Acetate correlates with disability and immune response in multiple sclerosis

Introduction

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) causing inflammation, demyelination, and neurodegeneration (Compston & Coles, 2008). Although its etiology remains unclear, several environmental and genetic risk factors seem to be involved in its development (Belbasis et al., 2015). Recently, gut microbiota has been added to this list.

The gut microbiota is considered as the pool of bacteria, archaea, fungi, eukaryotes and viruses that reside in mucosal surfaces of the intestine. The gut bacteria mainly include six major phyla: Actinomycetes, Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria and Verrucomicrobia (Eckburg et al., 2005).

After many years of common development, the gut bacteria have reached a symbiotic state with the human body. Gut microbiota may affect the CNS and participate in the regulation of nervous system function. The CNS also plays an important role in regulating the gut function and homeostasis. This complex relationship may be included in the new concept: the microbiota gut-brain axis (Zhu et al., 2017).

When the mutualistic relationship between the host and microbiota is disrupted, the gut microbiota can cause or contribute to different pathologies, such as MS (Littman & Pamer, 2011). During the last years, several groups studied the dysbiosis of the gut microbiota in MS. Two main approaches have been addressed. The first one was based in the experimental autoimmune encephalomyelitis (EAE) model. The alteration of the gut microbiota diminished the EAE frequency and mitigated the severity of the disease (Ochoa-Repáraz et al., 2011).

In the second approach, the microbiota of MS patients was studied by several groups that showed the presence of gut microbial dysbiosis in MS compared with healthy controls (Lee et al., 2011).

Some microorganism-derived molecules as short-chain fatty acids (SCFA) can have a role in the microbiota gut-brain axis (Zhu et al., 2017). They are produced from the dietary fibers fermented by gut microorganisms in the large intestine. The most abundant are butyrate, propionate, and acetate (95% of SCFA) (Wong et al., 2006). Bacteria of the Bacteroidetes phylum produce high levels of acetate and propionate, whereas bacteria of the Firmicutes phylum produce high amounts of butyrate (Macfarlane & Macfarlane, 2003). Then, it is logical to think that the levels of SCFAs could be modified in MS patients compared with healthy population, considering that both phylum Bacteroidetes and Firmicutes are altered in these patients and have important immunoregulatory functions (Tremlett et al., 2016).

Previous metabolomic studies performed in MS patients showed higher levels of acetate in the serum of MS patients in comparison with healthy controls (Moussallieh et al., 2014). Furthermore, a previous pilot study performed by our group showed significantly higher levels of acetate among MS patients with higher scores in the Expanded Disability Status Scale (EDSS) in comparison to those MS patients with lower EDSS scores (results are shown in the Table S1; original data are available in a Supplemental File). Based on these previous results, we designed the current study to analyze the possible differences in the levels of circulating SCFA in two groups of MS patients (those without disability, EDSS ≤ 1.5, and those with severe disability, EDSS ≥ 4.5) and in healthy donors (HD). To study the immunoregulatory functions of the SCFA, we also analyzed their possible correlations with blood immune cell subsets and if this function was the same or not in MS patients and healthy controls. Since immunomodulatory and immunosuppressive treatments modify cell subsets, around 50% of MS patients recruited in each group were without treatment to perform cell studies by flow-cytometry. The last aim of the study was to analyze possible correlations between circulating SCFA levels and different clinical and demographic characteristics.

Materials & Methods

Statistical analysis

Continuous variables were expressed as median (25th, 75th percentile) and categorical variables as percentages. We described the association between different demographical and clinical variables to the levels of acetate, propionate and butyrate in plasma. For this purpose, the continuous variables between groups were compared using the Mann–Whitney U-test and the Spearman’s rank correlation coefficient was used when studying the relationship between two continuous variables, included the percentages of the different immune populations. In case of multiple comparisons, the Bonferroni correction was applied. Subjects with missing data were omitted from the corresponding analyses. P-values ≤ 0.05 were referred to as statistically significant in the text. All analyses were performed using SPSS for Windows (Ver. 15.0) software (SPSS Inc, Chicago, IL, USA).

Results

Discussion

The SCFA are bacterial metabolites mainly produced during the colonic fermentation of undigested carbohydrates. There are evidences for their regulatory role in host metabolism: they are ligands for free fatty acid receptor 2 and 3 (FFAR 2/3), found on a wide range of cell types, (Morrison & Preston, 2016), and have been recognized as mediators of immune responses (Asarat et al., 2016).

Acetate production pathways are widely distributed among bacterial groups whereas pathways for propionate and butyrate production appear more highly conserved and substrate specific (Morrison & Preston, 2016). Changes in the circulating levels of these metabolites could reflect variations in the bacteria composition of the gut microbiota. Dysbiosis has been observed in different pathologies, including MS (Jangi et al., 2016).

