Neutrophils In Wild-type Mice Research Articles

NET formation is independent of gasdermin D and pyroptotic cell death.

Neutrophil extracellular traps (NETs) are DNA scaffolds coated with granule proteins that are released by neutrophils to ensnare and kill bacteria. NET formation occurs in response to many stimuli through independent molecular pathways. Although NET release has been equated to a form of lytic cell death, live neutrophils can rapidly release antimicrobial NETs. Gasdermin D (GSDMD), which causes pyroptotic death in macrophages, is thought to be required for NET formation by neutrophils. Through experiments with known physiological activators of NET formation and ligands that activate canonical and noncanonical inflammasome signaling pathways, we demonstrated that Gsdmd-deficient mouse neutrophils were as competent as wild-type mouse neutrophils in producing NETs. Furthermore, GSDMD was not cleaved in wild-type neutrophils during NET release in response to inflammatory mediators. We found that activation of both canonical and noncanonical inflammasome signaling pathways resulted in GSDMD cleavage in wild-type neutrophils but was not associated with cell death. Moreover, NET formation as a result of either pathway of inflammasome activation did not require GSDMD. Together, these data suggest that NETs can be formed by viable neutrophils after inflammasome activation and that this function does not require GSDMD.

IF1 inactivation attenuates experimental colitis through downregulation of neutrophil infiltration in colon mucosa

IF1 is a mitochondrial protein involved in the regulation of ATP synthase activity. The role of IF1 remains to be established in inflammatory bowel diseases (IBD). In this study, we report that IF1 gene inactivation generated protection against IBD in the dextran sodium sulfate (DSS) model. IF1 gene knockout (IF1-KO) mice developed less severe colitis than the wild type (WT) mice as judged by parameters including disease activity index (DAI), body weight loss, inflammatory cytokines, leukocyte infiltration and bacterial invasion in the colon tissue. The intestinal barrier integrity was protected in the colon tissue of IF1-KO mice through a reduction in apoptosis and inflammasomal activity. The protection was abolished in the KO mice after substitution of the immune cells with the wild type cells following bone marrow transplantation. Depletion of neutrophils with anti-Gr-1 antibody abolished the protection from colitis in IF1-KO mice. Neutrophil number was decreased in the peripheral blood of IF1-KO mice, which was associated with a reduction in LC3A/B proteins in the KO neutrophils in Rapamycin-induced autophagy response. Inhibition of autophagy with the lysosome inhibitor Chloroquine (CQ) decreased the absolute number of neutrophils in WT mice and protected the mice from colitis. Taken together, these findings suggest that IF1 may contribute to the pathogenesis of IBD through acceleration of neutrophil autophagy. The activity is attenuated in the IF1-KO mice through reduction of autophagy in neutrophils leading to resistance to IBD.

Marginal Zone B Cells Assist With Neutrophil Accumulation to Fight Against Systemic Staphylococcus aureus Infection.

