Neutrophil-depleted Mice Research Articles

Investigation of Allogeneic Neutrophil Transfusion in Improving Survival Rates of Severe Infection Mice.

The management of granulocytopenia-associated infections is challenging, and a high mortality rate is associated with traditional supportive therapies. Neutrophils-the primary defenders of the human immune system-have potent bactericidal capabilities. Here, we investigated the dynamic in vivo distribution of neutrophil transfusion and their impact on the treatment outcome of severe granulocytopenic infections. We transfused 89Zr-labeled neutrophils in the C57BL/6 mice and observed the dynamic neutrophil distribution in mice for 24 h using the micro-positron emission tomography (Micro-PET) technique. The labeled neutrophils were predominantly retained in the lungs and spleen up to 4 h after injection and then redistributed to other organs, such as the spleen, liver, and bone marrow. Neutrophil transfusion did not elicit marked inflammatory responses or organ damage in healthy host mice. Notably, allogeneic neutrophils showed rapid chemotaxis to the infected area of the host within 1 h. Tail vein infusion of approximately 107 neutrophils substantially bolstered host immunity, ameliorated the inflammatory state, and increased survival rates in neutrophil-depleted and infected mice. Overall, massive allogeneic neutrophil transfusion had a therapeutic effect in severe infections and can have extensive applications in the future.

Platelet-derived chemokines promote skeletal muscle regeneration by guiding neutrophil recruitment to injured muscles

Skeletal muscle regeneration involves coordinated interactions between different cell types. Injection of platelet-rich plasma is circumstantially considered an aid to muscle repair but whether platelets promote regeneration beyond their role in hemostasis remains unexplored. Here, we find that signaling via platelet-released chemokines is an early event necessary for muscle repair in mice. Platelet depletion reduces the levels of the platelet-secreted neutrophil chemoattractants CXCL5 and CXCL7/PPBP. Consequently, early-phase neutrophil infiltration to injured muscles is impaired whereas later inflammation is exacerbated. Consistent with this model, neutrophil infiltration to injured muscles is compromised in male mice with Cxcl7-knockout platelets. Moreover, neo-angiogenesis and the re-establishment of myofiber size and muscle strength occurs optimally in control mice post-injury but not in Cxcl7ko mice and in neutrophil-depleted mice. Altogether, these findings indicate that platelet-secreted CXCL7 promotes regeneration by recruiting neutrophils to injured muscles, and that this signaling axis could be utilized therapeutically to boost muscle regeneration.

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 ...

Cutting Edge: Neutrophils License the Maturation of Monocytes into Effective Antifungal Effectors.

Neutrophils are critical for the direct eradication of Aspergillus fumigatus conidia, but whether they mediate antifungal defense beyond their role as effectors is unclear. In this study, we demonstrate that neutrophil depletion impairs the activation of protective antifungal CCR2+ inflammatory monocytes. In the absence of neutrophils, monocytes displayed limited differentiation into monocyte-derived dendritic cells, reduced formation of reactive oxygen species, and diminished conidiacidal activity. Upstream regulator analysis of the transcriptional response in monocytes predicted a loss of STAT1-dependent signals as the potential basis for the dysfunction seen in neutrophil-depleted mice. We find that conditional removal of STAT1 on CCR2+ cells results in diminished antifungal monocyte responses, whereas exogenous administration of IFN-γ to neutrophil-depleted mice restores monocyte-derived dendritic cell maturation and reactive oxygen species production. Altogether, our findings support a critical role for neutrophils in antifungal immunity not only as effectors but also as important contributors to antifungal monocyte activation, in part by regulating STAT1-dependent functions.

The Actin-Binding Protein Cortactin Promotes Sepsis Severity by Supporting Excessive Neutrophil Infiltration into the Lung.

Sepsis is a systemic infection that can lead to multi-organ failure. It is characterised by an uncontrolled immune response with massive neutrophil influx into peripheral organs. Neutrophil extravasation into tissues depends on actin remodeling and actin-binding proteins such as cortactin, which is expressed ubiquitously, except for neutrophils. Endothelial cortactin is necessary for proper regulation of neutrophil transendothelial migration and recruitment to sites of infection. We therefore hypothesised that cortactin plays a crucial role in sepsis development by regulating neutrophil trafficking. Using a murine model of sepsis induced by cecal ligation and puncture (CLP), we showed that cortactin-deficient (KO) mice survive better due to reduced lung injury. Histopathological analysis of lungs from septic KO mice revealed absence of oedema, reduced vascular congestion and mucus deposition, and better-preserved alveoli compared to septic wild-type (WT) mice. Additionally, sepsis-induced cytokine storm, excessive neutrophil infiltration into the lung and oxidative stress were significantly reduced in KO mice. Neutrophil depletion 12 h after sepsis improved survival in WT mice by averting lung injury, similar to both neutrophil-depleted and non-depleted KO mice. Our findings highlight a critical role of cortactin for lung neutrophil infiltration and sepsis severity.

