Fulminant Lung Fibrosis in Non-Resolvable Covid-19 Requiring Transplantation

BACKGROUND: Coronavirus Disease 2019 (COVID-19) can lead to the development of acute respiratory distress syndrome (ARDS). In some patients with non-resolvable (NR) COVID-19, lung injury can progress rapidly to the point that lung transplantation is the only viable option for survival. This fatal progression of lung injury involves a rapid fibroproliferative response and takes on average 15 weeks from initial symptom presentation. Little is known about the mechanisms that lead to this fulminant lung fibrosis (FLF) in NR-COVID-19. METHODS: Using a pre-designed unbiased PCR array for fibrotic markers, we analyzed the fibrotic signature in a subset of NR-COVID-19 lungs. We compared the expression profile against control lungs (donor lungs discarded for transplantation), and explanted tissue from patients with idiopathic pulmonary fibrosis (IPF). Subsequently, RT-qPCR, Western blots and immunohistochemistry were conducted to validate and localize selected pro-fibrotic targets. A total of 23 NR-COVID-19 lungs were used for RT-qPCR validation. FINDINGS: We revealed a unique fibrotic gene signature in NR-COVID-19 that is dominated by a hyper-expression of pro-fibrotic genes, including collagens and periostin. Our results also show a significantly increased expression of Collagen Triple Helix Repeat Containing 1(CTHRC1) which co-localized in areas rich in alpha smooth muscle expression, denoting myofibroblasts. We also show a significant increase in cytokeratin (KRT) 5 and 8 expressing cells adjacent to fibroblastic areas and in areas of apparent epithelial bronchiolization. INTERPRETATION: Our studies may provide insights into potential cellular mechanisms that lead to a fulminant presentation of lung fibrosis in NR-COVID-19. FUNDING: National Institute of Health (NIH) Grants R01HL154720, R01DK122796, R01DK109574, R01HL133900, and Department of Defense (DoD) Grant W81XWH2110032 to H.K.E. NIH Grants: R01HL138510 and R01HL157100, DoD Grant W81XWH-19-1-0007, and American Heart Association Grant: 18IPA34170220 to H.K.-Q. American Heart Association: 19CDA34660279, American Lung Association: CA-622265, Parker B. Francis Fellowship, 1UL1TR003167-01 and The Center for Clinical and Translational Sciences, McGovern Medical School to X.Y

Functional Variant in the Autophagy-Related 5 Gene Promotor is Associated with Childhood Asthma

Rationale and Objective: Autophagy is a cellular process directed at eliminating or recycling cellular proteins. Recently, the autophagy pathway has been implicated in immune dysfunction, the pathogenesis of inflammatory disorders, and response to viral infection. Associations between two genes in the autophagy pathway, ATG5 and ATG7, with childhood asthma were investigated. Methods: Using genetic and experimental approaches, we examined the association of 13 HapMap-derived tagging SNPs in ATG5 and ATG7 with childhood asthma in 312 asthmatic and 246 non-allergic control children. We confirmed our findings by using independent cohorts and imputation analysis. Finally, we evaluated the functional relevance of a disease associated SNP. Measurements and Main Results: We demonstrated that ATG5 single nucleotide polymorphisms rs12201458 and rs510432 were associated with asthma (p = 0.00085 and 0.0025, respectively). In three independent cohorts, additional variants in ATG5 in the same LD block were associated with asthma (p,0.05). We found that rs510432 was functionally relevant and conferred significantly increased promotor activity. Furthermore, Atg5 expression was increased in nasal epithelium of acute asthmatics compared to stable asthmatics and non-asthmatic controls. Conclusion: Genetic variants in ATG5, including a functional promotor variant, are associated with childhood asthma. Thes

Switching-Off Adora2b in Vascular Smooth Muscle Cells Halts the Development of Pulmonary Hypertension

International audienceBackground: Pulmonary hypertension (PH) is a devastating and progressive disease characterized by excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) and remodeling of the lung vasculature. Adenosine signaling through the ADORA2B receptor has previously been implicated in disease progression and tissue remodeling in chronic lung disease. In experimental models of PH associated with chronic lung injury, pharmacological or genetic inhibition of ADORA2B improved markers of chronic lung injury and hallmarks of PH. However, the contribution of ADORA2B expression in the PASMC was not fully evaluated. Hypothesis: We hypothesized that adenosine signaling through the ADORA2B receptor in PASMC mediates the development of PH. Methods: PASMCs from controls and patients with idiopathic pulmonary arterial hypertension (iPAH) were characterized for expression levels of all adenosine receptors. Next, we evaluated the development of PH in ADORA2Bf/f-Transgelin (Tagln)cre mice. These mice or adequate controls were exposed to a combination of SUGEN (SU5416, 20 mg/kg/b.w. IP) and hypoxia (10% O2) for 28 days (HX-SU) or to chronic low doses of bleomycin (BLM, 0.035U/kg/b.w. IP). Cardiovascular readouts including right ventricle systolic pressures (RVSPs), Fulton indices and vascular remodeling were determined. Using PASMCs we identified ADORA2B-dependent mediators involved in vascular remodeling. These mediators: IL-6, hyaluronan synthase 2 (HAS2) and tissue transglutaminase (Tgm2) were determined by RT-PCR and validated in our HX-SU and BLM models. Results: Increased levels of ADORA2B were observed in PASMC from iPAH patients. ADORA2Bf/f-Taglncre mice were protected from the development of PH following HX-SU or BLM exposure. In the BLM model of PH, ADORA2Bf/f- Taglncre mice were not protected from the development of fibrosis. Increased expression of IL-6, HAS2 and Tgm2 was observed in PASMC in an ADORA2B-dependent manner. These mediators were also reduced in ADORA2Bf/f- Taglncre mice exposed to HX-SU or BLM. Conclusions: Our studies revealed ADORA2B-dependent increased levels of IL-6, hyaluronan and Tgm2 in PASMC, consistent with reduced levels in ADORA2Bf/f- Taglncre mice exposed to HX-SU or BLM. Taken together, our data indicates that ADORA2B on PASMC mediates the development of PH through the induction of IL-6, hyaluronan and Tgm2. These studies point at ADORA2B as a therapeutic target to treat PH

Identification of association between asthma and <i>ATG5</i> SNPs.

