Increase in inflammasome activation in acute exacerbation of idiopathic pulmonary fibrosis

Introduction: Acute exacerbation (AE) of IPF is defined as acute periods of disease acceleration of IPF. Clinic and pathophysiology of acute exacerbations of IPF resembles Acute Respiratory Distress Syndrome (ARDS). Several studies demonstrated an increase in pulmonary and circulatory IL-1β levels in ARDS. Increased IL-1β production may play a detrimental role in ARDS. Inflammasomes are intracellular immune receptors which are triggered by PAMPs and DAMPs. Recent studies also indicate the essential role of inflammasome activation and IL-1β in mouse models of pulmonary fibrosis. Rationale: The aim of our study was to test whether inflammasome activation of human alveolar macrophages (AM) is increased in acute exacerbation of IPF and to study potentially involved mechanism.

Methods: The study consisted of a group of 26 patients with IPF and a group with 29 HD. Bronchoalveolar lavage was performed and alveolar macrophages (AM) were isolated. Inflammasome activation was introduced either by ATP or by Nigericin in combination with LPS. AM were stimulated for 6h with ATP and Nigericin w/o LPS. Activation of the inflammasome was analyzed by measurement of active caspase-1p10 subunit by Western blot, NLRP3 mRNA expression and IL1-β production by ELISA. Furthermore, role of efferocytosis of 10Gy radiated apoptotic A549 cells in inflammasome activation was tested and the role of ROS production and Cathepsins by respective inhibitors studied. Results: Our data show an increase in inflammasome activation and inflammasome mediated IL-1β production of AM from IPF patients in comparison to AM from HD (p=0.002[LPS+ATP];p=0.0008[LPS+Nig]). We found increased expression of the active caspase1p10 subunit by Western blot and significantly increased IL1-β production in IPF versus HD(p=0.002). Inflammasome activation was further increased in acute exacerbation. Contact with apoptotic epithelial cells primed inflammasome activation and IL-1β production in AM in vitro (p<0.04). Further experiments revealed an increased relative NLRP3 mRNA expression in AM which were primed with apoptotic A549 cells. This priming effect on NLRP3 expression was blocked by ROS inhibitors (DPI) as well as Cathepsin inhibitors(E64D). Furthermore, inflammasome mediated IL1-β production was decreased in the presence of either ROS inhibitors or Cathepsin inhibitors. Conclusion: Our data show an increase in inflammasome activation and inflammasome mediated IL-1β production of AM from patients with IPF and particularly of AM derived from patients with acute exacerbation of IPF. We propose that phagocytosis of apoptotic alveolar epithelial cells may prime inflammasome activation in IPF.