Decreased Neutrophil Recruitment Research Articles

The lipid-metabolism enzyme ECI2 reduces neutrophil extracellular traps formation for colorectal cancer suppression

Abnormalities in ether lipid metabolism as well as the formation of neutrophil extracellular traps have recently been recognized as detrimental factors affecting tumorigenesis and progression. However, the role of abnormal ether lipid metabolism in colorectal cancer (CRC) evolution has not been reported. Here we show that the lipid metabolism-related gene enoyl-CoA δ-isomerase 2 (ECI2) plays a tumor-suppressor role in CRC and is negatively associated with poor prognosis in CRC patients. We mechanistically demonstrate that ECI2 reduces ether lipid-mediated Interleukin 8 (IL-8) expression leading to decreased neutrophil recruitment and neutrophil extracellular traps formation for colorectal cancer suppression. In particular, ECI2 inhibits ether lipid production in CRC cells by inhibiting the peroxisomal localization of alkylglycerone phosphate synthase (AGPS), the rate-limiting enzyme for ether lipid synthesis. These findings not only deepen our understanding of the role of metabolic reprogramming and neutrophil interactions in the progression of CRC, but also provide ideas for identifying potential diagnostic markers and therapeutic targets for CRC.

The LPS-inactivating enzyme acyloxyacyl hydrolase protects the brain from experimental stroke

Blood-brain-barrier (BBB) disruption is a pathological hallmark of ischemic stroke, and inflammation occurring at the BBB contributes to the pathogenesis of ischemic brain injury. Lipopolysaccharide (LPS), a cell wall component of Gram-negative bacteria, is elevated in patients with acute stroke. The activity of LPS is controlled by acyloxyacyl hydrolase (AOAH), a host enzyme that deacylates LPS to inactivated forms. However, whether AOAH influences the pathogenesis of ischemic stroke remain elusive. We performed in vivo experiments to explore the role and mechanism of AOAH on neutrophil extravasation, BBB disruption, and brain infarction. We found that AOAH was upregulated in neutrophils in peri-infarct areas from mice with transient focal cerebral ischemia. AOAH deficiency increased neutrophil extravasation into the brain parenchyma and proinflammatory cytokine production, broke down the BBB and worsened stroke outcomes in mice. These effects require Toll-like receptor 4 (TLR4) because absence of TLR4 or pharmacologic inhibition of TLR4 signaling prevented the exacerbated inflammation and BBB damage in Aoah−/− mice after ischemic stroke. Importantly, neutrophil depletion or inhibition of neutrophil trafficking by blocking LFA-1 integrin dramatically reduced stroke-induced BBB breakdown in Aoah−/− mice. Furthermore, virus-mediated overexpression of AOAH induced a substantial decrease in neutrophil recruitment that was accompanied by reducing BBB damage and stroke volumes. Our findings show the importance of AOAH in regulating neutrophil-dependent BBB breakdown and cerebral infarction. Consequently, strategies that modulate AOAH may be a new therapeutic approach for treatment of ischemic stroke.

