Plasma cytokines, chemokines and cellular immune responses in pre-school Nigerian children infected with Plasmodium falciparum

Abstract

Background: Malaria is a major cause of morbidity and mortality worldwide with over one million deaths annually, particularly in children under five years. This study was the first to examine plasma cytokines, chemokines and cellular immune responses in pre-school Nigerian children infected with Plasmodium falciparum from four semi-urban villages near Ile-Ife, Osun State, Nigeria.

Methods: Blood was obtained from 231 children (aged 39-73 months) who were classified according to mean P. falciparum density per μl of blood (uninfected (n = 89), low density (<1,000, n = 51), medium density (1,000-10,000, n = 65) and high density (>10,000, n = 22)). IL-12p70, IL-10, Nitric oxide, IFN-γ, TNF, IL-17, IL-4 and TGF-β, C-C chemokine RANTES, MMP-8 and TIMP-1 were measured in plasma. Peripheral blood mononuclear cells were obtained and examined markers of innate immune cells (CD14, CD36, CD56, CD54, CD11c AND HLA-DR). T-cell sub-populations (CD4, CD3 and γδTCR) were intracellularly stained for IL-10, IFN-γ and TNF following polyclonal stimulation or stimulated with malaria parasites. Ascaris lumbricoides was endemic in these villages and all data were analysed taking into account the potential impact of bystander helminth infection. All data were analysed using SPSS 15 for windows and in all tests, p <0.05 was deemed significant.

Results: The level of P. falciparum parasitaemia was positively associated with plasma IL-10 and negatively associated with IL-12p70. The percentage of monocytes was significantly decreased in malaria-infected individuals while malaria parasitaemia was positively associated with increasing percentages of CD54+, CD11c+ and CD56+ cell populations. No association was observed in cytokine expression in mitogen-activated T-cell populations between groups and no malaria specific immune responses were detected. Although A. lumbricoides is endemic in these villages, an analysis of the data showed no impact of this helminth infection on P. falciparum parasitaemia or on immune responses associated with P. falciparum infection.

Conclusions: These findings indicate that Nigerian children infected with P. falciparum exhibit immune responses associated with active malaria infection and these responses were positively associated with increased P. falciparum parasitaemia.

Figure 1

Plasmodium falciparum parasitaemia was positively associated with IL-10 and negatively associated with IL-12p70 levels in the plasma of infected children. Mean plasma levels of (A) IL-10 and (B) IL-12p70 were determined by ELISA for each group (low (<1,000; n=51), medium (1,000-10,000; n=65) and high (>10,000; n=22) mean parasite density (per μl of blood) and compared to endemic controls (EC) (n=89). ^*, p ≤0.05; ^**, p ≤0.01 and ***, p ≤0.001 (ANOVA). Parasitaemia was plotted against (C) IL-10 and (D) IL-12p70 and association between variables was assessed using regression analysis. p <0.05 was deemed significant.

Figure 2

Malaria infection was associated with decreased CD14⁺ percentages and enhanced percentages of CD11c⁺, CD54⁺, CD56 but not HLA-DR⁺ and CD36⁺ cell populations. PBMCs were stained extracellularly for CD14 (A), CD54 (B) CD11c (C) HLA-DR (D), CD36 (E) and CD56 (F) cell expression for each group (low (<1,000; n=51), medium (1,000-10,000; n=65) and high (>10,000; n=22) mean parasite density (per μl of blood)) was determined by flow cytometry and compared to endemic controls (EC). ^**p ≤0.01 (ANOVA).

Figure 3

Malaria infection was not correlated with an increase in the secretion of IFN-γ, TNF, IL-10 and IL-2 in mitogen-activated T-cell populations. PBMCs were stimulated for 4 h with PMA and ionomycin. Cells were washed and stained for cell surface expression of γδTCR, CD3 and CD4 and were intracellular stained for IFN-γ, IL-2, IL-10 or TNF. Appropriate isotype controls were included to define parameter gates. PBMCs were gated on the lymphocyte population and the percentage of positive cells for each group (low (<1,000; n = 51), medium (1,000-10,000; n = 65) and high (>10,000; n = 22) mean parasite density (per μl of blood) were also compared to uninfected endemic controls (EC).

Figure 4

PBMCs from infected individuals did not secrete antigen specific immune responses. PBMCs (1x10⁶ cells/ml) from malaria infected (n=23) and non-infected (n=7) children were stimulated with medium (Med), mycoplasma free P. falciparum parasites (at a ratio of 1:5) (malaria antigen) and PMA/anti-CD3. After three days IFN-γ, IL-10, and IL-5 were measured in supernatant by commercial assay.

Figure 5

Ascaris lumbricoides infection did not impact upon Plasmodium falciparum parasitaemia. Mean P. falciparum density (per μl of blood) was measured from malaria infected (MAL) and Ascaris and malaria co-infected (ASC+MAL) children by Giemsa stained blood slides (n = 109 for MAL; n = 32 for ASC+MAL) NS = non-significant.

Figure 6

Ascaris lumbricoides infection did not alter the percentage of CD14⁺ CD11c⁺, CD54⁺, CD56 HLA-DR⁺ and CD36⁺ cell populations in-co-infected individuals. PBMCs were stained extracellularly for CD14 (A), CD54 (B) CD11c (C), HLA-DR (D), CD36 (E) and CD56 (F) cell expression for each group Ascaris infection only (ASC), Malaria infection only (MAL) and co-infected (ASC + MAL) was determined by flow cytometry and compared to endemic controls (EC). ^*p ≤0.05; ^**p ≤0.01 (ANOVA) and MAL group was compared to the ASC + MAL group (no significant differences).

Figure 7

Ascaris lumbricoides infection did not alter IFN-γ, TNF, IL-10 and IL-2 expression in mitogen-activated T-cell populations. PBMCs were stimulated for 4 h with PMA and ionomycin. Cells were washed and stained for cell surface expression of γδTCR, CD3 and CD4 and were intracellular stained for IFN-γ, IL-2, IL-10 or TNF-a. Appropriate isotype controls were included to define parameter gates. PBMCs were gated on the lymphocyte population and the percentage of positive cells for each group Ascaris infection only (ASC), Malaria infection only (MAL) and co-infected (ASC + MAL) was determined by flow cytometry and compared to uninfected endemic controls (EC) and MAL group was compared to the (ASC + MAL).