT and Robo302-N177I transfected cells. Only culture supernatants of Robo302WT and Robo302-N177I RNA transfected cells were positive for RV, suggestive of the virion release into the culture medium. Further, detection of intracellular RV E1 and newly released virion-associated E1 was possible only from cells previously incubated with Robo302-N177I and Robo302WT culture supernatants, suggesting that N177I substituted virus retained infectivity. These results suggest that while glycosylation at N177 is not critical, N76I and N209I mutations are lethal to RV viability.

While reactive oxygen species (ROS) are important for normal cellular activities, deviant production of ROS, or diminished capacity to scavenge excessive ROS, leads to an imbalance in the redox environment of the cell. Because of the dual role of ROS in cells in the production and removal of cellular ROS, a greater understanding of oxidative stress, under both normal and disease-causing conditions, and the involvement of cell organelle (mitochondrial, endoplasmic reticulum) ROS in global regulation of gene expression can illuminate the contribution of mitochondria and other cell organelles in the development of disease and may lead to the advancement of new and novel therapeutic modalities that exploit oxidative stress in treating many diseases. Understanding the role of ROS signaling and redox biology in pathophysiological conditions is reflected by the wised range of topics covered in this special issue. T. Peng et al. underscore dual phase of mitochondrial respiratory chain defective cells harboring less mitochondrial stress due to low mitochondrial respiratory chain activity during mitochondrial ROS-mediated mitochondrial Ca2+ stress during severe oxidative insult. N. V. Gorbunov et al. propose that the cell survival mechanisms activated in lipopolysaccharide-treated mesenchymal stromal cells in vitro could be a part of adaptive responses employed by stromal cells under septic conditions. F. Tseng et al. provided a platform for an in vitro assay to characterize the effects of bone marrow mesenchymal stem cells on lipopolysaccharide-stimulated microglia. A powerful cell culture tool for investigating the molecular and cellular changes in microglia are bone marrow mesenchymal stem cells cocultures. R. Dumitrascu et al. have shown that obstructive sleep apnea is an independent risk factor for cardiovascular disease such as arterial hypertension, heart failure, and stroke. The results clearly show that radical flux exerts direct cytotoxic effects, decreases NO bioavailability, enhances lipid peroxidation, increases sympathetic activity, and activates the proinflammatory transcription factor NF-κB leading to the well-known clinical manifestations of obstructive sleep apnea in the cardiovascular disease system. C. Tronel et al. demonstrated the involvement of Fe2+ in brain ROS production and the deleterious effects of heme-oxygenase-1 expression in vivo neuroinflammatory model linked to a hyperproduction of ROS, itself promoted by Fe2+ liberation. M. Godinez-Rubi et al. have reviewed on the role of nitric oxide donors as possible neuroprotective therapeutic agents for ischemia/reperfusion treatment. S. Whelan and B. S. Zuckerbraun manuscript reviews on the mitochondria signaling to other components of stress response via ROS, the unfolded protein response, mitochondrial autophagy, and biogenesis. The avenues of mitochondrial signaling were discussed in this review. Y. Zhou et al. discuss how ROS regulates different steps in vascular development, including smooth muscle cell differentiation, angiogenesis, endothelial progenitor cells recruitment, and vascular cell migration, while Y. J. H. J. Taverne et al. review focuses on the function of ROS in cardiovascular pathology and on the effects of antioxidants on cardiovascular outcomes with emphasis on the so-called oxidative paradox. A. J. Lepedda et al. suggest the presence of a more pronounced oxidative environment in unstable plaques. Identifying specific oxidative modifications and understanding their effects on protein function could provide further insight into the relevance of oxidative stress in atherosclerosis. E. Menshchikova et al. data appear to indicate a possible role of hydrogen peroxide in intercellular communication during organization, maturation, and “dissociation” of granulomas in the dynamics of the process. X. Zhan et al. study eventually addresses the mechanisms and biological functions of tyrosine nitration in pituitary tumorigenesis and will discover nitro protein biomarkers for pituitary adenomas and targets for drug design for pituitary adenoma therapy. A. V. Ermakov et al. provided in their in vitro data suggesting that the oxidized DNA is a stress signal released in response to oxidative stress in the cultured cells, and, possibly, in the human body; in particular it might contribute to systemic abscopal effects of localized irradiation treatments. M. Tsai et al. study shows that enhanced prostacyclin synthesis reduced glial activation and ameliorated motor dysfunction in hemiparkinsonian rats. Prostacyclin may have a neuroprotective role in modulating the inflammatory response in degenerating nigrastriatal pathway. J. Espino et al. underlie the antioxidant and immune enhancing actions displayed by melatonin, thereby providing evidence for the potential application of this indoleamine as a “replacement therapy” to limit or reverse some of the effects of the changes that occur during immunosenescence. M. Jerkic et al. indicate that eNOS-derived ROS contributes to endothelial dysfunction and likely predisposes to disease manifestations in several organs of hereditary hemorrhagic telangiectasia patients. B. Song et al. review aims are to briefly describe the mechanisms, functional consequences, and detection methods of mitochondrial dysfunction. They describe the advantages and limitations of the Cys-targeted redox proteomics method with alternative approaches. Finally, they discuss various applications of this method in studying oxidatively modified mitochondrial proteins in extrahepatic tissues or different subcellular organelles and translational research. K. E. Al-Otaibiet et al. results suggest a significant role of oxidative stress, proinflammatory myeloperoxidase, and vasoregulatory nitric oxide in the pathogenesis of contrast-induced nephropathy. G. Aliev et al. provide a review discussing the link between cancer and Alzheimer disease via oxidative stress induced by nitric oxide-dependent mitochondrial DNA over proliferation and deletion. S. Miriyala et al. provided a therapeutic approach showing that a novel tetra peptide derivative exhibits in vitro inhibition of neutrophil-derived reactive oxygen species and lysosomal enzymes release. A. S. Kunt and M. H. Andac have shown a clinical study proving that persistent oxidative stress during reperfusion may lead to depressed myocardial function resulting in low cardiac output syndrome necessitating inotropic or intra-aortic balloon counterpulsation support. Besides total antioxidant capacity decreases during operation in a significant proportion of patients undergoing isolated coronary artery bypass which is more prominent and serious. These manuscripts represent an exciting and insightful snapshot of current oxidative stress biology. State of the art, existing challenges and emerging future topics are highlighted in this special issue, which may inspire the reader and help advance the present redox biology.