Furthermore, it has been suggested that the microbiota ability to produce SCFA, rather than the microbiota “composition”, is essential to generate the benefits linked to fibres (Mithieux, 2018). The plasma SCFA levels of MS patients have not been fully addressed yet. We explored them in our cohort and did not find significant differences in the levels of propionate and butyrate between MS patients and controls. However, MS patients showed significantly higher plasma acetate levels than HD (p = 0.003). These levels also correlated with the degree of disability, being significantly increased in patients with high disability than in patients with low or no disability (p = 0.029) and in HD (2.97e−4). Moreover, we showed a correlation between plasma acetate levels and the degree of disability, both with the EDSS (r-value = 0.387, p-value = 1.08e−4) and the MSSS (r-value = 0.265, p-value = 0.011). These results do not agree with a recent publication (Duscha et al., 2020). The authors find lower levels of propionate in MS patients than in HD, but they do not find any significant difference for acetate or butyrate. A possible explanation for this discordant results could be found in the patients selection. In this study, based on previous results of our group, we selected two groups of MS patients: those without disability, EDSS ≤1.5, and those with severe disability, EDSS ≥ 4.5, while in the study of Duscha et al. patients were not selected by their EDSS. As we have previously mentioned, a statistical significant difference was found between HD and patients with EDSS ≥ 4.5 (2.97e−4), but we did not find any statistical significant difference when we compared acetate levels between HD and patients with lower EDSS.

To study the possible mechanisms by which SCFA could contribute to disability we studied different PBMC subsets. In spite of not having found differences in SCFA levels among patients differently treated, we only performed flow cytometry analysis in samples from non-treated patients, because it has been recently described that some treatments, such as dimethyl fumarate and glatiramer acetate, could affect microbiota composition (Sand et al., 2019). Increases in plasma acetate levels correlated with a decrease of CD4+ naïve cells and with higher CD8+ T cells producing IL-17. By contrast, the only association that remained significant in control group was an inverse correlation between plasma propionate levels and memory B cells. CD8+ T cells are the most frequent immune cells present in MS lesions, and they have been involved in inflammation and axonal damage (Bar-Or et al., 2010; Salou et al., 2015). In addition it has been postulated that CD8+ IL-17+ T cells play an important role in MS pathology by inducing inflammation, tissue destruction and proinflammatory cytokines production (Tzartos et al., 2008). This can contribute to increase MS activity.

Although several studies claim that SCFA may have an antiinflamatory effect, a controversy exists. It has been described that butyrate suppresses demyelination and enhances remyelination in mice with cuprizone-induced demyelination (Chen et al., 2019) and oral propionate can suppress the potential deleterious activities of T helper cells while Treg frequencies in all treated individuals increased significantly (Haghikia et al., 2016). However, as we have mentioned above, previous metabolomic studies performed in MS patients have also shown higher levels of acetate in the serum of MS patients in comparison with healthy controls (Moussallieh et al., 2014). Furthermore, it has been recently published (Qiu et al., 2019) that ex vivo acetate treatment increases IFN-γ production by exhausted T cells; thus, hyporesponsive T cells can be epigenetically remodeled and reactivated by acetate. Therefore, acetate, although it is a SCFA like propionate and butyrate, does not seem to share with them their beneficial effects.

Furthermore, other studies support a dual effect for them: depending on its levels and the immunological context of the host, SCFA could modulate T cells differentiation into both regulatory and effector cells (Park et al., 2015; Park et al., 2016; Mizuno et al., 2017). Recently, it has been published that SCFAs can induce CD4+ effector T cells, which are highly inflammatory, when transferred into mice. The authors suggest that in contrast to the moderate protective effect of SCFA, mice deficient in their receptors are more resistant to EAE pathogenesis. Thus, SCFA and their receptors could have the potential to regulate autoimmune CNS inflammation both positively and negatively (Park et al., 2019).

One limitation of the study could be the lack of information about the diet of patients, since diet can modify the composition of the microbiota. However, our aim was to describe, for the first time, plasma SCFA levels in MS patients and to study a possible relationship of these levels with immune cells, regardless of the cause or origin of SCFA levels. In future studies, we will take dietary data into account in order to go further investigating the role of SCFA and microbiota in MS. Another limitation is the absence of SCFA measurements in the CSF; it would be very interesting to know if SCFA levels in serum correlate with those in CSF to be able to understand the role that these SCFA could have in the interaction / modulation of the CNS cells.

Conclusions

To sum up, our study shows that there are increased acetate levels in the plasma of MS patients; furthermore, they correlate with higher EDSS and MSSS. Higher values of acetate, propionate and butyrate also correlate with augmented CD8+ IL-17+ T cells, but only in MS patients. These results show that inflammatory background should be taken into account when exploring the association of microbiota metabolites and the immune system, and those basal patient conditions may change the influence of microbiota in the immune cells. Finally, our results strongly suggest that these bacterial metabolites can be part of the mechanisms by which microbiota may influence MS pathology. Future studies will completely elucidate the role of these molecules in this disease.

Supplemental Information