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Table of Contents
ORIGINAL ARTICLE
Year : 2021  |  Volume : 7  |  Issue : 4  |  Page : 467-476

Experimental autoimmune encephalomyelitis inhibited by huangqi guizhi wuwu decoction via th2 cytokine enhancement


1 Department of Neurology, Affiliated First Hospital of Hunan Traditional Chinese Medical College, Zhuzhou, China
2 Department of Biochemistry and Molecular Biology, School of Life Sciences, Central South University, Changsha, Hunan, China

Date of Submission12-Oct-2020
Date of Acceptance23-Feb-2021
Date of Web Publication21-Oct-2021

Correspondence Address:
Dr. Yong Peng
Department of Neurology, Affiliated First Hospital of Hunan Traditional Chinese Medical College, Renmin Road 571, Zhuzhou, Hunan 412000
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2311-8571.328617

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  Abstract 


Background: Huangqi Guizhi Wuwu decoction (HQGZWW) exhibits good effects when administered to treat multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Understanding the precise mechanism of this decoction is thus important. Based on the findings of our previous study, the aim of the present study was to understand the role of antigen-specific CD8+ T-cells on the pathogenesis of MS/EAE when HQGZWW is administered as treatment. Methods: Myelin oligodendrocyte glycoprotein (MOG) 35-55-induced mice were administered distilled water, prednisone, and high dose or low dose HQGZWW. After purified CD4+ and CD8+ T-cells were stimulated with the MOG35-55 peptide, proliferation and cytokine secretion assays were performed. To establish the adoptive transfer EAE model, naïve mice were injected with MOG35-55 -CD8+ or CD4+ T-cells. Results: Significant improvements in EAE score and pathology were observed in the high dose HQGZWW and prednisone groups. Compared to the low dose HQGZWW and distilled water groups, lower antigen-specific responses, lower levels of interferon-gamma, and higher levels of interleukin (IL)-4 and IL-10 from CD8+ and CD4+ T cells were observed in the high dose HQGZWW and prednisone groups. Finally, the EAE score was observed to be similar between the high dose HQGZWW group and prednisone group; however, this finding was not observed in the low dose HQGZWW group. Conclusion: Our findings suggest that high dose HQGZWW has similar effects on cell proliferation, cytokine secretion, and EAE score to prednisone, while low dose HQGZWW does not have such effect. The protective role of HQGZWW against EAE might thus depend on the Th2 cytokine secretion profile induced by either MOG35-55 specific CD8+ or CD4+ T-cells.

Keywords: CD8-Positive T-lymphocytes, experimental autoimmune encephalomyelitis, multiple sclerosis, myelin-oligodendrocyte glycoprotein, Th2 cytokine, traditional Chinese medicine


How to cite this article:
Peng Y, Zhu FZ, Deng X, Zhou JX, Gao S, Chen ZX, Yang SS, Gan L, Li ZL, Liu QQ. Experimental autoimmune encephalomyelitis inhibited by huangqi guizhi wuwu decoction via th2 cytokine enhancement. World J Tradit Chin Med 2021;7:467-76

How to cite this URL:
Peng Y, Zhu FZ, Deng X, Zhou JX, Gao S, Chen ZX, Yang SS, Gan L, Li ZL, Liu QQ. Experimental autoimmune encephalomyelitis inhibited by huangqi guizhi wuwu decoction via th2 cytokine enhancement. World J Tradit Chin Med [serial online] 2021 [cited 2021 Nov 29];7:467-76. Available from: https://www.wjtcm.net/text.asp?2021/7/4/467/328617




  Introduction Top


As an autoimmune disease of the central nervous system (CNS), multiple sclerosis (MS) occurs in the middle age individuals and is associated with a hefty social and economic burden.[1] Current modern conventional medicines are focused on acute relapse, disease-modifying therapies, and MS symptoms,[2] such as β-interferon (IFN), glatiramer acetate, mitoxantrone, teriflunamide, dimethyl fumarate, fingolimod, alemtuzumab, and natalizumab.[3] However, these therapies are associated with serious side-effects, including progressive multifocal leukoencephalopathy as well as serious viral and fungal infections,[4],[5] as they induce universal immunosuppression.[3]