Interplay of Trypanosome Lytic Factor and innate immune cells in the resolution of cutaneous Leishmania infection.

Trypanosome Lytic Factor (TLF) is a primate-specific high-density lipoprotein (HDL) complex that, through the cation channel-forming protein apolipoprotein L-1 (APOL1), provides innate immunity to select kinetoplastid parasites. The immunoprotective effects of TLF have been extensively investigated in the context of its interaction with the extracellular protozoan Trypanosoma brucei brucei, to which it confers sterile immunity. We previously showed that TLF could act against an intracellular pathogen Leishmania, and here we dissected the role of TLF and its synergy with host-immune cells. Leishmania major is transmitted by Phlebotomine sand flies, which deposit the parasite intradermally into mammalian hosts, where neutrophils are the predominant phagocytes recruited to the site of infection. Once in the host, the parasites are phagocytosed and shed their surface glycoconjugates during differentiation to the mammalian-resident amastigote stage. Our data show that mice producing TLF have reduced parasite burdens when infected intradermally with metacyclic promastigotes of L. major, the infective, fly-transmitted stage. This TLF-mediated reduction in parasite burden was lost in neutrophil-depleted mice, suggesting that early recruitment of neutrophils is required for TLF-mediated killing of L. major. In vitro we find that only metacyclic promastigotes co-incubated with TLF in an acidic milieu were lysed. However, amastigotes were not killed by TLF at any pH. These findings correlated with binding experiments, revealing that labeled TLF binds specifically to the surface of metacyclic promastigotes, but not to amastigotes. Metacyclic promastigotes of L. major deficient in the synthesis of surface glycoconjugates LPG and/or PPG (lpg1- and lpg5A-/lpg5B- respectively) whose absence mimics the amastigote surface, were resistant to TLF-mediated lysis. We propose that TLF binds to the outer surface glycoconjugates of metacyclic promastigotes, whereupon it kills the parasite in the acidic phagosome of phagocytes. We hypothesize that resistance to TLF requires shedding of the surface glycoconjugates, which occurs several hours after phagocytosis by immune cells, creating a relatively short-lived but effective window for TLF to act against Leishmania.

Small extracellular vesicles obtained from hypoxic mesenchymal stromal cells have unique characteristics that promote cerebral angiogenesis, brain remodeling and neurological recovery after focal cerebral ischemia in mice

Obtained from the right cell-type, mesenchymal stromal cell (MSC)-derived small extracellular vesicles (sEVs) promote stroke recovery. Within this process, microvascular remodeling plays a central role. Herein, we evaluated the effects of MSC-sEVs on the proliferation, migration, and tube formation of human cerebral microvascular endothelial cells (hCMEC/D3) in vitro and on post-ischemic angiogenesis, brain remodeling and neurological recovery after middle cerebral artery occlusion (MCAO) in mice. In vitro, sEVs obtained from hypoxic (1% O2), but not ‘normoxic’ (21% O2) MSCs dose-dependently promoted endothelial proliferation, migration, and tube formation and increased post-ischemic endothelial survival. sEVs from hypoxic MSCs regulated a distinct set of miRNAs in hCMEC/D3 cells previously linked to angiogenesis, three being upregulated (miR-126-3p, miR-140-5p, let-7c-5p) and three downregulated (miR-186-5p, miR-370-3p, miR-409-3p). LC/MS–MS revealed 52 proteins differentially abundant in sEVs from hypoxic and ‘normoxic’ MSCs. 19 proteins were enriched (among them proteins involved in extracellular matrix–receptor interaction, focal adhesion, leukocyte transendothelial migration, protein digestion, and absorption), and 33 proteins reduced (among them proteins associated with metabolic pathways, extracellular matrix–receptor interaction, focal adhesion, and actin cytoskeleton) in hypoxic MSC-sEVs. Post-MCAO, sEVs from hypoxic MSCs increased microvascular length and branching point density in previously ischemic tissue assessed by 3D light sheet microscopy over up to 56 days, reduced delayed neuronal degeneration and brain atrophy, and enhanced neurological recovery. sEV-induced angiogenesis in vivo depended on the presence of polymorphonuclear neutrophils. In neutrophil-depleted mice, MSC-sEVs did not influence microvascular remodeling. sEVs from hypoxic MSCs have distinct angiogenic properties. Hypoxic preconditioning enhances the restorative effects of MSC-sEVs.

