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CBD Suppression of EAE Is Correlated with Early Inhibition of Splenic IFN-γ + CD8+ T Cells and Modest Inhibition of Neuroinflammation

Abstract

In this study cannabidiol (CBD) was administered orally to determine its effects and mechanisms in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). We hypothesized that 75 mg/kg of oral CBD given for 5 days after initiation of disease would reduce EAE severity through suppression of either the early peripheral immune or late neuroimmune response. EAE was induced in C57BL/6 mice at two different magnitudes, and peripheral inflammatory and neuroinflammatory responses were measured at days 3, 10, and 18. Th1, Th17, Tc1, Tc17, Tregs, and myeloid derived suppressor cells (MDSC) were identified from the lymph nodes and spleens of each mouse to determine if CBD altered the suppressor cell or inflammatory cell populations in secondary lymphoid tissues. Additionally, neuroinflammation was identified in brain and spinal cord tissues using various immunohistochemical techniques and flow cytometry. Early treatment of EAE with oral CBD reduced clinical disease at the day 18 timepoint which correlated with a significant decrease in the percentage of MOG35–55 specific IFN-γ producing CD8+ T cells in the spleen at day 10. Analysis of both T cell infiltration and lesion size within the spinal cord also showed a moderate reduction in neuroinflammation within the central nervous system (CNS). These results provide evidence that oral CBD suppressed the peripheral immune response that precedes neuroinflammation; however, analysis of the neuroinflammatory endpoints also suggest that the modest reduction in neuroinflammation was only partially responsible for CBD’s neuroprotective capability.

CBD was administered orally for the first 5 days following initiation of EAE. CBD attenuated clinical disease, and we found that CBD suppressed IFN-γ producing CD8+ T cells in the spleen at day 10. There was also modest suppression of neuroinflammation. Together these data demonstrate that early, oral administration of CBD protected mice from disease, but the modest effects on neuroinflammation suggest other mechanisms participate in CBD’s neuroprotective effect in EAE.

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Abbreviations

THC:

Δ9-tetrahydrocannabinol

BSA:

Bovine serum albumin

CBD:

Cannabidiol

CB1/CB2:

Cannabinoid receptors

CO:

Corn oil

DAB:

Diaminobenzidine

EAE:

Experimental Autoimmune Encephalomyelitis

FVD:

Fixable Viability Dye

FC buffer:

Flow cytometry buffer

GFAP:

Glial Fibrillary Acidic Protein

HKMT:

Heat-killed Mycobacterium tuberculosis H37Ra

H&E:

Hematoxylin and eosin

IBD:

Inflammatory bowel disease

i.p.:

Intraperitoneal

MS:

Multiple Sclerosis

MBP:

Myelin basic protein

MOG:

Myelin oligodendrocyte protein

MDSC:

Myeloid derived suppressor cell

NBF:

Neutral buffered formalin

PBST:

PBS containing 0.05% Tween 20

PTX:

Pertussis toxin

PMA/Io:

Phorbol 12-myristate-13-acetate and ionomycin

PBS:

Phosphate buffered saline

PLP:

Proteolipid protein

RA:

Rheumatoid arthritis

TMEV:

Theiler’s murine encephalomyelitis virus

Tx:

Triton X-100

VCAM-1:

Vascular cell-adhesion molecule-1

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Acknowledgments

The authors thank Dr. T. Graham Rosser for help with illustrations. Funding was provided by National Institutes of Health training grant T35OD010432, Center of Biomedical Research Excellence (COBRE) grant P20GM103646 and Institutional Development Award (IDeA) grant P20GM103476.

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Corresponding author

Correspondence to Barbara L. F. Kaplan.

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Experiments were approved by Mississippi State University Institutional Animal Care and Use Committee (IACUC).

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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The authors declare that they have no competing interests.

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Supplemental Fig. 1
figure 10

Gating strategy for identifying Tc1, Tc17, Th1, and Th17cells. CD8+IFN-γ+, CD8+IL17A+, CD4+IFN-γ+, CD4+IL-17A+ lymphocytes were identified in the live singlet population. (PNG 1007 kb)

Supplemental Fig. 2
figure 11

Cell counts of inflammatory T cells from ex vivo restimulated splenocytes. These cell counts correspond to the percentages of inflammatory T cells given in Fig. 3 of the main body of the paper. * p < 0.05 differences between SAL/CO and EAE/CO; ‡ p < 0.05 difference between SAL/CO and Mild EAE/CO; # p < 0.05 difference between EAE/CO and EAE/CBD; @p < 0.05 difference between EAE/CO and Mild EAE/CO € p < 0.05 difference between EAE/CBD and Mild EAE/CBD. (PNG 305 kb)

Supplemental Fig. 3
figure 12

Cell counts of inflammatory T cells from ex vivo restimulated cells isolated from lymph nodes. These cell counts correspond to the percentages of inflammatory T cells given in Fig. 4 of the main body of the paper. * p < 0.05 differences between SAL/CO and EAE/CO; ‡ p < 0.05 difference between SAL/CO and Mild EAE/CO; § p < 0.05 difference between Mild EAE/CO and Mild EAE/CBD. (PNG 375 kb)

Supplemental Fig. 4
figure 13

Gating strategy for identifying MDSCs and Tregs. Tregs were identified as CD4+CD25+FoxP3+ Lymphocytes in the singlet population. Granulocytic MDSC are identified as CD11b+Ly6CloLy6G+ cells and monocytic MDSC are identified as CD11b+Ly6C+Ly6G cells in the singlet population. (PNG 1288 kb)

Supplemental Fig. 5
figure 14

Cell counts of regulatory cells from secondary lymphoid organs. These cell counts correspond to the percentages of regulator cells given in Fig. 2 of the main body of the paper. * p < 0.05 differences between SAL/CO and EAE/CO; ‡ p < 0.05 difference between SAL/CO and Mild EAE/CO; # p < 0.05 difference between EAE/CO and EAE/CBD; @p < 0.05 difference between EAE/CO and Mild EAE/CO € p < 0.05 difference between EAE/CBD and Mild EAE/CBD. (PNG 409 kb)

Supplemental Fig. 6
figure 15

Cell counts of T cells isolated from the spinal cord of EAE mice. These cell counts correspond to the percentages of T cells given in Fig. 9D of the main body of the paper. (PNG 236 kb)

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Nichols, J.M., Kummari, E., Sherman, J. et al. CBD Suppression of EAE Is Correlated with Early Inhibition of Splenic IFN-γ + CD8+ T Cells and Modest Inhibition of Neuroinflammation. J Neuroimmune Pharmacol 16, 346–362 (2021). https://doi.org/10.1007/s11481-020-09917-8

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Keywords

  • Multiple sclerosis
  • IFN-γ
  • Experimental autoimmune encephalomyelitis
  • Cannabidiol
  • Neuroinflammation