PMN Recruitment Research Articles

Abstract 108: Tumor-derived complement C3 is overexpressed in STK11 mutant non-small cell lung cancer and contributes to an immunosuppressive tumor microenvironment in a syngeneic mouse model

Abstract Mutations in STK11 that lead to loss of its protein LKB1 occur in 15-20% of non-squamous non-small cell lung cancer (NSCLC) and are frequently co-mutated with oncogenic KRAS mutations. KRAS and STK11 co-mutant NSCLC is associated with poor prognosis, low PD-L1 expression, low T-cell infiltration, and poor response to immune-checkpoint inhibitors (ICI). Loss of LKB1 leads to altered transcriptional programs with the activation of CREB-dependent gene transcription and changes in the tumor microenvironment including recruitment of suppressive myeloid cells. We used the syngeneic Kras mutant murine CMT167 tumor cell line that is wild-type for Tp53 and Stk11 and sensitive to ICI and generated isogeneic Stk11 knockout cell lines (CMT167-Stk11-KO) by CRISPR/Cas-9 deletion. Loss of Stk11 recapitulated LKB1-loss transcriptional signatures and rendered CMT167 tumors resistant to ICI. Single-cell RNA sequencing (10x) of implanted tumors demonstrated significantly less CD8 T-cells and increased neutrophils (PMN) in Stk11-KO versus Stk11-WT tumors. Analysis of differentially expressed genes in the tumor compartment identified increased expression of complement pathway genes including the central mediator C3 in CMT-167-Stk11-KO tumors. Examination of a well characterized clinical cohort of human KRAS-mutant NSCLC patient samples with and without STK11 mutation confirmed the increased expression of complement C3 in STK11-mutant tumors. Analysis of a human NSCLC cell line panel demonstrated that C3 expression was modulated by LKB1. Knockout of C3 in CMT167-Stk11-KO tumor resulted in dramatic inhibition of tumor growth and re-sensitized CMT167-Stk11-KO tumors to ICI treatment in WT mice. Using C3-/- knockout mice, this effect was determined to be reliant on tumor-derived C3 and dependent on adaptive immunity as no difference in growth of CMT167-Stk11-KO tumors occurred in nude mice or in WT mice after CD8 T-cell depletion. We then compared the transcriptome of Stk11-KO tumor cells with and without C3 deletion, and observed that C3 upregulates the expression of Cxcl1, Cxcl2 and Cxcl3 which mediate PMN recruitment and activation. Since these chemokines ligate Cxcr2, we asked whether Cxcr2 mediated tumor growth and ICI resistance in Stk11-KO tumors. While treatment with single-agent Cxcr2 inhibitor and anti-PD-1 therapy had no effect on tumor growth, the combination resulted in significant suppression of tumor growth in vivo. These results support a role for tumor-derived C3 suppressing CD8 T-cell immunity, potentially indirectly through recruitment of PMN or inducing PMN suppressor function. Our results also provide rationale for targeting tumor-derived complement and inhibiting Cxcr2 to enhance ICI efficacy as novel therapeutic approaches in patients with STK11-mutant NSCLC. Citation Format: Sora Suzuki, Catrina Ting, Bojidar Kandar, Te-An Chen, Anm Nazmul Khan, Thejaswini Giridharan, Brahm Segal, Edwin H. Yau. Tumor-derived complement C3 is overexpressed in STK11 mutant non-small cell lung cancer and contributes to an immunosuppressive tumor microenvironment in a syngeneic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 108.

Uridine Diphosphate Glucose (UDP-G) Activates Oxidative Stress and Respiratory Burst in Isolated Neutrophils.

The extracellular purinergic agonist uridine diphosphate glucose (UDP-G) activates chemotaxis of human neutrophils (PMN) and the recruitment of PMN at the lung level, via P2Y14 purinergic receptor signaling. This effect is similar to the activation of PMN with N-formyl-methionyl-leucyl-phenylalanine (fMLP), a mechanism that also triggers the production of superoxide anion and hydrogen peroxide via the NADPH oxidase system. However, the effects of UDP-G on this system have not been studied. Defects in the intracellular phagocyte respiratory burst (RB) cause recurrent infections, immunodeficiency, and chronic and severe diseases in affected patients, often with sepsis and hypoxia. The extracellular activation of PMN by UDP-G could affect the RB and oxidative stress (OS) in situations of inflammation, infection and/or sepsis. The association of PMNs activation by UDP-G with OS and RB was studied. OS was evaluated by measuring spontaneous chemiluminescence (CL) of PMNs with a scintillation photon counter, and RB by measuring oxygen consumption with an oxygen Clark electrode at 37 °C, in non-stimulated cells and after activation (15 min) with lipopolysaccharides (LPS, 2 µg/mL), phorbol myristate acetate (PMA, 20 ng/mL), or UDP-G (100 μM). The stimulation index (SI) was calculated in order to establish the activation effect of the three agonists. After stimulation with LPS or PMA, the activated PMNs (0.1 × 106 cells/mL) showed an increase in CL (35%, p < 0.05 and 56%, p < 0.01, SI of 1.56 and 2.20, respectively). Contrariwise, the stimulation with UDP-G led to a decreased CL in a dose-dependent manner (60%, 25 μM, p < 0.05; 90%, 50-150 μM, p < 0.001). Nonetheless, despite the lack of oxidative damage, UDP-G triggered RB (SI 1.8) in a dose-dependent manner (38-50%, 100-200 μM, p < 0.0001). UDP-G is able to trigger NADPH oxidase activation in PMNs. Therefore, the prevention of OS and oxidative damage observed upon PMN stimulation with UDP-G indicates an antioxidant property of this molecule which is likely due to the activation of antioxidant defenses. Altogether, LPS and UDP-G have a synergistic effect, suggesting a key role in infection and/or sepsis.

