Research ArticleIMMUNOLOGY

Blood-stage malaria parasites manipulate host innate immune responses through the induction of sFGL2

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Science Advances  26 Feb 2020:
Vol. 6, no. 9, eaay9269
DOI: 10.1126/sciadv.aay9269
  • Fig. 1 sFGL2 was markedly induced by malaria blood-stage infection and promoted parasite development.

    (A) sFGL2 levels in the sera of P. vivax– and P. falciparum–infected patients from the China-Myanmar border and healthy donors as measured by enzyme-linked immunosorbent assay (ELISA). NER, nonendemic healthy residents; ER, endemic healthy residents; Pf, patients with acute P. falciparum infection; Pv, patients with acute P. vivax infection. (B) Correlation between parasitemia and sFGL2 levels in patients with malaria. r, Pearson’s correlation coefficient. (C to E) The time courses of the sFGL2 levels in sera (red line) measured by ELISA and levels of parasitemia (blue line) determined by Giemsa staining in mice (n = 5) infected with P. chabaudi (C), P. yoelii (D), and P. berghei (E). (F and G) Parasitemias of wild-type (WT) and FGL2−/− mice (n = 6) infected with P. chabaudi (F) or P. yoelii (G). (H) In vivo images of parasite burden at day 6 in WT and FGL2−/− mice (n = 4) infected with P. berghei luciferase (left), and the parasite loads between the WT and FGL2−/− mice (n = 4) at the indicated times after infection (right) were compared. (I to K) The effect of recombinant FGL2 administration (+rFGL2) on the parasitemia of FGL2−/− mice (n = 5) infected with P. chabaudi (I), P. yoelii (J), and P. berghei (K). Triplicate experiments were performed. Data represent the means ± SD. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 2 sFGL2 promotes parasite growth through impeding the production of IFN-γ secreted by NK/NKT cells.

    (A) The levels of IFN-γ in the sera of WT and FGL2−/− mice (n = 4) at the indicated time points after infection with P. chabaudi as measured by ELISA. (B) The parasitemia of the parasite-infected WT mice and FGL2−/− mice (n = 4) depleted with or without anti–IFN-γ monoclonal antibody (mAb). (C to E) Representative fluorescence-activated cell sorting (FACS) analyses (left) and statistical analyses (right) of IFN-γ production capacity by splenic total NK (C), NKT (D), and γδ T (E) cells from both WT and FGL2−/− mice (n = 4) at the indicated time points after infection with P. chabaudi. d6 p.i., day 6 post-infection. (F) The parasitemia of the infected WT mice (n = 5) and FGL2−/− mice (n = 5) with both NK and NKT cells depleted with or without anti-NK1.1 mAb. (G) The depletion efficacies of NK (NK1.1+ CD3) and NKT (NK1.1+ CD3+) cells in FGL2−/− mice (n = 4) as determined by FACS. Data represent three separate experiments with at least four mice per group. Numbers represent the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 3 sFGL2 reduces IFN-γ secretion by suppressing MCP-1–mediated recruitment of NK/NKT cells.

    (A) The level of IFN-γ secreted by a single NK (left) or NKT (right) cell from P. chabaudi–infected WT and FGL2−/− mice (n = 4) was analyzed by FACS at the indicated time points. (B) The numbers of NK (left) and NKT (right) cells in the spleens of P. chabaudi–infected WT and FGL2−/− mice (n = 4) were statistically analyzed at the indicated time after infection. (C) The level of MCP-1 in the serum of P. chabaudi–infected WT and FGL2−/− mice (n = 4) was analyzed by ELISA at the indicated time points. (D and E) The percentages (left) and numbers (right) of IFN-γ–secreting NK cells (D) or NKT cells (E) in the spleens of both P. chabaudi–infected WT and FGL2−/− mice (n = 4) were analyzed by flow cytometry, at the indicated time points after MCP-1 was depleted. (F) The IFN-γ levels in the sera of parasite-infected WT and FGL2−/− mice (n = 5) were measured by ELISA after MCP-1 was depleted. (G) The parasitemia of the parasite-infected WT mice and FGL2−/− mice (n = 5) with or without MCP-1 depletion was determined. Each experiment was repeated three times with at least four mice per group. Data represent the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 4 sFGL2 inhibits parasite-stimulated macrophages from secreting MCP-1 through blocking JNK activation.

    (A) Representative FACS (left) and statistical (right) analyses of the production of MCP-1 by macrophages from the spleens of both WT and FGL2−/− mice (n = 4) at the indicated time points. (B) The levels of MCP-1 (left) and IFN-γ (right) in the serum of the parasite-infected WT mice and FGL2−/− mice (n = 5) with or without macrophages depleted by clodronate liposomes at day 6 after infection. (C) The parasitemia of both parasite-infected WT mice and FGL2−/− mice (n = 5) with or without macrophage depletion by clodronate liposomes. (D) MCP-1 levels in the supernatants of pRBC lysate–stimulated RAW264.7 macrophages (left) and bone marrow–derived macrophages (BMDMs) (right) treated with or without rFGL2 for the indicated times as detected by ELISA. (E) MCP-1 levels released by WT and TLR2−/− BMDMs incubated with the pRBC lysate preparation as measured by ELISA (left), and the MCP-1 levels produced by the pRBC lysate–stimulated macrophages pretreated with or without the TLR9 antagonist ODN2088 as determined by ELISA (right). (F) MCP-1 levels in the supernatants of pRBC lysate–stimulated macrophages pretreated with or without nuclear factor κB (NF-κB) inhibitor BAY 11-7082, p38 inhibitor SB203580, c-Jun N-terminal kinase (JNK) inhibitor SP600125, and extracellular signal–regulated kinase 1/2 (ERK1/2) inhibitor SCH772984. (G) Western blots of the phosphorylation of TAK1, MKK4, MKK7, JNK, ERK1/2, and p65 in pRBC lysate–stimulated RAW264.7 cells pretreated with or without rFGL2. nRBC, normal red blood cell. Two to four independent experiments were performed. Data represent the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. The symbol “†” indicates the time point when all mice died.

