Disruption of the SP-A/SP-R210L (MYO18Aα) pathway prolongs gestation and reduces fetal survival during lipopolysaccharide-induced parturition in late gestation.

Prolonged labor can lead to infection, fetal distress, asphyxia, and life-threatening harm to both the mother and the baby. Surfactant protein A (SP-A) was shown to contribute to the maintenance of pregnancy and timing of term labor. SP-A modulates the stoichiometric expression of the SP-R210L and SP-R210S isoforms of the SP-R210 receptor on alveolar macrophages (AMs). Lack of SP-R210L dysregulates macrophage inflammatory responses. We asked whether SP-A alters normal and inflammation-induced parturition through SP-R210 using SP-A- and SP-R210L-deficient mice. Labor and delivery of time-pregnant mice were monitored in real time using a time-lapse infrared camera. Intrauterine injection with either vehicle or Escherichia coli lipopolysaccharide (LPS) on embryonic (E) day 18.5 post coitus was used to assess the effect of gene disruption in chorioamnionitis-induced labor. We report that either lack of SP-A or disruption of SP-R210L delays parturition by 0.40 and 0.55 days compared with controls, respectively. LPS induced labor at 0.60, 1.01, 0.40, 1.00, and 1.31 days earlier than PBS controls in wild type (WT), SP-A-deficient, littermate controls, heterozygous, and homozygous SP-R210L-deficient mice, respectively. Lack of SP-A reduced litter size in PBS-treated mice, whereas the total number of pups delivered was similar in all LPS-treated mice. The number of live pups, however, was significantly reduced by 50%-70% in SP-A and SP-R210L-deficient mice compared with controls. Differences in gestational length were not associated with intrauterine growth restriction. The present findings support the novel concept that the SP-A/SP-R210 pathway modulates timely labor and delivery and supports fetal lung barrier integrity during fetal-to-neonatal transition in term pregnancy.NEW & NOTEWORTHY To our knowledge, this study is the first to report that SP-A prevents delay of labor and inflammation-induced stillbirth through the receptor SP-R210L.

SP-R210 isoforms of Myosin18A modulate endosomal sorting and recognition of influenza A virus infection in macrophages.

Influenza A virus (IAV) infection causes acute and often lethal inflammation in the lung. The role of macrophages in this adverse inflammation is partially understood. The surfactant protein A receptor 210 (SP-R210) consists of two isoforms, a long (L) SP-R210L and a short (S) SP-R210S isoform encoded by alternative splicing of the myosin 18A gene. We reported that disruption of SP-R210L enhances cytosolic and endosomal antiviral response pathways. Here, we report that SP-R210L antagonizes type I interferon ß (IFNß), as depletion of SP-R210L potentiates IFNß secretion. SP-R210 antibodies enhance and attenuate IFNß secretion in SP-R210L replete and deficient macrophages, respectively, indicating that SP-R210 isoform stoichiometry alters macrophage function intrinsically. This reciprocal response is coupled to unopposed and restricted expression of viral genes in control and SP-R210L-deficient macrophages, respectively. Human monocytic cells with sub-stoichiometric expression of SP-R210L resist IAV infection, whereas alveolar macrophages with increased abundance of SP-R210L permit viral gene expression similar to murine macrophages. Uptake and membrane binding studies show that lack of SP-R210 isoforms does not impair IAV binding and internalization. Lack of SP-R210L, however, results in macropinocytic retention of the virus that depends on both SP-R210S and interferon-inducible transmembrane protein-3 (IFITM3). Mass spectrometry and Western blot analyses indicate that SP-R210 isoforms modulate differential recruitment of the Rho-family GTPase RAC1 and guanine nucleotide exchange factors. Our study suggests that SP-R210 isoforms modulate RAC-dependent macropinosomal sorting of IAV to discrete endosomal and lysosomal compartments that either permit or prevent endolysosomal escape and inflammatory sensing of viral genomes in macrophages.

Comparison of whole blood cytokine immunoassays for rapid, functional immune phenotyping in critically ill patients with sepsis.

