Precision engineered peptide targeting leukocyte extracellular traps mitigate acute kidney injury in Crush syndrome.

Crush syndrome induced by skeletal muscle compression causes fatal rhabdomyolysis-induced acute kidney injury (RIAKI) that requires intensive care, including hemodialysis. However, access to crucial medical supplies is highly limited while treating earthquake victims trapped under fallen buildings, lowering their chances of survival. Developing a compact, portable, and simple treatment method for RIAKI remains an important challenge. Based on our previous finding that RIAKI depends on leukocyte extracellular traps (ETs), we aimed to develop a novel medium-molecular-weight peptide to provide clinical treatment of Crush syndrome. We conducted a structure-activity relationship study to develop a new therapeutic peptide. Using human peripheral polymorphonuclear neutrophils, we identified a 12-amino acid peptide sequence (FK-12) that strongly inhibited neutrophil extracellular trap (NET) release in vitro and further modified it by alanine scanning to construct multiple peptide analogs that were screened for their NET inhibition ability. The clinical applicability and renal-protective effects of these analogs were evaluated in vivo using the rhabdomyolysis-induced AKI mouse model. One candidate drug [M10Hse(Me)], wherein the sulfur of Met10 is substituted by oxygen, exhibited excellent renal-protective effects and completely inhibited fatality in the RIAKI mouse model. Furthermore, we observed that both therapeutic and prophylactic administration of M10Hse(Me) markedly protected the renal function during the acute and chronic phases of RIAKI. In conclusion, we developed a novel medium-molecular-weight peptide that could potentially treat patients with rhabdomyolysis and protect their renal function, thereby increasing the survival rate of victims affected by Crush syndrome.

Inhibitory effect of aspirin on inflammation-induced lung metastasis of cancer cells associated with neutrophil infiltration.

PURPOSE: Systematic inflammation has been reported to contribute to cancer progression through various mechanisms; however, the exact mechanism is still the subject of research. In this study, we evaluated the influence of systematic inflammation on lung metastasis, using a murine abdominal sepsis model, and assessed its relationship with pneumonia after curative esophagectomy in patients with esophageal cancer. METHODS: We used a murine abdominal sepsis model given highly metastatic osteosarcoma, to reveal the mechanism of systematic inflammation and its potential for lung metastasis. The therapeutic effect of aspirin (ASA) in preventing distant metastasis was also investigated. Subsequently, we analyzed, retrospectively, the relationship between pneumonia and lung metastasis after esophagectomy in patients who underwent esophagectomy at Keio University between January, 2007 and October, 2020. RESULTS: Abdominal sepsis provoked lung injury in the acute phase. ASA inhibited the recruitment of neutrophils triggered by the lung injury, and it also suppressed lung metastasis. Our retrospective study revealed that lung metastasis was more frequent in patients with postoperative pneumonia. CONCLUSIONS: Postoperative acute lung injury is associated with a higher risk of lung metastasis. ASA may be a potential preoperative treatment for inhibiting lung metastasis by preventing the recruitment of neutrophils.

FcγR engagement reprograms neutrophils into antigen cross-presenting cells that elicit acquired anti-tumor immunity.

Classical dendritic cells (cDC) are professional antigen-presenting cells (APC) that regulate immunity and tolerance. Neutrophil-derived cells with properties of DCs (nAPC) are observed in human diseases and after culture of neutrophils with cytokines. Here we show that FcγR-mediated endocytosis of antibody-antigen complexes or an anti-FcγRIIIB-antigen conjugate converts neutrophils into nAPCs that, in contrast to those generated with cytokines alone, activate T cells to levels observed with cDCs and elicit CD8+ T cell-dependent anti-tumor immunity in mice. Single cell transcript analyses and validation studies implicate the transcription factor PU.1 in neutrophil to nAPC conversion. In humans, blood nAPC frequency in lupus patients correlates with disease. Moreover, anti-FcγRIIIB-antigen conjugate treatment induces nAPCs that can activate autologous T cells when using neutrophils from individuals with myeloid neoplasms that harbor neoantigens or those vaccinated against bacterial toxins. Thus, anti-FcγRIIIB-antigen conjugate-induced conversion of neutrophils to immunogenic nAPCs may represent a possible immunotherapy for cancer and infectious diseases.

Inhibitory affinity modulation of FcγRIIA ligand binding by glycosphingolipids by inside-out signaling.

