Serum anti-PAD4 autoantibodies are present in cystic fibrosis children and increase with age and lung disease severity.

Cystic fibrosis (CF) lung disease begins early in childhood and is characterized by neutrophilic inflammation of the airways. Neutrophil extracellular traps (NETs) represent one mechanism by which neutrophils contribute to lung damage. The enzyme peptidylarginine deiminase 4 (PAD4) is required for NET formation. Our overall concept is that NET formation delivers PAD4 outside the neutrophil resulting in autoantibody generation, and this autoimmunity may be a novel mechanism contributing to CF lung disease progression. The aim of this study was to investigate clinical predictors of serum anti-PAD4 autoantibody (PAD4 Ab) levels in CF subjects with a wide range of ages from early childhood through middle age. We measured PAD4 Ab levels in sera from 104 CF subjects. PAD4 Abs were detectable among CF children as young as one year of age and elevated compared to paediatric healthy controls. PAD4 Ab levels increased significantly with age (r = 0.584, p <.001) and correlated with lower lung function (r = -0.481, n = 99, p <.001). PAD4 Abs were elevated in subjects with chronic Pseudomonas aeruginosa airways infection (p <.001), but not with other key clinical CF co-variates including sex, CFTR genotype, sweat chloride, pancreatic enzyme use, nutritional status, recent pulmonary exacerbations, Staphylococcus aureus, or CF-related diabetes. PAD4 Ab levels were also correlated with serum anti-double-stranded DNA IgA autoantibodies, which have similarly been shown to be elevated in CF subjects and associated with lung damage. In multivariable analysis, age and lung function remained correlated with PAD4 Ab levels. In summary, we describe novel findings of anti-PAD4 autoantibodies in CF that are present early in childhood, increase over time with age, and correlate with lung disease severity. Autoimmunity to antigens extruded by NETs appears to be an early event in CF lung disease, and airway autoimmunity related to NET formation is a potential mechanism of lung disease progression in CF.HighlightsSerum anti-PAD4 autoantibodies are detected in paediatric CF serum and are elevated compared to healthy paediatric controlsAnti-PAD4 autoantibodies increase with ageAnti-PAD4 autoantibodies correlate with lower lung function, Pseudomonas aeruginosa airway infection and anti-dsDNA IgA autoantibodies, but not with other key clinical CF co-variatesAge and lung function remain correlated with anti-PAD4 autoantibodies in multivariable analysis.

Influenza-induced Tpl2 expression within alveolar epithelial cells is dispensable for host viral control and anti-viral immunity.

Tumor progression locus 2 (Tpl2) is a serine/threonine kinase that regulates the expression of inflammatory mediators in response to Toll-like receptors (TLR) and cytokine receptors. Global ablation of Tpl2 leads to severe disease in response to influenza A virus (IAV) infection, characterized by respiratory distress, and studies in bone marrow chimeric mice implicated Tpl2 in non-hematopoietic cells. Lung epithelial cells are primary targets and replicative niches of influenza viruses; however, the specific regulation of antiviral responses by Tpl2 within lung epithelial cells has not been investigated. Herein, we show that Tpl2 is basally expressed in primary airway epithelial cells and that its expression increases in both type I and type II airway epithelial cells (AECI and AECII) in response to influenza infection. We used Nkx2.1-cre to drive Tpl2 deletion within pulmonary epithelial cells to delineate epithelial cell-specific functions of Tpl2 during influenza infection in mice. Although modest increases in morbidity and mortality were attributed to cre-dependent deletion in lung epithelial cells, no alterations in host cytokine production or lung pathology were observed. In vitro, Tpl2 inhibition within the type I airway epithelial cell line, LET1, as well as genetic ablation in primary airway epithelial cells did not alter cytokine production. Overall, these findings establish that Tpl2-dependent defects in cells other than AECs are primarily responsible for the morbidity and mortality seen in influenza-infected mice with global Tpl2 ablation.

Granulocytic Myeloid-Derived Suppressor Cells in Cystic Fibrosis.