Background Co-stimulation through the CD40- CD40L axis has been implicated in the pathogenesis of RA including T cell mediated responses, B cell driven autoantibodies, adhesion molecule expression, synovial hyperplasia, and pannus formation, in addition to the secretion of proinflammatory cytokines and MMPs. Circulating CD40L levels are elevated in RA patients versus healthy controls. BI 655064 is a humanized antagonistic anti-CD40 monoclonal antibody which blocks the CD40/ CD40L interaction in in vitro CD40L induced B cell and APC activation assays with IC50 of Objectives To characterize the effect of BI 655064 on cellular and protein biomarkers in RA patients with prior inadequate response to a stable dose MTX ≥15 mg over a 12 wk period of treatment. Methods In this double-blinded, randomized trial RA patients were treated with either 120 mg BI 655064 or placebo q1w for 12 wks as add-on to MTX. Inclusion criteria were ≥6 swollen and ≥6 tender joints, CRP >8 mg/L or ESR>28 mm/1h. The primary efficacy endpoint was ACR20 response at wk 12.Select biomarkers were assessed in whole blood and serum pre and post treatment with BI 655064. Results Treatment with BI 655064 resulted in minimal changes in median %CD19+ B cell population but larger median decreases in select %CD95+ activated B cell subsets, specifically class switched (CD19+IgD-CD27+CD95+), pre-switched (CD19+IgD+CD27+CD95+) and double negative (CD19+IgD-CD27-CD95+) cells and decreases in autoantibodies (IgG-RF and IgA-RF), total IgG and IgM levels in RA patients vs placebo at 12 wks. Median decreases in IL-6 levels (important for B cell and monocyte differentiation) and select bone resorption biomarkers (MMP-3 and RANKL) were also observed in the BI 655064 treated patients vs placebo through 12 wks. Baseline levels of IgG RF, IgM RF, TNFa and COMP correlated with improvement in swollen joint counts at 12 wks and IgA RF levels correlated with improvement in DAS-28 CRP scores at 12 wks in patients treated with BI 655064. Conclusions These results demonstrate that treatment of RA patients with BI 655064 decreases activated B cell subsets, inhibits the production of pathogenic autoantibodies, and reduces the level of circulaiting inflammatory and bone resorption biomarkers at 12 wks. Baseline levels of select biomarkers significantly correlated with clinical response to treatment with BI 655064 at 12 wks. Disclosure of Interest S. Visvanathan Employee of: Boehringer Ingelheim, M. Ramanujam Employee of: Boehringer Ingelheim, C. Schoelch Employee of: Boehringer Ingelheim, R. Vinisko Employee of: Boehringer Ingelheim, U. Mueller-Ladner Consultant for: Boehringer Ingelheim, S. Padula Employee of: Boehringer Ingelheim, J. Fine Employee of: Boehringer Ingelheim, J. Steffgen Employee of: Boehringer Ingelheim

