Butyrate suppresses cuprizone-induced demyelination

To investigate the effect of intestinal dysbiosis on the function of oligodendrocytes, we induced a cuprizone-induced demyelinating mouse model, which lacks infiltrating peripheral T and B lymphocytes, to eliminate the potential effect of immunological mechanisms on demyelination. We started the oral administration of a non-absorbing antibiotic mixture (kanamycin, colistin, and vancomycin in drinking water) at 1 week before treatment with cuprizone. After 3 weeks of cuprizone treatment, we evaluated the demyelination of the corpus callosum. The myelinated areas of the corpus callosum were significantly reduced in the brains from antibiotic-treated mice compared with those from non-treated mice (Fig. 1a, b). This result indicated that intestinal dysbiosis induced by the antibiotics affected the cuprizone-induced demyelination. Next, we examined the effect of major metabolites of microbiota, SCFAs, on the cuprizone-induced demyelination. We administered SCFAs (acetate, propionate, or butyrate; 200 mM each in drinking water) to mice at 1 week before treatment with cuprizone and evaluated the demyelination of the corpus callosum after 3 weeks of cuprizone treatment. The myelinated areas of the corpus callosum were significantly reduced in the brains from cuprizone-treated mice compared with those from control mice (Fig. 1c, d). The myelinated areas of the corpus callosum in brains from butyrate-treated mice were significantly ameliorated compared with those in mice treated with cuprizone alone. Oral treatment with acetate showed mild improvement, but the difference did not reach statistical significance. Oral treatment with propionate did not ameliorate demyelination. These data suggested that gut microbiota may affect cuprizone-induced demyelination via their metabolites.

Fig. 1 Oral treatment with antibiotics exacerbates cuprizone-induced demyelination and oral treatment of SCFAs suppresses demyelination in the corpus callosum. a Black-Gold staining of brains from cuprizone and antibiotic-treated mice. Scale bar, 100 μm. b Myelinated area of the corpus callosum (n = 8 mice per group, pooled from two independent experiments). c Black-Gold staining of brains from cuprizone and SCFA-treated mice. Scale bar, 100 μm. d Myelinated area of the corpus callosum (n = 12 mice per group, pooled from four independent experiments). The box plot indicates the first and third quartiles and the middle line indicates the median. Whiskers indicate the minimum and maximum. *p < 0.05, ***p < 0.001 Full size image

Microglia are not affected by SCFA treatment in cuprizone-induced demyelination

In the cuprizone-induced demyelination model, microglia affect demyelination through cytokine production or phagocytosis [34, 35]. Recently, the oral treatment of SCFAs was reported to restore the immature phenotype of microglia in germ-free mice [31]. To analyze the effect of SCFA treatment on microglia during cuprizone-induced demyelination, we evaluated microglial accumulation into the demyelinating corpus callosum and cerebral cortex using immunohistochemistry. In the corpus callosum, cuprizone-treated mice had significantly increased microglial numbers compared with control mice (Fig. 2a, c). In SCFA-treated mice, the microglial number in the corpus callosum tended to be decreased by oral treatment with acetate, but the difference did not reach statistical significance compared with mice treated with cuprizone alone (Fig. 2a, c). The microglial number in propionate- or butyrate-treated mice was not decreased compared with mice treated with cuprizone alone (Fig. 2a, c). We also measured the number of microglia in the cerebral cortex to evaluate microglial accumulation in a mild demyelinated area. The cuprizone-treated mice had a slightly increased number of microglia in the cerebral cortex compared with control mice. The microglial number in the cortex of SCFA-treated mice was similar to that of cuprizone-treated mice (Fig. 2b, d). Morphologically, microglia in cuprizone-treated mice appeared to have an ameboid shape in the corpus callosum and cortex, whereas microglia in control mice had a ramified shape. Microglia in SCFA-treated mice also displayed an ameboid shape similar to mice treated with cuprizone alone (Fig. 2a, b). We also analyzed the proportion of CD68+-activated microglia in the corpus callosum and cortex. The proportion of activated microglia in the corpus callosum and cerebral cortex was not affected by oral treatment with SCFAs (Fig. 2e, f). Our data suggest that oral treatment with SCFAs did not reduce the number or activation status of microglia in demyelinated lesions. Therefore, the amelioration of demyelination by butyrate treatment did not appear to be related to the modulation of microglia.

