Cystic fibrosis macrophage function and clinical outcomes after elexacaftor/tezacaftor/ivacaftor.

BACKGROUND: Abnormal macrophage function caused by dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) is a critical contributor to chronic airway infections and inflammation in people with cystic fibrosis (PWCF). Elexacaftor/tezacaftor/ivacaftor (ETI) is a new CFTR modulator therapy for PWCF. Host-pathogen and clinical responses to CFTR modulators are poorly described. We sought to determine how ETI impacts macrophage CFTR function, resulting effector functions and relationships to clinical outcome changes. METHODS: Clinical information and/or biospecimens were obtained at ETI initiation and 3, 6, 9 and 12 months post-ETI in 56 PWCF and compared with non-CF controls. Peripheral blood monocyte-derived macrophages (MDMs) were isolated and functional assays performed. RESULTS: ETI treatment was associated with increased CF MDM CFTR expression, function and localisation to the plasma membrane. CF MDM phagocytosis, intracellular killing of CF pathogens and efferocytosis of apoptotic neutrophils were partially restored by ETI, but inflammatory cytokine production remained unchanged. Clinical outcomes including increased forced expiratory volume in 1 s (+10%) and body mass index (+1.0 kg·m-2) showed fluctuations over time and were highly individualised. Significant correlations between post-ETI MDM CFTR function and sweat chloride levels were observed. However, MDM CFTR function correlated with clinical outcomes better than sweat chloride. CONCLUSION: ETI is associated with unique changes in innate immune function and clinical outcomes.

Disruption of Nurse-like Cell Differentiation as a Therapeutic Strategy for Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia, but, despite advances in treatment, many patients still experience relapse. CLL cells depend on interactions with supportive cells, and nurse-like cells (NLCs) are the major such cell type. However, little is known about how NLCs develop. Here, we performed DNA methylation analysis of CLL patient-derived NLCs using the 850K Illumina array, comparing CD14+ cells at day 1 (monocytes) versus day 14 (NLCs). We found a strong loss of methylation in AP-1 transcription factor binding sites, which may be driven by MAPK signaling. Testing of individual MAPK pathways (MEK, p38, and JNK) revealed a strong dependence on MEK/ERK for NLC development, because treatment of patient samples with the MEK inhibitor trametinib dramatically reduced NLC development in vitro. Using the adoptive transfer Eµ-TCL1 mouse model of CLL, we found that MEK inhibition slowed CLL progression, leading to lower WBC counts and to significantly longer survival time. There were also lower numbers of mouse macrophages, particularly within the M2-like population. In summary, NLC development depends on MEK signaling, and inhibition of MEK leads to increased survival time in vivo. Hence, targeting the MEK/ERK pathway may be an effective treatment strategy for CLL.

Caspase-4/11 exacerbates disease severity in SARS-CoV-2 infection by promoting inflammation and immunothrombosis.

Severe acute respiratory syndrome coronavirus 2 (SARS­CoV-2) is a worldwide health concern, and new treatment strategies are needed. Targeting inflammatory innate immunity pathways holds therapeutic promise, but effective molecular targets remain elusive. Here, we show that human caspase-4 (CASP4) and its mouse homolog, caspase-11 (CASP11), are up-regulated in SARS­CoV-2 infections and that CASP4 expression correlates with severity of SARS­CoV-2 infection in humans. SARS­CoV-2­infected Casp11−/− mice were protected from severe weight loss and lung pathology, including blood vessel damage, compared to wild-type (WT) mice and mice lacking the caspase downstream effector gasdermin-D (Gsdmd−/−). Notably, viral titers were similar regardless of CASP11 knockout. Global transcriptomics of SARS­CoV-2­infected WT, Casp11−/−, and Gsdmd−/− lungs identified restrained expression of inflammatory molecules and altered neutrophil gene signatures in Casp11−/− mice. We confirmed that protein levels of inflammatory mediators interleukin (IL)-1ß, IL-6, and CXCL1, as well as neutrophil functions, were reduced in Casp11−/− lungs. Additionally, Casp11−/− lungs accumulated less von Willebrand factor, a marker for endothelial damage, but expressed more Kruppel-Like Factor 2, a transcription factor that maintains vascular integrity. Overall, our results demonstrate that CASP4/11 promotes detrimental SARS­CoV-2­induced inflammation and coagulopathy, largely independently of GSDMD, identifying CASP4/11 as a promising drug target for treatment and prevention of severe COVID-19.

