Host response aimed at eliminating the infecting pathogen, as well as the pathogen itself, can cause tissue injury. Tissue injury leads to the release of a myriad of cellular components including mitochondrial DNA, which the host senses through pattern recognition receptors. How the sensing of tissue injury by the host shapes the anti-pathogen response remains poorly understood. In this study, we utilized mice that are deficient in toll-like receptor-9 (TLR9), which binds to unmethylated CpG DNA sequences such as those present in bacterial and mitochondrial DNA. To avoid direct pathogen sensing by TLR9, we utilized the influenza virus, which lacks ligands for TLR9, to determine how damage sensing by TLR9 contributes to anti-influenza immunity. Our data show that TLR9-mediated sensing of tissue damage promotes an inflammatory response during early infection, driven by the epithelial and myeloid cells. Along with the diminished inflammatory response, the absence of TLR9 led to impaired viral clearance manifested as a higher and prolonged influenza components in myeloid cells including monocytes and macrophages rendering them highly inflammatory. The persistent inflammation driven by infected myeloid cells led to persistent lung injury and impaired recovery in influenza-infected TLR9-/- mice. Further, we show elevated TLR9 activation in the plasma samples of patients with influenza and its association with the disease severity in hospitalized patients, demonstrating its clinical relevance. Overall, we demonstrate an essential role of damage sensing through TLR9 in promoting anti-influenza immunity and inflammatory response AUTHOR SUMMARY: Tissue damage is an inevitable outcome of clinically relevant lung infections, but the host mechanisms for detecting such damage during infection are not well understood. We investigated the role of Toll-like receptor 9 (TLR9) in sensing tissue damage caused by influenza. Since influenza lacks TLR9 ligands, we hypothesized that TLR9 signaling is driven by tissue damage molecules like mitochondrial DNA (mtDNA). Our data indicate that TLR9 reduces early inflammatory lung injury but impairs viral clearance, resulting in extensive immune cell infection, persistent inflammation, and delayed recovery. Myeloid-specific TLR9 deletion ameliorated late-stage inflammatory responses. In humans, influenza-infected individuals exhibited elevated TLR9 activity and mtDNA levels in plasma compared to healthy controls, with higher TLR9 activation potential correlating with severe disease requiring ICU admission. These findings suggest that TLR9-mediated damage sensing triggers both inflammatory tissue injury and viral clearance. These data indicate that TLR9 activity can serve as a crucial biomarker and therapeutic target to limit influenza induced tissue injury.

Tracheal replacement using tissue engineering technologies offers great potential to improve previously intractable clinical interventions, and interest in this area has increased in recent years. Many engineered airway constructs currently rely on decellularized native tracheas to serve as the scaffold for tissue repair. Yet, mechanical failure leading to airway narrowing and collapse remains a major cause of morbidity and mortality following clinical implantation of decellularized tracheal grafts. To understand better the factors contributing to mechanical failure in vivo, we characterized the histo-mechanical properties of tracheas following two different decellularization protocols, including one that has been used clinically. All decellularized tracheas deviated from native mechanical behavior, which may provide insights into observed in vivo graft failures. We further analyzed protein content by western blot and analyzed microstructure by histological staining and found that the specific method of decellularization resulted in significant differences in the depletion of proteoglycans and degradation of collagens I, II, III, and elastin. Taken together, this work demonstrates that the heterogeneous architecture and mechanical behavior of the trachea is severely compromised by decellularization. Such structural deterioration may contribute to graft failure clinically and limit the potential of decellularized native tracheas as viable long-term orthotopic airway replacements.

