Bacillus Calmette–Guérin Tokyo-172 vaccine provides age-related neuroprotection in actively induced and spontaneous experimental autoimmune encephalomyelitis models

Abstract

Multiple sclerosis is the most common immune-mediated disorder affecting the central nervous system in young adults but still has no cure. Bacillus Calmette–Guérin (BCG) vaccine is reported to have non-specific anti-inflammatory effects and therapeutic benefits in autoimmune disorders including multiple sclerosis. However, the precise mechanism of action of BCG and the host immune response to it remain unclear. In this study, we aimed to investigate the efficacy of the BCG Tokyo-172 vaccine in suppressing experimental autoimmune encephalomyelitis (EAE). Groups of young and mature adult female C57BL/6J mice were BCG-vaccinated 1 month prior or 6 days after active EAE induction using myelin oligodendrocyte glycoprotein (MOG)_35–55 peptide. Another group of 2D2 TCR^MOG transgenic female mice was BCG-vaccinated before and after the onset of spontaneous EAE. BCG had an age-associated protective effect against active EAE only in wild-type mice vaccinated 1 month before EAE induction. Furthermore, the incidence of spontaneous EAE was significantly lower in BCG vaccinated 2D2 mice than in non-vaccinated controls. Protection against EAE was associated with reduced splenic T-cell proliferation in response to MOG_35–55 peptide together with high frequency of CD8⁺ interleukin-10-secreting T cells in the spleen. In addition, microglia and astrocytes isolated from BCG-vaccinated mice showed polarization to anti-inflammatory M2 and A2 phenotypes, respectively. Our data provide new insights into the cell-mediated and humoral immune mechanisms underlying BCG vaccine-induced neuroprotection, potentially useful for developing better strategies for the treatment of MS.

BCG Tokyo-172 vaccine confers neuroprotection in active and spontaneous EAE, via inducing CD8 interleukin-10-secreting T-cells in the periphery and a differentiation of glial cells toward an anti-inflammatory phenotype.

Graphical Abstract

Graphical Abstract.

Introduction

Multiple sclerosis (MS) is the most widespread immune-mediated disorder affecting the central nervous system (CNS) in young adults, with an estimated 2.8 million cases worldwide [1]. Although the etiology and pathogenic mechanisms of MS are still not completely understood, the interplay between genetic and environmental factors seems to affect the risk of developing this disease [2]. While there is still no cure for MS, the success rates of different immunotherapies targeting T and/or B cells indicate the involvement of both these cell types in its pathogenesis [3].

Several studies suggest that vaccines can alter adaptive immune cell populations and heterologous immune responses, having non-specific effects and therapeutic benefits in unrelated infections and autoimmune diseases [4]. Bacillus Calmette–Guérin (BCG) vaccine is a non-pathogenic strain of live attenuated Mycobacterium bovis. It was developed in 1921 at the Pasteur Institute for Tuberculosis Prevention in France, and different sub-strains of this vaccine are currently used, including BCG Tokyo-172 [5]. Several lines of evidence indicate that BCG vaccine, in addition to protecting against tuberculosis transmission, also has non-specific effects, such as anti-inflammatory effects and therapeutic benefits in autoimmune disorders including MS [6, 7]. Retrospective studies have shown that the humoral response elicited by BCG in MS patients is significantly lower than that in patients with neuromyelitis optica spectrum disorders and in healthy individuals [8]. Clinical trials demonstrated that the administration of BCG vaccine significantly reduced magnetic resonance (MRI) activity in patients with relapsing-remitting MS [9]. MRI disease activity, defined as the identification of T1 gadolinium-enhancing lesions and new/enlarging T2 lesions, is a useful tool used in the diagnosis and monitoring of the progression of MS, as well as in determining the impact of disease-modifying therapies (DMTs) [9]. BCG vaccination seems to modulate the immune response in a similar way to the results obtained in patients with relapsing-remitting MS treated with DMTs [9], reducing the frequency of MRI-detected inflammatory lesions, thus promoting the repair mechanism.

It was also able to delay the second demyelinating episode over a 5-year period in patients with clinically isolated syndrome [10]. In the laboratory, it has been shown in C57BL/6 female mice that BCG vaccination and infection suppress immune responses and reduce the severity of myelin oligodendrocyte glycoprotein (MOG)_35–55-induced experimental autoimmune encephalomyelitis (EAE) [11, 12].