CFTR Modulators Restore Acidification of Autophago-Lysosomes and Bacterial Clearance in Cystic Fibrosis Macrophages.

Cystic fibrosis (CF) human and mouse macrophages are defective in their ability to clear bacteria such as Burkholderia cenocepacia. The autophagy process in CF (F508del) macrophages is halted, and the underlying mechanism remains unclear. Furthermore, the role of CFTR in maintaining the acidification of endosomal and lysosomal compartments in CF cells has been a subject of debate. Using 3D reconstruction of z-stack confocal images, we show that CFTR is recruited to LC3-labeled autophagosomes harboring B. cenocepacia. Using several complementary approaches, we report that CF macrophages display defective lysosomal acidification and degradative function for cargos destined to autophagosomes, whereas non-autophagosomal cargos are effectively degraded within acidic compartments. Notably, treatment of CF macrophages with CFTR modulators (tezacaftor/ivacaftor) improved the autophagy flux, lysosomal acidification and function, and bacterial clearance. In addition, CFTR modulators improved CFTR function as demonstrated by patch-clamp. In conclusion, CFTR regulates the acidification of a specific subset of lysosomes that specifically fuse with autophagosomes. Therefore, our study describes a new biological location and function for CFTR in autophago-lysosomes and clarifies the long-standing discrepancies in the field.

Z-form extracellular DNA is a structural component of the bacterial biofilm matrix.

Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental stresses and, in many species, incorporates extracellular DNA (eDNA) and DNABII proteins for structural integrity throughout biofilm development. Here, we present evidence that this eDNA-based architecture relies on the rare Z-form. Z-form DNA accumulates as biofilms mature and, through stabilization by the DNABII proteins, confers structural integrity to the biofilm matrix. Indeed, substances known to drive B-DNA into Z-DNA promoted biofilm formation whereas those that drive Z-DNA into B-DNA disrupted extant biofilms. Importantly, we demonstrated that the universal bacterial DNABII family of proteins stabilizes both bacterial- and host-eDNA in the Z-form in situ. A model is proposed that incorporates the role of Z-DNA in biofilm pathogenesis, innate immune response, and immune evasion.

The extracellular innate-immune effector HMGB1 limits pathogenic bacterial biofilm proliferation.

Herein, we describe an extracellular function of the vertebrate high-mobility group box 1 protein (HMGB1) in the proliferation of bacterial biofilms. Within host cells, HMGB1 functions as a DNA architectural protein, similar to the ubiquitous DNABII family of bacterial proteins; despite that, these proteins share no amino acid sequence identity. Extracellularly, HMGB1 induces a proinflammatory immune response, whereas the DNABII proteins stabilize the extracellular DNA-dependent matrix that maintains bacterial biofilms. We showed that when both proteins converged on extracellular DNA within bacterial biofilms, HMGB1, unlike the DNABII proteins, disrupted biofilms both in vitro (including the high-priority ESKAPEE pathogens) and in vivo in 2 distinct animal models, albeit with induction of a strong inflammatory response that we attenuated by a single engineered amino acid change. We propose a model where extracellular HMGB1 balances the degree of induced inflammation and biofilm containment without excessive release of biofilm-resident bacteria.

Antibody-Mediated Protection against Staphylococcus aureus Dermonecrosis: Synergy of Toxin Neutralization and Neutrophil Recruitment.

The staphylococcal α-hemolysin is critical for the pathogenesis of Staphylococcus aureus skin and soft tissue infection. Vaccine and infection-elicited α-hemolysin-specific antibodies protect against S. aureus‒induced dermonecrosis, a key feature of skin and soft tissue infection. Many interactions between α-hemolysin and host cells have been identified that promote tissue damage and modulate immune responses, but the mechanisms by which protective adaptive responses cross talk with innate responses at the tissue level are not clear. Using an established mouse model of skin and soft tissue infection and a newly developed histopathologic scoring system, we observed pathologic correlates early after infection, predicting protection against dermonecrosis by anti-α-hemolysin antibody. Protection was characterized by robust neutrophilic inflammation and compartmentalization of bacteria into discrete abscesses, which led to the attenuation of dermonecrosis and enhancement of bacterial clearance later in the infection. The ultimate outcome of infection was driven by the recruitment of neutrophils within the first day after infection but not later. Antibody-mediated protection was dependent on toxin neutralization rather than on enhanced opsonophagocytic killing by neutrophils or protection against toxin-mediated neutrophil lysis. Together, these findings advance our understanding of the mechanisms by which the early synergism between antibody-mediated toxin neutralization and tissue-specific neutrophilic inflammation preserve tissue integrity during infection.

