Figure 4.

Hydroxychloroquine (HCQ) Prevents the Induction of Trained Immunity

(A) Schematic representation of the interaction between the lysosome and the AKT/mTOR signaling pathway.

(B) Schematic representation of the trained immunity assay.

(C–E) PBMCs were stimulated for 24 h with HKCA with or without specified inhibitors or RPMI as a control. After a 5-day resting period, cells were restimulated for 24 h, and cytokine production was measured in the supernatant.

(C) HCQ and chloroquine prevent the induction of a trained immune response to LPS (B, n = 7–17).

(D and E) HCQ and chloroquine prevent the induction of a trained immune response to poly I:C (D, n = 5), IFNα (E, n = 5), IFNβ (E, n = 5), and IFNγ (E, n = 5-8, n.d., not detectable).

(F) mTOR inhibition prevents the induction of a trained immunity response to LPS (n = 11).

(G) Inhibiting lysosome acidification with bafilomycin A prevents the induction of a trained immune response to LPS (n = 4–9).

(H and I) PBMCs were stimulated with HKCA, HKCA+HCQ, or RPMI as a control for 24 h. Subsequently, monocytes were purified and transcriptome analysis was performed. (n = 3 for each treatment group)

(H) Heatmap of most significantly changing genes between HKCA-stimulated and control PBMCs.

(I) Pathway analysis of most significant genes identified (FDR, <0.05) between HKCA-treated cells and HKCA+HCQ-treated cells. Normalized enrichment score is shown for HKCA+HCQ and HKCA, with positive values showing pathways upregulated in HKCA and negative values indicating pathways upregulated in HKCA+HCQ.

Data are presented as mean ± SEM; ˆp < 0.06, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; one-way ANOVA with Dunnett’s post-test.