To reduce the side effects of treatments, more ideal treatment options should be discovered more ideal results must be achieved.[6],[7] Traditional Chinese medicine (TCM) provides a new possibility for MS treatment. Several TCM prescriptions, such as (1) Buyang Huanwu decoction;[8],[9] (2) Bushen Yisui Fang, formerly known as Erhuang Fang;[3] (3) Liuwei Dihuang Pills, Zuogui pills, and Yougui pills;[10] (4) Catalpol, an extraction from the root of Rehmannia;[11],[12] (5) Hyungbangpaedok- san, a classic formula in TCM called Jingfangbaidu San;[13] and (6) Huangqi Guizhi Wuwu decoction (HQGZWW),[3] are used to treat MS and its animal model, experimental autoimmune encephalomyelitis (EAE). Of these prescriptions, the mechanism of HQGZWW in MS has been identified to be close to an immune-protection mechanism. However, this precise mechanism is currently unclear. HQGZWW is a common TCM formula for arthromyodynia, consisting of five types of herbs: Astragalus mongholicus, Radix Paeoniae Rubra, Cassia twig, Ginger, and 4 Fructus Ziziphi Jujubae. HQGZWW was reported to be effective when administered to patients with MS, as well as the EAE model.[14]

MS is well-known as a typical CD4+ T-cell-mediated disease;[15] however, CD8+ T-cells also play a critical role in the pathogenesis of MS/EAE.[16] As reported previously, both myelin oligodendrocyte glycoprotein (MOG) 35-55-specific CD4+ and CD8+ autoreactive T-cells from EAE mice are encephalitogenic. Further, CD8+ T-cells act independently in the pathogenicity of EAE without CD4+ T-cells.[17] However, CD8+ T-cells display weaker proliferation response, and less IFN-gamma secretion and encephalitogenic than CD4+ T-cells.[17] Based on previous clinical and animal studies, HQGZWW plays a protective role against MS/EAE; however, the precise mechanism is unclear. To our knowledge, this is the first study to assess the role of MOG35-55 CD8+ T-cells in MS/EAE following treatment with HQGZWW. Herein, MOG 35-55-specific CD8+ T-cells were isolated from MOG35-55 induced active-induced EAE Models (aEAE) after the administration of different doses of HQGZWW and prednisone. Thereafter, the functions of CD8+ T cells, such as antigen-specific, cytokine secretion profile, and adoptive transfer, were examined.


  Methods Top


Ethical approval

All experiments were performed at the School of Life Sciences, Central South University. All experimental steps were evaluated and approved by the Ethics Committee of Central South University (No. 2019sydw0005). Animal treatment was carried out in accordance with the National Institute of Health's Guide for the Care and Use of Laboratory Animals. All steps were taken to minimize animal pain and suffering.

Animals

Specific pathogen-free female C57BL/6 mice (age 8–10 weeks) were purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd. (Changsha, Hunan, China) (License No. SCXK [Hunan] 2016-0002) and maintained at the Center of Experimental Animal, Xiangya Medical College, Central South University (License No. SCXK [Hunan] 2015-0017). Mice were housed in a single cage with an independent ventilation system at a temperature of 18°C–22°C and relative humidity of 40%–60%, with free access to food and water. The sample size prediction matched the requirements of the experimental design.

Preparation of Huangqi Guizhi Wuwu decoction

HQGZWW was prepared using the following herbs: A. mongholicus 9 g, Radix Paeoniae Rubra 9 g, Cassia twig 9 g, Ginger 18 g, and Fructus Ziziphi Jujubae 4 g. All herbs were dried decoction pieces (i.e., TCMs prepared in ready-to-use forms) purchased from Honghua Decoction pieces company (Changsha, Hunan, China). All dry decoction pieces were immersed in distilled water for 30 min and boiled for 2 h. The inspissation of the filtered solution was carried out under reduced pressure at 70°C and was ready-to-use as the stock solution.

Active-induced experimental autoimmune encephalomyelitis models and adoptive-transferred experimental autoimmune encephalomyelitis models

Mice were randomly divided into the following four groups (n = 8 per group): (1) Distilled water; (2) high dose HQGZWW (stock solution); (3) low dose HQGZWW (1:10 of stock solution diluted with distilled water); and (4) prednisone (6 mg/kg). The aEAE and tEAE models were established according to our previous studies.[17] Briefly, for the aEAE, a subcutaneous injection of 200 μL of an emulsion containing 200 μg MOG35–55 peptide purified by high-performance liquid chromatography with a purity of >95% (amino acids 35–55 of bovine MOG; Sigma-Aldrich, St. Louis, MO, USA) and complete Freund's adjuvant (Sigma-Aldrich, St. Louis, MO, USA) was distributed over six sites at the tail base and on the flank. At 0 and 24 h after immunization, mice were intraperitoneally administered pertussis toxin (250 ng per mice, List Biological, Campbell, CA, USA). For tEAE, unless stated otherwise, recipient mice were injected via the caudal vein with 5 × 106 MOG35-55 -specific CD8+ or CD4+ enriched T-cells prepared as described previously in 0.2 mL phosphate buffer saline.[17]