BACKGROUND: Sepsis is characterized by highly heterogeneous immune responses associated with a spectrum of disease severity. Methods that rapidly and sensitively profile these immune responses can potentially personalize immune-adjuvant therapies for sepsis. We hypothesized that the ELLA microfluidic approach to measure cytokine production from the whole blood of septic and critically ill patients would deliver faster, more precise results than the existing optic-driven ELISpot quantification. We tested our hypothesis by measuring ex vivo-stimulated production of TNF and IFNγ in critically ill and septic patients (n = 22), critically ill and non-septic patients (n = 10), and healthy volunteers (n = 10) through both ELLA and ELISpot immunoassays. Blood samples were subjected to one of three stimulants for 4 h or 18 h durations during days 1, 7-10, and 14 of critical illness. Stimulants for lymphocytes included anti-CD3/anti-CD28 and phorbol 12-myristate 13-acetate (PMA), whereas LPS was used for monocytes. Stimulated TNF and IFNγ concentrations were then associated with 30-day mortality. RESULTS: Both ELISpot and ELLA immunoassays showed substantial agreement in TNF concentrations post 4 h and 18 h LPS stimulation, with concordance correlation coefficients at 0.62 and 0.60, respectively. ELLA had a broad dynamic measurement range and provided accurate TNF and IFNγ readings at both minimal and elevated cytokine concentrations (with mean coefficients of variation between triplicate readings at 2.1 ± 1.4% and 4.9 ± 7.2%, respectively). However, there was no association between the ELLA-determined cytokine concentrations on the first day of critical illness and 30-day mortality rate. In contrast, using the ELISpot for cytokine quantification revealed that non-survivors had reduced baseline TNF levels at 18 h, decreased LPS-induced TNF levels at 18 h, and diminished TNF levels post 4 h/18 h anti-CD3/28 stimulation. CONCLUSIONS: Our study affirms the feasibility of obtaining dependable immune phenotyping data within 6 h of blood collection from critically ill patients, both septic and non-septic, using the ELLA immunoassay. Both ELLA and ELISpot can offer valuable insights into prognosis, therapeutic strategies, and the underlying mechanisms of sepsis development.

Surfactant protein A alters endosomal trafficking of influenza A virus in macrophages.

Influenza A virus infection (IAV) often leads to acute lung injury that impairs breathing and can lead to death, with disproportionate mortality in children and the elderly. Surfactant Protein A (SP-A) is a calcium-dependent opsonin that binds a variety of pathogens to help control pulmonary infections by alveolar macrophages. Alveolar macrophages play critical roles in host resistance and susceptibility to IAV infection. The effect of SP-A on IAV infection and antiviral response of macrophages, however, is not understood. Here, we report that SP-A attenuates IAV infection in a dose-dependent manner at the level of endosomal trafficking, resulting in infection delay in a model macrophage cell line. The ability of SP-A to suppress infection was independent of its glycosylation status. Binding of SP-A to hemagglutinin did not rely on the glycosylation status or sugar binding properties of either protein. Incubation of either macrophages or IAV with SP-A slowed endocytic uptake rate of IAV. SP-A interfered with binding to cell membrane and endosomal exit of the viral genome as indicated by experiments using isolated cell membranes, an antibody recognizing a pH-sensitive conformational epitope on hemagglutinin, and microscopy. Lack of SP-A in mice enhanced IFNß expression, viral clearance and reduced mortality from IAV infection. These findings support the idea that IAV is an opportunistic pathogen that co-opts SP-A to evade host defense by alveolar macrophages. Our study highlights novel aspects of host-pathogen interactions that may lead to better understanding of the local mechanisms that shape activation of antiviral and inflammatory responses to viral infection in the lung.

All trans-retinoic acid modulates hyperoxia-induced suppression of NF-kB-dependent Wnt signaling in alveolar A549 epithelial cells.

INTRODUCTION: Despite recent advances in perinatal medicine, bronchopulmonary dysplasia (BPD) remains the most common complication of preterm birth. Inflammation, the main cause for BPD, results in arrested alveolarization. All trans-retinoic acid (ATRA), the active metabolite of Vitamin A, facilitates recovery from hyperoxia induced cell damage. The mechanisms involved in this response, and the genes activated, however, are poorly understood. In this study, we investigated the mechanisms of action of ATRA in human lung epithelial cells exposed to hyperoxia. We hypothesized that ATRA reduces hyperoxia-induced inflammatory responses in A549 alveolar epithelial cells. METHODS: A549 cells were exposed to hyperoxia with or without treatment with ATRA, followed by RNA-seq analysis. RESULTS: Transcriptomic analysis of A549 cells revealed ~2,000 differentially expressed genes with a higher than 2-fold change. Treatment of cells with ATRA alleviated some of the hyperoxia-induced changes, including Wnt signaling, cell adhesion and cytochrome P450 genes, partially through NF-κB signaling. DISCUSSION/CONCLUSION: Our findings support the idea that ATRA supplementation may decrease hyperoxia-induced disruption of the neonatal respiratory epithelium and alleviate development of BPD.

