Antibodies as tools in cytokine discovery and usage for diagnosis and therapy of inflammatory diseases.

Polyclonal antisera from patients have been at the basis of the description of autoimmune diseases and today monoclonal antibodies are widely used in the therapy of cancer and many inflammatory diseases. How antisera and antibodies in combination with traditional in vitro and in vivo biological test systems have been instrumental reagents for the discovery of new cytokines is illustrated here for interleukin-1, -6 and -8. Furthermore, widely used immunological detection/quantification systems, such as ELISAs and multiplex assays, based on the use of either polyclonal or monoclonal antibodies, are often fraught with misinterpretations, because the results are affected by the possible occurrence of posttranslational modifications (PTMs) of the analytes. Cytokines and chemokines are present in vivo as mixtures of proteoforms with different amino- or carboxytermini or carrying heterogeneous glycan chains and possibly also being subject to citrullination, pyroglutamination and other PTMs. Increased knowledge about the specificities of antibody (cross)reactivities with cytokine ligands have improved diagnosis and treatment of many diseases, with inflammatory processes, including cancer-associated inflammation, at the frontline.

Matrix metalloproteinases in arthritis: towards precision medicine.

Proteolysis of structural molecules of the extracellular matrix (ECM) is an irreversible post-translational modification in all arthropathies. Common joint disorders, including osteoarthritis and rheumatoid arthritis, have been associated with increased levels of matrix remodelling enzymes, including matrix metalloproteinases (MMPs). MMPs, in concert with other host proteinases and glycanases, destroy proteoglycans, collagens and other ECM molecules. MMPs may also control joint remodelling indirectly by signalling through cell-surface receptors or by proteolysis of cytokines and receptor molecules. After synthesis as pro-forms, MMPs can be activated by various types of post-translational modifications, including proteolysis. Once activated, MMPs are controlled by general and specific tissue inhibitors of metalloproteinases (TIMPs). In rheumatoid arthritis, proteolysis of the ECM results in so-called remnant epitopes that enhance and perpetuate autoimmune processes in susceptible hosts. In osteoarthritis, the considerable production of MMP-13 by chondrocytes, often concurrent with mechanical overload, is a key event. Hence, information about the regulation, timing, localization and activities of MMPs in specific disease phases and arthritic entities will help to develop better diagnostics. Insights into beneficial and detrimental effects of MMPs on joint tissue inflammation are also necessary to plan and execute (pre)clinical studies for better therapy and precision medicine with MMP inhibitors. With the advances in proteomics and single-cell transcriptomics, two critical points need attention: neglected neutrophil MMP biology, and the analysis of net proteolytic activities as the result of balances between MMPs and their inhibitors.

Acute-serum amyloid A and A-SAA-derived peptides as formyl peptide receptor (FPR) 2 ligands.

Originally, it was thought that a single serum amyloid A (SAA) protein was involved in amyloid A amyloidosis, but in fact, SAA represents a four-membered family wherein SAA1 and SAA2 are acute phase proteins (A-SAA). SAA is highly conserved throughout evolution within a wide range of animal species suggestive of an important biological function. In fact, A-SAA has been linked to a number of divergent biological activities wherein a number of these functions are mediated via the G protein-coupled receptor (GPCR), formyl peptide receptor (FPR) 2. For instance, through the activation of FPR2, A-SAA has been described to regulate leukocyte activation, atherosclerosis, pathogen recognition, bone formation and cell survival. Moreover, A-SAA is subject to post-translational modification, primarily through proteolytic processing, generating a range of A-SAA-derived peptides. Although very little is known regarding the biological effect of A-SAA-derived peptides, they have been shown to promote neutrophil and monocyte migration through FPR2 activation via synergy with other GPCR ligands namely, the chemokines CXCL8 and CCL3, respectively. Within this review, we provide a detailed analysis of the FPR2-mediated functions of A-SAA. Moreover, we discuss the potential role of A-SAA-derived peptides as allosteric modulators of FPR2.