Despite the progress made in recent years, knowledge of the precise mechanism of action of BCG vaccine and host immune response to it remains limited. In this study, we investigated the non-specific effects of BCG vaccine on suppressing neuroinflammation, using two different EAE models. First, we studied the efficacy of BCG vaccine against active EAE in relation to age and time of administration. Second, we investigated the impact of BCG on spontaneous EAE, focusing on peripheral immune cells and glial cells in the CNS. Our findings may open new avenues for development of new BCG vaccines for therapeutic use in neurological diseases, for example by combining it with other therapies, exploiting its systemic impact on adaptive immune system T cells such as regulatory T cells, which may be key immunomodulators alleviating the neuroinflammatory response.

Material and methods

Mice

Groups of young (8 weeks old) and mature adult (6 months old) female wild-type C57BL/6 mice and young female 2D2 C57BL/6 TCR^MOG (2D2 TCR^MOG) transgenic mice (Charles River Laboratories Japan, Inc.) were used in the experiments (N =30/group).

All mice were raised under specific-pathogen-free conditions on a 12-h-light/dark cycle, and BCG-vaccinated mice were maintained at biosafety level (BSL)-2 Animal Facility of Juntendo University School of Medicine. The animals were randomly housed during the experiments to adequately blind the animal caregivers and investigators. The animals were randomly selected for outcome assessment, which was the same in all groups. All animal experiments were approved by the Institutional Animal Care and Use Committee of Juntendo University School of Medicine (No. 290238) and were carried out in accordance with the Guidelines for Animal Experimentation of Juntendo University School of Medicine.

BCG treatments and active and spontaneous EAE induction

BCG Tokyo-172 vaccine (Japan BCG Laboratory, Tokyo, Japan) stock was diluted in sterile phosphate-buffered saline (PBS) to prepare a suspension with a final concentration of 10⁷ colony-forming units (CFU)/ml. After anesthesia by intraperitoneal injection of sodium pentobarbital, mice were inoculated with 100 µl (10⁶ CFU/dose) of BCG vaccine intradermally in the flank (wild-type C57BL/6 and 2D2 TCR^MOG) or intraperitoneally (wild type C57BL/6) using a 1-ml syringe (26-gauge needle). Wild-type mice received one dose of vaccine 1 month before EAE induction, whereas the other group received the same dose of vaccine 6 days after EAE induction. Sterile PBS was administrated to the control group.

For active EAE induction, wild-type mice were immunized subcutaneously with 200 μg of MOG₃₅₋₅₅ peptide (MEVGWYRSPFSRVVHLYRNGK; BEX CO., LTD, Japan) emulsified in incomplete Freund’s adjuvant (BD Diagnosis) supplemented with 400 μg/ml Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI, USA). The antigenic determinants of H37Ra promote the overall polarization toward T helper (Th)1 response in MOG_35-55 epitope-specific CD4⁺ T cells [13]. At the time of immunization and 48 h after immunization, the mice received 200 ng of pertussis toxin (PTX) (Difco) intraperitoneally.

2D2 TCR^MOG mice can develop spontaneous EAE at different frequencies as well as spontaneous optic neuritis without evidence of clinical or histological EAE [14]. Therefore, to induce a more severe conventional EAE, 2D2 TCR^MOG mice and controls received an intraperitoneal dose of 200 ng PTX 1 month after BCG vaccination [15]. PTX treatment increases blood–brain barrier permeability, thereby facilitating pathogenic T-cell migration to the CNS [15].

All mice were monitored daily for clinical symptoms of the disease and blindly scored as follows: (1) flaccid tail, (2) impaired righting reflex and hind limb weakness, (3) complete hind limb paralysis, (4) complete hind limb paralysis with partial fore limb paralysis, and (5) moribund.

Due to the severity of EAE, humane endpoints were used to prevent or minimize animal pain and distress: body weight loss exceeding 20%, clinical score ≥4.0, absence of righting reflex at score 3, animal not eating or drinking for 2 consecutive days.

Splenic lymphocyte proliferation assay

This assay was performed using ³H-thymidine incorporation (PerkinElmer, Waltham, MA, USA) according to a previously reported method [16]. Briefly, spleen cells (4 × 10⁵ cells/well) from MOG_35–55 immunized or 2D2 TCR^MOG mice were cultured for 2 days with 50 μg/mL MOG_35–55 in the presence of gamma-irradiated (3000 rad) accessory spleen cells syngeneic to the responding T cells at 1 × 10⁶ cells/ml. Cell proliferation during the last 18 h was determined by measuring the radioactivity of the incorporated-³H-thymidine using a microplate scintillation counter (MicroBeta TriLux, PerkinElmer). The proliferative response was expressed as a stimulation index (counts per minute [cpm] of cells with test peptides/cpm of cells with no stimulated cells) from triplicate determinations.

