Elevated IP-10 at the Protein and Gene Level Associates With Pulmonary TB

Abstract

There is an urgent need for accurate and sensitive diagnostic tools that can overcome the current challenge to distinguish individuals with latent tuberculosis infection (LTBI) from individuals with active tuberculosis (TB). Recent literature has suggested that a group of cytokines may serve as biomarkers of TB disease progression. Using a multiplex ELISA, we quantified 27 circulatory markers present within the unstimulated plasma of individuals in Durban, South Africa who were healthy (n=20), LTBI (n=13), or had active TB (n=30). RT-qPCR was performed to measure gene expression of the cytokines of interest, using RNA isolated from healthy (n=20), LTBI (n=20), or active TB (n=30). We found that at the protein level, IL-1RA, IL-6, and IP-10 were significantly more abundant in participants with active TB (p< 0.05) compared to those with LTBI individuals. IP-10 also showed the strongest association with active TB compared to healthy and LTBI at mRNA level. Our data shows that these proteins may serve as biomarkers of TB at both the protein and gene level.

Keywords: IL-1RA; IP-10; TB diagnosis; biomarkers; disease progression; latent TB; tuberculosis.

Figure 1

Principal component analysis (PCA) of the association between circulatory cytokines and TB disease states. (A) Circulatory cytokines separate according to whether individuals are healthy, latent TB infected, or with active TB disease. (B) PC1 accounted for 38.38% of the variance observed in the data set. (C) PC2 accounted for 16.12% of the variance observed in the data set. PC2 was used to define the association between circulatory cytokine and whether individuals were healthy, LTBI, or those who had active TB. Each dot represents a participant score on the loading components.

Figure 2

Plasma IL-RA, IL-6, and IP-10 are elevated in TB patients. Quantification of various cytokines in the plasma of healthy (n=20) individuals compared to LTBI (n=13) and TB (n=30) patients. Each dot represents a participant sample. (A) IL-1RA, (B) IL-6, and (C) IP-10 were quantified and measured in pg/ml. Significant differences were identified as those values that were less than p<0.05. Data was analyzed using the one-way ANOVA and followed by a multiple comparisons test using Tukey’s test. Tukey’s p-values have been reported in the above figure and illustrate the differences observed between groups.

Figure 3

IP-10 is upregulated in whole blood of TB patients. Gene expression of (A) IL-1RA, (B) IL-6, and (C) IP-10 in the whole blood of healthy (n=20), LTBI (n=20), and TB (n=30) participants normalized to a house-keeping gene, RPLP1. P< 0.05 was considered statistically significant. Data were analyzed using the one-way ANOVA and followed by a multiple comparisons test using Tukey’s test. Tukey’s p-values have been reported in the above figure and illustrate the differences observed between groups.

Figure 4

ROC curve characteristics for IP-10 protein and IL-1RA protein as well as IP-10 mRNA, as discriminatory markers for TB. AUC, specificity, sensitivity, PPV, and NPV indicate the power of (A) IL-1RA protein abundance, (B) IP-10 protein abundance, and (C) IP-10 mRNA expression which are reported and indicate the power of these biomarkers for discriminating between those who are healthy and those with TB.