Th1 and Th17 Cells and Associated Cytokines Discriminate among Clinically Isolated Syndrome and Multiple Sclerosis Phenotypes

Abstract

Multiple sclerosis (MS) is a chronic, inflammatory, and demyelinating disease of the central nervous system. It is a heterogeneous pathology that can follow different clinical courses, and the mechanisms that underlie the progression of the immune response across MS subtypes remain incompletely understood. Here, we aimed to determine differences in the immunological status among different MS clinical subtypes. Blood samples from untreated patients diagnosed with clinically isolated syndrome (CIS) (n = 21), different clinical forms of MS (n = 62) [relapsing-remitting (RRMS), secondary progressive, and primary progressive], and healthy controls (HCs) (n = 17) were tested for plasma levels of interferon (IFN)-γ, IL-10, TGF-β, IL-17A, and IL-17F by immunoanalysis. Th1 and Th17 lymphocyte frequencies were determined by flow cytometry. Our results showed that IFN-γ levels and the IFN-γ/IL-10 ratio were higher in CIS patients than in RRMS patients and HC. Th1 cell frequencies were higher in CIS and RRMS than in progressive MS, and RRMS had a higher Th17 frequency than CIS. The Th1/Th17 cell ratio was skewed toward Th1 in CIS compared to MS phenotypes and HC. Receiver operating characteristic statistical analysis determined that IFN-γ, the IFN-γ/IL-10 ratio, Th1 cell frequency, and the Th1/Th17 cell ratio discriminated among CIS and MS subtypes. A subanalysis among patients expressing high IL-17F levels showed that IL-17F and the IFN-γ/IL-17F ratio discriminated between disease subtypes. Overall, our data showed that CIS and MS phenotypes displayed distinct Th1- and Th17-related cytokines and cell profiles and that these immune parameters discriminated between clinical forms. Upon validation, these parameters might be useful as biomarkers to predict disease progression.

Keywords: Th17 cells; Th1 cells; biomarker; clinical isolated syndrome; cytokines; multiple sclerosis; progressive multiple sclerosis; relapsing–remitting multiple sclerosis.

Figure 1

Individual cytokine production in patients with clinically isolated syndrome (CIS) and different multiple sclerosis (MS) phenotypes. Secretion of cytokines was determined in plasma samples from CIS, relapsing–remitting MS (RRMS), secondary progressive MS (SPMS), primary progressive MS (PPMS) patients, and healthy control (HC) individuals by ELISA. Some patients were found below the detection limit in all patient groups. (A) Interferon (IFN)-γ (HC, n = 17; CIS, n = 20; RRMS, n = 34; SPMS, n = 11; PPMS, n = 17), (B) IL-10 (HC, n = 17; CIS, n = 14; RRMS, n = 19; SPMS, n = 8; PPMS, n = 14), (C) TGF-β (HC, n = 15; CIS, n = 11; RRMS, n = 21; SPMS, n = 10; PPMS, n = 10), (D) IL-17A (HC, n = 17; CIS, n = 17; RRMS, n = 28; SPMS, n = 10; PPMS, n = 17), (E) IL-17F (HC, n = 13; CIS, n = 21; RRMS, n = 34; SPMS, n = 11; PPMS, n = 17), and (F) a subgroup of CIS and MS patients with IL-17F levels above 250 pg/ml was analyzed separately (HC, n = 13; CIS, n = 4; RRMS, n = 10; SPMS, n = 4; PPMS, n = 7). Horizontal line represents the median of each patient group and HC. ^#IL-17F levels of HC were significantly lower than each patient group: p = 0.0038 (HC vs CIS and SPMS), p < 0.0001 (HC vs RRMS), and p = 0.0004 (HC vs PPMS). *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 2

Plasma cytokine ratios in patients with clinically isolated syndrome (CIS) and different multiple sclerosis (MS) phenotypes. The relative production of cytokines was calculated in patients with CIS (n = 21), relapsing–remitting MS (RRMS, n = 34), secondary progressive MS (SPMS, n = 11), primary progressive MS (PPMS, n = 17), and healthy control (HC, n = 17) individuals. (A) Interferon (IFN)-γ/IL-17F ratio, (B) IFN-γ/IL-17F ratio in a subgroup of CIS and MS patients with high IL-17F levels, and (C) IFN-γ/IL-10 ratio. Dotted horizontal line represents the median of the respective cytokine ratio in HC. The y-axis in each graph is represented by logarithmic scale (*p < 0.05, **p < 0.01, and ***p < 0.001).

Figure 3

Th1 and Th17 cell frequencies and their ratios in patients with clinically isolated syndrome (CIS) and multiple sclerosis (MS) phenotypes. Purified peripheral blood mononuclear cells from CIS and MS patients and healthy control (HC) individuals were ex vivo stimulated with 1 µg/ml anti-CD3/CD28 antibodies (CD3/CD28) for 72 h. Intracellular expression of interferon (IFN)-γ and IL-17A was determined by flow cytometry. The results are shown as the median of the percentage of CD4⁺ T lymphocytes producing (A) IFN-γ (Th1) or (B) IL-17A (Th17). (C) The Th1/Th17 cell ratio was expressed as the ratio of the percentage of CD4⁺ T lymphocytes producing IFN-γ to those of IL-17A of each patient and HC. Dotted horizontal line represents the median of the Th1/Th17 ratio of HC. CIS (n = 17), relapsing–remitting MS (RRMS, n = 17), secondary progressive MS (SPMS, n = 10), primary progressive MS (PPMS, n = 15), and HCs (n = 7). In a few patients, it was not possible to obtain sufficient blood sample to analyze both plasma cytokine levels and cell frequencies, and therefore, they were not included in the Th1 and Th17 analyses (*p < 0.05 and **p < 0.01).