IL-4 and helminth infection downregulate MINCLE-dependent macrophage response to mycobacteria and Th17 adjuvanticity

Abstract

The myeloid C-type lectin receptor (CLR) MINCLE senses the mycobacterial cell wall component trehalose-6,6'-dimycolate (TDM). Recently, we found that IL-4 downregulates MINCLE expression in macrophages. IL-4 is a hallmark cytokine in helminth infections, which appear to increase the risk for mycobacterial infection and active tuberculosis. Here, we investigated functional consequences of IL-4 and helminth infection on MINCLE-driven macrophage activation and Th1/Th17 adjuvanticity. IL-4 inhibited MINCLE and cytokine induction after macrophage infection with Mycobacterium bovis bacille Calmette-Guerin (BCG). Infection of mice with BCG upregulated MINCLE on myeloid cells, which was inhibited by IL-4 plasmid injection and by infection with the nematode Nippostrongylus brasiliensis in monocytes. To determine the impact of helminth infection on MINCLE-dependent immune responses, we vaccinated mice with a recombinant protein together with the MINCLE ligand trehalose-6,6-dibehenate (TDB) as adjuvant. Concurrent infection with N. brasiliensis or with Schistosoma mansoni promoted T cell-derived IL-4 production and suppressed Th1/Th17 differentiation in the spleen. In contrast, helminth infection did not reduce Th1/Th17 induction by TDB in draining peripheral lymph nodes, where IL-4 levels were unaltered. Upon use of the TLR4-dependent adjuvant G3D6A, N. brasiliensis infection impaired selectively the induction of splenic antigen-specific Th1 but not of Th17 cells. Inhibition of MINCLE-dependent Th1/Th17 responses in mice infected with N. brasiliensis was dependent on IL-4/IL-13. Thus, helminth infection attenuated the Th17 response to MINCLE-dependent immunization in an organ- and adjuvant-specific manner via the Th2 cytokines IL-4/IL-13. Taken together, our results demonstrate downregulation of MINCLE expression on monocytes and macrophages by IL-4 as a possible mechanism of thwarted Th17 vaccination responses by underlying helminth infection.

Keywords: C-type lectin receptors; Nippostrongylus brasiliensis; Schistosoma mansoni; Th cell differentiation; co-infection; immunology; infectious disease; inflammation; microbiology; mouse; mycobacterial cord factor.

Figure 1.. IL-4 impairs upregulation of MINCLE and other DECTIN-2 family C-type lectin receptor (CLR) in macrophages stimulated with bacille Calmette-Guerin (BCG).

(A–D) C57BL/6 bone marrow-derived macrophages (BMM) were stimulated as indicated in presence or absence of IL-4 for 6, 24, or 48 hr. BCG was used at MOI 10. MINCLE (A), DECTIN-2 (B), MCL (C), and DECTIN-1 (D) mRNA expression was determined by quantitative real-time PCR (qRT-PCR) shown as fold change calibrated to unstimulated control. Data are depicted as mean + SD from two independent experiments performed in biological duplicates. (E–G) BMM were stimulated with BCG at the indicated MOI in the presence or absence of IL-4 for 24 hr, followed by staining for MINCLE (E), DECTIN-2 (F), or DECTIN-1 (G) expression. Representative stainings are shown as histogram overlay (left panel), for quantification (right panel) the median fluorescence intensity of the isotype control staining was subtracted from the respective CLR signal to obtain the ΔMFI. Each point represents one mouse, pooled from at least two independent experiments. *p<0.05, **p<0.01, ***p<0.001 in Wilcoxon signed rank test.

Figure 2.. IL-4 does not affect phagocytosis of bacille Calmette-Guerin (BCG) but inhibits cytokine production.

(A) C57BL/6 bone marrow-derived macrophages (BMMs) were infected with different MOI of fluorescent BCG-Dsred co-treated with IL-4 or not as indicated. Phagocytic uptake was measured via flow cytometry. (A) Representative histograms show phagocytosis of BCG by detection of fluorescent DsRed signal in BMMs. Quantitative analysis of phagocytosis based on percentage of BCG-positive cells at 6 and 24 hr post infection. Data is depicted from two to three independent experiments performed in biological duplicates. (B) C57BL/6 (WT), MINCLE knockout (Clec4e^-/-), and FcRγ (Fcer1g^-/-) BMM were stimulated with BCG for 24 hr. Production of G-CSF and TNF was measured from cell culture supernatants via ELISA. No significant cytokine production was detected from non-stimulated BMM. Data is depicted from three independent experiments performed in biological duplicates. (C) Phagocytosis of fluorescent BCG is unaltered in BMM deficient in MINCLE (Clec4e^-/-) or FcRγ (Fcer1g^-/-). Mean percentages of one representative experiment out of two (Fcer1g^-/-) and three (Clec4e^-/-) performed. *p<0.05, **p<0.01, ***p<0.001.

Figure 3.. Overexpression of IL-4 or co-infection with Nippostrongylus brasiliensis impair MINCLE upregulation on peritoneal monocytes but does not reduce phagocytosis upon bacille Calmette-Guerin (BCG) infection.