Abstract 5328: Dual role of Kupffer cell secreted CXCL5 in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is hallmarked by inflammatory cell infiltration. Chemokines secreted during inflammation are crucial at directing immune cell infiltration during cancer development. We screened patient dataset from different etiologies including HBV, HCV, alcohol and non-alcoholic steatohepatitis (NASH) and identified CXCL5 as the only chemokine consistently upregulated in HCC. Immunohistochemistry (IHC) of patient liver tumor tissues further showed the production of CXCL5 by tumor surrounding non parenchymal cells. Using a mouse model (Pte loxP/loxP; Alb-Cre+, Pten null) which recapitulates human liver disease progression from non-alcoholic fatty liver disease (NAFLD) to NASH to liver cancer, we found that CXCL5 was expression was gradually increased during disease progression. Further analysis using IHC and flow cytometry analysis showed that CXCL5 is predominantly expressed by Kupffer cells in Pten null livers bearing tumors. Furthermore, we found that LPS stimulated the expression and secretion of CXCL5 in Kupffer cells, but not other macrophages including peritoneal macrophage and macrophage cell line Raw264.7 cells. We hypothesized that the Kupffer cell-secreted CXCL5 in response to LPS promotes tumor progression by promoting HCC cell proliferation and regulating HCC associated immune cell infiltration. To investigate whether CXCL5 increases tumor cell proliferation, we treated wild type and Pten null hepatocytes with various concentrations of CXCL5. Our results showed that CXCL5 increased hepatocytes viability in a dose dependent manner. BrdU incorporation assay further confirmed that CXCL5 treatments induces HCC cell proliferation through its receptor CXCR2. We further demonstrated that CXCR2 knockdown led to reduced xenograft tumor growth using HepG2 cells. Additionally, we injected Pten null mice with CXCR2 inhibitor to block CXCL5 signal and observed partial response. We analyzed the immune cell profile of the responder mice vs. non-responder mice and showed that tumor inhibition is correlated with T cell infiltration. To test whether Kupffer cells regulate T cells through CXCL5, we isolated primary Kupffer cells from murine livers. We performed transwell migration assay using immune cells from murine spleens. Our results indicate that LPS stimulation in Kupffer cells reduced the ratio of migrated CD4+ T cells, while blocking CXCL5 using a CXCL5 antibody significantly increased CD4+ T cell recruitment to the LPS-stimulated Kupffer cells and moderately decreased neutrophil recruitment. Consistently, we found that recombinant CXCL5 protein reduces CD4+ T cell migration independent of neutrophils. In summary, our study suggests that Kupffer cell secreted CXCL5 reduces liver CD4+ T cell recruitment and increases liver cancer cell proliferation. Citation Format: Taojian Tu, Handan Hong, Lina He, Mario Alba, Tang Qi, Curtis T. Okamoto, Bangyan L. Stiles. Dual role of Kupffer cell secreted CXCL5 in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5328.

Trichinella-derived protein ameliorates colitis by altering the gut microbiome and improving intestinal barrier function

BackgroundInflammatory bowel disease (IBD) encompasses Crohn’s Disease and Ulcerative Colitis. Reports have highlighted the potential use of helminths or their byproducts as a possible treatment for IBD; however, the mechanisms underlying their ability to modulate inflammation remain incompletely understood. In the present study, we analyze the possible mechanism of a serine protease inhibitor from adult T. spiralis excretion–secretion products (rTsSPI) on the improvement of colitis. MethodsThe immune protective effect of rTsSPI was studied by using DSS or Salmonella-induced colitis in female C56BL/6 mice. The effect of rTsSPI on the immune and inflammatory responses, gut microbiota, permeability of colon epithelium and junction proteins was analyzed. ResultsTreating mice with rTsSPI induced type 2 immunity and significantly attenuated clinical symptoms, macroscopical and histological features of DSS or bacteria-induced colonic inflammation. This was accompanied by decreasing neutrophil recruitment in the colonic lamina propria, and reducing TNF-α mRNA levels in the colon; in contrast, the recruitment of M2 macrophages, the expression level of IL-10 and adhesion molecules increased in the colon tissue. Moreover, treatment with rTsSPI led to an improvement in gut microbiota diversity, as well as an increase in the abundance of the bacterial genera Bifidobacterium and Ruminclostridium 5. ConclusionsCollective findings suggest that pretreatment with rTsSPI can ameliorate colitis in mice by inducing a Th2-type response with M2 macrophages. Data also indicate that immunotherapy with rTsSPI represents an additional strategy to ameliorate inflammatory processes in IBD by enhancing probiotic colonization and maintaining intestinal epithelial barrier function.

Acidic extracellular pH drives accumulation of N1-acetylspermidine and recruitment of protumor neutrophils.