Treatment and experimental autoimmune encephalomyelitis score

EAE mice were administered distilled water, prednisone, high dose or low dose HQGZWW via oral gavage, starting 1 h after immunization once per day for 14 days. EAE was then scored on a scale ranging from 0 to 5 as follows: 0, no obvious changes in the motor functions of immunized mice compared to non-immunized mice; 1, limp tail; 2, limp tail and weakness of the hind limbs; 3, limp tail and complete paralysis of the hind limbs (most common) or limp tail with paralysis of one front and one hind limb; 4, complete hind limb and partial front limb paralysis; and 5, death or sacrificed due to severe paralysis.[3]

Preparation of myelin oligodendrocyte glycoprotein35–55-specific T-cells

The procedure used to prepare MOG35–55-specific T cells was a modified version of our previously published protocol.[17] On day 14 after immunization, EAE mice were sacrificed via overdose with sodium pentobarbital (150 mg/kg, Sinopharm Chemical Reagent Co., Beijing, China). T cells were then isolated from the spleen of mice via passage through a nylon wool column (Kisker, Steinfurt, Germany). Thereafter, 1 × 107 cells in 2 mL RPMI 1640 medium per well in a six-well plate (Costar; Corning, Corning, NY, USA) were stimulated with 20 μg/mL MOG35–55 in the presence of 1 × 107 mitomycin C (MCE, Monmouth Junction, NJ, USA)-treated syngeneic spleen cells as antigen-presenting cells (APCs). After 2 days, the activated lymphoblasts were isolated via density gradient centrifugation (Lymphocyte Separation, Tianjin, China) and cultured in RPMI 1640 medium containing IL-2 (USCN Co., Wuhan, Hubei, China, 10 ng/mL).

Proliferation assay

Based on a modified version of our previously published protocol,[17] CD8 or CD4 enriched T-cells from MOG35–55-immunized B6 mice were prepared and seeded at 4 × 105 cells/well in 96-well plates. The cells were cultured at 37°C for 48 h in 200 μL medium with or without MOG35–55 in the presence of mitomycin C-treated syngeneic spleen APCs (1 × 105 cells/well). [3H] thymidine incorporation during the last 8 h was assessed using a microplate scintillation counter (Packard; PerkinElmer, Meriden, CT, USA). The proliferative response is expressed as the mean counts per minute (cpm) ± standard deviation (SD) of triplicate measurements.

Purification of CD4+ and CD8+ T-cells

According to a modified version of our previously published protocol,[17] purified CD4+ and CD8+ T-cells were prepared from spleens using CD4 and CD8 isolation kits (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Briefly, the spleen cells were incubated in 10 μL of CD8 (TIL) MicroBeads and 90 μL buffer per 1 × 10⁷ cells for 15 min in a refrigerator (2°C − 8°C). Buffer was added to a final volume of 500 μL for up to 5 × 10⁷ cells. The cells were then separated into bound and unbound cells on a magnetic separator column (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and washed with 15 mL medium, according to the manufacturer's protocol. The flow-through fraction containing CD4- or CD8-enriched cells was collected, and the purity of the isolated cell fraction was determined by flow cytometry.

Flow cytometry

Based on a modified version of our previously published protocol,[17] aliquots of 2 × 105 cells were double stained with combinations of APC-, FITC-, or PE-conjugated monoclonal antibodies against mouse CD3, CD4, or CD8 (Biolegend, San Diego, CA, USA). Data collection and analyses were performed using the flow cytometer, Dxp Athena (Cytek Biosciences Inc., Fremont, CA, USA). The data were analyzed using FlowJo software (FlowJo Co., San Diego, CA, USA).

Enzyme-linked immunosorbent assays

According to a modified version of our previously published protocol,[17] 1 × 106 cells in 1 mL RPMI 1640 medium per well in a 24-well plate (Costar; Corning, Corning, NY, USA) were stimulated with 20 μg/mL MOG35–55 in the presence of mitomycin C-treated syngeneic spleen cells as APCs. After 24 h, the secretion of IFN-gamma, interleukin (IL)-4, and IL-10 from the supernatant of cultured CD4+ and CD8+ T-cells was measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits (USCN Co., Wuhan, Hubei, China).

Histology

Based on a modified version of our previously published protocol,[17] spinal cord sections were prepared from the tissues collected from aEAE animals or tEAE animals that were adoptively transferred MOG35–55-specific CD8 or CD4 enriched T-cells obtained from aEAE animals. Spinal cord tissues were fixed in ice-cold 4% paraformaldehyde/methanol and embedded in paraffin before the preparation of microtome sections (5 μm thick) for staining with hematoxylin and eosin (HE), and Luxol fast blue (LFB) for myelin staining.

