A gender-related action of IFNbeta-therapy was found in multiple sclerosis

Abstract

Background: Understanding how sexual dimorphism affects the physiological and pathological responses of the immune system is of considerable clinical importance and could lead to new approaches in therapy. Sexual dimorphism has already been noted as an important factor in autoimmune diseases: the aim of this study was to establish whether sexual dimorphism in autoimmune diseases is the result of differing pathways being involved in the regulation of T-helper (Th) cell network homeostasis.

Methods: We focused on sexually dimorphic changes in the immune response in multiple sclerosis (MS) patients in order to ascertain how these alterations relate to the pathway regulation of the cytokine homeostasis and the Th cell networks. We studied antigen presenting cell (APC)-dependent T cell activation in groups of healthy subjects, in patients under interferon (IFN) β-therapy and untreated. Cytokines, soluble (s) CD30 and the expanded disability status scale (EDSS) were used as biomarkers for T cell differentiation and neurological deficit.

Results: The data confirm our belief that sexual dimorphism in autoimmune diseases is the result of differing pathways that regulate Th cell network homeostasis: interleukin (IL) 6 pathways in women and IFNγ pathways in men. Given the increased susceptibility of women to MS and the significance of IL6 in the autoimmune process compared to IFNγ, it is logical to assume that IL6 pathways are in some way implicated in the prevalence of autoimmune diseases in women. Indeed, our data indicate that IL6 pathways are also involved in T regulatory (Treg) cell imbalance and an increase in neurological deficit in both men and women groups of MS patients, underlining the autoimmune etiology of multiple sclerosis. In further support of differing cytokine pathways in men and women, we noted that the efficacy of IFNβ-treatment in the re-establishment of Th-network balance and in the delaying of the neurological disability progression is linked to the IL6 pathway in women, but to the IFNγ pathway in men. Lastly, we also identified specific gender biomarkers for the use in therapy.

Conclusions: The identification of gender-specific drugs is of considerable importance in translational medicine and will undoubtedly lead to more appropriate therapeutic strategies and more successful treatment.

Figure 1

APCs regulate Th cell differentiation and Th cell network homeostasis in both men and women, and this effect appears to be exerted through IFNγ production in men, but through IL6 production in women. No significant relationships were observed in either the 24 h (early regulation) or 72 h (late regulation) cultures in resting conditions (b-cytokines) in men. In women, relationships were found between production of IL2-IL4 and IFNγ-IL6 Th1/Th2 cytokines in resting conditions (b-cytokines). The IL2 and IL4 inter-regulation in women was significant in both the 24 h and 72 h culture supernatant cytokine assays, while the inter-regulation between IL6 and IFNγ levels is only significant in the 72 h assay. Significant relationships emerged in cell cytokine production in activated (+PHA ) conditions in both, men and women. In the 24 h (early regulation) PHA culture: positive linked production between IL6 and IL4, and IFNγ and IL10 cytokines (p-cytokines) were found in men, and negative linked production between IL6 and IL10 cytokines (p-cytokines) were found in women. In the 72 h (late regulation) PHA culture: positive linked production between IFNγ and IL4 were found in men, while positive linked production between IFNγ and IL6 were found in women (p-cytokines).

Figure 2

Gender dimorphism of Th cytokine pathways and regulation of Th cell network homeostasis are normally present in the immune response. The multivariate statistical analysis of the correlation between the levels of Th cytokines (Table 1) confirms the cytokine regulation of immune response cell phases through gender specific pathways. APC cytokines regulate the resting and activated (PHA) cell phases of immune response, through gender specific pathways: IL6 pathways is the gender specific pathways in women, but IFNγ pathways is the gender specific pathways in in men. Consequently, autoimmune disease susceptibility in women could be attributed to the influence of ΙL6, since IL6 prevents the conversion of naive Th into Treg cells in vivo, by switching Th cell differentiation from Treg to Th17, which plays a key role in autoimmune diseases. The greater probability of men developing the primary progressive form, conversely, could be the result of the influence of IFNγ on Th9 cell inhibition, since co-expression of IL-9 and IL-17 was identified as a novel Th17 function in mediating autoimmune tissue destruction, including the CNS.