An acidic tumor microenvironment plays a critical role in tumor progression. However, understanding of metabolic reprogramming of tumors in response to acidic extracellular pH has remained elusive. Using comprehensive metabolomic analyses, we demonstrated that acidic extracellular pH (pH 6.8) leads to the accumulation of N1-acetylspermidine, a protumor metabolite, through up-regulation of the expression of spermidine/spermine acetyltransferase 1 (SAT1). Inhibition of SAT1 expression suppressed the accumulation of intra- and extracellular N1-acetylspermidine at acidic pH. Conversely, overexpression of SAT1 increased intra- and extracellular N1-acetylspermidine levels, supporting the proposal that SAT1 is responsible for accumulation of N1-acetylspermidine. While inhibition of SAT1 expression only had a minor effect on cancer cell growth in vitro, SAT1 knockdown significantly decreased tumor growth in vivo, supporting a contribution of the SAT1-N1-acetylspermidine axis to protumor immunity. Immune cell profiling revealed that inhibition of SAT1 expression decreased neutrophil recruitment to the tumor, resulting in impaired angiogenesis and tumor growth. We showed that antineutrophil-neutralizing antibodies suppressed growth in control tumors to a similar extent to that seen in SAT1 knockdown tumors in vivo. Further, a SAT1 signature was found to be correlated with poor patient prognosis. Our findings demonstrate that extracellular acidity stimulates recruitment of protumor neutrophils via the SAT1-N1-acetylspermidine axis, which may represent a metabolic target for antitumor immune therapy.

Sfxn5 Regulation of Actin Polymerization for Neutrophil Spreading Depends on a Citrate-Cholesterol-PI(4,5)P2 Pathway.

Cell spreading is an initial and critical step in neutrophil adhesion and migration, leading to neutrophil recruitment to inflammatory tissues. Sideroflexin (Sfxn) family proteins are metabolite transporters located in the mitochondrial membrane. Recombinant SFXN5 protein is a citrate transporter invitro; however, whether Sfxn5 regulates any cellular behavior or function remains unknown. In this study, we found that small interfering RNA transfection or morpholino injection achieving Sfxn5 deficiency in neutrophils significantly decreased neutrophil recruitment in mice and zebrafish, respectively. Sfxn5 deficiency impaired neutrophil spreading and spreading-associated cellular phenotypes, such as cell adhesion, chemotaxis, and ROS production. Actin polymerization is critical for neutrophil spreading, and we found that actin polymerization in spreading neutrophils was partially inhibited by Sfxn5 deficiency. Mechanistically, we observed that the levels of cytosolic citrate and its downstream metabolic products, acetyl-CoA and cholesterol, were decreased in Sfxn5-deficient neutrophils. The levels of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a mediator for the regulation of actin polymerization by cholesterol, were reduced in the plasma membrane of Sfxn5-deficient neutrophils. Exogenous supplementation with citrate or cholesterol partially reversed the reduction in PI(4,5)P2 levels, defective neutrophil actin polymerization, and cell spreading. Altogether, we demonstrated that Sfxn5 maintains cytosolic citrate levels and ensures the synthesis of sufficient cholesterol to promote actin polymerization in a PI(4,5)P2-dependent manner during neutrophil spreading, which is essential for the eventual inflammatory recruitment of neutrophils. Our study revealed the importance of Sfxn5 in neutrophil spreading and migration, thus identifying, to our knowledge, for the first time, the physiological cellular functions of the Sfxn5 gene.