Histopathology of the brain and spinal cord

For histological analyses, mice were euthanized by intraperitoneal injection of sodium pentobarbital overdose during the peak (days 14–16) of EAE. The brain and spinal cord were removed and fixed in 4% paraformaldehyde phosphate buffer solution (Nakalai Tesque, Kyoto, Japan), and the tissue sections were processed and sectioned as previously described [16]. Histological examination was performed on sections of formalin-fixed paraffin-embedded tissues stained with hematoxylin and eosin for inflammatory foci detection, and the Kluver–Barrera method was used to determine the severity of demyelination. Immunohistochemical staining was performed as previously reported [16]. A list of primary antibodies used is provided in Supplementary Table S1. Immunohistochemistry was performed using biotinylated secondary antibodies biotin–avidin-peroxidase complex, and diaminobenzidine (DAB) as the developing agent (all from Vector Laboratories). Mayer’s hematoxylin was used to counterstain cell nuclei.

Flow cytometry

For flow cytometry analysis, single-cell suspensions of spleen cells (1 × 10⁶ cells) were stained with live/dead marker (Zombie NIR Fixable Viability Kit, Biolegend, USA) for 15 min at room temperature (24°C), then incubated with FcBlock (FcγRII-RIII) for 10 min at 4°C, and stained with antibodies (Supplementary Table S1) according to previously reported methods [13, 16]. Data were acquired using BD FACSCelesta™ (BD Biosciences, USA) and analyzed using FlowJo software (v.10 Flow Jo Company). Concentration-matched isotype antibodies were used as negative controls.

Isolation of microglia and blood-borne macrophages

Cerebral cortex dissected from mice and transcardially perfused with PBS was resuspended in Hanks’ balanced salt solution without CaCl₂⁺/MgSO₄⁺ (Sigma) containing activated papain (>50 units) and DNase (>250 units) (both from Worthington, NJ, USA) and incubated with rocking for 30 min at 37°C. To stop enzymatic digestion, the samples were diluted with cold Hanks’ balanced salt solution. Tissues were then homogenized using BioMasher tissue grinder (Nippi, Japan) and passed through a 70-μm nylon cell strainer (Merck, Germany). The resulting homogenates were centrifuged at 300 × g for 10 min, and the cell pellets were resuspended in 1 ml 70% isotonic Percoll (Sigma). Two milliliters of 50% isotonic Percoll were gently layered on top of a 1 ml 70% layer, and then 1 ml of 1× PBS was layered on top of the 50% Percoll layer. The density gradient was centrifuged at 1200 × g for 45 min (minimum acceleration and brake) at 20°C. Microglia were collected, washed, and surface-stained for subsequent analysis of M1/M2-like protein expression using flow cytometry.

Analysis of cytokine responses in activated CD4⁺ and CD8⁺ T cells

CD8⁺ or CD4⁺ responder T cells were isolated from the spleen of EAE mice through immunomagnetic negative selection using MojoSort Mouse CD4 or CD8 T-Cell Isolation Kit (BioLegend) according to the manufacturer’s protocol. Post-sort analysis showed that the cells were >99% pure. CD8⁺ and/or CD4⁺ T cells (1 × 10⁵ cells/well) were activated with 50 μg/ml MOG_35–55 in the presence of gamma-irradiated accessory cells (1 × 10⁶ cells per well) and cultured for 4 days at 37°C. Activated T cells were then re-stimulated in complete RPMI-1640 medium containing phorbol 12-myristate 13-acetate (PMA)/ionomycin for 3 h, and GolgiStop (BD Biosciences) solution was added to the culture at the beginning of the last 2 h. Cytokine concentrations (Supplementary Table S1) were determined using an intracellular fixation and permeabilization buffer set (eBioscience).

Statistical analysis

EAE clinical scores were analyzed using two-way analysis of variance (ANOVA) with post hoc Bonferroni’s test for multiple comparisons. Student’s t-test was used for comparison of histological and flow cytometric analysis results between two groups. Data are presented as mean ± SD. All statistical analyses were performed using the Prism software (v.9 GraphPad company). Statistical significance was set at P < 0.05.

