Type I Interferons Regulate Immune Responses in Humans with Blood-Stage Plasmodium falciparum Infection

Abstract

The development of immunoregulatory networks is important to prevent disease. However, these same networks allow pathogens to persist and reduce vaccine efficacy. Here, we identify type I interferons (IFNs) as important regulators in developing anti-parasitic immunity in healthy volunteers infected for the first time with Plasmodium falciparum. Type I IFNs suppressed innate immune cell function and parasitic-specific CD4⁺ T cell IFNγ production, and they promoted the development of parasitic-specific IL-10-producing Th1 (Tr1) cells. Type I IFN-dependent, parasite-specific IL-10 production was also observed in P. falciparum malaria patients in the field following chemoprophylaxis. Parasite-induced IL-10 suppressed inflammatory cytokine production, and IL-10 levels after drug treatment were positively associated with parasite burdens before anti-parasitic drug administration. These findings have important implications for understanding the development of host immune responses following blood-stage P. falciparum infection, and they identify type I IFNs and related signaling pathways as potential targets for therapies or vaccine efficacy improvement.

Keywords: CD4(+) T cells; Th1 cells; Tr1 cells; immune regulation; malaria; type I interferons.

Figure 1. Blood-Stage P. falciparum Infection Induces Parasite-Specific CD4⁺ T Cell Responses

(A) Volunteers were injected with 1,800 pRBCs/mL, and blood parasitemia was monitored by PCR beginning day 4 p.i. until day 35 p.i. (cohort 6; n = 7). Drug treatment commenced on day 7 p.i. (as shown by the arrow). (B) PBMCs were isolated from participants throughout infection and cultured in the presence of nRBCs or pRBCs as indicated for 72 hr, and then cell culture supernatants were harvested and IFNγ levels were measured (cohorts 6–8; n = 16). (C) PBMCs were isolated from participants 7 days p.i. and cultured in the presence of pRBCs and anti-HLADR antibody or its isotype control for 72 hr, and then cell culture supernatants were harvested and IFNγ levels were measured (cohorts 9 and 10; n = 13). (D) The frequencies of CD4⁺ and CD8⁺ T cells in PBMCs isolated from volunteers at day 0 (D0) and day 7 (D7) p.i. were measured by flow cytometry (cohorts 1–5; n = 33). Mean ± SEM (A, B, and D), median + min and max (C). ***p < 0.001, **p < 0.01, Wilcoxon matched-pairs signed rank test. See also Figure S1.

Figure 2. Blood-Stage P. falciparum Induces IFNα Production upon First Exposure and Is Produced by Various Cell Types

(A) PBMCs from volunteers at days 0, 7, and 14 p.i. were cultured in the presence of nRBCs or pRBCs for 72 hr, and then cell culture supernatants were harvested and IFNα levels were measured (cohort 10; n = 7). (B) Cryopreserved PBMCs from volunteers at day 0 (D0) or 7 (D7) p.i. were cultured in the presence of nRBCs or pRBCs for 6 hr (as indicated), and then cellular sources of IFNα was measured by intracellular cytokine staining (cohorts 9–10; n = 6). Total IFNα⁺ events were gated first, and then cell types were gated as shown in Figure S2, using CpG as a positive control for IFNα staining. Not all samples from these cohorts were used in this assay, because a limited number of samples were cryopreserved. Median + min and max. *p < 0.05, Wilcoxon matched-pairs signed rank test.

Figure 3. Blood-Stage P. falciparum Induces a Type I IFN Response that Suppresses IFNγ Production

(A) PBMCs were isolated from volunteers 7 days p.i. and cultured in the presence of nRBCs, pRBCs, or pRBCs + anti-IFNAR antibody or its isotype control for 72 hr, and then IFNγ levels were measured in cell culture supernatants (cohorts 6 and 7; n = 12). (B) PBMCs were isolated from participants 7 days p.i. and cultured in the presence of nRBCs, pRBCs, pRBCs + anti-IFNAR, or pRBCs + anti-IFNAR + anti-HLA-DR antibody, as well as the relevant isotype controls, for 72 hr, and then IFNγ levels were measured (cohorts 6, 7, 9, and 10; n = 25). (C) Linear regression analysis was performed to determine the correlation between log-transformed IFNγ levels and blood parasitemia using the AUC values from qPCR measurements of parasite numbers (cohorts 6–10; n = 29). D7, day 7. p < 0.0001, linear regression function (goodness of fit). (D) PBMCs were isolated from volunteers 7 days p.i. and cultured in the presence of pRBCs + anti-IFNAR antibody or its isotype control for 72 hr, and then IFNγ levels measured by intracellular cytokine staining, in which the IFNγ MFI on IFNγ⁺ CD4⁺ and CD8⁺ T cells, CD56⁺ CD3⁻ (NK) cells, CD56⁺ CD3⁺ cells, and CD3⁻ CD56⁻ (other) cells was determined using the GeoMean function in FlowJo (cohorts 7 and 10; n = 12). Median + min and max (A, B, and D). ***p < 0.001, **p < 0.01, *p < 0.05, Wilcoxon matched-pairs signed rank test.

