Project Progress Report
Remyelination and the gut microbiota
Chris McMurran and Robin Franklin, University of Cambridge | 28th May 2019
Introduction
The aim of this project has been to explore the gut bacteria as a potential therapeutic target for treating myelin diseases.
Demyelination describes the destruction of myelin sheaths, which normally insulate and protect the neurons in our brains. For patients with diseases like multiple sclerosis, demyelination can lead to problems including disability, cognitive difficulties and bladder and bowel dysfunction. Our lab work focuses on ways to enhance the regeneration of myelin sheaths after they are damaged – a process known as remyelination. The hope is that by optimising remyelination we can reduce the devastating consequences of these diseases.
With generous support from the British Trust for the Myelin Project, one area that we have now explored in some detail is the gut-brain axis. As humans, our gut is colonised by trillions of bacteria and other microorganisms, and growing body of evidence has linked these microbial residents (our “microbiota”) to other processes in the body, including the immune system. Previous work from our group and others had shown that the immune system is important for coordinating remyelination, as immune cells can clear up myelin debris and produce various pro-regenerative factors. Our hypothesis was that by modulating the dynamics of our gut bacteria, we could tune the immune system in a way that promotes
efficient remyelination (Fig 1).
Figure 1 – Our hypothesis for how the microbiota might influence remyelination in the central nervous system.
Experiments and results so far
Antibiotics study
As the gut-brain axis is a whole-body phenomenon, it is difficult to replicate in vitro, so the majority of our experiments have been carried out in mice, whose nervous systems contain similar structures and cells to our own.
Firstly, we investigated whether the depletion of gut bacteria (by feeding mice high doses of antibiotics) would affect remyelination. Following two months of antibiotic treatment, Microbiota Immune response remyelination was assessed. We found that antibiotic treatment caused changes in the immune cells during remyelination, and that myelin debris was not cleared up so efficiently.
We then examined the oligodendrocyte progenitor cells (OPCs) in antibiotics-treated mice. Oligodendrocytes are the cells that make myelin and an important step in remyelination is the creation of new oligodendrocytes from OPCs. The antibiotics-treated mice did not produce as many oligodendrocytes during remyelination as control mice (Fig 2).
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Figure 2 - Oligodendrocytes in the CNS of antibiotics-treated mice.
A) Immunohistochemistry was used to detect oligodendrocytes (green with red centre) and OPCs (just red).
B) 14 days after demyelination, there were fewer oligodendrocytes in the antibiotics-treated groups.
Finally, another group of mice were treated with antibiotics, but then received a faecal microbial transplant (FMT) to restore their gut microbes. This restored some aspects of the immune response but did not reverse the low levels of oligodendrocyte production (Fig 2).
In summary, depletion of gut microbes using a combination of antibiotics inhibited the immune response and resulted in inefficient myelin debris clearance with lower levels of oligodendrocyte production by OPCs.
Germ-free study
One difficulty in using antibiotics to deplete the microbiota is that the antibiotics themselves might have other effects in the body. Indeed, some antibiotics (though not those used in our study) are well known to pass into the brain and inhibit immune cells directly. It was therefore difficult to tell how much our results reflected changes in the microbiota versus other effects of the antibiotics used.
Our solution to this was to conduct a supporting experiment assessing remyelination in germfree mice. Germ-free mice live from birth in a completely sterile environment; thus, their microbiota is depleted without the need to expose them to high levels of antibiotics.
Just like depleting the microbiota with antibiotics, germ-free (GF) mice had an impaired immune response during remyelination (Fig 3A, B). A group of “ex-GF” mice were born into sterile conditions, but as young adults were introduced to conventional mice and acquired a microbiota. The immune response during remyelination returned to normal in this ex-GF group.
Despite these changes in the immune response, OPC activity was unaffected in GF mice and ex-GF mice. OPCs were able to produce normal numbers of oligodendrocytes, and myelin sheaths were restored to the same extent (Fig 3C, D).
