Echinococcus multilocularis specific antibody, systemic cytokine, and chemokine levels, as well as antigen-specific cellular responses in patients with progressive, stable, and cured alveolar echinococcosis: A 10-year follow-up

Abstract

Background: The infestation with Echinococcus multilocularis larvae may persist in humans for up to decades without evident clinical symptoms. Longitudinal investigations are needed to understand the dynamic immunological processes in alveolar echinococcosis (AE) patients associated with an active and progressive, a stable or a regressive course of disease.

Methodology/principal findings: This study evaluated the E. multilocularis specific antibody responses, systemic cytokine, and chemokine serum levels over a 10-year follow-up period, as well as cellular responsiveness in AE patients. Our results demonstrate a rapid decrease in antibodies against E. multilocularis specific antigen Em2+. Especially in cured patients, these antibodies remained negative, making them a significant predictor for cured AE. E. multilocularis specific IgG4, and indirect hemagglutination IHA decreased later in time, after around 5 years. While total IgE did not show significant dynamics over the course of disease, E. multilocularis specific IgE decreased after one to two years, and increasing levels were a significant predictor of progressive disease. There was no significant change in systemic IL-8, IL-9, CCL18 or CCL20 serum levels over time. Univariate analysis across groups indicated lower IL-8 levels in cured patients; however, this result could not be confirmed by multivariate analysis. Levels of CCL17 decreased during treatment, especially in cured patients, and thus might serve as a predictive or risk factor for progressive disease. Levels of IL-10 and CCL13 decreased during disease, especially after five and ten years of intervention. The E. multilocularis antigen (EmAg) inducible cellular productions of MCP1(CCL13), TARC(CCL17) and PARC(CCL18) were lowest in patients with cured AE and infection-free controls, while the EmAg inducible cellular production of IFN-γ increased after cure. Significant positive cytokine and chemokine correlations were observed in AE patients for IL-9, IL-10, CCL13(MCP-4), CCL17(TARC) and CCL20(LARC)(for all p<0.001). E. multilocularis specific IgG4 response correlated positively with TARC (p<0.001). Both markers enhanced over time in progressive disease and decreased after cure. The levels of IL-8, IL-10, MCP4 and LARC enhanced with AE regression.

Conclusions/significance: Repeated biomarker surveys are advisable to evaluate progression or regression of disease during longitudinal follow-up and such analyses can support imaging techniques and improve staging of AE patients.

Fig 1. The E. multilocularis antigen-specific IgG reactivity (OD) in alveolar echinococcosis (AE) patients and infection-free controls.

The AE patients were staged into those with progressive AE, with stable (<60y), stable (≥60) and cured AE. For the determination of age dependent EmAg specific IgG4 responses, the patients were grouped in five age groups. Blood samples from AE patients were collected at three time points in 2005 (year 1) in 2010 (year 5) and in 2016 (year10). Controls were echinococcosis infection-free healthy blood donors. In (A) the EmAg specific IgG1, in (B) the EmAg specific IgG3 and in (C) the EmAg specific IgG4 responses are shown as mean optical densities (OD) with the 95% confidence intervals for the means (diamonds). The data presented in box plots show the median OD per group with the 25% and 75% quartiles and the 1,5x of the interquartile range with outlier as individual points. In (D) the IgG4 reactivity in AE patients to EmAg is shown as mean OD per age group with the 95% confidence intervals.

Fig 2. The E. multilocularis antigen inducible cellular production by PBMC of chemokines and cytokines.

In (A) MCP-1(CCL2), MCP-3(CCL7), MCP-4(CCL13), in (B) cytokines IL-10 and IFN-γ and in (C) chemokines CCL17(TARC) and CCL18(PARC) were investigated in patients with progressive, stable, and cured AE, and in E. multilocularis infection-free controls. Freshly isolated and in vitro cultured PBMC (2.5x10⁶/ml) were stimulated with E. multilocularis antigen (EmAg, 12 μg/ml) or remained without stimulation (baseline) for 48 hours. Cytokines and chemokines secreted into cell culture supernatants were quantified with specific ELISA. The concentrations are indicated as mean (net) amounts in pg/ml (with the 5% upper and 95% lower confidence interval) of cytokine or chemokine released from stimulated PBMC minus the cellular production of unstimulated (baseline) PBMC. MCP-1(CCL2), MCP-3(CCL7) and MCP-4(CCL13) production by PBMC was quantified in AE patients with progressive (n = 7) stable (n = 29) and cured disease (n = 10) disease. For IL-10, IFN-γ and PARC(CCL18) and TARC(CCL17) production was investigated in AE patients with progressive (n = 19), stable (n = 27) and cured disease (n = 14), and in E. multilocularis infection-free controls (n = 6).** p<0.01 versus cured or control.