BPA modulates the neutrophil response and cytokine environment in a model of tail fin regeneration

Abstract The ER-α and β ligand bisphenol A (BPA) is used in the production of polycarbonate plastic and epoxy resins. BPA presents a potential environmental challenge to both human and wildlife immunity due to its widespread use and its resulting presence in surface water and wastewater runoff. We observed that BPA exposure downregulates the expression of a neutrophil-recruiting chemokine in both mice (cxcl1) and zebrafish (cxcl8) and that BPA exposure decreases neutrophil recruitment to a tail injury in zebrafish. Neutrophils are essential for the removal of damaged tissue and invading microbes, thus facilitating tissue regeneration of the tail fin. To investigate BPA’s effects on larval zebrafish tail regeneration, we induced a tail injury at day 3 post fertilization (dpf) and found that fish exposed to 100 ng/ml BPA regenerate a smaller tail fin area at 3 dpi (p-value < 0.01). We hypothesized that neutrophil recruitment could be tied to regeneration by altering the expression of inflammation- and regeneration-related genes. 3 dpf embryos exposed to BPA show modulated levels of background wnt10aand il-10expression compared to controls. BPA exposure reduces the 3 hour post-injury inflammatory response by decreasing both il-1and il-6expression. By modifying the cytokine environment both prior to and following infection and injury, BPA impacts both the inflammatory response and tissue regeneration. Multiple cytokine and chemokine genes are regulated by the transcription factor NF-kB and Wnt plays a key role in tissue regeneration; these two proteins also cross regulate one another. The impact of BPA on wnt10aand nfkbexpression following tissue injury and LPS exposure will be discussed. Supported by Carleton College.

Glutamine prevents acute kidney injury by modulating oxidative stress and apoptosis in tubular epithelial cells.

Acute kidney injury (AKI) represents a common complication in critically ill patients that is associated with increased morbidity and mortality. In a murine AKI model induced by ischemia/reperfusion injury (IRI), we show that glutamine significantly decreases kidney damage and improves kidney function. We demonstrate that glutamine causes transcriptomic and proteomic reprogramming in murine renal tubular epithelial cells (TECs), resulting in decreased epithelial apoptosis, decreased neutrophil recruitment, and improved mitochondrial functionality and respiration provoked by an ameliorated oxidative phosphorylation. We identify the proteins glutamine gamma glutamyltransferase 2 (Tgm2) and apoptosis signal-regulating kinase (Ask1) as the major targets of glutamine in apoptotic signaling. Furthermore, the direct modulation of the Tgm2-HSP70 signalosome and reduced Ask1 activation resulted in decreased JNK activation, leading to diminished mitochondrial intrinsic apoptosis in TECs. Glutamine administration attenuated kidney damage in vivo during AKI and TEC viability in vitro under inflammatory or hypoxic conditions.

Efficacy of a Probiotic and Herbal Supplement in Models of Lung Inflammation.

Gut microbiome dysbiosis is associated with lung disease through the gut-lung axis. Abundant proteobacteria increase MMP-9 and contribute to tissue proteolysis followed by neutrophil recruitment, lung tissue injury, and perpetuation of chronic lung disease. We sought to determine if a scientifically formulated probiotic and herbal supplement could attenuate neutrophilic inflammation and improve lung structure and function in models of lung inflammation. For in vitro experiments, epithelial cells exposed to proteobacteria were treated with resB-a blend of three probiotic Lactobacillus strains and turmeric, holy basil, and vasaka herbal extracts. For in vivo experimentation, mice exposed to pulmonary proteobacteria-derived lipopolysaccharide were treated by gavage with resB. In vitro, the bacterial and herbal components of resB decreased activity of the MMP-9 pathway. Mice exposed to LPS and pre- and post-treated with resB had decreased neutrophil recruitment and inflammatory biomarkers in bronchoalveolar lavage fluid, serum, and lung tissue compared to untreated mice. This study describes the mechanisms and efficacy of probiotic and herbal blend in pre-clinical models of lung injury and inflammation.

Nexinhib20 Inhibits Neutrophil Adhesion and β2 Integrin Activation by Antagonizing Rac-1-Guanosine 5'-Triphosphate Interaction.

