Increased Neutrophil Infiltration Research Articles

Elevated Fecal Calprotectin Accompanied by Intestinal Neutrophil Infiltration and Goblet Cell Hyperplasia in a Murine Model of Multiple Sclerosis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system caused by myelin-specific autoreactive T cells. We previously demonstrated intestinal barrier disruption and signs of inflammation in experimental autoimmune encephalomyelitis (EAE), a model of MS. Fecal calprotectin is a disease activity biomarker in inflammatory bowel diseases, released by neutrophils in response to inflammation. We aimed to further investigate EAE manifestations in the gastrointestinal tract and to determine whether calprotectin is a useful biomarker of intestinal inflammation in EAE. Calprotectin was analyzed in feces, cecal contents, and plasma of EAE mice. Infiltrating neutrophils and goblet cells were investigated in different parts of the gastrointestinal tract before the onset of neurological symptoms and during established disease. We found increased calprotectin levels in feces, cecal content, and plasma preceding EAE onset that further escalated during disease progression. Increased neutrophil infiltration in the intestinal tissue concomitant with IL-17 expression and myeloperoxidase activity was found to correlate well with clinical activity. Increased goblet cells in the intestine, similar to irritable bowel syndrome (IBS), were also observed. The results suggest calprotectin as a good biomarker of gastrointestinal inflammation in EAE and the potential of this model as a useful animal model for IBS.

PECAM-1 mediates temsirolimus-induced increase in neutrophil transendothelial migration that leads to lung injury

Temsirolimus is a first-generation mTOR inhibitor commonly used in the clinical treatment of cancers that is associated with lung injury. However, the mechanism underlying this adverse effect remains elusive. Endothelial barrier dysfunction plays a pivotal role in the infiltration of neutrophils into the pulmonary alveoli, which eventually induces lung injury. The present study demonstrates that temsirolimus induces the aberrant expression of adhesion molecules in endothelial cells, leading to enhanced neutrophil infiltration and subsequent lung injury. Results of a mouse model revealed that temsirolimus disrupted capillary–alveolar barrier function and facilitated neutrophil transmigration across the endothelium within the alveolar space. Consistent with our in vivo observations, temsirolimus impaired intercellular barrier function within monolayers of human lung endothelial cells, resulting in increased neutrophil infiltration. Furthermore, we demonstrated that temsirolimus-induced neutrophil transendothelial migration was mediated by platelet endothelial cell adhesion molecule-1 (PECAM-1) in both in vitro and in vivo experiments. Collectively, these findings highlight that temsirolimus induces endothelial barrier dysfunction via PECAM-1-dependent pathway both in vitro and in vivo, ultimately leading to neutrophil infiltration and subsequent pulmonary injury.

How does the Immunological System Change during the SARS-COV-2 Attack? A Clue for the New Immunotherapy Discovery.

The COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-COV-2) is one of the biggest unsolved global problems of the 21st century for which there has been no definitive cure yet. Like other respiratory viruses, SARS-COV-2 triggers the host immunity dramatically, causing dysfunction in the immune system, both innate and adaptive, which is a common feature of COVID-19 patients. Evidence shows that in the early stages of COVID-19, the immune system is suppressed while it is overactive in severe patients characterized by excessive and prolonged inflammatory responses called "Cytokine Storm". There are many elements in the immune system that undergo alterations as the disease progresses. Some significant changes in the innate immune system following infection with SARS-COV-2 include delayed or inhibited interferon type 1 production by the infected cells leading to elevated virus replication, excessive recruitment of activated monocytes and macrophages, decrease in eosinophil population (eosinopenia), consequent decrease in CD8+T lymphocyte proliferation, natural killer (NK) cell dysfunction, and increase in neutrophil infiltration (neutrophilia) and neutrophil extracellular trap (NET) formation. Moreover, hallmark alterations in the adaptive immune system in this process cause an overall decrease in the T lymphocyte number (lymphopenia) and changes in the activity of some lymphocyte subsets and a number of B cells. This review delves into the mentioned changes in the immune system following SARS-COV-2 infection and the implications thereof to guide the development of immunotherapies for patients with COVID-19.