In addition to regulating immune responses by producing antibodies that confer humoral immunity, B cells can also affect these responses by producing cytokines. How B cells participate in the clearance of pathogenic infections via functions other than the production of pathogen-specific antibodies is still largely unknown. Marginal zone (MZ) B cells can quickly respond to bacterial invasion by providing the initial round of antibodies. After a bloodborne bacterial infection, neutrophils promptly migrate to the MZ. However, the mechanisms regulating neutrophil accumulation in the MZ during the initial phase of infection also remain obscure. Here, we found that MZ B cell-deficient mice are more susceptible to systemic Staphylococcus aureus (S. aureus) infection compared with wildtype mice. The expression levels of interleukin (IL)-6 and CXCL1/CXCL2 in MZ B cells increased significantly in mice at 3–4 h after infection with S. aureus, then decreased at 24 h post-infection. After systemic S. aureus infection, splenic neutrophils express increased CXCR2 levels. Our results from confocal microscopy imaging of thick-section staining demonstrate that neutrophils in wildtype mice form cell clusters and are in close contact with MZ B cells at 3 h post-infection. This neutrophil cluster formation shortly after infection was diminished in both MZ B cell-deficient mice and IL-6-deficient mice. Blocking the action of CXCL1/CXCL2 by injecting anti-CXCL1 and anti-CXCL2 antibodies 1 h before S. aureus infection significantly suppressed the recruitment of neutrophils to the MZ at 3 h post-infection. Compared with peptidoglycan stimulation alone, peptidoglycan stimulation with neutrophil co-culture further enhanced MZ B-cell activation and differentiation. Using a Förster resonance energy transfer by fluorescence lifetime imaging (FLIM-FRET) analysis, we observed evidence of a direct interaction between neutrophils and MZ B cells after peptidoglycan stimulation. Furthermore, neutrophil depletion in mice resulted in a reduced production of S. aureus-specific immunoglobulin (Ig)M at 24 h post-infection. Together, our results demonstrate that MZ B cells regulate the rapid neutrophil swarming into the spleen during the early phase of systemic S. aureus infection. Interaction with neutrophils assists MZ B cells with their differentiation into IgM-secreting cells and contributes to the clearance of systemic bacterial infections.

IL33 activates CD8+T and NK cells through MyD88 pathway to suppress the lung cancer cell growth in mice.

In this study, we observed the effects of IL-33 on tumor immune response in lung cancer-bearing mice using wild type and MyD88-/- mice respectively. Wild C57BL/6 (C57BL/6WT), MyD88 knockout C57BL/6 mice (C57BL/6 MyD88-/-) and Lewis cells were used in this study. Cell proliferation, cytokine release and cytotoxicity were detected. IL-33 could significantly up-regulate specific cellular immunity, inhibit tumor growth and improve survival time in wild type mice group, and it had dose dependent effect. However, IL-33 had no effect on cell immunity and tumor growth in MyD88-/- mice group. Compared with MyD88-/- mice, IL-33 could significantly increase the ratio of CD8+T cells to neutrophils in wild type mice, while the percentage of tumor infiltrating CD11b+ cells, Mo-MDSC, F4/80+ macrophages and mDC cells decreased significantly in wild type mice group. IL-33 could upregulate the expression of CD107a and IFN-γ in CD8+T cells and NK cells of wild type mice, while IL-33 could not upregulate them in MyD88-/- mice. IL-33 could upregulate the expression of CD40, CD80, CD86 and CD205 in DC cells in wild type mice, induce T cells to differentiate into Th1 cells and enhance tumor cell immunity. IL-33 could promote differentiation and maturation of DC cells through MyD88 pathway, up-regulate the tumor immunity of CD8+T cells and NK cells, and inhibit the proliferation of lung cancer cells.

The pro-inflammatory effects of miR-155 promote liver fibrosis and alcohol-induced steatohepatitis

Alcoholic liver disease (ALD) ranges from fatty liver to inflammation and cirrhosis. miRNA-155 is an important regulator of inflammation. In this study, we describe the in vivo role of miR-155 in ALD. Wild-type (WT) (C57/BL6J) or miR-155 knockout (KO) and TLR4 KO mice received Lieber DeCarli diet for 5weeks. Some mice received corn oil or CCl4 for 2 or 9weeks. We found that miR-155 KO mice are protected from alcohol-induced steatosis and inflammation. The reduction in alcohol-induced fat accumulation in miR-155 KO mice was associated with increased peroxisome proliferator-activated receptor response element (PPRE) and peroxisome proliferator-activated receptors (PPAR)α (miR-155 target) binding and decreased MCP1 production. Treatment with a miR-155 inhibitor increased PPARγ expression in naïve and alcohol treated RAW macrophages. Alcohol increased lipid metabolism gene expression (FABP4, LXRα, ACC1 and LDLR) in WT mice and this was prevented in KO mice. Alcohol diet caused an increase in the number of CD163(+) CD206(+) infiltrating macrophages and neutrophils in WT mice, which was prevented in miR-155 KO mice. Kupffer cells isolated from miR-155 KO mice exhibited predominance of M2 phenotype when exposed to M1 polarized signals and this was due to increased C/EBPβ. Pro-fibrotic genes were attenuated in miR-155 KO mice after alcohol diet or CCl4 treatment. Compared to WT mice, attenuation in CCl4 induced hydroxyproline and α-SMA was observed in KO mice. Finally, we show TLR4 signaling regulates miR-155 as TLR4 KO mice showed no induction of miR-155 after alcohol diet. Collectively our results demonstrated the role of miR-155 in alcohol-induced steatohepatitis and fibrosis in vivo.

