NLRP3 is a cytoplasmic receptor that, when activated, forms a protein complex called inflammasome, consisting of NLRP3, caspase-1 and an adapter protein (ASC), and responsible for acting in the release of IL-1b and IL-18. Thus, the NLRP3 inflammasome participates in the pathogenesis of many diseases, including allergic reactions such as asthma, atopic dermatitis, rhinitis and allergic conjunctivitis (AC). Studies show that the NLRP3/IL-1b axis plays an important role in the development of AC. Furthermore, the expression of the Gal-3 protein plays an important role in the activation of the NLRP3 inflammasome and in the production of IL-1b by macrophages, however, its relationship with NLRP3 activation has not been investigated in the allergic response. Thus, in this project, we will evaluate the role of Gal-3 in the allergic response and its possible relationship with the regulation of the NLRP3 inflammasome, using an experimental AC model and assays with cultured human keratinocytes stimulated with IL-4. In the experimental model of ovalbumin-induced AC (OVA), wild-type Balb/c (WT) and Gal-3 knockout (Gal-3-/-) mice will be used and the palpebral conjunctivae will be evaluated under the following conditionsexperimental (n = 5 animals/group): Sham (control) and AC. Under these conditions, we will analyze: i) histopathology of the eyelids and quantification of inflammatory cells; ii) localization and possible modulation of NLRP3, caspase-1 and IL-1b expression by means of immunohistochemistry; and iii) possible co-localization of NLRP3 in mast cells by immunofluorescence. Furthermore, we will evaluate, in vitro, the effect of administering Gal-3recombinant in human keratinocytes, HaCaT lineage, stimulated by IL-4. In cell culture, cell viability assays will be performed, as well as IL-1b dosage and investigation of the expression of NLRP3, caspase-1 and p38 kinase (MAPK14). In publicly available transcriptomes from the Gene Expression Omnibus (GEO) repository, we will assess transcriptional levels of LGALS3, NLRP3, CASP1 and MAPK14 in studies involving in vitro assays with mast cells and keratinocytes related to allergic responses. The results will contribute to a better understanding of the role of Gal-3 in the activation of the NLRP3 inflammasome in the allergic response, as well as may indicate possible therapeutic targets for ocular allergic processes.
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