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Human neutrophil peptides (HNPs) can induce cell proliferation and activation so their growth promoting activities may have potential clinical benefit. This study investigated the effects of HNPs on human dermal fibroblasts. Differential gene expression in HNP-treated cells and genes involved in regulating intracellular pathways were explored. Dermal fibroblasts were isolated from healthy neonatal foreskin and treated with HNPs in 2D and 3D cell culture systems. The expression of cell proliferation (Ki-67) gene and cell activation (COL1A1) gene plus their proteins was measured. Differential gene expression was determined using RNA-seq, and upregulated and downregulated genes were mapped onto intracellular pathways by KEGG analysis and Gene Ontology databases. HNPs significantly increased cell proliferation without cytotoxicity whilst HNP1 enhanced expression of COL1A1 and type I collagen production in 2D cells and 3D spheroids. RNA-sequencing analysis showed gene clustering with clear separation between HNP1-treated and control groups. A heatmap of top 50 differentially expressed genes was consistent among HNP1-treated samples. Most upregulated genes were associated with cell proliferation and activation as mapped into intracellular pathways whilst most downregulated genes belonged to steroid/arachidonic acid metabolism and inflammatory signaling pathways. HNP1 increased cell proliferation and activation but reduced lipid metabolism and inflammation., (© 2023. Springer Nature Limited.)

Renal ischemia is the most common cause of acute kidney injury (AKI) that might be exacerbate lupus activity through neutrophil extracellular traps (NETs) and apoptosis. Here, the renal ischemia reperfusion injury (I/R) was performed in Fc gamma receptor 2b deficient (Fcgr2b-/-) lupus mice and the in vitro experiments. At 24 h post-renal I/R injury, NETs in peripheral blood neutrophils and in kidneys were detected using myeloperoxidase (MPO), neutrophil elastase (NE) and citrullinated histone H3 (CitH3), as well as kidney apoptosis (activating caspase-3), which were prominent in Fcgr2b-/- mice more compared to wild-type (WT). After 120 h renal-I/R injury, renal NETs (using MPO and NE) were non-detectable, whereas glomerular immunoglobulin (Ig) deposition and serum anti-dsDNA were increased in Fcgr2b-/- mice. These results imply that renal NETs at 24 h post-renal I/R exacerbated the lupus nephritis at 120 h post-renal I/R injury in Fcgr2b-/- lupus mice. Furthermore, a Syk inhibitor attenuated NETs, that activated by phorbol myristate acetate (PMA; a NETs activator) or lipopolysaccharide (LPS; a potent inflammatory stimulator), more prominently in Fcgr2b-/- neutrophils than the WT cells as determined by dsDNA, PAD4 and MPO. In addition, the inhibitors against Syk and PAD4 attenuated lupus characteristics (serum creatinine, proteinuria, and anti-dsDNA) in Fcgr2b-/- mice at 120 h post-renal I/R injury. In conclusion, renal I/R in Fcgr2b-/- mice induced lupus exacerbation at 120 h post-I/R injury partly because Syk-enhanced renal NETs led to apoptosis-induced anti-dsDNA, which was attenuated by a Syk inhibitor., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Saisorn, Saithong, Phuengmaung, Udompornpitak, Bhunyakarnjanarat, Visitchanakun, Chareonsappakit, Pisitkun, Chiewchengchol and Leelahavanichkul.)

Juvenile systemic lupus erythematosus (JSLE) is one of the most common autoimmune diseases in children and can affect multiple organs and systems. The etiology remains unclear, and current management only suppresses rather than eliminates the disease. The pathogenesis is triggered by autoantigens that induce autoantibody production. Apoptotic neutrophils may be one source of autoantigens in JSLE, and increased numbers of apoptotic neutrophils in JSLE have been reported. This study aimed to determine if factor(s) in JSLE serum induce neutrophil apoptosis, to identify the most potent cytokine in delaying neutrophil apoptosis, and to investigate whether this cytokine can reverse the pro-apoptotic effects of JSLE serum. Blood neutrophils and sera were collected from JSLE patients, healthy children and adult controls. Neutrophils from healthy adult controls were incubated with 10 % serum from either JSLE patients or pediatric controls. Neutrophils from healthy adult controls were also incubated with 10 % JSLE serum with or without granulocyte-macrophage colony-stimulating factor (GM-CSF) supplementation. Neutrophil apoptosis was measured by flow cytometry (annexin-V/propidium iodide staining). Caspase-3, caspase-7 and caspase-8 protein expression was detected using Western blotting. Neutrophils incubated with JSLE sera had significantly increased apoptosis at 6 h compared to those incubated with control sera. Cleaved (active) forms of caspase-3, caspase-7 and caspase-8 were identified in neutrophils incubated with JSLE sera (that showed high rates of apoptosis) compared to control sera. GM-CSF had the most protective effect on neutrophil apoptosis, significantly preventing neutrophil apoptosis and caspase activation induced by JSLE serum. JSLE serum significantly induced neutrophil apoptosis in healthy adult neutrophils, activating the extrinsic pathway of apoptosis. The observation that GM-CSF prevents activation of apoptosis in response to JSLE serum should prompt further studies to evaluate the therapeutic potential of this cytokine for the treatment of JSLE.

Kritsanawan Sae-khow,1 Sasipha Tachaboon,2 Helen L Wright,3 Steven W Edwards,4 Nattachai Srisawat,2 Asada Leelahavanichkul,1 Direkrit Chiewchengchol1 1Translational Research in Inflammation and Immunology Research Unit, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; 2Excellence Center for Critical Care Nephrology, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand; 3Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK; 4Institute of Integrative Biology, University of Liverpool, Liverpool, UKCorrespondence: Direkrit Chiewchengchol Email cdirekrit@live.comBackground: Neutrophil function is essential for effective defence against bacterial infections but is defective in patients with sepsis. Ascorbate or vitamin C, which is low in the plasma of patients with sepsis, is stored inside human neutrophils and is essential for their normal function.Objective: This study aimed to determine if ascorbate treatment ex vivo improved neutrophil function in patients with sepsis.Patients and Methods: Human blood neutrophils were isolated from 20 patients with sepsis and 20 healthy age-matched controls. Neutrophils were incubated with or without ascorbate (1, 5, 10, 20 and 40 mM) for periods up to 2h. Chemotaxis was evaluated using a chemotactic chamber in response to the chemoattractant, fMLP. Phagocytosis (uptake of pHrodo red stained S. aureus) and apoptosis (annexin-V/propidium iodide staining) were measured by flow cytometry. Neutrophil extracellular trap (NET) formation was detected and quantified using DAPI, anti-myeloperoxidase and anti-neutrophil elastase immuno-fluorescence staining. Quantifluor detected the amount of dsDNA in NET supernatants, while quantitative PCR identified changes in expression of PADI4 gene.Results: Chemotactic and phagocytic activities were decreased in patients with sepsis but increased after treatment with the high concentrations of ascorbate. Apoptosis was increased in the sepsis patients but not altered by ascorbate treatment. Spontaneous NET formation was observed in patients with sepsis. A quantity of 1mM ascorbate decreased spontaneous NETosis to that of normal, healthy neutrophils, while high concentrations of ascorbate (> 10mM) further promoted NET formation.Conclusion: Dysregulated neutrophil function was observed in patients with sepsis which could contribute to disease pathology and outcomes. Exposure to ascorbate could reverse some of these changes in function. These novel discoveries raise the possibility that ascorbate treatment could be used as an adjunctive therapy that could result in improved neutrophil function during sepsis.Keywords: ascorbate, neutrophils, sepsis