Differential Contributions of the Complement Anaphylotoxin Receptors C5aR1 and C5aR2 to the Early Innate Immune Response against Staphylococcus aureus Infection.

The complement anaphylatoxin C5a contributes to host defense against Staphylococcus aureus. In this study, we investigated the functional role of the two known C5a receptors, C5aR1 and C5aR2, in the host response to S. aureus. We found that C5aR1−/− mice exhibited greater susceptibility to S. aureus bloodstream infection than wild type and C5aR2−/− mice, as demonstrated by the significantly higher bacterial loads in the kidneys and heart at 24 h of infection, and by the higher levels of inflammatory IL-6 in serum. Histological and immunohistochemistry investigation of infected kidneys at 24 h after bacterial inoculation revealed a discrete infiltration of neutrophils in wild type mice but already well-developed abscesses consisting of bacterial clusters surrounded by a large number of neutrophils in both C5aR1−/− and C5aR2−/− mice. Furthermore, blood neutrophils from C5aR1−/− mice were less efficient than those from wild type or C5aR2−/− mice at killing S. aureus. The requirement of C5aR1 for efficient killing of S. aureus was also demonstrated in human blood after disrupting C5a-C5aR1 signaling using specific inhibitors. These results demonstrated a role for C5aR1 in S. aureus clearance as well as a role for both C5aR1 and C5aR2 in the orchestration of the inflammatory response during infection.

T cell augments the antitumor activity of tumor-targeting Salmonella

Systemic administration of Salmonella to tumor-bearing mice leads to preferential accumulation within tumor sites and retardation of tumor growth. However, the detailed mechanism of Salmonella-induced antitumor immune response via host T cell remains uncertain. Herein, we used wild-type, CD4(+) T-cell-deficient, and CD8(+) T-cell-deficient mice to study the role of T cell in the antitumor immune responses induced by Salmonella enterica serovar Choleraesuis (Salmonella Choleraesuis). When systemically administered into mice bearing tumors, Salmonella Choleraesuis significantly inhibited tumor growth by 50%. In contrast, in T-cell-deficient mice, there was only 34-42% inhibition of tumor growth. We found that treatment with Salmonella Choleraesuis significantly upregulates interferon-γ in wild-type and CD8(+) T-cell-deficient mice, but not in CD4(+) T-cell-deficient mice. Furthermore, immunohistochemical staining of the tumors revealed more infiltration of macrophages and neutrophils in wild-type mice after Salmonella Choleraesuis treatment compared with those in T-cell-deficient mice. The antitumor therapeutic effect mediated by Salmonella Choleraesuis is associated with an inflammatory immune response at the tumor site and a tumor T helper 1-type immune response. In conclusion, these results suggest that tumor-targeted therapy using Salmonella Choleraesuis, which exerts tumoricidal effects and stimulates T cell activities, represents a potential strategy for the treatment of tumor.