Comparison of Rapid Cytokine Immunoassays for Functional Immune Phenotyping.

Background: Cell-based functional immune-assays may allow for risk stratification of patients with complex, heterogeneous immune disorders such as sepsis. Given the heterogeneity of patient responses and the uncertain immune pathogenesis of sepsis, these assays must first be defined and calibrated in the healthy population. Objective: Our objective was to compare the internal consistency and practicality of two immune assays that may provide data on surrogate markers of the innate and adaptive immune response. We hypothesized that a rapid turnaround, microfluidic-based immune assay (ELLA) would be comparable to a dual-color, enzyme-linked immunospot (ELISpot) assay in identifying tumor necrosis factor (TNF) and interferon (IFN)γ production following ex vivo whole blood stimulation. Design: This was a prospective, observational cohort analysis. Whole blood samples from ten healthy, immune-competent volunteers were stimulated for either 4 hours or 18 hours with lipopolysaccharide, anti-CD3/anti-CD28 antibodies, or phorbol 12-myristate 13-acetate with ionomycin to interrogate innate and adaptive immune responses, respectively. Measurements and Main Results: ELLA analysis produced more precise measurement of TNF and IFNγ concentrations as compared with ELISpot, as well as a four- to five-log10 dynamic range for TNF and IFNγ concentrations, as compared with a two-log10 dynamic range with ELISpot. Unsupervised clustering accurately predicted the ex vivo immune stimulant used for 90% of samples analyzed via ELLA, as compared with 72% of samples analyzed via ELISpot. Conclusions: We describe, for the first time, a rapid and precise assay for functional interrogation of the innate and adaptive arms of the immune system in healthy volunteers. The advantages of the ELLA microfluidic platform may represent a step forward in generating a point-of-care test with clinical utility, for identifying deranged immune phenotypes in septic patients.

Resistin production does not affect outcomes in a mouse model of acute surgical sepsis.

INTRODUCTION: Because of the strong correlation between the blood concentration of circulating resistin and the illness severity of septic patients, resistin has been proposed as a mediator of sepsis pathophysiology. In vitro data indicate that human resistin directly impairs neutrophil migration and intracellular bacterial killing, although the significance of these findings in vivo remain unclear. OBJECTIVE: The objectives of the present study were: (1) to validate the expression of human resistin in a clinically relevant, murine model of surgical sepsis, (2) to assess how sepsis-induced changes in resistin correlate with markers of infection and organ dysfunction, and (3) to investigate whether the expression of human resistin alters immune function or disease outcomes in vivo. METHODS: 107 male, C57BL/6 mice transgenic for the human resistin gene and its promoter elements (Retn+/-/-, or Retn+) were generated on a Retn-/- (mouse resistin knockout, or Rko) background. Outcomes were compared between age-matched transgenic and knockout mice. Acute sepsis was defined as the initial 24 h following cecal ligation and puncture (CLP). Physiologic and laboratory parameters correlating to the human Sequential Organ Failure Assessment (SOFA) Score were measured in mice, and innate immune cell number/function in the blood and peritoneal cavity were assessed. RESULTS: CLP significantly increased circulating levels of human resistin. The severity of sepsis-induced leukopenia was comparable between Retn+ and Rko mice. Resistin was associated with increased production of neutrophil reactive oxygen species, a decrease in circulating neutrophils at 6 h and an increase in peritoneal Ly6Chi monocytes at 6 h and 24 h post-sepsis. However, intraperitoneal bacterial growth, organ dysfunction and mouse survival did not differ with resistin production in septic mice. SIGNIFICANCE: Ex vivo resistin-induced impairment of neutrophil function do not appear to translate to increased sepsis severity or poorer outcomes in vivo following CLP.

Genomic and epigenomic adaptation in SP-R210 (Myo18A) isoform-deficient macrophages.

