Anti-Aspergillus human host defence relies on type 1 T helper (Th1), rather than type 17 T helper (Th17), cellular immunity

Abstract

Both interferon-gamma-producing type 1 T helper (Th1)- and interleukin-17 (IL-17)-producing Th17 cells have been proposed to be involved in anti-fungal host defence. Although invasive aspergillosis is one of the most severe human fungal infections, little is known regarding the relative importance of the Th1 versus Th17 cellular immune pathways for the human anti-Aspergillus host defence. Using human peripheral blood mononuclear cells and a system consisting of monocyte-derived macrophages with lymphocytes, we found that Aspergillus fumigatus is a weak inducer of human IL-17 but induces a strong Th1 response. These data were validated by the very low IL-17 levels in bronchoalveolar lavage fluid and serum of patients with invasive aspergillosis. Surprisingly, live A. fumigatus reduced IL-17 production induced by mitogenic stimuli. This effect was mediated through the propensity of A. fumigatus to metabolize tryptophan and release kynurenine, which modulates the inflammatory response through inhibition of IL-17 production. In conclusion, A. fumigatus does not stimulate production of IL-17 and human host defence against aspergillosis may not rely on potent Th17 responses.

Figure 1

(a, b) Interleukin-17 (IL-17) -inducing capacity of 10⁴–10⁶ micro-organisms/ml Aspergillus fumigatus in peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM)/lymphocytes systems over 7 days with Candida albicans as positive control. (c, d) Flow cytometry of MDM/lymphocytes stimulated by A. fumigatus and C. albicans, respectively. The cells were labelled with anti-CD4-allophycocyanin and anti-IL-17-fluorescein isothiocyanate. Density plots show surface staining of CD4 (x-axis) and intracellular staining of IL-17 (y-axis). Numbers on the plots represent percentage of cells bearing positivity for the respective markers. Results from a representative experiment are shown. (e, f) IL-17 and IL-6 induced by 10⁵–10⁷ micro-organisms/ml of heat-killed A. fumigatus components: resting conidia, germinating conidia and hyphae in PBMC. Stimulation studies are cumulative from three sets of experiments. *P < 0·05 compared with control (RPMI-1640). Aspergillus: live A. fumigatus conidia (unless otherwise stated), Candida: heat-killed C. albicans blastoconidia (as positive control), HK: heat-killed.

Figure 2

Interleukin-6 (IL-6), IL-10, interferon-γ (IFN-γ) and IL-1β production by monocyte-derived macrophages (MDM) following stimulation by live Aspergillus fumigatus at incremental concentrations. IFN--γ and IL-1β responses are robust compared with IL-6 and IL-10. *P < 0·05 compared with RPMI-1640 control, n = 8 subjects.

Figure 3

(a, b) Interferon-γ (IFN-γ) and interleukin-17 (IL-17) produced by monocyte-derived macrophages (MDM)/lymphocytes stimulated with anti-CD3/anti-CD28 beads alone and with incremental amounts of Aspergillus fumigatus or Candida albicans (positive control) over 3 days (c, d) In addition to anti-CD3/anti-CD28 beads, recombinant IL-6 (rIL-6) + rIL-1β + rIL-23 were added to MDM/lymphocytes stimulated with A. fumigatus or C. albicans (positive control) over 3 days. *P < 0·05 compared with stimulated MDM/lymphocytes but without pathogen, n = 6 subjects. Aspergillus: live A. fumigatus conidia, Candida: heat-killed C. albicans blastoconidia, aCD3CD28: anti-CD3/anti-CD28-coated beads.

Figure 4

Interferon-γ (IFN-γ) and interleukin-17 (IL-17) from monocyte-derived macrophages (MDM)/lymphocytes stimulated with anti-CD3/anti-CD28 beads and incremental amounts of Aspergillus fumigatus or Candida albicans (positive control) in the presence of anti-IFN-γ (a, b). *P < 0·05 compared with respective control (white bar) without anti-IFN-γ, n = 6 subjects. Aspergillus: live A. fumigatus conidia, Candida: heat-killed C. albicans blastoconidia (as positive control).

Figure 5

(a, b) Interferon-γ (IFN-γ) and interleukin-17 (IL-17) produced by anti-CD3/anti-CD28-stimulated monocyte-derived macrophages (MDM)/lymphocytes over 3 days in the presence of conditioned medium in which the respective concentrations of live Aspergillus fumigatus had previously been grown over 72 hr. *P < 0·05 compared with anti-CD3/anti-CD28-stimulated MDM/lymphocytes in the presence of conditioned medium in which no Aspergillus had been grown (control; first bar), n = 6 subjects. (c) IL-17 production by anti-CD3/anti-CD28-stimulated MDM/lymphocytes over 3 days in the presence of increasing concentrations of kynurenine compared with control (without kynurenine). *P < 0·05 compared with control, n = 6 subjects. Aspergillus: live A. fumigatus conidia, aCD3CD28: anti-CD3/anti-CD28-coated beads.

Figure 6

(a) Interleukin-17 (IL-17) levels in bronchoalveolar lavage of patients diagnosed with proven/probable invasive aspergillosis (IA) compared with a corresponding patient cohort without IA (No IA): n = 17 in IA group (eight proven IA cases and nine probable IA cases), n = 20 in No IA group. (b) IL-17 levels in serum of IA patients (IA) and corresponding patient controls (No IA): n = 19 in IA group (eight proven IA cases and 11 probable IA cases), and n = 15 in No IA group. *P < 0·05 (unpaired t-test).