Increase In Number Of Neutrophils Research Articles

IL-17 signaling pathway: A potential therapeutic target for reducing skeletal muscle inflammation

BackgroundThe interleukin-17 (IL-17) signaling pathway is intricately linked with immunity and inflammation; however, the association between the IL-17 signaling pathway and skeletal muscle inflammation remains poorly understood. The study aims to investigate the role of the IL-17 signaling pathway in skeletal muscle inflammation and to evaluate the therapeutic potential of anti-IL-17 antibodies in reducing muscle inflammation. MethodsA skeletal muscle inflammation model was induced by cardiotoxin (CTX) injection in C57BL6/J mice. Following treatment with an anti-IL-17 antibody, we conducted a comprehensive analysis integrating single-cell RNA sequencing (scRNA-seq), bioinformatics, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and Western blot techniques to elucidate underlying mechanisms. ResultsscRNA-seq analysis revealed a significant increase in neutrophil numbers and activity in inflamed skeletal muscle compared to other cell types, including macrophages, T cells, B cells, endothelial cells, fast muscle cells, fibroblasts, and skeletal muscle satellite cells. The top 30 differentially expressed genes within neutrophils, along with 55 chemokines, were predominantly enriched in the IL-17 signaling pathway. Moreover, the IL-17 signaling pathway exhibited heightened expression in inflamed skeletal muscle, particularly within neutrophils. Treatment with anti-IL-17 antibody resulted in the suppression of IL-17 signaling pathway expression, accompanied by reduced levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, as well as decreased numbers and activity of Ly6g+/Mpo+ neutrophils compared to CTX-induced skeletal muscle inflammation. ConclusionOur findings suggest that the IL-17 signaling pathway plays a crucial role in promoting inflammation within skeletal muscle. Targeting this pathway may hold promise as a therapeutic strategy for ameliorating the inflammatory micro-environment and reducing cytokine production.

Identification of purinergic system components in the venom of Bothrops mattogrossensis and the inhibitory effect of specioside extracted from Tabebuia aurea.

Snake bites are a severe problem in the countryside of Brazil and are usually attributed to snakes of the genera Bothrops, Crotalus, and Lachesis. Snake venom can release ectoenzymes and nucleotidases that modulate the purinergic system. In addition to serum therapy against snake poisoning, medicinal plants with anti-inflammatory activities, such as Tabebuia aurea, is empirically applied in accidents that occur in difficult-to-access areas. This study aimedwas to verify the presence and activity of nucleotidases in the crude venom of Bothrops mattogrossensis (BmtV) in vitro and characterize the modulation of purinergic components, myeloid differentiation, and inflammatory/oxidative stress markers by BmtV in vivo and in vitro. Moreover, our study assessed the inhibitory activities of specioside, an iridoid isolated from Tabebuia aurea, against the effects of BmtV. Proteomic analysis of venom content and nucleotidase activity confirm the presence of ectonucleotidase-like enzymes in BmtV. In in vivo experiments, BmtV altered purinergic component expression (P2X7 receptor, CD39 and CD73), increased neutrophil numbers in peripheral blood, and elevated oxidative stress/inflammatory parameters such as lipid peroxidation and myeloperoxidase activity. BmtV also decreased viability and increased spreading index and phagocytic activity on macrophages. Specioside inhibited nucleotidase activity, restored neutrophil numbers, and mediate the oxidative/inflammatory effects produced by BmtV. We highlight the effects produced by BmtV in purinergic system components, myeloid differentiation, and inflammatory/oxidative stress parameters, while specioside reduced the main BmtV-dependent effects.

ATG16L1 in myeloid cells limits colorectal tumor growth in ApcMin/+ mice infected with colibactin-producing Escherichia coli via decreasing inflammasome activation

ABSTRACT Escherichia coli strains producing the genotoxin colibactin, designated as CoPEC (colibactin-producing E. coli), have emerged as an important player in the etiology of colorectal cancer (CRC). Here, we investigated the role of macroautophagy/autophagy in myeloid cells, an important component of the tumor microenvironment, in the tumorigenesis of a susceptible mouse model infected with CoPEC. For that, a preclinical mouse model of CRC, the Apc Min/+ mice, with Atg16l1 deficiency specifically in myeloid cells (Apc Min/+ /Atg16l1[∆MC]) and the corresponding control mice (Apc Min/+ ), were infected with a clinical CoPEC strain 11G5 or its isogenic mutant 11G5∆clbQ that does not produce colibactin. We showed that myeloid cell-specific Atg16l1 deficiency led to an increase in the volume of colonic tumors in Apc Min/+ mice under infection with 11G5, but not with 11G5∆clbQ. This was accompanied by increased colonocyte proliferation, enhanced inflammasome activation and IL1B/IL-1β secretion, increased neutrophil number and decreased total T cell and cytotoxic CD8+ T cell numbers in the colonic mucosa and tumors. In bone marrow-derived macrophages (BMDMs), compared to uninfected and 11G5∆clbQ-infected conditions, 11G5 infection increased inflammasome activation and IL1B secretion, and this was further enhanced by autophagy deficiency. These data indicate that ATG16L1 in myeloid cells was necessary to inhibit colonic tumor growth in CoPEC-infected Apc Min/+ mice via inhibiting colibactin-induced inflammasome activation and modulating immune cell response in the tumor microenvironment. Abbreviation: AOM, azoxymethane; APC, APC regulator of WNT signaling pathway; ATG, autophagy related; Atg16l1[∆MC] mice, mice deficient for Atg16l1 specifically in myeloid cells; CASP1, caspase 1; BMDM, bone marrow-derived macrophage; CFU, colony-forming unit; CoPEC, colibactin-producing Escherichia coli; CRC, colorectal cancer; CXCL1/KC, C-X-C motif chemokine ligand 1; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; MC, myeloid cell; MOI, multiplicity of infection; PBS, phosphate-buffered saline; pks, polyketide synthase; qRT-PCR, quantitative real-time reverse-transcription polymerase chain reaction; siRNA, small interfering RNA; TME, tumor microenvironment; TNF/TNF-α, tumor necrosis factor.