Targeting the CD40-CD154 pathway to induce transplantation tolerance has been of interest to transplantation immunologists for over two decades. The efficacy of anti-CD154 antibody (Ab) observed in murine and non-human primate models for preventing allograft rejection and suppressing autoimmunity, generated great interest in the development of this antibody for clinical use. However, clinical trials with anti-CD154 Ab resulted in thromboembolic complications in patients as a result of CD154 expression by human platelets and limited the clinical application. To develop an alternative approach to anti-CD154 Ab, recent efforts have focused on targeting its receptor, CD40. In this study we evaluated the effects of a novel murine antagonistic antibody to CD40 (BI CD40-1) on alloreactive immune responses. Costimulation blockade consisting of donor splenocytes (DST) and BI CD40-1 reduced activation of alloreactive CD8 T cells as shown by inhibition of expansion, decreased cytokine production and decreased killing of allogeneic targets using an in vivo cytotoxicity assay. Moreover DST/BI CD40-1 treatment significantly prolonged skin graft survival in a BALB/c to B6 MHC mismatch model. The efficacy of BI CD40-1 in preventing alloreactive T cell responses was similar to anti-CD154 Ab. These results indicate that anti-CD40 Ab is an attractive candidate for translation to clinical application.

Targeting CD40-CD40L interactions has been an interesting drug concept for the treatment of autoimmune diseases given this pathway’s role in the development of both humoral and cell mediated immune responses. While preclinical blockade of CD40L is well documented in various autoimmune animal models of disease, limited data exists for CD40 blockade due to lack of a truly antagonistic anti-mouse CD40 tool antibody (Ab). Here we describe the in vitro and in vivo characterization of a fully antagonistic anti-mouse CD40 monoclonal Ab (BI CD40-1); a chimeric rat Fv anti-mouse CD40 mAb engineered with a mouse IgG2a Fc containing mutations to abrogate Fcγ receptor binding. BI CD40-1 blocks molecular CD40-CD40L interactions (IC50 = 0.25 nM) and exhibits potent binding to CD40 expressed on mouse B cells (EC50 = 0.42 nM ± 0.08). In vitro profiling of BI CD40-1 using a mouse splenocyte proliferation assay confirmed potent antagonistic activity (IC50 = 0.27 nM ± 0.09) as well as absence of any agonistic properties (stimulation index < 2 @ 67 nM). Administration of BI CD40-1 to mice prior to ovalbumin (OVA) immunization resulted in dose-dependent blockade of OVA-specific IgG responses (100, 100, 74, 0% inhibition at 10, 3, 1, and 0.3 MPK; day 13) correlating with similar dose dependent receptor occupancy and inhibition of B cell activation as measured by ex vivo CD54 upregulation. Prophylactic dosing in cGVHD model of scleroderma showed dose dependent protection in disease development as seen by decreased dermal thickness, myofibroblast counts and collagen deposition. BI CD40-1 represents a novel fully antagonistic anti-mouse CD40 mAb, an important tool for mechanistic understanding of the therapeutic value of targeting of CD40 in inflammatory diseases.