BACKGROUND: Disorders in pulmonary vascular integrity are a prominent feature in many lung diseases. Paracrine signaling is highly enriched in the lung and plays a crucial role in regulating vascular homeostasis. However, the specific local cell-cell crosstalk signals that maintain pulmonary microvascular stability in adult animals and humans remain largely unexplored. METHODS: In this study, we used single-cell RNA-sequencing–based computational pipelines to systematically profile ligand-receptor interactions within the lung microvascular niche and identified VEGF-D (vascular endothelial growth factor-D) as a key local factor with previously unrecognized barrier-protective properties in models of acute lung injury. RESULTS: Our single-cell RNA-sequencing data revealed that, under physiological conditions, soluble ligand-receptor interactions between mesenchymal cells, in particular alveolar fibroblasts, and microvascular endothelial cells are predominantly associated with pathways involved in maintaining vascular integrity as compared with all other cells. On treatment with top identified ligands, we found that VEGF-D significantly enhanced endothelial barrier function and conferred protection against inflammatory challenges induced by TNF-α (tumor necrosis factor-α), IL (interleukin)-1β, and thrombin. This barrier-protective effect of VEGF-D was significantly attenuated by inhibition of VEGFR2 (vascular endothelial growth factor receptor 2), either through small interfering RNA (siRNA) knockdown or pharmacological blockade using specific VEGFR2 inhibitors. Intravenous administration of recombinant VEGF-D in lipopolysaccharide-induced acute lung injury models significantly reduced vascular permeability (7339±2510 arbitrary unit [a.u.] [lipopolysaccharides] versus 5350±1821 a.u. [lipopolysaccharides+VEGF-D]; P <0.05), immune cell infiltration (0.791±0.199×10 ⁶ whole blood cells/mL [lipopolysaccharides] versus 0.540±0.190×10 ⁶ whole blood cells/mL [lipopolysaccharide+VEGF-D]; P <0.01), and the expression of proinflammatory markers TNF-α, IL-6, and keratinocyte chemoattractant in the lung tissue. This effect was abolished in VEGFR2 ^iECKO (VEGFR2 inducible endothelial cells knockout) mice, confirming that VEGF-D mediates its effects via VEGFR2-dependent signaling. CONCLUSIONS: This study demonstrates an unexpected protective role for VEGF-D in promoting lung endothelial barrier integrity and suggests that paracrine signaling from the alveolar fibroblast niche contributes critically to lung capillary homeostasis.

Analysis of pulmonary vascular dysfunction in various lung pathologies remains challenging due to the lack of functional ex vivo models. Paracrine signaling in the lung plays a critical role in regulating endothelial maturation and vascular homeostasis. Previously, we employed single-cell RNA-sequencing (scRNAseq) to systematically map ligand–receptor (L/R) interactions within the lung vascular niche. However, the functional impact of these ligands on endothelial biology remained unknown. Here, we systematically evaluated selected ligands in vitro to assess their effects on endothelial barrier integrity, anti-inflammatory responses, and phenotypic maturation. Among the top soluble ligands, we found that adrenomedulin (ADM) exhibited superior barrier enhancing effect on human pulmonary endothelial cell monolayers, as evidenced by electrical cell impedance sensing (ECIS) and XperT assays. ADM also exhibited anti-inflammatory properties, decreasing ICAM1 and increasing IkBa expression in a dose-dependent manner. Perfusion is commonly used in bioengineered vascular model systems. Shear stress (15 dynes/cm²) alone increased endothelial characteristics, including homeostatic markers such as CDH5, NOS3, TEK, and S1PR1. ADM treatment maintained the enhanced level of these markers under shear stress and further improved anti-coagulation by increasing THBD and decreasing F3 expression and synergistically enhanced the expression of the native lung aerocyte capillary endothelial marker EDNRB. This effect was completely attenuated by a blockade of ADM receptor, RAMP2. Together, these findings identify ADM/RAMP2 signaling as a key paracrine pathway that enhances vascular barrier integrity, anti-inflammatory phenotype, and endothelial homeostasis, providing a framework for improving the physiological relevance of engineered vascular models.

Therapeutic lung regeneration is predicated upon successful reconstitution of lung alveoli, the functional units of gas exchange. Here, we identify requisite multimodal cues that are critical to reconstructing the alveolar epithelium and alveoli in lung scaffolds. Alveolar reconstruction in vitro is divided into several distinct phases. In the first phase, endothelial cells coordinate with fibroblasts and select exogenous factors to promote alveolar scaffold population with surfactant-secreting alveolar epithelial type 2 cells (AEC2s). After formation of organized epithelial alveolar-like structures, subsequent withdrawal of Wnt and FGF agonism synergizes with tidal-level mechanical strain to induce differentiation of AEC2s to squamous type 1 AECs (AEC1s) in cultured alveoli, in situ . These results outline a rational strategy to engineer an alveolus of AEC2s and AEC1s contained within epithelial-mesenchymal-endothelial units, and reveal the critical interplay amongst biochemical, cellular, and mechanical niche cues within the reconstituting alveolus.