Pore-forming activity of S. pneumoniae pneumolysin disrupts the paracellular localization of the epithelial adherens junction protein E-cadherin.

Streptococcus pneumoniae, a common cause of community-acquired bacterial pneumonia, can cross the respiratory epithelial barrier to cause lethal septicemia and meningitis. S. pneumoniae pore-forming toxin pneumolysin (PLY) triggers robust neutrophil (PMN) infiltration that promotes bacterial transepithelial migration in vitro and disseminated disease in mice. Apical infection of polarized respiratory epithelial monolayers by S. pneumoniae at a multiplicity of infection (MOI) of 20 resulted in recruitment of PMNs, loss of 50% of the monolayer, and PMN-dependent bacterial translocation. Reducing the MOI to 2 decreased PMN recruitment two-fold and preserved the monolayer, but apical-to-basolateral translocation of S. pneumoniae remained relatively efficient. At both MOI of 2 and 20, PLY was required for maximal PMN recruitment and bacterial translocation. Co-infection by wild-type S. pneumoniae restored translocation by a PLY-deficient mutant, indicating that PLY can act in trans. Investigating the contribution of S. pneumoniae infection on apical junction complexes in the absence of PMN transmigration, we found that S. pneumoniae infection triggered the cleavage and mislocalization of the adherens junction (AJ) protein E-cadherin. This disruption was PLY-dependent at MOI of 2 and was recapitulated by purified PLY, requiring its pore-forming activity. In contrast, at MOI of 20, E-cadherin disruption was independent of PLY, indicating that S. pneumoniae encodes multiple means to disrupt epithelial integrity. This disruption was insufficient to promote bacterial translocation in the absence of PMNs. Thus, S. pneumoniae triggers cleavage and mislocalization of E-cadherin through PLY-dependent and -independent mechanisms, but maximal bacterial translocation across epithelial monolayers requires PLY-dependent neutrophil transmigration.

Inhibition of lysophosphatidic acid receptor 1 relieves PMN recruitment in CNS via LPA1/TSP1/CXCR2 pathway and alleviates disruption on blood-brain barrier following intracerebral haemorrhage in mice

BackgroudThe frequencies of morbidity and impairment associated with spontaneous intracerebral haemorrhage (ICH) are comparatively high. Blood-brain barrier (BBB) integrity was compromised due to subsequent brain injury induced by ICH, which is crucial for a poor prognosis. Polymorphonuclear leukocyte (PMN) strongly modulate the disruption of BBB in the central nervous system (CNS). The lysophosphatidic acid receptor 1 (LPA1) mediated thrombospondin-1 (TSP1) regulation in astrocytes, which induce macrophage inflammatory protein 2(MIP2) secretion. MIP2 enhance PMN recruitment through CXC chemokine type 2 (CXCR2) activation. The purpose of this study was to investigate whether the LPA1-mediated inhibition of PMN recruitment and BBB protection after ICH is regulated by TSP1 and CXCR2 networks.MethodsICH induction was performed in CD1 mice using collagenase administration. AM966, a targeted LPA1 antagonist, was orally administered 1 and 12 h following ICH. further identify possible LPA1-mediated BBB protection mechanisms, we intracerebroventricularly (ICV) administered a CXCR2 ligand MIP2, as well as TSP1 CRISPR activation (ACT) with AM966. Consequently, we performed neurobehavioral, brain water content (BWC), Evans blue staining (EBS), immunofluorescence (IF), and western blot (WB) analyses.ResultsAfter ICH, astrocytes showed signs of LPA1, which peaked after 24 h, while PMN\\ displayed evidence of CXCR2. The AM966-mediated LPA1 suppression relieved PMN recruitment, diminished brain oedema, demonstrated extravasation (as evidenced by EBS), protected BBB integrity, and enhanced neurologic activity following ICH. AM966 treatment strongly reduced TSP1, CXCR2, Occludin, and Claudin-5 expressions and PMN recruitment following ICH, and their expressions were restored by MIP2 and TSP1 CRISPR (ACT).ConclusionsThis study shows that LAP1 suppression reduced PMN recruitment after ICH in mice via TSP1/CXCR2 signalling, which minimized BBB disruption and improved the CNS’s neurobehavioral functioning. Hence, LPA1 is a strong candidate for therapy to reduce PMN recruitment and offer protection of BBB integrity after ICH.