157 Effect of Sheep Breed on Neutrophil Chemotaxis toHaemonchus Contortus Larval Antigen

Abstract Parasite-resistant St. Croix sheep generate a potent neutrophilic response to larval stages of Haemonchus contortus. Protective neutrophil responses can be best characterized by increased neutrophil infiltration to the abomasum within the first 3 days of infection. These data indicate breed differences in neutrophil chemotaxis and response to H. contortus infection. The purpose of this study was to determine differences in neutrophil chemotaxis by H. contortus third-stage larval (L3) antigen (HcLA). Suffolk (SUF) and St. Croix (STC) neutrophils were isolated and applied to matrigel-coated clearview inserts, and placed into a reservoir containing H. contortus HcLA, Interleukin-8 (IL-8), or complete media. Neutrophil chemotaxis plates were incubated (37°C, 5% CO2) in an Incucyte S3 live cell imaging system for 24 hours, and measurements taken hourly. Neutrophils from STC migrated towards HcLA quicker than SUF neutrophils, and this migration occurred as early as 3 hours (10% and 4% respectively, P < 0.001). The difference in breed chemotaxis increased over time, where 28% of STC neutrophils migrated at 14 hours compared with 13% of SUF cells at the same time. By 24 hours, 37% of STC neutrophils migrated towards HcLA while the percentage of migrating SUF neutrophils remained the same (13%). Positive control for neutrophil chemotaxis was IL-8, and 37% of STC neutrophils achieved migration at 7 hours compared with 26% SUF neutrophils. By 24 hours, IL-8 induced neutrophil chemotaxis was similar in both breeds (STC 47%, SUF 41%). Taken together, these data indicate that delayed responses in neutrophil chemotaxis may contribute to H. contortus establishment in parasite-susceptible hosts. Thus, indicating that neutrophil response to H. contortus infection is a critical component to full host protective immunity.

C-Kit signaling modulated group 3 innate lymphoid cell functions in mouse neutrophilic asthma model

Abstract Group 3 innate lymphoid cells (ILC3s) are involved in air pollution- and bacterial infection-associated neutrophilic asthma. Studies showed that asthmatic patients have higher stem cell factor (SCF) expression in airway and sera. Of note, ILC3s express SCF receptor, c-Kit, suggesting a potential role of SCF/c-Kit signaling in ILC3 functions and neutrophilic asthma. In this study, we aimed to determine whether SCF/c-Kit signaling could modulate ILC3 functions and neutrophilic asthma. Wild-type (WT) and c-Kit deficient mice (Kit w-sh) were intranasally treated with IL-1β/IL-23 or particulate matter (PM) 2.5 to induce neutrophilic asthma. While IL-1β/IL-23 or PM2.5 caused increases in neutrophil infiltration to bronchoalveolar lavage fluid (BALF) and airway hyperreactivity (AHR) in WT mice, Kit w-shmice had decreased infiltration of neutrophils and ameliorated AHR. Concomitantly, flow cytometry results showed a decreased number of IL-17+ ILC3 in the lung of Kit w-shmice compared to WT mice, which may be due to the reduced proliferation of c-Kit-deficient ILC3. In addition, we showed SCF enhanced IL-17 secretion from ILC3s in vitro. Based on re-analysis of published mouse whole lung cells scRNAseq data, we hypothesized fibroblasts are the source of SCF. Indeed, mice with SCF knockout in fibroblast exhibited decreased neutrophil infiltration and reduced IL-17+ ILC3 number in response to IL-1β/IL-23. Finally, administration of imatinib, a FDA-approved drug targeting c-Kit signaling, ameliorated AHR and ILC3 activation in the mouse neutrophilic asthma model. Taken together, our data suggested that inhibition of ILC3 activation by targeting c-Kit signaling may provide a potential therapeutic management for neutrophilic asthma.

Increased renal dysfunction and neutrophil infiltration in a mouse model of psoriasis