Chlorite-Oxidized Oxyamylose (COAM) Has Antibacterial Activity and Positively Affects Skin Wound Healing.

Purpose: To verify the antibacterial and immunomodulatory effects of the amylose derivative - chlorite-oxidized oxyamylose (COAM) - in a skin wound setting. Methods: In vitro antibacterial effects of COAM against opportunistic bacterial pathogens common to skin wounds, including Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA), were determined by cultivation methods. The effects of COAM on myeloid cell infiltration into full thickness skin wounds were investigated in wild-type and in transgenic CX3CR1-GFP mice. Results: On the basis of in vitro experiments, an antibacterial effect of COAM against Staphylococcus species including MRSA was confirmed. The minimum inhibitory concentration of COAM was determined as 2000 µg/mL against these bacterial strains. Control full thickness skin wounds yielded maximal neutrophil influxes and no additive effect on neutrophil influx was observed following topical COAM-treatment. However, COAM administration increased local CX3CR1 macrophage counts at days 3 and 4 and induced a trend towards better wound healing. Conclusion: Aside from its known broad antiviral impact, COAM possesses in vitro antibacterial effects specifically against Gram-positive opportunistic pathogens of the skin and modulates in vivo macrophage contents in mouse skin wounds.

Inhibition of renal fibrosis with a human CXCL9-derived glycosaminoglycan-binding peptide.

OBJECTIVES: Renal fibrosis accompanies all chronic kidney disorders, ultimately leading to end-stage kidney disease and the need for dialysis or even renal replacement. As such, renal fibrosis poses a major threat to global health and the search for effective therapeutic strategies to prevent or treat fibrosis is highly needed. We evaluated the applicability of a highly positively charged human peptide derived from the COOH-terminal domain of the chemokine CXCL9, namely CXCL9(74-103), for therapeutic intervention. Because of its high density of net positive charges at physiological pH, CXCL9(74-103) competes with full-length chemokines for glycosaminoglycan (GAG) binding. Consequently, CXCL9(74-103) prevents recruitment of inflammatory leucocytes to sites of inflammation. METHODS: CXCL9(74-103) was chemically synthesised and tested in vitro for anti-fibrotic properties on human fibroblasts and in vivo in the unilateral ureteral obstruction (UUO) mouse model. RESULTS: CXCL9(74-103) significantly reduced the mRNA and/or protein expression of connective tissue growth factor (CTGF), alpha-smooth muscle actin (α-SMA) and collagen III by transforming growth factor (TGF)-ß1-stimulated human fibroblasts. In addition, administration of CXCL9(74-103) inhibited fibroblast migration towards platelet-derived growth factor (PDGF), without affecting cell viability. In the UUO model, CXCL9(74-103) treatment significantly decreased renal α-SMA, vimentin, and fibronectin mRNA and protein expression. Compared with vehicle, CXCL9(74-103) attenuated mRNA expression of TGF-ß1 and the inflammatory markers/mediators MMP-9, F4/80, CCL2, IL-6 and TNF-α. Finally, CXCL9(74-103) treatment resulted in reduced influx of leucocytes in the UUO model and preserved tubular morphology. The anti-fibrotic and anti-inflammatory effects of CXCL9(74-103) were mediated by competition with chemokines and growth factors for GAG binding. CONCLUSIONS: Our findings provide a scientific rationale for targeting GAG-protein interactions in renal fibrotic disease.

Efficacy of B Cell Depletion Therapy with Rituximab in Refractory Chronic Recurrent Uveitis Associated with Vogt-Koyanagi-Harada Disease.

