Epithelial senescence in idiopathic pulmonary fibrosis is propagated by small extracellular vesicles

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease that affects 3 million people worldwide. Senescence and small extracellular vesicles (sEVs) have been implicated in the pathogenesis of IPF, although how sEVs promote disease remains unclear. Here, we profile sEVs from bronchial epithelial cells and determine small RNA (smRNA) content. METHODS: Conditioned media was collected and sEVs were isolated from normal human bronchial epithelial cells (NHBEs) and IPF-diseased human bronchial epithelial cells (DHBEs). RESULTS: Increased sEV release from DHBEs compared to NHBEs (n = 4; p < 0.05) was detected by nanoparticle tracking analysis. NHBEs co-cultured with DHBE-derived sEVs for 72 h expressed higher levels of SA-β-Gal and γH2AX protein, p16 and p21 RNA and increased secretion of IL6 and IL8 proteins (all n = 6-8; p < 0.05). sEVs were also co-cultured with healthy air-liquid interface (ALI) cultures and similar results were observed, with increases in p21 and p16 gene expression and IL6 and IL8 (basal and apical) secretion (n = 6; p < 0.05). Transepithelial electrical resistance (TEER) measurements, a reflection of epithelial barrier integrity, were decreased upon the addition of DHBE-derived sEVs (n = 6; p < 0.05). smRNA-sequencing identified nineteen significantly differentially expressed miRNA in DHBE-derived sEVs compared to NHBE-derived sEVs, with candidate miRNAs validated by qPCR (all n = 5; p < 0.05). Four of these miRNAs were upregulated in NHBEs co-cultured with DHBE-derived sEVs and three in healthy ALI cultures co-cultured with DHBE-derived sEVs (n = 3-4; p < 0.05). CONCLUSIONS: This data demonstrates that DHBE-derived sEVs transfer senescence to neighbouring healthy cells, promoting the disease state in IPF

The IL-33:ST2 axis is unlikely to play a central fibrogenic role in idiopathic pulmonary fibrosis

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease (ILD) with limited treatment options. Interleukin-33 (IL-33) is proposed to play a role in the development of IPF however the exclusive use of prophylactic dosing regimens means that the therapeutic benefit of targeting this cytokine in IPF is unclear. METHODS: IL-33 expression was assessed in ILD lung sections and human lung fibroblasts (HLFs) by immunohistochemistry and gene/protein expression and responses of HLFs to IL-33 stimulation measured by qPCR. In vivo, the fibrotic potential of IL-33:ST2 signalling was assessed using a murine model of bleomycin (BLM)-induced pulmonary fibrosis and therapeutic dosing with an ST2-Fc fusion protein. Lung and bronchoalveolar lavage fluid were collected for measurement of inflammatory and fibrotic endpoints. Human precision-cut lung slices (PCLS) were stimulated with transforming growth factor-β (TGFβ) or IL-33 and fibrotic readouts assessed. RESULTS: IL-33 was expressed by fibrotic fibroblasts in situ and was increased by TGFβ treatment in vitro. IL-33 treatment of HLFs did not induce IL6, CXCL8, ACTA2 and COL1A1 mRNA expression with these cells found to lack the IL-33 receptor ST2. Similarly, IL-33 stimulation had no effect on ACTA2, COL1A1, FN1 and fibronectin expression by PCLS. Despite having effects on inflammation suggestive of target engagement, therapeutic dosing with the ST2-Fc fusion protein failed to reduce BLM-induced fibrosis measured by hydroxyproline content or Ashcroft score. CONCLUSIONS: Together these findings suggest the IL-33:ST2 axis does not play a central fibrogenic role in the lungs with therapeutic blockade of this pathway unlikely to surpass the current standard of care for IPF

Proteolytic Processing of Interleukin-1 Family Cytokines: Variations on a Common Theme

Members of the extended interleukin-1 (IL-1) cytokine family, such as IL-1, IL-18, IL-33, and IL-36, play a pivotal role in the initiation and amplification of immune responses. However, deregulated production and/or activation of these cytokines can lead to the development of multiple inflammatory disorders. IL-1 family members share a broadly similar domain organization and receptor signaling pathways. Another striking similarity between IL-1 family members is the requirement for proteolytic processing in order to unlock their full biological potential. Although much emphasis has been put on the role of caspase-1, another emerging theme is the involvement of neutrophil- and mast cell-derived proteases in IL-1 family cytokine processing. Elucidating the regulation of IL-1 family members by proteolytic processing is of great interest for understanding inflammation and immunity. Here, we review the identity of the proteases involved in the proteolytic processing of IL-1 family cytokines and the therapeutic implications in inflammatory disease

The diversity of myeloid immune cells shaping wound repair and fibrosis in the lung

In healthy circumstances the immune system coordinates tissue repair responses in a tight balance that entails efficient inflammation for removal of potential threats, proper wound closure, and regeneration to regain tissue function. Pathological conditions, continuous exposure to noxious agents, and even ageing can dysregulate immune responses after injury. This dysregulation can lead to a chronic repair mechanism known as fibrosis. Alterations in wound healing can occur in many organs, but our focus lies with the lung as it requires highly regulated immune and repair responses with its continuous exposure to airborne threats. Dysregulated repair responses can lead to pulmonary fibrosis but the exact reason for its development is often not known. Here, we review the diversity of innate immune cells of myeloid origin that are involved in tissue repair and we illustrate how these cell types can contribute to the development of pulmonary fibrosis. Moreover, we briefly discuss the effect of age on innate immune responses and therefore on wound healing and we conclude with the implications of current knowledge on the avenues for future research

Modeling fibrotic alveolar transitional cells with pluripotent stem cell-derived alveolar organoids

Repeated injury of the lung epithelium is proposed to be the main driver of idiopathic pulmonary fibrosis (IPF). However, available therapies do not specifically target the epithelium and human models of fibrotic epithelial damage with suitability for drug discovery are lacking. We developed a model of the aberrant epithelial reprogramming observed in IPF using alveolar organoids derived from human-induced pluripotent stem cells stimulated with a cocktail of pro-fibrotic and inflammatory cytokines. Deconvolution of RNA-seq data of alveolar organoids indicated that the fibrosis cocktail rapidly increased the proportion of transitional cell types including the KRT5-/KRT17+ aberrant basaloid phenotype recently identified in the lungs of IPF patients. We found that epithelial reprogramming and extracellular matrix (ECM) production persisted after removal of the fibrosis cocktail. We evaluated the effect of the two clinically approved compounds for IPF, nintedanib and pirfenidone, and found that they reduced the expression of ECM and pro-fibrotic mediators but did not completely reverse epithelial reprogramming. Thus, our system recapitulates key aspects of IPF and is a promising system for drug discovery