Mechanistic Insights into Macrophages Regulation of Neutrophil Transendothelial Migration in Inflamed Mucosa

Rapid neutrophil (PMN) mobilization to sites of insult is critical for host defense and requires crossing of the vascular wall. PMN transendothelial migration (TEM) involves several well‐studied sequential adhesive interactions with vascular ECs, however what initiates or terminates this process is not well‐understood. Our findings identified a new mechanism where gut interstitial macrophages, which are rapidly recruited towards the vascular wall in response to inflammatory cues, were found to locally prime endothelial cells (ECs) responses to regulate PMN TEM. Using real‐time intravital microscopy (IVM) on lipopolysaccharides (LPS)‐inflamed intestines in anesthetized CX3CR1‐EGFP macrophage‐reporter mice, complemented by whole‐mount tissue imaging we demonstrate that macrophage presence was critical for the induction of PMN‐ECs adhesive interactions and subsequent PMN recruitment and accumulation in the intestinal mucosa. Anti CSFR‐1 antibody‐based macrophage depletion in the lamina propria and at the vessel wall significantly reduced PMN adhesion and TEM in inflamed intestines. We further observed that macrophages at the vessel wall localized specifically to regions of high ICAM‐1 intensity and their removal resulted in elimination of the ICAM‐1 “hot spots”, overall lowering the ECs ICAM‐1 expression. Mechanistically, using murine/human ECs‐macrophage and PMN co‐cultures we established that activated macrophages elevate PMN adhesion and TEM via TNFα‐dependent upregulation of ECs ICAM‐1. Antibody‐mediated neutralization of TNFα in macrophage co‐cultures with ECs and/or PMNs suppressed ICAM‐1 upregulation, decreasing PMN TEM. Further in vivo imaging studies of inflamed gut revealed high TNFα expression in macrophages and specific expression of TNFa receptor type II (TNFR II) but not type I in intestinal ECs. The use of bone marrow chimeras with TNFα knockout macrophages further confirmed the novel role of macrophages‐TNFα in regulating ECs adhesion molecule expression and PMN TEM. As such, our findings identify new, clinically relevant mechanism by which macrophages regulate PMN trafficking in inflamed mucosa.

The Pore-Forming Subunit C2IIa of the Binary Clostridium botulinum C2 Toxin Reduces the Chemotactic Translocation of Human Polymorphonuclear Leukocytes.

The binary C2 toxin of Clostridium (C.) botulinum consists of two non-linked proteins, the enzyme subunit C2I and the separate binding/transport subunit C2II. To exhibit toxic effects on mammalian cells, proteolytically activated C2II (C2IIa) forms barrel-shaped heptamers that bind to carbohydrate receptors which are present on all mammalian cell types. C2I binds to C2IIa and the toxin complexes are internalized via receptor-mediated endocytosis. In acidified endosomal vesicles, C2IIa heptamers change their conformation and insert as pores into endosomal membranes. These pores serve as translocation-channels for the subsequent transport of C2I from the endosomal lumen into the cytosol. There, C2I mono-ADP-ribosylates G-actin, which results in depolymerization of F-actin and cell rounding. Noteworthy, so far morphological changes in cells were only observed after incubation with the complete C2 toxin, i.e., C2IIa plus C2I, but not with the single subunits. Unexpectedly, we observed that the non-catalytic transport subunit C2IIa (but not C2II) alone induced morphological changes and actin alterations in primary human polymorphonuclear leukocytes (PMNs, alias neutrophils) from healthy donors ex vivo, but not macrophages, epithelial and endothelial cells, as detected by phase contrast microscopy and fluorescent microscopy of the actin cytoskeleton. This suggests a PMN selective mode of action for C2IIa. The cytotoxicity of C2IIa on PMNs was prevented by C2IIa pore blockers and treatment with C2IIa (but not C2II) rapidly induced Ca2+ influx in PMNs, suggesting that pore-formation by C2IIa in cell membranes of PMNs is crucial for this effect. In addition, incubation of primary human PMNs with C2IIa decreased their chemotaxis ex vivo through porous culture inserts and in co-culture with human endothelial cells which is closer to the physiological extravasation process. In conclusion, the results suggest that C2IIa is a PMN-selective inhibitor of chemotaxis. This provides new knowledge for a pathophysiological role of C2 toxin as a modulator of innate immune cells and makes C2IIa an attractive candidate for the development of novel pharmacological strategies to selectively down-modulate the excessive and detrimental PMN recruitment into organs after traumatic injuries.

Seminal plasma, and not sperm, induces time and concentration‐dependent neutrophil extracellular trap release in donkeys

In several mammalian species, acute endometritis driven by the recruitment of polymorphonuclear cells (PMN) occurs in response to semen. These PMNs release DNA to form neutrophil extracellular traps (NETs) in cattle, horse and human, leading to sperm entrapment. While there is no evidence of this phenomenon occurring in donkeys, artificial insemination (AI) with frozen-thawed semen, which results in very poor pregnancy rates, leads to a large PMN recruitment to the uterus. To investigate whether donkey semen can trigger NET release (NETosis) and if excessive NETosis occurs in response to frozen-thawed semen. In vitro experiments. Jenny PMNs were exposed to jackass fresh or frozen-thawed semen, isolated sperm or seminal plasma (SP), over the course of three experiments. NET formation in response to different treatments was assessed through manual quantification of stained slides. A one-way analysis of variance (ANOVA), followed by a post hoc Sidak test, was carried out to determine statistical significance. NET release occurred in a semen concentration- and incubation-time-dependent manner. Surprisingly, frozen-thawed donkey sperm did not increase NETosis rate in comparison with the control (23±2.5% vs. 31±3.7%; P>.05), whereas fresh semen exposure did (78±5.7% vs. 26±3.2%, P<.01). NETosis increased in the presence of SP, regardless of the presence or absence of sperm, in comparison with the control in both fresh (84±5.2% and 77±5.0% vs. 12±2.7%, respectively; P<.01) and frozen (95±2.2% and 94±2.9% vs. 14±3.8%, respectively; P<.01) samples. Moreover, exposure of PMN to viable and motile sperm, in the absence of SP, did not increase NETosis rates (P>.05). Donkey SP, and not sperm-intrinsic factors, is able to trigger NETosis in both time- and semen concentration-dependent manner. The physiological relevance of such response against semen in the donkey remains to be elucidated.

