Impaired Bacterial Clearance Research Articles

γδ T Cells Mediate Protection against Neutrophil-associated Lung Inflammation in Pulmonary Melioidosis.

Pulmonary melioidosis is a severe tropical infection caused by Burkholderia pseudomallei and is associated with high mortality, despite early antibiotic treatment. γδ T cells have been increasingly implicated as drivers of the host neutrophil response during bacterial pneumonia, but their role in pulmonary melioidosis is unknown. Here, we report that in patients with melioidosis, a lower peripheral blood γδ T-cell concentration is associated with higher mortality, even when adjusting for severity of illness. γδ T cells were also enriched in the lung and protected against mortality in a mouse model of pulmonary melioidosis. γδ T-cell deficiency in infected mice induced an early recruitment of neutrophils to the lung, independent of bacterial burden. Subsequently, γδ T-cell deficiency resulted in increased neutrophil-associated inflammation in the lung as well as impaired bacterial clearance. In addition, γδ T cells influenced neutrophil function and subset diversity in the lung after infection. Our results indicate that γδ T cells serve a novel protective role in the lung during severe bacterial pneumonia by regulating excessive neutrophil-associated inflammation.

Nociceptor sensory neurons promote pneumonic sepsis during carbapenem-resistant Klebsiella pneumoniae lung infection.

Abstract Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a leading cause of Gram-negative pneumonia and pneumonic sepsis for which limited therapeutic options are available. The lungs are densely innervated by nociceptive sensory neurons (nociceptors), which mediate breathing, cough, and bronchoconstriction. However, the role of these neurons in host defense against CRKP-induced pneumonic sepsis is unknown. We found that nociceptors play a host deleterious role to promote pneumonic sepsis. Nociceptor-ablated mice showed an enhanced survival and bacterial clearance after CRKP lung infection. Furthermore, nociceptors ablation resulted in the increased influx of neutrophils and Ly6Chi monocytes and cytokine induction. Depletion of Ly6Chi monocytes, but not of neutrophils, impaired lung bacterial clearance and bacterial dissemination in nociceptor-ablated mice, indicating that Ly6Chi monocytes are a critical cellular population downstream of nociceptors to regulate pneumonic sepsis. Moreover, nociceptive neuropeptide calcitonin gene-related peptide (CGRP) suppressed induction of reactive oxygen species and intracellular bacterial killing. Collectively, our data suggest that the nociceptor neuronal signaling could be a therapeutic target for treating multidrug-resistant CRKP lung infections and pneumonic sepsis. Supported by grants from NIH/NIGMS P20GM113117, American Lung Association, and JCRC Heart Research Award.

Neutrophil-mediated innate immune resistance to bacterial pneumonia is dependent on Tet2 function.

Individuals with clonal hematopoiesis of indeterminate potential (CHIP) are at increased risk of aging related health conditions and all-cause mortality, but whether CHIP affects risk of infection is much less clear. Using UK Biobank data, we revealed a positive association between CHIP and incident pneumonia in 438,421 individuals. We show that inflammation enhanced pneumonia risk, as CHIP carriers with a hypomorphic IL6 receptor polymorphism were protected. To better characterize the pathways of susceptibility, we challenged hematopoietic Tet Methylcytosine Dioxygenase 2-knockout (Tet2-/-) and floxed control mice (Tet2fl/fl) with Streptococcus pneumoniae. As with human CHIP carriers, Tet2-/- mice had hematopoietic abnormalities resulting in the expansion of inflammatory monocytes and neutrophils in peripheral blood. Yet, these cells were insufficient in defending against S. pneumoniae and resulted in increased pathology, impaired bacterial clearance, and higher mortality in Tet2-/- mice. We delineated the transcriptional landscape of Tet2-/- neutrophils and found that, while inflammation-related pathways were upregulated in Tet2-/- neutrophils, migration and motility pathways were compromised. Using live-imaging techniques, we demonstrated impairments in motility, pathogen uptake, and neutrophil extracellular trap (NET) formation by Tet2-/- neutrophils. Collectively, we show that CHIP is a risk factor for bacterial pneumonia related to innate immune impairments.

