Recruitment Of Polymorphonuclear Neutrophils Research Articles

IL-17RA-Mediated Epithelial Cell Activity Prevents Severe Inflammatory Response to Helicobacter pylori Infection.

Helicobacter pylori is a Gram-negative pathogen that colonizes the stomach, induces inflammation, and drives pathological changes in the stomach tissue, including gastric cancer. As the principal cytokine produced by Th17 cells, IL-17 mediates protective immunity against pathogens by inducing the activation and mobilization of neutrophils. Whereas IL-17A is largely produced by lymphocytes, the IL-17 receptor is expressed in epithelial cells, fibroblasts, and hematopoietic cells. Loss of the IL-17RA in mice results in impaired antimicrobial responses to extracellular bacteria. In the context of H. pylori infection, this is compounded by extensive inflammation in Il17ra-/- mice. In this study, Foxa3creIl17rafl/fl (Il17raΔGI-Epi) and Il17rafl/fl (control) mice were used to test the hypothesis that IL-17RA signaling, specifically in epithelial cells, protects against severe inflammation after H. pylori infection. The data indicate that Il17raΔGI-Epi mice develop increased inflammation compared with controls. Despite reduced Pigr expression, levels of IgA increased in the gastric wash, suggesting significant increase in Ag-specific activation of the T follicular helper/B cell axis. Gene expression analysis of stomach tissues indicate that both acute and chronic responses are significantly increased in Il17raΔGI-Epi mice compared with controls. These data suggest that a deficiency of IL-17RA in epithelial cells is sufficient to drive chronic inflammation and hyperactivation of the Th17/T follicular helper/B cell axis but is not required for recruitment of polymorphonuclear neutrophils. Furthermore, the data suggest that fibroblasts can produce chemokines in response to IL-17 and may contribute to H. pylori-induced inflammation through this pathway.

Crosstalk between keratinocytes and neutrophils shapes skin immunity against S. aureus infection.

Staphylococcus aureus (S. aureus) infection of the skin leads to a rapid initial innate immune response with keratinocytes in the epidermis as the initial sensors. Polymorphonuclear neutrophils (PMNs) are the first innate immune cells to infiltrate infection sites where they provide an effective first-line of defense. Previous work of our group showed that in inflamed skin a crosstalk between PMNs and keratinocytes results in enhanced S. aureus skin colonization. In this work, we used an in vitro co-culture model to studied the crosstalk between primary human keratinocytes (PHKs) and PMNs in a sterile environment and upon S. aureus infection. We investigated the influence of PHKs on PMN activation by analyzing PMN lifespan, expression of degranulation markers and induction of proinflammatory cytokines. Furthermore, we analyzed the influence of PMNs on the inflammatory response of PHKs. Finally, we investigated the influence of the skin microbiome on PMN-mediated skin inflammation. We show that co-culture of PMNs with PHKs induces activation and degranulation of PMNs and significantly enhances their lifespan compared to PMN cultivation alone by an IL-8 mediated mechanism and, furthermore, primes PMNs for enhanced activity after S. aureus infection. The prolonged incubation with PMNs also induces inflammatory responses in PHKs which are further exacerbated in the presence of S. aureus and induces further PMN recruitment thus fueling skin inflammation. Interestingly, infection of PHKs with the skin commensal S. epidermidis reduces the inflammatory effects of PMNs in the skin and exhibits an anti-inflammatory effect. Our data indicate that skin infiltrating PMNs and PHKs influence each other in such a way to enhance skin inflammation and that commensal bacteria are able to reduce the inflammatory effect.

Single-cell transcriptomics of immune cells in lymph nodes reveals their composition and alterations in functional dynamics during the early stages of bubonic plague.

Bubonic plague caused by Yersinia pestis is highly infectious and often fatal. Characterization of the host immune response and its subsequent suppression by Y. pestis is critical to understanding the pathogenesis of Y. pestis. Here, we utilized single-cell RNA sequencing to systematically profile the transcriptomes of immune cells in draining lymph nodes (dLNs) during the early stage of Y. pestis infection. Dendritic cells responded to Y. pestis within 2 h post-infection (hpi), followed by the activation of macrophages/monocytes (Mφs/Mons) and recruitment of polymorphonuclear neutrophils (PMNs) to dLNs at 24 hpi. Analysis of cell-to-cell communication suggests that PMNs may be recruited to lymph nodes following the secretion of CCL9 by Mφs/Mons stimulated through CCR1-CCL9 interaction. Significant functional suppression of all the three innate immune cell types occurred during the early stage of infection. In summary, we present a dynamic immune landscape, at single-cell resolution, of murine dLNs involved in the response to Y. pestis infection, which may facilitate the understanding of the plague pathogenesis of during the early stage of infection.

