Short-chain fatty acids ameliorate spinal cord injury recovery by regulating the balance of regulatory T cells and effector IL-17+ γδ T cells

Abstract

Spinal cord injury (SCI) causes motor, sensory, and autonomic dysfunctions. The gut microbiome has an important role in SCI, while short-chain fatty acids (SCFAs) are one of the main bioactive mediators of microbiota. In the present study, we explored the effects of oral administration of exogenous SCFAs on the recovery of locomotor function and tissue repair in SCI. Allen's method was utilized to establish an SCI model in Sprague-Dawley (SD) rats. The animals received water containing a mixture of 150 mmol/L SCFAs after SCI. After 21 d of treatment, the Basso, Beattie, and Bresnahan (BBB) score increased, the regularity index improved, and the base of support (BOS) value declined. Spinal cord tissue inflammatory infiltration was alleviated, the spinal cord necrosis cavity was reduced, and the numbers of motor neurons and Nissl bodies were elevated. Enzyme-linked immunosorbent assay (ELISA), real-time quantitative polymerase chain reaction (qPCR), and immunohistochemistry assay revealed that the expression of interleukin (IL)‍-10 increased and that of IL-17 decreased in the spinal cord. SCFAs promoted gut homeostasis, induced intestinal T cells to shift toward an anti-inflammatory phenotype, and promoted regulatory T (Treg) cells to secrete IL-10, affecting Treg cells and IL-17⁺ γδ T cells in the spinal cord. Furthermore, we observed that Treg cells migrated from the gut to the spinal cord region after SCI. The above findings confirm that SCFAs can regulate Treg cells in the gut and affect the balance of Treg and IL-17⁺ γδ T cells in the spinal cord, which inhibits the inflammatory response and promotes the motor function in SCI rats. Our findings suggest that there is a relationship among gut, spinal cord, and immune cells, and the "gut-spinal cord-immune" axis may be one of the mechanisms regulating neural repair after SCI.

Keywords: IL-17+ γδ T cells; Inflammation; Motor function recovery; Neuroprotection; Regulatory T cells; Short-chain fatty acids (SCFAs); Spinal cord injury (SCI).

Fig. 1. Improvement of locomotor recovery in SCI via exogenously administered oral SCFAs. (a) The BBB score of each group was recorded. (b) The typical "catwalk" image of each group at 21 d after SCI. (c) The regularity indexes of the different groups were analyzed. (d) The BOS value of the rat hind limb was determined. (e) Hematoxylin-eosin (HE) staining was used to assess injury in spinal cord slices from different groups. Representative images of Nissl staining for spinal motor neurons. (f) The proportion of the lesion cavity area to the total area was calculated. (g) The number of surviving motor neurons was counted after Nissl staining. Data were expressed as mean±standard deviation (SD), n=5. ^*** P<0.001, SCI vs. sham; ^## P<0.01, ^### P<0.001, SCI vs. SCI+SCFAs; ^†† P<0.01, ^††† P<0.001, SCI+SCFAs vs. sham. SCI: spinal cord injury; SCFAs: short-chain fatty acids; BBB: Basso, Beattie, and Bresnahan; BOS: base of support; LF: left forelimb; LH: left hindlimb; RF: right forelimb; RH: right hindlimb.

Fig. 2. Expression of IL-10 and IL-17 in the spinal cord after SCFA treatment. (a, b) The expression of IL-10 and IL-17 was detected by ELISA. (c, d) The relative expression of IL-10 and IL-17 mRNAs was detected by qPCR. (e) IL-10 and IL-17 were stained by IHC in the spinal cord. (f) The IHC scores of IL-10 and IL-17 of the spinal cord. Data were expressed as mean±standard deviation (SD), n=5. ^* P<0.05, ^*** P<0.001, SCI vs. sham; ^# P<0.05, ^## P<0.01, ^### P<0.001, SCI vs. SCI+SCFAs; ^† P<0.05, ^††† P<0.001, SCI+SCFAs vs. sham. IL: interleukin; SCI: spinal cord injury; SCFA: short-chain fatty acid; ELISA: enzyme-linked immunosorbent assay; mRNA: messenger RNA; qPCR: real-time quantitative polymerase chain reaction; IHC: immunohistochemistry.