Statistical analyses

Statistical analysis was carried out using the SPSS 21.0 statistical software (IBM, Almonk, NY, USA). All the data were analyzed using the one-way analysis of variance, followed by the least significant difference test for multiple comparisons. Data are expressed as mean ± SD, including data from the proliferation assay, ELISA, EAE score, and flow cytometry. Each experiment was repeated at least 3 times. A P valued 0.05 was considered to indicate significance.


  Results Top


Remarkable improvement in the experimental autoimmune encephalomyelitis clinical signs in the high dose Huangqi Guizhi Wuwu decoction group

To determine the role of HQGZWW in the pathogenesis of EAE, we randomly divided EAE mice into four groups: distilled water, high dose HQGZWW, low dose HQGZWW, and prednisone. Thereafter, we measured the clinical signs of mice based on the EAE score obtained through daily checks and histopathology.[3] As shown in [Figure 1]a, from day 0 to day 21 after MOG35Ger peptide immunization, we measured the EAE scores. As a result, we found significant improvements in the EAE score in both the high dose HQGZWW and prednisone groups (2.52 ± 0.09 and 2.46 ± 0.04, respectively, P = 0.001) compared to the distilled water group (3.98 ± 0.04); however, only a slight improvement was observed in the low dose HQGZWW group (3.35 ± 0.09, P = 0.616). Compared to the distilled water group, a greater improvement was observed in the high dose HQGZWW group and prednisone group (P = 0.569); however, there was no significant improvement in EAE score in the low dose HQGZWW group (P = 0.001). Based on the pathological examination of the spinal cord, compared to naïve mice [HE, [Figure 1]b and LFB, [Figure 1]g], severe inflammation and demyelination were observed in mice administered distilled water [HE, [Figure 1]c and LFB, [Figure 1]h] or low dose HQGZWW [HE, [Figure 1]e and LFB, [Figure 1]j]. Interestingly, slight inflammation and demyelination were observed in mice administered high dose HQGZWW (HE, [Figure 1]d and LFB, [Figure 1]i) or prednisone [HE, [Figure 1]f and LFB, [Figure 1]k].
Figure 1: Great improvement of EAE clinical course in HQGZWW high dose and prednisone groups. AEAE mice which were induced by an emulsion containing 200 μg MOG35-55 and CFA plus pertussis toxin, were treated with four groups: distilled water, HQGZWW high dose, HQGZWW low dose or prednisone by oral gavages started on 1 hour after immunization once a day for 14 days. EAE was scored on a 0–5 scale, MOG35-55-induced B6 EAE mice, and measurement of clinical signs of EAE score was assessed by daily check (1a) and histopathology (Spinal cord, HE&LFB). Naïve mice (1b,1g), distilled water group (1c,1h), HQGZWW high dose group (1d,1i), HQGZWW low dose group (1e,1j) or prednisone group (1f,1k). **: P < 0.01, (HQGZWW high dose and prednisone groups vs distilled water group); myelin oligodendrocyte glycoprotein (MOG); antigen presenting cell (APC); standard deviation (SD); Figure 1B, 1D, 1F&1H (HE, 50X); Figure 1C, 1E, 1G&1I (LFB, 50X); aEAE: Active-induced EAE Models; myelin oligodendrocyte glycoprotein (MOG); standard deviation (SD); hematoxylin and eosin (HE); Luxol fast blue (LFB), Huangqi Guizhi Wuwu Decoction (HQGZWW).

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Altogether, these findings suggest that the aEAE stage was evidently inhibited in the high dose HQGZWW and prednisone groups, as expected. To identify the precise mechanism for the marked improvement of the EAE clinical signs in the high dose HQGZWW group, we opted to focus on the role of the MOG35–55 specificity of purified CD8+ and CD4+ T cells on EAE.

Myelin oligo-dendrocyte glycoprotein35–55 specificity of purified CD8+ and CD4+ T-cells