Inhibition of Recruitment and Activation of Neutrophils by Pyridazinone-Scaffold-Based Compounds.

In inflammatory diseases, polymorphonuclear neutrophils (PMNs) are known to produce elevated levels of pro-inflammatory cytokines and proteases. To limit ensuing exacerbated cell responses and tissue damage, novel therapeutic agents are sought. 4aa and 4ba, two pyridazinone-scaffold-based phosphodiesterase-IV inhibitors are compared in vitro to zardaverine for their ability to: (1) modulate production of pro-inflammatory mediators, reactive oxygen species (ROS), and phagocytosis; (2) modulate degranulation by PMNs after transepithelial lung migration. Compound 4ba and zardaverine were tested in vivo for their ability to limit tissue recruitment of PMNs in a murine air pouch model. In vitro treatment of lipopolysaccharide-stimulated PMNs with compounds 4aa and 4ba inhibited the release of interleukin-8, tumor necrosis factor-α, and matrix metalloproteinase-9. PMNs phagocytic ability, but not ROS production, was reduced following treatment. Using a lung inflammation model, we proved that PMNs transmigration led to reduced expression of the CD16 phagocytic receptor, which was significantly blunted after treatment with compound 4ba or zardaverine. Using the murine air pouch model, LPS-induced PMNs recruitment was significantly decreased upon addition of compound 4ba or zardaverine. Our data suggest that new pyridazinone derivatives have therapeutic potential in inflammatory diseases by limiting tissue recruitment and activation of PMNs.

Effect of neutrophil extracellular traps on tumor lymph nodes.

4033 Background: In most human cancers, regional lymph nodes (LNs) are the first sites of metastasis. In addition to being an important part of the tumor staging system, with the advent of novel therapies, lymph node metastasis has become a crucial clinical intervention point before distant metastasis, the leading cause of cancer-associated deaths. To initiate metastasis, the conditions of LNs need to be optimized for tumor cell deposition and growth. This process is believed to be mediated by the activation of immune cells including polymorphonuclear neutrophils (PMNs). However, the cellular mechanism is not well defined. Our early observations suggest that PMNs and neutrophil extracellular traps (NETs), DNA comprising structures that are extruded in response to inflammatory cues, are associated with adverse oncologic outcomes. Thus, one potential mechanism of increased LN metastasis is that tEVs recruit PMNs and propend NETs formation. Methods: Human tissue micro-arrays (TMAs) of gastroesophageal (GEA) cancer patients were stained with PMN and NETs markers and quantified by HALO software. C57BL/6 or pad4-/- mice were injected with B16F10 or H59 cells alone or treated with neutrophil elastase inhibitor (NEi) or PMN depletion antibody. LN sections were stained with NETs markers and quantified by ImageJ (NIH). Results: In the study of 175 GEA cancer patients, lymphatic NET deposition was observed in both tumor infiltrated lymph nodes and tumor negative lymph nodes. We also demonstrated high LN NETs deposition was associated with reduced survival, even in the absence of overt metastasis ( p=0.03). Next, we sought to investigate the dynamic and the consequence of LN NETs deposition using animal models. We found that LN Neutrophil Recruitment and NET deposition happens in a pre-metastatic manner. Moreover, LN metastasis was abrogated through different kinds of NETs inhibition (neutrophil depletion, pad4 knockout and NEi treatment, n=10, p<0.001), demonstrating the consequences of LN NETs deposition and its potential as a treatment target. Finally, we showed that the LN PMN recruitment and NETs formation was mediated by increased production of IL-8 by Lymphatic Endothelial Cells (LEC). Conclusions: Together, we demonstrated that NETs can contribute to LN metastasis, and can serve as a potential therapeutic targets. By further investigating the detailed mechanism, this project will lead to major advances in the management of cancer patients.