The chemerin-CMKLR1 axis in keratinocytes impairs innate host defense against cutaneous Staphylococcus aureus infection.

The skin is the most common site of Staphylococcus aureus infection, which can lead to various diseases, including invasive and life-threatening infections, through evasion of host defense. However, little is known about the host factors that facilitate the innate immune evasion of S. aureus in the skin. Chemerin, which is abundantly expressed in the skin and can be activated by proteases derived from S. aureus, has both direct bacteria-killing activity and immunomodulatory effects via interactions with its receptor CMKLR1. Here, we demonstrate that a lack of the chemerin/CMKLR1 axis increases the neutrophil-mediated host defense against S. aureus in a mouse model of cutaneous infection, whereas chemerin overexpression, which mimics high levels of chemerin in obese individuals, exacerbates S. aureus cutaneous infection. Mechanistically, we identified keratinocytes that express CMKLR1 as the main target of chemerin to suppress S. aureus-induced IL-33 expression, leading to impaired skin neutrophilia and bacterial clearance. CMKLR1 signaling specifically inhibits IL-33 expression induced by cell wall components but not secreted proteins of S. aureus by inhibiting Akt activation in mouse keratinocytes. Thus, our study revealed that the immunomodulatory effect of the chemerin/CMKLR1 axis mediates innate immune evasion of S. aureus in vivo and likely increases susceptibility to S. aureus infection in obese individuals.

Impact of age on the host response to sepsis in a murine model of fecal-induced peritonitis

IntroductionDespite older adults being more vulnerable to sepsis, most preclinical research on sepsis has been conducted using young animals. This results in decreased scientific validity since age is an independent predictor of poor outcome. In this study, we explored the impact of aging on the host response to sepsis using the fecal-induced peritonitis (FIP) model developed by the National Preclinical Sepsis Platform (NPSP).MethodsC57BL/6 mice (3 or 12 months old) were injected intraperitoneally with rat fecal slurry (0.75 mg/g) or a control vehicle. To investigate the early stage of sepsis, mice were culled at 4 h, 8 h, or 12 h to investigate disease severity, immunothrombosis biomarkers, and organ injury. Mice received buprenorphine at 4 h post-FIP. A separate cohort of FIP mice were studied for 72 h (with buprenorphine given at 4 h, 12 h, and then every 12 h post-FIP and antibiotics/fluids starting at 12 h post-FIP). Organs were harvested, plasma levels of Interleukin (IL)-6, IL-10, monocyte chemoattract protein (MCP-1)/CCL2, thrombin-antithrombin (TAT) complexes, cell-free DNA (CFDNA), and ADAMTS13 activity were quantified, and bacterial loads were measured.ResultsIn the 12 h time course study, aged FIP mice demonstrated increased inflammation and injury to the lungs compared to young FIP mice. In the 72 h study, aged FIP mice exhibited a higher mortality rate (89%) compared to young FIP mice (42%) (p < 0.001). Aged FIP non-survivors also exhibited a trend towards elevated IL-6, TAT, CFDNA, CCL2, and decreased IL-10, and impaired bacterial clearance compared to young FIP non-survivors.ConclusionTo our knowledge, this is the first study to investigate the impact of age on survival using the FIP model of sepsis. Our model includes clinically-relevant supportive therapies and inclusion of both sexes. The higher mortality rate in aged mice may reflect increased inflammation and worsened organ injury in the early stage of sepsis. We also observed trends in impaired bacterial clearance, increase in IL-6, TAT, CFDNA, CCL2, and decreased IL-10 and ADAMTS13 activity in aged septic non-survivors compared to young septic non-survivors. Our aging model may help to increase the scientific validity of preclinical research and may be useful for identifying mechanisms of age-related susceptibility to sepsis as well as age-specific treatment strategies.