Neutrophils are critical for mediating inflammatory responses. Inhibiting neutrophil recruitment is an attractive approach for preventing inflammatory injuries, including myocardial ischemia-reperfusion (I/R) injury, which exacerbates cardiomyocyte death after primary percutaneous coronary intervention in acute myocardial infarction. In this study, we found out that a neutrophil exocytosis inhibitor Nexinhib20 inhibits not only exocytosis but also neutrophil adhesion by limiting β2 integrin activation. Using a microfluidic chamber, we found that Nexinhib20 inhibited IL-8-induced β2 integrin-dependent human neutrophil adhesion under flow. Using a dynamic flow cytometry assay, we discovered that Nexinhib20 suppresses intracellular calcium flux and β2 integrin activation after IL-8 stimulation. Western blots of Ras-related C3 botulinum toxin substrate 1 (Rac-1)-GTP pull-down assays confirmed that Nexinhib20 inhibited Rac-1 activation in leukocytes. An in vitro competition assay showed that Nexinhib20 antagonized the binding of Rac-1 and GTP. Using a mouse model of myocardial I/R injury, Nexinhib20 administration after ischemia and before reperfusion significantly decreased neutrophil recruitment and infarct size. Our results highlight the translational potential of Nexinhib20 as a dual-functional neutrophil inhibitory drug to prevent myocardial I/R injury.

Human PD-1 agonist treatment alleviates neutrophilic asthma by reprogramming T cells

Neutrophilic asthma is associated with disease severity and corticosteroid insensitivity. Novel therapies are required to manage this life-threatening asthma phenotype. Programmed cell death protein-1 (PD-1) is a key homeostatic modulator of the immune response for T-cell effector functions. We sought to investigate the role of PD-1 in the regulation of acute neutrophilic inflammation in a murine model of airway hyperreactivity (AHR). House dust mite was used to induce and compare neutrophilic AHR in wild-type and PD-1 knockout mice. Then, the therapeutic potential of a human PD-1 agonist was tested in a humanized mouse model in which the PD-1 extracellular domain is entirely humanized. Single-cell RNA sequencing and flow cytometry were mainly used to investigate molecular and cellular mechanisms. PD-1 was highly induced on pulmonary T cells in our inflammatory model. PD-1 deficiency was associated with an increased neutrophilic AHR and high recruitment of inflammatory cells to the lungs. Consistently, PD-1 agonist treatment dampened AHR, decreased neutrophil recruitment, and modulated cytokine production in a humanized PD-1 mouse model. Mechanistically, we demonstrated at the transcriptional and protein levels that the inhibitory effect of PD-1 agonist is associated with the reprogramming of pulmonary effector T cells that showed decreased number and activation. PD-1 agonist treatment is efficient in dampening neutrophilic AHR and lung inflammation in a preclinical humanized mouse model.

IL-34 and protein-tyrosine phosphatase receptor type-zeta-dependent mechanisms limit arthritis in mice

AbstractMyeloid cell mediated mechanisms regulate synovial joint inflammation. IL-34, a macrophage (Mø) growth and differentiation molecule, is markedly expressed in neutrophil and Mø-rich arthritic synovium. IL-34 engages a newly identified independent receptor, protein-tyrosine phosphatase, receptor-type, zeta (PTPRZ), that we find is expressed by Mø. As IL-34 is prominent in rheumatoid arthritis, we probed for the IL-34 and PTPRZ-dependent myeloid cell mediated mechanisms central to arthritis using genetic deficient mice in K/BxN serum-transfer arthritis. Unanticipatedly, we now report that IL-34 and PTPRZ limited arthritis as intra-synovial pathology and bone erosion were more severe in IL-34 and PTPRZ KO mice during induced arthritis. We found that IL-34 and PTPRZ: (i) were elevated, bind, and induce downstream signaling within the synovium in arthritic mice and (ii) were upregulated in the serum and track with disease activity in rheumatoid arthritis patients. Mechanistically, IL-34 and PTPRZ skewed Mø toward a reparative phenotype, and enhanced Mø clearance of apoptotic neutrophils, thereby decreasing neutrophil recruitment and intra-synovial neutrophil extracellular traps. With fewer neutrophils and neutrophil extracellular traps in the synovium, destructive inflammation was restricted, and joint pathology and bone erosion diminished. These novel findings suggest that IL-34 and PTPRZ-dependent mechanisms in the inflamed synovium limit, rather than promote, inflammatory arthritis.