Psoriasis is a chronic inflammatory skin disease affecting 2-5% of the global population. As an autoimmune disease, psoriasis is characterized by T cell-mediated hyperproliferation of epidermal keratinocytes. Interestingly, most of the morbidity and mortality from psoriasis is related to increased risk of cardiovascular and renal disease. For example, moderate to severe psoriasis is associated with an approximately 1.5-fold increased risk of chronic kidney disease. Neutrophils are increased in the skin and circulation of patients with psoriasis, and markers of neutrophil activation are associated with extra-cutaneous manifestations of psoriasis including vascular inflammation. However, the molecular mechanisms linking psoriasis and kidney disease remain unclear. We hypothesized that increased neutrophil infiltration of the kidney through enhanced granulopoiesis promotes renal dysfunction in psoriasis. Using a KC-Tie2 mouse model of psoriasis with keratinocyte-specific overexpression of the angiopoietin receptor Tie2, we found that KC-Tie2 mice compared to littermate controls exhibit significantly increased urine albumin, suggestive of renal dysfunction. KC-Tie2 mice also exhibit significantly increased kidney/body weight and heart/body weight ratios compared to littermate controls. To determine a mechanism for the increased renal dysfunction in these mice, we assessed skin and renal immune cell content using flow cytometry. KC-Tie2 mice had increased neutrophils in the skin, as well as an approximately 10-fold increase in neutrophils in the kidneys. This increase in renal neutrophils in KC-Tie2 mice was relatively selective as there were no differences in the number of renal dendritic cells, lymphocytes, or monocytes and a modest but significant increase in macrophages. We also observed a selective increase in neutrophils in the spleen of KC-Tie2 mice. Given these findings and prior reports of increased circulating neutrophils in KC-Tie2 mice, we assessed mediators of granulopoiesis in the serum and found a significant increase in granulocyte colony stimulating factor (GCSF) in KC-Tie2 mice. Taken together, these results provide evidence for increased renal dysfunction in a mouse model of psoriasis coincident with marked increases in renal neutrophils that may represent a novel mechanism linking skin inflammation to renal dysfunction through enhanced granulopoiesis. MRA is funded by NIH K08HL153786-01. JM is funded by 2T32GM007569. NW is funded by NIH R01 AR075777, NIH R01 AR073196, NIH P50 CORT AR070590, and National Psoriasis Foundation. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

ELMO1 Deficiency Reduces Neutrophil Chemotaxis in Murine Peritonitis.

Peritoneal inflammation remains a major cause of treatment failure in patients with kidney failure who receive peritoneal dialysis. Peritoneal inflammation is characterized by an increase in neutrophil infiltration. However, the molecular mechanisms that control neutrophil recruitment in peritonitis are not fully understood. ELMO and DOCK proteins form complexes which function as guanine nucleotide exchange factors to activate the small GTPase Rac to regulate F-actin dynamics during chemotaxis. In the current study, we found that deletion of the Elmo1 gene causes defects in chemotaxis and the adhesion of neutrophils. ELMO1 plays a role in the fMLP-induced activation of Rac1 in parallel with the PI3K and mTORC2 signaling pathways. Importantly, we also reveal that peritoneal inflammation is alleviated in Elmo1 knockout mice in the mouse model of thioglycollate-induced peritonitis. Our results suggest that ELMO1 functions as an evolutionarily conserved regulator for the activation of Rac to control the chemotaxis of neutrophils both in vitro and in vivo. Our results suggest that the targeted inhibition of ELMO1 may pave the way for the design of novel anti-inflammatory therapies for peritonitis.