Antiviral epithelial-macrophage crosstalk permits secondary bacterial infections.

Miscommunication of antiviral and antibacterial immune signals drives worsened morbidity and mortality during respiratory viral-bacterial coinfections. Extracellular vesicles (EVs) are a form of intercellular communication with broad implications during infection, and here we show that epithelium-derived EVs released during the antiviral response impair the antibacterial activity of macrophages, an innate immune cell crucial for bacterial control in the airway. Macrophages exposed to antiviral EVs display reduced clearance of Staphylococcus aureus as well as altered inflammatory signaling and anti-inflammatory metabolic reprogramming, thus revealing EVs as a source of dysregulated epithelium-macrophage crosstalk during coinfection. As effective epithelium-macrophage communication is critical in mounting an appropriate immune response, this novel observation of epithelium-macrophage crosstalk shaping macrophage metabolism and antimicrobial function provides exciting new insight and improves our understanding of immune dysfunction during respiratory coinfections.

In vivo impact of tubulin polymerization promoting protein (Tppp) knockout to the airway inflammatory response

Microtubule dysfunction has been implicated as a mediator of inflammation in multiple diseases such as disorders of the cardiovascular and neurologic systems. Tubulin polymerization promoting protein (Tppp) facilitates microtubule elongation and regulates tubulin acetylation through inhibition of cytosolic deacetylase enzymes. Pathologic alterations in microtubule structure and dynamics have been described in cystic fibrosis (CF) and associated with inflammation, however the causality and mechanism remain unclear. Likewise, Tppp has been identified as a potential modifier of CF airway disease severity. Here we directly assess the impact of microtubule dysfunction on infection and inflammation by interrogating wild type and a Tppp knockout mouse model (Tppp − / −). Mice are challenged with a clinical isolate of Pseudomonas aeruginosa-laden agarose beads and assessed for bacterial clearance and inflammatory markers. Tppp − / − mouse model demonstrate impaired bacterial clearance and an elevated inflammatory response compared to control mice. These data are consistent with the hypothesis microtubule dysregulation is sufficient to lead to CF-like airway responses in mice.

Amphipathic Liponecrosis Impairs Bacterial Clearance and Causes Infection During Sterile Inflammation

Although transient bacteremia is common during dental and endoscopic procedures, infections developing during sterile diseases like acute pancreatitis (AP) can have grave consequences. We examined how impaired bacterial clearance may cause this transition. Blood samples from patients with AP, normal controls, and rodents with pancreatitis or those administered different nonesterified fatty acids (NEFAs) were analyzed for albumin-unbound NEFAs, microbiome, and inflammatory cell injury. Macrophage uptake of unbound NEFAs using a novel coumarin tracer were done and the downstream effects-NEFA-membrane phospholipid (phosphatidylcholine) interactions-were studied on isothermal titration calorimetry. Patients with infected AP had higher circulating unsaturated NEFAs; unbound NEFAs, including linoleic acid (LA) and oleic acid (OA); higher bacterial 16S DNA; mitochondrial DNA; altered β-diversity; enrichment in Pseudomonadales; and increased annexin V-positive myeloid (CD14) and CD3-positive T cells on admission. These, and increased circulating dead inflammatory cells, were also noted in rodents with unbound, unsaturated NEFAs. Isothermal titration calorimetry showed progressively stronger unbound LA interactions with aqueous media, phosphatidylcholine, cardiolipin, and albumin. Unbound NEFAs were taken into protein-free membranes, cells, and mitochondria, inducing voltage-dependent anion channel oligomerization, reducing ATP, and impairing phagocytosis. These were reversed by albumin. Invivo, unbound LA and OA increased bacterial loads and impaired phagocytosis, causing infection. LA and OA were more potent for these amphipathic interactions than the hydrophobic palmitic acid. Release of stored LA and OA can increase their circulating unbound levels and cause amphipathic liponecrosis of immune cells via uptake by membrane phospholipids. This impairs bacterial clearance and causes infection during sterile inflammation.