  • Fig. 5 sFGL2 attenuates parasite-stimulated macrophages from releasing MCP-1 dependent on FcγRIIB receptors.

    (A) RAW264.7 cells (left) and BMDMs (right) were pretreated with anti-FcγRIII/FcγRII for the indicated time and then incubated with pRBC lysate and rFGL2, and MCP-1 levels in the supernatants were measured by ELISA. (B) BMDMs isolated from control and FcγRIIBfl/fl Lyz2-Cre mice were incubated with pRBC lysate and rFGL2, and MCP-1 levels in the supernatants were detected by ELISA. (C) Western blots of JNK activation in pRBC lysate–stimulated control and FcγRIIB-deficient BMDMs preincubated with or without rFGL2. (D) The parasitemia levels of control and FcγRIIBfl/fl Lyz2-Cre mice (n = 5) infected with P. chabaudi were determined by Giemsa staining. (E) The MCP-1 levels in the sera of control and FcγRIIBfl/fl Lyz2-Cre mice (n = 4) after infection with P. chabaudi were detected by ELISA. (F) The IFN-γ levels in the serum of the infected control and FcγRIIBfl/fl Lyz2-Cre mice (n = 4) were determined by ELISA. (G) MCP-1 levels in the supernatants of P. falciparum lysate–stimulated human THP-1–derived macrophages treated with or without rFGL2 and anti-FcγRIII/FcγRII 2 hours before parasite stimulation were detected by ELISA. (H) Western blots of JNK activation in P. falciparum lysate–stimulated macrophages pretreated with or without rFGL2. Each experiment was repeated three times. Data represent the means ± SD. *P < 0.05, **P < 0.01.

  • Fig. 6 sFGL2 is secreted mainly by CD4+Foxp3+CD25+ Tregs and promotes the growth of malaria blood stage.

    (A) The expansion of CD4+Foxp3+CD25+ Tregs in mice (n = 4) infected with P. chabaudi was analyzed at the indicated time points by flow cytometry (left), and a statistical analysis was also performed (right). (B) The transcript levels of FGL2 in CD4, CD4+CD25+, and CD4+CD25 T cells were determined by real-time polymerase chain reaction (PCR). (C) ELISA of the level of sFGL2 in the serum of P. chabaudi–infected mice (n = 4) after Tregs were depleted with anti-CD25 mAb at −1, 1, 3, 5, and 7 days after infection. (D) The depletion of Tregs was determined by FACS. (E) The effects of the adoptive transfer of WT or FGL2−/− mice–derived Tregs on the parasitemia of FGL2−/− mice (n = 4) infected with P. chabaudi. Each experiment was performed three times, with at least four mice per group. Data represent the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/9/eaay9269/DC1

    Fig. S1. FGL2 deficiency has no significant effect on both fibrin deposition in the spleen and coagulation function of the parasite-infected mice.

    Fig. S2. sFGL2 has no effect on parasite-specific antibody production.

    Fig. S3. sFGL2 has no effect on parasite-specific CD4+ T cell activation.

    Fig. S4. sFGL2 has no effect on parasite-specific CD8+ T cell activation.

    Fig. S5. Full blots of the effect of rFGL2 administration on both mitogen-activated protein kinase and NF-κB activity in P. chabaudi lysate–stimulated macrophages by rFGL2.

    Fig. S6. Full blots of the FcγRIIB-dependent inhibition of JNK in P. chabaudi lysate–activated macrophages by sFGL2.

    Fig. S7. Full blots of the inhibition of JNK in P. falciparum lysate–activated macrophages by rFGL2.

    Table S1. Detailed information of patients with malaria.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. FGL2 deficiency has no significant effect on both fibrin deposition in the spleen and coagulation function of the parasite-infected mice.
    • Fig. S2. sFGL2 has no effect on parasite-specific antibody production.
    • Fig. S3. sFGL2 has no effect on parasite-specific CD4+ T cell activation.
    • Fig. S4. sFGL2 has no effect on parasite-specific CD8+ T cell activation.
    • Fig. S5. Full blots of the effect of rFGL2 administration on both mitogen-activated protein kinase and NF-κB activity in P. chabaudi lysate–stimulated macrophages by rFGL2.
    • Fig. S6. Full blots of the FcγRIIB-dependent inhibition of JNK in P. chabaudi lysate–activated macrophages by sFGL2.
    • Fig. S7. Full blots of the inhibition of JNK in P. falciparum lysate–activated macrophages by rFGL2.
    • Table S1. Detailed information of patients with malaria.

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