Estradiol Augments the Production and Actions of Polymorphonuclear Cells to Promote Lymphangioleiomyomatosis (LAM) Progression

Affecting almost exclusively women, lymphangioleiomyomatosis (LAM) is a rare lung disease characterized by slowly growing, estrogen-sensitive metastatic smooth muscle cell-like adenomas that result in cystic lung changes and loss of pulmonary function. LAM tumors are caused by mutations in either TSC1 or TSC2 genes that induces defective inhibition of the mTORC1 pathway, leading to increased mTORC1 activity and augmented cell proliferation. We have previously reported that estrogen ablation in our uterine-specific Tsc2 knockout mouse, which grows tumors with characteristic LAM features and lung colonization potential, effects notable regression of tumors. Thus, estrogen is required for to maintain heightened mTORC1 activity and LAM-like tumor progression. Interestingly, the observed estrogen sensitivity in vivo is more markedly pronounced than that of our estrogen receptor-positive TSC2-null cells when stimulated with estradiol in vitro, suggesting that estradiol may act elsewhere—in mTORC1 independent manner—in vivo to promote LAM progression. Flow cytometry revealed large numbers of Ly-6Cint Ly-6Ghigh myeloid cells—polymorphonuclear cells or PMNs—in the blood and myometrial tumors of our uterine-specific Tsc2-null mice. Accordingly, we found that Tsc2-null tumors required PMNs for normal disease progression, as Gr-1 (Ly-6C/Ly-6G) depletion or inhibition of PMN recruitment reduced tumor growth. Therefore, we hypothesized that, in addition to direct effects of estrogen on tumor cells, estrogen might also stimulate tumor growth by promoting PMN production in the bone marrow and actions in the tumor microenvironment. Using bone marrow cultures, we found that estradiol is indeed a potent inducer of PMN production. This effect occurs equally in both male and female bone marrow. Employing both pharmacologic agents and bone marrow from ERα; knockout mice, we showed that ERα; is necessary for promoting a PMN fate for myeloid progenitors. Additionally, we have evidence implicating estrogen in the pro-tumorigenic function of PMNs co-cultured with TSC2-null cell lines. Overall, these data suggest that estradiol maybe facilitating crosstalk in LAM tumors, directly stimulating tumor cells while also promoting the production and actions of PMNs, which in turn promote tumor growth.

Update on the pathophysiology of transfusion-related acute lung injury.

The aim of this study was to discuss recent advances regarding the pathogenesis of transfusion-related acute lung injury (TRALI), which highlight the pathogenic role of macrophages. TRALI remains a leading cause of transfusion-related fatalities, despite the success of the mitigation strategy, and therapeutic approaches are unavailable. Neutrophils (PMNs) are recognized pathogenic cells in TRALI. Macrophages have previously also been suggested to be pathogenic in mice via binding of C5a to their C5a-receptor, producing reactive oxygen species (ROS), which damages the pulmonary endothelium. Recent work has further highlighted the role of macrophages in the TRALI-pathogenesis. It has been shown that the protein osteopontin (OPN) released by macrophages is critical for pulmonary PMN recruitment in mice suffering from TRALI and that targeting OPN prevents the occurrence of TRALI. Another recent study demonstrated the importance of M1-polarized alveolar macrophages in murine TRALI induction by showing that α1-antitrypsin (AAT) overexpression prevented TRALI in mice through decreasing the polarization of alveolar macrophages towards the M1 phenotype. Apart from PMNs, macrophages also appear to be important in the pathogenesis of TRALI. Targeting the pathogenic functions of macrophages may be a promising therapeutic strategy to explore in TRALI.

Loss of AC10 impairs neutrophil recruitment into the lung following Pseudomonas aeruginosa infection

