Body fluid from the parasitic worm Ascaris suum inhibits broad-acting pro-inflammatory programs in dendritic cells

Abstract

Dendritic cells (DCs) are essential for generating T-cell-based immune responses through sensing of potential inflammatory and metabolic cues in the local environment. However, there is still limited insight into the processes defining the resultant DC phenotype, including the type of early transcriptional changes in pro-inflammatory cues towards regulatory or type 2 immune-based cues induced by a variety of exogenous and endogenous molecules. Here we compared the ability of a selected number of molecules to modulate the pro-inflammatory phenotype of lipopolysaccharide (LPS) and interferon-γ (IFN-γ)-stimulated human monocyte-derived DCs towards an anti-inflammatory or regulatory phenotype, including Ascaris suum body fluid [helminth pseudocoelomic fluid (PCF)], the metabolites succinate and butyrate, and the type 2 cytokines thymic stromal lymphopoietin and interleukin-25. Our data show that helminth PCF and butyrate treatment suppress the T helper type 1 (Th1)-inducing pro-inflammatory DC phenotype through induction of different transcriptional programs in DCs. RNA sequencing indicated that helminth PCF treatment strongly inhibited the Th1 and Th17 polarizing ability of LPS + IFN-γ-matured DCs by down-regulating myeloid differentiation primary response gene 88 (MyD88)-dependent and MyD88-independent pathways in Toll-like receptor 4 signaling. By contrast, butyrate treatment had a strong Th1-inhibiting action, and transcripts encoding important gut barrier defending factors such as IL18, IL1B and CXCL8 were up-regulated. Collectively, our results further understanding of how compounds from parasites and gut microbiota-derived butyrate may exert immunomodulatory effects on the host immune system.

Keywords: Ascaris suum; dendritic cells; type 2 immune response.

Figure 1

Helminth pseudocoelomic fluid (PCF) and butyrate suppress lipopolysaccharide (LPS) + interferon‐γ (IFN‐γ)‐induced interleukin‐12p70 (IL‐12p70) secretion in monocyte‐derived dendritic cells (moDCs). (a) Overview of study design: (i) isolation of CD14⁺ monocytes from human blood peripheral blood mononuclear cells, (ii) differentiation of monocytes into dendritic cells (moDCs) using recombinant human IL‐4 and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) for 6 days, (iii) stimulation of moDCs with indicated compounds (1), (iv) collection and preparation of samples for RNA‐seq, flow cytometry and ELISA analyses. End‐point measures are the type of transcripts (mRNA; RNA‐seq, 4 hr of stimulation), surface marker expression (flow cytometry, 20 hr) and cytokines (proteins, 20 hr) produced by the stimulated moDCs. The figure also displays the specific molecules guiding naive T helper cell polarization into different effector T cells. GM‐CSF, granulocyte–macrophage colony‐stimulating factor; LPS, lipopolysaccharide; IFN‐γ, interferon‐γ; PCF, pseudocoelomic fluid; Th1, type 1 T helper cells; Th2, type 2 T helper cells; Th17, type 17 T helper cells; Tregs, regulatory T cells. (b) Concentration of secreted IL‐12p70 in cell‐free culture supernatant and expression of the co‐stimulatory molecules CD40 and CD86 in moDCs stimulated with the indicated compounds relative to LPS + IFN‐γ only. Final concentrations of the compounds were as follows: IL‐25: 25 ng/ml, TSLP: 25 ng/ml, PCF: 100 μg dry matter/ml, Butyrate: 2 mm. Levels of IL‐12p70, CD40 and CD86 in LPS + IFN‐γ‐stimulated moDCs were: IL‐12p70: 4641·8 ± 688·1 pg/ml, CD40: 1647 ± 361 MFI, CD86: 2424 ± 311 MFI. MFI, median fluorescence intensity, mean ± SD. The experiment was performed using moDCs from three different donors. *P < 0·05, ***P < 0·001 by one‐way analysis of variance and Dunnett's post‐test for multiple comparisons to LPS + IFN‐γ.

Figure 2

Helminth pseudocoelomic fluid (PCF) predominantly targets T helper type 1 (Th1) and Th17 polarizing abilities in pro‐inflammatory monocyte‐derived dendritic cells (moDCs). (a) Differentially expressed genes arranged by fold change in PCF + lipopolysaccharide (LPS) + interferon‐γ (IFN‐γ) ‐stimulated moDCs compared with LPS + IFN‐γ only. All genes (131) with a q‐value < 0·1 are shown. (b) Predicted phenotype of moDCs based on their transcriptional profile. The illustration focuses mainly on molecules from moDCs involved in the polarizing signals provided to naive T helper cells. Shown are the differentially regulated genes with a q‐value < 0·1. Black: genes up‐regulated by LPS + IFN‐γ (relative to unstimulated moDCs), Red: genes down‐regulated by PCF + LPS + IFN‐γ (relative to LPS + IFN‐γ only). Th1, type 1 T helper cells; NK, natural killer cells; Tcyto, cytotoxic T cells; Th17, type 17 T helper cells; Neu, neutrophils; Th2, type 2 T helper cells; Mo, monocytes; Tregs, regulatory T cells.

Figure 3

Helminth pseudocoelomic fluid (PCF) and butyrate modify gene expression in lipopolysaccharide (LPS) + interferon‐γ (IFN‐γ) ‐stimulated monocyte‐derived dendritic cells (moDCs) differently. (a) Venn diagram displaying unique and shared regulated genes (q value < 0·1) in helminth PCF + LPS + IFN‐γ‐ and butyrate + LPS + IFN‐γ‐stimulated moDCs relative to LPS + IFN‐γ only. (b) Distinct regulation of immune relevant genes in helminth PCF + LPS + IFN‐γ‐ and butyrate + LPS + IFN‐γ‐stimulated moDCs capable of modifying DC‐guided T helper cell polarization and immune cell trafficking. Black: genes down‐regulated by both helminth PCF and butyrate, Black in bold: genes up‐regulated by butyrate, but down‐regulated by PCF, Red: genes down‐regulated by PCF uniquely but not regulated by butyrate. Mo, monocytes; Neu, neutrophils; NK, natural killer cells; Tcyto, cytotoxic T cells; Th1, type 1 T helper cells; Th17, type 17 T helper cells; Th2, type 2 T helper cells; Tregs, regulatory T cells.

Figure 4

Succinate is unable to down‐regulate lipopolysaccharide (LPS) + interferon‐γ (IFN‐γ) ‐induced interleukin‐12p70 (IL‐12p70) secretion in monocyte‐derived dendritic cells (moDCs). Concentration of secreted IL‐12p70 in cell‐free culture supernatant and expression of co‐stimulatory molecules CD40 and CD86 on moDCs stimulated with helminth pseudocoelomic fluid (PCF) as in Fig. 1, and indicated succinate (suc) concentrations relative to LPS + IFN‐γ only. Graphs display mean ± SD.