Dysregulated neutrophils failed to control bacteria in an intradermal murine model of Orientia tsutsugamushi infection

Abstract Scrub typhus is a vector-borne febrile illness caused by the obligately intracellular bacterium, Orientia tsutsugamushi that are transmitted by the larvae of trombiculid mites. Approximately 1 million people are infected every year in an Asia-Pacific region. Patients without appropriate treatment may develop severe damage in various organs and lead to death. However, the host protective immunity against this infection is still not understood. Using an intradermal infection mouse model and CFSE-labeled bacteria, we found that neutrophils and monocytes were the majority of infected cells in the skin infection site at 4 and 8 hours post-infection (hpi). However, very few of them can migrate to local lymph node at 24 hpi. Instead, the infected langerhans cells were observed in local lymph nodes, but not in other organs including lung, bone marrow and spleen, as well as blood. To investigate the role of neutrophils in Orientia infection, we treated the infected mice with Anti-Ly6G antibody daily from −1 to 3 dpi. However, the bodyweight loss was comparable between control and neutrophil-depleted mice until 14 dpi. No significant difference of bacterial loads were found in organs including lung and brain. Our in vitro study showed that Orientia infection induced an immunosuppressive neutrophil phenotype and increased neutrophil death. Collectively, our data suggest that infected-neutrophil may undergo the functional dysregulation and not be able to control bacteria efficiently, while antigen-presenting cells may contribute to bacterial dissemination from skin to systemic infection in scrub typhus.

Neutrophils Are Required During Immunization With the Pneumococcal Conjugate Vaccine for Protective Antibody Responses and Host Defense Against Infection.

Neutrophils can shape adaptive immunity; however, their role in vaccine-induced protection against infections in vivo remains unclear. Here, we tested their role in the clinically relevant polysaccharide conjugate vaccine against Streptococcus pneumoniae (pneumococcus). We antibody depleted neutrophils during vaccination, allowed them to recover, and 4 weeks later challenged mice with pneumococci. We found that while isotype-treated vaccinated controls were protected against an otherwise lethal infection in naive mice, full protection was lost upon neutrophil depletion. Compared to vaccinated controls, neutrophil-depleted mice had higher lung bacterial burdens, increased incidence of bacteremia, and lower survival rates. Sera from neutrophil-depleted mice had less antipneumococcal IgG2c and IgG3, were less efficient at inducing opsonophagocytic killing of bacteria by neutrophils in vitro, and were worse at protecting naive mice against pneumococcal pneumonia. In summary, neutrophils are required during vaccination for optimal host protection, which has important implications for future vaccine design against pneumococci and other pathogens.

Therapeutic hypothermia rescues severe acute lung injury (ALI) by IL-1β-induced neutrophil extracellular traps (NETs) formation through down-regulation of Gasdermin D

Abstract ARDS is a serious ALI defined by the acute onset of non-cardiogenic pulmonary edema and hypoxemia. We have previously published that NLRP3 inflammasome is required to develop hypoxemia in LPS plus mechanical ventilator (MV) two-hit-induced ALI. We initially observed that, despite equal neutrophil infiltration, severe alveolar leakage and hypoxemia were not observed in Il1r1KO mice, confirming that NLRP3 inflammasome is critical in this two-hit induced hypoxemia. However, this hypoxemia was significantly attenuated in neutrophil-depleted mice. Furthermore, we found that NETs formations were significantly attenuated in Il1r1KO mice. These data demonstrated that IL-1R signaling plays a regulating role in neutrophil functionality in the alveoli, and not their infiltration. We next examined whether NETs formation contributed to develop hypoxemia in two-hit ALI. by Pad4 inhibitor and DNase I. Both treatments improved the development of hypoxemia. In the course of experiments, we serendipitously found that body temperature is a critical factor regulating severe hypoxemia. Therapeutic hypothermia at 32°C during MV rescued development of hypoxemia with significantly attenuated alveolar leakage, reduced IL-1β secretion, and less NETs formation. 32°C condition inhibited IL-1β secretion by Mϕ as well as NETs induction. Unexpectedly, we observed similar Caspase-1 activities in LPS-primed Mϕ stimulated with ATP or nigericin at 32°C while IL-1βsecretion was significantly reduced. We found decreased GSDMD expression and unsecreted mature IL-1β at 32 °C treatment. Overall our data suggests that hypothermia suppresses ALI development by inhibiting IL-1β secretion and NETs formation through down-regulation of GSDMD.