Neutrophil-Macrophage Imbalance Drives the Development of Renal Scarring during Experimental Pyelonephritis.

BACKGROUND: In children, the acute pyelonephritis that can result from urinary tract infections (UTIs), which commonly ascend from the bladder to the kidney, is a growing concern because it poses a risk of renal scarring and irreversible loss of kidney function. To date, the cellular mechanisms underlying acute pyelonephritis-driven renal scarring remain unknown. METHODS: We used a preclinical model of uropathogenic Escherichia coli-induced acute pyelonephritis to determine the contribution of neutrophils and monocytes to resolution of the condition and the subsequent development of kidney fibrosis. We used cell-specific monoclonal antibodies to eliminate neutrophils, monocytes, or both. Bacterial ascent and the cell dynamics of phagocytic cells were assessed by biophotonic imaging and flow cytometry, respectively. We used quantitative RT-PCR and histopathologic analyses to evaluate inflammation and renal scarring. RESULTS: We found that neutrophils are critical to control bacterial ascent, which is in line with previous studies suggesting a protective role for neutrophils during a UTI, whereas monocyte-derived macrophages orchestrate a strong, but ineffective, inflammatory response against uropathogenic, E. coli-induced, acute pyelonephritis. Experimental neutropenia during acute pyelonephritis resulted in a compensatory increase in the number of monocytes and heightened macrophage-dependent inflammation in the kidney. Exacerbated macrophage-mediated inflammatory responses promoted renal scarring and compromised renal function, as indicated by elevated serum creatinine, BUN, and potassium. CONCLUSIONS: These findings reveal a previously unappreciated outcome for neutrophil-macrophage imbalance in promoting host susceptibility to acute pyelonephritis and the development of permanent renal damage. This suggests targeting dysregulated macrophage responses might be a therapeutic tool to prevent renal scarring during acute pyelonephritis.

A Bacterial Epigenetic Switch in Non-typeable Haemophilus influenzae Modifies Host Immune Response During Otitis Media.

Non-typeable Haemophilus influenzae (NTHi) causes multiple diseases of the human airway and is a predominant bacterial pathogen of acute otitis media and otitis media in which treatment fails. NTHi utilizes a system of phase variable epigenetic regulation, termed the phasevarion, to facilitate adaptation and survival within multiple sites of the human host. The NTHi phasevarion influences numerous disease-relevant phenotypes such as biofilm formation, antibiotic resistance, and opsonization. We have previously identified an advantageous selection for a specific phasevarion status, which significantly affects severity and chronicity of experimental otitis media. In this study, we utilized pure cultures of NTHi variants in which modA was either locked ON or locked OFF, and thus modA was unable to phase vary. These locked variants were used to assess the progression of experimental otitis media and define the specific immune response induced by each subpopulation. Although the initial disease caused by each subpopulation was similar, the immune response elicited by each subpopulation was unique. The modA2 OFF variant induced significantly greater activation of macrophages both in vitro and within the middle ear during disease. In contrast, the modA2 ON variant induced a greater neutrophil extracellular trap response, which led to greater killing of the modA2 ON variant. These data suggest that not only does the NTHi phasevarion facilitate adaptation, but also allows the bacteria to alter immune responses during disease. Understanding these complex bacterial-host interactions and the regulation of bacterial factors responsible is critical to the development of better diagnostic, treatment, and preventative strategies for these bacterial pathogens.

The TRPM2 Ion Channel Regulates Inflammatory Functions of Neutrophils During Listeria monocytogenes Infection.

