Amelioration of nephritis in receptor for advanced glycation end-products (RAGE)-deficient lupus-prone mice through neutrophil extracellular traps.

The receptor for advanced glycation end-products (RAGE) is a pattern recognition receptor that regulates inflammation, cell migration, and cell fate. Systemic lupus erythematosus (SLE) is a chronic multiorgan autoimmune disease. To understand the function of RAGE in SLE, we generated RAGE-deficient (Ager-/-) lupus-prone mice by backcrossing MRL/MpJ-Faslpr/J (MRL-lpr) mice with Ager-/- C57BL/6 mice. In 18-week-old Ager-/- MRL-lpr, the weights of the spleen and lymph nodes, as well as the frequency of CD3+CD4-CD8- cells, were significantly decreased. Ager-/- MRL-lpr mice had significantly reduced urine albumin/creatinine ratios and markedly improved renal pathological scores. Moreover, neutrophil infiltration and neutrophil extracellular trap formation in the glomerulus were significantly reduced in Ager-/- MRL-lpr. Our study is the first to reveal that RAGE can have a pathologic role in immune cells, particularly neutrophils and T cells, in inflammatory tissues and suggests that the inhibition of RAGE may be a potential therapeutic strategy for SLE.

Thiocarbazate building blocks enable the construction of azapeptides for rapid development of therapeutic candidates.

Peptides, polymers of amino acids, comprise a vital and expanding therapeutic approach. Their rapid degradation by proteases, however, represents a major limitation to their therapeutic utility and chemical modifications to native peptides have been employed to mitigate this weakness. Herein, we describe functionalized thiocarbazate scaffolds as precursors of aza-amino acids, that, upon activation, can be integrated in a peptide sequence to generate azapeptides using conventional peptide synthetic methods. This methodology facilitates peptide editing-replacing targeted amino acid(s) with aza-amino acid(s) within a peptide-to form azapeptides with preferred therapeutic characteristics (extending half-life/bioavailability, while at the same time typically preserving structural features and biological activities). We demonstrate the convenience of this azapeptide synthesis platform in two well-studied peptides with short half-lives: FSSE/P5779, a tetrapeptide inhibitor of HMGB1/MD-2/TLR4 complex formation, and bradykinin, a nine-residue vasoactive peptide. This bench-stable thiocarbazate platform offers a robust and universal approach to optimize peptide-based therapeutics.

Understanding the contextual functions of C1q and LAIR-1 and their applications.

The importance of the complement component C1q has been highlighted by its involvement in autoimmunity, infection, inflammatory diseases, and tumors. The unique tulip-like structure of C1q has both a collagen-like stalk (C1q tail) and heterotrimeric globular head (gC1q), each with different binding specificities, and the binding of these components to their respective receptors leads to functional complexities in the body and bridges innate and adaptive immunity. This review describes the fundamental roles of C1q in various microenvironments and focuses on the importance of the interactions of C1q and its receptors with the inhibitory receptor LAIR-1 in maintaining homeostasis. Current therapeutic opportunities modulating LAIR-1 are also discussed.

The collagen structure of C1q induces wound healing by engaging discoidin domain receptor 2.

BACKGROUND: C1q has been reported to reveal complement-independent roles in immune and non-immune cells. C1q binds to its specific receptors to regulate distinct functions that rely on the environment and cell types. Discoidin domain receptor 2 (DDR2) is activated by collagen and functions in wound healing by controlling matrix metalloproteinase (MMP) expression. Since C1q exhibits a collagen-like structure, we hypothesized that C1q might engage DDR2 to regulate wound healing and extracellular matrix (ECM) remodeling. METHODS: Cell-based assay, proximity ligation assay, ELISA, and surface plasmon analysis were utilized to investigate DDR2 and C1q binding. We also investigate the C1q-mediated in vitro wound healing ability using the human fibrosarcoma cell line, HT1080. RESULTS: C1q induced the phosphorylation of DDR2, p38 kinase, and ERK1/2. C1q and DDR2 binding improved cell migration and induced MMP2 and MMP9 expression. DDR2-specific shRNA reduced C1q-mediated cell migration for wound healing. CONCLUSIONS: C1q is a new DDR2 ligand that promotes wound healing. These findings have therapeutic implications in wound healing-related diseases.

HIF-1α is a negative regulator of interferon regulatory factors: Implications for interferon production by hypoxic monocytes.