PURPOSE: To evaluate the efficacy of B cell depletion therapy with the chimeric mouse/human anti-CD20 monoclonal antibody rituximab for refractory chronic recurrent granulomatous uveitis associated with Vogt-Koyanagi-Harada (VKH) disease. METHODS: Retrospective study of 9 patients (18 eyes) who failed to respond to conventional combination immunosuppressive therapy. RESULTS: All the patients received 3 rituximab infusions. The follow-up period after initiation of rituximab therapy ranged from 9 to 36 months (mean ±SD, 19.2 ± 10.1). All patients achieved remission and visual acuity significantly improved (p < .001). Rituximab provided corticosteroid-sparing effect along with control of inflammation. No rituximab-related complications were observed. CONCLUSIONS: Rituximab is effective for the treatment of refractory chronic recurrent granulomatous uveitis associated with VKH disease.

Proteoform Analysis of Matrix Metalloproteinase-9/Gelatinase B and Discovery of Its Citrullination in Rheumatoid Arthritis Synovial Fluids.

Objectives: To explore posttranslational modifications (PTMs), including proteolytic activation, multimerization, complex formation and citrullination of gelatinases, in particular of gelatinase B/MMP-9, and to detect in gelatin-Sepharose affinity-purified synovial fluids, the presence of specific MMP proteoforms in relation to arthritis. Methods: Latent, activated, complexed and truncated gelatinase-A/MMP-2 and gelatinase B/MMP-9 proteoforms were detected with the use of zymography analysis to compare specific levels, with substrate conversion assays, to test net proteolytic activities and by Western blot analysis to decipher truncation variants. Citrullination was detected with enhanced sensitivity, by the use of a new monoclonal antibody against modified citrullines. Results: All MMP-9 and MMP-2 proteoforms were identified in archival synovial fluids with the use of zymography analysis and the levels of MMP-9 versus MMP-2 were studied in various arthritic diseases, including rheumatoid arthritis (RA). Secondly, we resolved misinterpretations of MMP-9 levels versus proteolytic activities. Thirdly, a citrullinated, truncated proteoform of MMP-9 was discovered in archival RA synovial fluid samples and its presence was corroborated as citrullinated hemopexin-less MMP-9 in a small prospective RA sample cohort. Conclusion: Synovial fluids from rheumatoid arthritis contain high levels of MMP-9, including its truncated and citrullinated proteoform. The combination of MMP-9 as analyte and its PTM by citrullination could be of clinical interest, especially in the field of arthritic diseases.

New Perspectives on the Immunopathogenesis and Treatment of Uveitis Associated With Vogt-Koyanagi-Harada Disease.

Uveitis associated with Vogt-Koyanagi-Harada (VKH) disease is a bilateral, chronic, granulomatous autoimmune disease associated with vitiligo, poliosis, alopecia, and meningeal and auditory manifestations. The disease affects pigmented races with a predisposing genetic background. Evidence has been provided that the clinical manifestations are caused by a T-lymphocyte-mediated autoimmune response directed against antigens associated with melanocytes in the target organs. Alongside of T lymphocytes, autoreactive B cells play a central role in the development and propagation of several autoimmune diseases. The potential role of B lymphocytes in the pathogenesis of granulomatous uveitis associated with VKH disease is exemplified within several studies. The early initial-onset acute uveitic phase typically exhibits granulomatous choroiditis with secondary exudative retinal detachment and optic disc hyperemia and swelling, subsequently involving the anterior segment if not adequately treated. The disease eventually progresses to chronic recurrent granulomatous anterior uveitis with progressive posterior segment depigmentation resulting in "sunset glow fundus" appearance and chorioretinal atrophy if not properly controlled. Chronically evolving disease is more refractory to treatment and, consequently, vision-threatening complications have been recognized to occur in the chronic recurrent phase of the disease. Conventional treatment with early high-dose systemic corticosteroids is not sufficient to prevent chronic evolution. Addition of immunomodulatory therapy with mycophenolate mofetil as first-line therapy combined with systemic corticosteroids in patients with acute initial-onset disease prevents progression to chronic evolution, late complications, vitiligo, and poliosis. Furthermore, patients under such combined therapy were able to discontinue treatment without relapse of inflammation. These findings suggest that there is a therapeutic window of opportunity for highly successful treatment during the early initial-onset acute uveitic phases, likely because the underlying disease process is not fully matured. It is hypothesized that early and aggressive immunosuppressive therapy will prevent remnant epitope generation in the initiation of the autoimmune process, the so-called primary response. B cell depleting therapy with the anti-CD20 monoclonal antibody rituximab is effective in patients with refractory chronic recurrent granulomatous uveitis. The good response after rituximab therapy reinforces the idea of an important role of B cells in the pathogenesis or progression of chronic recurrent uveitis associated with VKH disease.

