There is a paucity of information about potential molecular brakes on the activation of fibroblasts that drive tissue fibrosis. The transcription factor Kruppel-like factor 4 (KLF4) is best known as a determinant of cell stemness and a tumor suppressor. We found that its expression was diminished in fibroblasts from fibrotic lung. Gain- and loss-of-function studies showed that KLF4 inhibits fibroblast proliferation, collagen synthesis, and differentiation to myofibroblasts, while restoring their sensitivity to apoptosis. Conditional deletion of KLF4 from fibroblasts potentiated the peak degree of pulmonary fibrosis and abrogated the subsequent spontaneous resolution that follows in a model of transient fibrosis. A small molecule inducer of KLF4 was able to restore its expression in fibrotic fibroblasts and elicit resolution in an experimental model characterized by more clinically relevant persistent pulmonary fibrosis. These data identify KLF4 as a pivotal brake on fibroblast activation whose induction represents a new therapeutic approach in fibrosis of the lung, and perhaps other organs.
Loka Raghu Kumar Penke, Jennifer M. Speth, Steven K. Huang, Sean M. Fortier, Jared Baas, Marc Peters-Golden
Usual Interstitial Pneumonia (UIP) is a histological pattern characteristic of Idiopathic Pulmonary Fibrosis (IPF). The UIP pattern is patchy with histologically normal lung adjacent to dense fibrotic tissue. At this interface, fibroblastic foci (FF) are present and are sites where myofibroblasts and extracellular matrix (ECM) accumulate. Utilizing laser capture microdissection coupled mass spectrometry (LCM-MS), we interrogated the FF, adjacent mature scar, and adjacent alveoli in 6 fibrotic (UIP/IPF) specimens plus 6 non-fibrotic alveolar specimens as controls. The data were subject to qualitative and quantitative analysis, and histologically validated. We found that the fibrotic alveoli protein signature is defined by immune deregulation as the strongest category. The fibrotic mature scar classified as end-stage fibrosis whereas the FF contained an overabundance of a distinctive ECM compared to non-fibrotic control. Furthermore, the FF is positive for both TGFB1 and TGFB3, whereas the aberrant basaloid cell lining of the FF is predominantly positive for TGFB2. In conclusion, spatial proteomics demonstrated distinct protein compositions in the histologically defined regions of UIP/IPF tissue. These data revealed that the FF is the main site of collagen biosynthesis and that the adjacent alveoli are abnormal. This new and essential information will inform future mechanistic studies on fibrosis progression.
Jeremy A. Herrera, Lewis A. Dingle, M. Angeles Montero Fernandez, Rajamiyer V. Venkateswaran, John F. Blaikley, Craig Lawless, Martin A. Schwartz
CD4+ T cells drive the immunopathogenesis of chronic beryllium disease (CBD), and their recruitment to the lung heralds the onset of granulomatous inflammation. We have shown that regulatory CD4+ T cells (Tregs) control granuloma formation in an HLA-DP2 transgenic (Tg) model of CBD. In these mice, Be oxide (BeO) exposure resulted in the accumulation of three distinct CD4+ T cell subsets in the lung with the majority of tissue-resident memory cells expressing FoxP3. The amount of Be regulated the number of total and antigen-specific CD4+ T cells and Tregs in the lungs of HLA-DP2 Tg mice. Depletion of Tregs increased the number of IFN-γ-producing CD4+ T cells and enhanced lung injury while mice treated with IL2/αIL-2 complexes had increased Tregs and reduced inflammation and Be-responsive T cells in the lung. BeO-experienced resident Tregs suppressed anti-CD3-induced proliferation of CD4+ T cells in a contact-dependent manner. CLTLA-4 and ICOS blockade as well as addition of LPS to BeO-exposed mice increased the Teff/Treg ratio and enhanced lung injury. Collectively, these data show that the protective role of tissue-resident Tregs is dependent on quantity of Be exposure and is overcome by blocking immune regulatory molecules or additional environmental insults.
Shaikh M. Atif, Douglas G. Mack, Allison K. Martin, Andrew P. Fontenot
Current treatments fail to modify the underlying pathophysiology and disease progression of chronic obstructive pulmonary disease (COPD), necessitating alternative therapies. Here, we show that COPD subjects have increased IL-36γ and decreased IL-36 receptor antagonist (IL-36Ra) in bronchoalveolar and nasal fluid compared to control subjects. IL-36γ is derived from small airway epithelial cells (SAEC) and further induced by a viral mimetic, whereas IL-36RA is derived from macrophages. IL-36γ stimulates release of the neutrophil chemoattractants CXCL1 and CXCL8, as well as elastolytic matrix metalloproteinases (MMPs) from small airway fibroblasts (SAF). Proteases released from COPD neutrophils cleave and activate IL-36γ thereby perpetuating IL-36 inflammation. Transfer of culture media from SAEC to SAF stimulated release of CXCL1, that was inhibited by exogenous IL-36RA. The use of a therapeutic antibody that inhibits binding to the IL-36 receptor (IL-36R) attenuated IL-36γ driven inflammation and cellular cross talk. We have demonstrated a mechanism for the amplification and propagation of neutrophilic inflammation in COPD and that blocking this cytokine family via a IL-36R neutralizing antibody could be a promising new therapeutic strategy in the treatment of COPD.