Patients with severe COVID-19 infection exhibit a low level of oxygen in affected tissue and blood. To understand the pathophysiology of COVID-19 infection, it is therefore necessary to understand cell function during hypoxia. We investigated aspects of human monocyte activation under hypoxic conditions. HMGB1 is an alarmin released by stressed cells. Under normoxic conditions, HMGB1 activates interferon regulatory factor (IRF)5 and nuclear factor-κB in monocytes, leading to expression of type I interferon (IFN) and inflammatory cytokines including tumor necrosis factor α, and interleukin 1ß, respectively. When hypoxic monocytes are activated by HMGB1, they produce proinflammatory cytokines but fail to produce type I IFN. Hypoxia-inducible factor-1α, induced by hypoxia, functions as a direct transcriptional repressor of IRF5 and IRF3. As hypoxia is a stressor that induces secretion of HMGB1 by epithelial cells, hypoxia establishes a microenvironment that favors monocyte production of inflammatory cytokines but not IFN. These findings have implications for the pathogenesis of COVID-19.

The Immune Tolerance Role of the HMGB1-RAGE Axis.

The disruption of the immune tolerance induces autoimmunity such as systemic lupus erythematosus and vasculitis. A chromatin-binding non-histone protein, high mobility group box 1 (HMGB1), is released from the nucleus to the extracellular milieu in particular environments such as autoimmunity, sepsis and hypoxia. Extracellular HMGB1 engages pattern recognition receptors, including Toll-like receptors (TLRs) and the receptor for advanced glycation endproducts (RAGE). While the HMGB1-RAGE axis drives inflammation in various diseases, recent studies also focus on the anti-inflammatory effects of HMGB1 and RAGE. This review discusses current perspectives on HMGB1 and RAGE's roles in controlling inflammation and immune tolerance. We also suggest how RAGE heterodimers responding microenvironments functions in immune responses.

The Transmembrane Adaptor Protein LIME Is Essential for Chemokine-Mediated Migration of Effector T Cells to Inflammatiory Sites.

Lck-interacting transmembrane adaptor 1 (LIME) has been previously identified as a raft-associated transmembrane protein expressed predominantly in T and B lymphocytes. Although LIME is shown to transduce the immunoreceptor signaling and immunological synapse formation via its tyrosine phosphorylation by Lck, a Src-family kinase, the in vivo function of LIME has remained elusive in the previous studies. Here we report that LIME is preferentially expressed in effector T cells and mediates chemokine-mediated T cell migration. Interestingly, in LIME-/- mice, while T cell receptor stimulation-dependent proliferation, differentiation to effector T cells, cytotoxic T lymphocyte (CTL) function and regulatory T lymphocyte (Treg) function were normal, only T cell-mediated inflammatory response was significantly defective. The reduced inflammation was accompanied by the impaired infiltration of leukocytes and T cells to the inflammatory sites of LIME-/- mice. More specifically, the absence of LIME in effector T cells resulted in the reduced migration and defective morphological polarization in response to inflammatory chemokines such as CCL5 and CXCL10. Consistently, LIME-/- effector T cells were found to be defective in chemokine-mediated activation of Rac1 and Rap1, and dysregulated phosphorylation of Pyk2 and Cas. Taken together, the present findings show that LIME is a critical regulator of inflammatory chemokine-mediated signaling and the subsequent migration of effector T cells to inflammatory sites.

HMGB1 in Systemic Lupus Erythematosus.

The high-mobility group box 1 (HMGB1) has been shown to exert proinflammatory effects on many cells of the innate immune system. Originally identified as a nuclear protein, HMGB1 has been found to play an important role in mediating inflammation when released from apoptotic or necrotic cells as a damage-associated molecular pattern (DAMP). Systemic lupus erythematosus (SLE) is a disease of non-resolving inflammation, characterized by the presence of autoantibodies and systemic inflammation involving multiple organ systems. SLE patients have impaired clearance of apoptotic debris, which releases HMGB1 and other DAMPs extracellularly. HMGB1 activity is implicated in multiple disease phenotypes in SLE, including lupus nephritis and neuropsychiatric lupus. Elucidating the various properties of HMGB1 in SLE provides a better understanding of the disease and opens up new opportunities for designing potential therapeutics.

Immunological Significance of HMGB1 Post-Translational Modification and Redox Biology.