The interaction of the human FcγRIIA with immune complexes (ICs) promotes neutrophil activation and thus must be tightly controlled to avoid damage to healthy tissue. Here, we demonstrate that a fungal-derived soluble ß-1,3/1,6-glucan binds to the glycosphingolipid long-chain lactosylceramide (LacCer) to reduce FcγRIIA-mediated recruitment to immobilized ICs under flow, a process requiring high-affinity FcγRIIA-immunoglobulin G (IgG) interactions. The inhibition requires Lyn phosphorylation of SHP-1 phosphatase and the FcγRIIA immunotyrosine-activating motif. ß-glucan reduces the effective 2D affinity of FcγRIIA for IgG via Lyn and SHP-1 and, in vivo, inhibits FcγRIIA-mediated neutrophil recruitment to intravascular IgG deposited in the kidney glomeruli in a glycosphingolipid- and Lyn-dependent manner. In contrast, ß-glucan did not affect FcγR functions that bypass FcγR affinity for IgG. In summary, we have identified a pathway for modulating the 2D affinity of FcγRIIA for ligand that relies on LacCer-Lyn-SHP-1-mediated inhibitory signaling triggered by ß-glucan, a previously described activator of innate immunity.

Group A Streptococcus establishes pharynx infection by degrading the deoxyribonucleic acid of neutrophil extracellular traps.

Group A Streptococcus (GAS) secretes deoxyribonucleases and evades neutrophil extracellular killing by degrading neutrophil extracellular traps (NETs). However, limited information is currently available on the interaction between GAS and NETs in the pathogenicity of GAS pharyngitis. In this study, we modified a mouse model of GAS pharyngitis and revealed an essential role for DNase in this model. After intranasal infection, the nasal mucosa was markedly damaged near the nasal cavity, at which GAS was surrounded by neutrophils. When neutrophils were depleted from mice, GAS colonization and damage to the nasal mucosa were significantly decreased. Furthermore, mice infected with deoxyribonuclease knockout GAS mutants (∆spd, ∆endA, and ∆sdaD2) survived significantly better than those infected with wild-type GAS. In addition, the supernatants of digested NETs enhanced GAS-induced cell death in vitro. Collectively, these results indicate that NET degradation products may contribute to the establishment of pharyngeal infection caused by GAS.

Humanised effector-null FcγRIIA antibody inhibits immune complex-mediated proinflammatory responses.

OBJECTIVE: Immune complexes (ICs) play a critical role in the pathology of autoimmune diseases. The aim of this study was to generate and characterise a first-in-class anti-FcγRIIA antibody (Ab) VIB9600 (previously known as MEDI9600) that blocks IgG immune complex-mediated cellular activation for clinical development. METHODS: VIB9600 was humanised and optimised from the IV.3 Ab. Binding affinity and specificity were determined by Biacore and ELISA. Confocal microscopy, Flow Cytometry-based assays and binding competition assays were used to assess the mode of action of the antibody. In vitro cell-based assays were used to demonstrate suppression of IC-mediated inflammatory responses. In vivo target suppression and efficacy was demonstrated in FcγRIIA-transgenic mice. Single-dose pharmacokinetic (PK)/pharmacodynamic study multiple dose Good Laboratory Practice (GLP) toxicity studies were conducted in non-human primates. RESULTS: We generated a humanised effector-deficient anti-FcγRIIA antibody (VIB9600) that potently blocks autoantibody and IC-mediated proinflammatory responses. VIB9600 suppresses FcγRIIA activation by blocking ligand engagement and by internalising FcγRIIA from the cell surface. VIB9600 inhibits IC-induced type I interferons from plasmacytoid dendritic cells (involved in SLE), antineutrophil cytoplasmic antibody (ANCA)-induced production of reactive oxygen species by neutrophils (involved in ANCA-associated vasculitis) and IC-induced tumour necrosis factor α and interleukin-6 production (involved in rheumatoid arthritis). In FcγRIIA transgenic mice, VIB9600 suppressed antiplatelet antibody-induced thrombocytopaenia, acute anti-GBM Ab-induced nephritis and anticollagen Ab-induced arthritis. VIB9600 also exhibited favourable PK and safety profiles in cynomolgus monkey studies. CONCLUSIONS: VIB9600 is a specific humanised antibody antagonist of FcγRIIA with null effector function that warrants further clinical development for the treatment of IC-mediated diseases.

Cis interaction between sialylated FcγRIIA and the αI-domain of Mac-1 limits antibody-mediated neutrophil recruitment.

