Fig 2: Ifnar1-Deficient Mice Have Improved Parasite Clearance and an Improved Inflammatory Response(A and B) Ifnar1-/- (open circles and columns) and control B6 WT (closed circles and columns) mice were infected with L. donovani, and liver and spleen parasite burdens (A) and organ weights (B) were measured on days 7, 14, 28, and 56 p.i., as indicated.(C and D) Serum cytokine (IFN?, TNF, and IL-10) levels were also measured (C), as well as cytokine production by splenic mononuclear cells isolated from infected mice on day 14 p.i., after 72 h in culture with fixed L. donovani amastigotes (D).n = 4–7 mice per group. Each experiment was conducted 3–5 times. Mean ± SEM; *p < 0.05 and **p < 0.01; significance assessed by one-way ANOVA or Mann-Whitney test, as appropriate.

Fig 3: Type I IFNs Act on DCs to Suppress Development of Antigen-Specific CD4+ T Cells(A) A 90:10 mix of B6.CD45.1/CD45.2 and Ifnar1-/- (CD45.2) BM cells was engrafted into lethally irradiated CD45.1 recipients.(B) Following 10–12 weeks of engraftment, chimeric mice were infected with L. donovani, and 14 days later, spleen and liver mononuclear cells were isolated to assess the activation status of control B6 (CD45.1/CD45.2) and Ifnar1-/- (CD45.2) CD4+ T cells from the same mouse using the gating strategy shown.(C) The frequency of splenic and hepatic CD4+ T cells recently activated by parasite antigen (CD11ahi/CD49dhi), Th1 cells, and Tr1 cells (gated as shown in Figure S2) was measured by flow cytometry, as indicated.(D) Ifnar1?DC mice generated by crossing Cd11c-Cre mice with Ifnar1-floxed animals were infected with L. donovani, and 14 days later, Th1 cell number and frequency as well as parasite burden in the spleen and liver were measured, as indicated.n = 4–10 mice per group. Experiments were conducted 3 times. Mean ± SEM (C and D), *p < 0.05; significance assessed by Mann-Whitney test.

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.