Most extracellular proteins are secreted via the classical endoplasmic reticulum (ER)/Golgi-dependent secretion pathway; however, some proteins, including a few danger-associated molecular patterns (DAMPs), are secreted via non-classical ER/Golgi-independent secretion pathways. The evolutionarily conserved high mobility group box1 (HMGB1) is a ubiquitous nuclear protein that can be released by almost all cell types. HMGB1 lacks signal peptide and utilizes diverse non-canonical secretion mechanisms for its extracellular export. Although the post-translational modifications of HMGB1 were demonstrated, the oxidation of HMGB1 and secretion mechanisms are not highlighted yet. We currently investigated that peroxiredoxins I and II (PrxI/II) induce the intramolecular disulfide bond formation of HMGB1 in the nucleus. Disulfide HMGB1 is preferentially transported out of the nucleus by binding to the nuclear exportin chromosome-region maintenance 1 (CRM1). We determined the kinetics of HMGB1 oxidation in bone marrow-derived macrophage as early as a few minutes after lipopolysaccharide treatment, peaking at 4 h while disulfide HMGB1 accumulation was observed within the cells, starting to secrete in the late time point. We have shown that HMGB1 oxidation status, which is known to determine the biological activity in extracellular HMGB1, is crucial for the secretion of HMGB1 from the nucleus. This review summarizes selected aspects of HMGB1 redox biology relevant to the induction and propagation of inflammatory diseases. We implicate the immunological significance and the need for novel HMGB1 inhibitors through mechanism-based studies.

HMGB1-C1q complexes regulate macrophage function by switching between leukotriene and specialized proresolving mediator biosynthesis.

Macrophage polarization is critical to inflammation and resolution of inflammation. We previously showed that high-mobility group box 1 (HMGB1) can engage receptor for advanced glycation end product (RAGE) to direct monocytes to a proinflammatory phenotype characterized by production of type 1 IFN and proinflammatory cytokines. In contrast, HMGB1 plus C1q form a tetramolecular complex cross-linking RAGE and LAIR-1 and directing monocytes to an antiinflammatory phenotype. Lipid mediators, as well as cytokines, help establish a milieu favoring either inflammation or resolution of inflammation. This study focuses on the induction of lipid mediators by HMGB1 and HMGB1 plus C1q and their regulation of IRF5, a transcription factor critical for the induction and maintenance of proinflammatory macrophages. Here, we show that HMGB1 induces leukotriene production through a RAGE-dependent pathway, while HMGB1 plus C1q induces specialized proresolving lipid mediators lipoxin A4, resolvin D1, and resolvin D2 through a RAGE- and LAIR-1-dependent pathway. Leukotriene exposure contributes to induction of IRF5 in a positive-feedback loop. In contrast, resolvins (at 20 nM) block IRF5 induction and prevent the differentiation of inflammatory macrophages. Finally, we have generated a molecular mimic of HMGB1 plus C1q, which cross-links RAGE and LAIR-1 and polarizes monocytes to an antiinflammatory phenotype. These findings may provide a mechanism to control nonresolving inflammation in many pathologic conditions.

Constitutive Vagus Nerve Activation Modulates Immune Suppression in Sepsis Survivors.

Patients surviving a septic episode exhibit persistent immune impairment and increased mortality due to enhanced vulnerability to infections. In the present study, using the cecal ligation and puncture (CLP) model of polymicrobial sepsis, we addressed the hypothesis that altered vagus nerve activity contributes to immune impairment in sepsis survivors. CLP-surviving mice exhibited less TNFα in serum following administration of LPS, a surrogate for an infectious challenge, than control-operated (control) mice. To evaluate the role of the vagus nerve in the diminished response to LPS, mice were subjected to bilateral subdiaphragmatic vagotomy at 2 weeks post-CLP. CLP-surviving vagotomized mice exhibited increased serum and tissue TNFα levels in response to LPS-challenge compared to CLP-surviving, non-vagotomized mice. Moreover, vagus nerve stimulation in control mice diminished the LPS-induced TNFα responses while having no effect in CLP mice, suggesting constitutive activation of vagus nerve signaling in CLP-survivors. The percentage of splenic CD4+ ChAT-EGFP+ T cells that relay vagus signals to macrophages was increased in CLP-survivors compared to control mice, and vagotomy in CLP-survivors resulted in a reduced percentage of ChAT-EGFP+ cells. Moreover, CD4 knockout CLP-surviving mice exhibited an enhanced LPS-induced TNFα response compared to wild-type mice, supporting a functional role for CD4+ ChAT+ T cells in mediating inhibition of LPS-induced TNFα responses in CLP-survivors. Blockade of the cholinergic anti-inflammatory pathway with methyllcaconitine, an α7 nicotinic acetylcholine receptor antagonist, restored LPS-induced TNFα responses in CLP-survivors. Our study demonstrates that the vagus nerve is constitutively active in CLP-survivors and contributes to the immune impairment.