During infection, phagocytic cells pursue homeostasis in the host via multiple mechanisms that control microbial invasion. Neutrophils respond to infection by exerting a variety of cellular processes, including chemotaxis, activation, phagocytosis, degranulation and the generation of reactive oxygen species (ROS). Calcium (Ca2+) signaling and the activation of specific Ca2+ channels are required for most antimicrobial effector functions of neutrophils. The transient receptor potential melastatin-2 (TRPM2) cation channel has been proposed to play important roles in modulating Ca2+ mobilization and oxidative stress in neutrophils. In the present study, we use a mouse model of Listeria monocytogenes infection to define the role of TRPM2 in the regulation of neutrophils' functions during infection. We show that the susceptibility of Trpm2-/- mice to L. monocytogenes infection is characterized by increased migration rates of neutrophils and monocytes to the liver and spleen in the first 24 h. During the acute phase of L. monocytogenes infection, Trpm2-/- mice developed septic shock, characterized by increased serum levels of TNF-α, IL-6, and IL-10. Furthermore, in vivo depletion of neutrophils demonstrated a critical role of these immune cells in regulating acute inflammation in Trpm2-/- infected mice. Gene expression and inflammatory cytokine analyses of infected tissues further confirmed the hyperinflammatory profile of Trpm2-/- neutrophils. Finally, the increased inflammatory properties of Trpm2-/- neutrophils correlated with the dysregulated cytoplasmic concentration of Ca2+ and potentiated membrane depolarization, in response to L. monocytogenes. In conclusion, our findings suggest that the TRPM2 channel plays critical functional roles in regulating the inflammatory properties of neutrophils and preventing tissue damage during Listeria infection.

Whole-blood transcriptomic responses to lumacaftor/ivacaftor therapy in cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) remains without a definitive cure. Novel therapeutics targeting the causative defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are in clinical use. Lumacaftor/ivacaftor is a CFTR modulator approved for patients homozygous for the CFTR variant p.Phe508del, but there are wide variations in treatment responses preventing prediction of patient responses. We aimed to determine changes in gene expression related to treatment initiation and response. METHODS: Whole-blood transcriptomics was performed using RNA-Seq in 20 patients with CF pre- and 6 months post-lumacaftor/ivacaftor (drug) initiation and 20 non-CF healthy controls. Correlation of gene expression with clinical variables was performed by stratification via clinical responses. RESULTS: We identified 491 genes that were differentially expressed in CF patients (pre-drug) compared with non-CF controls and 36 genes when comparing pre-drug to post-drug profiles. Both pre- and post-drug CF profiles were associated with marked overexpression of inflammation-related genes and apoptosis genes, and significant under-expression of T cell and NK cell-related genes compared to non-CF. CF patients post-drug demonstrated normalized protein synthesis expression, and decreased expression of cell-death genes compared to pre-drug profiles, irrespective of clinical response. However, CF clinical responders demonstrated changes in eIF2 signaling, oxidative phosphorylation, IL-17 signaling, and mitochondrial function compared to non-responders. Top overexpressed genes (MMP9 and SOCS3) that decreased post-drug were validated by qRT-PCR. Functional assays demonstrated that CF monocytes normalized calcium (increases MMP9 expression) concentrations post-drug. CONCLUSIONS: Transcriptomics revealed differentially regulated pathways in CF patients at baseline compared to non-CF, and in clinical responders to lumacaftor/ivacaftor.

Caspase-11 Mediates Neutrophil Chemotaxis and Extracellular Trap Formation During Acute Gouty Arthritis Through Alteration of Cofilin Phosphorylation.

Gout is characterized by attacks of arthritis with hyperuricemia and monosodium urate (MSU) crystal-induced inflammation within joints. Innate immune responses are the primary drivers for tissue destruction and inflammation in gout. MSU crystals engage the Nlrp3 inflammasome, leading to the activation of caspase-1 and production of IL-1ß and IL-18 within gout-affected joints, promoting the influx of neutrophils and monocytes. Here, we show that caspase-11-/- mice and their derived macrophages produce significantly reduced levels of gout-specific cytokines including IL-1ß, TNFα, IL-6, and KC, while others like IFNγ and IL-12p70 are not altered. IL-1ß induces the expression of caspase-11 in an IL-1 receptor-dependent manner in macrophages contributing to the priming of macrophages during sterile inflammation. The absence of caspase-11 reduced the ability of macrophages and neutrophils to migrate in response to exogenously injected KC in vivo. Notably, in vitro, caspase-11-/- neutrophils displayed random migration in response to a KC gradient when compared to their WT counterparts. This phenotype was associated with altered cofilin phosphorylation. Unlike their wild-type counterparts, caspase-11-/- neutrophils also failed to produce neutrophil extracellular traps (NETs) when treated with MSU. Together, this is the first report demonstrating that caspase-11 promotes neutrophil directional trafficking and function in an acute model of gout. Caspase-11 also governs the production of inflammasome-dependent and -independent cytokines from macrophages. Our results offer new, previously unrecognized functions for caspase-11 in macrophages and neutrophils that may apply to other neutrophil-mediated disease conditions besides gout.