Unfractionated T-cells were isolated using a CD8 or CD4 kit. As shown in [Figure 2], approximately 90% CD8+ CD3+ cells were found in the fraction of T-cells in the four groups after CD8 bead enrichment and approximately 90% CD4+ CD3+ cells were found in the fraction of T-cells after CD4 bead enrichment in the four treatment groups. These CD8+ CD3+ and CD4+ CD3+ cells were highly pure and appropriate for the subsequent functional experiments.
Figure 2: Lowest percentage of CD8+ T-cells and lowest CD8/CD4 ratio in total CD3+ cells of unfractionated T-cells in distilled water group. The phonotype and CD8/CD4 ratio in four groups were studied. T-cells were isolated from spleen cells of active-induced experimental autoimmune encephalomyelitis models mice of 4 groups by passing through a nylon wool column, then purified by CD4 and CD8 isolation kits, finally the purity of the isolated cell fraction was determined by flow cytometric analysis with APC-, FITC- or PE-conjugated anti-CD3, anti-CD4 and anti-CD8 antibodies. Four groups indicated as distilled water [after nylon wood (2a), after CD8 beads (2b), or after CD4 beads (2c)]; HQGZWW high dose [after nylon wood (2d), after CD8 beads (2e), or after CD4 beads (2f)]; HQGZWW low dose [after nylon wood (2g), after CD8 beads (2h), or after CD4 beads (2i)]; Predinose [after nylon wood (2j), after CD8 beads (2k), or after CD4 beads (2l)]; **: P < 0.01, (Huangqi Guizhi Wuwu decoction high dose and prednisone and Huangqi Guizhi Wuwu decoction low dose groups vs distilled water group); SSC: Side scatter, FSC: Forward scatter, aEAE: Active-induced experimental autoimmune encephalomyelitis Models

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The purified CD8+ and CD4+ T-cells were examined to determine their antigen-specific functions via a proliferation assay. The representative results shown in [Figure 3] indicate that the MOG35–55 peptide had a strong stimulatory effect on both CD8+ and CD4+ T-cells. Although the purified CD8+ T-cells had a generally lower response to MOG35–55 than CD4+ T-cells, the response of CD8+ T-cells did not always depend CD4+ T-cells. In fact, up to the highest MOG35–55 concentration (20 μg/mL shown in [Figure 3]a, lower antigen-specific responses of CD8+ T-cells were observed in the high dose HQGZWW and prednisone groups (10510.67 ± 1189.87 cpm and 9575.33 ± 1064.00 cpm, respectively, P = 0.001) compared to the distilled water group (21711.33 ± 1589.42 cpm). However, this was not observed in the low dose HQGZWW group (19665.33 ± 1553.61 cpm, P = 0.104). Antigen-specific responses of CD8+ T-cells were observed in the high dose HQGZWW group compared to the low dose HQGZWW low dose group (P = 0.001). Further, this observation in the high dose group was similar to that found in the prednisone group (P = 0.427).
Figure 3: MOG35–55 specificity of purified CD8+ and CD4+T Cells. To compare of the response of the purified CD4 and CD8 T cell populations to MOG35–55, CD8 or CD4 enriched T cells from MOG35–55-immunized wild-type B6 mice of 4 groups were prepared and seeded at 4 × 105 cells/well in 96-well plates and cultured at 37°C for 48 h in a total volume of 200 μL of medium, with or without MOG35–55, in the presence of mytomycin C-treated syngeneic spleen APCs (1 × 105), and [3H] thymidine incorporation during the last 8 h was assessed. The proliferative response is expressed as the mean counts per minute ± SD of triplicate determinations (3a: cpm of CD8+ T Cells; 3b: cpm of CD4+ T Cells;). **: P < 0.01, (HQGZWW high dose and prednisone groups vs distilled water group); myelin oligodendrocyte glycoprotein (MOG)

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As shown in [Figure 3]b, a similar pattern of CD4+ T-cell proliferation was observed among the treatment groups. Compared to the distilled water group (64557.67 ± 2547.57 cpm), a significant decrease in T-cell proliferation was observed in the high dose HQGZWW group, prednisone group, and low dose HQGZWW group (21281.67 ± 1739.08 cpm, 20104.00 ± 1364.82 cpm, and 50799.33 ± 2449.17 cpm, respectively, P = 0.001). Further, antigen-specific responses of CD8+ T-cells were observed in the high-dose HQGZWW group compared to the low-dose HQGZWW group (P = 0.001); these responses in the high dose group were similar to those found in the prednisone group (P = 0.509). Such findings suggest that the antigen response of CD8+ or CD4+ T-cells in the four groups was consistent with the EAE score and histology results presented in [Figure 1].

Evidently, the proliferation of CD8+ T-cells, which were stimulated by the induced antigen, was inhibited in the high dose HQGZWW and prednisone groups. To determine whether the pathogenic function of CD8+ T-cells was reduced, cytokine profile and adoptive transfer experiments were carried out.