Macrophages play an important role in maintaining tissue homeostasis, from regulating the inflammatory response to pathogens to resolving inflammation and aiding tissue repair. The surfactant protein A (SP-A) receptor SP-R210 (MYO18A) has been shown to affect basal and inflammatory macrophage states. Specifically, disruption of the longer splice isoform SP-R210L/MYO18Aα renders macrophages hyper-inflammatory, although the mechanism by which this occurs is not well understood. We asked whether disruption of the L isoform led to the hyper-inflammatory state via alteration of global genomic responses. RNA sequencing analysis of L isoform-deficient macrophages (SP-R210L(DN)) revealed basal and influenza-induced upregulation of genes associated with inflammatory pathways, such as TLR, RIG-I, NOD, and cytoplasmic DNA signaling, whereas knockout of both SP-R210 isoforms (L and S) only resulted in increased RIG-I and NOD signaling. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis showed increased genome-wide deposition of the pioneer transcription factor PU.1 in SP-R210L(DN) cells, with increased representation around genes relevant to inflammatory pathways. Additional ChIP-seq analysis of histone H3 methylation marks showed decreases in both repressive H3K9me3 and H3K27me3 marks with a commensurate increase in transcriptionally active (H3K4me3) histone marks in the L isoform deficient macrophages. Influenza A virus (IAV) infection, known to stimulate a wide array of anti-viral responses, caused a differential redistribution of PU.1 binding between proximal promoter and distal sites and decoupling from Toll-like receptor regulated gene promoters in SP-R210L(DN) cells. These finding suggest that the inflammatory differences seen in SP-R210L-deficient macrophages are a result of transcriptional differences that are mediated by epigenetic changes brought about by differential expression of the SP-R210 isoforms. This provides an avenue to explore how the signaling pathways downstream of the receptor and the ligands can modulate the macrophage inflammatory response.

IL-4Rα signaling by CD8α+ dendritic cells contributes to cerebral malaria by enhancing inflammatory, Th1, and cytotoxic CD8+ T cell responses.

Persistent high levels of proinflammatory and Th1 responses contribute to cerebral malaria (CM). Suppression of inflammatory responses and promotion of Th2 responses prevent pathogenesis. IL-4 commonly promotes Th2 responses and inhibits inflammatory and Th1 responses. Therefore, IL-4 is widely considered as a beneficial cytokine via its Th2-promoting role that is predicted to provide protection against severe malaria by inhibiting inflammatory responses. However, IL-4 may also induce inflammatory responses, as the result of IL-4 action depends on the timing and levels of its production and the tissue environment in which it is produced. Recently, we showed that dendritic cells (DCs) produce IL-4 early during malaria infection in response to a parasite protein and that this IL-4 response may contribute to severe malaria. However, the mechanism by which IL-4 produced by DCs contributing to lethal malaria is unknown. Using Plasmodium berghei ANKA-infected C57BL/6 mice, a CM model, we show here that mice lacking IL-4Rα only in CD8α+ DCs are protected against CM pathogenesis and survive, whereas WT mice develop CM and die. Compared with WT mice, mice lacking IL-4Rα in CD11c+ or CD8α+ DCs showed reduced inflammatory responses leading to decreased Th1 and cytotoxic CD8+ T cell responses, lower infiltration of CD8+ T cells to the brain, and negligible brain pathology. The novel results presented here reveal a paradoxical role of IL-4Rα signaling in CM pathogenesis that promotes CD8α+ DC-mediated inflammatory responses that generate damaging Th1 and cytotoxic CD8+ T cell responses.

Dual targeting of MTOR as a novel therapeutic approach for high-risk B-cell acute lymphoblastic leukemia.

Children of Hispanic/Latino ancestry have increased incidence of high-risk B-cell acute lymphoblastic leukemia (HR B-ALL) with poor prognosis. This leukemia is characterized by a single-copy deletion of the IKZF1 (IKAROS) tumor suppressor and increased activation of the PI3K/AKT/mTOR pathway. This identifies mTOR as an attractive therapeutic target in HR B-ALL. Here, we report that IKAROS represses MTOR transcription and IKAROS' ability to repress MTOR in leukemia is impaired by oncogenic CK2 kinase. Treatment with the CK2 inhibitor, CX-4945, enhances IKAROS activity as a repressor of MTOR, resulting in reduced expression of MTOR in HR B-ALL. Thus, we designed a novel therapeutic approach that implements dual targeting of mTOR: direct inhibition of the mTOR protein (with rapamycin), in combination with IKAROS-mediated transcriptional repression of the MTOR gene (using the CK2 inhibitor, CX-4945). Combination treatment with rapamycin and CX-4945 shows synergistic therapeutic effects in vitro and in patient-derived xenografts from Hispanic/Latino children with HR B-ALL. These data suggest that such therapy has the potential to reduce the health disparity in HR B-ALL among Hispanic/Latino children. The dual targeting of oncogene transcription, combined with inhibition of the corresponding oncoprotein provides a paradigm for a novel precision medicine approach for treating hematological malignancies.

