Autoantibodies targeting α6β4 integrin have been identified in individual patients with mucous membrane pemphigoid (MMP). Reactivity against α6 integrin has been associated with oral lesions, while anti-β4 integrin reactivity has been linked to ocular involvement. However, the pathogenic effects of these antibodies have not been fully elucidated. Here, we investigated the pathogenic potential of anti-α6 and anti-β4 integrin IgG both in vitro and in vivo. Immune complexes of anti-α6 and anti-β4 integrin induced the release of reactive oxygen species from normal human leukocytes and stimulated CXCL2 secretion in cultured murine C5N keratinocytes. In vivo, repeated injections of IgG against a recombinant fragment of β4 integrin into C57BL/6 mice led to palpebral conjunctival swelling and mild oral lesions. The latter was observed following injection of IgG against a recombinant fragment of α6 integrin. Histopathological analysis revealed subepithelial inflammatory infiltrates without evidence of split formation. Direct immunofluorescence microscopy showed linear deposits of IgG at the basement membrane zone in most tissues, whereas C3 deposition was largely absent. This lack of complement activation was corroborated by a complement fixation assay, which confirmed that IgG against α6 and β4 integrin failed to induce C3 deposition in normal murine conjunctivae, buccal mucosa, or skin. Collectively, these findings indicate that IgG autoantibodies against α6 and β4 integrin exhibit pathogenic activity in vitro and induce mild disease in vivo, possibly due in part to relatively inefficient complement activation in this model.

Rheumatoid arthritis is characterized by chronic synovial inflammation and recurrent flares, which are thought to involve joint-specific inflammatory memory. Synovial fibroblasts (SFs) contribute to this process through altered responses to repeated inflammatory stimulation. However, pharmacological targeting of these state-dependent responses remains limited, partly due to the lack of screening systems that distinguish responses under initial and repeated stimulation conditions. Here, we established a quantitative in vitro system using primary murine SFs. Repeated tumor necrosis factor-α stimulation enhanced interleukin-6 (IL-6) production, recapitulating key features previously reported in human SFs. This system enabled direct comparison of drug effects under single and repeated stimulation conditions, revealing distinct response patterns and identifying compounds that selectively modulate the enhanced response induced by repeated stimulation. Notably, a complement receptor antagonist attenuated the additional increase in IL-6 production observed after repeated stimulation, accompanied by the attenuation of glycolysis-related gene expression, while showing no significant effect under single stimulation conditions. These findings highlight the importance of state-dependent pharmacological evaluation and demonstrate the utility of this experimental framework for analyzing differential responses associated with repeated inflammatory stimulation.

The molecular details of macrophage-fibroblast crosstalk during the onset and resolution of inflammatory disease remain incompletely understood. Here, we apply a bioinformatic modeling approach based on single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin using sequencing to map heterocellular signaling circuits of synovial macrophage and synovial fibroblast (SF) subsets during various stages of inflammatory arthritis. While SFs function as key pacemakers of synovial inflammation, individual subsets of synovial macrophages support both the perpetuation and the resolution of arthritis. Pro-inflammatory Il1b+ macrophages dominate the early stages of inflammation and retain a substantial intrinsic plasticity that is characterized by chromatin remodeling and an eventual differentiation into Spp1+ macrophages. These cells display a terminally differentiated phenotype, suppress the activation of pro-inflammatory SFs, and initiate the resolution of arthritis by secretion of regulatory mediators, including osteopontin. Our data highlight the dichotomous character of macrophage-fibroblast crosstalk and define the cellular and molecular checkpoints that control the onset and resolution of immune-mediated inflammatory diseases.

Tumor-associated macrophages (TAMs) dynamically influence anti-tumor immunity. Understanding TAM function is therefore critical to design immunotherapies. By combining syngeneic models of colorectal and pancreatic cancer with cell type-specific deletion of the epithelial-to-mesenchymal transition driver Zeb1, which is expressed in subsets of TAMs, we discovered that ZEB1 is an intrinsic regulator of TAM-controlled T cell trafficking and anti-tumor immune responses. ZEB1 supports secretion of a subset of chemokines via the constitutive pathway, including CXCL10, CCL2 and CCL22, by regulating their biosynthesis, vesicular transport and release. This elevates cytotoxic T cell (CTL) recruitment in vitro and fosters immunosurveillance by CTLs in tumors and metastases as well in an organotypic model for therapeutic CD8 + T cell addition. Our study identifies ZEB1 in TAMs as a facilitator of anti-tumor immunity, suggests a window of opportunity for cytokine-guided CTL tropism and reinforces the importance of onco-immunological context, particularly in the design of macrophage- and/or cytokine-depleting strategies.