The crosstalk between microbial sensors ELMO1 and NOD2 shape intestinal immune responses

ABSTRACT Microbial sensors play an essential role in maintaining cellular homoeostasis. Our knowledge is limited on how microbial sensing helps in differential immune response and its link to inflammatory diseases. Recently we have confirmed that ELMO1 (Engulfment and Cell Motility Protein-1) present in cytosol is involved in pathogen sensing, engulfment, and intestinal inflammation. Here, we show that ELMO1 interacts with another sensor, NOD2 (Nucleotide-binding oligomerization domain-containing protein 2), that recognizes bacterial cell wall component muramyl dipeptide (MDP). The polymorphism of NOD2 is linked to Crohn’s disease (CD) pathogenesis. Interestingly, we found that overexpression of ELMO1 and mutant NOD2 (L1007fs) were not able to clear the CD-associated adherent invasive E. coli (AIEC-LF82). The functional implications of ELMO1-NOD2 interaction in epithelial cells were evaluated by using enteroid-derived monolayers (EDMs) from ELMO1 and NOD2 KO mice. Subsequently we also assessed the immune response in J774 macrophages depleted of either ELMO1 or NOD2 or both. The infection of murine EDMs with AIEC-LF82 showed higher bacterial load in ELMO1-KO, NOD2 KO EDMs, and ELMO1 KO EDMs treated with NOD2 inhibitors. The murine macrophage cells showed that the downregulation of ELMO1 and NOD2 is associated with impaired bacterial clearance that is linked to reduce pro-inflammatory cytokines and reactive oxygen species. Our results indicated that the crosstalk between microbial sensors in enteric infection and inflammatory diseases impacts the fate of the bacterial load and disease pathogenesis.

Formyl Peptide Receptor Type 2 Deficiency in Myeloid Cells Amplifies Sepsis-Induced Cardiac Dysfunction

Using a global formyl peptide receptor (Fpr) 2 knockout mouse colony, we have reported the modulatory properties of this pro-resolving receptor in polymicrobial sepsis. Herein, we have used a humanized FPR2 (hFPR2) mouse colony, bearing an intact or a selective receptor deficiency in myeloid cells to dwell on the cellular mechanisms. hFPR2 mice and myeloid cell-specific hFPR2 KO (KO) mice were subjected to cecal ligation and puncture (CLP)-induced polymicrobial sepsis. Compared with hFPR2 mice, CLP caused exacerbated cardiac dysfunction (assessed by echocardiography), worsened clinical outcome, and impaired bacterial clearance in KO mice. This pathological scenario was paralleled by increased recruitment of pro-inflammatory monocytes and reduced M2-like macrophages within the KO hearts. In peritoneal exudates of KO mice, we quantified increased neutrophil and MHC II+ macrophage numbers but decreased monocyte/macrophage and MHC II- macrophage recruitment. hFPR2 upregulation was absent in myeloid cells, and local production of lipoxin A4 was reduced in septic KO mice. Administration of the FPR2 agonist annexin A1 (AnxA1) improved cardiac function in hFPR2 septic mice but had limited beneficial effects in KO mice, in which the FPR2 ligand failed to polarize macrophages toward an MHC II- phenotype. In conclusion, FPR2 deficiency in myeloid cells exacerbates cardiac dysfunction and worsens clinical outcome in polymicrobial sepsis. The improvement of cardiac function and the host immune response by AnxA1 is more effective in hFPR2-competent septic mice.

