Verbenalin alleviates acute lung injury induced by sepsis and IgG immune complex through GPR18 receptor.

Acute lung injury is significantly associated with the aberrant activation and pyroptosis of alveolar macrophages. Targeting the GPR18 receptor presents a potential therapeutic approach to mitigate inflammation. Verbenalin, a prominent component of Verbena in Xuanfeibaidu (XFBD) granules, is recommended for treating COVID-19. In this study, we demonstrate the therapeutic effect of verbenalin on lung injury through direct binding to the GPR18 receptor. Verbenalin inhibits the activation of inflammatory signaling pathways induced by lipopolysaccharide (LPS) and IgG immune complex (IgG IC) via GPR18 receptor activation. The structural basis for verbenalin's effect on GPR18 activation is elucidated through molecular docking and molecular dynamics simulations. Furthermore, we establish that IgG IC induces macrophage pyroptosis by upregulating the expression of GSDME and GSDMD through CEBP-δ activation, while verbenalin inhibits this process. Additionally, we provide the first evidence that IgG IC promotes the formation of neutrophil extracellular traps (NETs), and verbenalin suppresses NETs formation. Collectively, our findings indicate that verbenalin functions as a "phytoresolvin" to promote inflammation regression and suggests that targeting the C/EBP-δ/GSDMD/GSDME axis to inhibit macrophage pyroptosis may represent a novel strategy for treating acute lung injury and sepsis.

The presence of activating IgG Fc receptors in macrophages aggravates the development of experimental abdominal aortic aneurysm. / La presencia de receptores Fc de IgG activadores en macrófagos agrava el desarrollo de aneurisma aórtico abdominal experimental.

INTRODUCTION: Abdominal aortic aneurysm (AAA) is a multifactorial, degenerative disease characterized by progressive aortic dilation and chronic activation of inflammation, proteolytic activity, and oxidative stress in the aortic wall. The immune response triggered by antibodies against antigens present in the vascular wall participates in the formation and progression of AAA through mechanisms not completely understood. This work analyses the function of specific IgG receptors (FcγR), especially those expressed by monocytes/macrophages, in the development of experimental AAA. METHODS: In the elastase-induced AAA model, the abdominal aortas from wildtype and FcγR deficient mice with/without macrophage adoptive transfer were analysed by histology and quantitative PCR. In vitro, mouse macrophages were transfected with RNA interference of FcγRIV/CD16.2 or treated with Syk kinase inhibitor before stimulation with IgG immune complexes. RESULTS: Macrophage adoptive transfer in FcγR deficient mice increased the susceptibility to AAA development. Mice receiving macrophages with functional FcγR exhibited higher aortic diameter increase, higher content of macrophages and B lymphocytes, and upregulated expression of chemokine CCL2, cytokines (TNF-α and IL-17), metalloproteinase MMP2, prooxidant enzyme NADPH oxidase-2, and the isoforms FcγRIII/CD16 and FcγRIV/CD16.2. In vitro, both FcγRIV/CD16.2 gene silencing and Syk inhibition reduced cytokines and reactive oxygen species production induced by immune complexes in macrophages. CONCLUSIONS: Activation of macrophage FcγR contributes to AAA development by inducing mediators of inflammation, proteolysis, and oxidative stress. Modulation of FcγR or effector molecules may represent a potential target for AAA treatment.

Skin testing might have a diagnostic role in immune complex-mediated hypersensitivity reactions.

Clinical applications of skin testing are known to help diagnose IgE-mediated and T-cell-mediated delayed cutaneous reactions. By contrast, drug-induced immune complex-mediated vasculitis is primarily diagnosed based on medical history, clinical setting and laboratory evidence of immune-complex formation, as there are no proven methods to identify the suspect culprit. We report three cases of drug- or biologic-induced immune complex-mediated vasculitis, in which the culprit agents could be confirmed by a positive intradermal test with later reading (between 12 and 24 h after the test), with verification by immunohistochemical or immunofluorescent results. The findings of our study suggest that skin tests with a delayed reading could have a potential role in diagnosing some instances of immune complex-mediated hypersensitivity reactions.

