Role Of NLRP7 Inflammasome In Host Defense Against Mycobacterium Bovis In THP-1 Macrophages

Mycobacterium bovis, a member of Mycobacterium tuberculosis complex (MTBC), is the causative agent of bovine tuberculosis which is chronic, wasting and infectious disease. It is estimated to infect more than 50 million cattle per annum with concomitant economic losses of approximately$3 billion worldwide. It is able to cross the species barrier and infects a variety of mammals, including human beings. About 2% of human tuberculosis cases are caused by M. bovis. It badly intimidates the public health and also imposes limitations on the potential for its eradication as well as prevention and control.Macrophages are considered the first line of host defense against invasive microbes. Upon mycobacterial infection, they initiate inflammatory responses by releasing cytokines. In vio and in vitro studies shows that IL-1β is a potent mediator, playing a critical role against mycobacteria. M. bovis bears lipopeptide in the cell wall and membrane, and secrets lipoproteins, including MPB70/80. NLRP7 inflammasome is composed of NLRP7, ASC and caspase-1, triggers caspase-1 maturation and IL-1βsecretion by sensing lipopeptide. However, whether NLRP7 inflammasome is activated following M. bovis infection in THP-1 macrophages and its role in host defense are still unclear. Here we stimulated THP-1 macrophages for 2 hours, then the cells were washed using PBS to remove the extracellular bacteria. The results showed that proIL-1βwas markedly enhanced at the protein level 3 hours postinfection, and IL-1βsecretion was increased in a dose-dependent manner between 3 and 50 hours postinfection. M. bovis also induced caspase-1 maturation in a dose-dependent way at MOIs ranging from 0.1 to 100, and led to release of IL-1β into the supernatant in a dose-dependent fashion at MOIs ranging from 0.1 to 10, but IL-1β secretion was not enhanced further at an MOI of 100. Caspase-1 maturation was indispensable to IL-1βsecretion by M. bovis-infected cells. Internalized M. bovis was required for caspase-1 maturation and IL-1β secretion. M. bovis infection induced aggregation of NLRP7, ASC and caspase-1, increased mRNA level of NLRP7, and colocalization with ASC and caspase-1. NLRP7 is involved in IL-1β secretion by THP-1 macrophages because NLRP7-or ASC-silencing resulted in obvious decrease of IL-1β. NLRP7 inflammasome activation by M. bovis contributed to TNF-α CCL3 and IL-1βexpression at the mRNA level, and also caspase-1 dependent pyroptosis. NLRP7-or ASC-silencing led to increased survival rates of intracellular bacteria, indicating that M. bovis survival is inhibited by NLRP7 inflammasome. Inflammasome is regulated by multiple factors. To clarify the regulating factors in NLRP7 inflammasome activation in M .bovis-infected THP-1 macrophages, we explored the roles of high concentration of potassium, de novo protein synthesis, deubiquitination and generation of mROS using the respective inhibitors:high concentration of potassium, cycloheximide, PR-619 and NAC. Cells were treated with each inhibitor and M. bovis, or Pam3CSK4, a NLRP7 inflammasome agonist. IL-1βsecretion and transcriptional levels of all NLRP7 inflammasome components were detected. The results indicated that NLRP7 inflammasome activation by M. bovis was inhibited by high concentration of potassium, and requires de novo protein synthesis. Generation of mROS was necessary to IL-1(3 secretion by infected macrophages, but made no difference to Pam3CSK4-stimulated cells, suggesting that generation of mROS is not involved in NLRP7 inflammasome activation by M. bovis. Deubiquitination promoted IL-1β secretion by M. feovw-infected macrophages, but inhibited NLRP7 inflammasome activation. Only change of ASC transcriptional level is consistent with that of IL-1βsecretion.Taken together, M. bovis induces NLRP7 inflammasome activation in THP-1 macrophages, which is regulated by high concentration of potassium and de novo protein synthesis, and not affected by generation of mROS. Deubiquitination inhibits NLRP7 inflammasome activation, which down-regulates IL-1βsecretion induced by M. bovis.