High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation.

High-mobility group box 1 (HMGB1), a well-known danger-associated molecular pattern molecule, acts as a pro-inflammatory molecule when secreted by activated immune cells or released after necrotic cell damage. HMGB1 binds to immunogenic bacterial components and augments septic inflammation. In this study, we show how HMGB1 mediates complement activation, promoting sterile inflammation. We show that HMGB1 activates the classical pathway of complement system in an antibody-independent manner after binding to C1q. The C3a complement activation product in human plasma and C5b-9 membrane attack complexes on cell membrane surface are detected after the addition of HMGB1. In an acetaminophen (APAP)-induced hepatotoxicity model, APAP injection reduced HMGB1 levels and elevated C3 levels in C1q-deficient mouse serum samples, compared to that in wild-type (WT) mice. APAP-induced C3 consumption was inhibited by sRAGE treatment in WT mice. Moreover, in a mouse model of brain ischemia-reperfusion injury based on middle cerebral arterial occlusion, C5b-9 complexes were deposited on vessels where HMGB1 was accumulated, an effect that was suppressed upon HMGB1 neutralization. We propose that the HMGB1 released after cell necrosis and in ischemic condition can trigger the classical pathway of complement activation to exacerbate sterile inflammation.

DNA-Mediated Interferon Signature Induction by SLE Serum Occurs in Monocytes Through Two Pathways: A Mechanism to Inhibit Both Pathways.

A primary mechanism for activation of innate immunity is recognition of damage or pathogen associated molecular patterns by pattern recognition receptors (PRRs). Nucleic acid is a damage associated molecular pattern molecule that when internalized into a monocyte and recognized by intracellular nucleic acid sensing toll like receptors will cause production of type 1 interferon. The process by which DNA or RNA is delivered into the cytosol of monocytes in systemic lupus erythematosus remains incompletely understood, and therapeutic approaches to prevent DNA-mediated monocyte activation are needed. We identified two mechanisms for internalization of DNA by monocytes. IgG-bound DNA was internalized by interacting with Fc gamma receptor IIa, while high-mobility group box-1 protein-bound DNA was internalized by interacting with the receptor for advanced glycation end products. Both pathways contribute to an inflammatory phenotype in monocytes exposed to serum from patients with SLE. Moreover, both of these pathways can be inhibited by a pentapeptide, DWEYS, which is a DNA mimetope. In one instance DWEYS directly competes with DNA for antibody binding and in the other DWEYS binds high-mobility group box-1 and blocks its interaction with RAGE. Our data highlight distinct pathways involved in nucleic acid enters monocytes in SLE, and identify a potential therapeutic to prevent nucleic acid internalization in SLE.

Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression.

C1q collagen-like region (CLR) engaging and activating the LAIR-1 inhibitory immunoreceptor represents a non-complement mechanism for maintaining immune quiescence. Given the binding promiscuity of C1q's globular region (gC1q), we hypothesized that C1q concurrently associates with distinct inhibitory immunoreceptors to produce C1q-mediated modulatory networking. Like LAIR-1, CD33 inhibitory immunoreceptors are highly expressed on monocytes. Binding CD33 restricts cell activation/differentiation; however, natural ligands for CD33 remain elusive. CD33 has IgC2-like domains potentially recognized by gC1q. Thus, we asked whether C1q binds to CD33 and if C1q mediates CD33/LAIR-1 crosslinking. Our findings demonstrate that C1q and gC1q interact with CD33 to activate its inhibitory motifs, while CLR does not. Whole C1q is required to crosslink CD33 and LAIR-1 and concurrently activate CD33/LAIR-1 inhibitory motifs. While C1q binds CD33C2 domains, decreased C1q-CD33 interactions resulting from sialic acid masking of CD33C2 domains suggests a process for regulating C1q-CD33 activity. Consistent with defective self-tolerance, CD33/LAIR-1 expression is reduced in systemic lupus erythematosus (SLE) myelomonocytes. The anti-inflammatory cytokine M-CSF, but not DC growth factors, sustains CD33/LAIR-1 expression on both healthy and SLE cells suggesting further biological control of C1q-CD33/LAIR-1 processes.

C1q and HMGB1 reciprocally regulate human macrophage polarization.