The 5th International Symposium on Regulatory Autoantibodies Targeting GPCR (RAB-GPCRs) advanced the understanding of the significant role played by autoantibodies targeting G-protein-coupled receptors (GPCRs) in various human diseases. Once considered passive markers, RAB-GPCRs are now recognized as active modulators of cellular signaling, immune regulation, and inflammation. The symposium highlighted their involvement in multiple prominent pathologies, including autoimmune diseases, cardio- and cerebrovascular diseases, and neuroimmunologic disorders such as myalgic encephalomyelitis/chronic fatigue syndrome and post-COVID-19 syndrome (ME/CFS/PCS), as well as solid organ and hematopoietic stem cell transplantation (SOT/HSCT). Experts from rheumatology, immunology, and neurology presented interdisciplinary discussions on the potential of RAB-GPCRs as biomarkers and therapeutic targets. Advances in screening methods, biomarker identification, and therapeutic strategies were shared, emphasizing their diagnostic potential and application in novel therapeutic interventions. This report summarizes key insights from the symposium, particularly focusing on the modulatory properties of RAB-GPCRs and their relevance in both immune-mediated diseases and other pathologies (e.g., vascular, degenerative) that are traditionally not considered primarily immune-mediated. Ongoing research is expected to further establish these autoantibodies as crucial components in disease modulation and systems biology contexts, offering new opportunities for precision medicine and improved clinical outcomes in immune-related disorders.

Background: Chronic inflammatory diseases (CIDs) are characterized by hyperactivated local tissue. This tissue sensitization is caused by tissue-resident structural cells such as fibroblasts or epithelial cells that become metabolically and functionally primed during frequent or long-lasting episodes of inflammation. This cellular maladaptation is often driven by the activation of intracellular complement components (also known as the complosome), which consequently metabolically invigorates cells and permits them to fully utilize their inflammatory potential. Methods: Primary human synovial fibroblasts (SFs) derived from the joints of individuals with established rheumatoid arthritis (RA) or SFs from healthy joints primed by repeated in vitro-stimulation with TNFα and ARPE-19 retinal epithelial cells were treated with compstatin derivatives. Uptake and subcellular localization of compstatins were analyzed by confocal microscopy employing antibodies against compstatin derivatives and cell organelles. Functional and metabolic profiling was performed in the presence or absence of human serum (providing an external source of C3) and with/without a C3-blocking antibody. Compstatin-induced transcriptomic changes in SFs were assessed by bulk RNA-Seq and downstream pathway analysis. Results: Compstatins were readily taken up into fibroblasts and epithelial cells and partially colocalized with intracellular C3 in endosomes, autophagsosomes, and the endoplasmatic reticulum. Compstatins inhibited fibroblast function associated with inflammatory tissue priming in arthritis, such as adherence, matrix invasion, cytokine production, and migration. Gene set enrichment analysis suggested a compstatin-induced reduction of SF proliferation and activation, and changes in polyamines and cellular metabolism, which were reflected by metabolic flux analyses. In ARPE-19 cells, compstatins inhibited production of reactive oxygen species and generation of C3a. Antibody-mediated blockade of cell-secreted C3 inhibited uptake of compstatins and their capacity to blunt pathogenic cellular function. Conclusion: Peptide-based C3- targeting therapeutics such as compstatins are attractive candidates for regulation of overshooting tissue inflammation in CIDs such as Rheumatoid arthritis and Age-related macular degeneration. Locally administered compstatins would block the activation of endogenously generated C3 in tissue-occupying cells, thus inhibiting or rolling back inflammatory maladaptation of tissues.

