THE ROLE OF INFLAMASSOME IN RECOGNITION OF THE INFECTION BY THE INTRACELLULAR BACTERIA COXIELLA BURNETII

Dario Simões Zamboni (FMRP-USP); Larissa Dias da Cunha (FMRP-USP)

Coxiella burnetii, the etiological agent of a severe pneumonia called Q-fever, is a Gram-negative obligate intracellular pathogen. The infection occurs via aerosols and the primary target cells are alveolar macrophages. Although Coxiella is highly adapted to avoid vertebrate immune responses, the infection is readily controlled by a competent innate immune response, suggesting that appropriate responses do occur. Herein we aimed to determine if the molecular platforms composed by caspase-1 and specific Nod-like receptors, so called inflammasome, play a role in recognition and restriction of C. burnetii infection. ELISA essays revealed that bone marrow-derived macrophages infected with C. burnetii fail to secrete IL-1B, a cytokine known to be secreted in a caspase-1-dependent pathway. In addition, we did not detect caspase-1 activation by western blot. Alveolar and bone marrow-derived macrophages deficient for caspase-1 do not fail to control replicative vacuole formation and bacterial multiplication, as determined by real time PCR and estimative of large parasitophorous vacuole formation. To investigate whether the absence of caspase-1 activation was due to a subversion of macrophages functions by Coxiella we performed co-infections with Coxiella and Legionella pneumophila, a pathogen known to trigger caspase-1 activation. The activation of caspase-1 and IL-1B secretion in response to Legionella  were not  blocked by the presence of Coxiella. Furthermore, Coxiella does not seem to directly inhibit caspase-1, as the bacteria fail to block caspase-1 autocatalytic activity in a HEK293 cell expression system (transfected with vectors encoding pro-caspase-1). In addition, our data suggests that the bacterium does not interfere with inflammasome activation in response to LPS + ATP, which are potent activators of the inflammasome. Interestingly, cells infected with Coxiella and treated with ATP do not activate caspase-1. In contrast, macrophages treated with heat killed Coxiella + ATP trigger a robust inflammasome response. Regardless to the participation of caspase-1, we found that caspase-3 was required for innate immune responses, as macrophages from caspase-3-/- mice were highly susceptible to Coxiella infection. In conclusion, we found that whereas caspase-3 is unequivocally required to generation of appropriate responses against Coxiella, the caspase-1 inflammasome seems to play no role in the recognition and control of Coxiella infection.