Luke I. Jones

I. Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by augmented transforming growth factor-β (TGF-β) signaling leading to excessive extracellular matrix (ECM) deposition. The fibroblast-to-myofibroblast-transition (FMT) and metabolic reprogramming of lung fibroblasts (HLFs) are essential to IPF pathogenesis, yet the connection between nutrient metabolism and fibrogenesis remains poorly defined. The O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is a nutrient-sensitive enzyme that adds O-GlcNAc moieties to substrates. We previously showed that loss of OGT reverses bleomycin-induced pulmonary fibrosis in mice. Here, using unbiased kinomics, we show that pharmacologic inhibition of OGT suppressed non-canonical TGF-β-induced mitogen-activated protein kinase (MAPK) signaling. Molecular confirmation revealed that TGF-β-induced phosphorylation of p38, but not ERK or JNK, was reduced by OGT blockade. Furthermore, p38 itself was O-GlcNAc-modified, which enhanced its phosphorylation and promoted downstream phosphorylation of the NADPH oxidase subunit, p47 ^phox . Inhibition of OGT, p38, or p47 ^phox reduced reactive oxygen species (ROS) in HLFs, revealing a previously unknown role of OGT-p38-p47 ^phox signaling in ROS production. Collectively, this work establishes that O-GlcNAc-modified p38 enhances p47 ^phox -dependent H ₂ O ₂ production. Highlights: Using PamChip STK arrays, we show that OGT inhibition causes broad kinomic remodeling, including suppression of non-canonical TGF-β MAPKs and multiple CDKs. OGT blockade selectively attenuates p38 phosphorylation, despite TGF-β-induced substrate redundancy with ERK and JNK. We provide evidence that p38 MAPK undergoes O-GlcNAcylation in human lung fibroblasts, a modification not previously reported. The study identifies a new signaling axis where O-GlcNAc modification of p38 modulates the phosphorylation of p47 ^phox , therefore regulating NOX-dependent H ₂ O ₂ production. Blocking OGT or inhibiting p38/p47 ^phox dramatically reduces TGF-β-driven H ₂ O ₂ production in human lung fibroblasts. Graphical

I. Background: Pulmonary arterial hypertension (PAH) is a debilitating cardiopulmonary disease characterized by progressive remodeling of the pulmonary vasculature. Pathologic transforming growth factor-β (TGF-β) signaling is an essential driver of vascular remodeling in PAH. While global inhibitors of TGF-β exist, their clinical application is limited by systemic adverse effects. Therefore, a critically unmet need in PAH is to identify pulmonary vascular-specific regulators of the TGF-β axis, which would selectively enhance clinical efficacy while minimizing adverse effects. As the clinical care of PAH largely promotes vasodilation, and only one FDA-approved agent targets vascular remodeling, this study aimed to identify selective, therapeutically targetable regulators of the TGF-β axis in the PAH pulmonary vasculature. Methods: CD248 was identified via liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics in human lungs. CD248 levels were assessed across human, rat, and mouse lung tissues using western blotting, RTqPCR, and/or immunofluorescence techniques. CD248-null (CD248 ^-/- ) mice were used to study the contribution of CD248 to hypoxia-sugen (H/S)-induced PAH. The mechanistic role of CD248 in PAH vascular remodeling and TGF-β signaling was assessed by genetic (siRNA knockdown; overexpression) and pharmacologic (Ontuxizumab) manipulation of primary human pulmonary vascular cells. Results: LC-MS/MS proteomics coupled with pathway enrichment analysis of human lung tissue identified CD248 as a putative mediator of vascular remodeling that is elevated in PAH lungs. CD248 was elevated in PAH pulmonary artery smooth muscle cells (PASMCs) across human, rat, and mouse lung tissue. CD248 ^-/- mice were protected from H/S-induced elevations in right ventricular (RV) systolic pressure (RVSP), RV hypertrophy, and pulmonary artery muscularization. CD248 knock-down reduced cell proliferation and migration of primary PAH PASMCs. CD248 was essential for phospho-activation of TGF-β receptor I (TβRI) at S165 and canonical phosphorylation of SMAD3 at S423/425. CD248 loss blunted TGF-β-induced gene expression (FN1, Col1α1, α-SMA) and activated expression of the vasoprotective matrix metalloprotease, MMP-8. Mechanistically, CD248 interacted with and enhanced de novo phosphorylation and stability of TβRI, blocking its ubiquitin-mediated proteasomal degradation. Ontuxizumab promoted TβRI instability and attenuated the production of FN1, Col1α1, and α-SMA in primary PAH PASMCs. Conclusions: This work identifies CD248 as a previously unrecognized co-activator of TβRI in PAH. As CD248 is largely quiescent in most adult tissues yet pathologically upregulated in the PAH pulmonary vasculature, this study supports the potential of anti-CD248 therapy as a novel pulmonary vascular-specific alternative to systemic TGF-β inhibition.