Laboratory Testing for Heparin-Induced Thrombocytopenia and Vaccine-Induced Immune Thrombotic Thrombocytopenia Antibodies: A Narrative Review.

Heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombotic thrombocytopenia (VITT) are highly prothrombotic (thrombosis frequency ≥50%). Both are caused by platelet-activating anti-platelet factor 4 (PF4) antibodies, forming PF4/IgG-containing immune complexes that engage platelet FcγIIa receptors, producing strong platelet activation. In HIT, heparin crosslinks several PF4 molecules, whereas in VITT, anti-PF4 antibodies alone crosslink PF4. Sufficient levels of circulating anti-PF4 antibodies are needed to create the pathogenic immune complexes on platelet surfaces; this explains why certain serum (plasma)-based assays are highly sensitive for detecting HIT/VITT antibodies. Accordingly, HIT and VITT are "clinical-pathological" disorders, that is, positive testing for such antibodies-together with a compatible clinical picture-is integral for diagnosis. Heparin (low concentrations) enhances HIT antibody-induced platelet activation, but platelet activation by VITT sera is usually inhibited by heparin. For both HIT and VITT, high sensitivity (>99% and >95%, respectively) characterizes PF4-dependent enzyme immunoassays (EIAs) and PF4-enhanced platelet activation assays; in contrast, certain rapid immunoassays have high sensitivity for HIT (>90-97%) but poor sensitivity (<25%) for VITT. HIT and VITT antibodies are directed at distinct sites on PF4: solid-phase EIAs and platelet activation assays are indifferent to these distinct antigen targets, but rapid immunoassays are not. We discuss a conceptual model where PF4 is viewed as a "globe," with the heparin-binding site the "equator"; in this model, HIT antibodies are primarily directed at antigen site(s) at the north and south "poles" of PF4 (formed when PF4 binds to heparin), whereas VITT antibodies recognize sites on the equator.

A case of immune complex type hemolytic anemia induced by initial micafungin administration.

We report the first case of immune complex type hemolytic anemia by initial micafungin administration that was given as prophylaxis to a 42-year-old Japanese man receiving chemotherapy for primary amyloidosis. The few cases found in the literature were associated with secondary administration causing immune hemolytic attacks. Despite its rarity, the present case calls for increased awareness of micafungin-induced hemolytic anemia upon initial administration.

The COVID Complex: A Review of Platelet Activation and Immune Complexes in COVID-19.

Coronavirus disease 2019 (COVID-19) is a highly prothrombotic viral infection that primarily manifests as an acute respiratory syndrome. However, critically ill COVID-19 patients will often develop venous thromboembolism with associated increases in morbidity and mortality. The cause for this prothrombotic state is unclear but is likely related to platelet hyperactivation. In this review, we summarize the current evidence surrounding COVID-19 thrombosis and platelet hyperactivation. We highlight the fact that several studies have identified a soluble factor in COVID-19 patient plasma that is capable of altering platelet phenotype in vitro. Furthermore, this soluble factor appears to be an immune complex, which may be composed of COVID-19 Spike protein and related antibodies. We suggest that these Spike-specific immune complexes contribute to COVID-19 platelet activation and thrombosis in a manner similar to heparin-induced thrombocytopenia. Understanding this underlying pathobiology will be critical for advancement of future research and therapeutic options.

Fcγ receptor activation mediates vascular inflammation and abdominal aortic aneurysm development.

