Fig 2: PBMCs from VL Patients Have a Type I IFN Gene Signature(A and B) RNA isolated from peripheral blood mononuclear cells (PBMCs) from VL patients upon admission to the clinic (active VL, n = 7–9), 3–6 months post-treatment (post, n = 6–7), and from ECs (n = 6) was subjected to qPCR to measure IFNA1 and IFNB1 (A) and IFNAR1 and IFNAR2 (B) mRNA transcripts.(C) IFNA1 and IFNB1 mRNA levels were also measured by qPCR in CD14+, CD19+, and CD1c+ cells isolated from VL patient PBMCs (n = 10) by magnetic activated cell sorting (MACS) as well as the flow through cells.(D) Antigen-specific IFN? production was measured in whole-blood cells from VL patients upon admission to the clinic (n = 17), cultured for 24 h with soluble leishmania antigen (SLA) with control, anti-IFNaR1, or anti-HLA-DR antibodies as indicated.Median + minimum and maximum; *p < 0.05, **p < 0.01, and ***p < 0.001; significance assessed by Wilcoxon matched-pairs signed-rank test or Mann-Whitney test, as appropriate.

Fig 3: Induction of TNF-a following neuronal ZIKV infection is a major regulatory mechanism that alters expression of genes relevant to neuronal function. A, B Expression values of candidate genes previously shown to be either downregulated (A) or upregulated (B) by ZIKV infection were measured via qRT-PCR in neuronal cultures treated for 24 h with 10 pg/ml exogenous cytokines: IFN-ß, IL-6, or TNF-a. n = 6. C–E Volcano plots depicting mean z-score and -log10FDR of representative DEGs in neurons treated with IFN-ß (C), IL-6 (D), or TNF-a (E). Significant differences (p < 0.05) are noted in red (upregulated DEGs) or blue (downregulated DEGs). n = 6. F, G) Concentrations of TNF-a in supernatants of in vitro neuronal cell cultures infected with ZIKV for 24 h (n = 4) (F) and brains harvested following in vivo intracranial infection (n = 5) (G). Cytokine concentrations were quantified via ELISA assay. H–M) qRT-PCR analysis was performed for known TNF-a transcriptional target genes, Cd69 (H), Ccl2 (I), Ccl5 (J), Stat1 (K), and TNF-a receptor genes, Tnfrsf1a (L) and Tnfrsf1b (M), in 8-week-old adult mouse brains following ZIKV-MR766 infection at 2 or 4 dpi. n = 5. N, O qRT-PCR analysis of representative DEGs associated with neurological functions (Chac1, Nr4a2, Lhx6, Dpp6, Cacna1e, and Tor3a) following pretreatment with neutralizing antibodies against IFN a/ß receptor (IFNAR) (N) or TNFR1 (O) and subsequent 24 h infection with ZIKV-MR766. n = 3–6 biological replicates per group. Data in (A) and (B) represent normalized and z-transformed values of qRT-PCR expression data. ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. Bars represent group means

Fig 4: Type I IFN Signaling Blockade Acts Synergistically with Ambisome to Improve Anti-parasitic Immunity(A–D) B6 WT mice were infected with L. donovani and, 14 days later, treated with either vehicle control, a single suboptimal dose of Ambisome (Amblo, 1 mg/kg) with or without 0.1 mg anti-IFNaR1 mAb every 3 days for 14 days (A), or with or without 60 mg/kg ruxolitinib (Rux) twice a day for 5 days (B). Parasite burdens in the liver and spleen were measured on day 28 p.i., as indicated, and a full dose of Ambisome (Ambhi, 10 mg/kg) was used as a positive control for drug efficacy. Ifnar1-/- (open columns) and control B6 WT (closed columns) mice were infected with L. donovani for 14 days and then treated with 60 mg/kg Rux twice a day for 5 days. Parasite burden (C) and number of parasite-specific CD4+ T cells (gated as in Figure 4A) (D) were measured on day 28 p.i., as indicated. n = 4–7 mice per group. Experiments were conducted twice. Mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001; significance assessed by one-way ANOVA or Mann-Whitney test, as appropriate.(E) Antigen-specific IFN-? production was measured in whole-blood cells from VL patients upon admission to the clinic and, 3 days after Ambisome treatment (n = 13), cultured for 24 h with SLA with vehicle control or Rux (1 µM), as indicated. Median + minimum and maximum; *p < 0.05; significance assessed by Wilcoxon matched-pairs signed-rank test.

Fig 5: Improved Control of Parasite Growth in the Absence of Type I IFNs Required CD4+ T Cells and IFN?(A and B) Ifnar1-/- (open columns) and control B6 WT (closed columns) mice were infected with L. donovani and treated with a depleting anti-CD4 mAb (A) or a blocking anti-IFN? mAb (B). Parasite burdens were measured in the liver and spleen, as indicated, on day 14 post-infection (p.i.) and compared with mice treated with control antibodies.(C) The number of MZ macrophages (MZMs) per square millimeter of spleen tissue was determined on day 14 p.i., as indicated. Representative images show nucleated cells (blue, DAPI) and MZMs (indicated by uptake of fluorescein isothiocyanate (FITC)-dextran, green) (objective, 20×; scale bars, 500 µm).n = 4–7 mice per group. Experiments were conducted 2–3 times. Mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001; significance assessed by one-way ANOVA or Mann-Whitney test, as appropriate.