Human monocyte-derived dendritic cells exposed to microorganisms involved in hypersensitivity pneumonitis induce a Th1-polarized immune response

Abstract

Hypersensitivity pneumonitis (HP) is an immunoallergic disease characterized by a prominent interstitial infiltrate composed predominantly of lymphocytes secreting inflammatory cytokines. Dendritic cells (DCs) are known to play a pivotal role in the lymphocytic response. However, their cross talk with microorganisms that cause HP has yet to be elucidated. This study aimed to investigate the initial interactions between human monocyte-derived DCs (MoDCs) and four microorganisms that are different in nature (Saccharopolyspora rectivirgula [actinomycetes], Mycobacterium immunogenum [mycobacteria], and Wallemia sebi and Eurotium amstelodami [filamentous fungi]) and are involved in HP. Our objectives were to determine the cross talk between MoDCs and HP-causative agents and to determine whether the resulting immune response varied according to the microbial extract tested. The phenotypic activation of MoDCs was measured by the increased expression of costimulatory molecules and levels of cytokines in supernatants. The functional activation of MoDCs was measured by the ability of MoDCs to induce lymphocytic proliferation and differentiation in a mixed lymphocytic reaction (MLR). E. amstelodami-exposed (EA) MoDCs expressed higher percentages of costimulatory molecules than did W. sebi-exposed (WS), S. rectivirgula-exposed (SR), or M. immunogenum-exposed (MI) MoDCs (P < 0.05, Wilcoxon signed-rank test). EA-MoDCs, WS-MoDCs, SR-MoDCs, and MI-MoDCs induced CD4(+) T cell proliferation and a Th1-polarized immune response. The present study provides evidence that, although differences were initially observed between MoDCs exposed to filamentous fungi and MoDCs exposed to bacteria, a Th1 response was ultimately promoted by DCs regardless of the microbial extract tested.

Fig 1

Expression of the costimulatory molecules CD80, CD86, and CD40 by immature MoDCs (iMoDCs) and MoDCs exposed to LPS (positive control), S. rectivirgula (SR), M. immunogenum (MI), W. sebi (WS), or E. amstelodami (EA). White peaks, isotypes; gray peaks, corresponding antibodies. The mean fluorescence intensity (MFI) is indicated for each condition.

Fig 2

Levels of IL-8, IL-10, and IL-23 (means ± SDs) in supernatants of EA-MoDCs, WS-MoDCs, SR-MoDCs, and MI-MoDCs, in six separate experiments. EA-MoDCs and WS-MoDCs both produced significantly higher levels of IL-10 and IL-23 than did SR-MoDCs (*, P < 0.05, Wilcoxon signed-rank test) and MI-MoDCs (●, P < 0.05, Wilcoxon signed-rank test). iMoDCs did produce significantly lower levels of IL-8 than EA-MoDCs, WS-MoDCs, MI-MoDCs, and SR-MoDCs (#, P < 0.05, Wilcoxon signed-rank test). NS, not significant.

Fig 3

Percentages of proliferation of CD4⁺ (green) and CD8⁺ (blue) lymphocytes after cultivation of total allogeneic T lymphocytes (LT) (MLR negative control) and after cocultivation of T lymphocytes plus LPS-exposed MoDCs, T lymphocytes plus SR-MoDCs, T lymphocytes plus MI-MoDCs, T lymphocytes plus WS-MoDCs, and T lymphocytes plus EA-MoDCs. Red, lymphocytes neither CD4⁺ nor CD8⁺. Greater proliferation of PBLs was observed in the case of stimulation with bacteria (S. rectivirgula and M. immunogenum) (P = 0.028, Wilcoxon signed-rank test). FSC, forward scatter; SSC, side scatter.

Fig 4

Standardized mRNA expression of the FoxP3, T-bet, Gata-3, and RORc genes after exposure of MoDCs to LPS, E. amstelodami, W. sebi, S. rectivirgula, or M. immunogenum, calculated with the ΔΔC_q method, with GAPDH as the reference gene. The data are presented as means ± SEMs from three separate experiments. In three separate experiments, levels of T-bet produced by T lymphocytes (Lt) after cocultivation with LPS-exposed MoDCs, EA-MoDCs, WS-MoDCs, SR-MoDCs, or MI-MoDCs were significantly higher (P < 0.05, Wilcoxon signed-rank test) than those produced by T lymphocytes alone and also were higher than levels of the three other genes investigated (FoxP3, RORc, and Gata-3). Transcription factor mRNA levels are presented for the four polarization controls (Th1, Th2, Th17, and Treg) at the right.

Fig 5

Intracellular staining for IL-17a, IL-4, IFN-γ, and TNF-α in allogeneic T lymphocytes (LT) and naive CD4⁺ T cells cocultured with MoDCs that had been previously exposed to LPS, S. rectivirgula, M. immunogenum, W. sebi, or E. amstelodami. Polarization controls for Th1, Th2, Th17, and Treg are presented at the right. Specific CD25/FoxP3 staining was added for the Treg polarization control.