The Pronounced Th17 Profile in Systemic Sclerosis (SSc) Together with Intracellular Expression of TGFβ and IFNγ Distinguishes SSc Phenotypes

Contains fulltext : 81194.pdf (publisher's version ) (Open Access)BACKGROUND: Systemic sclerosis (SSc) is an autoimmune disease where controversy on Th1/Th2 balance dominates. We investigated whether the recently discovered Th17 pattern was present in SSc. METHODOLOGY AND PRINCIPAL FINDINGS: Patients were subdivided as having limited cutaneous SSc (lcSSc, n = 12) or diffuse cutaneous SSc (dcSSc, n = 24). A further arbitrary subdivision was made between early dcSSc (n = 11) and late dcSSc (n = 13) based upon the duration of disease. As a comparator group 14 healthy controls were studied. CD3+ cells were isolated using FACS and subsequently studied for the expression of CD4, CD8, CD25, CD45Ro, CD45Ra, IL-23, GITR, CD69 and intracellular expression of IL-17, TGFbeta and IFNgamma using flow cytometry. Levels of IL-17, IL-6, IL-1alpha and IL-23 were measured using Bioplex assays. SSc patients had more and more activated CD4+ cells. In addition, CD4, CD45Ro and CD45Ra cells from all SSc patients highly expressed the IL23R, which was associated with a higher IL-17 expression as well. In contrast, IFNgamma and TGFbeta were selectively up regulated in SSc subsets. In line with these observation, circulating levels of IL-17 inducing cytokines IL-6, IL-23 and IL-1alpha were increased in all or subsets of SSc patients. CONCLUSION AND SIGNIFICANCE: The combination of IL-17, IFNgamma and TGFbeta levels in CD45Ro and CD45Ra cells from SSc patients is useful to distinguish between lSSc, ldSSc or edSSc. Blocking Th17 inducing cytokines such as IL-6 and IL-23 may provide a useful tool to intervene in the progression of SSc

Cytokine expression pattern in SSc clinical phenotypes.

<p>Cytokine expression pattern in SSc clinical phenotypes.</p

SSc patients express high levels of IL-17-positive T cells and the co-presence of IL-17 with either IFNγ, IFNα or TGFβ reflects SSc phenotype.

<p>Panel A presents the percentage of IL-23R positive cells in the CD4+ positive pool of T cells (left panel) and CD45Ro and CD45Ra positive cells (right panel) from healthy controls (n = 14), and SSc patients with the lcSSc (n = 12), ldcSSc (n = 11) and edcSSc (n = 13) phenotype. Panel B shows the mean intracellular expression level of IL-17, IFNγ and TGFβ in CD4+ cells from each a representative individual from each tested group (left side). On the right side, the mean percentage of CD45Ro-positive or CD45Ra-positive cells that express IL-17, IFNγ or TGFβ and the mean intensity thereof are presented for the whole group of healthy controls (n = 14), and SSc patients with the lcSSc (n = 12), ldcSSc (n = 11) and edcSSc (n = 13) phenotype. Panel C depicts the percentage of CD25^high cells that co-express IL-17 in healthy controls and different SSc phenotypes (* represents a p-value<0.01). Panel D represents the level of cytokines (IL-17 and IFNγ) spontaneously secreted by CD3+ T cells from healthy controls and SSc patients after 24 hrs of incubation. * <i>P</i>-value<0.0001, ** <i>P</i>-value<0.002, *** <i>P</i>-value<0.004.</p

SSc patients have more and more activated CD4+ T cells compared to healthy controls.

<p>Panel A depicts the percentage of CD4+ and CD8+ cells in the whole T cell pool (CD3+ cells) from healthy controls (n = 14), and SSc patients with the lcSSc (n = 12), ldcSSc (n = 11) and edcSSc (n = 13) phenotype. The CD3+ cells were isolated using MACS bead isolation after which CD4 and CD8 positivity was analyzed using flow cytometry. Panel B (left) depicts the percentage of CD4+ and CD8+ cells that were double-positive for the T cell activation markers CD69 and GITR. For this aim a representative individual from each group was selected. In the right panel, the percentage of CD4-CD69, CD8-CD69, CD4-GITR, CD8-GITR, CD45Ro-CD69 and CD45Ra-CD69 double positive cells is presented over the whole group of healthy donors (n = 14) and/or SSc patients (n = 36).</p

Clinical characteristics of patients included in in vitro assays.

*<p>0.03.</p

Increased level of Th17 inducing cytokines in the circulation of SSc patients.

<p>Panel A depicts the presence of IL-17 in the circulation of SSc patients and healthy controls. Panel B, C and D represents the levels of IL-6, IL-1α and IL-23, respectively. The circulating levels of IL-17, IL-1α and IL-23 was measured by ELISA whereas IL-6 was studied by Bioplex assays.</p