BACKGROUND: Abdominal aortic aneurysm (AAA), a degenerative vascular pathology characterized by permanent dilation of the aorta, is considered a chronic inflammatory disease involving innate/adaptive immunity. However, the functional role of antibody-dependent immune response against antigens present in the damaged vessel remains unresolved. We hypothesized that engagement of immunoglobulin G (IgG) Fc receptors (FcγR) by immune complexes (IC) in the aortic wall contributes to AAA development. We therefore evaluated FcγR expression in AAA lesions and analysed whether inhibition of FcγR signaling molecules (γ-chain and Syk kinase) influences AAA formation in mice. METHODS: FcγR gene/protein expression was assessed in human and mouse AAA tissues. Experimental AAA was induced by aortic elastase perfusion in wild-type (WT) mice and γ-chain knockout (γKO) mice (devoid of activating FcγR) in combination with macrophage adoptive transfer or Syk inhibitor treatment. To verify the mechanisms of FcγR in vitro, vascular smooth muscle cells (VSMC) and macrophages were stimulated with IgG IC. RESULTS: FcγR overexpression was detected in adventitia and media layers of human and mouse AAA. Elastase-perfused γKO mice exhibited a decrease in AAA incidence, aortic dilation, elastin degradation, and VSMC loss. This was associated with (1) reduced infiltrating leukocytes and immune deposits in AAA lesions, (2) inflammatory genes and metalloproteinases downregulation, (3) redox balance restoration, and (4) converse phenotype of anti-inflammatory macrophage M2 and contractile VSMC. Adoptive transfer of FcγR-expressing macrophages aggravated aneurysm in γKO mice. In vitro, FcγR deficiency attenuated inflammatory gene expression, oxidative stress, and phenotypic switch triggered by IC. Additionally, Syk inhibition prevented IC-mediated cell responses, reduced inflammation, and mitigated AAA formation. CONCLUSION: Our findings provide insight into the role and mechanisms mediating IgG-FcγR-associated inflammation and aortic wall injury in AAA, which might represent therapeutic targets against AAA disease.

GILZ Regulates the Expression of Pro-Inflammatory Cytokines and Protects Against End-Organ Damage in a Model of Lupus.

Glucocorticoid-induced leucine zipper (GILZ) mimics many of the anti-inflammatory effects of glucocorticoids, suggesting it as a point of therapeutic intervention that could bypass GC adverse effects. We previously reported that GILZ down-regulation is a feature of human SLE, and loss of GILZ permits the development of autoantibodies and lupus-like autoimmunity in mice. To further query the contribution of GILZ to protection against autoimmune inflammation, we studied the development of the lupus phenotype in Lyn-deficient (Lyn-/-) mice in which GILZ expression was genetically ablated. In Lyn-/- mice, splenomegaly, glomerulonephritis, anti-dsDNA antibody titres and cytokine expression were exacerbated by GILZ deficiency, while other autoantibody titres and glomerular immune complex deposition were unaffected. Likewise, in patients with SLE, GILZ was inversely correlated with IL23A, and in SLE patients not taking glucocorticoids, GILZ was also inversely correlated with BAFF and IL18. This suggests that at the onset of autoimmunity, GILZ protects against tissue injury by modulating pro-inflammatory pathways, downstream of antibodies, to regulate the cycle of inflammation in SLE.

Antibody Complexes.

Monoclonal based therapeutics have always been looked at as a futuristic natural way we could take care of pathogens and many diseases. However, in order to develop, establish and realize monoclonal based therapy we need to understand how the immune system contains or kill pathogens. Antibody complexes serve the means to decode this black box. We have discussed examples of antibody complexes both at biochemical and structural levels to understand and appreciate how discoveries in the field of antibody complexes have started to decoded mechanism of viral invasion and create potential vaccine targets against many pathogens. Antibody complexes have made advancement in our knowledge about the molecular interaction between antibody and antigen. It has also led to identification of potent protective monoclonal antibodies. Further use of selective combination of monoclonal antibodies have provided improved protection against deadly diseases. The administration of newly designed and improved immunogen has been used as potential vaccine. Therefore, antibody complexes are important tools to develop new vaccine targets and design an improved combination of monoclonal antibodies for passive immunization or protection with very little or no side effects.