Complement alone drives efficacy of a chimeric antigonococcal monoclonal antibody.

Multidrug-resistant Neisseria gonorrhoeae is a global health problem. Monoclonal antibody (mAb) 2C7 recognizes a gonococcal lipooligosaccharide epitope that is expressed by >95% of clinical isolates and hastens gonococcal vaginal clearance in mice. Chimeric mAb 2C7 (human immunoglobulin G1 [IgG1]) with an E430G Fc modification that enhances Fc:Fc interactions and hexamerization following surface-target binding and increases complement activation (HexaBody technology) showed significantly greater C1q engagement and C4 and C3 deposition compared to mAb 2C7 with wild-type Fc. Greater complement activation by 2C7-E430G Fc translated to increased bactericidal activity in vitro and, consequently, enhanced efficacy in mice, compared with “Fc-unmodified” chimeric 2C7. Gonococci bind the complement inhibitors factor H (FH) and C4b-binding protein (C4BP) in a human-specific manner, which dampens antibody (Ab)-mediated complement-dependent killing. The variant 2C7-E430G Fc overcame the barrier posed by these inhibitors in human FH/C4BP transgenic mice, for which a single 1 μg intravenous dose cleared established infection. Chlamydia frequently coexists with and exacerbates gonorrhea; 2C7-E430G Fc also proved effective against gonorrhea in gonorrhea/chlamydia-coinfected mice. Complement activation alone was necessary and sufficient for 2C7 function, evidenced by the fact that (1) “complement-inactive” Fc modifications that engaged Fc gamma receptor (FcγR) rendered 2C7 ineffective, nonetheless; (2) 2C7 was nonfunctional in C1q−/− mice, when C5 function was blocked, or in C9−/− mice; and (3) 2C7 remained effective in neutrophil-depleted mice and in mice treated with PMX205, a C5a receptor (C5aR1) inhibitor. We highlight the importance of complement activation for antigonococcal Ab function in the genital tract. Elucidating the correlates of protection against gonorrhea will inform the development of Ab-based gonococcal vaccines and immunotherapeutics.

Neutrophils promote the development of reparative macrophages mediated by ROS to orchestrate liver repair

Phagocytes, including neutrophils and macrophages, have been suggested to function in a cooperative way in the initial phase of inflammatory responses, but their interaction and integration in the resolution of inflammation and tissue repair remain unclear. Here we show that neutrophils have crucial functions in liver repair by promoting the phenotypic conversion of pro-inflammatory Ly6ChiCX3CR1lo monocytes/macrophages to pro-resolving Ly6CloCX3CR1hi macrophages. Intriguingly, reactive oxygen species (ROS), expressed predominantly by neutrophils, are important mediators that trigger this phenotypic conversion to promote liver repair. Moreover, this conversion is prevented by the depletion of neutrophils via anti-Ly6G antibody, genetic deficiency of granulocyte colony-stimulating factor, or genetic deficiency of NADPH oxidase 2 (Nox2). By contrast, adoptive transfer of WT rather than Nox2−/− neutrophils rescues the impaired phenotypic conversion of macrophages in neutrophil-depleted mice. Our findings thus identify an intricate cooperation between neutrophils and macrophages that orchestrate resolution of inflammation and tissue repair.

Neutrophil Cathepsin G Regulates Dendritic Cell Production of IL-12 during Development of CD4 T Cell Responses to Antigens in the Skin.