Autoimmune blistering diseases (AIBDs) involve autoantibodies targeting proteins in the epidermal/epithelial desmosome (pemphigus) or basement membrane zone (pemphigoid). Despite widespread antigen distribution, lesions exhibit a scattered involvement pattern. This study maps the frequency/severity of AIBD lesions on various body parts and investigates whether differential antigen expression contributes to specific predilection sites. We analyzed affected sites presenting blisters/erosions, erythematous/urticarial lesions, and mucosal lesions in bullous pemphigoid (BP-cohort 1, n = 65; BP-cohort 2, n = 119), pemphigus vulgaris (PV, n = 67), and pemphigus foliaceus (PF, n = 20) patients. To assess antigen expression, we conducted indirect immunofluorescence (IF) staining of 11 AIBD antigens from 13 anatomical sites of 10 body donors without AIBD. In BP, blisters/erosions and erythematous/urticarial lesions predominantly affected arms and legs, while PV/PF patients exhibited frequent involvement of buccal mucosa and back, respectively. IF staining identified significant regional differences in BP180, BP230, and integrin β4 expression, although these variations did not correlate with a higher lesion frequency/severity. Other antigens showed consistent expression across all regions. Our findings suggest that predilection sites for BP and PV/PF are largely unaffected by regional variations in antigen expression but may be influenced by factors like microbiota, mechanical stress, sunlight exposure, local immunity, or genetics.

Tumor-associated macrophages (TAMs) shape the tumor microenvironment (TME) and exert a decisive impact on anti-tumor immunity. Understanding TAM function is therefore critical to understand anti-tumor immune responses and to design immunotherapies. Here, we describe the transcription factor ZEB1, a well-known driver of epithelial-to-mesenchymal transition, as an intrinsic regulator of TAM function in adaptive anti-tumor immunity. By combining cell type-specific deletion of Zeb1 with syngeneic models of colorectal and pancreatic cancer, we discovered an unexpected function of ZEB1 in the TAM-mediated control of T cell trafficking. ZEB1 supports secretion of a subset of chemokines including CCL2 and CCL22 by promoting their transcription and translation as well as by safeguarding protein processing. ZEB1 thereby elevates cytotoxic T cell (CTL) recruitment in vitro and in vivo and fosters immunosurveillance during tumor as well as lung metastatic outgrowth. Our study spotlights ZEB1 as a crucial facilitator of adaptive anti-tumor immunity and uncovers a potential therapeutic window of opportunity for cytokine-guided enhancement of CTL infiltration into tumors and metastases.### Competing Interest StatementThe authors have declared no competing interest.

Due to optimised treatment strategies and the availability of new therapies during the last decades, formerly devastating chronic inflammatory diseases such as rheumatoid arthritis or systemic sclerosis (SSc) have become less menacing. However, in many patients, even state-of-the-art treatment cannot induce remission. Moreover, the risk for flares strongly increases once anti-inflammatory therapy is tapered or withdrawn, suggesting that underlying pathological processes remain active even in the absence of overt inflammation. It has become evident that tissues have the ability to remember past encounters with pathogens, wounds and other irritants, and to react more strongly and/or persistently to the next occurrence. This priming of the tissue bears a paramount role in defence from microbes, but on the other hand drives inflammatory pathologies (the Dr Jekyll and Mr Hyde aspect of tissue adaptation). Emerging evidence suggests that long-lived tissue-resident cells, such as fibroblasts, macrophages, long-lived plasma cells and tissue-resident memory T cells, determine inflammatory tissue priming in an interplay with infiltrating immune cells of lymphoid and myeloid origin, and with systemically acting factors such as cytokines, extracellular vesicles and antibodies. Here, we review the current state of science on inflammatory tissue priming, focusing on tissue-resident and tissue-occupying cells in arthritis and SSc, and reflect on the most promising treatment options targeting the maladapted tissue response during these diseases.

