Protein biomarkers discriminate Leishmania major-infected and non-infected individuals in areas endemic for cutaneous leishmaniasis

Abstract

Background: A successful host immune response to infection is dependent upon both innate and adaptive immune effector mechanisms. Cutaneous leishmaniasis results in an adaptive Th1 CD4(+) T cell response that efficiently clears the parasite, but may also result in scaring. However the role of innate mechanisms during parasite clearance remains less well defined.

Methods: We examined a unique cohort of individuals, living in a Leishmania major endemic region, that were stratified among 3 distinct clinical groups in a cross-sectional study. Specifically, patients were classified either as healed (n = 17), asymptomatic (23), or naïve to infection (18) based upon the classical Leishmanin Skin Test (LST) and the presence or absence of scars. Utilizing a multiplexed immunoassay approach we characterized the induced cytokine and chemokine response to L. major.

Results: A subset of innate immune molecules was induced in all groups. By contrast, T cell-associated cytokines were largely induced in exposed groups as compared to L. major-infection naïve individuals. Two exceptions were IL-17A and IL-12p70, induced and not induced, respectively, in naïve individuals. In addition, GM-CSF was more strongly induced in healed patients as compared to the other two groups. Surprisingly an IL-13 response was the best cytokine for classifying previously infected donors.

Conclusions: Exploratory data analysis, utilizing principle component analysis (PCA), revealed distinct patient clusters of the healed and naïve groups based on the most differentially induced proteins. Asymptomatic previously infected individuals were more difficult to assign to a particular cluster based on these induced proteins. Analysis of these proteins may enable the identification of biomarkers associated with disease, leading to a better understanding of the protective mechanisms of immune response against leishmaniasis.

Keywords: Asymptomatic infection; Leishmaniasis; Protein biomarkers.

Fig. 1

Characterization of the adaptive immune response in LST+ individuals. Secreted cytokines, (a) IFN-γ, (b) IL-2, (c) IL-18, (d) IL-12p70, and (e) IL-13 measured by Luminex assays following Leishmania parasite stimulation of PBMCs from donors subdivided into 3 clinical groups, according to the Leishmanin skin test response (LST+/−) and presence or absence of specific scars (SCAR+/−). (***) denotes p < 0.001 as determined by Wilcoxon paired T test, samples with no measurable value were given a value that was half of the lowest detectable dose of the assays)

Fig. 2

Shared induced innate immune responses across all 3 clinical groups. Secreted cytokines significantly induced (Wilcoxon paired T test, p < 0.05) (a) MIP-1α, (b) IL-1β, (c) M-CSF, (d) IL-10, (e) IL-17A, (f) TNFα, (g) IL-5, and (h) GMCSF measured by Luminex assays following Leishmania parasite stimulation of PBMCs from donors subdivided into 3 clinical groups, according to the Leishmanin skin test response (LST+/−) and presence or absence of specific scars (SCAR+/−). (*** denotes p < 0.001, ** p < 0.01, * p < 0.05 as determined by Kruskal Wallis test with Dunn’s multiple testing between the 3 groups, samples with no measurable value were given a value that was half of the lowest detectable dose of the assays)

Fig. 3

Lack of induced response to Leishmania parasite for key inflammatory molecules. Cytokines (a) IL-6, (b) IL-8 with no differences as measured by Luminex assays following parasite stimulation of PBMCs from donors subdivided into 3 clinical groups, according to the Leishmanin skin test response (LST+/−) and presence or absence of specific scars (SCAR+/−)

Fig. 4

Multivariate protein analysis. Principal component analysis (PCA) of LST + SCAR+ (blue), LST + SCAR- (yellow), and LST-SCAR- (pink) donors (a) based on the 10 most significantly differential proteins as identified by multi ANOVA (q < 0.01), (b) heat map overlay of IFN-γ expression. PCA of (c) LST + SCAR+ (blue) and LST-SCAR- (pink) donors, (d) LST + SCAR- (yellow) and LST-SCAR- (pink) donors. Numbers represent percentage of variance captured by each principal component. (e) List of most differential proteins with p and q values as determined by multi ANOVA testing