CD31 Acts as a Checkpoint Molecule and Is Modulated by FcγR-Mediated Signaling in Monocytes.

Monocytes and macrophages express FcγR that engage IgG immune complexes such as Ab-opsonized pathogens or cancer cells to destroy them by various mechanisms, including phagocytosis. FcγR-mediated phagocytosis is regulated by the concerted actions of activating FcγR and inhibitory receptors, such as FcγRIIb and SIRPα. In this study, we report that another ITIM-containing receptor, PECAM1/CD31, regulates FcγR function and is itself regulated by FcγR activation. First, quantitative RT-PCR and flow cytometry analyses revealed that human monocyte FcγR activation leads to a significant downregulation of CD31 expression, both at the message level and at surface expression, mainly mediated through FcγRIIa. Interestingly, the kinetics of downregulation between the two varied, with surface expression reducing earlier than the message. Experiments to analyze the mechanism behind this discrepancy revealed that the loss of surface expression was because of internalization, which depended predominantly on the PI3 kinase pathway and was independent of FcγR internalization. Finally, functional analyses showed that the downregulation of CD31 expression in monocytes by small interfering RNA enhanced FcγR-mediated phagocytic ability but have little effect on cytokine production. Together, these results suggest that CD31 acts as a checkpoint receptor that could be targeted to enhance FcγR functions in Ab-mediated therapies.

AR-13 reduces antibiotic-resistant bacterial burden in cystic fibrosis phagocytes and improves cystic fibrosis transmembrane conductance regulator function.

BACKGROUND: There are no effective treatments for Burkholderia cenocepacia in patients with cystic fibrosis (CF) due to bacterial multi-drug resistance and defective host killing. We demonstrated that decreased bacterial killing in CF is caused by reduced macrophage autophagy due to defective cystic fibrosis transmembrane conductance regulator (CFTR) function. AR-12 is a small molecule autophagy inducer that kills intracellular pathogens such as Francisella. We evaluated the efficacy of AR-12 and a new analogue AR-13 in reducing bacterial burden in CF phagocytes. METHODS: Human CF and non-CF peripheral blood monocyte-derived macrophages, neutrophils, and nasal epithelial cells were exposed to CF bacterial strains in conjunction with treatment with antibiotics and/or AR compounds. RESULTS: AR-13 and not AR-12 had growth inhibition on B. cenocepacia and methicillin-resistantStaphylococcus aureus (MRSA) in media alone. There was a 99% reduction in MRSA in CF macrophages, 71% reduction in Pseudomonas aeruginosa in CF neutrophils, and 70% reduction in non-CF neutrophils using AR-13. Conversely, there was no reduction in B. cenocepacia in infected CF and non-CF macrophages using AR-13 alone, but AR-13 and antibiotics synergistically reduced B. cenocepacia in CF macrophages. AR-13 improved autophagy in CF macrophages and CF patient-derived epithelial cells, and increased CFTR protein expression and channel function in CF epithelial cells. CONCLUSIONS: The novel AR-12 analogue AR-13, in combination with antibiotics, reduced antibiotic-resistant bacterial burden in CF phagocytes, which correlated with increased autophagy and CFTR expression. AR-13 is a novel therapeutic for patients infected with B. cenocepacia and other resistant organisms that lack effective therapies.

Dysregulated Calcium Homeostasis in Cystic Fibrosis Neutrophils Leads to Deficient Antimicrobial Responses.

