Fig 1.
Naïve individuals are High- or Low- IFN-γ producers upon stimulation with Leishmania braziliensis promastigotes.
PBMCs were cultured with L. braziliensis for 72h (Lb) or left unstimulated (Unst). Supernatants were collected and IFN-γ levels were determined by ELISA. Data from one representative experiment are shown individually for High Producers (HP) (n = 4) and Low Producers (LP) (n = 5). Samples from three HP (orange) and three LP (blue) individuals, depicted as triangle, diamond and circle were used in subsequent experiments.
Fig 2.
Genes modulated in High- or Low- IFN-γ producers following stimulation with L. braziliensis.
PBMCs from High IFN-γ Producers (HPs) (n = 3) and from Low IFN-γ Producers (LPs) (n = 3) were stimulated with L. braziliensis for 72h. Gene expression was determined using total RNA and arrays covering Th1-Th2-Th3 responses, IFN-γ and receptors, chemokines and receptors and TLRs. (A) Venn diagram depicting the total number of genes differentially modulated in HPs only, LPs only or modulated in both categories, as evidenced by arrays analysis (see materials and methods). (B) Fold expression of the 12 genes differentially modulated in both HPs and LPs, as evidenced by array analysis (see materials and methods).
Fig 3.
qRT-PCR validation of genes commonly modulated in High- and Low- IFN-γ producers.
PBMCs from High Producers (HPs) (n = 3) and Low Producers (LPs) (n = 3) were stimulated with L. braziliensis for 72h. Relative expression of IFNG, CXCL10, IFI27, IL6 and IRF1 was evaluated by qRT-PCR. Gene expression is represented as fold change of stimulated over unstimulated cultures, normalized to a housekeeping gene. Bars represent the mean ± SEM.
Fig 4.
Expression profile of genes uniquely modulated in High- IFN-γ producers.
(A) PBMCs from High IFN-γ Producers (HPs) (n = 3) were stimulated with L. braziliensis for 72h. Gene expression was determined using total RNA and immune gene arrays. Fold expression of the genes modulated in HPs or in LPs only, as evidenced by PCR array (see materials and methods). (B) Top canonical pathways identified in HPs by Ingenuity Pathway Analysis. Levels of significance are given by the right-tailed Fisher exact test. The negative log P value (blue bars), along the x-axis, increases as a pathway is more significantly associated with the set of genes expressed in HPs. The ratio (orange line) indicates the proportion of upregulated genes relative to all the genes present in a pathway.
Fig 5.
Expression profile of genes uniquely modulated in Low- IFN-γ producers.
(A) PBMCs from Low IFN-γ Producers (LPs) (n = 3) were stimulated with L. braziliensis for 72h. Gene expression was determined using total RNA and immune gene arrays. Fold expression of the genes modulated in HPs or in LPs only, as evidenced by PCR array (see materials and methods). (B) Top canonical pathways identified in LPs by Ingenuity Pathway Analysis. Levels of significance are given by the right-tailed Fisher exact test. The negative log P value (blue bars), along the x-axis, increases as a pathway is more significantly associated with the set of genes expressed in LPs. The ratio (orange line) indicates the proportion of upregulated genes relative to all the genes present in a pathway.
Fig 6.
Genes modulated in High- and Low- IFN-γ producers discriminate subclinical L. braziliensis infection from active CL.
(A) PBMCs from CL patients (n = 5) and SC individuals (n = 8) were stimulated with L. braziliensis for 72h. Relative expression of IFI27, IFIT1, TLR2, IRF1, JAK2 and IL6 was evaluated by qRT-PCR. Gene expression is represented as fold change of stimulated over unstimulated cultures, normalized to a housekeeping gene and each symbol represents one individual. (B) A heat map was designed to depict the pattern of gene expression [shown in (A)] of SC individuals) vs. active CL and two-way hierarchical cluster analysis (Ward’s method) of differentially expressed genes was performed. Expression scale for each gene represents the log2-fold change from the mean. (C) Principal component analysis of the differentially expressed genes [depicted in (A)] showing PC1 (x axis), PC2 (y axis) and PC3 (z axis).
Fig 7.
Genes modulated in High- IFN-γ producers are upregulated in CL lesions.
The number of transcripts for each gene was quantified in Data sets GSM1341365 [12] and GSM1560512 [13]. Data were normalized using RMA (Robust Multichip Average) for each dataset, log-transformed expression for each gene is shown as box and whiskers (displaying quartile range) and outliers (identified by Tukey’s test). (t-test, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05).