Leukotrienes Are Dispensable for Vaginal Neutrophil Recruitment as Part of the Immunopathological Response During Experimental Vulvovaginal Candidiasis

Recruitment of polymorphonuclear neutrophils (PMNs) into the vaginal lumen is the hallmark of an acute immunopathologic inflammatory response during vulvovaginal candidiasis (VVC) caused by Candida albicans. Recurrent VVC (RVVC) remains a chronic health burden in affected women worldwide despite the use of antifungal therapy. Based on the role leukotrienes (LTs) play in promoting inflammation, leukotriene receptor antagonists (LTRAs) targeted for LTB4 (etalocib) or LTC4, LTD4, and LTE4 (zafirlukast or montelukast) have been shown to reduce inflammation of epithelial tissues. An open-label pilot study using long-term regimens of zafirlukast in women with RVVC indicated the potential for some relief from recurrent episodes. To investigate this clinical observation further, we evaluated the effects of LT antagonistic agents and LT deficiency on the immunopathogenic response in a mouse model of VVC. Results showed that mice given daily intraperitoneal injections of individual LTRAs, starting 2days prior to vaginal inoculation with C. albicans and continuing through 14days post-inoculation, had no measurable reduction in PMN migration. The LTRAs were also ineffective in reducing levels of the hallmark vaginal inflammatory markers (S100A8, IL-1β) and tissue damage (LDH) associated with the immunopathogenic response. Finally, LT-deficient 5-lipoxygenase knockout mice showed comparable levels of vaginal fungal burden and PMN infiltration to wild-type mice following inoculation with a vaginal (ATCC 96113) or laboratory (SC5314) C. albicans isolate. These results indicate that despite some clinical evidence suggestive of off-target efficacy of LTRAs in RVVC, LTs and associated signaling pathways appear to be dispensable in the immunopathogenesis of VVC.

Differential Early in vivo Dynamics and Functionality of Recruited Polymorphonuclear Neutrophils After Infection by Planktonic or Biofilm Staphylococcus aureus.

Staphylococcus aureus is a human pathogen known for its capacity to shift between the planktonic and biofilm lifestyles. In vivo, the antimicrobial immune response is characterized by the recruitment of inflammatory phagocytes, namely polymorphonuclear neutrophils (PMNs) and monocytes/macrophages. Immune responses to planktonic bacteria have been extensively studied, but many questions remain about how biofilms can modulate inflammatory responses and cause recurrent infections in live vertebrates. Thus, the use of biologically sound experimental models is essential to study the specific immune signatures elicited by biofilms. Here, a mouse ear pinna model of infection was used to compare early innate immune responses toward S. aureus planktonic or biofilm bacteria. Flow cytometry and cytokine assays were carried out to study the inflammatory responses in infected tissues. These data were complemented with intravital confocal imaging analyses, allowing the real-time observation of the dynamic interactions between EGFP + phagocytes and bacteria in the ear pinna tissue of LysM-EGFP transgenic mice. Both bacterial forms induced an early and considerable recruitment of phagocytes in the ear tissue, associated with a predominantly pro-inflammatory cytokine profile. The inflammatory response was mostly composed of PMNs in the skin and the auricular lymph node. However, the kinetics of PMN recruitment were different between the 2 forms in the first 2 days post-infection (pi). Two hours pi, biofilm inocula recruited more PMNs than planktonic bacteria, but with decreased motility parameters and capacity to emit pseudopods. Inversely, biofilm inocula recruited less PMNs 2 days pi, but with an “over-activated” status, illustrated by an increased phagocytic activity, CD11b level of expression and ROS production. Thus, the mouse ear pinna model allowed us to reveal specific differences in the dynamics of recruitment and functional properties of phagocytes against biofilms. These differences would influence the specific adaptive immune responses to biofilms elicited in the lymphoid tissues.

Abstract 2863: Cancer extracellular vesicles induce lymph node metastasis via neutrophil extracellular traps