Leukemia inhibitory factor receptor inhibition reduces stenosis grade and total serum cholesterol levels in a mouse model for atherosclerosis

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Dutch Heart Foundation Atherosclerosis is characterized by the accumulation of lipids and immune cells in the arterial wall and it is the main underlying pathology causing cardiovascular diseases (CVD). Cytokines are involved in all stages of atherosclerosis and most cytokines contribute to disease progression. For instance, IL-6 cytokine family members, such as IL-6, oncostatin-M and cardiotrophin-1, are closely related to CVDs. However, the role of leukemia inhibitory factor (LIF), a member of the IL-6 cytokine family, and its receptor (LIFR) in atherosclerosis remains unknown. Therefore, the aim of this study is to assess the contribution of LIFR signaling to atherosclerosis development by systemically inhibiting LIFR in an atherosclerotic mouse model. First, the expression of LIF and LIFR was examined using single-cell RNA sequencing data of human carotid artery plaques. Mast cells highly and exclusively express LIF, whereas LIFR was specifically expressed by activated endothelial cells. To validate LIFR signaling in vivo, female western-type diet fed Ldlr-/- mice were treated with LIFR inhibitor EC359 (5 mg/kg s.c., n=15) or control solvent (n=15) three times per week for eight weeks. During the experiment, weights of the mice did not differ between groups, whereas total serum cholesterol levels were significantly reduced in EC359 treated mice (p=0.03). In particular, EC359 treatment reduced VLDL levels. Additionally, EC359 treatment significantly reduced Sr1, Abca1 and Lrp1 expression in the liver, suggesting an altered lipid metabolism in EC359 treated mice. Furthermore, LIFR inhibition affected innate immunity, both systemically and locally in the plaque. Neutrophil numbers were significantly increased in the circulation (ctrl 18.7±1.7%; EC359 28.0±2.0%; p=0.002) and aortic plaques (ctrl 3.9±0.8%; EC359 9.9±1.9%; p=0.003) of mice treated with EC359 compared to control mice. Moreover, LIFR inhibition affected circulating monocytes resulting in less inflammatory monocytes. The adaptive immune cells, such as T and B cells, remained largely unaffected. Collectively, the reduction of serum total cholesterol and increase in neutrophil numbers in EC359 treated mice resulted in a trend towards reduced atherosclerotic stenosis grade compared to control mice (39.8±1.4% versus 34.9±1.9%; p=0.054), but it did not affect plaque collagen and monocyte/macrophage content in the aortic root. Conclusively, LIFR signaling inhibition reduces atherosclerotic stenosis grade, lowers plasma total cholesterol levels and affects innate immunity.

#1116 Hyperuricemia worsens bacterial sepsis by suppressing neutrophil functions in CKD

Abstract Background and Aims Patients with kidney disease, especially those on dialysis, are more susceptible to infections such as peritonitis, sepsis, pneumonia, and are at a higher risk for infection-related mortality. Pathogenic mechanisms underlying the secondary immunodeficiency in kidney disease include gut dysbiosis and barrier dysfunction, persistent inflammation, immune paralysis due to increased levels of immunoregulatory metabolites (e.g. uric acid) and proteins (e.g. FGF23) [1]. Currently, it is unknown whether CKD-related hyperuricemia may contribute to the immune paralysis in this context. We hypothesized that hyperuricemia (HU) may aggravate the innate immune response to infection in CKD in mice. Method Mono- and polybacterial sepsis was induced by LPS injection or cecal ligation and puncture in Alb-creERT2;Glut9lox/lox mice with HU and/or CKD, and in Glut9lox/lox (healthy) control mice. After 24 hours, systemic inflammation was evaluated by measuring serum cytokine levels, immune cell activation and recruitment, and neutrophil extracellular trap release. Tissue injury in liver, heart, and kidney was assessed by performing RT-qPCR and immunohistochemistry. In addition, kidney function was determined by measuring the glomerular filtration rate (GFR). Additionally, mice were treated with urate-lowering therapy to reduce serum uric acid levels and the immune response and functional outcomes after sepsis is quantified. Results We found that both mono- and polybacterial sepsis caused systemic inflammation as indicated by an increased number of blood neutrophils (see Figure), neutrophil activation and pro-inflammatory cytokine levels as compared to the PBS-treated or sham-operated control groups in Glut9lox/lox healthy mice, respectively. Mice with sepsis also displayed a slightly worsened kidney function. Interestingly, HU significantly increased neutrophil numbers but decreased their activation status and the ability of neutrophils to form neutrophil extracellular traps in Alb-creERT2;Glut9lox/lox mice with sepsis as compared to the control groups, which was further aggravated in mice with CKD-related HU (CKD+HU). This impaired inflammatory response was partially reversible by lowering serum uric acid with febuxostat. Conclusion Our results identify hyperuricemia related or unrelated to kidney disease as immune regulator in bacterial infection by suppressing neutrophil functions in mice. Specifically targeting uric acid may help to overcome the secondary immunodeficiency related to kidney disease during infection but enhances sterile inflammation [2].