Thus, in contrast to antibiotics-treated mice, GF mice had no deficits in OPC responses, and remyelinated just as well as conventional mice.
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Figure 3 – Immune response and remyelination in the CNS of germ-free mice treated with cuprizone.
A) Immunohistochemistry was used to detect CD68+ (green) immune cells.
B) During demyelination and remyelination, germ-free (GF) mice had lower numbers of immune cells, whereas ex-GF mice had similar levels to the control group.
C) 5000x magnified electron microscope images showing individual axons surrounded by dark myelin sheaths.
D) After remyelination, there was no difference between groups in the density of axons with a myelin sheath, suggesting similar efficiency of remyelination.
Probiotic Study
The most recent study we have completed as part of this project looked at whether a probiotic might be able to enhance remyelination. Whereas the antibiotics and germ-free studies investigated a “loss-of function”, in which the microbiota was depleted, this study involved boosting the microbiota using a mixture of live bacteria (known as a probiotic).
In contrast to the previous studies, probiotic treatment boosted the immune response during demyelination. Higher numbers of immune cells were seen in probiotic-mice compared to control mice (Fig 4). However, this was not associated with changes in OPC activity, and remyelination was no different following the probiotic treatment.
Figure 4 – Immune response during remyelination in probiotic-treated mice
A)Immunohistochemistry was used to detect CD68+ (green) immune cells.
B) During remyelination, probiotic-treated mice had higher numbers of immune cells.
Conclusions and future work
By looking at the results of these three studies together, we can draw some conclusions about the interactions between the gut microbiota and remyelination. Firstly, in all cases the immune response that was observed during remyelination depended on the microbiota. The two interventions that depleted the microbiota (antibiotics, germ-free) put the brakes on the immune response, whilst supplementing the microbiota with a probiotic boosted the numbers of immune cells.
However, the link between microbiota and OPC responses was less clear. Germ-free mice and probiotic-treated mice had normal OPC responses, and myelin sheaths were restored at a normal rate. In contrast, antibiotics impaired the generation of new oligodendrocytes from OPCs. This suggests that the effects observed in the antibiotics study could indeed be an “offtarget” effect of high combined doses of antibiotics, rather than a direct consequence of depleting the microbiota.
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Figure 5 – ADF enhances remyelination in aged rats partially through the restoration of OPC differentiation capacity.
(A), Schematic of the fasting experiment. ADF animals had access to food on alternate days only. Control animals had free access to food (ad libitum). Fasting was initiated at 12 months of age for 6 months. White matter demyelination was induced by focal injection of ethidium bromide (EB) into the caudal cerebellar peduncule (CCP).
(B), Remyelination was assessed 50 days post-lesion induction in semi-thin resin sections stained with toluidine blue. Remyelination is evident as dark circles surrounding a pale grey axon. Myelinated axons, that have not undergone demyelination, are surrounded by thick, dark myelin. Demyelinated axons have poorly discernible borders. Scale bars =100μm.
(C), Electron micrographs from areas within the lesion center. Scale bars = 5μm. (D), Quantification of the remyelination data. Each dot represents a single animal. The rank corresponds to the extent of remyelination, where a higher rank indicates better remyelination (n≥6 for each group, Mann-Whitney-U test).
Recently, in work not directly funded by the BTMP, we have shown that restricting calorie intake (i.e. calorie restriction - or CR) has a profoundly beneficial effect on reversing the age associated decline in remyelination (Fig 5). It is now imperative to understand how this effect is achieved. One very plausible hypothesis is that long term CR leads to profound and sustained changes in the microbiota (more than we have been able to achieve in our experiments so far).
There is currently much excitement around the idea that changes in the microbiota might contribute to MS developing in the first place. As researchers begin to develop therapies that alter the microbiota with a therapeutic intention, it will be certainly be important to understand how this could impact subsequent remyelination.