The Chemokine-Based Peptide, CXCL9(74-103), Inhibits Angiogenesis by Blocking Heparan Sulfate Proteoglycan-Mediated Signaling of Multiple Endothelial Growth Factors.

Growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and epidermal growth factor (EGF) are important angiogenesis-mediating factors. They exert their effects not only through their respective receptor tyrosine kinases (RTKs), but they also require molecular pairing with heparan sulfate proteoglycans (HSPGs). Angiogenic growth factors and their signaling pathways are commonly targeted in current anti-angiogenic cancer therapies but have unfortunately insufficient impact on patient survival. Considering their obvious role in pathological angiogenesis, HS-targeting drugs have become an appealing new strategy. Therefore, we aimed to reduce angiogenesis through interference with growth factor-HS binding and downstream signaling using a CXCL9-derived peptide with a high affinity for glycosaminoglycans (GAGs), CXCL9(74-103). We showed that CXCL9(74-103) reduced EGF-, VEGF165- and FGF-2-mediated angiogenic processes in vitro, such as endothelial cell proliferation, chemotaxis, adhesion and sprouting, without exerting cell toxicity. CXCL9(74-103) interfered with growth factor signaling in diverse ways, e.g., by diminishing VEGF165 binding to HS and by direct association with FGF-2. The dependency of CXCL9(74-103) on HS for binding to HMVECs and for exerting its anti-angiogenic activity was also demonstrated. In vivo, CXCL9(74-103) attenuated neovascularization in the Matrigel plug assay, the corneal cauterization assay and in MDA-MB-231 breast cancer xenografts. Additionally, CXCL9(74-103) reduced vascular leakage in the retina of diabetic rats. In contrast, CXCL9(86-103), a peptide with low GAG affinity, showed no overall anti-angiogenic activity. Altogether, our results indicate that CXCL9(74-103) reduces angiogenesis by interfering with multiple HS-dependent growth factor signaling pathways.

The Role of Post-Translational Modifications of Chemokines by CD26 in Cancer.

Chemokines are a large family of small chemotactic cytokines that fulfill a central function in cancer. Both tumor-promoting and -impeding roles have been ascribed to chemokines, which they exert in a direct or indirect manner. An important post-translational modification that regulates chemokine activity is the NH2-terminal truncation by peptidases. CD26 is a dipeptidyl peptidase (DPPIV), which typically clips a NH2-terminal dipeptide from the chemokine. With a certain degree of selectivity in terms of chemokine substrate, CD26 only recognizes chemokines with a penultimate proline or alanine. Chemokines can be protected against CD26 recognition by specific amino acid residues within the chemokine structure, by oligomerization or by binding to cellular glycosaminoglycans (GAGs). Upon truncation, the binding affinity for receptors and GAGs is altered, which influences chemokine function. The consequences of CD26-mediated clipping vary, as unchanged, enhanced, and reduced activities are reported. In tumors, CD26 most likely has the most profound effect on CXCL12 and the interferon (IFN)-inducible CXCR3 ligands, which are converted into receptor antagonists upon truncation. Depending on the tumor type, expression of CD26 is upregulated or downregulated and often results in the preferential generation of the chemokine isoform most favorable for tumor progression. Considering the tight relationship between chemokine sequence and chemokine binding specificity, molecules with the appropriate characteristics can be chemically engineered to provide innovative therapeutic strategies in a cancer setting.