Inhibitory Effects of <italic>Ssanghwa-tang</italic> on Lung Injury and Muscle Loss in a Cigarette Smoke Extract and Lipopolysaccharide-induced Chronic Obstructive Pulmonary Disease Mouse Model

Objectives: This study evaluated the effects of <i>Ssanghwa-tang</i> (SHT) on lung injury and muscle loss in a COPD mouse model.Methods: C57BL/6 mice were challenged with cigarette smoke extract and lipopolysaccharide, and then treated with two concentrations of SHT (250 and 500 mg/kg). After sacrifice, the bronchoalveolar lavage fluid (BALF) or lung tissue was analyzed by cytospin, ELISA, real-time PCR, flow cytometry analysis, and H&E and Masson’s trichrome staining. The grip strength of COPD mice was measured using a grip strength meter. The running time of COPD mice was measured by a treadmill test. Muscle tissue of the quadriceps was stained with H&E and Masson’s trichrome staining.Results: SHT significantly inhibited the increase in neutrophil numbers in BALF and significantly decreased immune cell activity in BALF and lung tissue. It also significantly inhibited the increase in TNF-α, IL-17, and MIP2 in BALF. Real-time PCR analysis revealed that the mRNA expression of TNF-α, IL-17, MIP2, and TRPV1 in lung tissue showed a significant decrease compared with the control group. Lung tissue damage was significantly reduced in the histological analysis. The grip strength and running time of the COPD mice showed a significant decrease compared with the control group. In histological staining, SHT was found to reduce the damage to muscle tissue.Conclusions: This study indicates that SHT can be used as a therapeutic agent for COPD patients by inhibiting lung injury and muscle loss.

Helicobacter pylori CagA Promotes the Formation of Gallstones by Increasing the Permeability of Gallbladder Epithelial Cells.

The formation of gallstones is often accompanied by chronic inflammation, and the mechanisms underlying inflammation and stone formation are not fully understood. Our aim is to utilize single-cell transcriptomics, bulk transcriptomics, and microbiome data to explore key pathogenic bacteria that may contribute to chronic inflammation and gallstone formation, as well as their associated mechanisms. scRNA-seq data from a gallstone mouse model were extracted from the Gene Expression Omnibus (GEO) database and analyzed using the FindCluster() package for cell clustering analysis. Bulk transcriptomics data from patients with gallstone were also extracted from the GEO database, and intergroup functional differences were assessed using GO and KEGG enrichment analysis. Additionally, 16S rRNA sequencing was performed on gallbladder mucosal samples from asymptomatic patients with gallstone (n = 6) and liver transplant donor gallbladder mucosal samples (n = 6) to identify key bacteria associated with stone formation and chronic inflammation. Animal models were constructed to investigate the mechanisms by which these key pathogenic bacterial genera promote gallstone formation. Analysis of scRNA-seq data from the gallstone mouse model (GSE179524) revealed seven distinct cell clusters, with a significant increase in neutrophil numbers in the gallstone group. Analysis of bulk transcriptomics data from patients with gallstone (GSE202479) identified chronic inflammation in the gallbladder, potentially associated with dysbiosis of the gallbladder microbiota. 16S rRNA sequencing identified Helicobacter pylori as a key bacterium associated with gallbladder chronic inflammation and stone formation. Dysbiosis of the gallbladder mucosal microbiota is implicated in gallstone disease and leads to chronic inflammation. This study identified H. pylori as a potential key mucosal resident bacterium contributing to gallstone formation and discovered its key pathogenic factor CagA, which causes damage to the gallbladder mucosal barrier. These findings provide important clues for the prevention and treatment of gallstones.

Do alternative tobacco products induce less adverse respiratory risk than cigarettes?