A healthy immune system results from a balance of stimulatory and inhibitory pathways that allow effective responses to acute insults, without descending into chronic inflammation. Failed homeostasis is characteristic of autoimmune diseases such as systemic lupus erythematosus. Although HMGB1 induces proinflammatory M1-like macrophage differentiation, we describe a mechanism by which C1q modulates this activity and collaborates with HMGB1 to induce the differentiation of monocytes to anti-inflammatory M2-like macrophages. These anti-inflammatory macrophages are unresponsive to dendritic cell induction factors, effectively removing them from participation in an adaptive immune response. This pathway is mediated through a complex with RAGE and LAIR-1 and depends on relative levels of C1q and HMGB1. Importantly, these data provide insight into a homeostatic mechanism in which C1q and HMGB1 can cooperate to terminate inflammation, and which may be impaired in C1q-deficient patients with autoimmune disease.

SLE-associated risk factors affect DC function.

Numerous risk alleles for systemic lupus erythematosus (SLE) have now been identified. Analysis of the expression of genes with risk alleles in cells of hematopoietic origin demonstrates them to be most abundantly expressed in B cells and dendritic cells (DCs), suggesting that these cell types may be the drivers of the inflammatory changes seen in SLE. DCs are of particular interest as they act to connect the innate and the adaptive immune response. Thus, DCs can transform inflammation into autoimmunity, and autoantibodies are the hallmark of SLE. In this review, we focus on mechanisms of tolerance that maintain DCs in a non-activated, non-immunogenic state. We demonstrate, using examples from our own studies, how alterations in DC function stemming from either DC-intrinsic abnormalities or DC-extrinsic regulators of function can predispose to autoimmunity.

Fundamental role of C1q in autoimmunity and inflammation.

C1q, historically viewed as the initiating component of the classical complement pathway, also exhibits a variety of complement-independent activities in both innate and acquired immunity. Recent studies focusing on C1q's suppressive role in the immune system have provided new insight into how abnormal C1q expression and bioactivity may contribute to autoimmunity. In particular, molecular networks involving C1q interactions with cell surface receptors and other ligands are emerging as mechanisms involved in C1q's modulation of immunity. Here, we discuss the role of C1q in controlling immune cell function, including recently elucidated mechanisms of action, and suggest how these processes are critical for maintaining tissue homeostasis under steady-state conditions and in preventing autoimmunity.

C1q-mediated repression of human monocytes is regulated by leukocyte-associated Ig-like receptor 1 (LAIR-1).

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by abnormal function of both the innate and the adaptive immune system, leading to a loss of tolerance to self-antigens. Monocytes are a key component of the innate immune system and are efficient producers of multiple cytokines. In SLE, inappropriate activation of monocytes is thought to contribute to the loss of self-tolerance. In this study, we demonstrate that type 1 interferon (IFN) production by CpG-challenged monocytes can be suppressed by C1q through activating leukocyte-associated Ig-like receptor-1 (LAIR-1), which contains immunoreceptor tyrosine-based inhibition motifs (ITIMs). The phosphorylation of LAIR-1 and the interaction of LAIR-1 with SH2 domain-containing protein tyrosine phosphatase-1 (SHP-1) were enhanced after LAIR-1 engagement by C1q. Moreover, engagement of LAIR-1 by C1q inhibited nuclear translocation of interferon regulatory factor (IRF)-3 and IRF5 in CpG-stimulated monocytes. These data suggest a model in which LAIR-1 engagement by C1q helps maintain monocyte tolerance, specifically with respect to Toll-like receptor-9-mediated monocyte activation.

Anti-DNA antibodies cross-react with C1q.

Systemic lupus erythematosus (SLE) is an autoimmune disorder that involves multiple organ systems and typically presents as a chronic inflammatory disease. Antibodies to double-stranded (ds) DNA are present in approximately 70% of patients and form nucleic acid containing immune complexes which activate dendritic cells through engagement of toll-like receptors, leading to a pro-inflammatory, pro-immunogenic milieu. In addition, anti-dsDNA antibodies deposit in kidneys to initiate glomerulonephritis. Antibodies to C1q have also been implicated in lupus nephritis and are found in 30-50% of patients. C1q is a known suppressor of immune activation and C1q deficiency is the strongest risk factor for SLE. We previously identified a subset of anti-DNA antibodies that binds the N-methyl-D-aspartate receptor. We now show that both mouse and human anti-DNA antibodies with this specificity bind C1q. These antibodies bind to Clq in glomeruli and exhibit decreased glomerular deposition in the absence of C1q. We propose that this subset of anti-DNA antibodies participates in lupus pathogenesis through direct targeting of C1q on glomeruli and also through removal of soluble C1q thereby limiting the ability of C1q to mediate immune homeostasis.