Cystic Fibrosis (CF) is a genetic disease that causes chronic and severe lung inflammation and infection associated with high rates of mortality. In CF, disrupted ion exchange in the epithelium results in excessive mucus production and reduced mucociliary clearance, leading to immune system exacerbation and chronic infections with pathogens such as P. aeruginosa and S. aureus. Constant immune stimulation leads to altered immune responses including T cell impairment and neutrophil dysfunction. Specifically, CF is considered a Th17-mediated disease, and it has been proposed that both P. aeruginosa and a subset of neutrophils known as granulocytic myeloid suppressor cells (gMDSCs) play a role in T cell suppression. The exact mechanisms behind these interactions are yet to be determined, but recent works demonstrate a role for arginase-1. It is also believed that P. aeruginosa drives gMDSC function as a means of immune evasion, leading to chronic infection. Herein, we review the current literature regarding immune suppression in CF by gMDSCs with an emphasis on T cell impairment and the role of P. aeruginosa in this dynamic interaction.

PI3K/ NF-κB-dependent TNF-α and HDAC activities facilitate LPS-induced RGS10 suppression in pulmonary macrophages.

Regulator of G-protein signaling 10 (RGS10) is a member of the superfamily of RGS proteins that canonically act as GTPase activating proteins (GAPs). RGS proteins accelerate GTP hydrolysis on the G-protein α subunits and result in termination of signaling pathways downstream of G protein-coupled receptors. Beyond its GAP function, RGS10 has emerged as an anti-inflammatory protein by inhibiting LPS-mediated NF-κB activation and expression of inflammatory cytokines, in particular TNF-α. Although RGS10 is abundantly expressed in resting macrophages, previous studies have shown that RGS10 expression is suppressed in macrophages following Toll-like receptor 4 (TLR4) activation by LPS. However, the molecular mechanism by which LPS induces Rgs10 silencing has not been clearly defined. The goal of the current study was to determine whether LPS silences Rgs10 expression through an NF-κB-mediated proinflammatory mechanism in pulmonary macrophages, a unique type of innate immune cells. We demonstrate that Rgs10 transcript and RGS10 protein levels are suppressed upon LPS treatment in the murine MH-S alveolar macrophage cell line. We show that pharmacological inhibition of PI3K/ NF-κB/p300 (NF-κB co-activator)/TNF-α signaling cascade and the activities of HDAC (1-3) enzymes block LPS-induced silencing of Rgs10 in MH-S cells as well as microglial BV2 cells and BMDMs. Further, loss of RGS10 generated by using CRISPR/Cas9 amplifies NF-κB phosphorylation and inflammatory gene expression following LPS treatment in MH-S cells. Together, our findings strongly provide critical insight into the molecular mechanism underlying RGS10 suppression by LPS in pulmonary macrophages.

Dual oxidase 1 promotes antiviral innate immunity.

Dual oxidase 1 (DUOX1) is an NADPH oxidase that is highly expre-ssed in respiratory epithelial cells and produces H2O2 in the airway lumen. While a line of prior in vitro observations suggested that DUOX1 works in partnership with an airway peroxidase, lactoperoxidase (LPO), to produce antimicrobial hypothiocyanite (OSCN-) in the airways, the in vivo role of DUOX1 in mammalian organisms has remained unproven to date. Here, we show that Duox1 promotes antiviral innate immunity in vivo. Upon influenza airway challenge, Duox1-/- mice have enhanced mortality, morbidity, and impaired lung viral clearance. Duox1 increases the airway levels of several cytokines (IL-1ß, IL-2, CCL1, CCL3, CCL11, CCL19, CCL20, CCL27, CXCL5, and CXCL11), contributes to innate immune cell recruitment, and affects epithelial apoptosis in the airways. In primary human tracheobronchial epithelial cells, OSCN- is generated by LPO using DUOX1-derived H2O2 and inactivates several influenza strains in vitro. We also show that OSCN- diminishes influenza replication and viral RNA synthesis in infected host cells that is inhibited by the H2O2 scavenger catalase. Binding of the influenza virus to host cells and viral entry are both reduced by OSCN- in an H2O2-dependent manner in vitro. OSCN- does not affect the neuraminidase activity or morphology of the influenza virus. Overall, this antiviral function of Duox1 identifies an in vivo role of this gene, defines the steps in the infection cycle targeted by OSCN-, and proposes that boosting this mechanism in vivo can have therapeutic potential in treating viral infections.