MP34-13 SYNERGY BETWEEN THE HOST IMMUNE SYSTEM AND BACTERIOPHAGES SUPPORTS PHAGE THERAPY FOR URINARY TRACT INFECTION

You have accessJournal of UrologyCME1 Apr 2023MP34-13 SYNERGY BETWEEN THE HOST IMMUNE SYSTEM AND BACTERIOPHAGES SUPPORTS PHAGE THERAPY FOR URINARY TRACT INFECTION Kathrin Bausch Kathrin BauschKathrin Bausch More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003268.13AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Urinary tract infections (UTI) are one of the most common bacterial infections affecting >150 million people world-wide, every year. Globally, extraintestinal pathogenic Escherichia coli (ExPEC), which include uropathogenic E. coli, increasingly display resistance to antibiotics. One ExPEC clonal group, sequence type 131 (ST131), has emerged as a pandemic multidrug-resistant pathogen. To continue to be able to effectively treat UTI, despite increasing rates of antibiotic resistance, bacteriophage therapy is attracting renewed interest. Although phage therapy has been used for decades, mechanisms underlying phage-mediated bacterial clearance in UTI remain largely unknown. METHODS: To address this question, we investigated phage treatment for UTI caused by the ST131 strain JJ2528 and its effects on the bladder immune response in a mouse model. RESULTS: Using this model, we found that female C57BL/6 mice infected with JJ2528 had significantly reduced bladder bacterial burdens compared to untreated mice when they received phage HP3 either intraperitoneally or intravesically. Additionally, we found phage therapy may protect memory responses. Our previous work demonstrates that a non-sterilizing adaptive immune response arises following an acute UTI, but elimination of bacteria by antibiotics ablates development of immune memory. We tested whether phage-treated mice, in which bacteria are cleared from the bladder with a similar kinetic to antibiotic treatment, had an impaired memory response. Surprisingly, bacterial burden in challenged animals was similar between control and phage-treated animals, suggesting that immune memory is intact. While bacteriophage therapy alone did not induce inflammation in the bladder, phage therapy in the context of infection induced a significant increase in neutrophil infiltration compared to control-treated mice. To determine the role of neutrophils in phage treatment, we used two models of neutrophil depletion and treated infected mice with phages. Neutrophil-depleted mice had a significantly higher bacterial burden compared to non-depleted mice, supporting that neutrophil infiltration is necessary for phage treatment efficacy. A possible mechanism behind this may be that the administration of phages and bacteria leads to increased ROS production and cell death in human neutrophils compared to bacteria alone. CONCLUSIONS: Our data demonstrate that phage treatment is effective in reducing a primary UTI and does not impair the development of immune memory to rUTI. Phage treatment induces a neutrophil infiltration that is essential for phage efficacy mediated by increased ROS production and “cell death”. This study supports that phage treatment of UTI is a potentially viable alternative to antibiotics for this very common infection. Source of Funding: none © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e464 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kathrin Bausch More articles by this author Expand All Advertisement PDF downloadLoading ...

389 Neutrophils: Novel Contributors to Estrogen-Dependent Intracranial Aneurysm Rupture Via Neutrophil Extracellular Trap Formation

INTRODUCTION: Intracranial aneurysms (IAs) are more prevalent in women than men and aneurysmal subarachnoid hemorrhage disproportionately affects postmenopausal women. These sex differences suggest estrogen protects against IA progression that can lead to rupture, but the underlying mechanisms are not fully understood. Although studies have demonstrated estrogen regulates inflammatory processes that contribute to IA pathogenesis, the role of neutrophils remains to be characterized. METHODS: We compared neutrophil infiltration in C57BL/6 mice that develop IAs to those with a normal circle of Willis. Next, we investigated the role of neutrophils in female estrogen-deficient mice, female estrogen-intact mice, and male mice using a neutrophil depletion antibody. Finally, we studied the role of neutrophil extracellular trap formation (NETosis) as an underlying mechanism of aneurysm progression. RESULTS: Mice that developed aneurysms had increased neutrophil infiltration compared to those with a normal circle of Willis. In female estrogen-deficient mice, both neutrophil depletion and NETosis inhibition decreased aneurysm rupture, but not formation, and had no effect on symptom-free survival. In female estrogen-intact mice, neither neutrophil depletion nor NETosis inhibition affected IA formation, rupture, or symptom-free survival. In male mice, neutrophil depletion decreased IA formation but did not affect rupture or symptom-free survival. NETosis inhibition in male mice did not affect IA formation, rupture, or symptom-free survival. CONCLUSIONS: Neutrophils contribute to aneurysm rupture in the setting of estrogen deficiency. NETosis appears to be an underlying mechanism for neutrophil-mediated IA rupture in estrogen deficiency. In males, neutrophils contribute to IA formation, but not rupture. Targeting NETosis may lead to the development of novel therapeutics to protect against IA progression, particularly IA rupture in post-menopausal women.

MLKL and other necroptosis-related genes promote the tumor immune cell infiltration, guiding for the administration of immunotherapy in bladder urothelial carcinoma