Cystic fibrosis (CF) is an autosomal recessive multisystem disorder caused by mutations in the cystic fibrosis conductance regulator (CFTR) anion channel. In the lungs specifically, CFTR mutations lead to changes in mucus viscosity and defective mucociliary clearance. Moreover, people with CF (pwCF) mount an insufficient immune response to invading pathogens, which predisposes individuals to chronic airway disease associated with chronic inflammation, colonization, and recurrent infections by mainly opportunistic pathogens. These chronic infections in the CF lung are typically polymicrobial and frequently harbour multidrug-resistant pathogens, making both treatment and eradication very challenging. During the last decade, the development of highly effective CFTR modulator therapy (HEMT) has led to a breakthrough in treatment options for pwCF. While the majority of pwCF now live longer and have fewer CF exacerbations, colonisation with common respiratory pathogens persists, thereby contributing to chronic inflammation and infection. Interestingly, there are limited reports examining the lung microbiome in the post-modulator era. Since ETI treatment is still quite novel and has only been used for about five years by now, this review will be one of the first discussing the current literature on the effect of ETI on CF pathogens. In addition, we will identify unanswered questions that remain from the effect of HEMT on the CF microbiome.

The number of adults living with cystic fibrosis (CF) has already increased significantly because of drastic improvements in life expectancy attributable to advances in treatment, including the development of highly effective modulator therapy. Chronic airway inflammation in CF contributes to morbidity and mortality, and aging processes like inflammaging and cell senescence influence CF pathology. Our results show that single-cell RNA sequencing data, human primary bronchial epithelial cells from non-CF and CF donors, a CF bronchial epithelial cell line, and Cftr -knockout ( Cftr ^–/– ) rats all demonstrated increased cell senescence markers in the CF bronchial epithelium. This was associated with upregulation of fibroblast growth factor receptors (FGFRs) and mitogen-activated protein kinase (MAPK) p38. Inhibition of FGFRs, specifically FGFR4 and to some extent FGFR1, attenuated cell senescence and improved mucociliary clearance, which was associated with MAPK p38 signaling. Mucociliary dysfunction could also be improved using a combination of senolytics in a CF ex vivo model. In summary, FGFR/MAPK p38 signaling contributes to cell senescence in CF airways, which is associated with impaired mucociliary clearance. Therefore, attenuation of cell senescence in the CF airways might be a future therapeutic strategy improving mucociliary dysfunction and lung disease in an aging population with CF.

Rationale: The role of MUC5B mucin expression in IPF pathogenesis is unknown. Bleomycin-exposed rodent models do not exhibit sustained fibrosis or airway remodeling. Unlike mice, ferrets have human-like distribution of MUC5B expressing cell types and natively express the risk-conferring variant that induces high MUC5B expression in humans. We hypothesized that ferrets would consequently exhibit aberrant repair to propagate fibrosis similar to human IPF. Methods: Bleomycin (5U/kg) or saline-control was micro-sprayed intratracheally then wild-type ferrets were evaluated through 22 wks. Clinical phenotype was assessed with lung function. Fibrosis was assessed with µCT imaging and comparative histology with Ashcroft scoring. Airway remodeling was assessed with histology and quantitative immunofluorescence. Results: Bleomycin ferrets exhibited sustained restrictive physiology including decreased inspiratory capacity, decreased compliance, and shifted Pressure-Volume loops through 22 wks. Volumetric µCT analysis revealed increased opacification of the lung bleomycin-ferrets. Histology showed extensive fibrotic injury that matured over time and MUC5B-positive cystic structures in the distal lung suggestive of honeycombing. Bleomycin ferrets had increased proportion of small airways that were double-positive for CCSP and alpha-tubulin compared to controls, indicating an aberrant ‘proximalization’ repair phenotype. Notably, this aberrant repair was associated with extent of fibrotic injury at the airway level. Conclusions: Bleomycin-exposed ferrets exhibit sustained fibrosis through 22 wks and have pathologic features of IPF not found in rodents. Ferrets exhibited proximalization of the distal airways and other pathologic features characteristic of human IPF. MUC5B expression through native cell types may play a key role in promoting airway remodeling and lung injury in IPF.

Increased levels of allergen-specific T-cells have been documented in the peripheral blood of patients with atopic dermatitis (AD) compared with nonatopic controls. However, little is known about how these relate to disease severity. This study sought to examine if frequencies of circulating allergen-specific T cells correlate with changes in clinical disease severity in a cohort of seven adults with AD who were positive for human leucocyte antigen DRB1*1501. We found that frequencies of allergen-specific CD4+ T cells across the study group were not significantly (P > 0.05) associated with clinical disease severity; however, longitudinal changes within an individual did correlate significantly (P < 0.01) with changes in disease severity. These findings support a role for allergen-specific T-cells in disease pathogenesis.