Cystic fibrosis (CF), one of the most common human genetic diseases worldwide, is caused by a defect in the CF transmembrane conductance regulator (CFTR). Patients with CF are highly susceptible to infections caused by opportunistic pathogens (including Burkholderia cenocepacia), which induce excessive lung inflammation and lead to the eventual loss of pulmonary function. Abundant neutrophil recruitment into the lung is a key characteristic of bacterial infections in CF patients. In response to infection, inflammatory neutrophils release reactive oxygen species and toxic proteins, leading to aggravated lung tissue damage in patients with CF. The present study shows a defect in reactive oxygen species production by mouse Cftr-/- , human F508del-CFTR, and CF neutrophils; this results in reduced antimicrobial activity against B. cenocepacia Furthermore, dysregulated Ca2+ homeostasis led to increased intracellular concentrations of Ca2+ that correlated with significantly diminished NADPH oxidase response and impaired secretion of neutrophil extracellular traps in human CF neutrophils. Functionally deficient human CF neutrophils recovered their antimicrobial killing capacity following treatment with pharmacological inhibitors of Ca2+ channels and CFTR channel potentiators. Our findings suggest that regulation of neutrophil Ca2+ homeostasis (via CFTR potentiation or by the regulation of Ca2+ channels) can be used as a new therapeutic approach for reestablishing immune function in patients with CF.

Angiotensin II receptor I blockade prevents stenosis of tissue engineered vascular grafts.

We previously developed a tissue-engineered vascular graft (TEVG) made by seeding autologous cells onto a biodegradable tubular scaffold, in an attempt to create a living vascular graft with growth potential for use in children undergoing congenital heart surgery. Results of our clinical trial showed that the TEVG possesses growth capacity but that its widespread clinical use is not yet advisable due to the high incidence of TEVG stenosis. In animal models, TEVG stenosis is caused by increased monocytic cell recruitment and its classic ("M1") activation. Here, we report on the source and regulation of these monocytes. TEVGs were implanted in wild-type, CCR2 knockout ( Ccr2-/-), splenectomized, and spleen graft recipient mice. We found that bone marrow-derived Ly6C+hi monocytes released from sequestration by the spleen are the source of mononuclear cells infiltrating the TEVG during the acute phase of neovessel formation. Furthermore, short-term administration of losartan (0.6 g/L, 2 wk), an angiotensin II type 1 receptor antagonist, significantly reduced the macrophage populations (Ly6C+/-/F480+) in the scaffolds and improved long-term patency in TEVGs. Notably, the combined effect of bone marrow-derived mononuclear cell seeding with short-term losartan treatment completely prevented the development of TEVG stenosis. Our results provide support for pharmacologic treatment with losartan as a strategy to modulate monocyte infiltration into the grafts and thus prevent TEVG stenosis.-Ruiz-Rosado, J. D. D., Lee, Y.-U., Mahler, N., Yi, T., Robledo-Avila, F., Martinez-Saucedo, D., Lee, A. Y., Shoji, T., Heuer, E., Yates, A. R., Pober, J. S., Shinoka, T., Partida-Sanchez, S., Breuer, C. K. Angiotensin II receptor I blockade prevents stenosis of tissue engineered vascular grafts.

The expression of Mirc1/Mir17-92 cluster in sputum samples correlates with pulmonary exacerbations in cystic fibrosis patients.

INTRODUCTION: Cystic fibrosis (CF) is a multi-organ disorder characterized by chronic sino-pulmonary infections and inflammation. Many patients with CF suffer from repeated pulmonary exacerbations that are predictors of worsened long-term morbidity and mortality. There are no reliable markers that associate with the onset or progression of an exacerbation or pulmonary deterioration. Previously, we found that the Mirc1/Mir17-92a cluster which is comprised of 6 microRNAs (Mirs) is highly expressed in CF mice and negatively regulates autophagy which in turn improves CF transmembrane conductance regulator (CFTR) function. Therefore, here we sought to examine the expression of individual Mirs within the Mirc1/Mir17-92 cluster in human cells and biological fluids and determine their role as biomarkers of pulmonary exacerbations and response to treatment. METHODS: Mirc1/Mir17-92 cluster expression was measured in human CF and non-CF plasma, blood-derived neutrophils, and sputum samples. Values were correlated with pulmonary function, exacerbations and use of CFTR modulators. RESULTS: Mirc1/Mir17-92 cluster expression was not significantly elevated in CF neutrophils nor plasma when compared to the non-CF cohort. Cluster expression in CF sputum was significantly higher than its expression in plasma. Elevated CF sputum Mirc1/Mir17-92 cluster expression positively correlated with pulmonary exacerbations and negatively correlated with lung function. Patients with CF undergoing treatment with the CFTR modulator Ivacaftor/Lumacaftor did not demonstrate significant change in the expression Mirc1/Mir17-92 cluster after six months of treatment. CONCLUSIONS: Mirc1/Mir17-92 cluster expression is a promising biomarker of respiratory status in patients with CF including pulmonary exacerbation.