STAT4 Is Largely Dispensable for Systemic Lupus Erythematosus-like Autoimmune- and Foreign Antigen-Driven Antibody-Forming Cell, Germinal Center, and Follicular Th Cell Responses.

Genome-wide association studies identified variants in the transcription factor STAT4 gene and several other genes in the STAT4 signaling pathway, such as IL12A, IL12B, JAK2, and TYK2, which are associated with an increased risk of developing systemic lupus erythematosus (SLE) and other autoimmune diseases. Consistent with the genome-wide association studies data, STAT4 was shown to play an important role in autoimmune responses and autoimmunity development in SLE mouse models. Despite such important role for STAT4 in SLE development in mice and humans, little is known whether and how STAT4 may regulate extrafollicular Ab-forming cell (AFC) and follicular germinal center (GC) responses, two major pathways of autoreactive B cell development and autoantibody production. To our surprise, we found STAT4 to be largely dispensable for promoting autoimmune AFC and GC responses in various autoimmune- and SLE-prone mouse models, which strongly correlated with autoantibody production, and immune complex deposition and immune cell infiltration in the kidney. We further observed that STAT4 deficiency had no effects on AFC, GC, and Ag-specific Ab responses during protein Ag immunization or influenza virus infection. Additionally, CD4+ effector and follicular Th cell responses in autoimmune- and SLE-prone mice and protein Ag-immunized and influenza virus-infected mice were intact in the absence of STAT4. Together, our data demonstrate a largely dispensable role for STAT4 in AFC, GC, and Ab responses in SLE mouse models and in certain foreign Ag-driven responses.

Cytokine Panels and Pediatric Acute Respiratory Distress Syndrome: A Translational Investigation.

OBJECTIVES: To identify and compare serum and lower respiratory tract fluid biomarkers of lung injury using well-characterized mouse models of lung injury. To explore the relationship between these preclinical biomarkers and clinical outcomes in a discovery cohort of pediatric patients with acute respiratory failure from pneumonia. DESIGN: Prospective, observational cohort study. SETTING: A basic science laboratory and the PICU of a tertiary-care children's hospital. PATIENTS: PICU patients intubated for respiratory failure from a suspected respiratory infection. INTERVENTIONS: Prospective enrollment and collection of lower respiratory tract fluid samples. MEASUREMENTS AND MAIN RESULTS: C57BL6/J mice were intranasally inoculated with escalating doses of influenza A virus or toll-like receptor agonists to simulate varying degrees of lung injury. Serum and bronchoalveolar lavage fluid were measured for the presence of cytokines using commercially available multiplex cytokine assays. Elevated levels of C-C motif chemokine ligand 7 at the peak of inflammation in both bronchoalveolar lavage fluid and serum correlated with lethality, with the bronchoalveolar lavage fluid ratio of C-C motif chemokine ligand 7:C-C motif chemokine ligand 22 providing the best prediction in the mouse models. These preclinical biomarkers were examined in the plasma and lower respiratory tract fluid of a discovery cohort of pediatric patients with acute respiratory failure from pneumonia. The primary clinical outcome measure was ventilator-free days, with secondary outcomes of pediatric acute respiratory distress syndrome severity and mortality. Elevation in peak lower respiratory tract fluid C-C motif chemokine ligand 7:C-C motif chemokine ligand 22 ratios demonstrated a significant negative correlation with ventilator-free days (r = -0.805; p < 0.02). CONCLUSIONS: This study provides evidence that lung immune profiling via lower respiratory tract fluid cytokine analysis is feasible and may provide insight into clinical outcomes. Further validation of markers, including the C-C motif chemokine ligand 7:C-C motif chemokine ligand 22 ratio in this limited study, in a larger cohort of patients is necessary.

Lower respiratory tract delivery, airway clearance, and preclinical efficacy of inhaled GM-CSF in a postinfluenza pneumococcal pneumonia model.

