Bacillus amyloliquifaciens- Supplemented Camel Milk Suppresses Neuroinflammation of Autoimmune Encephalomyelitis in a Mouse Model by Regulating Inflammatory Markers

Abstract

Multiple sclerosis (MS), a distinct autoimmune neuroinflammatory disorder, affects millions of people worldwide, including Saudi Arabia. Changes in the gut microbiome are linked to the development of neuroinflammation via mechanisms that are not fully understood. Prebiotics and probiotics in camel milk that has been fermented have a variety of health benefits. In this study, Bacillus amyloliquefaciens-supplemented camel milk (BASY) was used to assess its preventive effect on MS symptoms in a myelin oligodendrocyte glycoprotein (MOG)-immunized C57BL6J mice model. To this end, MOG-induced experimental autoimmune encephalomyelitis (EAE) was established and the level of disease index, pathological scores, and anti-inflammatory markers of BASY-treated mice using macroscopic and microscopic examinations, qPCR and immunoblot were investigated. The results demonstrate that BASY significantly reduced the EAE disease index, increased total microbial load (2.5 fold), and improved the levels of the short-chain fatty acids propionic, butyric and caproic acids in the diseased mice group. Additionally, myeloperoxidase (MPO) proinflammatory cytokines (IL-1β, IL-6, IL-17, TNF-α) and anti-inflammatory cytokines (TGF-β) were regulated by BASY treatment. Significant suppression of MPO and VCAM levels were noticed in the BASY-treated group (from 168 to 111 µM and from 34 to 27 pg/mL, respectively), in comparison to the EAE group. BASY treatment significantly reduced the expression of inflammatory cytokines, inflammatory progression related transcripts, and inflammatory progression protein markers. In conclusion, BASY significantly reduced the symptoms of EAE mice and may be used to develop a probiotic-based diet to promote host gut health. The cumulative findings of this study confirm the significant neuroprotection of BASY in the MOG-induced mice model. They could also suggest a novel approach to the treatment of MS-associated disorders.

Keywords: Bacillus amyloliquifaciens; VCAM signals; fermented camel's milk; multiple sclerosis; probiotics.

Figure 1

BASY effectiveness on different parameters of MOG-induced EAE mice. The mice were immunized with MOG 35-33 (emulsified with CFA using a T connector). BASY induced the tolerance against EAE induction in mice: (A) Body weight (BW) of mice (EAE, EAE+BASY and BASY-treated mice). (B) Clinical symptoms recorded post-MOG immunization resulted in gradual increase in paralysis and hind limb inactivity, noted from 11 days of MOG immunization, which became stable after the 18th day post immunization. (C) The total microbial load in pre-administered BASY for 25 days of pre-immunization and MOG-immunized mice after the 16th day. The total bacterial count in fecal samples before and after BASY treatment (Group 1: untreated control group; Group 2: MOG-immunized mice (EAE); Group 3: BASY treated with MOG immunization (EAE+BASY); Group 4: BASY–only treated mice). The uncharacterized colonies were counted using the colony counter and recorded as 10⁴ colonies/gram of fecal samples. (D) SCFAs, butyric acid, caproic acid, and propionic acid were quantified using Biovision biochemical kits in the ileal content. The values are measured using a microplate reader and expressed as mM/mg of ileal content. Data were pooled from three independent experiments and shown as mean ± SD. * p < 0.05, ** p < 0.01. (A) microbial load compared using a student t-test. (B) SCFA content was compared using a one-way ANOVA.

Figure 2

BASY ameliorated the histopathological hallmarks in the CNS and colon of EAE mice. On the 24th day of MOG post-immunization, spinal cords were euthanized from EAE mice, fixed in 10% formaldehyde following paraffin embedding and sectioned into 4 µm thick sections. (A) Demyelination was assessed in the spinal cords using H&E, and the pathological scores were recorded at 200× magnification power. (B) Infiltration and inflammation scores were noted. (C) Intestinal integrity and mucin damage were assessed using H&E of the proximal colon of EAE mice. (D) Infiltration of T-lymphoid cells, neutrophils, and inflammation scores were noted on the 24th day of MOG post immunization. Data were pooled from three independent experiments and shown as mean ± SD. * p < 0.05. ** p < 0.01.

Figure 3

Effect of BASY on inflammatory markers (IL-1B, IL-6, IL-17 and TNF-α), oxidative damage (MPO) and cell adhesion (VCAM) markers in CNS of MOG-induced EAE mice. These markers were quantified in MOG-induced EAE mice and BASY-treated mice after 24 days of the experiment. The protein markers were extracted from the CNS of MOG-immunized mice. The tissues were homogenized after 21 days of induction using RIPA lysis buffer. The cytokines and chemokines were quantified using Invitrogen and Cayman ELISA kits. (A) IL-1B was quantified using an Invitrogen kit. The TMB optical variation was quantified at 450 nm by the microplate reader, and values were expressed in pg/mL. (B–D) IL-6, IL-17 and TNF-alpha were quantified using the Cayman kit. (E,F) The oxidative stress marker MPO and adhesion molecule VCAM were quantified in the periphery of the spinal cord tissues, respectively. All data were collected from three individual experiments, pooled, and expressed as mean ± SD (p < 0.05). * p < 0.05 and ** p < 0.01 represents significance compared to the MOG vs. MOG+ BASY group. IL—interleukin; TNF-α—tumor necrosis factor alpha; MPO—myeloperoxidase; VCAM—vascular cell adhesion molecule.