DUOX1 in mammalian disease pathophysiology.

Dual oxidase 1 (DUOX1) is a member of the protein family of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. DUOX1 has several normal physiological, immunological, and biochemical functions in different parts of the body. Dysregulated oxidative metabolism interferes with various disease pathologies and numerous therapeutic options are based on targeting cellular redox pathways. DUOX1 forms an important enzymatic source of biological oxidants, and DUOX1 expression is frequently dysregulated in various diseases. While this review shortly addresses the biochemical and cellular properties and proposed physiological roles of DUOX1, its main purpose is to summarize the current knowledge with respect to the potential role of DUOX1 enzyme in disease pathology, especially in mammalian organisms. Although DUOX1 is normally prominently expressed in epithelial lineages, it is frequently silenced in epithelial-derived cancers by epigenetic mechanisms. While an abundance of information is available on DUOX1 transcription in different diseases, an increasing number of mechanistic studies indicate a causative relationship between DUOX1 function and disease pathophysiology. Additionally, specific functions of the DUOX1 maturation factor, DUOXA1, will also be addressed. Lastly, urgent and outstanding questions on the field of DUOX1 will be discussed that could provide valuable new diagnostic tools and novel therapeutic options.

Neutrophil extracellular traps are present in the airways of ENaC-overexpressing mice with cystic fibrosis-like lung disease.

BACKGROUND: Neutrophils are key components of the exacerbated inflammation and tissue damage in cystic fibrosis (CF) airways. Neutrophil extracellular traps (NETs) trap and kill extracellular pathogens. While NETs are abundant in the airways of CF patients and have been hypothesized to contribute to lung damage in CF, the in vivo role of NETs remains controversial, partially due to lack of appropriate animal models. The goal of this study was to detect NETs and to further characterize neutrophil-mediated inflammation in the airways of mice overexpressing the epithelial sodium channel (ßENaC-Tg mice on C57BL/6 background) in their lung with CF-like airway disease, in the absence of any apparent bacterial infections. METHODS: Histology scoring of lung tissues, flow cytometry, multiplex ELISA, immunohistochemistry and immunofluorescence were used to characterize NETs and the airway environment in uninfected, ßENaC-Tg mice at 6 and 8 weeks of age, the most chronic time points so far studied in this model. RESULTS: Excessive neutrophilic infiltration characterized the lungs of uninfected, ßENaC-Tg mice at 6 and 8 weeks of age. The bronchoalveolar lavage fluid (BALF) of ßENaC-Tg mice contains increased levels of CF-associated cytokines and chemokines: KC, MIP-1α/ß, MCP-1, G-CSF, IL-5, and IL-6. The BALF of ßENaC-Tg mice contain MPO-DNA complexes, indicative of the presence of NETs. Immunofluorescence and flow cytometry of BALF neutrophils and lung tissues demonstrated increased histone citrullination, a NET-specific marker, in ßENaC-Tg mice. CONCLUSIONS: NETs are detected in the airways of ßENaC-Tg mice, in the absence of bacterial infections. These data demonstrate the usefulness of the ßENaC-Tg mouse to serve as a model for studying the role of NETs in chronic CF airway inflammation.

Chronic infection during placental malaria is associated with up-regulation of cycloxygenase-2.