RationaleDue to the relatively short existence of alternative tobacco products, gaps exist in our current understanding of their long-term respiratory health effects. We therefore undertook the first-ever side-by-side comparison of the impact of chronic inhalation of aerosols emitted from electronic cigarettes (EC) and heated tobacco products (HTP), and combustible cigarettes (CC) smoke.ObjectivesTo evaluate the potential differential effects of alternative tobacco products on lung inflammatory responses and efficacy of vaccination in comparison to CC.MethodsMice were exposed to emissions from EC, HTP, CC, or air for 8 weeks. BAL and lung tissue were analyzed for markers of inflammation, lung damage, and oxidative stress. Another group was exposed for 12 weeks and vaccinated and challenged with a bacterial respiratory infection. Antibody titers in BAL and sera and pulmonary bacterial clearance were assessed.Main resultsEC- and HTP-aerosols significantly augmented lung immune cell infiltrates equivalent to that achieved following CC-exposure. HTP and CC significantly increased neutrophil numbers compared to EC. All products augmented numbers of B cells, T cells, and pro-inflammatory IL17A+ T cells in the lungs. Decreased lung antioxidant activity and lung epithelial and endothelial damage was induced by all products. EC and HTP differentially augmented inflammatory cytokines/chemokines in the BAL. Generation of immunity following vaccination was impaired by EC and HTP but to a lesser extent than CC, with a CC > HTP > EC hierarchy of suppression of pulmonary bacterial clearance.ConclusionsHTP and EC-aerosols induced a proinflammatory pulmonary microenvironment, lung damage, and suppressed efficacy of vaccination.

131 Alveolar Macrophage Degranulation Initiates the Spatially Restricted Innate Immune Response During Ventilator-Assisted Nanoparticle Inhalation

Abstract To visualize and measure in real-time the cellular pulmonary innate immune response elicited by inhaled NPs, we apply state-of-the-art intravital microscopy on the alveolar region of the murine lung. Fluorescent carboxyl-quantum-dot-NPs (deposited dose of 16 cm2/g) applied via ventilator-assisted inhalation accumulated within seconds as distinct fluorescent spots at the alveolar walls. Already at 30min, cQD-NPs elicited an increase in microvascular neutrophil numbers as compared to controls, which subsequently transmigrated into the alveolar space. This early immune response was not specific to cQD-NPs, since a comparable increase in neutrophil numbers was also observed upon inhalation of a bioequivalent dose of carbon black NPs, a typical component of urban air pollution. AMs actively accumulated cQD-NPs over time, starting few minutes after inhalation. Significantly more neutrophils were attracted in proximity to cQD-NP-laden AMs, as compared to NP-free AMs or control areas with epithelial associated cQD-NP. Prior airway application of a cellular degranulation inhibitor prevented cQD-NP and CNP induced neutrophil recruitment at 2h and 24 h after inhalation. Inhibition of Fcγ-receptor mediated AM cQD-NP uptake by prior application of anti-CD64 mAbs or neutralizing of TNFα by anti-TNFα mAbs in the airspace, abolished this early immune response. Importantly, initial ImageStream flow cytometry analysis of BAL samples indicated an increase of macrophage-derived microvesicles upon NP inhalation. Taken together, our data links NP-induced AM degranulation and cytokine release to the rapid and site-specific recruitment of neutrophils, suggesting this process to be a key event in mounting the immune response upon NP inhalation.

45 Behavior and Effect of Nanoparticles on Neutrophil Recruitment in the Pulmonary Microcirculation

Abstract NP exposure is frequently associated with adverse cardiovascular effects. A small fraction of NP deposited in lung has been shown to penetrate into the blood circulation. However, the interactions and effects of NP in the pulmonary microcirculation under healthy and pathophysiologic conditions such as LPS-induced acute lung injury are largely unknown. To visualize and quantify NP dynamics and pulmonary vascular innate immune responses elicited by different surface-modified NPs in real time, we applied state-of-the-art intra-vital microscopy in the alveolar region of murine lungs. I.v. injected polyethylene-glycol-amine (aPEG)-QDs and carboxyl-QDs exhibited a half-life time of 30 and 6 mins in the microcirculation and did not permanently interact with vessel wall. Blood flow velocity was significantly decreased after aPEG-QDs application in healthy mice, but did not further decrease the reduced microcirculatory perfusion after LPS treatment. In healthy mice, aPEG-QDs elicited a significant increase in neutrophil number in alveolar micro-vessels from 30 min upon application compared to controls, whereas cQDs induced only a slight neutrophil increment. Accordingly, the crawling velocity of neutrophils in pulmonary micro-vessels was significantly reduced after aPEG-QDs application. No effect of QDs on neutrophil number and crawling was detected in the LPS group. Importantly, neutrophil recruitment by aPEG-QDs was diminished by prior i.v. application of anti-TNFa, anti-E-selectin and anti-LFA-1 blocking antibodies. Taken together our data suggest that aPEG-QDs induced microvascular inflammation involving TNFa secretion, expression of the vascular cell adhesion molecule E-selectin and neutrophil integrin LFA-1, whereas QDs application did not alter LPS-dependent microvascular inflammation.