Bruton's tyrosine kinase (BTK) plays a critical role in B cell development and function. We recently described a selective BTK inhibitor, RN486, that blocks B cell receptor (BCR) and Fcγ receptor signaling and is efficacious in animal models of arthritis. The aim of this study was to examine the potential efficacy of BTK in systemic lupus erythematosus (SLE), using an NZB × NZW mouse model of spontaneous SLE.Mice received RN486 or its vehicle (administered in chow) at a final concentration of 30 mg/kg for 8 weeks, starting at 32 weeks of age.The administration of RN486 completely stopped disease progression, as determined by histologic and functional analyses of glomerular nephritis. The efficacy was associated with striking inhibition of B cell activation, as demonstrated by a significant reduction in CD69 expression in response to BCR crosslinking. RN486 markedly reduced the secretion of IgG anti-double-stranded DNA (anti-dsDNA) secretion, as determined by enzyme-linked immunosorbent and enzyme-linked immunospot assays. Flow cytometric analysis demonstrated depletion of CD138(high) B220(low) plasma cells in the spleen. RN486 inhibited secretion of IgG anti-dsDNA but not IgM anti-dsDNA, suggesting that pharmacologic blockade of BTK resembles the reported transgenic expression of low levels of endogenous BTK in B cells. In addition, RN486 may also impact the effector function of autoantibodies, as evidenced by a significant reduction in immune complex-mediated activation of human monocytes in vitro and down-regulation of the expression of macrophage-related and interferon-inducible genes in both the kidneys and spleens of treated mice.Collectively, our data suggest that BTK inhibitors may simultaneously target autoantibody-producing and effector cells in SLE, thus constituting a promising therapeutic alternative for this disease.

TNFα (TNF) is an important regulator of physiologic and inflammatory immune responses. Although TNF antagonists have remarkable therapeutic benefits in several autoimmune diseases including rheumatoid arthritis, psoriasis and Crohn’s disease, they also have been associated with the development of anti-nuclear antibodies and even clinical SLE, multiple sclerosis and other demyelinating diseases. TNF is protective in the early stages of SLE in some mouse models but is overexpressed in inflamed target organs in both mice and humans with SLE (1–2). Consistent with both protective and pro-inflammatory roles of TNF, blockade of TNF improved proteinuria in patients with refractory SLE nephritis but increased anti-DNA and anti-cardiolipin autoantibody titers (3). In this study, we determined the clinical effects of TNFR2-Ig in a mouse model of lupus nephritis in which an increase in serum levels of TNF and renal production of TNF occurs concomitantly with the onset of renal disease. TNF inhibition introduced after the development of autoantibodies and at the onset of clinical nephritis stabilized the nephritis and resulted in a long period of relapsing and remitting proteinuria that was associated with a marked improvement in survival. Mechanistic studies revealed that there was no effect of TNF inhibition on autoantibody production or lymphocyte activation in the spleen or renal autoantibody deposition, however there was a marked decrease in renal periglomerular and interstitial accumulation of F4/80hi renal macrophages. Real-time PCR analysis revealed a significant decrease in renal expression of chemokines CCL2, CCL5 and CCL9, the endothelial activation marker VCAM-1, genes involved in tissue remodeling and the markers of proximal tubule damage lipocalin-2 and HAVCR1 (KIM-1). We conclude that TNF inhibition decreases the renal inflammatory response to immune complex deposition. This is associated with decreased accumulation of periglomerular and interstitial renal macrophages that play a role in tissue remodeling, and decreased damage of renal tubular cells. In contrast, perivascular aggregates containing CD11chi dendritic cells as well as B and T cells accumulate in the kidneys in a TNF independent manner and are not sufficient to induce terminal renal damage.

