Idiopathic pulmonary fibrosis (IPF) is a devastating pulmonary disease with no curative treatment other than lung transplantation that results from maladaptive responses to lung epithelial injury; however, the underlying mechanisms remain unclear, and treatment options are limited. Here, we showed that deficiency in the innate immune receptor toll-like receptor 5 (TLR5) is associated with IPF in humans and with increased susceptibility to bleomycin-induced pulmonary fibrosis in mice and that activation of lung epithelial TLR5 through a synthetic flagellin analog protected mice from experimental fibrosis. Mechanistically, epithelial TLR5 activation induced antimicrobial gene expression and ameliorated lung dysbiosis after injury. In contrast, TLR5 deficiency in mice and patients with IPF was associated with lung dysbiosis. Elimination of the microbiome in mice through administration of antibiotics abolished the protective effect of TLR5, and reconstitution of the microbiome by fecal microbiota transplantation rescued the observed phenotype. In conclusion, these studies revealed that TLR5 protects against pulmonary fibrosis through effects on the lung microbiota, providing insight into therapeutic approaches that may ultimately benefit patients with IPF.

Airway pollutants, like the reactive oxygen species ozone, cause significant lung injury, which can lead to the development or exacerbation of lung diseases like asthma and chronic obstructive pulmonary disease (COPD), and drives worldwide morbidity and mortality. Altered epithelial function is a hallmark and trigger of ozone-induced lung injury, but its precise mechanisms are incompletely known. The extracellular matrix, and specifically its major component, hyaluronan (HA), plays a crucial role in cellular injury responses. We hypothesized that HA signaling mediates epithelial responses to ozone-induced injury. We exposed human and murine differentiated primary epithelia to ozone in vitro and evaluated epithelial integrity and transcriptomic responses. We used genetically deficient cells for cognate HA receptors cluster of differentiation 44 (CD44) and receptor for HA-mediated motility (RHAMM), and innate immune receptors toll-like receptor 4 (TLR4) and TLR5 to study signaling pathways, and evaluated high molecular weight HA (HMWHA) as a potential treatment for ozone-induced epithelial injury. In vitro ozone exposure caused significant reduction in epithelial integrity and very similar inflammatory changes in human and murine cells. CD44 deficiency led to decreased inflammation, while RHAMM deficiency exacerbated cell injury. HMWHA protected against ozone-induced epithelial injury, mediated by TLR4 and TLR5 but not CD44 or RHAMM. Our results identify novel contributions of HA signaling to ozone-induced epithelial injury and suggest that HMWHA protects epithelia via innate immune activation of TLR4 and TLR5.

Viral lung infections are a major cause of morbidity and mortality worldwide. Despite significant advances in vaccines and antivirals, there remains a tremendous need for broadly applicable treatments that can be utilized across viral infections. Before infecting epithelial cells, viruses interact with the epithelial glycocalyx, which contains high-molecular weight hyaluronan (HMWHA), a glycosaminoglycan that has beneficial effects in lung injury. In this study, we sought to determine the role of HMWHA in viral pneumonia. We infected mice with influenza or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and treated them with prophylactic or therapeutic doses of HMWHA or saline control. We performed in vitro experiments of infection with viruses of respiratory and nonrespiratory human and animal cells and evaluated the effect of HMWHA on infection. We analyzed existing databases for expression of hyaluronan and the transcription factor E2F1. Finally, we performed a clinical trial with HMWHA in patients with severe coronavirus disease (COVID-19). Exogenously applied HMWHA improved survival in SARS-CoV-2 and influenza infection in mice by ameliorating inflammation through the inhibition of E2F1. In a clinical study, inhaled HMWHA improved outcomes in patients with severe COVID-19. Furthermore, airway epithelia naturally express HMWHA, which is induced during viral infection and prevents infection through the macromolecular crowding of viruses. Our data provide a mechanistic justification for the use of HMWHA as a broadly effective prophylactic and therapeutic agent in viral airway infection.