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to hapten skin sensitization and challenge. Sensitization of wild-type (WT) mice induces hapten-reactive effector CD8 T cells producing IFN-γ and IL-17- and IL-4-producing CD4 T cells that cannot mediate CHS. Although CXCR2-dependent Ly6G+ (neutrophil) cell recruitment into hapten-challenged skin is required to direct effector CD8 T cell infiltration into the challenge site to elicit CHS, 2,4-dinitrofluorobenezene (DNFB) sensitization of CXCR2-/- mice and neutrophil-depleted WT mice induced both hapten-reactive CD4 and CD8 T cells producing IFN-γ and IL-17. CD4 T cell-mediated CHS responses were not generated during DNFB sensitization of neutrophil-depleted WT mice treated with anti-IL-12 mAb or neutrophil-depleted IL-12-/- mice. Neutrophil depletion during DNFB sensitization of WT mice markedly increased IL-12-producing hapten-primed dendritic cell numbers in the skin-draining lymph nodes. Sensitization of mice lacking the neutrophil serine protease cathepsin G (CG)-induced hapten-reactive CD4 and CD8 T cells producing IFN-γ and IL-17 with elevated and elongated CHS responses to DNFB challenge. Induction of CHS effector CD4 T cells producing IFN-γ in neutrophil-depleted WT mice was eliminated by s.c. injection of active, but not inactivated, CG during sensitization. Thus, hapten skin sensitization induces neutrophil release of CG that systemically inhibits hapten-presenting dendritic cell production of IL-12 and the development of hapten-reactive CD4 T cells to IFN-γ-producing CHS effector cells.

Neutrophils mitigate the systemic host response during endotoxemia in mice

SummaryRecent studies have suggested that neutrophils can exert anti‐inflammatory effects. To determine the role of neutrophils in the acute response to lipopolysaccharide (LPS), a component of the Gram‐negative bacterial cell wall, we challenged neutrophil‐depleted and control mice with LPS and analyzed the plasma concentrations of biomarkers indicative of the cytokine and chemokine network, activation of coagulation and the vascular endothelium, and cellular injury. We here show that neutrophils serve an anti‐inflammatory role upon LPS administration, as reflected by sustained elevations of multiple cytokines and chemokines, and enhanced release of nucleosomes in mice depleted of neutrophils, compared with control mice.

Neutrophil cathepsin G inhibits hapten-presenting dendritic cell production of IL-12 and skewing of hapten-reactive CD4 T Cell development to IFN-γ- and IL-17-producing effector cells in allergic contact sensitivity

Abstract Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to hapten skin sensitization and challenge. Sensitization of WT mice induces hapten-reactive CD8 cells producing CHS effector cytokines IFN-γ/IL-17 and IL-4-producing CD4 cells that cannot mediate CHS. Although CXCR2-dependent Gr-1+ cell recruitment into hapten challenged skin is required to direct effector CD8 T cell infiltration to elicit CHS, sensitization of CXCR2−/− mice or neutrophil-depleted WT mice induced both hapten-reactive CD4 and CD8 cells producing IFN-γ and IL-17. CD4 T cell-mediated CHS responses were not generated during hapten sensitization of neutrophil depleted WT mice treated with anti-IL-12 mAb or neutrophil depleted IL-12−/−mice. Neutrophil depletion during hapten sensitization of WT mice markedly increased numbers of IL-12-producing hapten-primed dendritic cells (DC), DC-derived p40, and T-bet-expressing CD4 T cells in the skin-draining lymph nodes. Sensitization of mice lacking the neutrophil serine protease cathepsin G induced hapten-reactive CD4 and CD8 T cells producing IFN-γ and IL-17 with elevated and elongated CHS responses to challenge. Few Gr-1+ cells were detected in the skin draining lymph nodes following senstization suggesting this regulation may not be mediated through direct Gr-1-DC interactions. In support of this, induction of CHS effector CD4 T cells producing IFN-γ in neutrophil-depleted WT mice was eliminated by subcutaneous injection of active, but not inactivated, cathepsin G during sensitization. Thus, hapten skin sensitization induces neutrophil release of cathepsin G that systemically inhibits hapten-presenting dendritic cell production of IL-12 and the development of CHS effector CD4 T cells.