<p>Negative log₁₀ p value of the associations in GCPCR, CAMP, CARE, and CINCY cohorts are presented as well as the meta analysis (METAL). Black circles represent genotyped SNPs and grey triangles represent imputed SNPs. The dotted line represents significance (p = 0.007 for GCPCR and METAL after correction for multiple comparisons, p = 0.05 for CAMP, CARE, CINCY).</p

Characteristics of the GCPCR population.

a<p>Indicates sample passing quality control.</p>b<p>Indicates significant differences (p<0.05) with non-allergic control children.</p

Asthma-associated rs510432 SNP G variant allele confers enhanced promotor activity.

<p>A. ATG5 promotor fragments generated from genomic DNA isolated from human peripheral blood mononuclear cells. B, C. Luciferase activity of promotor assay vectors. In the mutant the corresponding sites of the two strands of the DNA were mutated from A to G. Promotor activities (corrected for transfection efficiency) are presented as fold increase relative to empty vector (PGL4.20). The fold ratio of empty PGL4.20 Firefly Luciferase plasmid to PGL4.73 Renilla transfection control vector was normalized to 1. Mean ± SD, n = 3 independent experiments.</p

Genotyped SNPs and their minor allele frequencies (MAF) in the GCPCR cohort.

a<p>p = 0.00085; OR = 0.52, 95% CI, 0.36–0.77.</p>b<p>p = 0.0025; OR = 1.47, 95% CI, 1.14–1.88.</p

ATG5 expression enhanced in nasal mucosal samples from children with acute asthma.

<p>Data are presented as normalized expression from Affymetrix array data using (A) 202511_s_at and (B) 210639_s_at <i>ATG5</i> probesets. Intensities were normalized to the median expression level of the control samples.</p

LD plot and identification of haplotype block in the <i>ATG5</i> gene.

<p>The position of the 6 SNPs within the <i>ATG5</i> gene are shown above the plot. <i>D’</i> values, indicating extent of LD between SNPs, are noted on the squares. Higher color intensity of the squares indicates higher LD between SNPs. The inverted black triangle represents a single haplotype block (estimated by Gabriel’s 90% bounds on <i>D’</i>³⁰).</p

Table_3_Switching-Off Adora2b in Vascular Smooth Muscle Cells Halts the Development of Pulmonary Hypertension.DOCX

<p>Background: Pulmonary hypertension (PH) is a devastating and progressive disease characterized by excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) and remodeling of the lung vasculature. Adenosine signaling through the ADORA2B receptor has previously been implicated in disease progression and tissue remodeling in chronic lung disease. In experimental models of PH associated with chronic lung injury, pharmacological or genetic inhibition of ADORA2B improved markers of chronic lung injury and hallmarks of PH. However, the contribution of ADORA2B expression in the PASMC was not fully evaluated.</p><p>Hypothesis: We hypothesized that adenosine signaling through the ADORA2B receptor in PASMC mediates the development of PH.</p><p>Methods: PASMCs from controls and patients with idiopathic pulmonary arterial hypertension (iPAH) were characterized for expression levels of all adenosine receptors. Next, we evaluated the development of PH in ADORA2B^f/f-Transgelin (Tagln)^cre mice. These mice or adequate controls were exposed to a combination of SUGEN (SU5416, 20 mg/kg/b.w. IP) and hypoxia (10% O₂) for 28 days (HX-SU) or to chronic low doses of bleomycin (BLM, 0.035U/kg/b.w. IP). Cardiovascular readouts including right ventricle systolic pressures (RVSPs), Fulton indices and vascular remodeling were determined. Using PASMCs we identified ADORA2B-dependent mediators involved in vascular remodeling. These mediators: IL-6, hyaluronan synthase 2 (HAS2) and tissue transglutaminase (Tgm2) were determined by RT-PCR and validated in our HX-SU and BLM models.</p><p>Results: Increased levels of ADORA2B were observed in PASMC from iPAH patients. ADORA2B^f/f-Tagln^cre mice were protected from the development of PH following HX-SU or BLM exposure. In the BLM model of PH, ADORA2B^f/f- Tagln^cre mice were not protected from the development of fibrosis. Increased expression of IL-6, HAS2 and Tgm2 was observed in PASMC in an ADORA2B-dependent manner. These mediators were also reduced in ADORA2B^f/f- Tagln^cre mice exposed to HX-SU or BLM.</p><p>Conclusions: Our studies revealed ADORA2B-dependent increased levels of IL-6, hyaluronan and Tgm2 in PASMC, consistent with reduced levels in ADORA2B^f/f- Tagln^cre mice exposed to HX-SU or BLM. Taken together, our data indicates that ADORA2B on PASMC mediates the development of PH through the induction of IL-6, hyaluronan and Tgm2. These studies point at ADORA2B as a therapeutic target to treat PH.</p