Uptake of Plasmodium chabaudi hemozoin drives Kupffer cell death and fuels superinfections

Kupffer cells (KCs) are self-maintained tissue-resident macrophages that line liver sinusoids and play an important role on host defense. It has been demonstrated that upon infection or intense liver inflammation, KCs might be severely depleted and replaced by immature monocytic cells; however, the mechanisms of cell death and the alterations on liver immunity against infections deserves further investigation. We explored the impact of acute Plasmodium infection on KC biology and on the hepatic immune response against secondary infections. Similar to patients, infection with Plasmodium chabaudi induced acute liver damage as determined by serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) elevation. This was associated with accumulation of hemozoin, increased of proinflammatory response and impaired bacterial and viral clearance, which led to pathogen spread to other organs. In line with this, mice infected with Plasmodium had enhanced mortality during secondary infections, which was associated with increased production of mitochondrial superoxide, lipid peroxidation and increased free iron within KCs—hallmarks of cell death by ferroptosis. Therefore, we revealed that accumulation of iron with KCs, triggered by uptake of circulating hemozoin, is a novel mechanism of macrophage depletion and liver inflammation during malaria, providing novel insights on host susceptibility to secondary infections. Malaria can cause severe liver damage, along with depletion of liver macrophages, which can predispose individuals to secondary infections and enhance the chances of death.

Vaccination with Mycoplasma pneumoniae membrane lipoproteins induces IL-17A driven neutrophilia that mediates Vaccine-Enhanced Disease

Bacterial lipoproteins are an often-underappreciated class of microbe-associated molecular patterns with potent immunomodulatory activity. We previously reported that vaccination of BALB/c mice with Mycoplasma pneumoniae (Mp) lipid-associated membrane proteins (LAMPs) resulted in lipoprotein-dependent vaccine enhanced disease after challenge with virulent Mp, though the immune responses underpinning this phenomenon remain poorly understood. Herein, we report that lipoprotein-induced VED in a mouse model is associated with elevated inflammatory cytokines TNF-α, IL-1β, IL-6, IL-17A, and KC in lung lavage fluid and with suppurative pneumonia marked by exuberant neutrophilia in the pulmonary parenchyma. Whole-lung-digest flow cytometry and RNAScope analysis identified multiple cellular sources for IL-17A, and the numbers of IL-17A producing cells were increased in LAMPs-vaccinated/Mp-challenged animals compared to controls. Specific IL-17A or neutrophil depletion reduced disease severity in our VED model—indicating that Mp lipoproteins induce VED in an IL-17A-dependent manner and through exuberant neutrophil recruitment. IL-17A neutralization reduced levels of TNF-α, IL-1β, IL-6, and KC, indicating that IL-17A preceded other inflammatory cytokines. Surprisingly, we found that IL-17A neutralization impaired bacterial clearance, while neutrophil depletion improved it—indicating that, while IL-17A appears to confer both maladaptive and protective responses, neutrophils play an entirely maladaptive role in VED. Given that lipoproteins are found in virtually all bacteria, the potential for lipoprotein-mediated maladaptive inflammatory responses should be taken into consideration when developing vaccines against bacterial pathogens.

Ethanol Intoxication Impairs Respiratory Function and Bacterial Clearance and Is Associated With Neutrophil Accumulation in the Lung After Streptococcus pneumoniae Infection.

Alcohol consumption is commonplace in the United States and its prevalence has increased in recent years. Excessive alcohol use is linked to an increased risk of infections including pneumococcal pneumonia, mostly commonly caused by Streptococcus pneumoniae. In addition, pneumonia patients with prior alcohol use often require more intensive treatment and longer hospital stays due to complications of infection. The initial respiratory tract immune response to S. pneumoniae includes the production of pro-inflammatory cytokines and chemokines by resident cells in the upper and lower airways which activate and recruit leukocytes to the site of infection. However, this inflammation must be tightly regulated to avoid accumulation of toxic by-products and subsequent tissue damage. A majority of previous work on alcohol and pneumonia involve animal models utilizing high concentrations of ethanol or chronic exposure and offer conflicting results about how ethanol alters immunity to pathogens. Further, animal models often employ a high bacterial inoculum which may overwhelm the immune system and obscure results, limiting their applicability to the course of human infection. Here, we sought to determine how a more moderate ethanol exposure paradigm affects respiratory function and innate immunity in mice after intranasal infection with 104 colony forming units of S. pneumoniae. Ethanol-exposed mice displayed respiratory dysfunction and impaired bacterial clearance after infection compared to their vehicle-exposed counterparts. This altered response was associated with increased gene expression of neutrophil chemokines Cxcl1 and Cxcl2 in whole lung homogenates, elevated concentrations of circulating granulocyte-colony stimulating factor (G-CSF), and higher neutrophil numbers in the lung 24 hours after infection. Taken together, these findings suggest that even a more moderate ethanol consumption pattern can dramatically modulate the innate immune response to S. pneumoniae after only 3 days of ethanol exposure and provide insight into possible mechanisms related to the compromised respiratory immunity seen in alcohol consumers with pneumonia.