Figure 4. Type I IFNs Suppress IL-6, IL-1β, and IL-17, but Not TNF Production in Response to P. falciparum

(A–D) PBMCs were isolated from volunteers on days 0, 7, 14, and 28 p.i. and cultured in the presence of nRBCs, pRBCs, or pRBCs + anti-IFNAR antibody or its isotype control for 72 hr before measuring IL-6 (A), IL-1β (B), IL-17 (C), or TNF (D) levels. Data from cohorts 6 and 7; n = 12. Mean ± SEM. ***p < 0.001, *p < 0.05, Wilcoxon matched-pairs signed rank test.

Figure 5. Type I IFNs Suppress Monocyte-Derived IL-6 Production in Response to P. falciparum

(A) Flow cytometry gating strategy is shown for pDCs and mDCs (top row) and for monocytes (bottom row) (cohort 6; n = 7). (B–D) Isolated PBMCs were cultured in the presence of pRBCs + anti-IFNAR antibody or its isotype control for 24 hr, and then BFA added for the last 3 hr. IL-6 was measured by intracellular cytokine staining and flow cytometry. IL-6 MFI on pDCs (B), mDCs (C), and monocytes (D) at days 0 and 7 p.i. is shown (cohort 6; n = 7). Median + min and max. **p < 0.01, Wilcoxon matched-pairs signed rank test.

Figure 6. Blood-Stage P. falciparum Induces HLA-DR Restricted Tr1 Cells and IL-10 Production

(A) PBMCs were isolated from volunteers at days 0, 7, 14, and 28 p.i. and cultured in the presence of nRBCs or pRBCs for 72 hr, and then cell culture supernatants were harvested and IL-10 levels were measured (cohort 6; n = 7). (B) PBMCs were isolated from volunteers 14 days p.i. (7 days after drug treatment) and cultured in the presence of nRBCs, pRBCs, or pRBCs + anti-HLADR antibody or its isotype control for 72 hr. BFA, PMA, and ionomycin were added for the remaining 3 hr of culture. Representative gating strategy from left to right shows lymphocytes, live or viable cells, CD16⁻ CD56⁻ (exclusion of NK and γδ T cells), CD3⁺, CD4⁺, and IFNγ⁺ IL-10⁺ (cohorts 10 and 11; n = 10). (C and D) The frequency of Tr1 cells was measured by flow cytometry (C), and IL-10 levels were measured in cell culture supernatants (D) (cohorts 10 and 11; n = 10). Mean ± SEM (A), median + min and max (C and D). **p < 0.01, *p < 0.05, Wilcoxon matched-pairs signed rank test.

Figure 7. Type I IFNs Promote Tr1 Cell Development and IL-10 Production after Drug Treatment in Volunteers Infected with P. falciparum and Malaria Patients with Natural P. falciparum Exposure

(A) PBMCs were isolated from volunteers at day 14 p.i. (7 days after drug treatment) and cultured in the presence of pRBCs + anti-IFNAR antibody or its isotype control for 72 hr before measuring IL-10 levels (cohorts 6 and 7; n = 12). (B) PBMCs were isolated from volunteers 14 days p.i. (7 days after drug treatment) and cultured in the presence of nRBCs, pRBCs, or pRBCs + anti-IFNAR antibody or its isotype control for 72 hr. BFA, PMA, and ionomycin were added for the remaining 3 hr of culture before frequencies of Tr1 cells were measured by flow cytometry (cohort 7; n = 5). (C) Linear regression analysis was performed to determine the correlation between log-transformed IL-10 levels and blood parasitemia using the AUC values from qPCR measurements of parasite numbers (cohorts 6–10; n = 29). D14, day 14. p < 0.01, linear regression function (goodness of fit). (D and E) PBMCs were isolated from volunteers 14 days p.i. (7 days after drug treatment) and cultured in the presence of pRBCs + anti-IL-10 antibody or its isotype control for 72 hr, and then IFNγ levels (D) and TNF levels (E) were measured (cohorts 7 and 8; n = 9). (F) Cryopreserved PBMCs from malaria patients infected with P. falciparum from Timika, Indonesia, taken at time of admission into the clinic (day 0) and 7 days after commencement of drug treatment (day 7) were thawed and cultured in the presence of nRBCs, pRBCs, or pRBCs + anti-IFNAR antibody or its isotype control for 72 hr, and then IL-10 levels were measured. Median + min and max (A, B, and D–F). ***p < 0.001, **p < 0.01, *p < 0.05, Wilcoxon matched-pairs signed rank test.