Abstract In most human cancers, regional lymph nodes (LNs) are the first sites of metastasis. In addition to being an important part of the tumor staging system, with the advent of novel therapies, lymph node metastasis has become a crucial clinical intervention point before distant metastasis, the leading cause of cancer-associated deaths. To initiate metastasis, the conditions of LNs need to be optimized for tumor cell deposition and growth. This process is believed to be mediated by the activation of immune cells including polymorphonuclear neutrophils (PMNs), and tumor derived factors, such as extracellular vesicles (EVs). Indeed, tumor derived EVs (tEVs) were shown to prepare sentinel LNs for increased melanoma metastasis, however, the cellular mechanism is not well defined. Early observations suggest that PMNs and neutrophil extracellular traps (NETs), DNA comprising structures that are extruded in response to inflammatory cues, are associated with adverse oncologic outcomes. Moreover, PMNs exhibit considerable plasticity to tEVs, as gastric tEVs can polarize PMN toward a pro-tumor (N2) phenotype and induce NET formation. Thus, one potential mechanism of increased LN metastasis is that tEVs recruit PMNs and propend NETs formation. Here, we show that lymphatic PMN accumulation is associated with higher rates of LN metastasis in human esophageal cancer patients. Furthermore, we demonstrate that LN PMN accumulation is mediated by tEVs-lymphatic interaction both in vitro and in vivo. Finally, we demonstrate that lymphatic PMN facilitate metastasis through the accumulation of PMN prior to tumor ingress. Using Boyden chamber assays, we observed an increase in PMN migration towards tEVs educated lymphatic endothelial cells (LECs). Moreover, ELISA showed tEVs educated LEC increased secretion of the PMN chemoattractants CXCL4 and CXCL8. Additionally, through confocal microscopy and immunofluorescence, we observed that tEVs induced PMN recruitment to LNs and NETs released in vivo in a dose-dependent manner. Finally, using transgenic pad4-/- knockout mice, which are unable to generate NETs, we showed that the absence of NETs led to decreased LN metastasis. Together, these findings highlight the role of tumor derived tEVs both as PMN recruiters to LNs and NETs inducers. By further investigating the detailed mechanism and the efficiency of NETs targeting agents, this project will lead to major advances in the management of cancer patients. Citation Format: Xin SU, Ariane Brassard, Ramin Rohanizadeh, Iqraa Dhoparee-Doomah, Betty Giannias, France Bourdeau, Veena Sangwan, Roni F. Rayes, Jonathan D. Spicer, Lorenzo E. Ferri, Jonathan J. Cools-Lartigue. Cancer extracellular vesicles induce lymph node metastasis via neutrophil extracellular traps [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2863.

Peptidoglycan Switches Off the TLR2-Mediated Sperm Recognition and Triggers Sperm Localization in the Bovine Endometrium.

In mammals, the uterine mucosal immune system simultaneously recognizes and reacts to most bacteria as well as allogenic sperm mainly through the Toll-like receptors (TLR)2/4 signaling pathway. Here, we characterized the impact of pathogen-derived TLR2/4 ligands (peptidoglycan (PGN)/lipopolysaccharide (LPS)) on the immune crosstalk of sperm with the bovine endometrial epithelium. The real-time PCR analysis showed that the presence of low levels of PGN, but not LPS, blocked the sperm-induced inflammatory responses in bovine endometrial epithelial cells (BEECs) in vitro. Immunoblotting analysis revealed that PGN prevented the sperm-induced phosphorylation of JNK in BEECs. Activation or blockade of the TLR2 system in the endometrial epithelium verified that TLR2 signaling acts as a commonly-shared pathway for PGN and sperm recognition. The impairment of endometrial sperm recognition, induced by PGN, subsequently inhibited sperm phagocytosis by polymorphonuclear neutrophils (PMNs). Moreover, using an ex vivo endometrial explant that more closely resembles those in vivo conditions, showed that sperm provoked a mild and reversible endometrial tissue injury and triggered PMN recruitment into uterine glands, while PGN inhibited these events. Of note, PGN markedly increased the sperm attachment to uterine glands, and relatively so in the surface epithelium. However, addition of the anti-CD44 antibody into a PGN-sperm-explant co-culture completely blocked sperm attachment into glands and surface epithelia, indicating that the CD44 adhesion molecule is involved in the PGN-triggered sperm attachment to the endometrial epithelium. Together, these findings demonstrate that, the presence of PGN residues disrupts sperm immune recognition and prevents the physiological inflammation induced by sperm in the endometrial epithelium via the MyD88-dependent pathway of TLR2 signaling, possibly leading to impairment of uterine clearance and subsequent embryo receptivity.

Absence of interleukin-10 reduces progression of shiga toxin-induced hemolytic uremic syndrome.

