T-bet, GATA-3, and Foxp3 expression and Th1/Th2 cytokine production in the clinical outcome of human infection with Leishmania (Viannia) species

Abstract

Background: T cell differentiation determines susceptibility and resistance to experimental cutaneous leishmaniasis, yet mixed T1/Th2 responses characterize the clinical spectrum of human infection with Leishmania (Viannia) species.

Materials and methods: To discern the interrelationship of T cell differentiation and outcome of human infection, we examined factors that regulate T cell differentiation and Th1/Th2 cytokine responses in asymptomatic infection, active and historical chronic and recurrent cutaneous leishmaniasis. T-bet, GATA-3, Foxp3, and cytokine gene expression were quantified by real-time polymerase chain reaction and correlated with interleukin 2, interferon gamma, tumor necrosis factor alpha, interleukin 4, interleukin 13, and interleukin 10 secretion during in vitro response to live Leishmania panamensis.

Results: Higher GATA-3 expression than T-bet expression occurred throughout the 15 days of coculture with promastigotes; however, neither transcription nor secretion of interleukin 4 was detected. A sustained inverse correlation between GATA-3 expression and secretion of proinflammatory cytokines interferon gamma and tumor necrosis factor alpha was observed in asymptomatic infection. In contrast, higher T-bet expression and a higher ratio of T-bet to GATA-3 characterized active recurrent disease. Down-regulation of T-bet and GATA-3 expression and increased interleukin 2 secretion, compared with control subjects, was directly correlated with Foxp3 expression and interleukin 13 secretion in chronic disease.

Conclusions: Regulation of the inflammatory response rather than biased Th1/Th2 response distinguished asymptomatic and recalcitrant outcomes of infection with Leishmnania viannia species.

Figure 1

Schematic summary of the research strategy to evaluate transcription factor expression and cytokine response profile in relation with outcome of infection with Leishmania (Viannia) species.

Figure 2

T-bet and GATA-3 expression in PBMCs from patients with active and historic cutaneous leishmaniasis, asymptomatically infected residents of endemic areas and healthy controls co-cultured during 8 hours, 24 hours, and 6 days with live promastigotes of L. (V) panamensis. All data were normalized to b-actin. P values were calculated using Kruskal Wallis (K-W) Test and Dunn procedure, *P < .05. Active Chronic (AC); Historic Chronic (HC); Active Recurrent (AR); Historic Recurrent (HR); Asymptomatic (AS); Healthy Control (C).

Figure 3

Inter-relation of Foxp3 expression, IL-2 and IL-13 production by PBMCs in cutaneous Leishmaniasis. A) Foxp3 expression at 24 hours in PBMCs from patients with Active Chronic (AC), Active Recurrent (AR) disease, asymptomatically infected individuals (AS) and Healthy Controls (C) co-cultured with live promastigotes of L.(V) panamensis; Kruskal-Wallis Tests, Dunn *P < .05. Data were normalized to β-actin. B) Net IL-2 secretion C) Correlation between IL-2 secretion and Foxp3 expression, D) Correlation between IL-2 at 24 hours and IL-13 secretion at 72 hours.

Figure 4

Net Cytokine secretion by PBMCs from patients with Active and Historic Cutaneous Leishmaniasis, Asymptomatically infected residents of endemic area and Healthy controls co-cultured during 72 hours with live promastigotes of L. V. panamensis. P values were calculated using Kruskal Wallis Test and Dunn procedure, *P < .05. Net production was calculated by subtraction of background (PBMC plus medium) from cytokine production in the presence of live promastigotes. Active Chronic (AC); Historic Chronic (HC); Active Recurrent (AR); Historic Recurrent (HR); Asymptomatic (AS); Healthy Control (C).

Figure 5

Inverse correlation between ratio GATA-3/B-actin expression with TNF-α and IFN-γ in Asymptomatic infection with Leishmania (Viannia) species. A) Regression analysis of TNF-α secretion (pg/mL), vs GATA-3 expression at 8 hours, 24 hours, 6 and 15 days of co-culture of PBMCs with live promastigotes of L. (V) panamensis, B) Regression analysis of IFN-γ secretion (pg/mL) at 8 hours, 24 hours, 6 and 15 days of co-culture of PBMCs with live promastigotes of L. (V) panamensis.

Figure 6

Correlation between IL-2 (pg/mL) secretion at 24 hours and T-bet expression at 24 hours, 6 days, and 15 days of co-culture of PBMCs from patients with active chronic cutaneous leishmaniasis