(A) IL-4 concentration in serum of mice injected with IL-4 minicircle. C57BL/6 mice were hydrodynamically injected with 0.25 or 0.5 µg of IL-4 plasmid (i.v.) or Ringer solution. Five days (0.5 µg) or 7 days later (0.25 µg) mice were sacrificed and serum IL-4 levels were determined via ELISA (n=5–6 mice per group). n.d.=not detectable. (B, C) 0.25 µg of IL-4 plasmid or Ringer solution was hydrodynamically injected into C57BL/6 wildtype mice. Two days later mice were infected i.p. with 40×10⁶ CFU of Mycobacterium bovis BCG. (B) Generic gating strategy for flow cytometry data. Monocytes were characterized as lineage^-CD11b⁺SiglecF^-Ly6C⁺ cells. Neutrophils were characterized as lineage^-CD11b⁺SiglecF^-Ly6G⁺ cells. Lineage marker: CD3, CD19, NK1.1. (C) Histograms depict MINCLE surface expression on Ly6C^hi monocytes and neutrophils 24 hr p.i. analysed via flow cytometry. Quantitative analysis of MINCLE surface expression shown as median fluorescence intensity (MFI). Fluorescence minus one control (FMO) was substracted. Infected MINCLE-/- mice were used as staining controls to exclude unspecific binding of 4A9 antibody. Data is depicted from two independent experiments (2–7 mice per group in total, each dot corresponding to one mouse). **p0.01; ns = not significant. (D–E) C57BL/6 mice were s.c. infected with N. brasiliensis or left uninfected followed by M. bovis BCG-Dsred infection (40×10⁶ CFU/mouse) on day 10 p.i. Twenty-four hr later mice were sacrificed. (D) Representative histograms of eosinophil population of BCG-infected and N. brasiliensis co-infected mouse. Bar graphs show relative percentage of myeloid cell populations as indicated. (E) MINCLE surface expression was analysed on peritoneal Ly6C^hi monocytes and neutrophils via flow cytometry. Quantitative analysis of MINCLE surface expression shown as MFI normalized to BCG-infected mice. FMO was substracted. Data is depicted from three independent experiments (11–13 mice per group in total). *p<0.05, ** p<0.01, ***p<0.001. (F) C57BL/6 mice were s.c. infected with N. brasiliensis followed by M. bovis BCG-Dsred infection (40×10⁶ CFU/mouse) or non-fluorescent BCG (nf) on day 10 p.i. Twenty-four hr after BCG infection, phagocytosis was measured by detection of PE signal in monocytes or neutrophils. Quantitative analysis of the percentage of BCG-positive monocytes.

Figure 4.. Co-infection with Schistosoma mansoni (S.m.) suppresses Th1/Th17 induction by a MINCLE-dependent adjuvant in the spleen but not in the draining lymph node.

(A) Scheme of experimental procedure. Cercariae of S.m. were injected s.c. into C57BL/6 mice. Eight to 9 weeks p.i. mice were immunized with CAF01 (B, C, D) or CpG ODN (E, F). Seven days after immunization, mice were sacrificed and inguinal lymph node cells and spleen cells were re-stimulated with H1 in vitro. Draining inguinal lymph node cells or splenocytes were re-stimulated with H1 or anti-CD3 for 96 hr. IL-17, IFNγ, IL-10, and IL-4 production was measured from cell culture supernatants by ELISA. Data is shown from three independent experiments (n=18 mice per group in total) for IFNγ, IL-17, and IL-10 (C, D) and from two independent experiments (n=13 mice) for IL-4 (B). (E, F) Data is shown from one experiment (n=5 mice per group in total). *p<0.05, **p<0.01, ***p<0.001. Dashed horizontal lines indicate the limit of detection for each cytokine.

Figure 5.. Co-infection with Nippostrongylus brasiliensis suppresses Th1/Th17 induction by a MINCLE-dependent adjuvant in the spleen but not in the draining lymph node.

(A) Scheme of experimental procedure. C57BL/6 mice were infected subcutaneously in the flank with 500 L3 larvae of N. brasiliensis in 200 µl PBS. Five days p.i. mice were immunized with H1/CAF01 (B–F) or H1/G3D6A (G). Seven days after immunization, mice were sacrificed, and inguinal and popliteal lymph node cells and spleen cells were re-stimulated with H1 in vitro. (B) Footpad swelling was measured over a period of 1 week after immunization with H1/CAF01 until mice were sacrificed (day 7 after immunization). The increase in footpad swelling is shown as mean + SD for the indicated time points (n=10–11 mice for each data point). (C) Absolute cell number of draining inguinal and popliteal lymph node cells (LN) and spleen on day 7 after H1/CAF01. (D–G) Draining inguinal and popliteal lymph node cells or splenocytes were re-stimulated with H1, anti-CD3, or left untreated (mock) for 96 hr, followed by cytokine determination by ELISA. (D) IL-4 levels produced by draining lymph node cells (left) or splenocytes (right). (E, F) IL-17, IFNγ, and IL-10 cytokine production by draining lymph node cells (E) and splenocytes (F). (G) Immunization of C57BL/6 mice with H1 in G3D6A adjuvant. Seven days after immunization mice were sacrificed and splenocytes were treated as described in (D, E). All data is shown from two (B–F) or three (G) independent experiments (n=9–14 mice per group in total). p<0.05, **p<0.01, ***p<0.001. Dashed horizontal lines indicate the limit of detection for each cytokine.

Figure 6.. Inhibition of Th1/Th17 induction in Nippostrongylus brasiliensis infection depends on IL-4 or IL-13.

C57BL/6 or of Il4^-/-Il13^-/- mice were infected with N. brasiliensis or not as indicated and immunized s.c. with H1/CAF01 5 days later. On day 7 after immunization, mice were sacrificed and splenocytes were re-stimulated with H1 or anti-CD3 for 96 hr. Cytokines were measured by ELISA. (A) IFNγ, (B) IL-17, (C) IL-10, (D) IL-4. Data shown is pooled from four independent experiments (n=15–17 mice per group in total). p<0.05, **p<0.01, ***p<0.001. Dashed horizontal lines indicate the limit of detection for each cytokine.

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