Epithelial cell-specific role of HMGB1 in murine lungs with muco-obstructive lung disease

Abstract Introduction Mucoobstructive lung diseases are characterized by defective mucociliary clearance, mucin hypersecretion, and mucoobstruction. High mobility group box 1 protein (HMGB1) plays a key role as an inflammatory mediator. However, the role of HMGB1 in mucoobstructive lung diseases remains unclear. Using Scnn1b-Tg+ mice, a mouse model of cystic fibrosis, we hypothesized that the airway epithelial cell-specific HMGB1 acts as an anti-inflammatory mediator in the Scnn1b-Tg+ lungs. Methods Airway epithelial cell-specific HMGB1-deficient (CCSP-iCre+/Hmgb1flox/flox) and control (CCSP-iCre− /Hmgb1flox/flox) mice were generated. The bronchoalveolar lavage fluid (BALF) and lung tissue were collected for endpoints analyses. Results Compared with wild-type (WT) mice, HMGB1 expression was significantly increased in BALF and airway epithelial cells of Scnn1b-Tg+ mice. Unlike HMGB1-sufficient Scnn1b-Tg+ mice, HMGB1-deficient Scnn1b-Tg+ mice exhibited enhanced lung inflammation evident from the elevated total protein and dsDNA contents in the BALF. HMGB1 deletion in airway epithelial cells in Scnn1b-Tg+ mice also exaggerated immune cell recruitment, which was accompanied by increased levels of chemokines and cytokines, including KC, G-CSF, MCP-1, MIP-2, MIP-1α, MIP-1β, and IP-10. Furthermore, HMGB1 deletion in airway epithelial cells in Scnn1b-Tg+ mice enhanced the type 2 inflammation manifested by increased levels of Th2 cytokines such as IL-4 and IL-5. Additionally, HMGB1-deficient Scnn1b-Tg+ mice showed increased airway mucus plugging and impaired bacterial clearance. Conclusion Epithelial cell-derived HMGB1 may play an anti-inflammatory role in murine lungs with mucoobstructive lung disease. Supported by NIEHS (RO1, ES030125)

Trauma-induced heme release increases susceptibility to bacterial infection.

Infection is a common complication of major trauma that causes significantly increased morbidity and mortality. The mechanisms, however, linking tissue injury to increased susceptibility to infection remain poorly understood. To study this relationship, we present a potentially novel murine model in which a major liver crush injury is followed by bacterial inoculation into the lung. We find that such tissue trauma both impaired bacterial clearance and was associated with significant elevations in plasma heme levels. While neutrophil (PMN) recruitment to the lung in response to Staphylococcus aureus was unchanged after trauma, PMN cleared bacteria poorly. Moreover, PMN show > 50% less expression of TLR2, which is responsible, in part, for bacterial recognition. Administration of heme effectively substituted for trauma. Finally, day 1 trauma patients (n = 9) showed similar elevations in free heme compared with that seen after murine liver injury, and circulating PMN showed similar TLR2 reduction compared with volunteers (n = 6). These findings correlate to high infection rates.