From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8.

With ELISAs one detects the ensemble of immunoreactive molecules in biological samples. For biomolecules undergoing proteolysis for activation, potentiation or inhibition, other techniques are necessary to study biology. Here we develop methodology that combines immunosorbent sample preparation and nano-scale liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) for proteoform analysis (ISTAMPA) and apply this to the aglycosyl chemokine CXCL8. CXCL8, the most powerful human chemokine with neutrophil chemotactic and -activating properties, occurs in different NH2-terminal proteoforms due to its susceptibility to site-specific proteolytic modification. Specific proteoforms display up to 30-fold enhanced activity. The immunosorbent ion trap top-down mass spectrometry-based approach for proteoform analysis allows for simultaneous detection and quantification of full-length CXCL8(1-77), elongated CXCL8(-2-77) and all naturally occurring truncated CXCL8 forms in biological samples. For the first time we demonstrate site-specific proteolytic activation of CXCL8 in synovial fluids from patients with chronic joint inflammation and address the importance of sample collection and processing.

Endogenous modification of the chemoattractant CXCL5 alters receptor usage and enhances its activity toward neutrophils and monocytes.

The inflammatory human chemokine CXCL5 interacts with the G protein-coupled receptor CXCR2 to induce chemotaxis and activation of neutrophils. CXCL5 also has weak agonist activity toward CXCR1. The N-terminus of CXCL5 can be modified by proteolytic cleavage or deimination of Arg9 to citrulline (Cit), and these modifications can occur separately or together. Here, we chemically synthesized native CXCL5(1-78), truncated CXCL5 [CXCL5(9-78)], and the citrullinated (Cit9) versions and characterized their functions in vitro and in vivo. Compared with full-length CXCL5, N-terminal truncation resulted in enhanced potency to induce G protein signaling and ß-arrestin recruitment through CXCR2, increased CXCL5-initiated internalization of CXCR2, and greater Ca2+ signaling downstream of not only CXCR2 but also CXCR1. Citrullination did not affect the capacity of CXCL5 to activate classical or alternative signaling pathways. Administering the various CXCL5 forms to mice revealed that in addition to neutrophils, CXCL5 exerted chemotactic activity toward monocytes and that this activity was increased by N-terminal truncation. These findings were confirmed by in vitro chemotaxis and Ca2+ signaling assays with primary human CD14+ monocytes and human THP-1 monocytes. In vitro and in vivo analyses suggested that CXCL5 targeted monocytes through CXCR1 and CXCR2. Thus, truncation of the N-terminus makes CXCL5 a more potent chemoattractant for both neutrophils and monocytes that acts through CXCR1 and CXCR2.

Interferons and other cytokines, genetics and beyond in COVID-19 and autoimmunity.

Interferons are the best antiviral agents in vitro against SARS-CoV-2 so far and genetic defects in their signaling cascade or neutralization of alfa-interferons by autoantibodies come with more severe COVID-19. However, there is more, as the SARS-CoV-2 dysregulates not only innate immune mechanisms but also T and B cell repertoires. Most genetic, hematological and immunological studies in COVID-19 are at present phenomenological. However, these and antecedent studies contain the seed grains to resolve many unanswered questions and a whole range of testable hypotheses. What are the links, if existing, between genetics and the occurrence of interferon-neutralizing antibodies? Are NAGGED (neutralizing and generated by gene defect) antibodies involved or not? Is the autoimmune process cause or consequence of virus infection? What are the roles played by cytokine posttranslational modifications, such as proteolysis, glycosylation, citrullination and others? How is systemic autoimmunity linked with type 1 interferons? These questions place cytokines and growth factors at pole positions as keys to unlock basic mechanisms of infection and (auto)immunity. Related to cytokine research, (1) COVID-19 patients develop neutralizing autoantibodies, mainly against alpha interferons and it is not yet established whether this is the consequence or cause of virus replication. (2) The glycosylation of recombinant interferon-beta protects against breaking tolerance and the development of neutralizing antibodies. (3) SARS-CoV-2 induces severe inflammation and release of extracellular proteases leading to remnant epitopes, e.g. of cytokines. (4) In the rare event of homozygous cytokine gene segment deletions, observed neutralizing antibodies may be named NAGGED antibodies. (5) Severe cytolysis releases intracellular content into the extracellular milieu and leads to regulated degradation of intracellular proteins and selection of antibody repertoires, similar to those observed in patients with systemic lupus erythematosus. (6) Systematic studies of novel autoimmune diseases on single cytokines will complement the present picture about interferons. (7) Interferon neutralization in COVID-19 constitutes a preamble of more studies about cytokine-regulated proteolysis in the control of autoimmunity. Here we reformulate these seven conjectures into testable questions for future research.