Hemolytic Uremic Syndrome (HUS), a disease triggered by Shiga toxin (Stx), is characterized by hemolytic anemia, thrombocytopenia and renal failure. The inflammatory response mediated by polymorphonuclear neutrophils (PMNs) and monocytes is essential to HUS onset. Still, the role of anti-inflammatory cytokines is less clear. The deficiency of IL-10, an anti-inflammatory cytokine, leads to severe pathology in bacterial infections but also to beneficial effects in models of sterile injury. The aim of this work was to analyze the role of IL-10 during HUS. Control and IL-10 lacking mice (IL-10-/-) were intravenously injected with Stx type 2 (Stx2) and survival rate was evaluated. PMN and circulating and renal pro- and anti-inflammatory factors were analyzed by FACS and enzyme-linked immunosorbent assay (ELISA) respectively. IL-10-/- mice showed a higher survival associated with lower renal damage reflected by reduced plasma urea and creatinine levels than control mice. Circulating PMN increased at 72 h in both mouse strains accompanied by an up-regulation of CD11b in control mice. In parallel, renal PMN were significantly increased only in control mice after toxin. Plasma TNF-α, IL-6 and corticosterone levels were higher increased in IL-10-/- than control mice. Simultaneously renal TNF-α raised constantly but was accompanied by increased TGF-β levels in IL-10-/- mice. These results demonstrate that the profile of circulating and renal cytokines after Stx2 differed between strains suggesting that balance of these factors could participate in renal protection. We conclude that IL-10 absence has a protective role in an experimental model of HUS by reducing PMN recruitment into kidney and renal damage, and increasing mice survival.

Periodontal Inflammation Primes the Systemic Innate Immune Response

The presence of periodontal diseases (PDs) often strongly correlates with other severe chronic inflammatory conditions, including cardiovascular disease, diabetes, and arthritis. However, the mechanisms through which these diseases interact are unclear. In PD, tissue and bone destruction in the mouth is driven by elevated recruitment of polymorphonuclear neutrophils (PMNs), which are primed and recruited from the circulation to sites of inflammation. We predicted that systemic effects on PMN mobilization or priming could account for the interaction between PD and other inflammatory conditions. We tested this using a mouse model of ligature-induced PD and found elevated PMN counts specifically in bone marrow, supporting a systemic effect of periodontal tissue inflammation on PMN production. In contrast, mice with induced peritonitis had elevated PMN counts in the blood, peritoneum, and colon. These elevated counts were further significantly increased when acute peritonitis was induced after ligature-induced PD in mice, revealing a synergistic effect of multiple inflammatory events on PMN levels. Flow cytometric analysis of CD marker expression revealed enhanced priming of PMNs from mice with both PD and peritonitis compared to mice with peritonitis alone. Thus, systemic factors associated with PD produce hyperinflammatory PMN responses during a secondary infection. To analyze this systemic effect in humans, we induced gingival inflammation in volunteers and also found significantly increased activation of blood PMNs in response to ex vivo stimulation, which reverted to normal following resolution of gingivitis. Together, these results demonstrate that periodontal tissue inflammation has systemic effects that predispose toward an exacerbated innate immune response. This indicates that peripheral PMNs can respond synergistically to simultaneous and remote inflammatory triggers and therefore contribute to the interaction between PD and other inflammatory conditions. This suggests larger implications of PD beyond oral health and reveals potential new approaches for treating systemic inflammatory diseases that interact with PD.

Characterization of neutrophil-neuronal co-cultures to investigate mechanisms of post-ischemic immune-mediated neurotoxicity

BackgroundImmune-mediated reperfusion injury is a critical component of post-ischemic central nervous system (CNS) damage. In this context, the activation and recruitment of polymorphonuclear neutrophils (PMNs) to the CNS induces neurotoxicity in part through the release of degradative enzymes, cytokines, and reactive oxygen species. However, the extent to which close-range interactions between PMNs and neurons contribute to injury in this context has not been directly investigated. New methodWe devised a co-culture model to investigate mechanisms of PMN-dependent neurotoxicity. Specifically, we established the effect of PMN dose, co-incident neuronal ischemia, lipopolysaccharide (LPS)-induced PMN priming, and the requirement for cell-cell contact on cumulative neuron damage. Results and comparison to existing method(s)Pre-exposure of day in vitro 10 primary cortical neurons to oxygen-glucose deprivation (OGD) enhanced PMN-dependent neuronal death. Likewise, LPS-induced priming of the PMN donor further increased PMN-induced toxicity in vitro compared to saline-injected controls. Compartmentalization of LPS-primed PMNs using net wells confirmed the requirement for close-range cell-cell interactions in the process of PMN-induced neuronal injury. Moreover, time-lapse imaging and quantitative neurite analyses implicate PMN-neurite interactions in this pathological response. These experiments establish a platform to investigate immune and neural factors that contribute to post-ischemic neurodegeneration. ConclusionsIschemic and immune priming enhance neurotoxicity in PMN-neuronal co-cultures. Moreover, cell-cell contact and neurite destruction are prominent features in the observed mechanism of post-ischemic neuronal death.