Inhaled granulocyte/macrophage colony-stimulating factor (GM-CSF) shows promise as a therapeutic to treat viral and bacterial pneumonia, but no mouse model of inhaled GM-CSF has been described. We sought to 1) develop a mouse model of aerosolized recombinant mouse GM-CSF administration and 2) investigate the protection conferred by inhaled GM-CSF during influenza A virus (IAV) infection against secondary bacterial infection with pneumococcus. To assess lower respiratory tract delivery of aerosolized therapeutics, mice were exposed to aerosolized fluorescein (FITC)-labeled dextran noninvasively via an aerosolization tower or invasively using a rodent ventilator. The efficiency of delivery to the lower respiratory tracts of mice was 0.01% noninvasively compared with 0.3% invasively. The airway pharmacokinetics of inhaled GM-CSF fit a two-compartment model with a terminal phase half-life of 1.3 h. To test if lower respiratory tract levels were sufficient for biological effect, mice were infected intranasally with IAV, treated with aerosolized recombinant mouse GM-CSF, and then secondarily infected with Streptococcus pneumoniae. Inhaled GM-CSF conferred a significant survival benefit to mice against secondary challenge with S. pneumoniae (P < 0.05). Inhaled GM-CSF did not reduce airway or lung parenchymal bacterial growth but significantly reduced the incidence of S. pneumoniae bacteremia (P < 0.01). However, GM-CSF overexpression during influenza virus infection did not affect lung epithelial permeability to FITC-dextran ingress into the bloodstream. Therefore, the mechanism of protection conferred by inhaled GM-CSF appears to be locally mediated improved lung antibacterial resistance to systemic bacteremia during IAV infection.

Structural and Functional Determinants of Rodent and Human Surfactant Protein A: A Synthesis of Binding and Computational Data.

Surfactant protein A (SP-A) provides surfactant stability, first line host defense, and lung homeostasis by binding surfactant phospholipids, pathogens, alveolar macrophages (AMs), and epithelial cells. Non-primates express one SP-A protein whereas humans express two: SP-A1 and SP-A2 with core intra- and inter-species differences in the collagen-like domain. Here, we used macrophages and solid phase binding assays to discern structural correlates of rat (r) and human (h) SP-A function. Binding assays using recombinant rSP-A expressed in insect cells showed that lack of proline hydroxylation, truncations of amino-terminal oligomerization domains, and site-directed serine (S) or alanine (A) mutagenesis of cysteine 6 (C6S), glutamate 195 (E195A), and glutamate 171 (E171A) in the carbohydrate recognition domain (CRD) all impaired SP-A binding. Replacement of arginine 197 with alanine found in hSP-A (R197A), however, restored the binding of hydroxyproline-deficient rSP-A to the SP-A receptor SP-R210 similar to native rat and human SP-A. In silico calculation of Ca++ coordination bond length and solvent accessibility surface area revealed that the "humanized" R197A substitution alters topology and solvent accessibility of the Ca++ coordination residues of the CRD domain. Binding assays in mouse AMs that were exposed to either endogenous SP-A or hSP-A1 (6A2) and hSP-A2 (1A0) isoforms in vivo revealed that mouse SP-A is a functional hybrid of hSP-A1 and hSP-A2 in regulating SP-A receptor occupancy and binding affinity. Binding assays using neonatal and adult human AMs indicates that the interaction of SP-A1 and SP-A2 with AMs is developmentally regulated. Furthermore, our data indicate that the auxiliary ion coordination loop encompassing the conserved E171 residue may comprise a conserved site of interaction with macrophages, and SP-R210 specifically, that merits further investigation to discern conserved and divergent SP-A functions between species. In summary, our findings support the notion that complex structural adaptation of SP-A regulate conserved and species specific AM functions in vertebrates.

17ß-Estradiol affects lung function and inflammation following ozone exposure in a sex-specific manner.