Fig. 2 Microglia are not affected by oral treatment with SCFAs. a Iba-1 staining of the corpus callosum. Scale bar, 100 μm. The lower row indicates a magnified figure of microglia. Scale bar, 20 μm. b Iba-1 staining of the corpus cerebral cortex. Scale bar, 100 μm. The lower row indicates a magnified figure of microglia. Scale bar, 20 μm. c The number of Iba-1+ cells in the corpus callosum (n = 6 mice per group, pooled from two independent experiments). d The number of Iba-1+ cells in the cerebral cortex (n = 6 mice per group, data are representative of two independent experiments). e Percentage of CD68+ activated microglia in the corpus callosum (n = 6 mice per group, pooled from two independent experiments). f Percentage of CD68+ activated microglia in the cerebral cortex (n = 6 mice per group, pooled from two independent experiments). The box plot indicates the first and third quartiles and the middle line indicates the median. Whiskers indicate the minimum and maximum. *p < 0.05, **p < 0.01 Full size image

Butyrate suppresses demyelination and enhances remyelination in an organotypic cerebellar slice culture

Recently, Treg cells were reported to facilitate remyelination in vivo [36]. In the cuprizone model, the possibility that butyrate enhances Treg cells infiltration cannot be excluded. To exclude the influence of Treg cells and assess the direct effect of butyrate on demyelination and remyelination, we used an organotypic cerebellar slice culture. We prepared the cerebellar slice culture from P9–10 mice and induced demyelination with LPC 6 days later. At the same time, we treated slice cultures with butyrate for 24 h. We then replaced the medium and cultured it for 72 h with normal medium. Cultured slices were fixed, and we evaluated the proportion of myelinated axons (Fig. 3a). Butyrate treatment significantly suppressed demyelination by a dose-dependent mechanism (Fig. 3b, c). To investigate the effect of butyrate on remyelination, we induced demyelination and then treated the demyelinated cerebellar slice culture with butyrate for 6 days. Cultured slices were fixed, and we evaluated the proportion of myelinated fibers present (Fig. 4a). Butyrate treatment significantly enhanced remyelination at all concentrations tested (Fig. 4b, c). These results indicated that butyrate directly suppressed demyelination and enhanced remyelination in vitro.

Fig. 3 Butyrate treatment ameliorates LPC-induced demyelination in vitro. a Schematic showing the experimental protocol for demyelination analysis. b MBP and NF200 immunocytochemical staining of organotypic cerebellar slice cultures. Scale bar, 100 μm. c Myelination index (MI) of organotypic cerebellar slice cultures (n = 12 slices per group, pooled from four independent experiments). The box plot indicates the first and third quartiles and the middle line indicates the median. Whiskers indicate the minimum and maximum. *p < 0.05, ***p < 0.001 Full size image

Fig. 4 Butyrate treatment enhances remyelination from LPC-induced demyelination in vitro. a Schematic showing the experimental protocol for remyelination analysis. b MBP and NF200 immunocytochemical staining of organotypic cerebellar slice cultures. Scale bar, 100 μm. c Myelination index (MI) of organotypic cerebellar slice cultures (n = 3 slices per group, pooled from four independent experiments). The box plot indicates the first and third quartiles and the middle line indicates the median. Whiskers indicate the minimum and maximum. *p < 0.05, **p < 0.01, ****p < 0.0001 Full size image

Microglia depletion does not affect the butyrate-mediated suppression of demyelination and enhancement of remyelination

To exclude the possibility that microglia affect the butyrate-mediated suppression of demyelination and enhancement of remyelination, we depleted microglia with PLX3397, an antagonist of colony-stimulating factor 1 receptor, through which signaling is necessary for microglial survival [37]. We confirmed that microglia in the cerebellar slice culture were almost completely depleted by the addition of PLX3397 (Fig. 5a). We treated cerebellar slice cultures with PLX3397 or vehicle during the demyelination phase (Fig. 5b) and observed that the depletion of microglia did not affect LPC-induced demyelination and suppression of demyelination mediated by butyrate (Fig. 5c, d). We also analyzed whether the depletion of microglia affected the butyrate-mediated enhancement of remyelination (Fig. 5e). No significant difference was observed between PLX3397 and vehicle treatment (Fig. 5f, g). To investigate whether butyrate treatment affects microglial phenotype, we analyzed the expression of M1 (inducible nitric oxide synthase: iNOS) and M2 (argnase-1 (Arg1)) markers in butyrate-treated or non-treated slice cultures using RT-qPCR. We found no difference in the expression of iNOS and Arg1 between butyrate-treated and non-treated slice cultures both in demyelination phase and remyelination phase (Fig. 5h). These data indicated that butyrate influenced demyelination and remyelination in the absence of microglia.