Figure 4

BASY activates the Treg cell population and the EAE condition by controlling CD4 polarization. (A–D) absolute number of infiltratedCD4, CD8, Treg and FOXP3 positive cells within the CNS of the control, EAE, EAE+BASY, and BASY pretreated MOG-immunized mice groups, respectively. The cells were sorted using the Miltenyi cell separation kit and the total population was counted using the Thermos cell counter. Incidence bars in each graph are expressed as the upper and lower limit of cell population in CNS of EAE and BASY with EAE mice groups. Data were pooled from three independent experiments and shown as mean ± SD. ** p < 0.01.

Figure 5

BASY augmented the level of neurotransmitters and neuronal hormones in MOG-immunized mice spinal tissues. (A) The peripheral crude lymphocytes were isolated, and total protein was extracted by the rapid freeze method. The melatonin was quantified at two intervals, on the 12th and 24th day of the MOG post-immunization period. The melatonin was quantified using an Abcam hormonal sensitivity kit. (B,C) Acetylcholine, a substrate of esterase, was quantified using the Sigma-Aldrich ELISA kit. Sirtuin protein was quantified using the Invitrogen Mouse ELISA kit. Data were pooled from three independent experiments and shown as mean ± SD. * p < 0.05. ** p < 0.01.

Figure 6

Peripheral and splenic lymphocytes were regulated by BASY administration in MOG- immunized EAE mice. The differential regulation of lymphocytes in MOG-immunized mice was evaluated by progressive inflammatory cytokine markers using an ELISA kit. CD4 and CD65L lymphocytes were isolated from peripheral lymph nodes of MOG-immunized mice and naïve mice. The isolated cells were restimulated with MOG of 1 µg/mL concentration for 18 h and cells were harvested for cytokine quantification. (A–F) GM-CSF, IFNγ, IL-8, IL-6, IL-17 and TGF-β were quantified in MOG-stimulated peripheral spinal lymph nodes CD4 cells. The isolated cells were re-stimulated with MOG of 1 µg/mL concentration for 12 h and cells were harvested for cytokine quantification. (G–L) GM-CSF, IFNγ, IL-8, IL-6, IL-17 and TGF-β were quantified in MOG-stimulated splenic CD4 cells. CD4 and CD65L lymphocytes were isolated from splenic lymphoid cells (SLC) of MOG -immunized mice and naïve mice. Data were pooled from three independent experiments and shown as mean ± SD. * p < 0.05. ** p < 0.01. GM -CSF—granulocyte-macrophage colony-stimulating factor; IFN-γ—interferon gamma; IL—interleukin.

Figure 7

The cell adhesion molecules regulated by BASY treatment in MOG-induced mice. The spinal cord and colon tissues were homogenized and endothelial cell adhesion molecules VCAM and ICAM were quantified using a protein specific ELISA Kit: (A,B) the differential expression of ICAM and VCAM in the spinal cord after MOG immunization was reduced by BASY treatment; (C,D) the expression and regulation of the invasion of lymphoid cells from the intestinal lamina propria to the peripheral lymph nodes of the spinal cord was quantified. Data were pooled from three independent experiments and shown as mean ± SD. * p < 0.05. ** p < 0.01 (n = 5). ICAM—intracellular adhesion molecule-1; VCAM—vascular cell adhesion molecule-1.

Figure 8

Various markers and neurotropic factors regulated by BASY in MOG-induced EAE mice: (A) mRNA expression of MBP, BDNF, and GABA in the spinal cord of MOG-immunized mice. (B) mRNA expression of COX-2 and VEGF in the spinal cord of MOG-immunized mice. (C) MBP, BDNF, GABA, COX-2, and VEGF protein expression was examined by immunoblot and densitometry analysis in the spinal cord and was normalized with laminin and actin as internal controls. (D) mRNA expression of MBP, BDNF, GABA, and COX-2, VEGF in the colon of MOG-immunized mice. (E,F) MBP, BDNF, GABA, COX-2, and VEGF protein expressions were examined by immunoblot and densitometry analysis in the colon and normalized with actin and laminin as internal controls. Results are presented as mean ± SEM (n = 3). When comparing MOG to MOG+BASY groups, * p < 0.05 denotes significance. MBP—myelin basic protein; BDNF—brain-derived neurotrophic factor; GABA—gamma-aminobutyric acid; COX-2—cyclooxygenase-2; and VEGF—vascular endothelial growth factor.