The involvement of necroptosis in the immunosuppressive tumor microenvironment has been established and has been shown to contribute to the growth of pancreatic ductal adenocarcinoma, indicating its role in promoting tumor development. However, the relationship between necroptosis and bladder urothelial carcinoma (BUC) has yet to be fully understood. To shed light on this issue, our study aimed to uncover the impact of necroptosis on immune cell infiltration and immunotherapy response in BUC patients. We conducted an analysis of 67 necroptosis genes to assess their expression and genomic changes across pan-cancer and identified 12 necroptosis genes that are prognostically relevant and associated with immune subtypes and tumor stemness in BUC. Using a public database of 1841 BUC samples, we then performed Unsupervised Cluster Analysis and discovered two distinct necroptotic phenotypes in BUC. These phenotypes showed significant differences in molecular subtypes, immune infiltration patterns, and gene mutation profiles. We confirmed this discovery in BUC through qPCR and WB experiments. To evaluate the impact of necroptosis on prognosis, chemotherapy sensitivity, and immunotherapy response (such as anti-PD-L1), we developed a principal component analysis model called NecroScore. Finally, we validated the effects of RIPK3 and MLKL through a nude mouse transplantation model for BUC. Our study has uncovered that necroptosis plays a role in shaping the tumor immune microenvironment in BUC. The high necroptosis phenotype (Cluster B) was characterized by a higher abundance of tumor immunosuppressive cells and more key biological processes driving tumor progression, while the low necroptosis group (Cluster A) had higher FGFR3 mutations. We found that the infiltration levels of immune cells, including CD8+ T cells, were significantly different between FGFR3 mutated and wild-type (WT) samples. Our results confirmed the reliability of NecroScore as a comprehensive assessment tool for evaluating the immunotherapeutic effect and prognosis of BUC patients, with high NecroScore values favoring basal-like differentiation and lower FGFR3 alterations. We also observed that high expression of MLKL had a significant inhibitory effect on tumor growth and increased neutrophil infiltration in vivo. In our study, we uncovered the regulation pattern of necroptosis in the tumor immune microenvironment of BUC. Additionally, we developed a scoring tool called NecroScore that can be utilized to predict the most suitable chemotherapy and immunotherapy strategy for bladder urothelial carcinoma patients. This tool can effectively guide the chemotherapy and immunotherapy regimens for patients with advanced BUC.

Transcriptomic Profiling Reveals Chemokine CXCL1 as a Mediator for Neutrophil Recruitment Associated With Blood-Retinal Barrier Alteration in Diabetic Retinopathy.

Intravitreal CXCL1 injection and diabetes result in increased retinal vascular permeability with neutrophil and monocyte recruitment. Ly6G antibody treatment for neutrophil depletion in both animal models showed decreased retinal permeability and decreased cytokine expression. CXCL1 is produced by retinal endothelial cells, pericytes, and astrocytes. CXCL1 level is significantly increased in serum samples of patients with diabetic retinopathy. CXCL1, through neutrophil recruitment, alters the blood-retinal barrier in diabetic retinopathy and, thus, may be used as a therapeutic target.

Single-cell RNA-seq Revealed that Altered Tumor Infiltrating Lymphocytes in Cirrhotic Liver Indicate Development of Hepatocellular Carcinoma.

Cirrhosis is the precursor lesion for most hepatocellular carcinoma (HCC) cases. However, no biomarker effectively predicted HCC initiation before diagnosis by imaging. We aimed to investigate the hallmarks of immune microenvironments in healthy, cirrhotic livers and HCC tumor tissues and to identify immune biomarkers of cirrhosis-HCC transition. Expression matrices of single-cell RNA sequencing studies were downloaded and integrated with Seurat package vignettes. Clustering was performed to analyze the immune cell compositions of different sample types. The cirrhotic liver and HCC tumors had distinct immune microenvironments, but the immune landscape of cirrhotic livers was not markedly modified compared with healthy livers. Two subsets of B cells and three subsets of T cells were identified in the samples. Among the T cells, naïve T cells were more prominent in the cirrhotic and healthy liver samples than in the HCC samples. In contrast, the neutrophil count was lower in cirrhotic livers. Two macrophage clusters were identified, one that actively interacted with T cells and B cells and was enriched in cirrhotic blood compared with HCC blood samples. Decreased naïve T cell infiltration and increased neutrophil infiltration in the liver may indicate the development of HCC in cirrhotic patients. Alterations in blood-resident immune cells may also be a sign of HCC development in cirrhotic patients. The dynamics of the immune cell subsets may serve as novel biomarkers to predict the transition from cirrhosis to HCC.

Interleukin 17 B regulates colonic myeloid cell infiltration in a mouse model of DSS-induced colitis.