The effect of mitochondria-targeted slow hydrogen sulfide releasing donor AP39-treatment on airway inflammation

Mitochondrial dysfunction has been shown to contribute to the pathophysiology of airway diseases. Therefore, mitochondria are targeted in the development of new therapeutic approaches. Hydrogen sulfide (H2S) has been shown to be involved in the pathophysiological processes of airway inflammation. We aimed to evaluate the effect of mitochondria-targeted slow H2S releasing donor AP39 [(10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol5yl)phenoxy)decyl)triphenylphosphoniumbromide)] on lipopolysaccharide (LPS)-induced airway inflammation in mice. LPS was applied to female Balb/c mice by intranasal (i.n.) route to induce airway inflammation and the subgroups of mice were treated with i.n. AP39 (250–1000 nmol/kg). 48 h after LPS administration airway reactivity was evaluated in vivo, then bronchoalveolar lavage (BAL) fluid and lungs were collected. LPS application led to bronchial hyperreactivity and neutrophil infiltration into the lung tissues along with increased TNF-α, IL-1β and IL-6 levels in BAL fluid. LPS also induced an increase in the rate of glycolysis, glycogenolysis and Krebs-cycle. AP39 treatment prevented the LPS-induced bronchial hyperreactivity and reversed the increase in TNF-α and IL-6 levels in BAL fluid. The increase in neutrophil numbers in BAL fluid was also prevented by AP39 treatment at the highest dose. Our results indicate that AP39 can prevent bronchial hyperreactivity and decrease airway inflammation. Targeting H2S to the mitochondria may be a new therapeutic approach in airway inflammation.

Chemistry and lung toxicity of particulate matter emitted from firearms

Smoke emissions produced by firearms contain hazardous chemicals, but little is known if their properties change depending on firearm and ammunition type and whether such changes affect toxicity outcomes. Pulmonary toxicity was assessed in mice exposed by oropharyngeal aspiration to six different types of smoke-related particulate matter (PM) samples; (1) handgun PM, (2) rifle PM, (3) copper (Cu) particles (a surrogate for Cu in the rifle PM) with and without the Cu chelator penicillamine, (4) water-soluble components of the rifle PM, (5) soluble components with removal of metal ions, and (6) insoluble components of the rifle PM. Gun firing smoke PM was in the respirable size range but the chemical composition varied with high levels of Pb in the handgun and Cu in the rifle smoke. The handgun PM did not induce appreciable lung toxicity at 4 and 24 h post-exposure while the rifle PM significantly increased lung inflammation and reduced lung function. The same levels of pure Cu particles alone and the soluble components from the rifle fire PM increased neutrophil numbers but did not cause appreciable cellular damage or lung function changes when compared to the negative (saline) control. Penicillamine treated rifle PM or Cu, slightly reduced lung inflammation and injury but did not improve the lung function decrements. Chelation of the soluble metal ions from the rifle fire PM neutralized the lung toxicity while the insoluble components induced the lung toxicity to the same degree as the rifle PM. The results show that different firearm types can generate contrasting chemical spectra in their emissions and that the rifle PM effects were mostly driven by water-insoluble components containing high levels of Cu. These findings provide better knowledge of hazardous substances in gun firing smoke and their potential toxicological profile.

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice.

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection or damage. Currently, the widely used mice models of sepsis include lipopolysaccharide (LPS)-induced endotoxemia, cecal ligation and puncture (CLP), and monobacterial infection model systems. This protocol describes a method to study the host responses during Salmonella Typhimurium infection-induced septic peritonitis in mice. S. Typhimurium, a Gram-negative intracellular pathogen, causes typhoid-like disease in mice. This protocol elaborates the culture preparation, induction of septic peritonitis in mice through intraperitoneal injection, and methods to study systemic host responses. Furthermore, the assessment of bacterial burden in different organs and the flow cytometric analysis of increased neutrophil numbers in the peritoneal lavage is presented. Salmonella Typhimurium-induced sepsis in mice leads to an increase in proinflammatory cytokines and rapid infiltration of neutrophils in the peritoneal cavity, leading to lower survival. Every step in this protocol has been optimized, resulting in high reproducibility of the pathogenesis of septic peritonitis. This model is useful for studying immunological responses during bacterial sepsis, the roles of different genes in disease progression, and the effects of drugs to attenuate sepsis.

Influenza infection reduces disease in a B-cell dependent EAE model of multiple sclerosis

Abstract Multiple sclerosis (MS) is an autoimmune disease characterized by demyelination of neurons and inflammation in the central nervous system. MS afflicts approximately 2.5 million people worldwide. Understanding how viral infection affects MS disease is of great importance because the immune-suppressive effects of some MS therapies can make patients more vulnerable to viral infections. The goal of our project is to elucidate the effect of respiratory viral infection in a B cell-driven experimental autoimmune encephalomyelitis (EAE), which models the immune pathology of MS. Here, we describe a comorbid animal model in which mice were immunized with hMOG1–125 to induce EAE and subsequently infected prior to disease onset with a sublethal dose of the H1N1 Influenza A mouse-adapted A/Puerto Rico/8/1934 virus (IAV). Our data show that comorbid mice have a less severe EAE paralysis phenotype than mice with EAE alone. Flow cytometry analyses show that the lungs of comorbid mice had increased neutrophil numbers and a reciprocal reduction in MHCII+ CD86+ inflammatory monocytes. Comorbid mice also had fewer germinal center B cells in the lungs and reduced levels of IAV-specific IgM and IgG antibodies compared to IAV-infected controls. Secondary IAV challenge of comorbid mice revealed deficits in immunological B cell memory. Overall, our data show that EAE disrupts the antiviral immune response, including by decreasing the IAV-specific B cell response. Paradoxically, viral infection decreased EAE severity, indicating that the antiviral immune response attenuates B cell-dependent neuroinflammation. Future studies will determine mechanisms by which inflammation induced by viral infection modulates neuroinflammatory diseases. Supported by grants from NIH (R01AI137047 and R01EY027346) A grant awarded to Dr. Kovats and Dr. Axtell from the Presbyterian Health Foundation