The turning away of serum amyloid A biological activities and receptor usage.

Serum amyloid A (SAA) is an acute-phase protein (APP) to which multiple immunological functions have been attributed. Regardless, the true biological role of SAA remains poorly understood. SAA is remarkably conserved in mammalian evolution, thereby suggesting an important biological function. Since its discovery in the 1970s, the majority of researchers have investigated SAA using recombinant forms made available through bacterial expression. Nevertheless, recent studies indicate that these recombinant forms of SAA are unreliable. Indeed, commercial SAA variants have been shown to be contaminated with bacterial products including lipopolysaccharides and lipoproteins. As such, biological activities and receptor usage (TLR2, TLR4) revealed through the use of commercial SAA variants may not reflect the inherent nature of this APP. Within this review, we discuss the biological effects of SAA that have been demonstrated through more solid experimental approaches. SAA takes part in the innate immune response via the recruitment of leucocytes and executes, through pathogen recognition, antimicrobial activity. Knockout animal models implicate SAA in a range of functions, such as regulation of T-cell-mediated responses and monopoiesis. Moreover, through its structural motifs, not only does SAA function as an extracellular matrix protein, but it also binds extracellular matrix proteins. Finally, we here also provide an overview of definite SAA receptor-mediated functions and highlight those that are yet to be validated. The role of FPR2 in SAA-mediated leucocyte recruitment has been confirmed; nevertheless, SAA has been linked to a range of other receptors including CD36, SR-BI/II, RAGE and P2RX7.

Biological Characterization of Commercial Recombinantly Expressed Immunomodulating Proteins Contaminated with Bacterial Products in the Year 2020: The SAA3 Case.

The serum amyloid A (SAA) gene family is highly conserved and encodes acute phase proteins that are upregulated in response to inflammatory triggers. Over the years, a considerable amount of literature has been published attributing a wide range of biological effects to SAAs such as leukocyte recruitment, cytokine and chemokine expression and induction of matrix metalloproteinases. Furthermore, SAAs have also been linked to protumorigenic, proatherogenic and anti-inflammatory effects. Here, we investigated the biological effects conveyed by murine SAA3 (mu rSAA3) recombinantly expressed in Escherichia coli. We observed the upregulation of a number of chemokines including CCL2, CCL3, CXCL1, CXCL2, CXCL6 or CXCL8 following stimulation of monocytic, fibroblastoid and peritoneal cells with mu rSAA3. Furthermore, this SAA variant displayed potent in vivo recruitment of neutrophils through the activation of TLR4. However, a major problem associated with proteins derived from recombinant expression in bacteria is potential contamination with various bacterial products, such as lipopolysaccharide, lipoproteins and formylated peptides. This is of particular relevance in the case of SAA as there currently exists a discrepancy in biological activity between SAA derived from recombinant expression and that of an endogenous source, i.e. inflammatory plasma. Therefore, we subjected commercial recombinant mu rSAA3 to purification to homogeneity via reversed-phase high-performance liquid chromatography (RP-HPLC) and re-assessed its biological potential. RP-HPLC-purified mu rSAA3 did not induce chemokines and lacked in vivo neutrophil chemotactic activity, but retained the capacity to synergize with CXCL8 in the activation of neutrophils. In conclusion, experimental results obtained when using proteins recombinantly expressed in bacteria should always be interpreted with care.