Pseudomonas aeruginosa is a gram‐negative, opportunistic bacteria and a common cause of nosocomial pneumonia in critically ill patients that can progress to acute respiratory distress syndrome (ARDS). These patients have impaired gas exchange leading to hypoxia, altered blood pH and elevated bicarbonate. Bicarbonate, delivered into the cell via sodium bicarbonate cotransporters (NBCs), activates adenylyl cyclase 10 (AC10) to increase intracellular cAMP. Indeed, bicarbonate activation of AC10 is required for LPS‐induced lung endothelial permeability. Neutrophil recruitment into the airspaces plays a central role in the progression of ARDS. Recently, AC10 has been implicated in transendothelial emigration of neutrophils from the blood into the underlying tissue via CD99, a transmembrane protein critical for neutrophil recruitment. We sought to determine whether AC10 is necessary for neutrophil recruitment into the airspaces following P. aeruginosa intratracheal inoculation. Increasing doses of bacteria were introduced directly into the lung of wild type and AC10 and NBCn2 knockout mice. Bronchoalveolar lavage fluid (BALF) was recovered 24 hours later and examined for recruitment of polymorphonuclear cells (PMNs) into the airspaces. The number of PMNs recruited into the airspace following Pseudomonas inoculation increased with bacterial number and reached a maximum at a dose of 1E5 bacteria after which 100% of cells in the BALF were PMNs. Thus, doses upto 1E5 bacteria were used to examine the role of AC10 and NBCn2 in PMN recruitment into the airspace between wild type and knockout animals. Our data reveal a decreased number of PMNs in the BALF of both AC10 and NBCn2 knockout female mice; however, no difference in the number of PMNs recruited into the airspace was detected between male wild type and knockout animals. In addition, we confirmed the expression of CD99 in pulmonary microvascular endothelial cells and whole lung from both rat and mouse. Thus, CD99 is expressed in pulmonary endothelial cells and female mice utilize AC10 and NBCn2 to facilitate neutrophil recruitment into the airspace following a model of P. aeruginosa pneumonia.Support or Funding InformationNIH R01HL121513

JMV5656, a short synthetic derivative of TLQP-21, alleviates acid-induced lung injury and fibrosis in mice

TLQP-21, a peptide encoded by the prohormone VGF, is expressed in neuroendocrine cells and can modulate inflammatory processes. Since TLQP-21 can bind the complement 3a receptor 1 on macrophages, interest has risen in this peptide as a potential drug for the treatment of Acute Respiratory Distress Syndrome (ARDS), whose hospital mortality can reach 35–46%. Since no effective pharmacologic therapies are available, our aim was to exploit the potential of a short analog of TLQP-21(JMV5656) in order to modulate the inflammatory process in ARDS and the progression to pulmonary fibrosis in an experimental model of unilateral acid aspiration in mice.Mice were divided in 2 treatment groups. In the acute protocol, mice received intra-peritoneal injection of either vehicle or 0.6 mg/kg JMV5656 on experimental days 1 and 2, and ARDS was induced on day 3 under deep anesthesia by instillation of HCl (1.5 ml/kg of 0.1 M HCl in 0.9% NaCl) into the right lung; all measurements were performed 24 h later. In the subacute protocol, mice were treated as previously, but treatment with vehicle or JMV5656 was repeated also on day 4 and measurements were made 2 weeks later. Twenty-four hours after acid instillation, the total number of immune cell in the BAL rose sharply due primarily to an increase in the PMN population which increased from 1% up to 58% of total cell numbers. JMV5656 significantly reduced PMN recruitment into the alveolar space, but had no effects on cytokine levels in BAL. Two weeks after acid injury, static compliance of the right lung was significantly higher in the JMV5656-treated group compared to vehicle-treated group. Treatment with JMV5656 also blunted the acid-induced collagen deposition in the right lung.These results suggest that JMV5656 can ameliorate mechanical compliance, and reduce collagen deposition in acid-injured lungs in mice. This effect was likely due to the ability of JMV5656 to inhibit PMN recruitment in the injured lung.

The Role of Autophagy in the Innate Immune Response to Fungal Keratitis Caused by Aspergillus fumigatus Infection.

PurposeTo determine the role of autophagy in the innate immune response to fungal keratitis (FK) caused by Aspergillus fumigatus infection.MethodsCorneal samples obtained from patients and mice with FK were visualized via transmission electron microscopy (TEM). Autophagy-related proteins LC3B-II, Beclin-1, LAMP-1, and p62 in A. fumigatus-infected corneas of C57BL/6 mice were tested by Western blot. After treatment with autophagy inhibitors 3-methyladenine (3-MA), chloroquine (CQ), or inducer rapamycin, autophagy-related proteins were detected by Western blot. Corneas were photographed with slit lamp microscopy and pathological changes were observed by hematoxylin and eosin staining. Polymorphonuclear neutrophilic leukocytes (PMNs) were assessed by immunofluorescent staining and observed under TEM. The levels of CXCL-1, IL-1β, HMGB1, IL-18, TNF-α, and IL-10 were tested by reverse transcription polymerase chain reaction and Western blot. The quantification of fungal loads was detected and photographed.ResultsThe accumulation of autophagosomes in corneas of patients and mice with FK was observed with TEM. The expression of LC3B-II, Beclin-1, and LAMP-1 was elevated in corneas after fungal infection, whereas p62 was reduced. Treatment with 3-MA or CQ upregulated clinical scores, pathological changes, and the expression of CXCL-1, IL-1β, HMGB1, IL-18, and TNF-α except IL-10. The morphology of PMNs was changed and PMN recruitment was increased in mice corneas treated with 3-MA or CQ, whereas rapamycin reduced the inflammatory response to keratitis. These results were statistically significant.Conclusions A. fumigatus infection increases the expression of autophagy in corneas. Autophagy plays an anti-inflammatory role in the innate immune response to A. fumigatus keratitis.