Aberrant fibroblast function plays a key role in the pathogenesis of idiopathic pulmonary fibrosis, a devastating disease of unrelenting extracellular matrix deposition in response to lung injury. Platelet-derived growth factor α–positive (Pdgfra+) lipofibroblasts (LipoFBs) are essential for lung injury response and maintenance of a functional alveolar stem cell niche. Little is known about the effects of lung injury on LipoFB function. Here, we used single-cell RNA-Seq (scRNA-Seq) technology and PdgfraGFP lineage tracing to generate a transcriptomic profile of Pdgfra+ fibroblasts in normal and injured mouse lungs 14 days after bleomycin exposure, generating 11 unique transcriptomic clusters that segregated according to treatment. While normal and injured LipoFBs shared a common gene signature, injured LipoFBs acquired fibrogenic pathway activity with an attenuation of lipogenic pathways. In a 3D organoid model, injured Pdgfra+ fibroblast–supported organoids were morphologically distinct from those cultured with normal fibroblasts, and scRNA-Seq analysis suggested distinct transcriptomic changes in alveolar epithelia supported by injured Pdgfra+ fibroblasts. In summary, while LipoFBs in injured lung have not migrated from their niche and retain their lipogenic identity, they acquire a potentially reversible fibrogenic profile, which may alter the kinetics of epithelial regeneration and potentially contribute to dysregulated repair, leading to fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating pulmonary disease with no curative treatment other than lung transplantation. IPF results from maladaptive responses to lung epithelial injury, but the underlying mechanisms remain unclear. Here, we show that deficiency in the innate immune receptor, toll-like receptor 5 (TLR5), is associated with IPF in humans and with increased susceptibility to epithelial injury and experimental fibrosis in mice, while activation of lung epithelial TLR5 through a synthetic flagellin analogue protects from experimental fibrosis. Mechanistically, epithelial TLR5 activation induces antimicrobial gene expression and ameliorates dysbiosis after lung injury. In contrast, TLR5 deficiency in mice and IPF patients is associated with lung dysbiosis. Elimination of the microbiome in mice through antibiotics abolishes the protective effect of TLR5 and reconstitution of the microbiome rescues the observed phenotype. In aggregate, TLR5 deficiency is associated with IPF and dysbiosis in humans and in the murine model of pulmonary fibrosis. Furthermore, TLR5 protects against pulmonary fibrosis in mice and this protection is mediated by effects on the microbiome.

Lung disease causes significant morbidity and mortality, and is exacerbated by environmental injury, for example through lipopolysaccharide (LPS) or ozone (O3). Toll-like receptors (TLRs) orchestrate immune responses to injury by recognizing pathogen- or danger-associated molecular patterns. TLR4, the prototypic receptor for LPS, also mediates inflammation after O3, triggered by endogenous hyaluronan. Regulation of TLR4 signaling is incompletely understood. TLR5, the flagellin receptor, is expressed in alveolar macrophages, and regulates immune responses to environmental injury. Using in vivo animal models of TLR4-mediated inflammations (LPS, O3, hyaluronan), we show that TLR5 impacts the in vivo response to LPS, hyaluronan and O3. We demonstrate that immune cells of human carriers of a dominant negative TLR5 allele have decreased inflammatory response to O3 exposure ex vivo and LPS exposure in vitro. Using primary murine macrophages, we find that TLR5 physically associates with TLR4 and biases TLR4 signaling towards the MyD88 pathway. Our results suggest an updated paradigm for TLR4/TLR5 signaling.

INTRODUCTION:Innate immune activation through exposure to indoor and outdoor pollutants is emerging as an important determinant of asthma severity. For example, household levels of the bacterial product lipopolysaccharide (LPS) are associated with increased asthma severity. We hypothesized that activation of the innate immune receptor TLR5 by its bacterial ligand flagellin will exacerbate airway inflammation and asthma symptoms.METHODS:We determined the effect of flagellin co-exposure with ovalbumin in a murine model of allergic asthma. We evaluated the presence of flagellin activity in house dust of asthma patients. Finally, we analyzed the association of a dominant-negative polymorphism in TLR5 (rs5744168) with asthma symptoms in patients with asthma.RESULTS:We showed that bacterial flagellin can be found in the house dust of patients with asthma and that this bacterial product exacerbates allergic airway inflammation in an allergen-specific mouse model of asthma. Furthermore, a dominant-negative genetic polymorphism in TLR5, the receptor for flagellin, is associated with decreased symptoms in patients with asthma.CONCLUSION:Together, our results reveal a novel genetic protective factor (TLR5 deficiency) and a novel environmental pollutant (microbial flagellin) that influence asthma severity. (Clinical trials NCT01688986 and NCT01087307).