Serum Amyloid A1 (SAA1) Revisited: Restricted Leukocyte-Activating Properties of Homogeneous SAA1.

Infection, sterile injury, and chronic inflammation trigger the acute phase response in order to re-establish homeostasis. This response includes production of positive acute phase proteins in the liver, such as members of the serum amyloid A (SAA) family. In humans the major acute phase SAAs comprise a group of closely related variants of SAA1 and SAA2. SAA1 was proven to be chemotactic for several leukocyte subtypes through activation of the G protein-coupled receptor FPRL1/FPR2. Several other biological activities of SAA1, such as cytokine induction, reported to be mediated via TLRs, have been debated recently. Especially commercial SAA1, recombinantly produced in Escherichia coli, was found to be contaminated with bacterial products confounding biological assays performed with this rSAA1. We purified rSAA1 by RP-HPLC to homogeneity, removing contaminants such as lipopolysaccharides, lipoproteins and formylated peptides, and re-assessed several biological activities attributed to SAA1 (chemotaxis, cytokine induction, MMP-9 release, ROS generation, and macrophage differentiation). The homogeneous rSAA1 (hrSAA1) lacked most cell-activating properties, but its leukocyte-recruiting capacity in vivo and it's in vitro synergy with other leukocyte attractants remained preserved. Furthermore, hrSAA1 maintained the ability to promote monocyte survival. This indicates that pure hrSAA1 retains its potential to activate FPR2, whereas TLR-mediated effects seem to be related to traces of bacterial TLR ligands in the E. coli-produced human rSAA1.

Remnant Epitopes Generating Autoimmunity: From Model to Useful Paradigm.

Autoimmune diseases are defined as pathologies of adaptive immunity by the presence of autoantibodies or MHC-restricted autoantigen-reactive T cells. Because autoreactivity is a normal process based on mechanisms producing repertoires of antibodies and T cell receptors, crucial questions about disease mechanisms and key steps for interference have been outstanding. We defined 25 years ago the 'remnant epitopes generate autoimmunity' (REGA)-model in which extracellular proteases from innate immune cells generate autoantigens. Here, we refine the REGA-model, tested in diseases ranging from organ-specific autoimmune diseases to systemic lupus erythematosus. It now constitutes a paradigm in which remnant epitopes generate, maintain, and regulate autoimmunity; are dependent on genetic and epigenetic influences; are produced in a disease phase-specific manner; and have therapeutic implications when targeted.

Induction of Chemokines by Hepatitis C Virus Proteins: Synergy of the Core Protein with Interleukin-1ß and Interferon-γ in Liver Bystander Cells.

Chronic hepatitis C virus (HCV) infection accounts for a large proportion of hepatic fibrosis and carcinoma cases observed worldwide. Mechanisms involved in HCV-induced hepatic injury have yet to be fully elucidated. Of particular interest is the capacity of HCV to regulate inflammatory responses. Here, we reveal modulation of cytokine activity by the HCV proteins non-structural protein 3 (NS3), glycoprotein E2, and core protein for their ability to induce chemokine expression in various liver bystander cells. Chemokines sustain chronic liver inflammation and relay multiple fibrogenic effects. CCL2, CCL3, CCL20, CXCL8, and CXCL10 were differentially expressed after treatment of monocytes, fibroblasts, or liver sinusoidal microvascular endothelial cells (LSECs) with HCV proteins. In comparison to NS3 and glycoprotein E2, core protein was a stronger inducer of chemokines in liver bystander cells. Interferon-γ (IFN-γ) and interleukin-1ß (IL-1ß) synergized with core protein to induce CCL2, CCL20, CXCL8, or CXCL10 in fibroblasts or LSECs. These findings reveal new mechanisms of hepatic injury caused by HCV.