Mechanical strength determines Ca2+ transients triggered by the engagement of β2 integrins to their ligands

Lymphocyte function-associated antigen-1 (LFA-1) and macrophage-1 antigen (Mac-1) are key adhesion receptors to mediate neutrophil (PMN) recruitment and intracellular calcium (Ca2+) signaling. Binding of LFA-1 and Mac-1 to their ligands is essential in triggering Ca2+ transients and activating Ca2+-dependent kinases involved in cytoskeletal remodeling and migratory function. While mechanical forces are critical in regulating integrin-mediated Ca2+ transients, it is still unclear how the bond strength of β2-integrin-ligand pair affects Ca2+ responses. Here three typical ligands with known mechanical features with LFA-1 and Mac-1 in our previous work were adopted to quantify their capabilities in inducing Ca2+ transients in adherent PMNs under shear flow. Data indicated that LFA-1 dominates Ca2+ transients in PMNs on intercellular adhesive molecule 1 (ICAM-1) and junctional adhesion molecule-A (JAM-A), while Mac-1 mediates Ca2+ transients induced by receptor for advanced glycation end products (RAGE), consistent with their corresponding bond strengths. These results link β2 integrin-ligand bond strength with Ca2+ transients in PMNs, suggesting high bond strength gives rise to strong Ca2+ response especially under physiological-like shear flow. The outcomes provide a new insight in understanding the mechanical regulatory mechanisms of PMN recruitment.

NADPH Oxidase Controls Neutrophil Infiltration in the Lung in Early Response to Fungal Cell Walls Via LTB4

The leukocyte NADPH oxidase generates superoxide, the precursor to reactive oxygen species important for host defense and immunoregulation. Genetic defects in NADPH oxidase result in chronic granulomatous disease (CGD), characterized by serious bacterial and fungal infections and aberrant inflammation. Aspergillus pneumonia is frequent, and associated with pyogranulomatous inflammation. Of note, even sterile fungal cell walls elicit increased neutrophilic inflammation in CGD mice, demonstrating that that NADPH oxidase can limit inflammation independent of its antimicrobial effects. Leukotriene B4 (LTB4) is a potent inflammatory mediator, acting as a chemoattractant and activating other polymorphonuclear leukocyte (PMN) functions. PMN themselves are prominent source of LTB4, and sense LTB4 through its receptor BLT1, which stimulates additional LTB4 synthesis by autocrine and paracrine routes. This leads to feed forward amplification of LTB4 production locally and formation of PMN clusters. Ca2+ acts as an important second messenger in PMN, including regulation of LTB4 synthesis. Activated human CGD PMN have increased intracellular Ca2+ levels compared to PMN from healthy donors due to electrogenic effects of the NADPH oxidase (Geiszt M. et al JBC 1997). Intracellular Ca2+ overload in PMN is speculated to contribute to increased PMN pro-inflammatory activity, but specific pathways and in vivo relevance are not well defined. Therefore, we therefore investigated the production of LTB4 by murine CGD PMN and the role of LTB4 in PMN recruitment and lung hyperinflammation following pulmonary challenge with zymosan, a sterile fungal cell wall preparation. Zymosan-stimulated PMN from Cybb-null mice, a model for X-linked CGD, produced higher LTB4 in vitro compared to wild type (WT), even in the presence of SOD and catalase to counteract oxidative degradation. Higher LTB4 production correlated with higher levels of intracellular Ca2+ in stimulated X-CGD PMN. The increased LTB4 produced by X-CGD PMN was dependent on cell density and the LTB4 receptor BLT1, consistent with a feed-forward loop that amplified LTB4 production. Zymosan-stimulated X-CGD PMN also formed larger and more numerous clusters in vitro compared to WT PMN. Cluster formation was abrogated by inhibiting LTB4 synthesis with the 5-lipoxygenase (5-LO) inhibitor zileuton or by the BLT1 receptor antagonist U75302, demonstrating that cluster formation was LTB4 and BLT1 dependent. We next examined whether LTB4 regulated the response to zymosan in the lung. Following intranasal administration of zymosan, X-CGD mice had higher PMN numbers in bronchoalveolar lavage (BAL), larger PMN foci by lung histology, and increased lung LTB4 as compared to WT mice. These differences were evident by 8 hr and progressed over the first 24 hr post-challenge, in contrast to WT mice where inflammation plateaued at 8 hr. Treatment with zileuton or U75302 just prior to zymosan significantly reduced lung PMN numbers for both WT and X-CGD mice at 8 hr post challenge, and lung PMN remained substantially reduced in X-CGD mice at 24 hrs. Moreover, delaying administration of zileuton to 8 hr after zymosan challenge also significantly decreased PMN inflammation in X-CGD but not WT mice at 24 hrs, indicating that ongoing synthesis of LTB4 otherwise continued to promote PMN recruitment in CGD mice. These results demonstrate that PMN production of LTB4 in response to fungal cell walls is limited by NADPH oxidase via its effects on intracellular Ca2+. In oxidase-deficient CGD mice, LTB4 plays a major role in driving excessive PMN recruitment to the lung in the early response to zymosan. This study is the first to implicate LTB4 in promoting neutrophilic lung inflammation in response to fungal cell walls in CGD, likely by an amplified feed-forward loop involving increased production of LTB4 by CGD PMN. LTB4 could be a potential therapeutic target to ameliorate CGD hyperinflammation. Disclosures No relevant conflicts of interest to declare.