Cytokine profiles of myelin oligo-dendrocyte glycoprotein35–55-specific CD8+ T and CD4+ T-cells

To determine the cytokine profiles of activated CD4+ and CD8+ autoreactive T-cells, ELISAs were carried out to measure the cytokine levels in the culture supernatants of activated CD4+ and CD8+ T-cells at 24 h poststimulation. As shown in [Figure 4]a, compared to the distilled water group (816.31 ± 42.88 pg/mL), a markedly lower IFN-γ secretion from the CD8+ T-cell supernatant was observed in the high dose HQGZWW and prednisone groups (531.66 ± 25.54 pg/mL and 484 ± 36.52 pg/mL, respectively, P = 0.001), and the low dose HQGZWW group (718.33 ± 29.81 pg/mL, P = 0.008). Furthermore, less IFN-γ secretion from CD8+ T-cells was observed in the high dose HQGZWW group relative to the low dose HQGZWW group (P = 0.001); the secretion level in the high dose group was similar to that found in the prednisone group (P = 0.127).
Figure 4: Cytokine profiles of Myelin oligodendrocyte glycoprotein35–55-specific CD8+ T and CD4+ T-Cells. Cytokine levels in the supernatants of activated CD4+ and CD+ 8 T-cells at 24–48 h poststimulation in vitro are measured by enzyme-linked immunosorbent assay. Stimulation means that CD4 enriched T-cells from Myelin oligodendrocyte glycoprotein35–55-immunized wild-type B6 mice were prepared and seeded at 8 × 105 cells/well in 24-well plates and cultured at 37°C for 24–48 h in a total volume of 500 μL of medium, with or without Myelin oligodendrocyte glycoprotein35–55, in the presence of mytomycin C-treated syngeneic spleen APCs (2 × 105). Cytokines indicated as IFN-γ (CD8+ T Cells: 4a, CD4+ T Cells: 4d), IL-4 (CD8+ T Cells: 4b, CD4+ T Cells:4e), IL-10 (CD8+ T Cells: 4c, CD4+ T Cells: 4f). The data are the mean ± standard deviation from three separate experiments. **: P < 0.01, (Huangqi Guizhi Wuwu decoction high dose and prednisone groups vs. distilled water group); MOG: Myelin oligodendrocyte glycoprotein, APC: Antigen presenting cell; SD: Standard deviation, IFN-γ: Interferon-gamma, IL-4: Interleukin-4, IL-10: Interleukin-10

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As shown in [Figure 4]d, compared to the distilled water group (1474.07 ± 90.43 pg/mL), a markedly lower IFN-γ secretion from the CD4+ T cell supernatant was observed in the high dose HQGZWW and prednisone groups (722.66 ± 64.50 pg/mL and 646 ± 46mL6 pg/mL, respectively, P = 0.001); this secretion level was also lower in the HQGZWW low dose group (1323.12 ± 79.50 pg/mL, P = 0.04). Further, less IFN-γ secretion from CD8+ T cells was observed in the high dose HQGZWW group relative to the low dose HQGZWW group (P = 0.001); the level in the high dose group was similar to that found in the prednisone group (P = 0.249).

As shown in [Figure 4]b, compared to the distilled water group (220.66 ± 56.51 pg/mL), a markedly higher IL-4 secretion from the CD8+ T cell supernatant was observed in the high dose HQGZWW and prednisone groups (762.33 ± 78.21 pg/mL and 954.06 ± 75.07 pg/mL, respectively, P = 0.001); however, this was not observed in the low dose HQGZWW group (293 ± 49.03 pg/mL, P = 0.215). Further, more IL-4 secretion from CD8+ T cells in the high dose HQGZWW group than the HQGZWW low dose group (P = 0.001) was observed; the secretion level in the high dose group was similar to that in the prednisone group (P = 0.07).

As shown in [Figure 4]e, compared to the distilled water group (324.33 ± 27.50 pg/mL), there was a markedly higher level of IL-4 secretion from the CD4+ T cell supernatant in the high dose HQGZWW and prednisone groups (620.33 ± 38.07 pg/mL and 707.66 ± 35.59 pg/mL, respectively, P = 0.001); however, this was not observed in the low dose HQGZWW group (355.13 ± 35.59 pg/mL, P = 0.349). Further, more IL-4 secretion from CD4+ T cells was observed in the high dose HQGZWW group relative to the low dose HQGZWW group (P = 0.001); the level in the high dose group was similar to that found in the prednisone group (P = 0.221).

Another important Th2 cytokine, IL-10, was detected in the present study. As shown in [Figure 4]c, compared to the distilled water group (830.33 ± 68.24 pg/mL), a markedly higher IL-10 secretion from the CD8+ T-cell supernatant was observed in the high dose HQGZWW and prednisone groups (2048.66 ± 2048.6 pg/mL and 2250.01 ± 67.55 pg/mL, respectively, P = 0.001); however, this was not reflected in the low dose HQGZWW group (1056.13 ± 114.89 pg/mL, P = 0.237). Moreover, we observed a higher IL-10 secretion from CD8+ T cells in the high dose HQGZWW group than the low dose HQGZWW group (P = 0.001); the secretion level in the high group was similar to that observed in the prednisone group (P = 0.12).