Results

Age and time-dependent effects of BCG vaccine on clinical course of active EAE

Groups of young and mature adult female C57BL/6J mice were vaccinated intradermally or intraperitoneally with BCG (10⁶ CFU/dose) 1 month prior to as well as 6 days after induction of active EAE using MOG_35–55 peptides. No significant differences were found between young and mature adult mice in locomotor functions and age-related behavioral changes.

As reported in Table 1, intradermal vaccination with BCG 1 month prior to EAE induction led to nearly 90% protection in young mice, which did not display any EAE symptoms compared with non-vaccinated control mice (Fig. 1A). During 30 days after the vaccination, no side effects or significant weight loss (20.8 ± 0.9 g vaccinated vs. 22.2 ± 1 g non-vaccinated) were observed in the vaccinated group compared with those in the control group.

Table 1.

Effect of age and time of administration on Bacillus Calmette–Guérin (BCG) vaccine effects in myelin oligodendrocyte glycoprotein (MOG)_35–55-induced experimental autoimmune encephalomyelitis (EAE)

Treatment before/after EAE induction	Mouse strain, and age	EAE incidence (%)	Mortality (%)	Mean day of onset	Peak clinical score	Cumulative disease score (30 d.p.i)
No vaccination	C57BL/6 (8 weeks)	100	0	10.3 ± 0.5	3.5 ± 0.3	42.2 ± 0.8
BCG vaccine injected ID 1 month before EAE induction BCG vaccine injected ID 6 days after EAE induction	C57BL/6 (8 weeks) C57BL/6 (8 weeks)	10=* 100	0 0	10 10.5 ± 0.5	0.3 ± 0.2* 2.7 ± 0.3*	3.8 ± 0.1* 42.7 ± 0.7
BCG vaccine injected IP 1 month before EAE induction BCG vaccine injected IP 6 days after EAE induction	C57BL/6 (8 weeks) C57BL/6 (8 weeks)	100 100	0 0	10.3 ± 0.5 10 ± 0.5	3.3 ± 0.2 3.5. ± 0.3	39.9 ± 0.9 44..9 ± 0.9
No vaccination	C57BL/6 (6 months)	100	0	11.3 ± 0.5	3.4 ± 0.4	44.2 ± 0.9
BCG vaccine injected ID 1 month before EAE induction BCG vaccine injected ID 6 days after EAE induction	C57BL/6 (6 months) C57BL/6 (6 months)	50* 100	0 0	11.6 ± 0.5 11.4 ± 0.5	2 ± 0.3* 2.8 ± 0.6*	29.9 ± 0.6* 42.2 ± 0.8
BCG vaccine injected IP 1 month before EAE induction BCG vaccine injected IP 6 days after EAE induction	C57BL/6 (6 months) C57BL/6 (6 months)	100 100	0 0	11 ± 0.5 11.3 ± 0.5	3.5 ± 0.3 3 ± 0.7	42.2 ± 0.9 40.3 ± 0.9

^*Statistically significant.

Figure 1.

Effect of Bacillus Calmette–Guérin (BCG) Tokyo-172 vaccine on induced active experimental autoimmune encephalomyelitis (EAE). Clinical score and course of the disease in young (8 weeks) and mature adult (6 months) wild-type BCG-vaccinated mice intradermally (A, B) or intraperitoneally (C, D) 1 month before or 6 months after myelin oligodendrocyte glycoprotein (MOG)_35–55 immunization. The data show combined results of three independent experiments with 30 mice per group. Data are expressed as mean ± SD calculated using analysis of variance (ANOVA) followed by Bonferroni’s post hoc test. (*P < 0.05).

BCG-vaccinated mature adult mice showed a 50% reduction in EAE incidence (Fig. 1B). The time of symptom onset in the vaccinated group was not significantly different from that in the control, whereas the peak of the acute phase and disease severity were significantly (P < 0.001) reduced in vaccinated mice (Fig. 1B).

Vaccination 6 days post EAE induction had no effect on disease development. All EAE-induced mouse groups, regardless of age, developed typical clinical EAE symptoms (Fig. 1A and B).

Further, BCG immunization by the intraperitoneal route seems to not prevent the development of EAE in all mice (Fig. 1C and D).

Impact of BCG vaccination on spontaneous EAE incidence

Phenotypic screening to identify the transgenic mice was routinely performed using blood sampling from the retro-orbital sinus (Fig. 2B) or spleen (Fig. 2C) and flow cytometry using antibodies specific to the T-cell receptor (TCR) Vβ11 or Vα3.2 [14]. 2D2 TCR transgenic founders were identified using polymerase chain reaction with specific primers for the transgene (The Jackson Laboratory genotyping protocol) and were then bred with wild-type C57BL/6 mice (Fig. 2D).