Jonathan R. Baker, Peter S. Fenwick, Carolin K. Koss, Harriet B. Owles, Sarah L. Elkin, Jay S. Fine, Matthew Thomas, Karim C. Kasmi, Peter J. Barnes, Louise E. Donnelly
Thick, viscous respiratory secretions are a major pathogenic feature of COVID-19, but the composition and physical properties of these secretions are poorly understood. We characterized the composition and rheological properties (i.e., resistance to flow) of respiratory secretions collected from intubated COVID-19 patients. We found the percentages of solids and protein content were greatly elevated in COVID-19 compared with heathy control samples and closely resembled levels seen in cystic fibrosis, a genetic disease known for thick, tenacious respiratory secretions. DNA and hyaluronan (HA) were major components of respiratory secretions in COVID-19 and were likewise abundant in cadaveric lung tissues from these patients. COVID-19 secretions exhibited heterogeneous rheological behaviors, with thicker samples showing increased sensitivity to DNase and hyaluronidase treatment. In histologic sections from these same patients, we observed increased accumulation of HA and the hyaladherin versican but reduced tumor necrosis factor–stimulated gene-6 staining, consistent with the inflammatory nature of these secretions. Finally, we observed diminished type I interferon and enhanced inflammatory cytokines in these secretions. Overall, our studies indicated that increases in HA and DNA in COVID-19 respiratory secretion samples correlated with enhanced inflammatory burden and suggested that DNA and HA may be viable therapeutic targets in COVID-19 infection.
Michael J. Kratochvil, Gernot Kaber, Sally Demirdjian, Pamela C. Cai, Elizabeth B. Burgener, Nadine Nagy, Graham L. Barlow, Medeea Popescu, Mark R. Nicolls, Michael G. Ozawa, Donald P. Regula, Ana E. Pacheco-Navarro, Samuel Yang, Vinicio A. de Jesus Perez, Harry Karmouty-Quintana, Andrew M. Peters, Bihong Zhao, Maximilian L. Buja, Pamela Y. Johnson, Robert B. Vernon, Thomas N. Wight, Stanford COVID-19 Biobank Study Group, Carlos E. Milla, Angela J. Rogers, Andrew J. Spakowitz, Sarah C. Heilshorn, Paul L. Bollyky
Chronic type 2 (T2) inflammatory diseases of the respiratory tract are characterized by mucus overproduction and disordered mucociliary function, which are largely attributed to the effects of IL-13 on common epithelial cell types (mucus secretory and ciliated cells). The role of rare cells in airway T2 inflammation is less clear, though tuft cells have been shown to be critical in the initiation of T2 immunity in the intestine. Using bulk and single cell RNA sequencing of airway epithelium and mouse modeling, we find that IL-13 expands and programs airway tuft cells towards eicosanoid metabolism, and that tuft cell deficiency leads to a reduction in airway prostaglandin E2 (PGE2)concentration. Allergic airway epithelia bear a signature of prostaglandin E2 activation, and PGE2 activation leads to CFTR-dependent ion and fluid secretion and accelerated mucociliary transport. Together these data reveal a role for tuft cells in regulating epithelial mucociliary function in the allergic airway.
Maya E Kotas, Camille M. Moore, Jose G. Gurrola II, Steven D. Pletcher, Andrew N. Goldberg, Raquel Alvarez, Sheyla Yamato, Preston E. Bratcher, Ciaran A. Shaughnessy, Pamela L. Zeitlin, Irene H Zhang, Yingchun Li, Michael T. Montgomery, Keehoon Lee, Emily K. Cope, Richard M. Locksley, Max A. Seibold, Erin D. Gordon
Nontuberculous mycobacteria (NTM) are an increasingly common cause of respiratory infection in people with cystic fibrosis (PwCF). Relative to those with no history of NTM infection (CF-NTMNEG), PwCF and a history of NTM infection (CF-NTMPOS) are more likely to develop severe lung disease and experience complications over the course of treatment. In other mycobacterial infections (e.g. tuberculosis), an overexuberant immune response causes pathology and compromises organ function; however, since the immune profiles of CF-NTMPOS and CF-NTMNEG airways are largely unexplored, it is unknown which if any immune responses distinguish these cohorts or concentrate in damaged tissues. Here we evaluated lung lobe-specific immune profiles of three cohorts (CF-NTMPOS, CF-NTMNEG, and non-CF adults) and found that CF-NTMPOS airways are distinguished by a hyper-inflammatory cytokine profile. Importantly, the CF-NTMPOS airway immune profile was dominated by B cells, classical macrophages and the cytokines which support their accumulation. These and other immunological differences between cohorts, including the near absence of NK cells and complement pathway members, were enriched in the most damaged lung lobes. The implications of these findings for our understanding of lung disease in PwCF are discussed, as are how they may inform the development of host-directed therapies to improve NTM disease treatment.