As shown in [Figure 4]f, compared to the distilled water group (853.66 ± 48.26 pg/mL), a markedly higher IL-10 secretion from the CD4+ T cell supernatant was observed in the high dose HQGZWW and prednisone groups (2242.33 ± 156.35 pg/mL and 2380.21 ± 131.73 pg/mL, respectively, P = 0.001); however, this was not observed in the low dose HQGZWW group (1055.33 ± 66.01 pg/mL, P = 0.247). Further, more IL-4 secretion from CD4+ T cells was observed in the high dose HQGZWW group relative to the low dose HQGZWW group (P = 0.001); the level in the high dose group was similar to that found in the prednisone group (P = 0.271).

In summary, the CD4+ and CD8+ T-cell supernatants were found to contain lower levels of IFN-γ and higher levels of IL-4 and IL-10 in the high dose HQGZWW and prednisone groups compared to the low dose HQGZWW and distilled water groups. Based on our data, a Th2 cytokine profile of CD4+ and CD8+ T cells was observed in the high dose HQGZWW and prednisone groups, thereby aligning with the results of EAE score, histological analysis presented in [Figure 1], and the proliferation assay displayed in [Figure 3]. In fact, as shown in [Figure 4], compared to the CD4+ T-cells, there were lower levels of IFN-γ and similar levels of IL-4 and IL-10 secretion in the CD8+ autoreactive T-cells among the four groups. Such finding is consistent with those of our previous EAE and experimental autoimmune uveitis (EAU) studies.[3]

Adoptive-transferred experimental autoimmune encephalomyelitis by adoptive transfer of myelin oligo-dendrocyte glycoprotein35–55 -specific CD8+ T-cells to naïve mice

To determine the role of MOG35–55-specific CD8+ T-cells in the pathogenesis of EAE, EAE was induced in wild-type B6 naïve mice through adoptive transfer of MOG35–55-specific T-cells from B6 aEAE mice administered four different treatments (distilled water, HQGZWW high dose, HQGZWW low dose, or prednisone). Thereafter, we measured the clinical signs based on the EAE score collected through daily checks and histopathology.[17]

As shown in [Figure 5]a, from day 0 to day 21 after MOG35Ger adoptive transfer, the EAE score was measured. Compared to the distilled water group (2.95 ± 0.09), there was a significant improvement in the EAE score in both the high dose HQGZWW and prednisone groups (2.10 ± 0.10 and 2.03 ± 0.04, respectively, P = 0.001), with only slight improvement in the low dose HQGZWW group (2.91 ± 0.07, P = 0.742). Further, high dose HQGZWW was found to have a markedly better effect on EAE mice than low dose HQGZWW (P = 0.001); the effect of the high dose was similar to that of prednisone (P = 0.631).
Figure 5: Adoptive transfer of Myelin oligodendrocyte glycoprotein35–55 -specific CD8+ T-Cells to naïve mice is able to induce tEAE. Attempts were made to induce disease in wild-type B6 naïve mice by adoptive transfer of Myelin oligodendrocyte glycoprotein35–55-specific CD8+ T-Cells from B6 aEAE mice of four groups upon four treatments (distilled water, Huangqi Guizhi Wuwu decoction high dose, Huangqi Guizhi Wuwu decoction low dose or prednisone), and measurement of clinical signs of experimental autoimmune encephalomyelitis score was assessed by daily check (a) and histopathology (Spinal cord, HE and LFB, ×50). Distilled water group (b and f), Huangqi Guizhi Wuwu decoction high dose group (c and g), Huangqi Guizhi Wuwu decoction low dose group (d and h) or prednisone group (e and i). The data are the mean ± SD from three separate experiments. aEAE: Activated experimental autoimmune encephalomyelitis, H and E: Hematoxylin and eosin, LFB: Luxol fast blue, MOG: Myelin oligodendrocyte glycoprotein, SD: Standard deviation, LFB: Luxol fast blue, HQGZWW: Huangqi Guizhi Wuwu Decoction

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Moderate inflammation and demyelination were observed in the distilled water group [HE, [Figure 5]b and LFB, [Figure 5]f] and the low dose HQGZWW group [HE, [Figure 5]d and LFB, [Figure 5]h] while slight inflammation and demyelination were observed in the high dose HQGZWW group [HE, [Figure 5]c and LFB, [Figure 5]g] and the prednisone group [HE, [Figure 5]e and LFB, [Figure 5]i]. These findings suggest that high dose HQGZWW had an effect that was similar to the typical conventional medicine, prednisone, on tEAE; however, no effect was observed with the low dose.