Figure 2.

Experimental autoimmune encephalomyelitis (EAE) clinical score and immunophenotype and genotype of 2D2 TCR^MOG transgenic mice. (A) Effect of Bacillus Calmette–Guérin (BCG) vaccine on spontaneous EAE. The data show clinical scores (combined results) of three independent experiments (n = 10 mice per group). Data are expressed as mean ± SD. Flow cytometric analysis of (B) orbital sinus blood, and (C) splenocytes from young 2D2 TCR^MOG or wild-type mice, stained with anti-CD4, CD8, Vβ11, and Vα3.2 antibodies. (D) Result of polymerase chain reaction products of mouse tail genomic DNA showing incorporation of transgene (675 bp) and the internal positive control (324 bp) gene. Data are expressed as mean ± SD. (*P < 0.05).

The EAE clinical data are summarized in Table 2. Approximately 27% of 2D2 TCR^MOG transgenic mice spontaneously developed clinical EAE signs within 2 months of birth with a mortality of 10%, whereas injections of suboptimal doses of PTX alone increased the overall incidence of spontaneous EAE by 80% with a mortality of 20%. Only 3 (10%) of the 2D2 TCR^MOG mice intradermally vaccinated with BCG showed clinical EAE symptoms (P < 0.001), whereas such symptoms appeared in 30% of BCG-vaccinated and PTX-treated mice, which showed peak disease and cumulative disease scores were significantly (P < 0.001) reduced (Fig. 2A).

Table 2:

Effect of Bacillus Calmette–Guérin (BCG) vaccine on disease course of spontaneous experimental autoimmune encephalomyelitis (EAE)

Treatment	Mouse strain, and age	EAE incidence (%)	Mortality (%)	Mean day of onset after pertussis toxin (PTX) injection	Peak clinical score	Cumulative disease score (30 days after EAE onset)
No treatment	2D2 MOGTCR (1.5 months)	30	10		3.7 ± 0.4	65.5 ± 0.7
PTX alone	2D2 MOGTCR (1.5 months)	80	20	5.2 ± 0.6	3.7 ± 0.3	63.3 ± 0.8
BCG vaccine injected ID	2D2 MOGTCR (1.5 months)	10*	0
BCG vaccine injected ID + PTX	2D2 MOGTCR (1.5 months)	30*	0	6.7 ± 0.6	2.8 ± 0.3 *	36.2 ± 0.6*

^*Statistically significant.

Reduced T-cell proliferative response to MOG_35–55 in BCG-vaccinated mice

BCG-vaccinated young mice developed less severe EAE accompanied by reduced CNS T-cell infiltration, which might have resulted from reduced T-cell proliferation. Therefore, we examined the proliferation of MOG_35–55-stimulated T cells isolated from the spleen of young mice at peak EAE. Preliminary assays indicated that MOG_35–55 had the strongest stimulatory effect on both CD8⁺ and CD4⁺ T cells at the highest concentration (50 μg/ml). In young wild-type mice with EAE, MOG_35–55 stimulation significantly (P < 0.0001) increased the proliferation of both CD4⁺ and CD8⁺ T cells (Fig. 3A), whereas it did not stimulate the proliferation of T lymphocytes isolated from BCG-vaccinated mice that did not develop any clinical EAE signs (Fig. 3B).

Figure 3.

T-cell response in the spleen of wild-type and 2D2 TCR^MOG transgenic Bacillus Calmette–Guérin (BCG)-vaccinated mice. CD4⁺ and CD8⁺ T cells were purified using immunomagnetic negative selection from the spleen, and the isolated T cells were cultured with myelin oligodendrocyte glycoprotein (MOG)_35–55 peptide. The cultures were pulsed with [³H] thymidine at 48 h, harvested, and analyzed at 56 h. The data show one experiment (n = 10 mice per group) of three independent ones. (A) wild-type experimental autoimmune encephalomyelitis (EAE) mice, (B) wild-type BCG-vaccinated mice, (C) 2D2 TCR^MOG EAE mice, and (D) 2D2 TCR^MOG BCG-vaccinated mice. Data are expressed as mean ± SD (*P < 0.05).