Don Hayes, Jr., Rajni Kant Shukla, Yizi Cheng, Emrah Gecili, Marlena R. Merling, Rhonda D. Szczesniak, Assem G Ziady, Jason C. Woods, Luanne Hall-Stoodley, Namal P.M. Liyanage, Richard T. Robinson
Studies have demonstrated the phenotypic heterogeneity of vascular endothelial cells (ECs) within a vascular bed; however, little is known about how distinct endothelial subpopulations in a particular organ respond to an inflammatory stimulus. We performed single cell RNA-sequencing of 35,973 lung ECs obtained during the baseline state as well as post-injury time points following inflammatory lung injury induced by lipopolysaccharide. Seurat clustering and gene expression pathway analysis identified two major subpopulations in the lung microvascular endothelium, a subpopulation enriched for expression of immune response genes such as major histocompatibility complex genes (immuneEC) and another defined by increased expression of vascular development genes such as Sox17 (devEC). The presence of immuneEC and devEC subpopulations was also observed in non-human primate lungs infected with SARS-CoV-2 and murine lungs infected with H1N1 influenza virus. Following the peak of inflammatory injury, we observed the emergence of a proliferative lung EC subpopulation. Overexpression of Sox17 prevented inflammatory activation in ECs. Thus, there appears to be a” division of labor” within the lung microvascular endothelium with some ECs showing propensity for inflammatory signaling and others for endothelial regeneration. These results provide underpinnings for the development of targeted therapies to limit inflammatory lung injury and promote regeneration.
Lianghui Zhang, Shang Gao, Zachary White, Yang Dai, Asrar B. Malik, Jalees Rehman
Idiopathic pulmonary fibrosis (IPF) is a fatal disease with limited treatment options. The role of the developmental transcription factor Sine Oculis homeobox homolog 1 (SIX1) in the pathophysiology of lung fibrosis is not known. IPF lung tissue samples and IPF-derived alveolar type II cells (AT2) showed a significant increase in SIX1 mRNA and protein levels, and the SIX1 transcriptional co-activators EYA1 and EYA2 were elevated. Six1 was also upregulated in bleomycin (BLM)-treated mice and in a model of spontaneous lung fibrosis driven by deletion of Telomeric Repeat Binding Factor 1 (Trf1) in AT2 cells. Conditional deletion of Six1 in AT2 cells prevented or halted BLM-induced lung fibrosis as measured by a significant reduction in histological burden of fibrosis, reduced fibrotic mediator expression and improved lung function. These effects were associated with increased macrophage migration inhibitory factor (MIF) in lung epithelial cells in vivo following SIX1 overexpression in BLM-induced fibrosis. A MIF promoter-driven luciferase assay demonstrated direct binding of Six1 to the 5’-TCAGG-3’ consensus sequence of the MIF promoter, identifying a likely mechanism of SIX1-driven MIF expression in the pathogenesis of lung fibrosis, and providing a novel pathway for targeting in IPF therapy.
Cory Wilson, Tinne C.J. Mertens, Pooja Shivshankar, Weizen Bi, Scott D. Collum, Nancy Wareing, Junsuk Ko, Tingting Weng, Ram P. Naikawadi, Paul J. Wolters, Pascal Maire, Soma S.K. Jyothula, Rajarajan A. Thandavarayan, Dewei Ren, Nathan D. Elrod, Eric J. Wagner, Howard J. Huang, Burton F. Dickey, Heide L. Ford, Harry Karmouty-Quintana
Sex/gender disparity in asthma is recognized, and suggests a modulatory role for sex-steroids, particularly estrogen. However, studies including our own show a dichotomous role for estrogen in airway remodeling, making it unclear whether sex hormones are protective or detrimental in asthma, and suggesting a need to explore mechanisms upstream or independent of estrogen. We hypothesize that Kisspeptin (Kp)/KISS1R signaling serves this role. Airway smooth muscle (ASM) is a key structural cell type that contributes to remodeling in asthma. We explored the role of Kp/KISS1R in regulating ASM proliferation. We report novel data that Kp and KISS1R are expressed in human airways, especially ASM, with lower expression in ASM from females compared to males, and asthmatics showing lowest expression compared to non-asthmatics. Proliferation studies showed that cleaved forms of Kp, particularly Kp-10 mitigates PDGF-induced ASM proliferation. Pharmacological inhibition and shRNA knockdown of KISS1R increased basal ASM proliferation, further amplified by PDGF. The anti-proliferative effect of Kp-10 in ASM was found to be mediated by inhibition of MAPK-ERK-Akt pathways, with altered expression of PCNA, C/EBP-alpha, Ki-67, Cyclin-D1, and Cyclin-E leading to cell-cycle arrest at G0/G1 phase. Overall, we demonstrate the importance of Kp/KISS1R signaling in regulating ASM proliferation and a potentially novel therapeutic avenue to blunt remodeling in asthma.
Niyati A. Borkar, Nilesh Sudhakar Ambhore, Rama Satyanarayana Raju Kalidhindi, Christina M. Pabelick, Y.S. Prakash, Venkatachalem Sathish
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