Antiphospholipid syndrome (APS) is a disease manifested by dysregulated clotting in both the arterial and venous systems and is caused by antibodies to phospholipids and their binding protein β2-glycoprotein I (1). The autoantibodies that cause APS are unresponsive to conventional immunosuppression, which suggests that they may be derived from a long-lived B cell subset. The (NZW × BXSB)F1 mouse is a murine model of spontaneous APS. The male offspring of this cross produce anticardiolipin antibodies (aCL) and anti-RNP antibodies by 12 weeks of age, and subsequently develop immune-mediated thrombocytopenia, inflammatory glomerulonephritis (GN), and a thrombotic vasculopathy of the small coronary arteries, which leads to myocardial infarcts, myocardial fibrosis, and a dilated cardiomyopathy. Full-thickness infarcts affecting the right ventricle are seen in the late stages of disease (2,3). APS in (NZW × BXSB)F1 mice is dependent upon both B and T cells and development of autoantibodies. Thrombocytopenia and nephritis are completely prevented by CD4 T cell depletion before 6–8 weeks of age (4). We have shown that the B7/CD28 antagonist CTLA-4Ig blocks class switching of antiphospholipid antibodies (aPL) to the IgG isotype and prevents both nephritis and coronary thromboses when given at 8 weeks of age, before the emergence of autoantibodies (5). However, administration of CTLA-4Ig at 12 weeks of age, after the development of autoantibodies, no longer prevents the disease (5). These findings indicate that the pathogenetic B cells in (NZW × BXSB)F1 mice arise in a CD4 T cell– and CD28-dependent manner but that B7/CD28 interactions are not required for disease perpetuation. Male (NZW × BXSB)F1 mice bear the Yaa gene, a reduplicated segment of the Y chromosome containing the genes for Toll-like receptor 7 (TLR-7) and TLR-8, intracellular single-stranded RNA–sensing TLRs expressed by plasmacytoid dendritic cells (DCs) and B cells (6). Activation of TLR-7 in plasmacytoid DCs induces the release of type I interferons that stimulate myeloid DCs to release BAFF, a tumor necrosis factor (TNF)–like cytokine that is crucial for B cell survival (for review, see refs. 7 and 8). BAFF expands the B cell compartment, leading to increased production of immune complexes that can further activate TLRs (9). We therefore hypothesized that BAFF blockade may be a successful therapeutic approach for APS, even in the later stages of disease. BAFF and its homologous molecule APRIL interact with 3 different BAFF receptors on B cells: TACI, BAFF-R, and BCMA. These interactions can be blocked by soluble Ig fusion proteins of the BAFF receptors TACI or BAFF-R. BAFF-R-Ig selectively blocks only BAFF, whereas TACI-Ig blocks both BAFF and APRIL (for review, see ref. 10). We found that blockade of BAFF alone is sufficient to prevent disease both in the early and late initiation stages.

Systemic lupus erythematosus (SLE) is a complex immune disorder in which loss of tolerance to nucleic acid antigens and other crossreactive antigens is associated with the development of pathogenic autoantibodies that damage target organs, including the skin, joints, brain and kidney. New drugs based on modulation of the immune system are currently being developed for the treatment of SLE. Many of these new therapies do not globally suppress the immune system but target specific activation pathways relevant to SLE pathogenesis. Immune modulation in SLE is complicated by differences in the immune defects between patients and at different disease stages. Since both deficiency and hyperactivity of the immune system can give rise to SLE, the ultimate goal for SLE therapy is to restore homeostasis without affecting protective immune responses to pathogens. Here we review recent immunological advances that have enhanced our understanding of SLE pathogenesis and discuss how they may lead to the development of new treatment regimens.

