Fig 2: HFD evokes glial activation in the nodose ganglia after 20 weeks on diet. Immunofluorescence images show the expression of GFAP (green), TLR4 (red), and Dapi (blue) in the nodose ganglia and relative immunolabeling quantification at weeks 6 and 20 (a–c) of the diet protocol. BDNF (green) and HuCD (red) expression in nodose ganglia and relative quantification is shown at the same time points (d–f). Data were analyzed by 2-way ANOVA and Dunnett post hoc. Results are expressed as average relative fluorescence units (RFU) ± SEM per area unit of n assessments. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001 versus relative SD group. Scale bar = 20 µm

Fig 3: Lipophosphoglycans (LPGs) from L. amazonensis and L. braziliensis are molecules responsible for inducing IL-32 production. LPGs induce IL-32 in a TLR4- and NOD2-dependent manner in human PBMCs. After LPG endocytosis by TLR4 it can escape the endosomes and activate NOD2. IL-32 can increase the production of pro-inflammatory cytokines such as IL-6 and IL-1ß induced by LPGs.

Fig 4: Induction of IL-32 by lipophosphoglycans from Leishmania spp. occurs in a TLR4-dependent manner. Peripheral blood mononuclear cells (5 x 105 cells/200 µL) were treated or not with LPS from Bartonella quintana (BartLPS, 100 ng/ml) for 1 h. After the incubation time, cells were stimulated with LPG (10 µg/ml) of (A) L. amazonensis (La) or from (B) L. braziliensis (Lb) or (C) LPS from E. coli (100 ng/mL), for an additional 24 h. Cell lysates were obtained for IL-32 evaluation by ELISA. IL-32 production was induced by LPG of L. amazonensis (A), L. braziliensis (B) and E. coli LPS (C). Data represent individual measurements and median values (n = 8). *p < 0.05 (vs. control) by Wilcoxon test.

Fig 5: Compressive forces and TLR4 blocking antibody modulate TLR4 production and MyD88 in primary PDL cells. (A) Protein levels of TLR4 and MyD88 were determined by Western blot in different conditions: HMGB1 (100 ng/mL), TLR4 blocking antibody (5 µg/mL) (TLR4: TLR4 blocking antibody) and compressive force (CF) 2 g/cm2 for 3 and 24 h. Three different donors showed similar patterns with reduction of TLR4 antibody. TLR4 production was upregulated under 3 h compression forces. MyD88 production was reduced in all conditions. (B) Quantification of three donors, normalized to the control with stain-free technology. Data were tested for normal distribution by Shapiro–Wilk test. Afterward, a one-way analysis of variance (ANOVA) followed by Tukeys’ post hoc test was performed.