In 2D2 mice with spontaneous EAE, MOG_35–55 had a significantly (P < 0.0001) stronger stimulatory effect on CD4⁺ T cells than on CD8⁺ T cells (Fig. 3C). Conversely, in BCG-vaccinated 2D2 mice without any clinical EAE signs, both purified CD4⁺ and CD8⁺ T cells had similar proliferative responses to MOG_35–55 peptide (Fig. 3D).

Association of neuroprotective effect of BCG vaccine with increased regulatory CD8⁺ T-cell frequency in the spleen and brain of 2D2 TCR^MOG mice

We analyzed the T-cell percentage in the spleen as well as in the brain of 2D2 TCR^MOG mice using flow cytometry. Four weeks after vaccination, BCG-vaccinated and PTX-treated 2D2 transgenic mice without EAE showed a significant increase in CD8⁺ T-cell frequency in their spleen and brain (P < 0.0001 for both) compared with non-vaccinated 2D2 TCR^MOG mice with PTX-induced EAE (Fig. 4A and B). Flow cytometric analysis also showed significantly lower CD4⁺ T-cell percentage in the spleen (P = 0.0004) and brain (P = 0.0002) of BCG-vaccinated mice than in those of non-vaccinated mice (Fig. 4A). Furthermore, because Th1 and Th17 cells are the major pathogenic cells that mediate EAE [17], and IL-10 plays a critical role in disease suppression [18], we stimulated CD8⁺ T cells isolated from the spleen of 2D2 TCR^MOG mice with MOG_35–55 peptides 1 month after BCG vaccination and analyzed them for interferon (IFN)-γ, IL-17A, and IL-10 production. Intracellular cytokine analysis showed that these CD8⁺ T-cells produced anti-inflammatory IL-10 in BCG-vaccinated and PTX-treated 2D2 TCR^MOG mice without any clinical EAE signs (Fig. 4C).

Figure 4.

Distinct T cells phenotype and cytokine expression profile in the spleen and brain of 2D2 TCR^MOG mice following Bacillus Calmette–Guérin (BCG) vaccination. Flow cytometric analyses from the quantification of T cells revealed increased frequency of CD8⁺ T cells in the (A) spleen and (B) brain cells isolated from 2D2 TCR^MOG BCG-vaccinated mice in comparison with that in non-vaccinated mice. CD8 T cells were purified using immunomagnetic negative selection from the spleen, and then cytokine concentrations were determined in myelin oligodendrocyte glycoprotein (MOG)_35–55-stimulated spleen cells. Expression of IL-10 by CD8 T-cell was significantly elevated in 2D2 TCR^MOG BCG-vaccinated mice (C). The data show combined results of three independent experiments (n = 30 mice per group). Data are expressed as mean ± SD calculated using Student’s t-test (*P < 0.05).

Different patterns of glial polarization in the midbrain and spinal cord of BCG-vaccinated mice

Flow cytometric analysis of mononuclear cells isolated from the brains of 2D2 TCR^MOG mice showed a cellular phenotype in BCG-vaccinated mice distinct from that in untreated mice (Fig. 5A). Compared with 2D2 TCR^MOG mice with EAE, BCG-vaccinated 2D2 TCR^MOG mice had significantly (P < 0.0001, P = 0.0133) lower number of CD45^lo/intCd11b⁺ resident microglia and CD45^highCD11b⁺ myeloid cells (Fig. 5B). Moreover, M2-polarized microglia were dominant in the BCG-vaccinated 2D2 TCR^MOG mice, whereas the M1 and a mixed M1/M2 phenotype were dominant in 2D2 TCR^MOG mice with PTX-induced EAE during the acute phase (Fig. 5C).

Figure 5.

Flow cytometric analyses of brains from 2D2 mice. (A) Gating strategy for different cell populations in mouse brain homogenate. (B) Graphs showing resident microglia, peripheral myeloid populations, and lymphocytes in the brains. (C) M1 pro-inflammatory microglia were considered CD45^intCD11b⁺CD86⁺CD206⁻, whereas M2 neuro-protective microglia were considered CD45^intCD11b⁺CD206⁺CD86⁻. The bar graphs represent the total number of cells in the brains (n = 5 mice per group). Data are expressed as mean ± SD calculated using Student’s t-test *P < 0.05.