Fig. 5 Depletion of microglia does not affect the butyrate-induced suppression of demyelination and enhancement of remyelination. a MBP, NF200, and Iba-1 immunocytochemical staining of PLX3397-treated or non-treated organotypic cerebellar slice cultures. Scale bar, 100 μm. b Schematic showing the experimental protocol of demyelination analysis. c Immunocytochemical staining of PLX3397-treated or non-treated organotypic cerebellar slice cultures with LPC and butyrate treatment. Scale bar, 100 μm. d Myelination index (MI) of organotypic cerebellar slice cultures (n = 12 slices per group, pooled from three independent experiments). e Schematic showing the experimental protocol of remyelination analysis. f Immunocytochemical staining of PLX3397-treated or non-treated organotypic cerebellar slice cultures with LPC and butyrate treatment. Scale bar, 100 μm. g MI of organotypic cerebellar slice cultures (n = 12 slices per group, pooled from three independent experiments). h relative expression of iNOS and Arg1 in organotypic slice cultures (n = 5 slice per group). The box plot indicates the first and third quartiles and the middle line indicates the median. Whiskers indicate the minimum and maximum. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Full size image

Butyrate enhances the maturation of oligodendrocytes

To explore the mechanism of the butyrate-induced enhancement of remyelination, we examined oligodendrocyte maturation. We induced demyelination in an organotypic cerebellar slice culture with LPC and analyzed the effect of butyrate on remyelination sequentially (Fig. 6a, b). At 3 days after LPC-induced demyelination, butyrate treatment slightly enhanced the remyelination. At day 5, butyrate treatment significantly enhanced remyelination in a dose-dependent manner. Although the remyelination progressed gradually in butyrate- and non-treated cultures at day 7, butyrate-treated slices still showed a significant enhancement of remyelination compared with non-treated slices (Fig. 6a, b). Next, we investigated the influence of butyrate on oligodendrocyte maturation. We stained slice cultures with anti-Olig2 antibody, anti-CC-1 antibody, and anti-PDGFRα antibody and measured the numbers of Olig2-positive and CC-1-positive mature oligodendrocytes, and PDGFRα-positive and Olig2-positive oligodendrocyte precursor cells (OPC) (Fig. 6c–e). We revealed that butyrate treatment did not affect the number of OPC in the cerebellar slice cultures (Fig. 6c, d). However, the number of Olig2+CC-1+ mature oligodendrocytes in the LPC-treated culture was reduced at day 3. At day 5, the number of mature oligodendrocytes in the butyrate-treated culture was significantly increased compared with the non-treated culture. At day 7, the butyrate-treated culture showed an increased tendency to contain mature oligodendrocytes compared with the non-treated culture (Fig. 6c, e). These data indicated that butyrate treatment enhanced remyelination by accelerating oligodendrocyte maturation rather than by enhancing OPC proliferation.

Fig. 6 Butyrate treatment enhances the differentiation of oligodendrocyte. a MBP and NF200 immunocytochemical staining of organotypic cerebellar slice cultures. Slice cultures were treated with LPC for 24 h and then cultured with normal medium. At 3, 5, and 7 days after LPC treatment, cultures were fixed and analyzed. Scale bar, 100 μm. b Myelination index (MI) of organotypic cerebellar slice cultures (n = 6 slices per group, pooled from two independent experiments). c Olig2, CC-1, and PDGFRα immunocytochemical staining of organotypic cerebellar slice cultures. Slice cultures were treated with LPC for 24 h and then cultured with normal medium. At 3, 5, and 7 days after LPC treatment, cultures were fixed and analyzed. Arrows indicate Olig2+CC-1+ mature oligodendrocytes. Arrowheads indicate Olig2+PDGFRα+ OPC. Scale bar, 100 μm. d OPC number in the organotypic cerebellar slice culture (n = 6 slices per group, pooled from two independent experiments). e Mature oligodendrocyte number in the organotypic cerebellar slice culture (n = 6 slices per group, pooled from two independent experiments). The box plot indicates the first and third quartiles and the middle line indicates the median. Whiskers indicate the minimum and maximum. *p < 0.05, **p < 0.01, ***p < 0.001 Full size image

HDAC inhibitor suppresses LPC-induced demyelination and enhances remyelination

Because butyrate is an HDAC inhibitor, we compared the effects of trichostatin A (TSA), an HDAC inhibitor, and butyrate on demyelination and remyelination. TSA treatment significantly suppressed LPC-induced demyelination similar to the butyrate treatment (Fig. 7a, b). In the remyelination phase, both butyrate and TSA treatment significantly improved remyelination from LPC-induced demyelination compared with the LPC-only treatment group (Fig. 7c, d). Our data revealed that the HDAC inhibitor suppressed demyelination and enhanced remyelination similar to butyrate and suggested that butyrate affects oligodendrocytes by acting as an HDAC inhibitor.