Attenuation of Immunogenicity in MOG-Induced Oligodendrocytes by the Probiotic Bacterium Lactococcus Sp. PO3

Abstract

Background and Objectives: Milk is healthy and includes several vital nutrients and microbiomes. Probiotics in milk and their derivatives modulate the immune system, fight inflammation, and protect against numerous diseases. The present study aimed to isolate novel bacterial species with probiotic potential for neuroinflammation. Materials and Methods: Six milk samples were collected from lactating dairy cows. Bacterial isolates were obtained using standard methods and were evaluated based on probiotic characteristics such as the catalase test, hemolysis, acid/bile tolerance, cell adhesion, and hydrophobicity, as well as in vitro screening. Results: Nine morphologically diverse bacterial isolates were found in six different types of cow's milk. Among the isolates, PO3 displayed probiotic characteristics. PO3 was a Gram-positive rod cell that grew in an acidic (pH-2) salty medium containing bile salt and salinity (8% NaCl). PO3 also exhibited substantial hydrophobicity and cell adhesion. The sequencing comparison of the 16S rRNA genes revealed that PO3 was Lactococcus raffinolactis with a similarity score of 99.3%. Furthermore, PO3 was assessed for its neuroanti-inflammatory activity on human oligodendrocyte (HOG) cell lines using four different neuroimmune markers: signal transducer and activator of transcription (STAT-3), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and GLAC in HOG cell lines induced by MOG. Unlike the rest of the evaluated neuroimmune markers, STAT-3 levels were elevated in the MOG-treated HOG cell lines compared to the untreated ones. The expression level of STAT-3 was attenuated in both PO3-MOG-treated and only PO3-treated cell lines. On the contrary, in PO3-treated cell lines, MBP, GFAP, and GLAC were significantly expressed at higher levels when compared with the MOG-treated cell lines. Conclusions: The findings reported in this article are to be used as a foundation for further in vivo research in order to pave the way for the possible use of probiotics in the treatment of neuroinflammatory diseases, including multiple sclerosis.

Keywords: Lactococcus; cow’s milk; neuroinflammatory diseases; oligodendrocyte; probiotics.

Figure 1

Bile salt hydrolase activity: The zone of hydrolase indicates enzyme reactivity against bile salt in MRS media. MRS media was prepared and supplemented with 0.8% bile salt for plate preparation. (A): The disc impregnated with different cow milk isolates (PN1, PN2, PO2, and PO3). The standard probiotics (STD-1) was Lactobacillus casei. (B): The BSH activity of the four bacterial isolates in terms of zone expression. The zone indicates the hydrolysis of bile sale and is expressed in mm. The data are shown as mean ± SD. Variation in lettering indicates statistical significance (p < 0.05). * p < 0.05 represents significance between the groups.

Figure 2

Hydrophobicity data on cholesterol assimilation ability are represented as the mean ± SD. The MRS media were supplemented with filter-sterilized cholesterol-polyethylene glycol (PEG) 600 (Sigma) at a final concentration of 100 µg mL⁻¹ and incubated anaerobically at 37 °C for 24 h. The degradation of cholesterol was quantified using left over MRS media using the formula. (A): the hydrophobicity percentages of the cow milk isolates (PN1, PN2, PO2, and PO3). (B): the assimilation of cholesterol by the bacterial isolates. The data are shown as mean ± SD. Variation in lettering indicates statistical significance (p < 0.05). * p < 0.05 represents significance between the groups. ** p < 0.01 represents significance between the groups.

Figure 3

Percentage of selected isolates adhering to HOG cell lines. (A): phase contrast inverted microscopic images of selected isolates that were enumerated by bacterial cultures and interpreted as the percentage of adherence relative to the control. (B): the percentage of cell adhesion for the cow milk isolates (PN1, PN2, PO2, and PO3). The data are shown as mean ± SD. * p < 0.05 represents significance between the groups.

Figure 4

Effect of PO3 on inflammatory markers of (A) nitric oxide and (B) IL-6, IFN-γ, and IL-1β cytokines in HOG cell lines induced by MOG. These markers were quantified in MOG-induced HOG cell lines and PO3 treated for 12 h. The treated cells were homogenized after 12 h of induction using RIPA lysis buffer. Invitrogen and Cayman ELISA kits were used for quantification of the cytokines and chemokines. Estimation of the quantity of NO was performed using a kit from Invitrogen. The optical variation was observed at 450 nm using a microplate reader, and values were expressed in µM. Cytokines IL-6 and IFN-γ were quantified using the Cayman kit. All data were collected from three individual experiments, pooled, and expressed as mean ± SD (p < 0.05). * p < 0.05 represents significance compared to the MOG vs. MOG + PO3 group. ** p < 0.01 represents significance between the groups.

Figure 5

Effect of PO3 on neurological immune markers (STAT-3, MBP, GFAP, and GALC in HOG cell lines induced by MOG (1 µg)). The expression levels of the markers were estimated in HOG cell lines treated with MOG and those treated with PO3 for 18 h. The treated cells were homogenized after 12 h of induction using RIPA lysis buffer. The cytokines and chemokines were quantified using Invitrogen and Cayman ELISA kits. mRNA expression was expressed as fold units. GAPDH was used as the internal control to nullify the expression of targets. All data were collected from three individual experiments, pooled, and expressed as mean ± SD (p < 0.05). * p < 0.05 represents significance compared to the MOG vs. MOG + PO3 group.

Figure 6

A phylogenetic tree based on the 16S rRNA gene sequences of PO (OQ914364)3 and the cluster showing a higher similarity sequence with Lactococcus genus groups from NCBI GenBank. The horizontal scale bar denoting the similarity between isolates expresses a 0.01 difference in nucleotide position.