Neutrophils predominate the immune signature of cerebral thrombi in COVID-19 stroke patients

Coronavirus disease 2019 (COVID-19) is associated with an increased risk of thrombotic events. Ischemic stroke in COVID-19 patients entails high severity and mortality rates. Here we aimed to analyze cerebral thrombi of COVID-19 patients with large vessel occlusion (LVO) acute ischemic stroke to expose molecular evidence for SARS-CoV-2 in the thrombus and to unravel any peculiar immune-thrombotic features. We conducted a systematic pathological analysis of cerebral thrombi retrieved by endovascular thrombectomy in patients with LVO stroke infected with COVID-19 (n = 7 patients) and non-covid LVO controls (n = 23). In thrombi of COVID-19 patients, the SARS-CoV-2 docking receptor ACE2 was mainly expressed in monocytes/macrophages and showed higher expression levels compared to controls. Using polymerase chain reaction and sequencing, we detected SARS-CoV-2 Clade20A, in the thrombus of one COVID-19 patient. Comparing thrombus composition of COVID-19 and control patients, we noted no overt differences in terms of red blood cells, fibrin, neutrophil extracellular traps (NETs), von Willebrand Factor (vWF), platelets and complement complex C5b-9. However, thrombi of COVID-19 patients showed increased neutrophil density (MPO+ cells) and a three-fold higher Neutrophil-to-Lymphocyte Ratio (tNLR). In the ROC analysis both neutrophils and tNLR had a good discriminative ability to differentiate thrombi of COVID-19 patients from controls. In summary, cerebral thrombi of COVID-19 patients can harbor SARS-CoV2 and are characterized by an increased neutrophil number and tNLR and higher ACE2 expression. These findings suggest neutrophils as the possible culprit in COVID-19-related thrombosis.Graphical

Systemic and local innate immune responses to surgical co-transplantation of mesenchymal stromal cells and biphasic calcium phosphate for bone regeneration

Bone regeneration from mesenchymal stromal cells (MSC) is attributed to comprehensive immune modulation mediated by the MSC. However, the temporal and spatial regulation of these immune responses has not yet been described. The aim of the present study was to assess the local and systemic innate immune responses to implantation of biphasic calcium phosphate biomaterial (BCP) alone, or with bone marrow derived MSC (BCP+MSC), in critical-sized calvarial bone defects of Lewis rats. Four weeks after implantation, flow cytometry analysis of innate immune cells revealed increased numbers of circulating classical monocyte-macrophages (MM) and decreased non-classical MM in the BCP+MSC group. At week 8, this differential systemic MM response was associated with an increased presence of local tissue anti-inflammatory macrophages expressing CD68 and CD163 markers (M2-like). In the BCP group without MSC, NK cells increased at weeks 1 and 4, and neutrophils increased in circulation at weeks 2 and 8. At week 8, the increase in number of neutrophils in circulation was associated with decreased local tissue neutrophils, in the BCP+MSC group. Gene expression analysis of tissue biopsies from defects implanted with BCP+MSC, in comparison to BCP alone, revealed upregulated expression of early osteogenesis genes along with macrophage differentiation-related genes at weeks 1 and 8 and neutrophil chemotaxis-related genes at week 1. This study is the first to demonstrate that surgical implantation of BCP or BCP+MSC grafts differentially regulate both systemic and local tissue innate immune responses which enhance bone formation. The results provide new insights into immune mechanisms underlying MSC-mediated bone regeneration. Statement of significanceThe suitability of biphasic calcium phosphate and mesenchymal stromal cell construct (BCP+MSC) transplantation is evident from their progress in clinical trials for treating challenging maxillofacial bone defects. But less is known about the overall immune response generated by this surgical process and how it later impacts the bone formation. To this end, it is crucial to understand for both clinicians and researchers, the systemic immune response to transplanting MSC in patients for ensuring both the safety and efficacy of cell therapies. In this study, we used rat calvarial bone defect model and showed that both systemic and local innate immunes responses (monocyte-macrophages and neutrophils) are favorably directed towards enhanced bone formation in BCP+MSC implanted defects, as compared to BCP alone.