FcγRIIb attenuates TLR4­mediated NF­κB signaling in B cells.

Toll­like receptors (TLRs) serve a vital role in activating the innate immune system by sensing conserved microbial products. Fc γ receptor IIb (FcγRIIb), the inhibitory Fc receptor, exerts its immune regulatory functions by binding to the immunoglobulin G Fc domain. Although the individual roles of TLRs and FcγRIIb have been studied intensively, the cross­talk between FcγRIIb and TLR4 on B cells remains unknown. The present study demonstrated that FcγRIIb ligation by the immune complex (IC) attenuated the TLR4­triggered nuclear factor (NF)­κΒ activation, and decreased the release of interleukin (IL)­6 from B cells, via enhancing LYN proto­oncogene (Lyn) phosphorylation. In addition, IC treatment protected mice from lethal endotoxic shock. Accordingly, IC decreased the LPS­induced serum levels of IL­6, as well as intracellular IL­6 production in B cells in vivo. However, these protective and inhibitory effects of IC were not observed in FcγRIIb­/­ mice. In conclusion, the present data demonstrated that FcγRIIb inhibited TLR4 signaling in B cells by activating Lyn phosphorylation and by inhibiting NF­κΒ signaling. The present study elucidated the mechanism associated with the TLR4 and FcγRIIb cross­talk in B cells.

Recombinant immune complexes as versatile and potent vaccines.

Immune complexes (IC) used as vaccines have the potential to enhance both antibody and cell-mediated immune responses over those obtained with antigen alone. However, difficulty of manufacture represents a significant hurdle to the widespread use of IC as vaccines. Recombinant IC (RIC) and their expression in plants enable manufacturing by the coordinate expression of immunoglobulin and antigen as a fusion protein. The use of a modular RIC system facilitates insertion of antigen genes and provides a broadly applicable platform that can be adapted for a wide variety of antigens.

Modulation of T cell responses by IL-2 and IL-2 complexes.

Interleukin-2 (IL-2) is a cytokine centrally involved in the regulation of immune tolerance and activation by its effects on CD4+ T regulatory (Treg) cells and cytotoxic effector lymphocytes, respectively. Due to these properties IL-2 immunotherapy has been used, as low-dose IL-2, in the treatment of autoimmune and chronic-inflammatory disorders; conversely, at high doses, IL-2 has shown efficacy in a subset of patients with metastatic cancer. Recent advances have highlighted the possibility of using improved IL-2-based therapies, such IL-2-antibody complexes (IL-2 complexes), able to selectively and potently stimulate either Treg cells or cytotoxic effector cells. This article discusses the properties and clinical implications of IL-2 and IL-2 complexes.

Do immune complexes play a role in hemolytic sequelae of intravenous immune globulin?

Intravenous immune globulin (IVIG) was developed initially as an immunoglobulin replacement therapy for primary humoral immunodeficiency, but is now widely used in the treatment of autoinflammatory and autoimmune pathologies. In a small number of patients, hemolytic sequelae have been observed after IVIG administration. The lack of a simple one-to-one correlation between measurable hemagglutinins and hemolysis has led to complicated hypotheses involving coincident necessary variables (e.g., a two-hit hypothesis) and also to the positing of causal factors other than hemagglutinins. One such hypothesis is that immune complexes (ICs) contained within IVIG lead to hemolysis. IVIG-mediated hemolysis was addressed at a recent meeting sponsored by the Food and Drug Administration; the Plasma Protein Therapeutics Association; and the National Heart, Lung, and Blood Institute. The primary literature was reviewed at this meeting followed by detailed discussion. Participants concluded that there is both a theoretical basis by which ICs could contribute to hemolysis after IVIG administration and some published data in support of such a possibility. However, the reported data contain substantial caveats, and the existing evidence does not rise to a level sufficient to either confirm or reject a role for ICs. More detailed and focused human studies will be required to further assess the potential role of ICs in IVIG induced hemolysis. This paper summarizes the relevant literature and expands upon the conclusions of this workshop.