Fig. 3. Effects of SCFAs on the gut homeostasis and intestinal T cells differentiation. (a) Assessment of terminal ileum homeostasis of different groups using hematoxylin-eosin (HE) staining. (b) The length of villus. (c) The depth of crypt. (d) The thickness of mucosa. (e) Flow cytometry was used to identify Treg cells in the small intestinal lamina propria of different groups. (f) The ratio of small intestinal lamina propria CD4 ⁺ T cells in the lymphocytes of different groups. (g) The ratio of small intestinal lamina propria Treg cells in CD4 ⁺ T cells of different groups. Data were expressed as mean±standard deviation (SD), n=5. ^* P<0.05, SCI vs. sham; ^# P<0.05, ^### P<0.001, SCI vs. SCI+SCFAs; ^† P<0.05, SCI+SCFAs vs. sham. SCI: spinal cord injury; SCFAs: short-chain fatty acids; SSC-A: side scatter-area; FSC-A: forward scatter-area; Treg: regulatory T; CD4: cluster of differentiation 4.

Fig. 4. Effects of SCFAs on the frequencies of Treg cells and IL-17 ⁺ γδ T cells in the spinal cord. (a) Representative flow cytometry plots to identify Treg cells and IL-17 ⁺ γδ T cells in the spinal cord of different groups. (b, c) The ratios of spinal cord Treg cells in CD4 ⁺ cells and IL-17 ⁺ γδ T cells in CD3 ⁺ cells of different groups. (d) Correlations between Treg cells and IL-17 ⁺ γδ T cells in the spinal cord. A negative correlation was found between Treg cells and IL-17 ⁺ γδ T cells. (e) Correlations of Treg cells between spinal cord and intestine. There was a positive correlation of Treg cells between spinal cord and intestine. Data were expressed as mean±standard deviation (SD), n=5. ^*** P<0.001, SCI vs. sham; ^### P<0.001, SCI vs. SCI+SCFAs; ^†† P<0.01, SCI+SCFAs vs. sham. Treg: regulatory T; SCI: spinal cord injury; SCFAs: short-chain fatty acids; SSC-A: side scatter-area; IL: interleukin; CD: cluster of differentiation; TCR: T cell receptor.

Fig. 5. Migration of Treg cells from the gut to the spinal cord region after SCI and the effect on the γδ T cells. (a) Experimental design of gut-derived Treg cell migration following SCI in rats. On the second day after SCI, fluorescently labeled Treg cells were injected into the Peyer's patches, and the labeled Treg cells in the spinal cord and blood were detected 24 h later. (b) Flow cytometric detection of labeled Treg cells in the spinal cord and blood 24 h after enteral microinjection of Peyer's patch microinjection. (c) Treg cells stained with CFSE. (d) Fluorescently labeled Treg cells from the gut were found in the SCI area and displayed in the horizontal sections of the spinal cord. (e) CCK-8 assays were used to detect the effects of varying SCFA concentrations (0‒2 mmol/L) on the proliferation of Treg cells. (f) The relative expression of IL-10 mRNA in Treg cells was detected by qPCR. (g) CCK-8 assays were used to measure the effects of varying IL-10 concentrations (0‒20 ng/mL) on the proliferation of γδ T cells. Data were expressed as mean±standard deviation (SD), n=5. ^* P<0.05, ^** P<0.01, ^*** P<0.001. Treg: regulatory T; SCI: spinal cord injury; CFSE: carboxyfluorescein succinimidyl amino ester; DAPI: 4',6-diamidino-2-phenylindole; SCFA: short-chain fatty acid; IL: interleukin; CCK-8: cell counting kit-8; mRNA: messenger RNA; qPCR: real-time quantitative polymerase chain reaction.

Fig. 6. Effects of SCFAs on the inflammatory response and the recovery of motor function in rats after SCI through regulation of Treg/IL-17 ⁺ γδ T cells. SCFAs promote gut homeostasis, induce intestinal T cells to shift toward an anti-inflammatory phenotype, promote Treg cells to secrete IL-10, and migrate from the gut to the spinal cord region after SCI to affect Treg cells and IL-17 ⁺ γδ T cells in the spinal cord. This exerts an inhibitory effect on the inflammatory response and promotes motor function in SCI rats. SCI: spinal cord injury; IL: interleukin; Treg: regulatory T; SCFAs: short-chain fatty acids.