Neutrophil Metabolic Rewiring in Severe and Mild COVID-19

▪SARS-CoV-2 infection can cause severe respiratory disease resulting in hospitalization and death in some people, but in others it causes a mild disease or is asymptomatic. The causes of severe COVID-19 are of profound public health importance. Severe COVID-19 is marked by an exacerbated innate immune response leading to hyperinflammation which damages host tissues. Broad inhibition of hyperinflammation by dexamethasone is a mainstay of severe COVID-19 treatment. However, more targeted therapeutic approaches to dampen hyperinflammation are lacking. Patients typically exhibit increased neutrophil numbers in the blood and lung, and increased formation of neutrophil extracellular traps. Previous work profiled the metabolome of plasma in severe COVID-19 patients and found several metabolic aberrations as compared to mild COVID-19 patients or healthy controls. However, the metabolome of immune cell populations in COVID-19 has not been characterized to our knowledge. In addition, it is not known if metabolic changes observed in severe COVID-19 mechanistically contribute to hyperinflammation and host tissue damage or if they are simply markers of pathology.To understand how metabolism can regulate the innate immune system in COVID-19 and to explore possible therapeutic targets, we carried out a comprehensive metabolomic study of neutrophils purified from healthy controls, mild/moderate COVID-19 patients, and severe COVID-19 patients. Severe COVID-19 patients were defined as having Acute Respiratory Distress and being treated in the intensive care unit. Plasma from the same patients and healthy controls was also collected and metabolomics was performed to study how COVID-19 impacts the neutrophil and plasma metabolome in tandem.Several metabolic pathways were significantly different between mild COVID-19 patients, severe COVID-19 patients, and healthy controls, consistent with rewired neutrophil and plasma metabolome in severe COVID-19. Differentially abundant metabolites belonged to central carbon metabolism, nucleotide metabolism, amino acid metabolism and other pathways. Variation in the PMN metabolome was largely independent from variation in the plasma metabolome. This suggests that processes internal to neutrophils drive changes in neutrophil metabolism. Metabolomics in 3 mouse models of hyperinflammation showed common or distinct metabolic features that change in inflammation compared to metabolomics of severe COVID-19 patients. Therefore changes in some metabolites comprise a general hyperinflammatory metabolic signature while others are more specific to severe COVID-19. Isotope tracing experiments were carried out to understand how nutrients were utilized in neutrophils in context of inflammation. We will describe mechanistic work testing whether metabolic features we observe in severe COVID-19 patients regulate the inflammatory response of human neutrophils. DisclosuresNo relevant conflicts of interest to declare.

The Effect of Lipoteichoic Acid from Lactobacillus plantarum on Dental Pulp Inflammation.

ObjectiveLactobacillus plantarum, a bacterium located in deep caries, has a virulence factor in the form of lipoteichoic acid (LTA), which is found in the bacterial cell wall. LTA is able to trigger a neutrophils response in the dental pulp inflammation process within the first 6 to 24 hours. The quantity of bacteria is one factor influencing the increase in number of neutrophils in addition to the quality of the bacteria. This study seeks to analyze the effect of lipoteichoic acid of Lactobacillus plantarum (LTA-Lp) in the dental pulp inflammation by observing the number of neutrophil cells in a histopathological view. Materials and Methods The LTA was isolated from L. plantarum. The left upper molar of Rattus novergicus was mechanically perforated under anesthesia to induce dental pulp inflammation. The perforated tooth was then induced by 10 and 15 µg/mL of LTA-Lp and then restored by a temporary filling. The perforated tooth in the control group was only restored by a temporary filling. After 24, 48, and 72 hours, the tooth was extracted and then stained with hematoxylins and eosin to observe the neutrophils in the dental pulp via a light microscope. Result The number of neutrophils in the dental pulp after induction by 15 µg/mL of LTA-Lp is higher than 10 µg/mL of LTA-Lp and both controls. There were significant differences in the number of neutrophils in the dental pulp, in each group on 24, 48, and 72 hours after LTA-Lp inducing ( p < 0.05). Conclusion The LTA-Lp dose of of 10 and 15 µg/mL affected the dental pulp inflammation by affecting the number of neutrophils.

CTLA4-FasL, the Hexameric Targeted FasL Fusion Protein, As Potential Novel Treatment for DLBCL