Neonatal Fc receptor promotes immune complex-mediated glomerular disease.

The neonatal Fc receptor (FcRn) is a major regulator of IgG and albumin homeostasis systemically and in the kidneys. We investigated the role of FcRn in the development of immune complex-mediated glomerular disease in mice. C57Bl/6 mice immunized with the noncollagenous domain of the α3 chain of type IV collagen (α3NC1) developed albuminuria associated with granular capillary loop deposition of exogenous antigen, mouse IgG, C3 and C5b-9, and podocyte injury. High-resolution imaging showed abundant IgG deposition in the expanded glomerular basement membrane, especially in regions corresponding to subepithelial electron dense deposits. FcRn-null and -humanized mice immunized with α3NC1 developed no albuminuria and had lower levels of serum IgG anti-α3NC1 antibodies and reduced glomerular deposition of IgG, antigen, and complement. Our results show that FcRn promotes the formation of subepithelial immune complexes and subsequent glomerular pathology leading to proteinuria, potentially by maintaining higher serum levels of pathogenic IgG antibodies. Therefore, reducing pathogenic IgG levels by pharmacologic inhibition of FcRn may provide a novel approach for the treatment of immune complex-mediated glomerular diseases. As proof of concept, we showed that a peptide inhibiting the interaction between human FcRn and human IgG accelerated the degradation of human IgG anti-α3NC1 autoantibodies injected into FCRN-humanized mice as effectively as genetic ablation of FcRn, thus preventing the glomerular deposition of immune complexes containing human IgG.

Cgnz1 allele confers kidney resistance to damage preventing progression of immune complex-mediated acute lupus glomerulonephritis.

Cgnz1 and Agnz1 on the distal region of mouse chromosome 1 are associated with chronic glomerulonephritis (cGN) and acute GN (aGN). NZM2328.Lc1R27 (R27) was generated by introgressing a C57L/J region where Cgnz1 is located to NZM2328. R27 female mice developed aGN mediated by immune complex (IC) deposition and complement activation without progression to cGN with severe proteinuria. End stage renal disease (ESRD) was not seen in R27 mice as old as 15 mo. Thus, aGN and cGN are under separate genetic control, and IC-mediated proliferative GN need not progress to cGN and ESRD. NZM2328 and R27 female mice have comparable immune and inflammatory parameters. In contrast to NZM2328, R27 mice were resistant to sheep anti-mouse GBM serum-induced nephritis, supporting the hypothesis that aGN is mediated by autoimmunity and resistance to the development of cGN is mediated by end organ resistance to damage. Thus, autoimmunity should be considered distinct from end organ damage. The Cgnz1 region has been mapped to a 1.34 MB region with 45 genes. Nine candidate genes were identified. Clinical relevance of these observations is supported by case studies. Clinical implications and the significance to human lupus and other diseases are presented.

Critical role for CCAAT/enhancer-binding protein ß in immune complex-induced acute lung injury.

C/EBPs, particularly C/EBPß and C/EBPδ, are known to participate in the regulation of many genes associated with inflammation. However, very little is known regarding the activation and functions of C/EBPß and C/EBPδ in acute lung inflammation and injury. In this study, we show that both C/EBPß and C/EBPδ activation are triggered in lungs and in alveolar macrophages following intrapulmonary deposition of IgG immune complexes. We further show that mice carrying a targeted deletion of the C/EBPß gene displayed significant attenuation of the permeability index (lung vascular leak of albumin), lung neutrophil accumulation (myeloperoxidase activity), total number of WBCs, and neutrophils in bronchoalveolar lavage fluids compared with wild-type mice. Moreover, the mutant mice expressed considerably less TNF-α, IL-6, and CXC/CC chemokine and soluble ICAM-1 proteins in bronchoalveolar lavage fluids, and corresponding mRNAs in the IgG immune complex-injured lung, compared with wild-type mice. These phenotypes were associated with a significant reduction in morphological lung injury. In contrast, C/EBPδ deficiency had no effect on IgG immune complex-induced lung injury. IgG immune complex-stimulated C/EBPß-deficient alveolar macrophages released significantly less TNF-α, IL-6, MIP-2, keratinocyte cell-derived chemokine, and MIP-1α compared with wild-type cells. Similar decreases in IgG immune complex-induced inflammatory mediator production were observed following small interfering RNA ablation of C/EBPß in a murine alveolar macrophage cell line. These findings implicate C/EBPß as a critical regulator of IgG immune complex-induced inflammatory responses and injury in the lung.