Histological analysis revealed inflammatory cell infiltration accompanied by widespread demyelination in the spinal cord of 2D2 TCR^MOG mice with EAE, whereas no evidence of inflammatory foci or demyelination was observed in BCG-vaccinated 2D2 TCR^MOG mice without EAE (Fig. 6A and B). Furthermore, histopathological analysis showed significant differences between BCG-vaccinated and non-vaccinated mice with respect to the number of CD4⁺ T cells (Fig. 6C and D 68⁺ monocytes (Fig. 6E), and TMEM119⁺ microglia (Fig. 6F). Moreover, we observed a high frequency of GFAP⁺C3⁺ (A1) pro-inflammatory astrocytes in the spinal cord of 2D2 TCR^MOG mice with EAE (Fig. 6G), whereas BCG-vaccinated 2D2 TCR^MOG mice showed upregulation of GFAP⁺C3⁺ (A2) anti-inflammatory astrocytes (Fig. 6H). The CD8⁺ T-cells histological score in the spinal cord of the two groups did not differ and was lower than the number of cells detected in the brain, possibly owing to a difference in lymphocyte recruitment and leukocyte-endothelial interactions in the brain compared with those in the spinal cord [19] (Fig. 6D).

Figure 6.

Histopathology and immunohistochemistry of spinal cords at day 20 after experimental autoimmune encephalomyelitis (EAE) induction. Spinal cord sections (4× magnification) were stained with (A) hematoxylin/eosin (HE) and (B) Klüwer–Barrera and for (C) CD4⁺, (D) CD8⁺, (E) CD68⁺, (F) TMEM119⁺, (G) GFAP⁺/C3⁺, and (H) GFAP⁺/S100A10⁺. Histological scores displayed at the bottom of the figure were calculated based on at least two sections per mouse (n = 5 mice per group). Data are representative of two independent experiments with similar results. Each graph represents the mean ± SD. *P < 0.05, vs. wild-type mice.

Discussion

We evaluated the effect of the BCG Tokyo-172 vaccine in two EAE models characterized by T-cell infiltration in the CNS associated with local inflammation. Overall, BCG vaccine conferred neuroprotection in both the models, decreasing the incidence and severity of the disease, but failed to attenuate disease progression or to modulate the recovery phase in both the models.

We first studied the impact of BCG vaccine administration on MOG_35–55-induced EAE, showing that the vaccine-induced age-specific immunity, offering greater protection to young mice than to adults, probably because age-related changes influence the host immune system [20]. Moreover, the beneficial effects of BCG were related to the time of administration, being effective only if administered 1 month before disease onset. Indeed, when BCG vaccine was administered during the disease in progress, it did not alleviate the clinical symptoms, suggesting that it cannot be used as a treatment at this stage.

Next, we investigated the effect of BCG vaccine using a spontaneous EAE model, detecting an increased number of CD8⁺ T cells in the spleen and brain of BCG-vaccinated 2D2 TCR^MOG mice that had not developed the disease. In addition, CD8⁺ T cells isolated from the spleen secreted IL-10 following stimulation with MOG_35–55 peptides. Thus, we hypothesized that BCG vaccine-mediated neuroprotection is associated with peripheral activation of CD8⁺ regulatory cells because the ratio between CD4⁺ and CD8⁺ lymphocytes is biased toward the CD4⁺ component in 2D2 TCR^MOG mice [14].

While CD4⁺ T cells mainly contribute to EAE, CD8⁺ T cells also have both a pathogenic and regulatory function in EAE [21]. With respect to the role of CD8⁺ T cells in BCG infection, live BCG vaccine is able to activate CD8⁺ regulatory T-cells in vitro [22], and studies on different mouse models have confirmed that α/β TCRs expressed by CD8⁺ T cells are necessary for the control of BCG infection [23]. In our study, we also found that BCG vaccine was able to modulate the glial phenotype, activate the M2 anti-inflammatory microglial phenotype, and subsequently induce the transformation of A1 to A2 reactive astrocytes.

Microglia and astrocytes express a wide variety of pattern recognition receptors, which, upon pathogen-associated molecular pattern engagement, activate various signaling pathways resulting in cytokine production against pathogen infection [24]. Peripheral immune cells such as CD8⁺ T cells may alter the cytokine milieu and thus reprogram microglia toward M1 or M2 phenotypes [25]. Furthermore, IL-10 secreted by CD8⁺ T cells can inhibit pro-inflammatory cytokine production by microglia, protecting astrocytes from excessive inflammation [26]; therefore, it plays a major role in the crosstalk between neurons and microglia or astrocytes. In the presence of IL-10, microglia differentiate toward the M2 neuroprotective phenotype, implicated in the resolution of inflammation, phagocytosis, and tissue repair [27].