Obesity Inhibits Innate Immune Functions During Pseudomonas aeruginosa Pneumonia

Abstract Obesity is a major global public health concern. Worldwide, 39% of the population is overweight and 13% of the population is obese. Obesity contributes to many diseases, such as heart disease, stroke, some cancers, and notably, type 2 diabetes (T2D). Although T2D itself is a non-communicable disease, many patients with diabetes are more susceptible to microbial infections and exhibit a higher burden of disease. Although obese animals have an increased production of myeloid cells, previous work from our labs has shown that a failure to heal diabetic wounds corresponds to elevated levels of prostaglandin E2 (PGE2) in the inflammatory macrophages that are recruited to the wound. Furthermore, past studies from our lab have demonstrated that PGE2 signaling can impair the innate immune functions of macrophages. Therefore, we hypothesized that diabetic obese mice would be more susceptible to a respiratory Pseudomonas aeruginosa strain PA01 infection due to impaired function of lung resident and recruited macrophages. Preliminary studies demonstrate that obese diabetic mice do have an increased P. aeruginosa burden in the lung 24 hours after infection. Additionally, we have determined that naïve obese diabetic mice have an increase in lung neutrophils compared to lean mice, however, after infection with P. aeruginosa, these mice have a trend of decreased neutrophil recruitment. Additionally, functional assays show that macrophages and neutrophils from obese diabetic mice have a defect in killing P. aeruginosa. These observations suggest that obese diabetic mice have not only a defect in innate immune cell recruitment, but also a defect in innate immune cell function during bacterial pneumonia. Supported by grants from NIH (T32 AI007528-21)

Nexinhib20 prevents myocardial ischemia‐reperfusion injury by inhibiting neutrophil adhesion and β2 integrin activation

Primary percutaneous coronary intervention (PPCI)—a therapy intended to restore blood flow after acute myocardial infarction—can also induce myocardial ischemia‐reperfusion (I/R) injury, exacerbating cardiomyocyte death. Neutrophil‐mediated cardiac tissue damage contributes prominently to the process of myocardial I/R injury. Therefore, limiting neutrophil recruitment and exocytosis may prevent myocardial I/R injury after PPCI. Here, we found out that a neutrophil exocytosis inhibitor Nexinhib20 inhibits both neutrophil adhesion and exocytosis and prevents myocardial I/R injury in a mouse model. Using a microfluidic chamber, we found that Nexinhib20 inhibited interleukin 8 (IL‐8)‐induced β2 integrin‐dependent human neutrophil adhesion under flow. Using dynamic flow cytometry assay, we discovered that Nexinhib20 suppresses intracellular calcium flux and β2 integrin activation after IL‐8 stimulation. Western blots of Rac‐1‐GTP pull‐down assay confirmed that Nexinhib20 inhibited Rac‐1 activation in leukocytes. In vitro competing assay showed that Nexinhib20 antagonized the binding of Rac‐1 and GTP. Using a mouse model of myocardial I/R injury, Nexinhib20 administration after ischemia and before reperfusion significantly decreased neutrophil recruitment and infarct size. Our results highlight the translational potential of Nexinhib20 as a dual‐functional neutrophil inhibitory drug to prevent myocardial I/R injury.