Interleukin-1 mediates thermal injury-induced lung damage through C-Jun NH2-terminal kinase signaling*

The molecular mechanisms of lung damage following thermal injury are not clear. The purpose of this study was to determine whether interleukin (IL)-1 mediates burn-induced inducible nitric oxide synthase (iNOS) expression, peroxynitrite production, and lung damage through c-Jun NH2-terminal kinase (JNK) signaling. Prospective, experimental study. Research laboratory at a university hospital. Thermal injury models in the mice. IL-1 receptor type 1 (IL-1R1) mice, Tnfrsf1a mice, and wild-type (WT) mice were subjected to 30% total body surface area third-degree burn. The JNK inhibitor, SP600125, was given to mice to study the involvement of the JNK pathway in thermal injury-induced lung damage. WT --> WT, WT --> IL-1R1, and IL-1R1 --> WT chimeric mice were generated to determine the role of hematopoietic cells in IL-1-mediated lung damage. Neutrophils were harvested and treated in vitro with N-formyl-methionyl-leucyl-phenylalanine (fMLP). IL-1R1 mice rather than Tnfrsf1a mice showed less thermal injury-induced lung damage. IL-1R1 mice displayed less lung JNK activity; intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), chemokine receptor 2 (CXCR2), and macrophage inflammatory protein-2 (MIP2), messenger RNA expression; myeloperoxidase activity; and neutrophil p38 mitogen-activated protein kinase (MAPK) phosphorylation after thermal injury. SP600125 significantly reduced thermal injury-induced blood dihydrorhodamine (DHR) 123 oxidation, iNOS expression, and lung permeability in WT mice but not in IL-1R1 mice. IL-1R1 --> WT chimeric mice rather than WT --> IL-1R1 chimeric mice showed less thermal injury-induced lung damage. fMLP increased reactive oxygen species (ROS) production of neutrophils in WT mice but not in IL-1R1 mice. SP600125 decreased ROS production of neutrophils in WT mice but not in IL-1R1 mice. Thermal injury-induced lung JNK activation; lung ICAM, VCAM, CXCR2, and MIP2 expression; and DHR 123 oxidation are IL-1 dependent. JNK inhibition decreases IL-1-mediated thermal injury-induced lung damage. Given that the IL-1 receptor is critical in thermal injury-induced p38 MAPK phosphorylation and ROS production of neutrophils, we conclude that IL-1 mediates thermal injury-induced iNOS expression and lung damage through the JNK signaling pathway.

P84, a New Gβγ-Activated Regulatory Subunit of the Type IB Phosphoinositide 3-Kinase p110γ

A variety of genetic and inhibitor studies have shown that phosphoinositide 3-kinase gamma (PI3Kgamma) plays an essential role in a number of physiological responses, including neutrophil chemotaxis, mast cell degranulation, and cardiac function []. PI3Kgamma is currently thought to be composed of a p110gamma catalytic subunit and a single regulatory subunit, p101. The binding of p110gamma to p101 dramatically increases the activation of the complex by Gbetagamma subunits and, hence, is thought to be critical for the coupling of PI3Kgamma to G protein coupled receptors []. Here, we characterize a new regulatory subunit for PI3Kgamma. p84 is present in human, mouse, chicken, frog, and fugu genomes and is located beside the p101 locus. It is broadly expressed in cells of the murine immune system. Both recombinant and endogenous p84 bind p110gamma specifically and with high affinity. Binding of p84 to p110gamma substantially increases the ability of Gbetagamma to stimulate phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) production both in vitro and in vivo. However, the p84/p110gamma heterodimer is approximately 4-fold less sensitive to Gbetagammas than p101/p110gamma. Endogenous murine p84 expression is substantially reduced in the absence of p110gamma expression. We conclude that p110gamma has two potential regulatory subunits in vivo, p84 and p101.