Background Costimulatory molecule pair CD40-CD40L plays a central role not only in immune cell interactions but also in immune-non immune cell activation. Targeting this pathway has generated great interest but early attempts to target CD40L failed mainly due to thrombotic complications observed in the clinic. In addition, developing a potent truly antagonistic CD40 antibody has proven to be challenging. Objectives Here we describe the preclinical characterization of BI 655064, an anti-human CD40 mAb that is being developed for the treatment of autoimmune disorders. BI 655064 is engineered as a human IgG1 molecule with a mutated Fc region to abrogate effector function. Methods Binding of BI 655064 to CD40 expressed on primary B cells was measured by flow cytometry. The ability of BI 655064 to block CD40L-induced B cell proliferation in vitro was measured by tritium uptake, while blockade of endothelial and DC activation was measured by inhibition of cytokine release. A subcutaneous PK/PD study in the cynomolgus monkey was performed to establish correlations between BI 655064 exposure, target coverage using a receptor occupancy assay and pharmacodynamic effects using an ex vivo CD54-induction assay. In vivo blockade of B cell function was also tested in a human-peripheral blood lymphocyte (huPBL) induced SCID mouse model of graft-versus-host disease (GvHD) and in cynomolgus monkeys immunized with keyhole limpet hemocyanin (KLH). Results In human whole blood, BI 655064 binds to CD40 on B cells (EC 90 of 6.85 nM ±0.74). Blockade of CD40L-induced B cell proliferation was observed at an average IC 50 of 0.4 nM. Inhibition of CD40L mediated cytokine release was observed in DCs (IC 50 =0.25 nM and 0.59 nM for TNFα and IL12/23p40, respectively) and to verify effects beyond immune cells, BI655064 was tested in CD40L stimulated endothelial cell cultures. Binding of BI 655064 to human platelets did not induce or augment platelet activation as determined by in vitro induction of CD62P. In the PK/PD study, cynomolgus monkeys dosed with greater than 1 mg/kg of BI 655064 exhibited complete blockade of ex vivo CD54 upregulation corresponding to full CD40 target coverage on B cells. In vivo , BI 655064 demonstrated clear effects on B cell function in the GvHD model where both human IgM and IgG responses were completely abrogated. As part of a repeat dose tolerability study, BI 655064 given to cynomolgus monkeys prior to immunization with KLH resulted in inhibition of KLH-specific IgM and IgG antibody responses. At doses of 5 mg/kg and above, germinal center size was decreased microscopically in Peyer9s patches, lymph nodes, spleens and tonsils in treated monkeys. Evaluation of platelet function in cynomolgus monkeys dosed with BI 655064 did not reveal any effects on ex vivo platelet activation or aggregation. Conclusions BI 655064 is a humanized antagonistic anti-CD40 mAb which is to be tested in human clinical trials for autoimmune disorders to establish safety and efficacy. BI 655064 demonstrated relevant pharmacologic in vitro and in vivo activity, blocking CD40-related functions at clinical dosing levels of >1 mg/kg in animal models. Disclosure of Interest K. Ralph Employee of: Boehringer Ingelheim Pharmaceuticals, A. Nicoletti Employee of: Boehringer Ingelheim Pharmaceuticals, E. Musvasva Employee of: Boehringer Ingelheim Pharmaceuticals, S. Cannan Employee of: Boehringer Ingelheim Pharmaceuticals, S. VanTongeren Employee of: Boehringer Ingelheim Pharmaceuticals, D. Blanset Employee of: Boehringer Ingelheim Pharmaceuticals, S. Brodeur Employee of: Boehringer Ingelheim Pharmaceuticals, J. Ahlberg Employee of: Boehringer Ingelheim Pharmaceuticals, H. Li Employee of: Boehringer Ingelheim Pharmaceuticals, S. Fogal Employee of: Boehringer Ingelheim Pharmaceuticals, S. Desai Employee of: Boehringer Ingelheim Pharmaceuticals, K. O9Shea Employee of: Boehringer Ingelheim Pharmaceuticals, R. Kroe-Barrett Employee of: Boehringer Ingelheim Pharmaceuticals, E. Mainolfi Employee of: Boehringer Ingelheim Pharmaceuticals, G. Nabozny: None declared, H. Wu Employee of: Boehringer Ingelheim Pharmaceuticals, G. Hansen Employee of: Boehringer Ingelheim Pharmaceuticals, K. Canada Employee of: Boehringer Ingelheim Pharmaceuticals, S. Singh Employee of: Boehringer Ingelheim Pharmaceuticals, X. Zhu Employee of: Boehringer Ingelheim Pharmaceuticals, M. Ramanujam Employee of: Boehringer Ingelheim Pharmaceuticals, C. Grimaldi Employee of: Boehringer Ingelheim Pharmaceuticals

B cells have multiple roles in immune activation and inflammation separate from their capacity to produce antibodies. B cell depletion is currently under intense investigation as a therapeutic strategy for autoimmune diseases. The TNF family members B cell-activating factor of the TNF family (BAFF) and its homolog A proliferation-inducing ligand (APRIL) are B cell survival and differentiation factors and are therefore rational therapeutic targets. We compared the effects of BAFF receptor-Ig, which blocks only BAFF, with those of transmembrane activator and calcium modulator ligand interactor-Ig, which blocks both BAFF and APRIL, in a murine SLE model. Both reagents prolonged the life of NZB/W F1 mice when given either before or after disease onset. Many immunologic effects of the 2 reagents were similar, including B cell and B cell subset depletion and prevention of the progressive T cell activation and dendritic cell accumulation that occurs with age in NZB/W mice without substantial effects on the emergence of the IgG anti-double-stranded DNA response. Furthermore, both reagents inhibited the T cell-independent marginal zone B cell response to particulate antigen delivered i.v., but not the B1 B cell response to the same antigen delivered i.p. In contrast, blockade of both BAFF and APRIL, but not blockade of BAFF alone, reduced the serum levels of IgM antibodies, decreased the frequency of plasma cells in the spleen, and inhibited the IgM response to a T cell-dependent antigen. The differences between selective and nonselective BAFF blockade are relevant to the choice of a BAFF blocking agent for the treatment of autoimmune and malignant diseases.