T lymphocytes and myeloid cells express the immunoglobulin-like glycoprotein cluster of differentiation (CD)101, notably in the gut. Here, we investigated the cell-specific functions of CD101 during dextran sulfate sodium (DSS)-induced colitis and Salmonella enterica Typhimurium infection. Similar to conventional CD101-/- mice, animals with a regulatory T cell-specific Cd101 deletion developed more severe intestinal pathology than littermate controls in both models. While the accumulation of T helper 1 cytokines in a CD101-deficient environment entertained DSS-induced colitis, it impeded the replication of Salmonella as revealed by studying CD101-/- x interferon-g-/- mice. Moreover, CD101-expressing neutrophils were capable to restrain Salmonella infection in vitro and in vivo. Both cell-intrinsic and -extrinsic mechanisms of CD101 contributed to the control of bacterial growth and spreading. The CD101-dependent containment of Salmonella infection required the expression of Irg-1 and Nox2 and the production of itaconate and reactive oxygen species. The level of intestinal microbial antigens in the sera of inflammatory bowel disease patients correlated inversely with the expression of CD101 on myeloid cells, which is in line with the suppression of CD101 seen in mice following DSS application or Salmonella infection. Thus, depending on the experimental or clinical setting, CD101 helps to limit inflammatory insults or bacterial infections due to cell type-specific modulation of metabolic, immune-regulatory, and anti-microbial pathways.

Caloric restriction and intermittent fasting prolong the lifespan and healthspan of model organisms and improve human health. The natural polyamine spermidine has been similarly linked to autophagy enhancement, geroprotection and reduced incidence of cardiovascular and neurodegenerative diseases across species borders. Here, we asked whether the cellular and physiological consequences of caloric restriction and fasting depend on polyamine metabolism. We report that spermidine levels increased upon distinct regimens of fasting or caloric restriction in yeast, flies, mice and human volunteers. Genetic or pharmacological blockade of endogenous spermidine synthesis reduced fasting-induced autophagy in yeast, nematodes and human cells. Furthermore, perturbing the polyamine pathway in vivo abrogated the lifespan- and healthspan-extending effects, as well as the cardioprotective and anti-arthritic consequences of fasting. Mechanistically, spermidine mediated these effects via autophagy induction and hypusination of the translation regulator eIF5A. In summary, the polyamine-hypusination axis emerges as a phylogenetically conserved metabolic control hub for fasting-mediated autophagy enhancement and longevity.

Introduction:Typical Western diet, rich in salt, contributes to autoimmune disease development. However, conflicting reports exist about the effect of salt on neutrophil effector functions, also in the context of arthritis.Methods:We investigated the effect of sodium chloride (NaCl) on neutrophil viability and functions in vitro, and in vivo employing the murine K/BxN-serum transfer arthritis (STA) model.Results and discussion:The effects of NaCl and external reactive oxygen species (H2O2) were further examined on osteoclasts in vitro. Hypertonic sodium-rich media caused primary/secondary cell necrosis, altered the nuclear morphology, inhibited phagocytosis, degranulation, myeloperoxidase (MPO) peroxidation activity and neutrophil extracellular trap (NET) formation, while increasing total ROS production, mitochondrial ROS production, and neutrophil elastase (NE) activity. High salt diet (HSD) aggravated arthritis by increasing inflammation, bone erosion, and osteoclast differentiation, accompanied by increased NE expression and activity. Osteoclast differentiation was decreased with 25 mM NaCl or 100 nM H2O2 addition to isotonic media. In contrast to NaCl, external H2O2 had pro-resorptive effects in vitro. We postulate that in arthritis under HSD, increased bone erosion can be attributed to an enhanced oxidative milieu maintained by infiltrating neutrophils, rather than a direct effect of NaCl.

Bromodomain- and extra-terminal domain (BET) proteins are epigenetic reader proteins that regulate transcription of their target genes by binding to acetylated histone side chains. Small molecule inhibitors, such as I-BET151, have anti-inflammatory properties in fibroblast-like synoviocytes (FLS) and in animal models of arthritis. Here, we investigated whether BET inhibition can also affect the levels of histone modifications, a novel mechanism underlying BET protein inhibition. On the one hand, FLSs were treated with I-BET151 (1 µM) for 24 h in absence and presence of TNF. On the other hand, FLSs were washed with PBS after 48 h of I-BET151 treatment, and the effects were measured 5 days after I-BET151 treatment or after an additional 24 h stimulation with TNF (5 d + 24 h). Mass spectrometry analysis indicated that I-BET151 induced profound changes in histone modifications, with a global reduction in acetylation on different histone side chains 5 days after treatment. We confirmed changes on acetylated histone side chains in independent samples by Western blotting. I-BET151 treatment reduced mean TNF-induced levels of total acetylated histone 3 (acH3), H3K18ac, and H3K27ac. In line with these changes, the TNF-induced expression of BET protein target genes was suppressed 5 d after I-BET151 treatment. Our data indicate that BET inhibitors not only prevent the reading of acetylated histones but directly influence overall chromatin organization, in particular after stimulation with TNF.