Lung disease causes significant morbidity and mortality, and is exacerbated by environmental injury, e.g. through lipopolysaccharide (LPS) or ozone (O 3 ). Toll-like receptors (TLRs) orchestrate immune responses to injury by recognizing pathogen- or danger-associated molecular patterns. TLR4, the prototypic receptor for LPS, also mediates inflammation after O 3 , triggered by endogenous hyaluronan. Regulation of TLR4 signaling is incompletely understood. TLR5, the flagellin receptor, is expressed in alveolar macrophages, and regulates immune responses to environmental injury. Using in vivo animal models of TLR4-mediated inflammations (LPS, O 3 , hyaluronan), we show that TLR5 impacts the in vivo response to LPS, hyaluronan and O 3 . We demonstrate that immune cells of human carriers of a dominant negative TLR5 allele have decreased inflammatory response to O 3 exposure ex vivo and LPS exposure in vitro . Using primary murine macrophages, we find that TLR5 physically associates with TLR4 and biases TLR4 signaling towards the MyD88 pathway. Our results suggest an updated paradigm for TLR4/TLR5 signaling.

Bone marrow-derived epithelial cells (BMDECs) are recruited to sites of chronic inflammation; however, their clinical significance has not been determined. Here, we used gender-mismatched allogeneic bone marrow transplantation (BMT) in the context of the classical multistage model for murine cutaneous carcinogenesis to probe the recruitment of BMDECs in skin tumors initiated with the carcinogen, dimethylbenz[a]anthracene (DMBA), and promoted with the phorbol ester, 12-O-tetradecanolyphorbol-13-acetate (TPA). We detected clusters of bone marrow-derived cells (BMDCs) in over 40% of papillomas, where they occupied 25% or more of the epithelial lesional area. In the dysplastic ulcers which arose in some irradiated DMBA/TPA treated mice, the magnitude of the recruitment was greater. The BMDCs clustered in the cutaneous epithelium where they became immunoreactive to epidermal keratins, and proliferated and stratified, thereby contributing to the lesions comparably with the progeny of hair follicle stem cells in engrafted Krt1-15Cre;R26R mice. Moreover, cytokeratins were detected by immunostaining and Q-PCR in plastic adherent bone marrow cells (BMCs) cultured in the presence of filter-separated epidermal keratinocytes (KCs). Cytokeratin production was enhanced by bone morphogenetic protein 5 (BMP5). Moreover, BMCs migrated towards the alarmin protein, High Mobility Group Box 1 (HMGB1), as well as towards epidermal KCs in ex vivo invasion assays. Furthermore, when naïve female mice received BMTs from donors previously treated with DMBA, several papillomas and a squamous cell carcinoma were observed after TPA promotion alone.We conclude that surprisingly large numbers of BMDECs are recruited to a subset of cutaneous papillomas and dysplastic ulcers and reflect a previously unrecognized systemic contribution to these lesions. We also conclude that carcinogen-exposed cells originating in the BMCs are sufficient to initiate a subset of benign and malignant lesions upon tumor promotion. Ultimately, these findings may provide novel targets for treatment of non-melanoma skin cancers as well as other cancers.Citation Format: Heuijoon Park, Sonali Lad, Kelsey Boland, Kelly Johnson, Nyssa Readio, Guangchun Jin, Samuel A sfaha, Kelly S. Patterson, Ashok Singh, Xiangdong Yang, Douglas Londono, Anupama Singh, Carol Trempus, Derek Gordon, Timothy C. Wang, Rebecca J. Morris. Chronic inflammation-mediated contribution of bone marrow-derived epithelial cells and hair follicle stem cells to development of cutaneous neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-039.

