Extracellular Vesicles from Pseudomonas aeruginosa Suppress MHC-Related Molecules in Human Lung Macrophages

Pseudomonas aeruginosa, a Gram-negative bacterium, is one of the most common pathogens colonizing the lungs of cystic fibrosis patients. P. aeruginosa secrete extracellular vesicles (EVs) that contain LPS and other virulence factors that modulate the host\u27s innate immune response, leading to an increased local proinflammatory response and reduced pathogen clearance, resulting in chronic infection and ultimately poor patient outcomes. Lung macrophages are the first line of defense in the airway innate immune response to pathogens. Proper host response to bacterial infection requires communication between APC and T cells, ultimately leading to pathogen clearance. In this study, we investigate whether EVs secreted from P. aeruginosa alter MHC Ag expression in lung macrophages, thereby potentially contributing to decreased pathogen clearance. Primary lung macrophages from human subjects were collected via bronchoalveolar lavage and exposed to EVs isolated from P. aeruginosa in vitro. Gene expression was measured with the NanoString nCounter gene expression assay. DNA methylation was measured with the EPIC array platform to assess changes in methylation. P. aeruginosa EVs suppress the expression of 11 different MHC-associated molecules in lung macrophages. Additionally, we show reduced DNA methylation in a regulatory region of gene complement factor B (CFB) as the possible driving mechanism of widespread MHC gene suppression. Our results demonstrate MHC molecule downregulation by P. aeruginosa-derived EVs in lung macrophages, which is consistent with an immune evasion strategy employed by a prokaryote in a host-pathogen interaction, potentially leading to decreased pulmonary bacterial clearance

<em>Trypanosoma cruzi</em> Coaxes Cardiac Fibroblasts into Preventing Cardiomyocyte Death by Activating Nerve Growth Factor Receptor TrkA

<div><p>Rationale</p><p>Cardiomyocytes express neurotrophin receptor TrkA that promotes survival following nerve growth factor (NGF) ligation. Whether TrkA also resides in cardiac fibroblasts (CFs) and underlies cardioprotection is unknown.</p> <p>Objective</p><p>To test whether CFs express TrkA that conveys paracrine signals to neighbor cardiomyocytes using, as probe, the Chagas disease parasite <i>Trypanosoma cruzi</i>, which expresses a TrkA-binding neurotrophin mimetic, named PDNF. <i>T cruzi</i> targets the heart, causing chronic debilitating cardiomyopathy in ∼30% patients.</p> <p>Methods and Results</p><p>Basal levels of TrkA and TrkC in primary CFs are comparable to those in cardiomyocytes. However, in the myocardium, TrkA expression is significantly lower in fibroblasts than myocytes, and vice versa for TrkC. Yet <i>T cruzi</i> recognition of TrkA on fibroblasts, preferentially over cardiomyocytes, triggers a sharp and sustained increase in NGF, including in the heart of infected mice or of mice administered PDNF intravenously, as early as 3-h post-administration. Further, NGF-containing <i>T cruzi-</i> or PDNF-induced fibroblast-conditioned medium averts cardiomyocyte damage by H₂O₂, in agreement with the previously recognized cardioprotective role of NGF.</p> <p>Conclusions</p><p>TrkA residing in CFs induces an exuberant NGF production in response to <i>T cruzi</i> infection, enabling, in a paracrine fashion, myocytes to resist oxidative stress, a leading Chagas cardiomyopathy trigger. Thus, PDNF-TrkA interaction on CFs may be a mechanism orchestrated by <i>T cruzi</i> to protect its heart habitat, in concert with the long-term (decades) asymptomatic heart parasitism that characterizes Chagas disease. Moreover, as a potent booster of cardioprotective NGF <i>in vivo</i>, PDNF may offer a novel therapeutic opportunity against cardiomyopathies.</p> </div

T cruzi infection upregulates NGF preferentially in primary cultures of cardiac fibroblasts compared to cardiomyocytes.