OBJECTIVE:We have recently shown that priming of synovial fibroblasts (SFs) drives arthritis flares. Pathogenic priming of SFs is essentially mediated by epigenetic reprogramming. Bromodomain and extraterminal motif (BET) proteins translate epigenetic changes into transcription. Here, we used a BET inhibitor (I-BET151) to target inflammatory tissue priming and to reduce flare severity in a murine experimental arthritis model.METHODS:BALB/c mice were treated by intraperitoneal injection or by local injection in the paw with I-BET151, which blocks the interaction of BET proteins with acetylated histones. We assessed the effects of I-BET151 on acute arthritis and/or inflammatory tissue priming in a model of repeated injections of monosodium urate crystals or zymosan into the mouse paw. I-BET151 was given before arthritis induction, at peak inflammation, or after healing of the first arthritis bout. We performed transcriptomic (RNA-Seq), epigenomic (ATAC-Seq), and functional (invasion, cytokine production, migration, senescence, metabolic flux) analyses of murine and human SFs treated with I-BET151 in vitro or in vivo.RESULTS:Systemic I-BET151 administration did not affect acute inflammation but abolished inflammatory tissue priming and diminished flare severity in both preventive and therapeutic treatment settings. I-BET151 was also effective when applied locally in the joint. BET inhibition also inhibited osteoclast differentiation, while macrophage activation in the joint was not affected. Flare reduction after BET inhibition was mediated, at least in part, by rolling back the primed transcriptional, metabolic, and pathogenic phenotype of SFs.CONCLUSION:Inflammatory tissue priming is dependent on transcriptional regulation by BET proteins, making them promising therapeutic targets for prevention of arthritis flares in previously affected joints.

Hyperactivated local tissue is a cardinal feature of immune-mediated inflammatory diseases of various organs such as the joints, the gut, the skin, or the lungs. Tissue-resident structural and stromal cells, which get primed during repeated or long-lasting bouts of inflammation form the basis of this sensitization of the tissue. During priming, cells change their metabolism to make them fit for the heightened energy demands that occur during persistent inflammation. Epigenetic changes and, curiously, an activation of intracellularly expressed parts of the complement system drive this metabolic invigoration and enable tissue-resident cells and infiltrating immune cells to employ an arsenal of inflammatory functions, including activation of inflammasomes. Here we provide a current overview on complement activation and inflammatory transformation in tissue-occupying cells, focusing on fibroblasts during arthritis, and illustrate ways how therapeutics directed at complement C3 could potentially target the complosome to unprime cells in the tissue and induce long-lasting abatement of inflammation.

In the last fifteen years it has become apparent that tissue-resident mesenchymal cells such as fibroblasts, which are the structural elements of all organs, play a cardinal role in the pathology of immune-mediated inflammatory diseases. We now know that all fibroblasts originate from universal pan-organ cellular ancestors and that they are diversified into more specific subsets according to the functional needs of their home tissue-and its activation state. In arthritis, a plethora of activated joint-resident and migrating fibroblast types have been recently described that are central for pathogenesis and persistence of inflammatory joint-disease. Here we provide a current overview on the multiple inflammatory and immune-related functions of fibroblasts and how they could be curbed to induce long-lasting abatement of disease.

Arthritis typically involves recurrence and progressive worsening at specific predilection sites, but the checkpoints between remission and persistence remain unknown. Here, we defined the molecular and cellular mechanisms of this inflammation-mediated tissue priming. Re-exposure to inflammatory stimuli caused aggravated arthritis in rodent models. Tissue priming developed locally and independently of adaptive immunity. Repeatedly stimulated primed synovial fibroblasts (SFs) exhibited enhanced metabolic activity inducing functional changes with intensified migration, invasiveness and osteoclastogenesis. Meanwhile, human SF from patients with established arthritis displayed a similar primed phenotype. Transcriptomic and epigenomic analyses as well as genetic and pharmacological targeting demonstrated that inflammatory tissue priming relies on intracellular complement C3- and C3a receptor-activation and downstream mammalian target of rapamycin- and hypoxia-inducible factor 1α-mediated metabolic SF invigoration that prevents activation-induced senescence, enhances NLRP3 inflammasome activity, and in consequence sensitizes tissue for inflammation. Our study suggests possibilities for therapeutic intervention abrogating tissue priming without immunosuppression.