Evaluation of Proteoforms of the Transmembrane Chemokines CXCL16 and CX3CL1, Their Receptors, and Their Processing Metalloproteinases ADAM10 and ADAM17 in Proliferative Diabetic Retinopathy.

The transmembrane chemokine pathways CXCL16/CXCR6 and CX3CL1/CX3CR1 are strongly implicated in inflammation and angiogenesis. We investigated the involvement of these chemokine pathways and their processing metalloproteinases ADAM10 and ADAM17 in the pathophysiology of proliferative diabetic retinopathy (PDR). Vitreous samples from 32 PDR and 24 non-diabetic patients, epiretinal membranes from 18 patients with PDR, rat retinas, human retinal Müller glial cells and human retinal microvascular endothelial cells (HRMECs) were studied by enzyme-linked immunosorbent assay, immunohistochemistry and Western blot analysis. In vitro angiogenesis assays were performed and the adherence of leukocytes to CXCL16-stimulated HRMECs was assessed. CXCL16, CX3CL1, ADAM10, ADAM17 and vascular endothelial growth factor (VEGF) levels were significantly increased in vitreous samples from PDR patients. The levels of CXCL16 were 417-fold higher than those of CX3CL1 in PDR vitreous samples. Significant positive correlations were found between the levels of VEGF and the levels of CXCL16, CX3CL1, ADAM10 and ADAM17. Significant positive correlations were detected between the numbers of blood vessels expressing CD31, reflecting the angiogenic activity of PDR epiretinal membranes, and the numbers of blood vessels and stromal cells expressing CXCL16, CXCR6, ADAM10 and ADAM17. CXCL16 induced upregulation of phospho-ERK1/2, p65 subunit of NF-κB and VEGF in cultured Müller cells and tumor necrosis factor-α induced upregulation of soluble CXCL16 and ADAM17 in Müller cells. Treatment of HRMECs with CXCL16 resulted in increased expression of intercellular adhesion molecule-1 (ICAM-1) and increased leukocyte adhesion to HRMECs. CXCL16 induced HRMEC proliferation, formation of sprouts from HRMEC spheroids and phosphorylation of ERK1/2. Intravitreal administration of CXCL16 in normal rats induced significant upregulation of the p65 subunit of NF-κB, VEGF and ICAM-1 in the retina. Our findings suggest that the chemokine axis CXCL16/CXCR6 and the processing metalloproteinases ADAM10 and ADAM17 might serve a role in the initiation and progression of PDR.

Local Cytokine Expression Profiling in Patients with Specific Autoimmune Uveitic Entities.

Purpose: To evaluate expression of cytokines GM-CSF, IL-11, IL-12p40, IL-12p70, IL-27p28, IL-35, APRIL, BAFF, TWEAK, and LIGHT in uveitis.Methods: Aqueous humor samples from patients with active uveitis associated with Behçet's disease (BD), sarcoidosis, HLA-B27-related inflammation, and Vogt-Koyanagi-Harada (VKH) disease and control patients were assayed with a multiplex assay.Results: Comparing all patients to controls, GM-CSF, IL-11, IL-12p40, APRIL, and BAFF were significantly increased, whereas LIGHT was significantly decreased. IL-11 and BAFF were the most strongly upregulated, being elevated 19.7-fold and 14.1-fold, respectively, compared with controls. IL-11 was significantly highest in HLA-B27 uveitis. GM-CSF, IL-11, and IL-12p40 were significantly higher in nongranulomatous uveitis (BD and HLA-B27) than in granulomatous uveitis (sarcoidosis and VKH), whereas APRIL and TWEAK were significantly higher in granulomatous uveitis.Conclusions: IL-11-driven immune responses might be more potent in nongranulomatous uveitis, particularly in HLA-B27 uveitis. BAFF and APRIL might contribute to B cell-driven autoimmune response in uveitis.