NZW “ x ” BXSB F1 mice develop SLE that is associated with an anti-phospholipid syndrome characterized by anti-cardiolipin antibodies, thrombocytopenia and small coronary artery thrombosis. This syndrome is immune mediated and, dependent, on CD4+T cells. To determine whether disease in these mice can be treated with blockade of T cell costimulation we treated them with the CD28 antagonist CTLA4Ig at 9 or 12 weeks of age. CTLA4Ig completely prevented both SLE nephritis and myocardial infarcts if it was given at 9 weeks of age, before anti-cardiolipin antibodies could be detected in the serum and prevented both B cell expansion and activation and the development of peripheral monocytosis. If treatment was delayed until 12 weeks of age after cardiolipin antibodies had arisen but before the onset of clinical disease, CTLA4Ig had very little effect on disease progression. These findings indicate that CD4+T cell activation through CD28 is critical for disease initiation in this model but plays little role in disease progression or tissue damage. These findings have relevance to the treatment of anti-phospholipid syndrome in humans.

Although the life span of patients with systemic lupus erythematosus (SLE) has improved considerably over the last several decades, the toxicities of chronic immunosuppressive therapy are major causes of morbidity and mortality. Safer and more effective therapies for SLE are clearly needed. SLE is characterized by excessive activation of both B and T lymphocytes. Activation of these cells requires both antigen engagement and co-stimulatory signals from interacting lymphocytes (Carreno, B.M. M. Collins, 2002, Annu. Rev. Immunol. 20: 29-53; Grewal, I.S. R.A. Flavell, 1998, Annu. Rev. Immunol. 16: 111-135). Thus, blockade of co-stimulatory signals offers a new therapeutic approach to SLE. Our short-term goal has been to understand the effect of co-stimulatory blocking reagents on the development, selection, and activation of pathogenic anti-dsDNA antibody producing B cells in mice genetically pre-determined to develop SLE and showing signs of either early or advanced disease activity. Our long-term goal is to use the knowledge we gain to design therapeutic regimens for humans that avoid the complications of long-term immunosuppression. As new co-stimulatory molecules are discovered, studying their mechanism of action in animal models and their clinical utility in human autoimmune disease should lead both to a new understanding of disease pathogenesis and also to safer and more effective therapies.

Antibodies to dsDNA are specific to SLE and are pathogenic, both due to their ability to deposit in tissues through a variety of mechanisms, and to their ability, when present in immune complexes, to activate inflammatory cells. The relationship of serum anti-dsDNA antibody levels to disease activity is a complex one and the factors that determine whether or not such antibodies will be pathogenic in an individual SLE patient are incompletely understood. Although anti-dsDNA antibodies can be made by naüve B cells and B cells belonging to the B1 and marginal zone subsets, pathogenic anti-dsDNA antibodies have the hallmarks of germinal center development and exposure to T cell help, including accumulation of somatic mutations and class switching to the IgG isotype. Epitope spreading may result in aquisition of cross-reactivities with multiple target organ antigens and aquisition of a memory phenotype will allow these B cells to acquire antigen presentation functions that amplify the autoreactive response. In the early stages of disease, or after remission induction protocols, autoreactive B cells may be susceptible to treatments that target T cell costimulation or that deplete or tolerize naüve and mature B cells. Therapeutic approaches targeting innate immune responses or regulatory T cells are starting to be tested in pre-clinical models. In later disease stages, memory and plasma cell accumulation may render patients more resistant to this type of therapeutic approach. Deposition of anti-dsDNA antibodies in target tissues can stimulate an inflammatory cascade that leads to tissue damage. A number of murine models have now been developed that show that interruption of this cascade can prevent or reverse such damage. This type of approach may be beneficial for individuals with established disease. As we learn more about the specific defects that cause SLE, it may become possible to individualize therapy based on patient specific biologic markers.