Inflammatory lung diseases affect men and women disproportionately, suggesting that fluctuations of circulating hormone levels mediate inflammatory responses. Studies have shown that ozone exposure contributes to lung injury and impairment of innate immunity with differential effects in men and women. Here, we hypothesized that 17ß-estradiol enhances inflammation and airway hyperresponsiveness (AHR), triggered by ozone exposure, in the female lung. We performed gonadectomy and hormone treatment (17ß-estradiol, 2 wk) in C57BL/6J female and male mice and exposed animals to 1 ppm of ozone or filtered air for 3 h. Twenty-four hours later, we tested lung function, inflammatory gene expression, and changes in bronchoalveolar lavage fluid (BALF). We found increased AHR and expression of inflammatory genes after ozone exposure. These changes were higher in females and were affected by gonadectomy and 17ß-estradiol treatment in a sex-specific manner. Gonadectomized male mice displayed higher AHR and inflammatory gene expression than controls exposed to ozone; 17ß-estradiol treatment did not affect this response. In females, ovariectomy reduced ozone-induced AHR, which was restored by 17ß-estradiol treatment. Ozone exposure also increased BALF lipocalin-2, which was reduced in both male and female gonadectomized mice. Treatment with 17ß-estradiol increased lipocalin-2 levels in females but lowered them in males. Gonadectomy also reduced ozone-induced expression of lung IL-6 and macrophage inflammatory protein-3 in females, which was restored by treatment with 17ß-estradiol. Together, these results indicate that 17ß-estradiol increases ozone-induced inflammation and AHR in females but not in males. Future studies examining diseases associated with air pollution exposure should consider the patient's sex and hormonal status.

Resistin directly inhibits bacterial killing in neutrophils.

BACKGROUND: Sepsis-induced immunosuppression is a key factor contributing to the morbidity and mortality of critically ill patients, and polymorphonuclear neutrophil dysfunction is believed to be a hallmark of this immunosuppression. Circulating myeloid cells produce the cytokine resistin (RETN), which has been associated with poor outcomes in sepsis/septic shock and can directly inhibit neutrophil function. We previously demonstrated that resistin caused a dose-dependent impairment in neutrophil migration, reactive oxygen species production, and bacterial clearance in neutrophil cell lines. However, the relative antimicrobial responses of other innate immune cells to Gram-positive and Gram-negative infections in the presence of elevated levels of resistin have not been evaluated. We hypothesized that resistin directly contributes to sepsis-induced immunosuppression by selectively targeting the neutrophil component of the innate cellular immune system. Thus, the goal of the present study was to compare the effect of resistin on bacterial killing using monocultures or co-cultures of monocyte and neutrophil cell lines, as well as to extend our findings to primary immune cells. RESULTS: Our results indicate that human resistin impairs the ability of neutrophils to kill the Gram-negative bacterium Pseudomonas aeruginosa and the Gram-positive bacterium Staphylococcus aureus. In contrast, with the exception of macrophages incubated with P. aeruginosa, resistin did not affect the ability of macrophages or monocytes to kill either Gram-positive or Gram-negative organisms. Furthermore, co-incubation of neutrophils with increasing proportions of monocytes did not enhance bacterial killing. Resistin blocked bactericidal activity through partial reduction of F-actin polymerization and suppression of the oxidative burst in neutrophils. CONCLUSIONS: Our studies indicate that resistin selectively impairs neutrophil bacterial killing. These findings further support the notion that resistin can mimic cell type-dependent immunosuppressive effects. This is consistent with its putative role in the pathogenesis of bacterial sepsis.

Modulation of the lung inflammatory response to ozone by the estrous cycle.

Emerging evidence suggests that sex differences exist in the control of lung innate immunity; however, the specific roles of sex hormones in the inflammatory response, and the mechanisms involved are unclear. Here, we investigated whether fluctuations in circulating hormone levels occurring in the mouse estrous cycle could affect the inflammatory response to air pollution exposure. For this, we exposed female mice (C57BL/6J, 8 weeks old) at different phases of the estrous cycle to 2 ppm of ozone or filtered air (FA) for 3 h. Following exposure, we collected lung tissue and bronchoalveolar lavage fluid (BAL), and performed lung function measurements to evaluate inflammatory responses and respiratory mechanics. We found a differential inflammatory response to ozone in females exposed in the luteal phase (metestrus, diestrus) versus the follicular phase (proestrus, estrus). Females exposed to ozone in the follicular phase had significantly higher expression of inflammatory genes, including Ccl2, Cxcl2, Ccl20, and Il6, compared to females exposed in the luteal phase (P < 0.05), and displayed differential activation of regulatory pathways. Exposure to ozone in the follicular phase also resulted in higher BAL neutrophilia, lipocalin levels, and airway resistance than exposure in the luteal phase (P < 0.05). Together, these results show that the effects of ozone exposure in the female lung are affected by the estrous cycle phase, and potentially hormonal status. Future studies investigating air pollution effects and inflammation in women should consider the menstrual cycle phase and/or circulating hormone levels.