Establishment and characterization of a murine model for allergic asthma using allergen-specific IgE monoclonal antibody to study pathological roles of IgE.

Allergen-specific IgE has long been regarded as a major molecular component of allergic asthma. Although IgE plays a central role in the early asthmatic response, its roles in the chronic phase, such as the late asthmatic response, airway hyperresponsiveness (AHR), and airway remodeling (goblet cell hyperplasia and subepithelial fibrosis) have not yet been defined well. In this study, we investigated the hypothesis that chronic responses could be induced by IgE-dependent mechanisms. BALB/c mice passively sensitized with an ovalbumin (OVA)-specific IgE monoclonal antibody (mAb) were repeatedly challenged with intratracheal administration of OVA. The first challenge induced early phase airway narrowing without any late response, but the fourth challenge caused not only an early but also a late phase response, AHR, and goblet cell hyperplasia. Macrophages, lymphocytes and neutrophils, but not eosinophils, were significantly increased in the lung 24h after the fourth challenge. Interestingly, levels of OVA-specific IgG1 in serum increased by multiple antigen challenges. A C3a receptor antagonist inhibited the late asthmatic response, AHR, and infiltration by neutrophils. In contrast, no late response, goblet cell hyperplasia, inflammatory cells, or production of IgG1 was observed in severe combined immunodeficient mice. On the other hand, seven challenges in BALB/c mice induced subepithelial fibrosis associated with infiltration by eosinophils. In conclusion, the allergic asthmatic responses induced by passive sensitization with IgE mAb can provide a useful model system to study the pathological roles of IgE in acute and chronic phases of allergic asthma.

Aggregation, immune complexes and immunogenicity.

The development of an immune response to a protein therapeutic may nullify its beneficial activity or result in adverse events. Immunogenicity is, therefore, a major concern for clinicians, regulatory authorities and the biopharmaceutical industry. These concerns are particularly acute for the treatment of chronic diseases, as opposed to cancer, that may require repeated exposure to therapeutic over extended cycles of remission/relapse. There are many parameters that may be contributory to immunogenicity; however, the "bête noire," for the past decade has been aggregation. ( 1-3).

Pathological role of tonsillar B cells in IgA nephropathy.

Although impaired immune regulation along the mucosa-bone marrow axis has been postulated to play an important role, the pathogenesis of IgA nephropathy (IgAN) is unknown; thus, no disease-specific therapy for this disease exists. The therapeutic efficacy of tonsillectomy or tonsillectomy in combination with steroid pulse therapy for IgAN has been discussed. Although randomized control trials for these therapies are ongoing in Japan, the scientific rationale for these therapies remains obscure. It is now widely accepted that abnormally glycosylated IgA1 and its related immune complex (IC) are probably key molecules for the pathogenesis, and are thus considered possible noninvasive biomarkers for this disease. Emerging evidence indicates that B cells in mucosal infections, particularly in tonsillitis, may produce the nephritogenic IgA. In this paper, we briefly summarize characteristics of the nephritogenic IgA/IgA IC, responsible B cells, and underlying mechanisms. This clinical and experimental information may provide important clues for a therapeutic rationale.