Effect of BPA on cytokine expression and regeneration in larval zebrafish

Abstract Bisphenol-A (BPA) binds to estrogen receptors ER-α and β and is used in the production of polycarbonate plastic and epoxy resins. BPA presents a potential environmental challenge to the immune system of both humans and wildlife due to its use in food containers and thermal receipts and its resulting presence in surface water and wastewater runoff. We observed that BPA exposure downregulates the expression of the chemokine that functions to recruit neutrophils in both mice (KC) and zebrafish (cxcl8) and that it decreases neutrophil recruitment to the injury site in MPO::GFP zebrafish. Neutrophils are essential for the removal of damaged tissue and invading microbes, thus facilitating tissue regeneration of the tail fin. To investigate BPA’s effects on larval zebrafish tail regeneration, we induced a tail injury at day 3 post fertilization (dpf) and measured tail fin growth at day 1 and 3 post injury (dpi). Fish exposed to 100 ng/ml BPA regenerate a smaller tail fin area at 3 dpi (p-value < 0.01). We hypothesized that BPA exposure and altered neutrophil recruitment could affect regeneration by directly altering the expression of regeneration-related genes wnt10a and il-10 or their downstream genes and/or indirectly by regulating inflammatory genes including il-1 and il-6. The expression of il-1, il-6, il-10, and wnt10a were detected in zebrafish embryos from 2–6 dpf using qPCR. BPA exposure reduces both il-1 and il-6 expression 3 hours post-injury, suggesting that BPA reduces the inflammatory response. To determine whether BPA alters gene expression of wnt10a, il-10, or their downstream-regulated genes qPCR will be performed at 24- and 72-hours post-injury. Supported by Carleton College.

Nexinhib20 inhibits neutrophil adhesion and β2 integrin activation to prevent myocardial ischemia-reperfusion injury

Abstract Primary percutaneous coronary intervention (PPCI)—a therapy intended to restore blood flow after acute myocardial infarction—can also induce myocardial ischemia-reperfusion (I/R) injury, exacerbating cardiomyocyte death. Neutrophil-mediated cardiac tissue damage contributes prominently to the process of myocardial I/R injury. Therefore, limiting neutrophil recruitment and exocytosis may prevent myocardial I/R injury after PPCI. Here, we found out that a neutrophil exocytosis inhibitor Nexinhib20 inhibits both neutrophil adhesion and exocytosis and prevents myocardial I/R injury in a mouse model. Using a microfluidic chamber, we found that Nexinhib20 inhibited interleukin 8 (IL-8)-induced β2 integrin-dependent human neutrophil adhesion under flow. Using dynamic flow cytometry assay, we discovered that Nexinhib20 suppresses intracellular calcium flux and β2 integrin activation after IL-8 stimulation. Western blots of Rac-1-GTP pull-down assay confirmed that Nexinhib20 inhibited Rac-1 activation in leukocytes. In vitro competing assay showed that Nexinhib20 antagonized the binding of Rac-1 and GTP. Using a mouse model of myocardial I/R injury, Nexinhib20 administration after ischemia and before reperfusion significantly decreased neutrophil recruitment and infarct size. Our results highlight the translational potential of Nexinhib20 as a dual-functional neutrophil inhibitory drug to prevent myocardial I/R injury. This research was supported by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute, USA (R01HL145454, R41HL156322, R44HL152710, and K99HL153678), a Career Development Award from American Heart Association (18CDA34110426), and a startup fund from UConn Health.

Inhibition of infection-induced vascular permeability modulates host leukocyte recruitment to Mycobacterium marinum granulomas in zebrafish.

