The prompt for the study came from MS patients: “We hear again and again from sufferers that they feel worse when they consume milk, cottage cheese or yogurt,” explains Stefanie Kürten from the Institute of Anatomy at University Hospital Bonn. “We are interested in the cause of this correlation.”
The professor of neuroanatomy is considered a renowned expert on multiple sclerosis. She began the study in 2018 at the University of Erlangen-Nuremberg. A year and a half ago, she moved to Bonn, where she continued the work together with her research group.
“We injected mice with different proteins from cow’s milk,” she says. “We wanted to find out if there was a constituent that they were responding to with symptoms of disease.”
And the researchers did indeed find what they were looking for: When they administered the cow’s milk constituent casein together with an effect enhancer to the animals, the mice went on to develop neurological disorders. Electron microscopy showed damage to the insulating layer around the nerve fibers, the myelin. The fat-like substance prevents short circuits and additionally significantly accelerates stimulus conduction.
Perforated myelin layer
In multiple sclerosis, the body’s immune system destroys the myelin sheath. The consequences range from paresthesia and vision problems to movement disorders. In extreme cases, patients need a wheelchair. The insulating sheath was also massively perforated in the mice – apparently triggered by casein administration.
“We suspected that the reason was a misdirected immune response, similar to that seen in MS patients,” explains Rittika Chunder, who is a postdoctoral fellow in Prof. Kürten’s research group. “The body’s defenses actually attack the casein, but in the process they also destroy proteins involved in the formation of myelin.”
Such cross-reactivity can occur when two molecules are very similar, at least in parts. The immune system then in a sense mistakes them for each other. “We compared casein to different molecules that are important for myelin production,” Chunder says. “In the process, we came across a protein called MAG. It looks markedly similar to casein in some respects – so much so that antibodies to casein were also active against MAG in the lab animals.”
This means that in the casein-treated mice, the body’s own defenses were also directed against MAG, destabilizing the myelin. But to what extent can the results be transferred to people with MS? To answer this question, the researchers added casein antibodies from mice to human brain tissue. These did indeed accumulate in the cells responsible for myelin production in the brain.
Self-test for antibodies against casein
Certain white blood cells, the B cells, are responsible for antibody production. The study found that the B cells in the blood of people with MS respond particularly strongly to casein. Presumably, the affected individuals developed an allergy to casein at some point as a result of consuming milk. Now, as soon as they consume fresh dairy products, the immune system produces masses of casein antibodies. Due to cross-reactivity with MAG, these also damage the myelin sheath around the nerve fibers.
However, this only affects MS patients who are allergic to cow’s milk casein. “We are currently developing a self-test with which affected individuals can check whether they carry corresponding antibodies,” says Kürten, who is also a member of the Cluster of Excellence ImmunoSensation2. “At least this subgroup should refrain from consuming milk, yogurt, or cottage cheese.”
It is possible that cow’s milk also increases the risk of developing MS in healthy individuals. Because casein can also trigger allergies in them – which is probably not even that rare. Once such an immune response exists, cross-reactivity with myelin can in theory occur.
However, this does not mean that hypersensitivity to casein necessarily leads to the development of multiple sclerosis, the professor emphasizes. This would presumably require other risk factors. This connection is nevertheless worrying,
Kürten says: “Studies indicate that MS rates are elevated in populations where a lot of cow’s milk is consumed.”
We present the first integrated characterization of gut microbiome alterations and host-microbiome interactions in MS patients compared to healthy control individuals using advanced multi-omics technologies and a longitudinal study design that offers insight into the stability of these factors over time.
We have confirmed prior findings that the gut microbiome community structure in MS patients largely resembles that of controls.9,10,37 We also extended the microbiome analyses by seeking factors that might drive microbiome variations between MS patients and controls, and validated the small impact from the disease itself. Interestingly, BMI contributed significantly to microbiome variation among all tested variables, and positively correlated with EDSS in MS patients. Hence, BMI should be considered in future inter-group microbiome comparisons in MS studies.
The under-representation of Faecalibacteria, Prevotella, Lachnospiraceae and Anaerostipes species in MS patients compared to controls aligns with previous findings,10 and is biologically plausible. Faecalibacteria, Lachnospiraceae, and Anaerostipes produce butyrate, which acts via G-protein coupled receptors activation and histone deacetylase inhibition to suppress CNS demyelination,50 the main pathological feature in MS.
Indeed, concentrations of fecal SCFAs (i.e., acetate, butyrate and propionate) were decreased in RRMS patients, compared to healthy controls.51, 52, 53 Blood SCFAs were significantly decreased in long-term active progressive MS patients.54 Propionic acid, but not butyrate and acetate, was significantly reduced in blood and stool in MS patients with all disease subtypes, particular after relapse. Supplementation of propionic acid promoted Treg cell function, and in long-term administration reduced relapse rate, disability and brain atrophy.55 In our study, we found a trend toward decreased concentrations of butyrate in the stools of MS patients, consistent with decreased SCFA-producing bacteria in MS. Notably, SCFA levels can also be affected by diet, and higher meat servings in MS patients may also contribute to the observed reduction of SCFAs.
Prevotella histicola reduces EAE severity by inducing FoxP3+ regulatory T cells and decreasing pro-inflammatory Th1 and Th17 cells in the CNS.15,56 We found reduced abundance of a different Prevotella species, P. copri, in stools of MS patients. P. copri is a dominant Prevotella species in healthy American adults,57 and is more prevalent in non-western populations.58 Moreover, P. copri was highly correlated with IL10+ memory B cells in our control group, providing a potential novel microbiome-driven immune pathway to test in future.