Neutrophils Provide a Favorable IL-1-Mediated Immunometabolic Niche that Primes GLUT4 Translocation and Performance in Skeletal Muscles

Metabolic immunomodulation involving IL-1 has been investigated for unfavorable metabolic effects, including obesity, but a potentially favorable role for IL-1 remains unclear. Here, we find mechanistic interactions between working skeletal muscles and locally recruited neutrophils expressing IL-1β, which supports muscle performance through priming exercise-dependent GLUT4 translocation. Thus, during exercise, both IL-1α/β-deficient and neutrophil-depleted mice similarly exhibit increased fatigability associated with impaired muscle glucose homeostasis due to GLUT4 dysregulation. Deficiency of IL-1-producing neutrophils results in intrinsic abnormalities represented by aberrant Rac1 signaling and irregular GLUT4-storage vesicles, suggesting that these properties are maintained by local IL-1 produced by recruited neutrophils upon exercise, possibly on a daily basis. We propose that neutrophils are highly engaged in skeletal muscle performance via IL-1 regulation, which coordinates favorable inflammatory microenvironments supporting muscle glucose metabolism.

Characterization of Stress and Innate Immunity Resistance of Wild-Type and Δp66 Borrelia burgdorferi.

Borrelia burgdorferi is a causative agent of Lyme disease, the most common arthropod-borne disease in the United States. B. burgdorferi evades host immune defenses to establish a persistent, disseminated infection. Previous work showed that P66-deficient B. burgdorferi (Δp66) is cleared quickly after inoculation in mice. We demonstrate that the Δp66 strain is rapidly cleared from the skin inoculation site prior to dissemination. The rapid clearance of Δp66 bacteria is not due to inherent defects in multiple properties that might affect infectivity: bacterial outer membrane integrity, motility, chemotactic response, or nutrient acquisition. This led us to the hypothesis that P66 has a role in mouse cathelicidin-related antimicrobial peptide (mCRAMP; a major skin antimicrobial peptide) and/or neutrophil evasion. Neither wild-type (WT) nor Δp66 B. burgdorferi was susceptible to mCRAMP. To examine the role of neutrophil evasion, we administered neutrophil-depleting antibody anti-Ly6G (1A8) to C3H/HeN mice and subsequently monitored the course of B. burgdorferi infection. Δp66 mutants were unable to establish infection in neutrophil-depleted mice, suggesting that the important role of P66 during early infection is through another mechanism. Neutrophil depletion did not affect WT B. burgdorferi bacterial burdens in the skin (inoculation site), ear, heart, or tibiotarsal joint at early time points postinoculation. This was unexpected given that prior in vitro studies demonstrated neutrophils phagocytose and kill B. burgdorferi These data, together with our previous work, suggest that despite the in vitro ability of host innate defenses to kill B. burgdorferi, individual innate immune mechanisms have limited contributions to controlling early B. burgdorferi infection in the laboratory model used.

IL-33 exacerbates liver sterile inflammation by amplifying neutrophil extracellular trap formation

Neutrophils and liver sinusoidal endothelial cells (LSECs) both contribute to sterile inflammatory injury during ischemia/reperfusion (I/R), a well-known liver surgical stress. Interleukin-33 (IL-33) has been shown to drive neutrophil infiltration during inflammatory responses through its receptor ST2. We recently reported that infiltrating neutrophils form neutrophil extracellular traps (NETs), which exacerbate sterile inflammatory injury in liver I/R. Here, we sought to determine the role of IL-33 in NET formation during liver sterile inflammation. Evaluation of IL-33 forming NETs was investigated using a partial liver I/R model to generate sterile injury in healthy WT, IL-33 and ST2 knockouts. Serum levels of IL-33 and myeloperoxidase (MPO)-DNA complex were measured in both humans and mice after the first surgery. Liver damage was assessed. Mouse neutrophil depletion was performed by intraperitoneal injection of anti-Ly6G antibody before I/R. Patients undergoing liver resection showed a significant increase in serum IL-33 compared to healthy volunteers. This coincided with higher serum MPO-DNA complexes. NET formation was decreased in IL-33 and ST2 knockout mice compared with control mice, after liver I/R. IL-33 or ST2 deficiency protected livers from I/R injury, whereas rIL-33 administration during I/R exacerbated hepatotoxicity and systemic inflammation. In vitro, IL-33 is released from LSECs to promote NET formation. IL-33 deficient LSECs failed to induce NETs. ST2 deficient neutrophils limited their capacity to form NETs in vitro and adoptive transfer of ST2 knockout neutrophils to neutrophil-depleted WT mice significantly decreased NET formation. Data establish that IL-33, mainly released from LSECs, causes excessive sterile inflammation after hepatic I/R by inducing NET formation. Therapeutic targeting of IL-33/ST2 might extend novel strategies to minimize organ damage in various clinical settings associated with sterile inflammation. Liver ischemia and reperfusion injury results in the formation of neutrophil extracellular traps, which contribute to organ damage in liver surgeries. Herein, we show that IL-33 is released from liver sinusoidal endothelial cells to promote NET formation during liver I/R, which exacerbates inflammatory cascades and sterile inflammation.