A Pyrrhic Victory: The PMN Response to Ocular Bacterial Infections.

Some tissues of the eye are susceptible to damage due to their exposure to the outside environment and inability to regenerate. Immune privilege, although beneficial to the eye in terms of homeostasis and protection, can be harmful when breached or when an aberrant response occurs in the face of challenge. In this review, we highlight the role of the PMN (polymorphonuclear leukocyte) in different bacterial ocular infections that invade the immune privileged eye at the anterior and posterior segments: keratitis, conjunctivitis, uveitis, and endophthalmitis. Interestingly, the PMN response from the host seems to be necessary for pathogen clearance in ocular disease, but the inflammatory response can also be detrimental to vision retention. This “Pyrrhic Victory” scenario is explored in each type of ocular infection, with details on PMN recruitment and response at the site of ocular infection. In addition, we emphasize the differences in PMN responses between each ocular disease and its most common corresponding bacterial pathogen. The in vitro and animal models used to identify PMN responses, such as recruitment, phagocytosis, degranulation, and NETosis, are also outlined in each ocular infection. This detailed study of the ocular acute immune response to infection could provide novel therapeutic strategies for blinding diseases, provide more general information on ocular PMN responses, and reveal areas of bacterial ocular infection research that lack PMN response studies.

An intranasally administered monoclonal antibody cocktail abrogates ricin toxin-induced pulmonary tissue damage and inflammation

ABSTRACT Ricin toxin, a plant-derived, mannosylated glycoprotein, elicits an incapacitating and potentially lethal inflammatory response in the airways following inhalation. Uptake of ricin by alveolar macrophages (AM) and other pulmonary cell types occurs via two parallel pathways: one mediated by ricin’s B subunit (RTB), a galactose-specific lectin, and one mediated by the mannose receptor (MR;CD206). Ricin’s A subunit (RTA) is a ribosome-inactivating protein that triggers apoptosis in mammalian cells. It was recently reported that a single monoclonal antibody (MAb), PB10, directed against an immunodominant epitope on RTA and administered intravenously, was able to rescue Rhesus macaques from lethal aerosol dose of ricin. In this study, we now demonstrate in mice that the effectiveness PB10 is significantly improved when combined with a second MAb, SylH3, against RTB. Mice treated with PB10 alone survived lethal-dose intranasal ricin challenge, but experienced significant weight loss, moderate pulmonary inflammation (e.g., elevated IL-1 and IL-6 levels, PMN influx), and apoptosis of lung macrophages. In contrast, mice treated with the PB10/SylH3 cocktail were essentially impervious to pulmonary ricin toxin exposure, as evidenced by no weight loss, no change in local IL-1 and IL-6 levels, retention of lung macrophages, and a significant dampening of PMN recruitment into the bronchoalveolar lavage (BAL) fluids. The PB10/SylH3 cocktail only marginally reduced ricin binding to target cells in the BAL, suggesting that the antibody mixture neutralizes ricin by interfering with one or more steps in the RTB- and MR-dependent uptake pathways.

Osteopontin mediates murine transfusion-related acute lung injury via stimulation of pulmonary neutrophil accumulation

AbstractTransfusion-related acute lung injury (TRALI) is one of the leading causes of transfusion-related fatalities and is characterized by the onset of acute respiratory distress within 6 hours upon blood transfusion. Specific therapies are unavailable. Preexisting inflammation is a risk factor for TRALI and neutrophils (polymorphonuclear neutrophils [PMNs]) are considered to be the major pathogenic cells. Osteopontin (OPN) is a multifunctional protein expressed at sites of inflammation and, for example, is involved in pulmonary disorders, can regulate cellular migration, and can function as a PMN chemoattractant. We investigated whether OPN is involved in TRALI induction by promoting PMN recruitment to the lungs. Using a previously established murine TRALI model, we found that in contrast to wild-type (WT) mice, OPN knockout (KO) mice were resistant to antibody-mediated PMN-dependent TRALI induction. Administration of purified OPN to the OPN KO mice, however, restored the TRALI response and pulmonary PMN accumulation. Alternatively, blockade of OPN in WT mice using an anti-OPN antibody prevented the onset of TRALI induction. Using pulmonary immunohistochemistry, OPN could be specifically detected in the lungs of mice that suffered from TRALI. The OPN-mediated TRALI response seemed dependent on macrophages, likely the cellular source of OPN and OPN polymerization, and independent from the OPN receptor CD44, interleukin 6 (IL-6), and other PMN chemoattractants including macrophage inflammatory protein-2 (MIP-2). These data indicate that OPN is critically required for induction of antibody-mediated murine TRALI through localization to the lungs and stimulation of pulmonary PMN recruitment. This suggests that anti-OPN antibody therapy may be a potential therapeutic strategy to explore in TRALI patients.