Vascular-deposited IgG immune complexes promote neutrophil recruitment, but how this process is regulated is still unclear. Here we show that the CD18 integrin Mac-1, in its bent state, interacts with the IgG receptor FcγRIIA in cis to reduce the affinity of FcγRIIA for IgG and inhibit FcγRIIA-mediated neutrophil recruitment under flow. The Mac-1 rs1143679 lupus-risk variant reverses Mac-1 inhibition of FcγRIIA, as does a Mac-1 ligand and a mutation in Mac-1's ligand binding αI-domain. Sialylated complex glycans on FcγRIIA interact with the αI-domain via divalent cations, and this interaction is required for FcγRIIA inhibition by Mac-1. Human neutrophils deficient in CD18 integrins exhibit augmented FcγRIIA-dependent recruitment to IgG-coated endothelium. In mice, CD18 integrins on neutrophils dampen IgG-mediated neutrophil accumulation in the kidney. In summary, cis interaction between sialylated FcγRIIA and the αI-domain of Mac-1 alters the threshold for IgG-mediated neutrophil recruitment. A disruption of this interaction may increase neutrophil influx in autoimmune diseases.

Macrophage extracellular trap formation promoted by platelet activation is a key mediator of rhabdomyolysis-induced acute kidney injury.

Rhabdomyolysis is a serious syndrome caused by skeletal muscle injury and the subsequent release of breakdown products from damaged muscle cells into systemic circulation. The muscle damage most often results from strenuous exercise, muscle hypoxia, medications, or drug abuse and can lead to life-threatening complications, such as acute kidney injury (AKI). Rhabdomyolysis and the AKI complication can also occur during crush syndrome, an emergency condition that commonly occurs in victims of natural disasters, such as earthquakes, and man-made disasters, such as wars and terrorism. Myoglobin released from damaged muscle is believed to trigger renal dysfunction in this form of AKI. Recently, macrophages were implicated in the disease pathogenesis of rhabdomyolysis-induced AKI, but the precise molecular mechanism remains unclear. In the present study, we show that macrophages released extracellular traps (ETs) comprising DNA fibers and granule proteins in a mouse model of rhabdomyolysis. Heme-activated platelets released from necrotic muscle cells during rhabdomyolysis enhanced the production of macrophage extracellular traps (METs) through increasing intracellular reactive oxygen species generation and histone citrullination. Here we report, for the first time to our knowledge, this unanticipated role for METs and platelets as a sensor of myoglobin-derived heme in rhabdomyolysis-induced AKI. This previously unknown mechanism might be targeted for treatment of the disease. Finally, we found a new therapeutic tool for prevention of AKI after rhabdomyolysis, which might rescue some sufferers of this pathology.

Lactoferrin Suppresses Neutrophil Extracellular Traps Release in Inflammation.

Neutrophils are central players in the innate immune system. They generate neutrophil extracellular traps (NETs), which protect against invading pathogens but are also associated with the development of autoimmune and/or inflammatory diseases and thrombosis. Here, we report that lactoferrin, one of the components of NETs, translocated from the cytoplasm to the plasma membrane and markedly suppressed NETs release. Furthermore, exogenous lactoferrin shrunk the chromatin fibers found in released NETs, without affecting the generation of oxygen radicals, but this failed after chemical removal of the positive charge of lactoferrin, suggesting that charge-charge interactions between lactoferrin and NETs were required for this function. In a model of immune complex-induced NET formation in vivo, intravenous lactoferrin injection markedly reduced the extent of NET formation. These observations suggest that lactoferrin serves as an intrinsic inhibitor of NETs release into the circulation. Thus, lactoferrin may represent a therapeutic lead for controlling NETs release in autoimmune and/or inflammatory diseases.

Immunomodulation with eicosapentaenoic acid supports the treatment of autoimmune small-vessel vasculitis.

Small-vessel vasculitis is a life-threatening autoimmune disease that is frequently associated with anti-neutrophil cytoplasmic antibodies (ANCAs). Conventional immunotherapy including steroids and cyclophosphamide can cause serious adverse events, limiting the efficacy and safety of treatment. Eicosapentaenoic acid (EPA), a key component of fish oil, is an omega-3 polyunsaturated fatty acid widely known to be cardioprotective and beneficial for vascular function. We report two elderly patients with systemic ANCA-associated vasculitis (AAV) in whom the administration of EPA in concert with steroids safely induced and maintained remission, without the use of additioal immunosuppressants. To explore the mechanisms by which EPA enhances the treatment of AAV, we employed SCG/Kj mice as a spontaneous murine model of AAV. Dietary enrichment with EPA significantly delayed the onset of crescentic glomerulonephritis and prolonged the overall survival. EPA-derived anti-inflammatory lipid mediators and their precursors were present in the kidney, plasma, spleen, and lungs in the EPA-treated mice. Furthermore, a decrease in ANCA production and CD4/CD8-double negative T cells, and an increase in Foxp3(+) regulatory T cells in the lymph nodes of the kidney were observed in the EPA-treated mice. These clinical and experimental observations suggest that EPA can safely support and augment conventional therapy for treating autoimmune small-vessel vasculitis.