Cytokines play vital roles in the pathogenesis of inflammatory bowel disease. IL17B is protective in the development of colitis. However, how IL17B regulates intestinal inflammation and what cells are regulated by IL17B is still unknown. Here, we aimed to illustrate the IL17B dependent cellular and molecular changes in colon tissue in a mouse colitis model. The results showed that IL17B expression in colon tissues was elevated in inflamed tissues than non-inflamed tissues of IBD patients. Wild type (WT) and Il17b deficient (Il17b -/-) mice were given 2.5% dextran sodium sulfate (DSS) water, and in some case, Il17b -/- mice were treated with recombinant mouse IL17B. IL17B deficiency resulted in severe DSS-induced colitis with exaggerated weight loss, shorter colon length, and elevated proinflammatory cytokines in colon. Reconstitution of Il17b -/- mice with recombinant IL17B alleviated the severity of DSS-induced colitis. Single cell transcriptional analyses of CD45+ immune cells in colonic lamina propria revealed that loss of IL17B resulted in an increased neutrophil infiltration and enhanced inflammatory cytokines in intestinal macrophages in colitis, which were confirmed by real-time PCR and flow cytometry. IL17B treatment also inhibited lipopolysaccharide-induced inflammation in bone marrow-derived macrophages and mice. IL17B inhibits colitis by regulating colonic myeloid cell response. It might represent a novel potential therapeutic approach to treat the colitis.

Abstract WMP112: Type 2 Diabetes Dampens Neuroprotection Against Ischemic Injury By Lipopolysaccharide-Induced Preconditioning

Introduction: Evidence suggests that lipopolysaccharide ( LPS ) preconditioning (PC) provides neuroprotection by inducing tolerance to subsequent lethal ischemia. Ischemic tolerance (IT) declines with age, yet it is unclear whether metabolic disease like type 2 diabetes (T2DM) also interfere with IT. The current study sought to determine whether T2DM reduces neuroprotection induced by LPS PC, and the molecular mechanisms underlying the dampened IT. Methods: Male and female diabetic db/db and control db/+ mice (8-10 months of age) were i.p. injected with Toll-like receptor (TLR)4 ligand LPS at 0.2 mg/kg or saline. Stroke was induced 3 days later by permanent distal occlusion of middle cerebral artery and temporary occlusion of ipsilateral common carotid artery for 60 min. Sensory and motor function was evaluated 1 day after stroke by a combination of tests. Infarct size was determined 3 days after stroke following TTC staining. The effect of LPS PC on the phenotypes of leukocytes in the brain parenchyma after stroke was quantified by Fluorescent activated cell sorter (FACS) 2 days after stroke with a panel of markers for subsets of myeloid cells. Transcriptome was determined by RNAseq of isolated monocytes from peripheral blood mononuclear cells. Differentially expressed genes were examined for distinct biological pathways via enrichment analysis. Results: LPS PC significantly reduced infarct in db/+ mice by one third and improved neurological scores, but it did not protect the db/db mice from stroke. FACS data showed that LPS PC reduced the number of Ly6G+ cells in db/+ but not db/db brains, suggesting that LPS PC curbed stroke-induced infiltration of neutrophils in the brain parenchyma. Pathway analysis suggests that diabetic mice exhibited down-regulated pathways in interferon signaling. The expression of Jak-Stat signaling (such as Stat1), MHC antigen presentation (H2A family), and Toll-like receptor (TLR) signaling (such as Irf7) were also reduced in diabetic mice after stroke. Conclusions: The dampened IT in the diabetic mice induced by LPS-PC is associated with increased neutrophil infiltration after stroke compared to control mice. Ongoing studies will seek toovercome the underlying defective molecular mechanisms in T2DM.

Quantitative tissue proteome profile reveals neutrophil degranulation and remodeling of extracellular matrix proteins in early stage gallbladder cancer.