Tumor ablation plus co-administration of CpG and saponin adjuvants affects IL-1 production and multifunctional T cell numbers in tumor draining lymph nodes

BackgroundTumor ablation techniques, like cryoablation, are successfully used in the clinic to treat tumors. The tumor debris remaining in situ after ablation is a major antigen depot, including neoantigens, which...

Progress in Model Systems of Cystic Fibrosis Mucosal Inflammation to Understand Aberrant Neutrophil Activity.

In response to recurrent infection in cystic fibrosis (CF), powerful innate immune signals trigger polymorphonuclear neutrophil recruitment into the airway lumen. Exaggerated neutrophil proteolytic activity results in sustained inflammation and scarring of the airways. Consequently, neutrophils and their secretions are reliable clinical biomarkers of lung disease progression. As neutrophils are required to clear infection and yet a direct cause of airway damage, modulating adverse neutrophil activity while preserving their pathogen fighting function remains a key area of CF research. The factors that drive their pathological behavior are still under investigation, especially in early disease when aberrant neutrophil behavior first becomes evident. Here we examine the latest findings of neutrophils in pediatric CF lung disease and proposed mechanisms of their pathogenicity. Highlighted in this review are current and emerging experimental methods for assessing CF mucosal immunity and human neutrophil function in the laboratory.

An Early Neutrophil Recruitment into the Infectious Site Is Critical for Bacterial Lipoprotein Tolerance-Afforded Protection against Microbial Sepsis.

Bacterial lipoprotein (BLP)-induced tolerance represents an essential regulatory mechanism during bacterial infection and has been shown to protect against microbial sepsis. This protection is generally attributed to BLP-tolerized monocytes/macrophages characterized by hyporesponsiveness in producing inflammatory cytokines and, simultaneously, an augmented antimicrobial activity. However, the contribution of polymorphonuclear neutrophils (PMNs), another major player in innate immunity against bacterial infection, to BLP tolerance-afforded protection against microbial sepsis has not been identified. In this study, we report that induction of BLP tolerance protected mice against cecal ligation and puncture-induced polymicrobial sepsis, with significantly improved survival. Importantly, BLP tolerization via i.p. injection triggered an early PMN recruitment even before bacterial infection and promoted further PMN influx into the infectious site (i.e., the peritoneal cavity upon cecal ligation and puncture-associated septic challenge). Notably, this early PMN influx was mediated by BLP tolerization-induced PMN chemoattractant CXCL2-formed concentration gradient between the circulation and peritoneal cavity. Critically, blockage of PMN influx with the CXCR2 antagonist SB225002 abolished BLP tolerance-afforded protection and rendered BLP-tolerized mice more vulnerable to microbial infection with impaired bacterial clearance and increased overall mortality. Thus, our results highlight that an early recruitment of PMNs in the infectious site, as an important cellular mechanism, contributes to BLP tolerance-afforded protection against microbial sepsis.

Impaired Peritoneal Macrophage Function and Delayed Neutrophil Recruitment are Secondary to a Leaky Gut in JAM‐A Deficient Mice

Junctional adhesion molecule‐A (JAM‐A) is a transmembrane glycoprotein expressed on leukocytes, endothelia and epithelia that regulates endothelial/epithelial barrier function as well as leukocyte transendothelial/transepithelial migration. While biological roles of JAM‐A have been extensively studied with JAM‐A knockout mice (Jam‐a KO), tissue‐specific JAM‐A deficient animals remain poorly documented. Therefore, we generated mice with tissue selective deletion of JAM‐A in myelomonocytic cells (LysM‐cre; Jam‐a fl/fl) and the intestinal epithelium (Villin‐cre; Jam‐a fl/fl). We then used an acute peritonitis model induced by intraperitoneal injection of zymosan or lipopolysaccharide (LPS) to study the role of tissue selective expression of JAM‐A in recruitment of polymorphonuclear neutrophils (PMN). Consistent with previous reports, infiltration of PMN into the peritoneum in response to zymosan or LPS was significantly reduced in Jam‐a KO versus Jam‐a WT mice. Surprisingly, in LysM‐cre; Jam‐a fl/fl, the number of PMN recruited into the peritoneum was not affected in response to zymosan nor LPS compared to control littermates, indicating that JAM‐A on PMN is not required for infiltration under such conditions. Importantly, intestinal epithelial‐selective loss of JAM‐A resulted in significant reduction of PMN infiltration in Villin‐cre; Jam‐a fl/fl mice, confirming that extrinsic factors, independent on JAM‐A expression on PMN are necessary for leukocyte recruitment to peritoneum under the conditions studied. We also observed that in response to zymosan or LPS, peritoneal macrophages from Jam‐a KO or Villin‐cre; Jam‐a fl/fl mice produced less of the chemokine CXCL1 and had reduced activity of the transcription factor NFkB. Conversely, peritoneal macrophages from LysM‐cre; Jam‐a fl/fl mice were unaffected compared to Jam‐a fl/fl mice. Given that loss of JAM‐A on intestinal epithelial cells resulted in increased intestinal permeability, we hypothesized that bacterial derived products may impair peritoneal macrophage function. Therefore, experiments were conducted with germ free Villin‐cre; Jam‐a fl/fl mice. We observed that macrophage responses as well as peritoneal recruitment of PMN in response to zymosan or LPS was not affected compared to control mice. These findings suggest that a leaky intestinal barrier and intestinal microbes are critical in regulating systemic neutrophil recruitment through effects on macrophage release of inflammatory mediators. Such observations highlight the functional link between the intestinal barrier, microbiota and the regulation of innate immune responses.Support or Funding InformationNIH grants 1R01DK097256 to TLD, DK59888, DK55679 to AN and DK072564, DK061379, and DK079392 to CP.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Midkine drives cardiac inflammation by promoting neutrophil trafficking and NETosis in myocarditis.