We used allogeneic bone marrow transplantation (BMT) and a mouse multistage cutaneous carcinogenesis model to probe recruitment of bone marrow-derived epithelial cells (BMDECs) in skin tumors initiated with the carcinogen, dimethylbenz[ a ]anthracene (DMBA), and promoted with 12- O -tetradecanolyphorbol-13-acetate (TPA). BMDECs clustered in the lesional epithelium, expressed cytokeratins, proliferated, and stratified. We detected cytokeratin induction in plastic-adherent bone marrow cells (BMCs) cultured in the presence of filter-separated keratinocytes (KCs) and bone morphogenetic protein 5 (BMP5). Lineage-depleted BMCs migrated towards High Mobility Group Box 1 (HMGB1) protein and epidermal KCs in ex vivo invasion assays. Naive female mice receiving BMTs from DMBA-treated donors developed benign and malignant lesions after TPA promotion alone. We conclude that BMDECs contribute to the development of papillomas and dysplasia, demonstrating a systemic contribution to these lesions. Furthermore, carcinogen-exposed BMCs can initiate benign and malignant lesions upon tumor promotion. Ultimately, these findings may suggest targets for treatment of non-melanoma skin cancers.

Acid (HCl) aspiration during anesthesia may lead to acute lung injury. There is no effective therapy. We hypothesized that HCl instilled intratracheally in C57BL/6 mice results in the formation of low-molecular weight hyaluronan (L-HA), which activates RhoA and Rho kinase (ROCK), causing airway hyperresponsiveness (AHR) and increased permeability. Furthermore, instillation of high-molecular weight hyaluronan (H-HA; Yabro) will reverse lung injury. We instilled HCl in C57BL/6 wild-type (WT), myeloperoxidase gene-deficient (MPO-/-) mice, and CD44 gene-deficient (CD44-/-) mice. WT mice were also instilled intranasally with H-HA (Yabro) at 1 and 23 h post-HCl. All measurements were performed at 1, 5, or 24 h post-HCl. Instillation of HCl in WT but not in CD44-/- resulted in increased inflammation, AHR, lung injury, and L-HA in the bronchoalveolar lavage fluid (BALF) 24 h post-HCl; L-HA levels and lung injury were significantly lower in HCl-instilled MPO-/- mice. Isolated perfused lungs of HCl instilled WT but not of CD44-/- mice had elevated values of the filtration coefficient ( Kf). Addition of L-HA on the apical surface of human primary bronchial epithelial cell monolayer decreased barrier resistance ( RT). H-HA significantly mitigated inflammation, AHR, and pulmonary vascular leakage at 24 h after HCl instillation and mitigated the increase of Kf and RT, as well as ROCK2 phosphorylation. Increased H- and L-HA levels were found in the BALF of mechanically ventilated patients but not in healthy volunteers. HCl instillation-induced lung injury is mediated by the L-HA-CD44-RhoA-ROCK2 signaling pathway, and H-HA is a potential novel therapeutic agent for acid aspiration-induced lung injury.

Allergic asthma is a major cause of morbidity in both pediatric and adult patients. Recent research has highlighted the role of hyaluronan (HA), an extracellular matrix glycosaminoglycan, in asthma pathogenesis. Experimental allergic airway inflammation and clinical asthma are associated with an increase of shorter fragments of HA (sHA), which complex with inter-α-inhibitor heavy chains (HCs) and induce inflammation and airway hyperresponsiveness (AHR). Importantly, the effects of sHA can be antagonized by the physiological counterpart high molecular weight HA (HMWHA). We used a mouse model of house dust mite-induced allergic airway inflammation and demonstrated that instilled HMWHA ameliorated allergic airway inflammation and AHR, even when given after the establishment of allergic sensitization and after challenge exposures. Furthermore, instilled HMWHA reduced the development of HA-HC complexes and the activation of Rho-associated, coiled-coil containing protein kinase 2. We conclude that airway application of HMWHA is a potential treatment for allergic airway inflammation.

Carol S. Trempus, Weifeng Song, Ahmed Lazrak, Zhihong Yu, Judy R. Creighton, Bethany M. Young, X Rebecca L. Heise, Yen Rei Yu, Jennifer L. Ingram, Robert M. Tighe, Sadis Matalon, and Stavros Garantziotis Matrix Biology Group, Immunity, Inflammation, and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina; Division of Molecular and Translational Biomedicine, Department of Anesthesiology and Perioperative Medicine, and Pulmonary Injury and Repair Center, School of Medicine, The University of Alabama at Birmingham, Birmingham, Alabama; Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, Virginia; and Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Duke University School of Medicine, Durham, North Carolina