Neutrophil Cathepsin G inhibits hapten-presenting dendritic cell production of IL-12 and skewing of hapten-reactive CD4 T cell development to IFN-γ- and IL-17-producing effector cells in allergic contact sensitivity

Abstract Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to hapten skin sensitization and challenge. Sensitization of wild-type (WT) mice induces hapten-reactive CD8 T cells that produce IFN-γ and IL-17 and mediate CHS and hapten-reactive CD4 T cells producing IL-4 that cannot mediate CHS. Although CXCR2-dependent neutrophil recruitment into the hapten challenged skin is required to direct effector CD8 T cell infiltration into the site to elicit CHS in WT mice, CHS was elicited following challenge of hapten sensitized CXCR2−/− mice. Sensitization of CXCR2−/− mice or antibody-mediated depletion of neutrophils during sensitization of WT mice generated hapten-reactive CD4 and CD8 T cells producing IFN-γ and IL-17 and either T cell population could mediate CHS independently of the other. The hapten-reactive CD4 cells producing IFN-γ and IL-17 and their CHS responses were not generated during hapten sensitization of IL-12−/− mice depleted of neutrophils or WT mice depleted of neutrophils and treated with anti-IL-12 mAb. Neutrophil depletion during hapten sensitization of WT mice markedly increased numbers of IL-12-producing dendritic cells in the skin draining lymph nodes. Sensitization of mice lacking the neutrophil serine protease cathepsin G induced hapten-reactive CD4 and CD8 T cells producing IFN-γ and IL-17 with elevated and elongated CHS responses to challenge. Induction of CHS effector CD4 T cells in neutrophil-depleted WT mice was eliminated by subcutaneous injection of recombinant cathepsin G during sensitization. Thus, hapten skin sensitization induces neutrophil production of cathepsin G that regulates hapten-presenting dendritic cell production of IL-12 and the development of CHS effector CD4 T cells.

757 A possible role for M2 macrophages in preservation of dermal collagen during UVB exposure

We previously reported that giving anti-TNFα antibodies (etanercept) to mice blocks UVB recruitment of neutrophils and macrophages into the dermis, but paradoxically, etanercept accelerates UVB-induced loss of dermal collagen. To examine an unsuspected role for neutrophils or macrophages in dermal collagen preservation during UVB exposure, we performed neutrophil depletion and quantification of macrophage subtypes. Of note, M2 macrophages are involved in tissue remodeling in other circumstances and produce TGFβ, an inducer of procollagen. C57BL/6J mice were treated with either murine etanercept (4mg/kg/d) or an anti-Ly6G-specific monoclonal antibody (400μg/d to deplete neutrophils) for 7 days i.p, beginning 2 days before UVB exposure. Mice were irradiated with UVB (100 mJ/cm2/d for 5 d) or sham-irradiated. Skin was stained for neutrophils, M2 macrophages, TGFβ by IHC, and collagen fibers using picrosirius red. Under circular polarized light, picrosirius red differentiates collagen fibers as red or yellow (mature thick type I fibers) versus green (thin type III fibers). Our data show that in UVB-irradiated mice red fibers were decreased compared with sham-irradiated controls. In UVB+etanercept-treated mice, red fibers were markedly decreased compared to UVB without etanercept (p=0.001). Neutrophil-depleted UVB-treated mice showed more red fibers relative to UVB+etanercept-treated mice (p=0.01) and were similar to that seen with sham-irradiated mice. We show that blocking TNFα significantly inhibited M2 macrophage recruitment and TGFβ accumulation in UVB-irradiated mice (p=0.01). Neutrophil depletion in UVB-treated mice did not inhibit M2 macrophage recruitment or TGFβ staining relative to UVB alone. In conclusion etanercept inhibits recruitment of M2 macrophages into skin, as well as TGFβ production, coinciding with decreased mature collagen in the dermis of UVB+etanercept–treated mice relative to neutrophil-depleted UVB-treated mice. Future studies will examine the effects of macrophage depletion on mature collagen in UV-irradiated murine models.