Cellular cytokine and chemokine responses to parasite antigens and fungus and mite allergens in children co-infected with helminthes and protozoa parasites

Abstract

Background: In sub-Saharan Africa poly-parasite infections are frequently observed in children, and with poly-parasitism modulating immune mechanisms, mediated by cytokines and chemokines, are required to prevent overwhelming inflammation and host tissue damage. We analyzed in children co-infected with helminthes and protozoan parasites their cellular production of regulatory and pro-inflammatory cytokines and chemokines in response to parasite antigens and allergens.

Methods: Intestinal and intravascular parasite infections were detected in stool and urines samples. The in vitro cellular cytokine and chemokine responses of peripheral blood mononuclear cells (PBMC) to parasite antigens and allergens were analysed in children (n = 87) with single and poly-parasite infection, and skin prick test reactivity to fungus and mite allergens was determined in singly and poly-parasitized children (n = 509).

Results: In children Entamoeba histolytica/dispar (62%), Necator americanus (31%), Schistosoma haematobium (28%), S. mansoni (21%), Hymenolepis nana (2%) and Strongyloides stercoralis (1%) were diagnosed. Singly infected were 37%, 47% were positive for 2 or more parasite species and 16% were infection-free. When PBMC were stimulated in vitro with parasite antigens and allergens, regulatory-type cytokine IL-27 and alarmin-type IL-33 enhanced with poly-parasite infections whilst IL-10 and pro-inflammatory MIP3-α/CCL20 and MIG/CXCL9 were produced in similar amounts in singly or poly-parasitized children. The co-stimulation in vitro of PBMC with mite allergens and Ascaris lumbricoides antigens depressed the allergen-induced pro-inflammatory IL-27, IL-33 and MIP3-α/CCL20 responses while regulatory IL-10 remained unaffected. Post albendazole and/or praziquantel treatment, the cellular release of IL-10, IL-33, MIP3-α/CCL20 and MIG/CXCL9 lessened significantly in all children infection groups. Skin prick test (SPT) reactivity to fungus Aspergillus fumigatus and mite Dermatophagoides pteronyssinus allergens was investigated in 509 children, and positive SPT responses were found in 23% of the infection-free, and in 47%, 53% and 56% of the singly, doubly and poly-parasite infected, respectively.

Conclusions: In children co-infected with helminthes and protozoan parasites a mixed cellular response profile of both inflammatory and regulatory chemokines and cytokines was stimulated by individual antigens and allergens, pro-inflammatory cytokines and chemokines enhanced with an increasing number of parasite infections, and in poly-parasitized children skin prick test reactivity to allergens extracts was highest.

Keywords: Allergen; Amoebiasis; Chemokine; Co-infection; Cytokine; Hookworm; Schistosomiasis; Skin prick test.

Figure 1

The inducible and spontaneous cellular production (in pg/ml) of interleukin 10 (IL-10) (Part A), Monocyte Inflammatory Protein 3-α (MIP-3α/CCL20) (Part B) and Monokine Inducible by Interferon-γ (MIG/CXCL9) (Part C) by peripheral blood mononuclear cells (PBMC) from children is shown. Cellular cytokine or chemokine production was quantified by specific ELISA in cell culture supernatants after stimulation of PBMC for 48 hours with parasite-specific antigens from Entamoeba histolytica (Eh), Plasmodium falciparum (Pf), Ascaris lumbricoides (Asc), Schistosoma mansoni (Sm), Echinococcus multilocularis (Em), bacterial lipopolysaccharid (LPS) from Escherichia coli or allergen extracts from Dermatophagoides farinae (Df), Dermatophagoides pteronyssinus (Dp), Aspergillus fumigatus (Af) and Candida albicans (Ca). The spontaneous cytokine or chemokine releases by PBMC, i.e. cell cultures without antigen or allergen activation, are shown as baseline (Bl), and the horizontal line indicates the mean spontaneous cytokine or chemokine release. Cellular production of IL-10, MIP-3α/CCL20 and MIG/CXCL9 are shown as means with the 95% upper and lower confidence interval.

Figure 2

The cellular production of interleukin 10 (IL-10) by peripheral blood mononuclear cells (PBMC) is shown in children without parasite infection (Group G0, n = 11), children with single infection (Group G1, n = 14) and children with poly-parasite infections (Group G3+, n = 7), and also, IL-10 production in children at 8 weeks post anti-parasite treatment (pT, n = 19) is given. The release of IL-10 by PBMC was quantified by cytokine specific ELISA in cell culture supernatant of PBMC activated with antigens from P. falciparum (Pf), with D. pteronyssinus (Dp) or with A. fumigatus (Af) is shown in G0, G1, G3+ children before and 8 weeks post anti-parasite treatment (pT). Cellular production of IL-10 is shown as means with the 95% upper and lower confidence interval. The level of significance was adjusted according to Bonferroni–Holm, and significant differences between groups are indicated as *P ≤ 0.002.

Figure 3

The cellular release of IL-27, IL-33, MIP3-a/CCL20 and MIG/CXCL9 in response to allergen extracts of Dermatophagoides pteronyssinus (Dp) and Aspergillus fumigatus (Af), in singly and poly-parasitized children; children without parasite infection (Group G0, n = 11), children with single infection (Group G1, n = 14) and children with poly-parasite infections (Group G3+, n = 7), and the cytokine and chemokine production in children at 8 weeks post anti-parasite treatment (pT, n = 19) is shown. The cytokine and chemokine responses to the extracts of D. pteronyssinus (Dp) and A. fumigatus (Af) are merged. The cellular productions of IL-10, IL-27, MIP-3α/CCL20 and MIG/CXCL9 are shown as means with the 95% upper and lower confidence interval. The level of significance was adjusted according to Bonferroni–Holm, and significant differences between groups are indicated as *P ≤ 0.002.

Figure 4

Cellular release (in pg/ml) of IL-10, IL-27, IL-33 and MIP3-a/CCL20 by PBMC activated alone with helminth-derived Ascaris lumbricoides (Asc) or allergen extracts from Dermatophagoides pteronyssinus (Dp) or after co-activation with Asc antigen and DP allergen. PBMC were isolated from singly and poly-parasitized children and cultured in vitro for 48 hours. Cellular production of IL-10, IL-27, IL-33, MIP-3α/CCL20 and MIG/CXCL9 are shown as means with the 95% upper and lower confidence interval. The level of significance was adjusted according to Bonferroni–Holm, and significant differences between groups are indicated as *P ≤ 0.002.

Figure 5

Skin prick test reactivity (positive/negative) in children (total n = 509) to Aspergillus fumigates and Dermatophagoides pteronysinus prick test solutions were evaluated in n = 287 non-infected (G0), in n = 137 singly (G1), in n = 58 doubly (G2) and n = 27 poly-parasitized (G3+) children. Allergens and positive histamine and negative saline controls were pricked onto the volar surface of the forearm, and reactions were recorded after 15 min. A skin prick test (SPT) weal cut-off diameters of 3 mm or larger was considered as a positive reaction. (p < 0.0001 between G0 and G1).