In this study, the MS group was compared to unrelated healthy controls, matched for age, gender and other important variables. An alternative approach would have been to obtain same household controls, to account for environmental influences.59 However, it is possible that the same household controls may decrease the sensitivity to detect MS associated microbes, because individuals from the same household tend to share gut microbes, and a shared microbe may still influence MS development in genetically predisposed individuals.
Changes in gut microbiota observed in MS participants in our study resembled previous studies conducted in different geographical locations, offering credence to our findings. MS-associated microbes are also over-represented in other autoimmune and metabolic diseases as well as cancer that are associated with inflammation.8,60,61 This finding argues against a unique microbiome signature for MS patients but supports a common microbiome dysbiosis indicator for extra-intestinal pathophysiology associated with inflammation.
Much work has shown that the immune system confines the gut microbiome within its physical niche and shapes microbial compositions in animal models,62 and our findings now link gut microbiome and systemic cellular immune profiles in healthy human adults.
Our data recapitulate in a human cohort the inter-relatedness of the gut microbiome, diet, immune system and host metabolome, a relation that has been reported mostly in germ-free mice.62 Specifically, we now demonstrate an association between the gut microbiome and peripheral immune phenotype in healthy participants, implying that healthy people with similar gut microbiome tend to have similar immune phenotype.
In contrast, gut microbiome-immune haemostatic interactions were disrupted in the MS cohort we studied. Immune cell phenotypes of MS patients significantly differed from healthy controls in our dataset. However, we wish to note that the degree of dysbiosis of gut microbes in MS was modest. The discordance of the changes in immune phenotypes and microbiome may explain the lack of correlation between the microbiome and peripheral blood immune profiles in MS.
It will be of great interest to elucidate the directionality and time-to-response of microbiome-immune regulation in MS. Future work might also be directed towards microbiota and immune response at the gut mucosa,63 as the site of the systemic changes that ultimately affect the CNS.
Interestingly, blood metabolites best differentiate MS patients from controls in our models. Indeed, MS patients experience metabolic alteration in different tissues,64,65 and dysfunctional lipid metabolism.66 The observed enrichment of circulating novel metabolites and multiple pathways in MS patients requires future validation to address their role in MS.
We found that methionine was significantly enriched in MS patients, which is consistent with higher meat consumption in the patients we studied. Methionine drives T cell proliferation and differentiation.67 Our data are also in accord with recent findings that methionine activates Th17 cells through epigenetic modification.68
Methionine is an essential metabolite for methyl donor SAM synthesis, and SAM promotes Th17 cell activation through methylation.68 Reduction of dietary methionine ameliorated EAE through reprogramming pathogenic Th17 cells. Our data, together with Roy et al., prompt the hypotheses that meat or methionine restriction might beneficially decrease the number of circulating inflammatory Th17 cells in MS patients. Future studies of metabolites in MS should consider specific dietary nutrients and gut microbiome derived metabolites, as these factors play large roles in ordaining human metabolism.
Global correlation among multi-omics provides a powerful tool to understand systemic interactions across organs. However, among hundreds or thousands of correlations, in the context of under-annotated features such as blood metabolites, mining biology from global correlations is challenging. Consequently, multi-omics has often been criticized for generating massive amount of data but providing little biology.
We highlighted the significant discovery power of multi-omics by deeply delving into a pathway linking meat serving, B. thetaiotaomicron, Th17 cell, and SAM. We not only confirmed the relationship between meat consumption, SAM, and Th17 cells,68 we identified their connections with a common gut commensal bacterium, B. thetaiotaomicron. This opens new research directions to elucidate regulatory pathways among diet, metabolites, microbiome, and immune response, and may identify therapeutic targets for MS.
Interestingly, routine dietary intake had small overall impact on gut microbiome variation, as is also reported in inflammatory bowel disease.69 However, differential microbiome responses to dietary intake in different individuals might explain the lack of strong correlation between diet and gut microbiome in human studies.70
We also found that food composition did not correlate with circulating immune phenotypes. Nonetheless, lack of systemic associations does not exclude the possibility of specific feature-feature correlations, as we have identified associations between food compositions and the microbiome, as well as food compositions and immune cell populations.
Feature-feature correlations were reported conservatively, and only significant associations after multiple comparison corrections were included in this final report, thereby strengthening confidence in the correlations.
Our longitudinal study design provided a unique opportunity to evaluate the stability of multi-omics data in MS patients over a period of six months, during which most DMTs take effect. Baseline and six-month follow-up measures for microbiome, metabolome components, immune-phenotypes and diet were similar, except for MS patients who began DMTs, which showed a decrease in memory Th17 cells and GM-CSF+ T cells at the six-month time point.
These findings suggest an overall stability of the different systems in this defined interval in humans without strong exogenous influences. In addition, the consistent results between baseline and six-months strengthen our findings related to differential features between MS and controls, suggesting that these differences are not likely spurious.
While our longitudinal study offers a highly textured view of microbial-host interactions in MS, we acknowledge several limitations. First, the relatively small sample size increases the risk of type II error. To avert this, we carefully chose analytical tools and statistical tests suitable for high dimension data analysis. Second, food diary in our study was self-recorded, which potentially pose selection bias since it is less likely that all food consumptions are completed by all participants.
Third, the majority of study participants are female because MS is more prevalent in women. It would be interesting to research next whether these findings still hold true in male MS patients. Lastly, we could not determine the causal connection or directionality of feature-feature interactions. Nonetheless, we provide data on multiple novel molecules that could be plausibly implicated in MS pathogenesis that obligate a more in-depth analysis in the future.
A larger study with multiple sampling points that capture both relapse and remitting stages of MS will demonstrate a more complete dynamic picture of multiple-omics interactions in MS.
reference link : https://www.thelancet.com/journals/ebiom/article/PIIS2352-3964(21)00592-2/fulltext
Source: University of Bonn