The bi-functional drug CTLA4-FasL (KAHR-102) is a fusion protein composed of the extracellular domains of CTLA4 and FasL. It naturally forms a stable homo-hexamer capable of targeting two different cellular receptors; the CTLA4 domains target B7 receptors (CD80 and CD86) on cells bearing these receptors, while the FasL domains target functional Fas-receptors (CD95) to induce apoptosis. Although CTLA4-FasL can induce apoptosis in all cells expressing functional Fas receptors, it is at least 100 times more potent on cells expressing both B7 and functional Fas receptors. The higher efficacy in cells expressing both receptors is adjoined by robust activation of pro-apoptotic intracellular pathways in conjunction to abrogation of anti-apoptotic ones. This unique combined activity cannot be detected in cells bearing just Fas receptors and can be blocked by preventing the binding of the protein to CD80 and CD86. Both B7 and functional Fas receptors are expressed on lymphoma cells, mainly of the B cell lineage, making them exceptionally sensitive to the CTLA4-FasL apoptotic effect.CTLA4-FasL was found to be highly efficient in inducing apoptosis in different resistant DLBCL cell-lines bearing translocations such as , FARAGE, OCI-LY7, OCI-LY19 and SUD-HL-4. Now we show that CTLA4-FasL specifically binds to CD80 and CD86 on these cells, hinders their viability with EC50 of 0.02-0.06 nM (MTS assay), and induces robust apoptosis in these cells (annexin V-PI assay). CTLA4-FasL's potency is correlated with CD80, CD86 and CD95 expression on the cell's surface.Importantly, CTLA4-FasL significantly improves mouse survival in a systemic lymphoma model induced by IV injection of FARAGE DLBCL cells into the tail vein of irradiated NUDE mice (4 SC injections, every other day). When given at a dose of 10mg/mice/injection its effect was comparable to that of 350 mg/mice of Rituximab.Previously, we evaluated CTLA4-FasL's toxicity in GLP studies in mice and cynomolgus monkeys. No gross pathology or adverse clinical symptoms were observed in mice (n=76) or monkeys (n=10) treated with the highest dose of CTLA4-FasL (0.5 mg/kg in mice, 0.125 mg/kg in monkeys). Transient and dose-dependent leukopenia was observed in monkeys only and this was resolves in 7 days. As leukopenia was observed in monkeys only and was not evident in mice treated with the same adjusted dose, we now compared Fas receptor level of expression and CTLA4-FasL 's binding to mice, monkeys and human PBMCs, and also CTLA4-FasL 's ability to induce apoptosis of the different PBMCs. Interestingly, the monkeys' cells express more Fas receptors and bind the protein better than both human and mice cells. In accordance with that, significantly more apoptosis of monkeys' PBMCs was evident when cells were treated with CTLA4-FasL.Though leukopenia was not observed in mice, it is still possible that some population of cells are affected by CTLA4-FasL. In order to test CTLA4-FasL's effect on different immune system cells in immune competent mice, BALB/C mice were treated with CTLA4-FasL and 24h later peripheral blood and spleens were harvested and cells were immuno-stained and analyzed by flow-cytometry. A significant decrease in percentage and actual counts of B and T lymphocytes (CD4 and CD8) as well as macrophages and monocytes was importantly accompanied by an increase in percentage and actual cell number of neutrophils and NK cells.In summary, the fusion protein CTLA4-FasL, with its unique hexameric structure and bi-functional mode of targeted action, was shown to be an effective treatment for human-mouse xenograft model of DLBCL. Transient and dose-dependent leukopenia was the only significant adverse effect, seen only in monkeys, and is maybe explained by the higher sensitivity of monkeys' cells to the protein's apoptotic effect. It might also be the result of the longer half-life of the protein in monkeys when compared to mice. The sparing and even increase in the number of neutrophils and NK cells might indicate that the innate immune system will be preserved after treatment with CTLA4-FasL. DisclosuresAronin:KAHR Medical: Employment. Ben Gigi-Tamir:KAHR Medical: Employment. Amsili:KAHR Medical: Employment, Other: Options. Sagiv:KAHR Medical: Employment. Gozlan:KAHR mEDICAL: Employment. Shani:KAHR Medical: Employment, Other: OPTIONS, Patents & Royalties. Dranitzki Elhalel:KAHR Medical: Consultancy, Other: Options, Patents & Royalties: PATENTS, Research Funding.

Inhibitory Effect of Paquinimod on a Murine Model of Neutrophilic Asthma Induced by Ovalbumin with Complete Freund's Adjuvant.

Background Quinoline-3-carboxamides have been used to treat autoimmune/inflammatory diseases in humans because they inhibit the functions of S100 calcium-binding protein A9 (S100A9), which participates in the development of neutrophilic inflammation in asthmatics and in an animal model of neutrophilic asthma. However, the therapeutic effects of these chemicals have not been evaluated in asthma. The purpose of this study was to evaluate the effect of paquinimod, one of the quinoline-3-carboxamides, on a murine model of neutrophilic asthma. Methods Paquinimod was orally administered to 6-week-old C57BL/6 mice sensitized and challenged with ovalbumin (OVA)/complete Freund's adjuvant (CFA) and OVA. Lung inflammation and remodeling were evaluated using bronchoalveolar lavage (BAL) and histologic findings including goblet cell count. S100A9, caspase-1, IL-1β, MPO, IL-17, IFN-γ, and TNF-α were measured in lung lysates using western blotting. Results Paquinimod restored the enhancement of airway resistance and the increases in numbers of neutrophils and macrophages of BAL fluids and those of goblet cells in OVA/CFA mice toward the levels of sham-treated mice in a dose-dependent manner (0.1, 1, 10, and 25 mg/kg/day, p.o.). Concomitantly, p20 activated caspase-1, IL-1β, IL-17, TNF-α, and IFN-γ levels were markedly attenuated. Conclusion These data indicate that paquinimod effectively inhibits neutrophilic inflammation and remodeling in the murine model of neutrophilic asthma, possibly via downregulation of IL-17, IFN-γ, and IL-1β.