СУБПОПУЛЯЦИОННЫЙ СОСТАВ ИНТРАЭПИТЕЛИАЛЬНЫХ ЛИМФОЦИТОВ У ПАЦИЕНТОВ С БОЛЕЗНЬЮ КРОНА

Crohn’s disease is an autoimmune inflammatory bowel disease characterized with the chronic relapsing-remitting inflammation, mainly in the terminal parts of the small intestine and colon, as a result of a genetic predisposition or environmental factors influence. It is associated with impaired bacterial clearance and changes in immunological parameters what resulted in disruption of the gastrointestinal tract ecosystem, formed by the microbiome and non-living components such as mucus, food metabolites or additives. Intraepithelial lymphocytes of the gastrointestinal tract play an important role in mucosal immunity and their phenotype and functional profile is largely determined by the microbiome composition and environmental factors affecting it (diet, smoking, seasonal changes, air pollution, hygiene, etc.). In this investigation the changes in intraepithelial lymphocytes phenotype of small and large intestine were shown in patient with Crohn’s disease as well as the correlation of intraepithelial lymphocytes phenotype with clinical data was established. Obtained data may be used as a hallmark of immune inflammation in the gut and make intraepithelial lymphocytes as ideal candidate for targeting in further immunoregulation of mucosal adaptive immune response against autoantigens.

Krueppel-Like Factor 4 Expression in Phagocytes Regulates Early Inflammatory Response and Disease Severity in Pneumococcal Pneumonia.

The transcription factor Krueppel-like factor (KLF) 4 fosters the pro-inflammatory immune response in macrophages and polymorphonuclear neutrophils (PMNs) when stimulated with Streptococcus pneumoniae, the main causative pathogen of community-acquired pneumonia (CAP). Here, we investigated the impact of KLF4 expression in myeloid cells such as macrophages and PMNs on inflammatory response and disease severity in a pneumococcal pneumonia mouse model and in patients admitted to hospital with CAP. We found that mice with a myeloid-specific knockout of KLF4 mount an insufficient early immune response with reduced levels of pro-inflammatory cytokines and increased levels of the anti-inflammatory cytokine interleukin (IL) 10 in bronchoalveolar lavage fluid and plasma and an impaired bacterial clearance from the lungs 24 hours after infection with S. pneumoniae. This results in higher rates of bacteremia, increased lung tissue damage, more severe symptoms of infection and reduced survival. Higher KLF4 gene expression levels in the peripheral blood of patients with CAP at hospital admission correlate with a favourable clinical presentation (lower sequential organ failure assessment (SOFA) score), lower serum levels of IL-10 at admission, shorter hospital stay and lower mortality or requirement of intensive care unit treatment within 28 days after admission. Thus, KLF4 in myeloid cells such as macrophages and PMNs is an important regulator of the early pro-inflammatory immune response and, therefore, a potentially interesting target for therapeutic interventions in pneumococcal pneumonia.

Gata2 heterozygous mutant mice exhibit reduced inflammatory responses and impaired bacterial clearance

SummaryInfectious diseases continually pose global medical challenges. The transcription factor GATA2 establishes gene networks and defines cellular identity in hematopoietic stem/progenitor cells and in progeny committed to specific lineages. GATA2-haploinsufficient patients exhibit a spectrum of immunodeficiencies associated with bacterial, viral, and fungal infections. Despite accumulating clinical knowledge of the consequences of GATA2 haploinsufficiency in humans, it is unclear how GATA2 haploinsufficiency compromises host anti-infectious defenses. To address this issue, we examined Gata2-heterozygous mutant (G2Het) mice as a model for human GATA2 haploinsufficiency. In vivo inflammation imaging and cytokine multiplex analysis demonstrated that G2Het mice had attenuated inflammatory responses with reduced levels of inflammatory cytokines, particularly IFN-γ, IL-12p40, and IL-17A, during lipopolysaccharide-induced acute inflammation. Consequently, bacterial clearance was significantly impaired in G2Het mice after cecal ligation and puncture-induced polymicrobial peritonitis. These results provide direct molecular insights into GATA2-directed host defenses and the pathogenic mechanisms underlying observed immunodeficiencies in GATA2-haploinsufficient patients.