Heart failure due to dilated cardiomyopathy is frequently caused by myocarditis. However, the pathogenesis of myocarditis remains incompletely understood. Here, we report the presence of neutrophil extracellular traps (NETs) in cardiac tissue of patients and mice with myocarditis. Inhibition of NET formation in experimental autoimmune myocarditis (EAM) of mice substantially reduces inflammation in the acute phase of the disease. Targeting the cytokine midkine (MK), which mediates NET formation in vitro, not only attenuates NET formation in vivo and the infiltration of polymorphonuclear neutrophils (PMNs) but also reduces fibrosis and preserves systolic function during EAM. Low-density lipoprotein receptor-related protein 1 (LRP1) acts as the functionally relevant receptor for MK-induced PMN recruitment as well as NET formation. In summary, NETosis substantially contributes to the pathogenesis of myocarditis and drives cardiac inflammation, probably via MK, which promotes PMN trafficking and NETosis. Thus, MK as well as NETs may represent novel therapeutic targets for the treatment of cardiac inflammation.

CD11b Regulates Fungal Outgrowth but Not Neutrophil Recruitment in a Mouse Model of Invasive Pulmonary Aspergillosis

Background and Aims: In immunosuppressed individuals Aspergillus (A.) fumigatus is a frequent cause of invasive pulmonary aspergillosis (IPA) which is highly associated with relevant morbidity and mortality. Moreover, it often occurs in patients suffering from leukocyte-adhesion deficiency type 1 (LAD1) which is triggered by a functional loss of CD18 in ß2 integrin receptors as these receptors consist of an alpha subunit (CD11a-CD11d) and CD18 as the common beta subunit. ß2 integrin receptors are differentially expressed by leukocytes, and are required for cell-cell interaction, transendothelial migration, uptake of opsonized pathogens, and cell signaling processes. Here, we asked for the importance of CD11b/CD18 also termed MAC-1 which is required for phagocytosis of opsonized A. fumigatus conidia by polymorphonuclear neutrophils (PMN) for control of pulmonary A. fumigatus infection.Methods: We used a murine IPA model (C57BL/6) and challenged CD11b deficient (CD11b-/-) or wild type (WT) mice with A. fumigatus conidia intratracheally. Afterwards, some mice were sacrificed 24h after infection. In these mice PMN recruitment and cytokine patterns were examined by analyzing bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) using flow cytometry, cytospin analysis, and cytometric bead array. Additionally, pulmonary fungal clearance and inflammation in murine lungs were analyzed by fungal culture assays and histopathologic examination using a scoring system. Furthermore, survival was studied with neutropenic animals serving as positive controls. To determine PMNs phagocytic activity and fungal killing capacity ex vivo, PMN were purified from bone marrow of CD11b-/- or WT mice by magnetic cell sorting using Ly6G specific antibodies. Afterwards, isolated PMN were stimulated with pacific blue-labeled tomato red-fluorescent modified A. fumigatus conidia and analyzed by flow cytometry.Results: We found that lung homogenates from CD11b-/- mice obtained 24h after infection showed an enhanced fungal burden as compared to lungs from WT mice. In contrast, lung tissue from infected CD11b-/- mice displayed impaired pulmonary inflammation as assessed by Hematoxilin-Eosin (HE) staining. Furthermore, the number of mucus-producing cells in bronchi of A. fumigatus infected CD11b-/- mice was decreased compared to cells in bronchi of WT mice. However, we observed markedly higher numbers of PMN in BALF of infected CD11b-/- mice compared to corresponding WT mice samples. In contrast, BALF derived from infected CD11b-/- mice contained lower levels of three different proinflammatory cytokines (TNF-α, IL-1α, IL-1β) compared to BALF from WT mice while levels of other cytokines and chemokines (IL-5, IL-6, IL-10, GM-CSF, CXCL1, CCL2) were comparable. In contrast, we measured higher levels of the chemokine CCL5 known as a relevant chemoattractant