The ModA2 Phasevarion of nontypeable Haemophilus influenzae Regulates Resistance to Oxidative Stress and Killing by Human Neutrophils.

Nontypeable Haemophilus influenzae (NTHI) is the causative agent of multiple respiratory tract infections. Several human pathogens, including NTHI, possess a novel genetic system, termed the phasevarion, which mediates a rapid and reversible change in the expression of many genes throughout the chromosome. This occurs by phase variation of a single gene (modA) that encodes a DNA methyltransferase and results in two phenotypically distinct subpopulations, ON and OFF. NTHI encounters many pressures within the various microenvironments of its human host as the disease course evolves from one of asymptomatic nasopharyngeal carriage to overt disease. These include oxidative stresses, which are present throughout the respiratory tract. To persist in the human nasopharynx and as a pathogen throughout the airways, NTHI must be able to mitigate toxic levels of oxidative stress. Here we show that expression of ModA2, modA2 ON status, resulted in increased sensitivity to oxidative stress. Furthermore, the modA2 ON status resulted in decreased resistance to neutrophil-mediated killing, which resulted in selection for the modA2 OFF subpopulation in an ex vivo survival assay. These findings highlight the importance of the ModA2 phasevarion in adaptation to innate host defences and reveal an additional microenvironmental pressure that selected for a specific ModA2 subpopulation.

Human Cystic Fibrosis Macrophages Have Defective Calcium-Dependent Protein Kinase C Activation of the NADPH Oxidase, an Effect Augmented by Burkholderia cenocepacia.

Macrophage intracellular pathogen killing is defective in cystic fibrosis (CF), despite abundant production of reactive oxygen species (ROS) in lung tissue. Burkholderia species can cause serious infection in CF and themselves affect key oxidase components in murine non-CF cells. However, it is unknown whether human CF macrophages have an independent defect in the oxidative burst and whether Burkholderia contributes to this defect in terms of assembly of the NADPH oxidase complex and subsequent ROS production. In this article, we analyze CF and non-CF human monocyte-derived macrophages (MDMs) for ROS production, NADPH assembly capacity, protein kinase C expression, and calcium release in response to PMA and CF pathogens. CF MDMs demonstrate a nearly 60% reduction in superoxide production after PMA stimulation compared with non-CF MDMs. Although CF MDMs generally have increased total NADPH component protein expression, they demonstrate decreased expression of the calcium-dependent protein kinase C conventional subclass α/ß leading to reduced phosphorylation of NADPH oxidase components p47 phox and p40 phox in comparison with non-CF MDMs. Ingestion of B. cenocepacia independently contributes to and worsens the overall oxidative burst deficits in CF MDMs compared with non-CF MDMs. Together, these results provide evidence for inherent deficits in the CF macrophage oxidative burst caused by decreased phosphorylation of NADPH oxidase cytosolic components that are augmented by Burkholderia These findings implicate a critical role for defective macrophage oxidative responses in persistent bacterial infections in CF and create new opportunities for boosting the macrophage immune response to limit infection.

Helminth-induced Ly6Chi monocyte-derived alternatively activated macrophages suppress experimental autoimmune encephalomyelitis.

Helminths cause chronic infections and affect the immune response to unrelated inflammatory diseases. Although helminths have been used therapeutically to ameliorate inflammatory conditions, their anti-inflammatory properties are poorly understood. Alternatively activated macrophages (AAMϕs) have been suggested as the anti-inflammatory effector cells during helminth infections. Here, we define the origin of AAMϕs during infection with Taenia crassiceps, and their disease-modulating activity on the Experimental Autoimmune Encephalomyelitis (EAE). Our data show two distinct populations of AAMϕs, based on the expression of PD-L1 and PD-L2 molecules, resulting upon T. crassiceps infection. Adoptive transfer of Ly6C+ monocytes gave rise to PD-L1+/PD-L2+, but not PD-L1+/PD-L2- cells in T. crassiceps-infected mice, demonstrating that the PD-L1+/PD-L2+ subpopulation of AAMϕs originates from blood monocytes. Furthermore, adoptive transfer of PD-L1+/PD-L2+ AAMϕs into EAE induced mice reduced disease incidence, delayed disease onset, and diminished the clinical disability, indicating the critical role of these cells in the regulation of autoimmune disorders.