Gallbladder cancer (GBC) is an aggressive malignancy of the gastrointestinal tract with a poor prognosis. It is important to understand the molecular processes associated with the pathogenesis of early stage GBC and identify proteins useful for diagnostic and therapeutic strategies. Here, we have carried out an iTRAQ-based quantitative proteomic analysis of tumor tissues from early stage GBC cases (stage I, n=7 and stage II, n=5) and non-tumor controls (n=6) from gallstone disease (GSD). We identified 357 differentially expressed proteins (DEPs) based on ≥ 2 unique peptides and ≥ 2 fold change with p value < 0.05. Pathway analysis using the STRING database showed, 'neutrophil degranulation' to be the major upregulated pathway that includes proteins such as MPO, PRTN3, S100A8, MMP9, DEFA1, AZU, and 'ECM organization' to be the major downregulated pathway that includes proteins such as COL14A1, COL1A2, COL6A1, COL6A2, COL6A3, BGN, DCN. Western blot and/or IHC analysis confirmed the elevated expression of MPO, PRTN3 and S100A8 in early stage of the disease. Based on the above results, we hypothesize that there is an increased neutrophil infiltration in tumor tissue and neutrophil degranulation leading to degradation of extracellular matrix (ECM) proteins promoting cancer cell invasion in the early stage GBC. Some of the proteins (MPO, MMP9, DEFA1) associated with 'neutrophil degranulation' showed the presence of 'signal sequence' suggesting their potential as circulatory markers for early detection of GBC. Overall, the study presents a protein dataset associated with early stage GBC.

Acinetobacter baumannii reinforces the pathogenesis by promoting IL-17 production in a mouse pneumonia model.

Interleukin-17 (IL-17) is involved in host defense against bacterial infection.Little is known about the role of IL-17 in A. baumannii-infected pneumonia. Our objective was to investigate the role of IL-17 in pulmonary A. baumannii infection in a mouse model. We infected C57BL/6 mice intra-tracheally (i.t.) with A. baumannii to establish pneumonia model and found A. baumannii infection elevated IL-17 expression in lungs. IL-17-deficient (Il17-/-) mice were resistant to pulmonary A. baumannii infection, showing improved mice survival, reduced bacteria burdens, and alleviated lung inflammation.Further, treatment of A. baumannii-infected Il17-/- mice with IL-17 exacerbated the severity of pneumonia. These data suggest a pathogenic role of IL-17 in pulmonary A. baumannii infection. Further, the infiltration and phagocytic function of neutrophils in broncho-alveolar lavage fluid were detected by flow cytometry. The results showed that Il17-/- mice had increased neutrophil infiltration and enhanced phagocytosis in neutrophils at the early time of infection. Treatment of mice with IL-17 suppressed phagocytic function of neutrophils. All data suggest that IL-17 promotes susceptibility of mice to pulmonary A. baumannii infection by suppressing neutrophil phagocytosis at early time of infection. Targeting IL-17 might be a potential therapeutic strategy in controlling the outcome of A. baumanniipneumonia.

Morphine mediated neutrophil infiltration in intestinal tissue play essential role in histological damage and microbial dysbiosis

ABSTRACT The gut microbial ecosystem exhibits a complex bidirectional communication with the host and is one of the key contributing factors in determining mucosal immune homeostasis or an inflammatory state. Opioid use has been established to induce gut microbial dysbiosis consistent with increased intestinal tissue inflammation. In this study, we investigated the role of infiltrated immune cells in morphine-induced intestinal tissue damage and gut microbial dysbiosis in mice. Results reveal a significant increase in chemokine expression in intestinal tissues followed by increased neutrophil infiltration post morphine treatment which is direct consequence of a dysbiotic microbiome since the effect is attenuated in antibiotics treated animals and in germ-free mice. Neutrophil neutralization using anti-Ly6G monoclonal antibody showed a significant decrease in tissue damage and an increase in tight junction protein organization. 16S rRNA sequencing on intestinal samples highlighted the role of infiltrated neutrophils in modulating microbial community structure by providing a growth benefit for pathogenic bacteria, such as Enterococcus, and simultaneously causing a significant depletion of commensal bacteria, such as Lactobacillus. Taken together, we provide the first direct evidence that neutrophil infiltration contributes to morphine-induced intestinal tissue damage and gut microbial dysbiosis. Our findings implicate that inhibition of neutrophil infiltration may provide therapeutic benefits against gastrointestinal dysfunctions associated with opioid use.

A comparative study of IL-33 and its receptor ST2 in a C57BL/6J mouse model of pulmonary Cryptococcus neoformans infection.