Since A2 astrocytes were upregulated in the spinal cords of 2D2 TCR^MOG mice after BCG vaccination, we suggest that BCG vaccine can exert neuroprotective effects in the CNS by modulating M1/M2 microglial polarization and A1/A2 astrocyte transformation.

The difference in the efficacy of BCG vaccine in young and adult mice may be because CD8⁺ T cells in young mice represent regulatory cells capable of producing IL-10, whereas, in old mice, they are mainly antigen-specific memory T cells [28]. These age-related differences in T-cell function may explain, in part, why BCG vaccine provided protection only in young mice, indicating that the CD8⁺ T cells were mostly memory and not regulatory cells in adult mice. T-cell proliferation experiments also suggested that the suppression of immune response by CD8⁺ T cells was not antigen-specific. Notably, in humans, BCG vaccine is usually administered around birth to prevent tuberculosis in neonates; however, mycobacterial immunity appears to decrease with advancing age [29]. Another possible explanation could be related to the effect of aging on the decline in thymic function, which contributes to a less efficient selection of T cells and a diminished ability for thymic self-antigen presentation [30].

Another important point is the effect of the timing of BCG vaccination, with the highest efficacy observed only when it was administered before EAE induction. In contrast, the immune response conferred by BCG vaccine was less likely to be protective under ongoing autoimmunity after EAE onset.

Finally, intradermal vaccination was more effective in stimulating BCG-specific immune response than intraperitoneal immunization, indicating that the immunization route mostly affects the targeted anatomical lymph node clusters, altering the traffic of encephalitogenic T cells from lymphoid ­organs [31].

Interestingly, it has been shown that oral immunization with Salmonella live vaccine protected against proteolipid protein (PLP)-induced EAE in female SJL mice, by enhancing multiple subsets of antigen-nonspecific IL-13 producing CD4⁺ regulatory T cells, which can be induced by vaccination in the absence of autoantigens [32]. Both Salmonella and BCG vaccines have important non-specific immunomodulatory effects; however, BCG appears to modulate immune responses to MOG peptide by antigen-specific IL-10 producing CD8⁺ regulatory T cells.

The main limitation of this study is the fact that findings from these animal models cannot be directly translated to humans and should be confirmed in a human study. Furthermore, in our study we used only BCG Tokyo-172; however, factors such as BCG strain manufacturing may influence the immunological mechanisms of vaccine-induced protection [33]. Finally, since we observed no significant sex-specific differences in the clinical course of active EAE as well as in the incidence of spontaneous EAE, we decided to use only female mice. However, sexual dimorphism in the immune system has been well demonstrated, including evidence that females generate a stronger immune response than males, just as women tend to develop autoimmune diseases more often than men [34]. Therefore, further research is needed to better assess the sex-specific response to BCG by using male mice as well.

Conclusions

Collectively, our findings confirm the potential use of BCG vaccine for the prevention of MS, opening fresh opportunities for development of new BCG-based vaccines for therapeutic use in neurological diseases. Future trials testing the efficacy of different strains of BCG vaccine in other disease models will help fully understand the precise mechanism of action by which BCG vaccine exerts such non-specific neuroprotective effects. Moreover, the population of CD8⁺ T cells requires additional subtype characterization and validation in regulating inflammatory responses, comparing the relationship between peripheral and CNS cytokines.

Glossary

Abbreviations

2D2 TCR^MOG

2D2 C57BL/6 TCR^MOG mice

BCG

Bacillus Calmette–Guérin

CNS

central nervous system

EAE

experimental autoimmune encephalomyelitis

ID

intradermal

IP

intraperitoneal

MOG

myelin oligodendrocyte glycoprotein

MS

multiple sclerosis

PTX

pertussis toxin

Ethical approval

All animal experiments were approved by the Institutional Animal Care and Use Committee of Juntendo University School of Medicine (No. 290238) and were carried out in accordance with the Guidelines for Animal Experimentation of Juntendo University School of Medicine. The animal research adheres to the ARRIVE guidelines.

Conflict of Interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI (grant number: JP 20K16468) and by Universita' degli Studi di Sassari (UNISS) FAR 2021 to Davide Cossu.

Data availability

The data underlying this article will be shared on reasonable request to the corresponding author.

Author contributions

DC designed and performed the experiments, analyzed the data, and wrote the manuscript; KY designed the experiments and provided critical feedback on the manuscript; TS supervised the flow cytometry experiments; and LAS, and NH critically revised the draft.