Fig 2: GSH attenuates OXA-aggravated hepatic oxidative stress and inflammation in NAFLD mice. (A-D) RT-PCR was employed to investigate the mRNA levels of (A) MDA, (B) ROS, (C) SOD and (D) GSH-Px in the liver tissues of each group 9 days after the first dose of OXA. (E-G) Flow cytometry detection of the intracellular cytokines (E) TNF-α, (F) IFN-γ and (G) IL-17 was performed on CD3+ T cells from each group 9 days after the first dose of OXA. (H) mRNA levels of TNF-α, IFN-γ and IL-17 in the liver tissues of each group 3 days after the final dose of OXA. The results are presented as the mean ± standard deviation from five mice in each group. *P<0.05 compared with the NA group; #P<0.05 compared with the NA + OXA group. GSH, reduced glutathione; OXA, oxaliplatin; NAFLD, non-alcoholic fatty liver disease; RT-PCR, reverse transcription-polymerase chain reaction; MDA, malondialdehyde; ROS, reactive oxygen species; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; TNF, tumor necrosis factor; IFN, interferon; IL, interleukin; NA, NAFLD control.

Fig 3: BoNT/A silencing of B16F10-innervating neurons decreases tumour growth.(a–e) Splenocytes-isolated CD8+ T cells from naive C57BL6J mice were cultured under Tc1-stimulating conditions (ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then exposed to BoNT/A (10–50 pg/µL) for 24h; effects on apoptosis, exhaustion, and activation were measured by flow cytometry. When compared to vehicle-exposed cells, BoNT/A did not affect the survival (a) of cultured cytotoxic CD8+ T cells, nor their relative expression of IFN?+ (b), TNF+ (c), IL-2+ (d) and PD-1+LAG3+TIM3+ (e). (f) B16F10 (1x105 cells) were cultured for 24h and subsequently exposed to BoNT/A (1.6-50 pg/µL) or its vehicle for an additional 24h. BoNT/A did not trigger B16F10 cells apoptosis, as measured by the mean fluorescence intensity of Annexin V. (g–n) One and three days prior to tumour inoculation (defined as prophylactic), the skin of 8-week-old male and female mice was injected with BoNT/A (25 pg/µL; i.d.) or its vehicle. One day after the last injection, orthotopic B16F10-mCherry-OVA (5x105 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. In another group of mice, BoNT/A was administered (25 pg/µL; i.d.) one and three days after the tumour reached a volume of ~200mm3 (defined as therapeutic). The effect of neuron silencing on tumour size and tumour-infiltrating CD8+ T cell exhaustion was measured. Nineteen days post tumour inoculation, we found that the tumour volume (g,h) and weight (i) were reduced in mice treated with BoNT/A (Prophylactic group). In parallel, we found that silencing tumour-innervating neurons increased the proportion of IFN?+ (k), TNF+ (l), and IL-2+ (m) CD8+ T cells. BoNT/A had no effect on the total number of intratumoral CD8 T cells (j) or the relative proportion of PD-1+LAG3+TIM3+ (n) CD8+ T cells. (o) One and three days prior to tumour inoculation, the skin of 8-week-old male and female sensory neuron-intact or ablated mice was injected with BoNT/A (25 pg/µL; i.d.) or its vehicle. One day following the last injection, orthotopic YUMMER1.7 cells (5×105 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. The effects of nociceptor neuron ablation on tumour size and volume were measured. Thirteen days post tumour inoculation, we found that the tumour growth was lower in mice treated with BoNT/A or in sensory neuron-ablated mice. BoNT/A had no additive effects when administered to sensory neuron-ablated mice. (p) One and three days prior to tumour inoculation, the skin of 8-week-old male and female mice was injected with BoNT/A (25 pg/µL; i.d.) or its vehicle. One day following the last injection, orthotopic B16F10-mCherry-OVA cells (5×105 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. On days 7, 10, 13 and 16 post tumour inoculation, the mice were exposed to aPD-L1 (6 mg/kg, i.p.) or its isotype control. Eighteen days post tumour inoculation, we found that neuron silencing using BoNT/A potentiated aPD-L1-mediated tumour reduction. Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25th to 75th percentile and the middle line indicates the median), for which individual data points are given (a–f; h–n) or as mean ± S.E.M (g,o,p). N are as follows: a-e: n = 5/groups, f: n = 3/groups, g–i: vehicle (n = 12), BoNT/A therapeutic (n = 12), BoNT/A prophylactic (n = 10), j: vehicle (n = 11), BoNT/A therapeutic (n = 12), BoNT/A prophylactic (n = 8), k–n: vehicle (n = 10), BoNT/A therapeutic (n = 12), BoNT/A prophylactic (n = 8), o: intact + vehicle (n = 9), ablated + vehicle (n = 8), intact + BoNT/A (n = 10), ablated + BoNT/A (n = 8), p: vehicle (n = 7), aPD-L1 (n = 8), aPD-L1 + BoNT/A (n = 7). Experiments were independently repeated two (a–f, o–p) or four (g–n) times with similar results. P-values are shown in the figure and determined by one-way ANOVA posthoc Bonferonni (a–f, h–n) or two-way ANOVA post-hoc Bonferroni (g,o,p). Source Data

Fig 4: Palmitic acid increases maximal cell metabolism and inflammation in macrophages in culture. (A) Schematic of innate immune memory in vitro models using bone marrow derived macrophages and 0.4 mM palmitic acid or control (DMSO). (B) Extracellular acidification rate (ECAR) and (C) oxygen consumption rate (OCR) during modified mitochondrial stress test by Seahorse metabolic analyzer after 6-day washout (D) 100 ng/mL LPS- induced TNF and IL-6 production by ELISA and normalized by protein concentration. (E) 100 ng/mL LPS-, 1 μg/mL LTA- or 1μg/mL P(I:C)- induced TNF and IL-6 production by ELISA and normalized by protein concentration. (F) 100 ng/mL LPS-induced TNF and IL-6 production by ELISA in WT or TLR4-KO mice and normalized by protein concentration. (G) 0.5 mM 5’-methylthioadenosine (MTA) or 10 μM metformin were added during the initial 24-hour treatment with palmitic acid. TNF and IL-6 production was measured by ELISA after 100 ng/mL LPS activation. Data are means ± SEM of 3-7 populations, representative of 2-3 independent experiments. *p < 0.05, **p < 0.01, *** p <0.001, ****p <.0001, NSD= no significant difference.

Fig 5: Transcriptional changes in adipose macrophages from weight loss (WL) and weight cycled (WC) mice suggest innate immune memory. (A) Weight cycling schematic. (B) Violin plot of inflammatory (Tnf, Il1b, Cd83) and metabolic genes (Ldha, Pkm) from lean, obese, weight loss (WL), and weight cycled (WC) macrophage clusters. (C) Pathway analysis of differentially expressed genes from WC vs. obese or WL vs. lean macrophage clusters. (D) Venn diagram of differentially expressed genes of obese, WL, and WC adipose macrophage clusters compared with lean and pathway analysis of the genes differentially expressed in both WL and WC groups.