Continuous time Bayesian networks identify Prdm1 as a negative regulator of TH17 cell differentiation in humans

Abstract

T helper 17 (TH17) cells represent a pivotal adaptive cell subset involved in multiple immune disorders in mammalian species. Deciphering the molecular interactions regulating TH17 cell differentiation is particularly critical for novel drug target discovery designed to control maladaptive inflammatory conditions. Using continuous time Bayesian networks over a time-course gene expression dataset, we inferred the global regulatory network controlling TH17 differentiation. From the network, we identified the Prdm1 gene encoding the B lymphocyte-induced maturation protein 1 as a crucial negative regulator of human TH17 cell differentiation. The results have been validated by perturbing Prdm1 expression on freshly isolated CD4(+) naïve T cells: reduction of Prdm1 expression leads to augmentation of IL-17 release. These data unravel a possible novel target to control TH17 polarization in inflammatory disorders. Furthermore, this study represents the first in vitro validation of continuous time Bayesian networks as gene network reconstruction method and as hypothesis generation tool for wet-lab biological experiments.

Figure 1. Inferred network of human TH17 cell differentiation.

Node size is proportional to the number of outgoing arcs.

Figure 2. Specific regulation mechanisms from the inferred network.

(A) Known pathway of negative regulation of SOCS3 on STAT3 (B) Regulatory interaction between PRDM1, SOCS3, SOCS1, BATF and CXCR5 as predicted by the inferred network.

Figure 3. Experimental validation through Prdm1 perturbation.

Prdm1 perturbation boosts IL-17 differentiation program. IL-17A (A) and Prdm1 mRNA levels (B) were assessed by ELISA and qPCR, respectively, in naïve CD4⁺ T cells and CD4⁺ T cells stimulated as follow: αCD3/αCD28, TH0 polarization for 72 h, or TH17 polarization for the indicated time. (C) qPCR was used to validate the siRNA-mediated reduction of Prdm1 expression levels upon siRNA-mediated perturbation. Graphs show the mean of 5 independent experiments. (D,F) IL-17A cytokine release (D,E) and mRNA (F) from siRNA-treated CD4⁺ T cells following TH17 polarization (48 h) was measured by ELISA and pPCR, respectively. In panel E results are shown as relative values normalized to the corresponding scramble control. Significance was calculated using two-tailed paired t-test (A,B,D) and one-sample t-test (C,E,F). *P value < 0.05; ***P value <0.001; n.s., non significant.

Figure 4. Effect of Prdm1 perturbation on IL-17A and IL-10 secretion.

Prdm1 perturbation increases IL-17A, but not IL-10, release. Secretion of IL-17A (upper) and IL-10 (lower) was measured in siRNA-treated CD4⁺T cells polarized towards TH0 (left) or TH17 (right) cells for 72 h. Two-tailed paired t-test has been performed. n.s., not significant; *P value < 0.05.