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Soumyava Basu, Nagaraj Kerur, Benjamin J Fowler, Jayakrishna Ambati; Mycobacterium tuberculosis secreted protein ESAT-6 activates the NLRP3 inflammasome in retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2878.
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To demonstrate role of early-secreted antigenic target protein 6 (ESAT-6) of Mycobacterium tuberculosis, in activation of nucleotide-binding domain, leucine-rich repeat containing protein 3 (NLRP3) inflammasome in retinal pigment epithelium (RPE)
Mouse RPE cell cultures from C57BL/6J and NLRP3 knock out mice were challenged with ESAT-6 antigen. Caspase-1 activation (in cell lysates) and IL-1beta (in cell culture supernatants) were evaluated by western blotting and ELISA respectively. Sub-retinal injections of ESAT-6 and PBS respectively, were administered to C57BL/6J mice and immunohistochemical staining done with rabbit antibody against NLRP3 (1:100, Sigma-Aldrich) and Caspase-1 (pre-diluted, AbCam). RPE/ choroid lysates from ESAT-6 injected eyes will be tested for IL-1beta, and caspase-1 cleavage by Western blotting.
Caspase-1 activation product (p20) and IL-1beta were noted in C57BL/6J but not in NLRP3 knock out RPE cell cultures, after ESAT-6 challenge. Immunohistochemical staining revealed constitutive expression of NLRP3 and Caspase-1 in mouse RPE. IL-1beta and caspase-1 activation were noted after sub-retinal injections of ESAT-6.
ESAT-6 induces NLRP3-dependent IL-1beta production in RPE
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