It has been reported that IL-33 receptor ST2 deficiency mitigates Cryptococcus neoformans (C. neoformans) pulmonary infection in BALB/c mice. IL-33 may modulate immune responses in ST2-dependent and ST2-independent manners. The host genetic background (i.e., BALB/c, C57BL/6J) influences immune responses against C. neoformans. In the present study, we aimed to explore the roles of IL-33 and ST2 in pulmonary C. neoformans-infected mice on a C57BL/6J genetic background. C. neoformans infection increased IL-33 expression in lung tissues. IL-33 deficiency but not ST2 deficiency significantly extended the survival time of C. neoformans-infected mice. In contrast, either IL-33 or ST2 deficiency reduced fungal burdens in lung, spleen and brain tissues from the mice following C. neoformans intratracheal inoculation. Similarly, inflammatory responses in the lung tissues were more pronounced in both the IL-33-/- and ST2-/- infected mice. However, mucus production was decreased in IL-33-/- infected mice alone, and the level of IL-5 in bronchoalveolar lavage fluid (BALF) was substantially decreased in the IL-33-/- infected mice but not ST2-/- infected mice. Moreover, IL-33 deficiency but not ST2 deficiency increased iNOS-positive macrophages. At the early stage of infection, the reduced pulmonary fungal burden in the IL-33-/- and ST2-/- mice was accompanied by increased neutrophil infiltration. Collectively, IL-33 regulated pulmonary C. neoformans infection in an ST2-dependent and ST2-independent manner in C57BL/6J mice.

Single-cell RNA-sequencing identifies anti-cancer immune phenotypes in the early lung metastatic niche during breast cancer.

Microenvironmental changes in the early metastatic niche may be exploited to identify therapeutic targets to inhibit secondary tumor formation and improve disease outcomes. We dissected the developing lung metastatic niche in a model of metastatic, triple-negative breast cancer using single-cell RNA-sequencing. Lungs were extracted from mice at 7-, 14-, or 21days after tumor inoculation corresponding to the pre-metastatic, micro-metastatic, and metastatic niche, respectively. The progression of the metastatic niche was marked by an increase in neutrophil infiltration (5% of cells at day 0 to 81% of cells at day 21) and signaling pathways corresponding to the hallmarks of cancer. Importantly, the pre-metastatic and early metastatic niche were composed of immune cells with an anti-cancer phenotype not traditionally associated with metastatic disease. As expected, the metastatic niche exhibited pro-cancer phenotypes. The transition from anti-cancer to pro-cancer phenotypes was directly associated with neutrophil and monocyte behaviors at these time points. Predicted metabolic, transcription factor, and receptor-ligand signaling suggested that changes in the neutrophils likely induced the transitions in the other immune cells. Conditioned medium generated by cells extracted from the pre-metastatic niche successfully inhibited tumor cell proliferation and migration in vitro and the in vivo depletion of pre-metastatic neutrophils and monocytes worsened survival outcomes, thus validating the anti-cancer phenotype of the developing niche. Genes associated with the early anti-cancer response could act as biomarkers that could serve as targets for the treatment of early metastatic disease. Such therapies have the potential to revolutionize clinical outcomes in metastatic breast cancer.

Empirical Study of Anti-Inflammatory Effects of Kecombrang (Etlingera elatior) in Mus musculus Sepsis Model

Mice used as experimental animals in the laboratory belong to the genus Mus, subfamily Murinae, family Muridae, superfamily Muroidea, order Rodentia, and class M. musculus , these mice are also referred to as home mice. Mice have been used as standard animals in toxicology, teratology, and carcinogenesis tests, even today, mice have also been used for behavioral, neurologic, nutritional, genetic, immunological, infectious, metabolic, and degenerative disease studies. Animal models of sepsis with intraperitoneal or intravenous injection of lipopolysaccharide (LPS) have been widely used for sepsis research. LPS induces systemic inflammation that mimics the early phase of sepsis. LPS injection causes kidney injury, including a decrease in glomerular filtration rate, an increase in blood urea nitrogen, and an increase in neutrophil infiltration in the kidney. The injectable dose of LPS can be titrated to mimic early sepsis without hemodynamic compromise, which has been useful for studying the systemic and renal responses. The response during the early phase of sepsis is that doses of LPS are usually used to induce systemic hypotension and decrease glomerular perfusion, whereas low doses of LPS do not cause systemic hypotension but still decrease glomerular perfusion. There are several advantages of LPS compared to others, namely, the method used is simple and the model is very controlled and standardized. The dose of endotoxin that causes 50% mortality in mice is 1–25 mg/kg. In this study, mice were given intraperitoneal injection of LPS at a dose of 0.3 mg/kg BW. LPS injection was given to the positive control group and treatment group 1, treatment group 2, and treatment group 3 at the start of the study.