BACKGROUND: Placental malaria (PM) is associated with poor foetal development, but the pathophysiological processes involved are poorly understood. Cyclooxygenase (COX) and lipoxygenase (LOX) which convert fatty acids to prostaglandins and leukotrienes, play important roles in pregnancy and foetal development. COX-2, currently targeted by specific drugs, plays a dual role as it associates with both pre-eclampsia pathology and recovery during infection. The role of COX during PM was questioned by quantifying at delivery COX-1, COX-2, 15-LOX, and IL-10 expression in two groups of malaria infected and uninfected placenta. METHODS: Placental biopsies were collected at delivery for mRNA isolation and quantification, using real time PCR. RESULTS: COX-2 and IL-10 mRNAs increased mainly during chronic infections (nine- and five-times, respectively), whereas COX-1 transcripts remained constant. COX-2 over-expression was associated with a higher birth weight of the baby, but with a lower rate of haemoglobin of the mother. It was associated with a macrophage infiltration of the placenta and with a low haemozoin infiltration. In the opposite way, placental infection was associated with lower expression of 15-LOX mRNA. A high degree of haemozoin deposition correlates with low birth weight and decreased expression of COX-2. CONCLUSION: These data provide evidence that COX-2 and IL-10 are highly induced during chronic infection of the placenta, but were not associated with preterm delivery or low birth weight. The data support the involvement of COX-2 in the recovery phase of the placental infection.

Malaria-induced murine pregnancy failure: distinct roles for IFN-gamma and TNF.

Although an important role for excessive proinflammatory cytokines in compromise of pregnancy has been established, an immunological basis for malaria-induced fetal loss remains to be demonstrated. In this study, the roles of IFN-gamma and TNF in Plasmodium chabaudi AS-induced fetal loss in mice were directly investigated. Pregnant IFN-gamma(-/-) mice experienced a more severe course of infection compared with intact C57BL/6 mice, characterized by high parasitemia, severe anemia, and marked weight loss. However, fetal loss was delayed in these mice relative to intact controls. Because IFN-gamma(-/-) mice exhibited sustained levels of plasma TNF, the role of this cytokine was examined. Whereas splenic tnf expression in C57BL/6 mice was highest 3 days before peak parasitemia, increased placental expression relative to uninfected mice was sustained, indicating that locally produced TNF may be important in malaria-induced pregnancy failure. Indeed, Ab neutralization of TNF resulted in preservation of embryos until day 12 of gestation, at which point all embryos were lost in untreated mice. Histological analysis revealed that TNF ablation preserved placental architecture whereas placentae from untreated infected mice had widespread hemorrhage and placental disruption, with fibrin thrombi in some maternal blood sinusoids. Consistent with a role for cytokine-driven thrombosis in fetal loss, expression of procoagulant tissue factor was significantly increased in the placentae of infected C57BL/6 mice but was reduced in mice treated with anti-TNF Ab. Together, these results suggest that IFN-gamma contributes to malaria-induced fetal loss and TNF is a critical factor that acts by inducing placental coagulopathy.

Accumulation of CVIET Pfcrt allele of Plasmodium falciparum in placenta of pregnant women living in an urban area of Dakar, Senegal.

OBJECTIVES: Chemoprophylaxis is recommended during pregnancy to reduce the risk of placental infection. However, in areas with increasing drug resistance, it can trigger selection of resistant parasites in the placenta and increase the frequency of placental malaria. The objective of this study was to analyse the selection of drug-resistant parasites in the placenta in an area where chloroquine was still recommended as prophylaxis. PATIENTS AND METHODS: We analysed the polymorphism of parasites from matched placental and venous blood samples at the time of delivery from women in Dakar. Polymorphism of the isolates was studied using nested PCR typing of MSA1 and MSA2 genes, and full sequence of PfCRT exon 2. RESULTS: Of 692 women recruited at delivery, 72 had placental malaria. Two Pfcrt exon 2 genotypes were found, and 86% of the placentas had monoallelelic CVIET infection compared with 39% that had peripheral blood infection. Mixed parasite populations of CVIET/CVMNK occurred in 53% of the peripheral blood samples but only in 7% of the infected placentas. This selection of CVIET in placenta was not related to a decreased polymorphism of the parasites, as a large diversity of MSA1 and MSA2 was found in both placenta and venous blood. This diversity confirms that a multiplicity of circulation isolates can occur at low parasite transmission. msp1 and msp2 genotyping revealed mostly distinct populations of parasites in venous and placental blood. CONCLUSIONS: These data suggest that, even in low transmission areas, diverse parasite populations can accumulate in the placenta during pregnancy despite strong selection at the PfCRT locus due to chemoprophylaxis with chloroquine.