Development of assays to evaluate inflammasomes’ activity in the peripheral blood and in alveolar macrophages in fibrotic lung diseases

Abstract

NOD-like receptors(NLRs) are key cytosolic components of the innate immune system. Upon activation they form the inflammasomes, that mediate the maturation and cleavage of IL-1b, a pro-inflammatory cytokine with fibrotic properties. NLRP3 activation is impaired in alveolar macrophages in Idiopathic Pulmonary Fibrosis (IPF) (Lasithiotaki, ERJ,2016), while decreased NLR pathway expression is associated with abundance of Streptococcus in BAL and decreased progression free survival. Conversely, upregulation of NLRC4 is correlated with worse survival(Salisbury, Curr Opin Pulm Med,2017) Aim of this study is to develop assays for comparison of NLRP3, NLRC4 and AIM2 inflammasomes’ activity at baseline and upon stimulation in alveolar macrophages(AMs) and in Peripheral blood leukocytes(PBMCs) in IPF, other fibrotic lung diseases and healthy subjects. PBMCs and AMs, obtained from blood and BAL of healthy volunteers and patients with lung fibrosis were primed with LPS, followed by inflammasome specific stimuli, including addition of ATP for NLRP3, or transection with Flagellin for NLRC4 and Poly(dA-dT) for AIM2. Stimulation of all three inflammasomes resulted in IL-1b release in PBMCs and AMs. Our preliminary data suggest that, LPS lead to significant IL-1b secretion in CPFE AMs, suggesting that inflammasome stimuli are present in the cells. NLPR3 stimulation resulted in significantly higher IL-1b secretion, in CPFE compared to other fibrotic diseases and IPF. AIM2 and NLRC4 also resulted in higher activation compared to other fibrotic diseases Our preliminary data suggests a particular role of NLRs and IL-1b in CPFE compared to other fibrotic lung diseases.