12/15-lipoxygenase supports inflammatory cytokine production and PMN recruitment during invasive aspergillosis

Abstract Eicosanoids are a family of inflammatory lipid mediators produced via the metabolism of arachidonic acid by a network of biosynthetic enzymes. We and others have recently shown that pathways within this network, specifically the cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) pathways, participate in induction of protective immune responses during invasive aspergillosis (IA). Based on the high degree of cooperation and interconnection within the eicosanoid network, we hypothesized that a third core enzyme, 12/15-lipoxygenase (12/15-LOX), also may support innate antifungal responses. We report here that mice deficient in the gene encoding 12/15-LOX, Alox15, exhibit decreased survival and increased fungal burden in a model of IA. These defects were associated with decreased PMN numbers in the lung and decreased levels of PMN chemokines (CXCL1, CXCL2, CCL3) in Alox15−/− mice, while fungal killing by PMNs in vitro was unaffected. Alox15−/− mice produced lower levels of the inflammatory cytokines IL-17A, IL-22, IL-1α and IL-6 during infection. iNKT cells, which we have reported to be a major cell source of IL-22 during IA, were present in decreased numbers in Alox15−/− mice. This reduction was associated with lower levels of IL-7, a cytokine we have recently shown to be required for optimal numbers of iNKT cells during IA. Decreased IL-22 levels were further associated with decreased levels of IL-23. Together, these data indicate that 12/15-LOX is a critical player in induction of multiple aspects of the innate immune response to A. fumigatus.

An Intranasally Administered Antibody Cocktail Prevents Ricin Toxin-Induced Lung Inflammation and Alveolar Macrophage Death

Abstract Ricin toxin, a plant-derived, mannosylated glycoprotein, elicits an incapacitating and potentially lethal, pulmonary inflammatory response in the airways following inhalation. Uptake by alveolar macrophages (AM) and other pulmonary cell types occurs via ricin’s B subunit (RTB), a galactose-specific lectin, and the mannose receptor (MR; CD206). Ricin’s A subunit (RTA) is a ribosome-inactivating protein capable of inducing apoptosis in all mammalian cell types. It was recently reported that a single monoclonal antibody (MAb), PB10, directed against an immunodominant epitope on RTA, is able rescue Rhesus macaques against lethal dose of ricin administered by aerosol. In this study, we now demonstrate in mice that the effectiveness PB10 is significantly improved when combined with a second MAb, SylH3, against RTB. Mice treated with PB10 alone survived lethal-dose intranasal ricin challenge, but experienced significant weight loss, moderate pulmonary inflammation (e.g., elevated IL-1 and IL-6 levels, PMN influx), and apoptosis of alveolar and tissue macrophages. In contrast, mice treated with the PB10/SylH3 cocktail were essentially impervious to pulmonary ricin toxin exposure, as evidenced by no weight loss, no change in local IL-1 and IL-6 levels, retention of alveolar and tissue macrophage numbers, and a significant dampening of PMN recruitment to the bronchoalveolar lavage (BAL) fluids. The PB10/SylH3 cocktail only marginally reduced ricin binding to target cells in the BAL, suggesting that the antibody mixture neutralizes ricin by interfering with one or more steps in the RTB- and MR-dependent uptake pathways.

Novel Junctional Adhesion Molecule‐Like (JAML) Regulates Neutrophil Transepithelial Migration During Intestinal Inflammation

Neutrophil (PMN) migration in the intestine is an essential component of host defense and wound repair. However, dysregulated PMN recruitment is a hallmark of inflammatory bowel disease (IBD). Disease flares in IBD are associated with robust PMN migrating across the intestinal mucosa resulting in crypt abscess formation and mucosal ulceration. Despite strong association of PMN infiltration into the gut with patient symptoms in IBD, the molecular basis of PMN transepithelial migration (TEpM) represents a significantly understudied area. It is now appreciated that PMN migration into intestinal mucosa requires multiple adhesive interactions between migrating PMN and intestinal epithelial cells (IECs), that represents a multi‐step process facilitated by complex interactions between PMN and IEC expressed proteins including members of the Junctional Adhesion Molecules (JAMs) family. We have reported that the novel JAM‐like protein termed JAML expressed on PMN, associates with the coxsackie and adenovirus receptor (CAR) expressed on epithelial cells. Binding of soluble JAML ectodomains to CAR has been shown to disrupt JAML‐CAR interactions and reduce PMN TEpM in‐vitro. To define the molecular mechanism of JAML regulation of PMN TEpM during intestinal inflammation in‐vivo, we generated JAML knockout mice (JAML−/−). PMN TEpM was evaluated using a small intestinal loop model, and PMN migration in response to the chemoattractant LTB4 (1nM) quantified by flow cytometry. While WT and JAML−/− mice had similar numbers of circulating leukocytes, we observed reduced PMN TEpM in‐vivo in JAML−/− mice compared to WT animals. This decrease in PMN traffic into the lumen was associated with increased retention of PMN within the intestinal epithelium in JAML−/− mice. In addition, epithelial accumulation of PMN in JAML−/− mice paralleled an increase in intestinal permeability to a 4KDa FITC‐dextran as detected by fluorescence intensity in serum. Finally, we studied the role of JAML on susceptibility to DSS induced colitis. Loss of JAML markedly increased disease activity index, inflammation and mucosal ulceration compared to WT mice. Overall, these observations suggest that JAML is an important adhesion molecule that regulates PMN TEpM in the intestinal mucosa. JAML thus represents a new therapeutic target to dampen excessive PMN migration during intestinal inflammation as seen in IBD.Support or Funding InformationCCFA CDA454814 and NIH 3R01 DK072564‐23S1This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.