*2.14. Statistical Analysis*

Prism 9 software (GraphPad Software Inc., San Diego, CA, USA) was used to perform the statistical analysis on all datasets. A repeated measures one-way ANOVA, with either Dunnett's or Šídák's post hoc test, as appropriate, was used for analysis of three or more datasets. A Paired Student's *t*-test was used for the analysis of only two datasets. The analysis of datasets with more than one variable were performed using a two-way ANOVA with Šídák's multiple comparisons post hoc test. Asterisks are used in the figures to denote *p* values < 0.05, which were considered significant.

### **3. Results**

#### *3.1. HPP and IP Upregulate HO-1 in Primary Human DC*

We have previously shown that HPP and IP are capable of inducing HO-1, as well as having immunomodulatory effects, in murine macrophages and glia [5]. Therefore, we sought to investigate if *T. brucei*-derived ketoacids have similar effects in primary human DC, as these are crucial immune cells linking the innate and adaptive immune system. DC were treated with HPP or IP (500–1000 μM) for 24 h (these concentrations are similar to the levels of aromatic ketoacids in circulation close to the peak of parasitaemia during trypanosomiasis and have been used in previously published studies [4,5,8,17]). Both HPP and IP were found to be non-toxic to human DC, having no effect on cell viability at these concentrations, and were confirmed to be endotoxin free (Supplementary Figures S1 and S2). HO-1 expression in HPP- and IP-treated DC was next examined by western blot at a range of timepoints (3, 6, and 24 h) and concentrations (250–1000 μM). Immature DC constitutively expressed HO-1 in line with previous reports [18–20], however, treatment with either HPP or IP resulted in a trend towards increased expression of HO-1 at all concentrations tested with a significance observed at 1000 μM in each case (Figure 1A). At this concentration, significant upregulation of HO-1 occurred within 3 h of HPP treatment, while significant induction of HO-1 occurred following 24 h treatment with IP (Figure 1B). Both IP and HPP also increased the total antioxidant capacity of DC and this was observed within 1 h of incubation with either compound (Figure 1C). The ketoacids also appear to be more potent antioxidants than the established HO-1 inducers, carnosol and curcumin (Figure 1C). These results indicate that IP and HPP rapidly enhance the total antioxidant capacity of human DC, while also upregulating the stress-response protein, HO-1.

#### *3.2. HPP and IP Induce HO-1 through Nrf2 Activation*

We have previously reported that HPP and IP activate Nrf2 in murine immune cells, and this is likely the mechanism through which they upregulate HO-1 [5]. We therefore sought to investigate if they have a similar mechanism of action in human DC. To test this, DC were treated with HPP or IP (1000 μM) for 6 and 24 h, and Nrf2 protein expression was measured by western blot. An increase in Nrf2 protein was observed in HPP-treated DC at 6 h (Figure 2A), while IP-treated DC showed increased Nrf2 protein expression (and therefore accumulation) at 24 h (Figure 2A). Both HPP and IP were also found to drive mRNA expression of the additional Nrf2-regulated genes, NQO1 and GSR, further confirming their ability to activate this transcription factor (Figure 2B). Finally, treatment of DC with the Nrf2 inhibitor ML385 (10 μM, a non-toxic dose, Supplementary Figure S3) for 1 h, prior to treatment with either HPP or IP (1000 μM) for 24 h, resulted in a significant decrease in the expression of HO-1 (Figure 2C), further confirming that ketoacid driven HO-1 expression is regulated by Nrf2.

**Figure 1.** Hydroxyphenylpyruvate (HPP) and indole pyruvate (IP) upregulate HO-1 in primary human dendritic cells (DC). (**A**) Primary human DC were left untreated (UT) or incubated with HPP or IP (250–1000 μM) for 24 h. HO-1 expression was detected by western blot. Densitometry results shown are mean ± SEM of the relative expression of HO-1: β-actin from five healthy donors. (**B**) DC were left UT or incubated with HPP or IP at 1000 μM for 3, 6, or 24 h. HO-1 expression was detected by western blot. Densitometry results shown are mean ± SEM of the relative expression of HO-1: β-actin from seven healthy donors. (**C**) Primary human DC were left UT or incubated with HPP or IP at 1000 μM, or carnosol or curcumin (both 10 μM), for 1 h. Total antioxidant capacity of the cells was determined and expressed as an equivalent concentration of Trolox (μM). Pooled data showing the mean (±SEM) from five healthy donors. Repeated measures one-way ANOVA, with Dunnett's multiple comparisons post hoc test, was used to determine statistical significance by comparing means of treatment groups against the mean of the control group (\*\* *p* < 0.01, \* *p* < 0.05). ImageLab (Bio-Rad) software was used to perform densitometric analysis.

**Figure 2.** HPP and IP induce HO-1 through Nrf2 activation. (**A**) Primary human DC were left untreated (UT) or incubated with HPP or IP at 1000 μM for 6 or 24 h. Nrf2 expression was measured by western blot. Densitometry results shown are mean ± SEM of the relative expression of Nrf2: β-actin from five healthy donors. (**B**) Primary human DC were left UT or incubated with HPP or IP at 1000 μM for 24 h. mRNA expression of the Nrf2-dependent genes, NQO-1 and GSR, were measured by RT-PCR. Results show mean (±SEM) for six healthy donors. (**C**) Primary human DC were pre-treated either with or without the Nrf2 inhibitor ML385 (10 μM) for 1 h, prior to incubation with HPP or IP at 1000 μM for 24 h. HO-1 expression was measured by western blot. Densitometry results shown are mean ± SEM of the relative expression of HO-1: β-actin from five healthy donors. (**A**) One-way ANOVA, with Dunnett's multiple comparisons post hoc test, was used to determine statistical significance. (**B**) Two-way ANOVA, with Šídák's multiple comparisons post hoc test, was used to determine statistical significance. (**C**) One-way ANOVA, with Šídák's multiple comparisons post hoc test, was used to determine statistical significance (\*\*\*\* *p* < 0.0001, \*\*\* *p* < 0.001, \*\* *p* < 0.01, \* *p* < 0.05). ImageLab (Bio-Rad) software was used to perform densitometric analysis.
