Phenotypic Characterization of Macrophage and Monocyte Populations in the NLRP3 KO DAHL/SS‐Nlrp3em2Mcwi Rat Model

Abstract

BackgroundThe NLR family pyrin domain containing 3 (NLRP3) inflammasome is a multimeric protein complex that is activated in response to pathogen and damage associated molecular patterns (PAMPs & DAMPs) to initiate inflammatory responses through IL‐1ß, IL‐18, and pyroptosis. Pharmacological inhibition of NLRP3 attenuates inflammatory responses after myocardial infarction and hypertension in rodents. However, the effect of NLRP3 gene loss on basal macrophage and monocyte populations in rats is unknown. We hypothesized that NLRP3 gene loss would not alter macrophage and monocyte phenotype under basal conditions.MethodsCRISPR‐Cas9 was used to induce a 14 base pair deletion in exon 1 of the NLRP3 gene in the DAHL/SSJrHsdMcwi strain to produce NLRP3 KO DAHL/SS‐Nlrp3em2Mcwi rats. NLRP3 KO male (n=11), NLRP3 KO female (n=10), wild type (WT) male (n=5), and WT female (n=10) were studied at 4‐5 mo. Teklad 7034 low salt (0.12% NaCl) diet was provided ad libitum. Proinflammatory M1 (CD11b+CD68+) and anti‐inflammatory M2 (CD11b+CD206+) macrophages (Mɸs) were quantified in CD45+ cells from left ventricle (LV), peripheral blood monocytes (PBMCs), bone marrow (BMDMs), and splenocytes using density gradient centrifugation and flow cytometry.ResultsSex (p<0.01) and genotype (p<0.01) main effects were observed in several morphological characteristics. NLRP3 KO and male rats exhibited significantly increased rat, kidney and LV weights, and LV/tibial length vs WT and female, respectively. Males also displayed significantly increased spleen length and visceral adipose weight vs females (p<0.01). In LV, the percent of CD11b+CD68+ cells was 28% greater in NLRP3 KO vs WT (n=1‐2/group). In PBMCs, the percent of CD11b+CD206+ cells was 13% greater in WT female vs males and NLRP3 KO (n=2‐4/group), while NLRP3 KO females exhibited ~20% less CD11b+CD68+ in splenocytes (n=1‐4/group). In BMDMs, ~60% were positive for CD11b+CD68+, while ~2% were CD11b+CD206+ across all groups (n=1‐2/group).ConclusionPreliminary data suggest that NLRP3 gene loss on a Dahl salt sensitive background is associated with increased proinflammatory M1 Mɸs in LV concomitant with increased LV mass, independent of sex. We hypothesize that NLRP3 may possess an inflammasome independent role in regulating resident LV macrophages under basal conditions.