Toll-like receptor agonists partially restore the production of pro-inflammatory cytokines and type I interferon in Sézary syndrome

Abstract

Sézary syndrome (SS) carries a poor prognosis, and infections represent the most frequent cause of death in SS patients. Toll-like receptors (TLRs) are a family of innate immune receptors that induce protective immune responses against infections. We sought to evaluate the ability of TLR agonists to induce inflammatory cytokine, Th2 cytokine, and type I interferon (IFN-I) production by peripheral blood mononuclear cells (PBMC) of untreated SS patients. We detected impaired IL-6, IL-10 and IL-13 secretion by PBMC induced by the agonists for TLR5, TLR3, TLR7 and TLR9 in SS patients, while it was partially recovered by TLR2/TLR4 and TLR7/8 agonists TNF secretion was restored following stimulation with TLR2/TLR4 agonists. IFN-γ was scarcely produced upon TLR activation in SS cells, albeit TLR 7/8 (CL097) enhanced their secretion at lower levels than the control group. TLR9 agonist efficiently induced IFN-I in SS patients, although this positive regulation was not observed for other cytokines, in direct contrast to the broad activity of CL097. Among the TLR agonists, TLR4 was able to induce pro-inflammatory, IL-10 and Th2 secretion, while TLR7-8 agonist induced the inflammatory cytokines, IFN-I and IFN-γ. These findings reveal a dysfunctional cytokine response upon both extracellular and intracellular TLR activation in SS patients, which was partially restored by TLRs agonists.

Keywords: Immune response; Immunity; Immunology and Microbiology Section; Toll-like receptor agonists; cytokines; innate immunity; type I interferon; Sézary syndrome.

Figure 1. Impaired IL-6, TNF and IFN-γ production by PBMC of SS patients induced by TLR activation

PBMC from SS patients (n = 10, closed circle) and healthy controls (HC, n = 13, open circle) were cultured in medium (unstimulated, UNS) or with TLR agonists (TLR2-TLR9) for 48 h. Supernatants were assessed for IL-6, TNF and IFN-γ secretion with a cytometric bead array. The results are shown as medians and interquartile ranges (IQRs).*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 compared with the HC group; #p ≤ 0.05, ##p ≤ 0.01, ###p≤ 0.001 compared with the unstimulated culture.

Figure 2. Altered IL-10 and IL-13 secretion by PBMC of SS patients induced by TLR agonists

PBMC from SS patients (n = 10, closed circle) and healthy controls (HC, n = 13, open circle, open circle) were cultured with medium (unstimulated, UNS) or stimulated for 48 h with TLR agonists (TLR2-TLR9). Supernatants were assessed for IL-10 and IL-13 secretion with a cytometric bead array. Horizontal line shows the detection limit for IL-13. The results are shown as medians and IQRs.*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 compared with the HC group; #p ≤ 0.05, ##p ≤ 0.01, ###p ≤ 0.001 compared with the unstimulated culture.

Figure 3. Agonists of TLR7/8 and TLR9 are able to induce IFN-I secretion in SS patient cells

A. PBMC from SS patients (n = 11, closed bar) and healthy controls (HC, n = 13, open bar) were cultured with medium (unstimulated, UNS) or stimulated for 48 h with agonists of TLR3, TLR7, TLR7/8 or TLR9. Supernatants were assessed for IFN-α secretion with a cytometric bead array. B. PBMC from SS patients (closed circle, n = 9) and HC (open circle, n = 12) were assessed for IFN-α, IFNR-I, IFN-λ and IFN-γ mRNA expression by qPCR. The results are presented as medians and IQRs.*p ≤ 0.05 compared with the HC group; #p ≤ 0.05, ##p ≤ 0.01 compared with the unstimulated culture.

Figure 4. Cytokine serum levels in SS patients

Sera from SS patients (n = 12, closed circle) and healthy controls (HC, n = 19, open circle) were assessed for cytokines using high-sensitivity flex sets for TNF, IL-6, IL-5, IL-10, IL-2 and IL-17A or a flex set for TGF-β1 by flow cytometry. The results are presented as medians and IQRs. **p ≤ 0.01, ***p ≤ 0.001 compared with the HC group.