GM-CSF overexpression after influenza a virus infection prevents mortality and moderates M1-like airway monocyte/macrophage polarization.

BACKGROUND: Influenza A viruses cause life-threatening pneumonia and lung injury in the lower respiratory tract. Application of high GM-CSF levels prior to infection has been shown to reduce morbidity and mortality from pathogenic influenza infection in mice, but the mechanisms of protection and treatment efficacy have not been established. METHODS: Mice were infected intranasally with influenza A virus (PR8 strain). Supra-physiologic levels of GM-CSF were induced in the airways using the double transgenic GM-CSF (DTGM) or littermate control mice starting on 3 days post-infection (dpi). Assessment of respiratory mechanical parameters was performed using the flexiVent rodent ventilator. RNA sequence analysis was performed on FACS-sorted airway macrophage subsets at 8 dpi. RESULTS: Supra-physiologic levels of GM-CSF conferred a survival benefit, arrested the deterioration of lung mechanics, and reduced the abundance of protein exudates in bronchoalveolar (BAL) fluid to near baseline levels. Transcriptome analysis, and subsequent validation ELISA assays, revealed that excess GM-CSF re-directs macrophages from an "M1-like" to a more "M2-like" activation state as revealed by alterations in the ratios of CXCL9 and CCL17 in BAL fluid, respectively. Ingenuity pathway analysis predicted that GM-CSF surplus during IAV infection elicits expression of anti-inflammatory mediators and moderates M1 macrophage pro-inflammatory signaling by Type II interferon (IFN-γ). CONCLUSIONS: Our data indicate that application of high levels of GM-CSF in the lung after influenza A virus infection alters pathogenic "M1-like" macrophage inflammation. These results indicate a possible therapeutic strategy for respiratory virus-associated pneumonia and acute lung injury.

SH3GLB2/endophilin B2 regulates lung homeostasis and recovery from severe influenza A virus infection.

New influenza A viruses that emerge frequently elicit composite inflammatory responses to both infection and structural damage of alveolar-capillary barrier cells that hinders regeneration of respiratory function. The host factors that relinquish restoration of lung health to enduring lung injury are insufficiently understood. Here, we investigated the role of endophilin B2 (B2) in susceptibility to severe influenza infection. WT and B2-deficient mice were infected with H1N1 PR8 by intranasal administration and course of influenza pneumonia, inflammatory, and tissue responses were monitored over time. Disruption of B2 enhanced recovery from severe influenza infection as indicated by swift body weight recovery and significantly better survival of endophilin B2-deficient mice compared to WT mice. Compared to WT mice, the B2-deficient lungs exhibited induction of genes that express surfactant proteins, ABCA3, GM-CSF, podoplanin, and caveolin mRNA after 7 days, temporal induction of CCAAT/enhancer binding protein CEBPα, ß, and δ mRNAs 3-14 days after infection, and differences in alveolar extracellular matrix integrity and respiratory mechanics. Flow cytometry and gene expression studies demonstrated robust recovery of alveolar macrophages and recruitment of CD4+ lymphocytes in B2-deficient lungs. Targeting of endophilin B2 alleviates adverse effects of IAV infection on respiratory and immune cells enabling restoration of alveolar homeostasis.

An Electrochemical Biosensor for Rapid Detection of Pediatric Bloodstream Infections.

Bloodstream infections are major contributing factors of morbidity and mortality among children. Precise and timely identification of causative agents can improve the clinical management and outcome of the infection, potentially saving lives. Electrochemical biosensors previously described by Gao et al. (2017) have the potential to deliver greater speed and discrimination. However, to date there are no data that determine whether the age of the host would cause bacteria to demonstrate different growth characteristics, or whether pediatric samples would behave differently using this electrochemical biosensor. The importance of this knowledge gap is clear: the preclinical testing phase of this line of research is limited by the relative lack of pediatric healthy blood volunteers to complete this work. Therefore, in this study we have applied this novel technology to diagnose bacteria spiked into pediatric blood and compared directly with adult blood samples. Only 180 µL of blood was utilized from both adult and pediatric volunteers and inoculated with Escherichia coli 67, and the signals generated at different time points were compared. We were able to demonstrate that the signals generated by adult and pediatric blood were not significantly different with this detection technology.