Mycobacterial granuloma formation involves significant stromal remodeling including the growth of leaky, granuloma-associated vasculature. These permeable blood vessels aid mycobacterial growth, as antiangiogenic or vascular normalizing therapies are beneficial host-directed therapies in preclinical models of tuberculosis across host-mycobacterial pairings. Using the zebrafish-Mycobacterium marinum infection model, we demonstrate that vascular normalization by inhibition of vascular endothelial protein tyrosine phosphatase (VE-PTP) decreases granuloma hypoxia, the opposite effect of hypoxia-inducing antiangiogenic therapy. Inhibition of VE-PTP decreased neutrophil recruitment to granulomas in adult and larval zebrafish, and decreased the proportion of neutrophils that extravasated distal to granulomas. Furthermore, VE-PTP inhibition increased the accumulation of T cells at M. marinum granulomas. Our study provides evidence that, similar to the effect in solid tumors, vascular normalization during mycobacterial infection increases the T cell:neutrophil ratio in lesions which may be correlates of protective immunity.

Targeting IL-36 improves age-related coronary microcirculatory dysfunction and attenuates myocardial ischemia/reperfusion injury in mice.

Following myocardial infarction (MI), elderly patients have a poorer prognosis than younger patients, which may be linked to increased coronary microvessel susceptibility to injury. Interleukin-36 (IL-36), a newly discovered proinflammatory member of the IL-1 superfamily, may mediate this injury, but its role in the injured heart is currently not known. We first demonstrated the presence of IL-36(α/β) and its receptor (IL-36R) in ischemia/reperfusion-injured (IR-injured) mouse hearts and, interestingly, noted that expression of both increased with aging. An intravital model for imaging the adult and aged IR-injured beating heart in real time in vivo was used to demonstrate heightened basal and injury-induced neutrophil recruitment, and poorer blood flow, in the aged coronary microcirculation when compared with adult hearts. An IL-36R antagonist (IL-36Ra) decreased neutrophil recruitment, improved blood flow, and reduced infarct size in both adult and aged mice. This may be mechanistically explained by attenuated endothelial oxidative damage and VCAM-1 expression in IL-36Ra–treated mice. Our findings of an enhanced age-related coronary microcirculatory dysfunction in reperfused hearts may explain the poorer outcomes in elderly patients following MI. Since targeting the IL-36/IL-36R pathway was vasculoprotective in aged hearts, it may potentially be a therapy for treating MI in the elderly population.

Dipeptidase-1 governs renal inflammation during ischemia reperfusion injury.

The mechanisms that drive leukocyte recruitment to the kidney are incompletely understood. Dipeptidase-1 (DPEP1) is a major neutrophil adhesion receptor highly expressed on proximal tubular cells and peritubular capillaries of the kidney. Renal ischemia reperfusion injury (IRI) induces robust neutrophil and monocyte recruitment and causes acute kidney injury (AKI). Renal inflammation and the AKI phenotype were attenuated in Dpep1−/− mice or mice pretreated with DPEP1 antagonists, including the LSALT peptide, a nonenzymatic DPEP1 inhibitor. DPEP1 deficiency or inhibition primarily blocked neutrophil adhesion to peritubular capillaries and reduced inflammatory monocyte recruitment to the kidney after IRI. CD44 but not ICAM-1 blockade also decreased neutrophil recruitment to the kidney during IRI and was additive to DPEP1 effects. DPEP1, CD44, and ICAM-1 all contributed to the recruitment of monocyte/macrophages to the kidney following IRI. These results identify DPEP1 as a major leukocyte adhesion receptor in the kidney and potential therapeutic target for AKI.

THE ROLE OF CXCR2 AND ITS LIGANDS IN ATTACHMENT, MIGRATION, AND INVASION OF MOUSE ENDOMETRIOSIS CELLS

Neutrophils are elevated in endometriosis and may play a role in attachment of lesions. The C-X-C Motif Chemokine Receptor 2 (CXCR2) is involved in neutrophil homeostasis. In mice, blocking CXCR2 decreases neutrophil recruitment and ligand production. CXC ligands (CXCL) 1,2,5 and 7 are known ligands for CXCR2. We hypothesize that CXCL 1, 2, 5, and 7 treatment will affect neutrophil recruitment that will alter attachment, migration, and invasion properties of mouse endometriosis cells (mEmLe) compared to normal mouse uterine epithelial cells (mUt).