Short-chain fatty acids are gut-bacteria-derived metabolites that execute important regulatory functions on adaptive immune responses, yet their influence on inflammation driven by innate immunity remains understudied. Here, we show that propionate treatment in drinking water or upon local application into the joint reduced experimental arthritis and lowered inflammatory tissue priming mediated by synovial fibroblasts. On a cellular level, incubation of synovial fibroblasts with propionate or a physiological mixture of short-chain fatty acids interfered with production of inflammatory mediators and migration and induced immune-regulatory fibroblast senescence. Our study suggests that propionate mediates its alleviating effect on arthritis by direct abrogation of local arthritogenic fibroblast function.

Excessive release of neutrophil extracellular traps (NETs) is associated with disease severity and contributes to tissue injury, followed by severe organ damage. Pharmacological or genetic inhibition of NET release reduces pathology in multiple inflammatory disease models, indicating that NETs are potential therapeutic targets. Here, we demonstrate using a preclinical basket approach that our therapeutic anti-citrullinated protein antibody (tACPA) has broad therapeutic potential. Treatment with tACPA prevents disease symptoms in various mouse models with plausible NET-mediated pathology, including inflammatory arthritis (IA), pulmonary fibrosis, inflammatory bowel disease and sepsis. We show that citrulline residues in the N-termini of histones 2A and 4 are specific targets for therapeutic intervention, whereas antibodies against other N-terminal post-translational histone modifications have no therapeutic effects. Because citrullinated histones are generated during NET release, we investigated the ability of tACPA to inhibit NET formation. tACPA suppressed NET release from human neutrophils triggered with physiologically relevant human disease-related stimuli. Moreover, tACPA diminished NET release and potentially initiated NET uptake by macrophages in vivo, which was associated with reduced tissue damage in the joints of a chronic arthritis mouse model of IA. To our knowledge, we are the first to describe an antibody with NET-inhibiting properties and thereby propose tACPA as a drug candidate for NET-mediated inflammatory diseases, as it eliminates the noxious triggers that lead to continued inflammation and tissue damage in a multidimensional manner.

The phagocyte NADPH oxidase (the NOX2 complex) generates superoxide, the precursor to reactive oxygen species (ROS). ROS possess both antimicrobial and immunoregulatory function. Inactivating mutations in alleles of the NOX2 complex cause chronic granulomatous disease (CGD), characterized by an enhanced susceptibility to infections and autoimmune diseases such as Systemic lupus erythematosus (SLE). The latter is characterized by insufficient removal of dead cells, resulting in an autoimmune response against components of the cell's nucleus when non-cleared apoptotic cells lose their membrane integrity and present autoantigenic molecules in an inflammatory context. Here we aimed to shed light on the role of the NOX2 complex in handling of secondary necrotic cells (SNECs) and associated consequences for inflammation and autoimmunity during lupus. We show that individuals with SLE and CGD display accumulation of SNECs in blood monocytes and neutrophils. In a CGD phenotypic mouse strain (Ncf1** mice) build-up of SNECs in Ly6CHI blood monocytes was connected with a delayed degradation of the phagosomal cargo and accompanied by production of inflammatory mediators. Treatment with H2O2 or activators of ROS-formation reconstituted phagosomal abundance of SNECs to normal levels. Induction of experimental lupus further induced increased antibody-dependent uptake of SNECs into neutrophils. Lupus-primed Ncf1** neutrophils took up more SNECs than wild type neutrophils, whereas SNEC-accumulation in regulatory Ly6C-/LO monocytes was lower in Ncf1**mice. We deduce that the inflammatory rerouting of immune-stimulatory necrotic material into inflammatory phagocyte subsets contributes to the connection between low ROS production by the NOX2 complex and SLE.