Intracellular IL-10 detection in T cells by flowcytometry: the use of protein transport inhibitors revisited

In the past two decades, interleukin-10 (IL-10) has gained much attention as an important regulatory cytokine involved in self-tolerance. Functional assessment of IL-10 producing immune cells is traditionally done by stimulation and measurement of cytokine production by flowcytometry. Thereby a protein transport inhibitor like monensin is used to accumulate the cytokine of interest intracellularly. In this study we elaborated on the monensin effect on cytokine detection and focused on IL-10 detection in human T cells. Peripheral blood mononuclear cells (PBMC) of 32 study subjects were isolated and stimulated with PMA/ionomycin, in the absence and presence of monensin, and stained intracellularly for IFN-gamma, IL-4, IL-10 and IL-17A. Our results re-established that detection of IFN-gamma+ and IL-4+ T cells benefited from the presence of monensin during stimulation. However, stimulation in the presence of monensin yielded lower proportions of IL-10+ T cells (0.45% (0.28-0.80) versus 0.80% (0.50-1.50) of CD4+ T cells, p<0.01), although monensin addition did result in an increased MFI (2431 (1273-4959) versus 1928 (1147-3760), p<0.01). Detectable fractions of IL-17A+ CD4+ T cells were not affected by monensin. A shorter incubation time, but not lower monensin concentrations, was effective in improving the detection of IL-10+ T cells. We found a strong correlation between the fraction of IL-10+ CD4+ T cells in the presence and absence of monensin (R=0.80 p<0.01). Next to this, also the detection of IL-10+ NK-T cells and IL-10+ monocytes, but not IL-10+ B cells, is impaired in the presence of monensin. This study shows that the effect of monensin on cytokine accumulation is time and cytokine dependent. Due to the use of monensin, previous research may have underestimated the number of IL-10+ leukocytes or may even have not been able to detect them at all. It is important to consider this for future research or when interpreting historical IL-10 data

Regulatory T cell function correlates with serum 25-hydroxyvitamin D, but not with 1,25-dihydroxyvitamin D, parathyroid hormone and calcium levels in patients with relapsing remitting multiple sclerosis

Vitamin D is a potent immune modulator in multiple sclerosis (MS), but was primarily identified for its effects on calcium homeostasis. It is uncertain whether these calcaemic functions of vitamin D are critically involved in its immune modulating potential. We earlier reported a correlation between serum 25-hydroxyvitamin D (25(OH)D) levels and regulatory T cell (Treg) function. In the present study, the correlation of serum levels of 1,25-dihydroxyvitamin D (1,25(OH)2D), intact parathyroid hormone (PTH), and total calcium with Treg number and functionality and the proportions of other T helper cell subsets was assessed in 29 relapsing remitting MS patients. In contrast to serum 25(OH)D levels, serum concentrations of neither 1,25(OH)2D, nor PTH and total calcium correlated significantly with Treg function or Th1/Th2 ratio. None of the parameters correlated with the relative and absolute number of Tregs. Interestingly, the serum levels of 1,25(OH)2D correlated positively with the proportion of T helper type 17 (Th17) cells. These results suggest that the serum levels of 1,25(OH)2D, PTH, and total calcium are not critically involved in the correlation between vitamin D status and T cell regulation

Reduction in IL-10 producing B cells (Breg) in multiple sclerosis is accompanied by a reduced naive/memory Breg ratio during a relapse but not in remission

In this study, we assessed B cell subsets, including Bregs, during stable and active disease in relapsing remitting multiple sclerosis (RRMS) patients and related B cell subsets to vitamin D status. We report that RRMS patients have a decreased percentage of both memory B cells and Bregs compared to healthy controls. During a relapse, the reduction in Bregs involved in particular naive Bregs. We found no correlation between vitamin D status and B cell subsets. An effect of vitamin D on Bregs cannot be ruled out, since it might be the function that is interfered with instead of relative numbers

Th17 expansion in MS patients is counterbalanced by an expanded CD39(+) regulatory T cell population during remission but not during relapse

AbstractIn this study, percentages of CD39+ Treg and Th17 cells were compared between relapsing-remitting MS patients and controls and were related to the vitamin D status. The Th17 cell population was expanded in about 40% of the MS patients. In MS patients in remission, but not during relapse, a positive association was found between Th17 cell and CD39+ Treg percentages (r=0.468, p=0.007). Since CD39+ Tregs have been shown to have Th17 suppressive capacities, we propose that a dysregulated Th17/CD39+ Treg balance might contribute to disease exacerbation. A clear role for vitamin D in this regulation could not be established

The contribution of disease severity, depression and negative affectivity to fatigue in multiple sclerosis: a comparison with ulcerative colitis

Background: Fatigue is one of the most common and troubling symptoms of multiple sclerosis (MS) and more severe and disabling than fatigue in other somatic populations. Although fatigue seems MS specific, its pathogenesis is still poorly understood. Objective: To study the disease specificity of fatigue in MS by comparing its level, its physical and psychological correlates to those of patients with ulcerative colitis (UC), a peripheral chronic auto-immune disease. We focused on the relative contribution of disease severity, depression and negative affectivity to fatigue in both patient samples. Methods: A total of 88 MS and 76 UC patients were included in this cross-sectional study. Fatigue, depression and negative affectivity were assessed respectively with the physical and mental fatigue subscales of the Multidimensional Fatigue Inventory, the depression subscale of the Hospital Anxiety and Depression Scale, and the neuroticism subscale of the Dutch NEO Five-Factor Inventory. The Expanded Disability Status Scale and the Colitis Activity Index were used to measure disease severity in MS and UC patients respectively. Results: While levels of both physical and mental fatigue were significantly higher in MS patients than in UC patients, there were no group differences in the contribution of disease severity, depression and negative affectivity to both physical and mental fatigue. Conclusion: Although levels of fatigue are higher for MS patients when compared with UC patients, the correlates of fatigue do not indicate MS specificity. As such our results support a transdiagnostic approach to fatigue in MS