LSP1 is an endothelial gatekeeper of leukocyte transendothelial migration

Leukocyte-specific protein 1 (LSP1), an F-actin binding protein and a major downstream substrate of p38 mitogen-activated protein kinase as well as protein kinase C, has been reported to be important in leukocyte chemotaxis. Although its distribution has been thought to be restricted to leukocytes, herein we report that LSP1 is expressed in endothelium and is essential to permit neutrophil emigration. Using intravital microscopy to directly visualize leukocyte rolling, adhesion, and emigration in postcapillary venules in LSP1-deficient (Lsp1 −/−) mice, we found that LSP1 deficiency inhibits neutrophil extravasation in response to various cytokines (tumor necrosis factor-α and interleukin-1β) and to neutrophil chemokine keratinocyte-derived chemokine in vivo. LSP1 deficiency did not affect leukocyte rolling or adhesion. Generation of Lsp1 −/− chimeric mice using bone marrow transplantation revealed that in mice with Lsp1 −/− endothelial cells and wild-type leukocytes, neutrophil transendothelial migration out of postcapillary venules is markedly restricted. In contrast, Lsp1 −/− neutrophils in wild-type mice were able to extravasate normally. Consistent with altered endothelial function was a reduction in vascular permeability to histamine in Lsp1 −/− animals. Western blot analysis and immunofluorescence microscopy examination confirmed the presence of LSP1 in wild-type but not in Lsp1 −/− mouse microvascular endothelial cells. Cultured human endothelial cells also stained positive for LSP1. Our results suggest that LSP1 expressed in endothelium regulates neutrophil transendothelial migration.

Neither neutrophils nor reactive oxygen species contribute to tissue damage during Pneumocystis pneumonia in mice.

Neutrophils are implicated in the damage of lung tissue in many disease states, including infectious diseases and environmental insults. These effects may be due to oxidative or nonoxidative functions of the neutrophil or both. We examined the role of neutrophils in pulmonary damage during infection with the opportunistic fungal pathogen Pneumocystis sp. in four mouse models of neutrophil dysfunction. These were (i) a knockout of the gp91(phox) component of NADPH oxidase, in which reactive oxygen species (ROS) production is greatly reduced; (ii) a double knockout of gp91(phox) and inducible nitric oxide synthase, in which ROS and nitric oxide production is greatly decreased; (iii) a knockout of the chemokine receptor CXCR2, in which accumulation of intra-alveolar neutrophils is severely diminished; and (iv) antibody depletion of circulating neutrophils in wild-type mice with the monoclonal antibody RB6. Surprisingly, in each case, indicators of pulmonary damage (respiratory rates, arterial oxygen partial pressures, and intra-alveolar albumin concentrations) were the same in knockout mice and comparable wild-type mice. Therefore, whereas neutrophils are a valid correlative marker of lung damage during Pneumocystis infection, neither neutrophils nor ROS appear to be the causative agent of tissue damage. We also show that there is no difference in Pneumocystis burdens between wild-type and knockout mice, which supports the idea that neutrophils do not have a major role in the clearance of this organism.

Identification of A3 receptor- and mast cell-dependent and -independent components of adenosine-mediated airway responsiveness in mice.

Adenosine-induced bronchoconstriction is a well-recognized feature of atopic asthma. Adenosine acts through four different G protein-coupled receptors to produce a myriad of physiological effects. To examine the contribution of the A(3) adenosine receptor to adenosine-induced bronchoconstriction and to assess the contribution of mast cells to this process, we quantified airway responsiveness to aerosolized adenosine in wild-type, A(3) receptor-deficient, and mast cell-deficient mice. Compared with the robust airway responses elicited by adenosine in wild-type mice, both A(3)-deficient and mast cell-deficient mice exhibited a significantly attenuated response compared with their respective wild-type controls. Histological examination of the airways 4 h after adenosine exposure revealed extensive degranulation of airway mast cells as well as infiltration of neutrophils in wild-type mice, whereas these findings were much diminished in A(3)-deficient mice and were not different from those in PBS-treated controls. These data indicate that the airway responses to aerosolized adenosine in mice occur largely through A(3) receptor activation and that mast cells contribute significantly to these responses, but that activation of additional adenosine receptors on a cell type(s) other than mast cells also contributes to adenosine-induced airway responsiveness in mice. Finally, our findings indicate that adenosine exposure can result in A(3)-dependent airway inflammation, as reflected in neutrophil recruitment, as well as alterations in airway function.