in innate and adaptive immune cells in BALF obtained from CD11b-/- mice. Interestingly, numbers of PMN, lymphocytes, and monocytes in the peripheral blood of A. fumigatus infected mice did not differ in a genotype-dependent manner. However, CD11b-/- mice PMN showed lower phagocytic activity than WT PMN, indicating an impaired capability to clear A. fumigatus. Nevertheless, CD11b-/- mice were finally characterized by similar long-term survival as WT mice after IPA induction while the PMN-depleted control mice died as expected.Conclusions: Our study demonstrates, that CD11b deficiency on myeloid cells affects the early course of IPA. This may be due to the importance of MAC-1 for PMN effector functions, and their interplay with DC and other leukocytes. Further work is necessary to elucidate the long-term course of IPA in CD11b-/- mice with regard to the interplay of PMN with dendritic cells, the efficacy of adaptive immune responses, and the potential pulmonary overexpression of CCL5. Beyond patients with LAD1 syndrome, we suggest our findings as being clinically relevant in other immunocompromised patients suffering from severe opportunistic infections. DisclosuresRadsak:Celgene: Honoraria, Other: Travel grant; Jazz Pharmaceuticals: Other: Travel grant; TEVA: Consultancy; Gilead: Other: Travel grant; Takeda: Consultancy; Novartis: Consultancy, Honoraria; Daiichi Sankyo: Honoraria, Other: Travel grant.

Analysis of leukocyte transepithelial migration using an in vivo murine colonic loop model.

Molecular mechanisms that control leukocyte migration across the vascular endothelium (transendothelial migration; TEndoM) have been extensively characterized in vivo, but details of leukocyte transepithelial migration (TEpM) and its dysregulation (a pathologic feature of many mucosal diseases) are missing due to the lack of suitable animal models. Here, we describe a murine model that utilizes a vascularized proximal colonic segment (pcLoop) and enables quantitative studies of leukocyte trafficking across colonic epithelium. Consistent with previous in vitro studies, intraluminal injection of antibodies against integrin CD11b/CD18 reduced recruitment of polymorphonuclear neutrophils (PMN) into the lumen of pcLoops, and it increased subepithelial accumulation of PMN. We extended studies using the pcLoop to determine contributions of Junctional Adhesion Molecule-A (JAM-A, or F11R) in PMN TEpM and confirmed that mice with total loss of JAM-A or mice with intestinal epithelial selective loss of JAM-A had increased colonic permeability. Furthermore, there was reduced PMN migration into the colonic lumen that paralleled subepithelial accumulation of PMN in global-KO mice, as well as in intestinal epithelial-targeted JAM-A-deficient mice. These findings highlight a potentially novel role for JAM-A in regulating PMN TEpM in vivo and demonstrate utility of this model for identifying receptors that may be targeted in vivo to reduce pathologic intestinal inflammation.

Mammary Dysbiosis

Mastitis can be an unwelcome and debilitating visitor to breastfeeding mothers. The mammary gland has its own microbiome that can be affected by reduced polymorphonuclear neutrophil recruitment during the first 3 months postpartum, as well as the receipt of antibiotics during the last trimester of pregnancy. This can leave the breast vulnerable to pathologic bacterial overgrowth. Mammary dysbiosis is a process whereby the population of potential pathogens increases at the expense of the normal mammary microbiota. Multiresistance to antibiotics plus tricky evasion techniques engaged in by bacterial agents can result in microbes that are elusive to antibiotic therapy. Therefore, new strategies are needed for the treatment of this threat to continued breastfeeding. Bacteriotherapy, targeting harmless bacteria to displace pathologic organisms, is an emerging therapeutic intervention that uses probiotics instead of antibiotics. Once more high-quality clinical trials of strain-specific probiotics have been conducted, bacteriotherapy may move into mainstream mastitis treatment.