NLRC4 and NLRP3 inflammasomes in experimental periodontal disease induced by Aggregatibacter actinomycetemcomitans

Abstract

Periodontal disease is the most prevalent lytic disease of bone in humans. It is initiated and maintained by microorganisms in the dental biofilm, but most of the tissue damage is mediated by the immune response to these microorganisms. It is thus, a good study model for host-microbial interactions and the information generated from these models can be useful for various conditions of infectious origin and also conditions characterized by a chronic inflammatory response. Inflammasomes are multi-protein complexes that include a nucleotide-oligomerization domain leucine-rich repeat containing receptor (NLR), caspase-1 and an adaptor protein. These complexes participate in the sensing of microbial (MAMPs) and tissue damage (DAMPs)-derived molecular patterns by the innate immunity. Caspase-1 is the central effector molecule in these protein complexes and participates in the processing of inflammatory cytokines and apoptosis. NLRP3 (Nalp3) has an essential role in caspase-1 activation in response to microbial and some non-microbial stimuli. Its relevance for immune regulation is evidenced by the association of genetic mutation of its protein components with various auto-immune diseases. NLRC4 (Ipaf) inflammasome is also expressed in myeloid cells and participates in the immune response to Gram-negative bacteria presenting a type III or IV secretory system; but its role on the bone resorption associated with periodontal infection has not been investigated, whereas there is scarse information derived from correlation studies in vivo on the role of NLRP3. Our hypothesis is that the NLRC4 and NLRP3 inflammasomes have important roles in the immune response associated with destructive periodontal disease and affects alveolar bone resorption. Preliminary experiments demonstrate a marked increase of the alveolar bone resorption induced by bacterial LPS in NLRC4 and Caspase-1 knockout mice. The fact that NLRC4 KO mice presenting normal Caspase-1 expression, which could be activated by other inflammasomes also showed aggravated bone loss indicate that this inflammasome have an important role in the immune response in periodontal tissues that is not redundant with that of other caspase-1 activating inflammasomes, such as NLRP1 and NLRP3. T-cell mediated inhibition of inflammasomes has been recently described as an endogenous control of the activation of innate immune cells, demonstrating the reciprocal regulation between the innate and adaptive arms of the host response. This inhibition of NLRP1 and NLRP3 inflammasomes involves not only Treg but also other subtypes of CD4+ T cells and particularly TNF-family genes, such as TNF-alpha and RANKL. Interestingly, activated CD4+ T cells were not able to inhibit the activity of NLRC4 inflammasome in macrophages. A better understanding of the role of the different inflammasomes in various disease models, as well as of the molecular mechanisms of inflammasome inhibition can provide novel opportunities for immune modulation therapies. To determine the role of NLRC4 and NLRP3 inflammasomes in periodontal disease and test our main hypothesis, we propose the following specific aim: - Determine the role of NLRC4and NLRP3 on the immune response in experimental periodontal disease. We will use genetically 'normal' mice (wild-type, WT) and deficient (knockout, KO) for NLRC4, NLRP3 and Caspase-1. Periodontal disease will be induced by injecting heat-killed Aggregatibacter actinomycetemcomitans into the palatal gingival tissues adjacent to the upper first molars. Immune response will be characterized by analyzing the production of inflammatory cytokines in the diseased gingival tissues using multiplex-based assays on the luminex platform. Descriptive, stereometric and immunohistochemistry analysis will also be used to study the inflammatory response in the periodontal microenvironment. Quantitation of alveolar bone loss will be performed by computer microtomography (uCT). (AU)