Cellular Interactions between Human Airway Epithelial Cells and Cystic Fibrosis Macrophages result in Elevated Inflammation and Impaired Multiciliogenesis

Introduction Patients with chronic lung diseases, such as cystic fibrosis (CF), are often characterized with chronic inflammation and impaired bacterial clearance. This suggests that their respiratory innate immune response is inadequate. Macrophages are the sentinel cells of the respiratory system acting as the first line of defense against invading pathogens, orchestrating wound healing, and maintaining tissue homeostasis. The phenotype and function of macrophages are governed by signals they receive from their microenvironment. Despite this they are often studied in isolation in vitro. We hypothesized that changes in the innate immune response in CF result from pathological signaling between macrophages and airway epithelium. Methods Primary lung macrophages, isolated from lung explants from CF patient and patients with no prior evidence of chronic lung disease, were co-cultured with human bronchial epithelial cell lines with or without mutations in the cystic fibrosis transmembrane regulator (CFTR), NuLi (control) and CuFi (CF). Cell lines were used for the initial experiments to reduce patient to patient variability due to factors such as treatment and bacterial colonization, allowing to focus on the specific role of CFTR mutations. A co-culture model at the air-liquid interface was developed to determine whether epithelial interactions with CFTR-mutant macrophages influenced basal stem cell differentiation. Tumor necrosis factor alpha (TNFα) and interleukins 6 and 8 (IL-6/8) were profiled by stimulating cultures with lipopolysaccharide (LPS) for 24 hours and analyzing the culture supernatants. Results Substantial differences in theinflammatory profile were observed in the presence of CFTR mutations in the co-culture model. A 2-fold increase in TNFα after LPS, was observed comparing CF to WT macrophage monocultures; this suggests they may maintain a hyperinflammatory physiological memory of the CF lung. The presence of epithelial cells suppresses TNFα release when compared to monoculture controls. In contrast, IL-6 is increased in the co-cultures. Additionally, CFTR mutant epithelial cells significantly increase IL-8 (3-fold increase, P<0.05), even in the absence of LPS, this response further exacerbated after LPS stimulation (1.5-fold increase, P<0.05), compared to WT controls. Finally, our data supports a role for macrophages in stimulating ciliogenesis in WT epithelium which is suppressed by the presence of CFTR-mutant macrophages (P<0.05). Conclusion This study highlights the importance of studying macrophages in a physiological microenvironment and supports a pathogenic role for CFTR-mutant epithelium in stimulating lung macrophages to a hyper-inflammatory phenotype. In addition, CF macrophages lose a capacity to promote efficient epithelial regeneration and homeostasis. This highlights the importance of understanding the cellular mechanisms regulated by macrophage-epithelial interactions, regulated by CFTR, to identify new therapeutic targets in CF.

Pyroptosis Mediates Neutrophil Extracellular Trap Formation during Bacterial Infection in Zebrafish

The formation of neutrophil extracellular trap (NET) is a critical host defense when neutrophils migrate to infection sites. Pyroptosis is a newly identified programmed cell death, which is tightly regulated by inflammasome activation. However, the mechanism of pyroptotic signaling participating in NET production remains to be elucidated. In this study, the zebrafish larvae otic vesicle microinjection model was used to infect larvae with hemolysin-overexpressing Edwardsiella piscicida (EthA+), and a rapid migration of neutrophils to infection sites was observed. Intriguingly, EthA+ infection effectively induced significant neutrophil membrane rupture in vivo, which was dependent on caspase-B (caspy2) and gasdermin Eb (GSDMEb) but not caspase-A or gasdermin Ea. Specifically, the EthA+ E. piscicida infection induced pyroptosis along with NETosis in vitro, and depletion of either caspy2 or GSDMEb impaired NET formation in vivo. Consequently, inhibition of the caspy2-GSDMEb axis-gated NETosis impaired bacterial clearance in vivo. Altogether, these data provide evidence that teleost fish innate immune cells, including neutrophils, express features of pyroptosis that are critical for NETosis in teleost innate immunity.