| Abstract |
The primary pathogenetic event of idiopathic pulmonary fibrosis (IPF) remains uncertain, despite increased clinical and scientific research in recent years. The median survival from the time of diagnosis is 3 years and explains why IPF is currently considered more lethal than many cancers .The radiologic and histopathologic pattern of IPF is that of usual interstitial pneumonia (UIP) . UIP, unlike the other collagen tissue disorders, is also the most frequent pattern when interstitial lung involvement occurs in patients with rheumatoid arthritis (RA-UIP) . RA-UIP and IPF present some rather intriguing similarities at the clinical and molecular level. Therefore, it is critical to identify initiating events that may drive the pathogenesis of lung fibrosis, both IPF and autoimmune lung fibrosis, with the histologic or radiologic appearance of UIP especially now that we have officially entered a new erawith two new agents able to slow IPF progression. Inflammasomes are multimolecular protein complexes responsible for caspase-1-driven activation of the pro-inflammatory cytokine interleukin (IL)-1β . They are involved in the innate immunity by recognising pathogen-associated molecular patterns (PAMPs; on bacteria, viruses and fungi), and intracellular and extracellular damage-associated molecular patterns. Furthermore, innate immunity mechanisms have been proposed to have a fundamental role in the initiation of IPF, mainly through the activation of Toll-like receptor (TLR)-9, which drives the rapid progression of fibrotic lung disease through the differentiation of pulmonary fibroblasts into myofibroblasts. NLRP3 activation could also be impaired in IPF, as is the innate immune response to bacteria and virus infections in IPF fibroblasts and macrophages.Previous studies in animal models have shown that stimulation with asbestos, bleomycin, silica and statins activates inflammasomes and ultimately the production of active IL-1β, which stimulates the production of transforming growth factor (TGF)-β, a central mediator of the development of lung fibrosis, via fibroblast proliferation, collagen production and inhibition of fibroblast apoptosis. Interestingly, it has been postulated that pharmacologically induced NLRP3 activation leads to interstitial lung disease.Caspase-1 activity has also been directly linked to systemic sclerosis-related fibrosis, which suggests that autocrine signalling mediated by IL-1β and IL-18 promotes the pro-fibrotic phenotype observed in these patients. Moreover, NLRP3 inflammasome activation has been linked to neutrophilic asthma ], while inflammasome-regulated cytokines are critical mediators of acute lung injury. Alternatively, a recent report suggests that the NLRP3 inflammasome pathway is not linked to the severity of stable chronic obstructive pulmonary disease following analyses in the bronchoalveolar lavage fluid (BALF) andcellular components. Recent evidence showing a possible activation of NLRP3 by cigarette smoking, another environmental risk factor for both IPF and rheumatoid arthritis, further supports the possible implication of inflammasomes in lung fibrosis. The role of IL-1β has emerged in the pathogenesis of cigarette smoke-induced emphysema and small airway remodelling.In the current study, we sought to investigate whether NLRP3 inflammasomes are involved in the pathogenesis of lung fibrosis in idiopathic (IPF) and autoimmune (RA-UIP) lung diseases. We used IL-1β and IL-18 cytokine expression in BALF and BALF macrophage cultures as surrogate markers of NLRP3 activation. Our findings suggest distinct inflammasome activation profiles between autoimmune lung fibrosis and IPF. The current study investigated the activation of the NLRP3 pathway in BALF from patients with IPF and RA-UIP at baseline and following NLRP3 inflammasome activation.Our findings suggest distinct inflammasome activation profiles between autoimmune lung fibrosis and IPF.Overall, our results suggest that inflammasome activation is severely impaired in IPF BALF macrophages. Furthermore, the inflammasome activation profile is distinct in RA-UIP, since IL-1β and IL-18 levels are already high at baseline, albeit upregulation is still possible, as shown by the further increase in the newly produced intracellular mature IL-1β levels following LPS plus ATP treatment. Inflammasomes are activated in RA-UIP lung. We observed that IL-1β and IL-18 levels were elevated in RA-UIP BALF. IL-1β and IL-18 are, at least in part, secreted by alveolar macrophages since we observed that the unstimulated macrophage cultures produced significantly higher levels of these cytokines as compared with controls. In addition, mRNA levels of other components of the NLRP3 pathway such as NLRP3 and caspase-1 were also upregulatedRA-UIP-derived BALF cells. Impaired activation of the NLRP3 inflammasome in IPF. In IPF, in contrast to RA-UIP, we observed only slightly higher levels of IL-1β in the BALF relative to controls, while cultured BALF cells secreted slightly lower IL-1β levels than controls. In both cases, however, IL-1β levels were significantly lower when compared with RA-UIP samples. Intriguingly, in IPF, similarly to RA-UIP samples, baseline NLRP3 and caspase-1 expression in cultured BALF macrophages as well as IL-18 and IL-6 levels were higher than in the controls.The main finding of this work is that alveolar macrophages from IPF patients exhibited impaired responses to LPS stimulation and LPS/ATP co-stimulation, leading to the hypothesis that both TLR-4 and NLRP3 inflammasome pathways are dysregulated. In particular, we observed no NLRP3-specific induction of either intracellular or secreted IL-1β in alveolar macrophage cultures in IPF, while the induction of IL-18 was significantly lower in IPF relative to RA-UIP and controls.Our observations are in agreement with cumulative evidence that the innate immune response is impaired in patients with IPF, including the reduced functional capacity of macrophages to kill bacteria . Our present findings suggest that deregulation of inflammasome activation in fibrotic patients could be crucial in the progression of the disease, in particular with respect to the occurrence of acute exacerbations. Immune mechanisms have been implicated in IPF and are also central to the development of rheumatoid lung. However, autoimmunity in IPF needs to be interpreted with caution, as it could provide a possible future therapeutic target. Our study provides further evidence for the association of innate immunity with IPF; however, this effect is more prominent, as expected, in rheumatoid lung.As the inflammasome plays a central role in host defence against wide variety of infectious and noninfectious agents, this finding further supports the dysregulation of the innate immune response of IPF patients which has been linked to the pathogenesis of IPF and in particular the inability of these patients to resolve lung infections linked to acute exacerbations.The importance of inflammation in initiation and progression of pulmonary fibrosis has already been established , while the initial pro-inflammatory scenario of inflammasome activation can rapidly progress to a fibrotic one. Our findings reveal a significant lapse in NLRP3 inflammasome activation in alveolar macrophages in IPF, while suggesting an impaired activation of NLRP3 in autoimmune fibrosis, compared with healthy macrophages. It is thus imperative to explore all possible pathological and therapeutic implications of inflammasomes in the fibrotic lung.
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