7.1 Discussion of results
Animal models suggest that fecal SCFA (and gut microbiota) might play a role in the pathogenesis of MS. Hitherto, there are only sparse data concerning alterations in the gut’s immune system in MS patients. In this study, we investigated fecal markers related to intestinal inflammation in RRMS patients and healthy controls.
There was a non-significant reduction in fecal SCFA concentrations in RRMS patients, especially for butyrate. Fecal calprotectin concentrations did not show any difference between RRMS patients and controls. Blood CRP concentrations, although not clinically elevated in any of the investigated subjects, correlated positively with fecal calprotectin concentrations which highlights the role of intestinal inflammation in MS. Constipation scores were significantly higher in RRMS patients and correlated with the disease activity. Immunosuppressive drugs, especially natalizumab and fingolimod, lead to significantly higher constipation scores. Notably, constipation is not a known side effect of these two drugs.
Our finding of descriptively reduced fecal SCFA concentrations in RRMS patients is in accordance with other studies in this field: Park et al. showed, that SCFA blood levels were roughly 50-65% reduced in MS patients (19). Fecal SCFA levels have already been reported to be reduced in MS in a Chinese cohort (18). Additionally, changes in the gut microbiota of MS patients have been published (16, 17, 15). Recently, the general relevance of SCFA for MS has been investigated in a clinical study by Duscha et al., who observed an enhancement of Treg-differentiation, a reduction of autoinflammation and amelioration of the disease course following oral administration of propionate (7). Nevertheless, it should be taken into account that oral administration of SCFA is unlikely to exert the same effects as SCFA produced by gut microbiota in the colon: orally administered SCFA are absorbed in the small intestine and act systemically, while SCFA produced in the colon by the gut microbiota mainly exert local effects and are unlikely to affect blood SCFA concentrations in a relevant way.
While Berg-Hansen et al. found elevated concentrations of calprotectin in the cerebrospinal fluid of MS patients in 2009 (25), fecal calprotectin concentrations have not been reported in MS patients so far. In spite of the assumption that there might be an intestinal inflammation in MS, we did not find elevated fecal calprotectin concentrations in our RRMS cohort. This might be due to the fact that we investigated fecal calprotectin in RRMS patients who were mostly under immunotherapy. Immunotherapies aimed at counteracting the inflammatory process in the CNS might affect systemic and enteric inflammation (either by direct mechanisms or via the gut brain axis) and consequently also fecal calprotectin concentrations. For natalizumab, the enteric anti-inflammatory effect is already known and therapeutically used in therapy of Crohn’s disease (26). Assuming that MS therapies affect gut-associated immunity, this effect might modulate the gut microbiota (as shown by Storm-Larsen et al. for dimethylfumarate (27)) and subsequently SCFA production as well. Since SCFA are known to enhance gut motility, an altered gut microbiota (and a subsequent reduction in fecal SCFA) might predispose to constipation. Indeed, we observed higher CSS scores in RRMS patients compared to age-matched controls. Future studies are needed to clarify whether an altered intestinal motility in MS is associated with an altered gut microbiota. In addition, longitudinal studies are necessary to distinguish between disease-immanent and therapeutic effects on the gut microbiota and intestinal inflammation in MS.
We are not able to draw conclusions on fecal calprotectin in drug-naïve MS patients as the vast majority of our RRMS cohort was under therapy. We suggest investigating intestinal inflammation in drug-naïve subjects patients and in MS relapses. Only such studies will be able to clarify the role of SCFA in the pathogenesis of MS.
There could also be a yet unknown pathogenic factor which leads to an altered gut microbiota and consecutively to a reduction of SCFA which finally initiates intestinal inflammation. Hypothetically, MS could be a second event caused by a lack of SCFA which leads to a lack of Tregs and overly active T cell immunity.
On the other hand, we cannot rule out the possibility of a confounder in constipation scores by MS itself. It is possible that several patients have lesions in vagal nuclei or in afferents to those, which might account for constipation. Additionally, reduced physical activity due to high EDSS might also account for higher constipation scores in these patients.
A surprising result of our study was the marked sex-associated difference in SCFA concentration between women and men. Fecal SCFA concentrations differed significantly between men and women – even within healthy controls. Sex-specific differences for the microbiota have been described (28), but there is still a lack of studies investigating sex-specific differences for fecal SCFA. Apart from branched-chain SCFAs, all fecal SCFA concentrations in our study were significantly reduced in women. The relevance and the reproducibility of this finding has to be determined. In addition, confounding factors like diet need to be considered. Fecal SCFA concentrations have already been subject of multiple studies, e.g. anorexia (29), obesity, diabetes and as a marker of risk for cardiometabolic disease (30). Neither Chen et al., Jangi et al. nor Miyake et al. reported a sex-specific effect on fecal SCFA. None of the above mentioned studies regarding gut microbiota and fecal SCFA concentrations reported a sex-specific difference for SCFA – maybe because this aspect was not explicitly examined. We found one gastroenterological study from 2013, in which Jakobsdottir et al. compared patients with microscopic colitis and celiac disease. Despite the fact that serum and fecal SCFA are not directly comparable, they also reported a significant sex-related difference of serum SCFA concentrations (31): women showed significantly reduced blood concentrations of SCFA. Mean acetate concentrations were about 12% reduced in women. In our study, median fecal acetate was 90.6% reduced in women. Yet, another Chinese study by Chen et al. could not reproduce a sex-related difference when analyzing serum SCFA levels (32).
The exact reasons for the higher female susceptibility for MS is not yet known. As SCFA are thought to modulate the immune system, SCFA might be pathophysiologic relevant for MS, especially if studies in drug-naïve MS patients should be able to reproduce this finding. We hypothesize, that reduced fecal SCFA concentrations in female subjects might be an additional risk factor for MS contributing to the higher susceptibility in comparison to men.
Limits of the Study
As the investigated RRMS patients were under different treatment regimes, we also analyzed subgroups of RRMS patients which were defined by the therapeutic regime. Yet, the number of subjects per subgroup was rather small and the study population did not represent the full spectrum of available MS therapies. Future studies should also focus on drug-naïve patients in order to identify therapy-related effects. As there is evidence that the gut microbiota is altered in MS, future studies should include the analysis of the gut microbiota Another relevant aspect might be to investigate alterations in the gut microbiota, microbial markers like SCFA and calprotectin related to MS relapse.
Since our control group was age-matched, but not sex-matched, this might be a confounder due to male predominance in the control group.