<p>A) T cruzi selectively upregulates NGF in cardiac fibroblasts. Primary cardiac fibroblasts (CFs) and cardiomyocytes (1°-CMs), and the cardiomyocyte cell line H9c2 were infected with T cruzi trypomastigotes (2×10⁵ per ml), and, at the indicated time points, their NGF mRNA was measured by qPCR (left panel). Similarly, culture supernatants were harvested and their NGF protein levels quantified by ELISA (right panel). Data are representative of three identical experiments; **, p<0.01, ***, p<0.001. B) Visualization of the preferential upregulation of NGF in cardiac fibroblast in T cruzi-infected co-culture of cardiac cells. Primary co-cultures (∼95% cardiac fibroblasts and 5% cardiomyocytes) were infected or not with 2×10⁵ T cruzi/mL for 24 h, then fixed. Leftmost panels: cells stained for NGF (red), and nuclei counterstained with DAPI (blue), scale bars = 100 µm. Right panels: high magnification of cells stained for NGF (red), vimentin (green), and MHC (blue), revealing upregulated NGF preferentially localized on cardiac fibroblasts in the T cruzi-infected co-cultures; scale bars = 10 µm.C) Cardiac fibroblasts secrete bioactive NGF in response to T cruzi infection. Primary cardiac fibroblasts were infected with T cruzi for 0 or 72 h and their conditioned media (CoM) were added to PC12 cell monolayers (left and center left panels). Note a robust neurite outgrowth produced by the 72-h CoM which was abolished by preincubating with a sheep antiserum against NGF (α-NGF) but not with normal sheep serum (NSS) (center right and right panels). Statistical significance is demonstrated in the bar-graphs, which represent the mean ± sd of >200 cells/well of triplicate wells, representative of three experiments; **, P<0.01; ***, P<0.001.</p

Safety of research bronchoscopy with BAL in stable adult patients with cystic fibrosis.

Data on adverse events from research bronchoscopy with bronchoalveolar lavage (BAL) in patients with cystic fibrosis (CF) is lacking. As research bronchoscopy with BAL is useful for isolation of immune cells and investigation of CF lung microbiome, we sought to investigate the safety of bronchoscopy in adult patients with CF. Between November 2016 and September 2019, we performed research bronchoscopies on CF subjects (32) and control subjects (82). Control subjects were nonsmokers without respiratory disease. CF subjects had mild or moderate obstructive lung disease (FEV1 > 50% predicted) and no evidence of recent CF pulmonary exacerbation. There was no significant difference in the age or sex of each cohort. Neither group experienced life threatening adverse events. The number of adverse events was similar between CF and control subjects. The most common adverse events were sore throat and cough, which occurred at similar frequencies in control and CF subjects. Fever and headache occurred more frequently in CF subjects. However, the majority of fevers were seen in CF subjects with FEV1 values below 65% predicted. We found that CF subjects had similar adverse event profiles following research bronchoscopy compared to healthy subjects. While CF subjects had a higher rate of fevers, this adverse event occurred with greater frequency in CF subjects with lower FEV1. Our data demonstrate that research bronchoscopy with BAL is safe in CF subjects and that safety profile is improved if bronchoscopies are limited to subjects with an FEV1 > 65% predicted

Intravenous administration of PDNF upregulates nerve growth factor in the heart selectively on cardiac fibroblasts.

<p><b>A</b>) Intravenous sPDNF upregulates NGF transcript in the heart of C57BL/6 mice. Mice (2–3/point) were injected with vehicle (PBS) or sPDNF intravenously (IV) (25 µg per mouse) daily for 2 or 6 days, sacrificed one day after the last injection, and their cardiac (atria) NGF transcript levels measured in duplicate by qPCR and compared to mice injected with IV PBS for 6 d. Results are representative of three similar experiments; *, P<0.05; ** P<0.01, ns, not statistically significant. <b>B</b>) Intravenous sPDNF upregulates NGF protein in the heart of C57BL/6 mice. Mice (2–3/point) were injected with sPDNF intravenously (25 µg per mouse) or vehicle for 6 d, sacrificed one day after the last injection, and their cardiac (atria) NGF protein levels ascertained by immunofluorescence using the NIH ImageJ software, n = 3–7, composite of two distinct experiments. <b>C</b>) NGF is preferentially localized in cardiac fibroblasts following intravenous injection of sPDNF. Representative images of heart (atria) tissue sections from the results presented in <b>B</b>) above. Note that iv sPDNF robustly increase NGF (leftmost panels) that co-localizes primarily with the cardiac fibroblasts (α-NGF/α-vimentin merge).</p

TrkA is targeted by T cruzi for upregulation of NGF in cardiac fibroblasts.

<p><b>A</b>) Antibodies against TrkA, but not against TrkB, TrkC or pan-neurotrophin receptor p75 (p75^NTR) significantly abrogate T cruzi-induced upregulation of NGF on cardiac fibroblasts. Primary cardiac fibroblasts were preincubated with the indicated antibodies (1 µg/ml), infected with T cruzi for 24 h, and the concentration of NGF in the culture overlays determined by ELISA. The results are the mean ± sd of triplicate points and represent the difference between NGF secreted by infected and uninfected cardiac fibroblasts; ***, P<0.001.<b>B</b>) shRNA against TrkA abrogates T cruzi-induced upregulation of NGF on cardiac fibroblasts. Cardiac fibroblasts were transfected with lentivirus encoding shRNA constructs against GFP, TrkA (two distinct vectors), or TrkC (one vector). Seven days later, fibroblasts were infected with T cruzi for 24 h, and NGF content in the culture overlays determined by ELISA. Results are the mean ± SEM of five separate experiments with similar results, and represent the difference between NGF secreted by infected and uninfected cardiac fibroblasts; *, P<0.05, **, P<0.005.</p

Conditioned media from T cruzi-infected or sPDNF-stimulated cardiac fibroblasts protect cardiomyocytes against oxidative stress.