Figure 3

APC analysis in MS untreated patients using IDC, DC and whole blood Th-cytokine data-driven computational models of disease state. Independent cohorts of MS untreated patients were evaluated, using IDC, DC and whole blood Th-cytokine data-driven computational models of disease state. We used the multivariate statistical procedure of principal component analysis (PCA) which allowed us to analyze the relationships between parameters (levels of cytokines, sCD30 and EDDS values) and the behaviour of this multicomponent system as a network. The levels of cytokines were used as biomarkers of Th differentiation (Treg, Th1, Th9 or Th17) and EDSS was used as biomarker for neurological deficit. Using the PCA analysis we plotted the network of vectors obtained by analyzing the data matrix of correlation coefficients. In these plots, the angle between vectors is inversely proportional to the degree of correlation between vectors: the same vector direction indicates a positive correlation/covariance, the opposite vector direction indicates a negative correlation/covariance. This allows a visualization of the situation under study and our results indicate that the IL6 pathway is involved in neurological deficit increase and Treg imbalance in resting (Figure 3a) and activated (Figure 3b) conditions in untreated, male and female patient groups. This we concluded from the fact that cytokine level increases of IDC/DC IL6 and TGFβ (the dual biomarker for an increase in Th17 cells and a fall in Treg cells) were positively related to increases in neurological deficit (EDSS) (Figures 3a and 3b).

Figure 4

The IL6 pathway alteration is involved in neurological deficit increase and Tregs imbalance in untreated male and female patient groups, underlining the autoimmune etiology of multiple sclerosis. Immunological alterations in IL6 pathways are responsible for the Treg imbalance and increase of EDSS in both sexes in untreated MS patients. In fact, IL6 together with TGFβ pathways, a dual biomarker for Treg/Th17-network imbalance, were involved in the EDSS increase in both untreated, men and women groups of MS patients (Figures 3c and 3d: the vectors of TGFβ IL6 and EDSS are positively related). Furthermore, in health, APC IL6 pathways regulate Th cell differentiation and Th cell network homeostasis by the positive linked production of IL6 and IL4 in men, and the negative linked production of IL6 and IL10 cytokines in women (Figure 1, p-cytokines). Whereas in untreated MS patients, the relationship between the production of IL6 and IL4 had changed from positive to negative in men, while the relationship between the production of IL6 and IL10 had changed from negative to positive in women (Figures 3c and 3d).

Figure 5

IL6 pathways regulate Th cell differentiation and Th cell network homeostasis in healthy men and women. In health APC IL6 pathways regulate Th cell differentiation and Th cell network homeostasis by the positive linked production of IL6 and IL4 in men, and the negative linked production of IL6 and IL10 cytokines in women. Instead (Figures 3 and 4), in MS untreated patients the relationship between the production of IL6 and IL4 changes from positive to negative in men, while the relationship between the production of IL6 and IL10 changes from negative to positive in women.

Figure 6

APC analysis in MS treated patients using IDC, DC and whole blood Th-cytokine data-driven computational models of disease state. Re-establishment of IL6 immunological pathways are responsible for the re-establishment of neurological (no increase of EDSS) and immunological (Th cell-network) homeostasis in treated patients and is induced by gender specific cytokine pathways. In fact , the IL6 vector in women, and the IFNγ vector in men, negatively opposed their respective EDSS vectors and the relationships between the production of IL6, IL4 and IL6, IL10 reverses to that found in healthy subjects, in both sexes.

Figure 7

The re-establishment of immunological (Th cell-network) and neurological (no increase of EDSS) homeostasis in treated patient is induced by gender specific cytokine pathways. The results confirm cytokine regulation of immune response cell phases through gender specific pathways. Autoimmune disease susceptibility in women could be attributed to the influence of ΙL6, which plays a key role in autoimmune diseases, since it is a T cell differentiation switch factor from T-regs to Th17 cells. The greater likelihood of men developing the primary progressive form, on the other hand, could be the results of the influence of IFNγ on Th9 cell inhibition. Targets and biomarkers for clinical monitoring and translational research for the development of gender specific therapy, were found. sCD30 -TGFβ is a dual clinical biomarker for both sexes, while IL6-IL10 is a dual clinical biomarker specific for women and IL6-IL4 together with IFNγ-IL10 are dual clinical biomarkers specific for men. Lastly, differences in the IL10 and IL12p40 cytokine levels in men and IL12p40 and IL12p70 cytokine levels in women, are also gender specific biomarkers.

Figure 8

Gender specific biomarkers for homeostasis in immunological, neurological and therapeutic pathways. Re-establishment of immunological and neurological homeostasis in treated patients induced by gender specific cytokine pathways, re-establishes the above relationships between the production of IL6 and IL4 (from negative, the condition of untreated patients, to positive, the condition of healthy subjects) in men; similarly in women homeostasis is re-established when the relationship between the production of IL6 and IL10 reverses (from positive, the condition of untreated patients, to negative, the condition of healthy subjects). Additionally, the significance of sCD30 and TGFβ as a dual clinical biomarker was confirmed for both sexes and gender specific new clinical biomarkers were identified.