<p><b>A</b>) Conditioned media produced by T cruzi infection of cardiac fibroblasts confer protection of cardiomyocytes against H₂O₂-induced death in an NGF-dependent manner. Primary cultures of cardiomyocytes were exposed to 150 µM H₂O₂ for 4 h following preincubation with unconditioned medium from control fibroblast cultures (Un-CoM) or conditioned medium obtained by infecting cardiac fibroblasts with T cruzi (2×10⁵/mL, 24 h) (CoM). Cardiomyocytes were also placed in medium unexposed to cells, with or without 50 ng/mL NGF. Lastly, CoM was mixed with an α-NGF-blocking sheep antiserum (α-NGF) or normal sheep serum (NSS). Panel on the right shows representative staining from each condition. Data are combined from three independent experiments. <b>B</b>) Conditioned media produced by the specific stimulation of cardiac fibroblast by sPDNF confer protection of cardiomyocytes against H₂O₂-induced death in an NGF-dependent manner. Cardiomyocytes were exposed to 150 µM H₂O₂ for 4 h following preincubation with unconditioned medium (Un-CoM) or conditioned media obtained by stimulating cardiac fibroblasts with sPDNF (50 ng/ml, 3 h) (CoM). Cardiomyocytes were also exposed in parallel to CoM preincubated with an α-NGF-blocking sheep antiserum (α-NGF) or normal sheep serum (NSS). Data are combined from three independent experiments; * p<0.05, **p<0.01 ***p<0.001.</p

Cardiomyocytes preferentially expresses TrkA and cardiac fibroblasts TrkC.

<p><b>A</b>) TrkA levels are significantly higher in cardiomyocytes than in cardiac fibroblasts in tissue sections of the heart. <b>Left panel</b> shows the quantification of TrkA, vimentin (cardiac fibroblast marker), and myosin heavy chain (MHC, cardiomyocyte marker) identified by immunofluorescence on sections of C57BL/6 mouse hearts. Vimentin and MHC staining is plotted as function of TrkA fluorescence showing that TrkA is expressed by both cardiac fibroblasts and myocytes and that expression is higher in the myocytes. <b>Right panel</b> is a plot of vimentin and MHC mean fluorescence on pixels where TrkA is >200 (n = 3); ****, p<0.0001. <b>B</b>) TrkC levels are significantly higher in cardiac fibroblasts than on cardiomyocytes in tissue sections of the heart. <b>Left panel</b> shows the quantification of TrkC, vimentin, and MHC identified by immunofluorescence on sections of C57BL/6 mouse hearts, showing a preferential association of TrkC with cardiac fibroblasts. <b>Right panel</b> is a plot of vimentin and MHC mean fluorescence on pixels where TrkC is >200 (n = 3);****, p<0.0001.<b>C</b>) Visualization of the preferential expression of TrkA on cardiomyocytes in the heart. Representative images from the results presented in (<b>A</b>) above. Note preferential merge of TrkA and cardiomyocyte staining, scale bar = 100 µm. <b>D</b>) Visualization of the preferential expression of TrkC in cardiac fibroblasts in the heart. Representative images from the results presented in (<b>B</b>) above. Of note, TrkC staining merges preferentially in vimentin-stained cardiac fibroblasts, typically surrounding cardiomyocyte bundles, scale bar = 100 µm.</p

Comparative effects of CFTR modulators on phagocytic, metabolic and inflammatory profiles of CF and nonCF macrophages

Abstract Macrophage dysfunction has been well-described in Cystic Fibrosis (CF) and may contribute to bacterial persistence in the lung. Whether CF macrophage dysfunction is related directly to Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in macrophages or an indirect consequence of chronic inflammation and mucostasis is a subject of ongoing debate. CFTR modulators that restore CFTR function in epithelial cells improve global CF monocyte inflammatory responses but their direct effects on macrophages are less well understood. To address this knowledge gap, we measured phagocytosis, metabolism, and cytokine expression in response to a classical CF pathogen, Pseudomonas aeruginosa in monocyte-derived macrophages (MDM) isolated from CF F508del homozygous subjects and nonCF controls. Unexpectedly, we found that CFTR modulators enhanced phagocytosis in both CF and nonCF cohorts. CFTR triple modulators also inhibited MDM mitochondrial function, consistent with MDM activation. In contrast to studies in humans where CFTR modulators decreased serum inflammatory cytokine levels, modulators did not alter cytokine secretion in our system. Our studies therefore suggest modulator induced metabolic effects may promote bacterial clearance in both CF and nonCF monocyte-derived macrophages