  Discussion Top


MS is a CNS disease characterized by inflammation, demyelination, and axonal damage. MS/EAE has been considered as an antigen-specific, CD4+ T helper (Th) cell-dominated disease for decades.[18] Cytokines play a critical role in the pathogenesis of MS/EAE. Further, treatments, such as Glatiramer acetate (Copaxone), IFN-γ and cyclophosphamide, have been demonstrated to alter the cytokine profiles.[3] There are two major cytokines secreted by Th cells: Th1-cytokines, such as IFN-γ, IL-2, and tumor necrosis factor-α (TNF-α); and Th2-cytokines, such as IL-4, IL-5, IL-10, and IL-13.[19]

Recently, there has been increasing evidence of the involvement of CD8+ T-cells in the pathogenesis of MS/EAE.[3] For example, CD8+ T-cells are present in the MS tissue at all stages of the disease and are characterized by their antigenic repertoire in MS and EAE.[3] The cytokine profiles produced by CD8+ T-cells involved in MS/EAE were previously examined.[20] Similar to their CD4 counterparts, pro-inflammatory cytokines include IFN-γ, IL-2, and TNF-α, as well as IL-17[21],[22] while immunosuppressive cytokines include IL-4 and IL-10.[3] For example, the IFN-γ and TNF-α secretion of auto-reactive CD8+ T-cells was detected in the peripheral blood through a response to their cognate antigen ex vivo.[23] Apoptotic T-cell-associated self-epitopes increased IFN-γ- and IL-17-producing CD8+ T-cells in the CNS.[24] However, in virus-induced encephalitis models, IL-10-producing CD8+ T-cells increased the expression of pro-inflammatory cytokines and chemokines, thereby resulting in reduced disease pathology.[25] Similar to our previous study, CD8+ autoreactive T-cells in EAE were found to have a lower encephalitogenic function but were identified to be unique and act independently on the pathogenicity of EAE instead of their CD4+ counterparts. Such finding might explain the generally lower response, less IFN-γ and IL-4 secretion from MOG35–55 caused by CD8+ T-cells relative to CD4+ T cells.[17] Our current data are consistent with our previous data with the EAU model and the PLP56–70-specific CD4+ autoreactive T cells of EAE in Biozi AB/H mice.[26],[27]

The aim of the current study was to determine the precise mechanism of HQGZWW in MS. Briefly, we used the strategy employed by a previous study to assess the pathological interrelationship between CD4 and CD8 autoreactive T-cells during different treatments.[17] As shown in [Figure 4], the Th2 cytokine profiles of CD4+ and CD8+ T-cells were observed in the high dose HQGZWW and prednisone groups, but not in the low dose HQGZWW and distilled water groups. Such findings align with the results of the EAE score, histological analysis presented in [Figure 1], the proliferation assay presented in [Figure 3], and the adoptive transfer displayed in [Figure 4].

Some reports have described the effects of HQGZWW on EAE models that support our current data. In fact, HQGZWW was demonstrated to increase the level of IL-35 in the peripheral blood of EAE rats.[28] Further, A. mongholicus, the major herb of HQGZWW, was demonstrated to have an effect on MOG35-55 T-cells in the EAE C57BL/6 mice model, ultimately reducing EAE score, inhibiting CNS inflammation and MOG35-55 T-cell proliferation, reducing IFN-γ IFN-γ, TNF-α and IL-17 secretion, and enhancing IL-4 secretion.[29] Recently, the major extraction of A. mongholicus, Huangqi glycoprotein (HQGP) treatment, was found to delay the onset of EAE; attenuate the clinical symptoms and inhibit CD68+ macrophage infiltration into the CNS; and downregulate the secretion of nitric oxide, TNF-αN and IL-6; and increase the secretion of IFN-γ. In addition, HQGP treatment effectively increased the numbers of CD4+ CD25+ T-cells, CD4+ IL-10+ T-cells, and CD4+ IFN-γ+ T-cells.[3] Another study by the same group revealed that HQGP effectively alleviated the EAE score, delayed the onset time, and decreased the numbers of CD4+ T-cells, CD68+ macrophages, and CD11b+ cells. Further studies demonstrated that HQGP could inhibit the secretion of IL-17, TNF-α, and IL-12 and promote the production of IL-4, IL-10, and IFN-γ.[30] The administration of HQGZWW, its major herb, and the extraction of its major herb, on CD4+ T-cells, macrophages, and dendritic cells of the EAE model was slightly investigated; however, the impact of treating CD8+ T-cells of MS/EAE with HQGZWW has never been assessed.


  Conclusion Top


Based on the findings of the present study, a high dosage of HQGZWW had effects that were similar to those of the typical conventional medicine, prednisone, on EAE; however, no effects were observed when a low dosage was administered. Such findings suggest that the CD8+ T-cells play a protective role in EAE when treated with HQGZWW by enhancing the Th2 cytokine profile. Moreover, several other mechanisms, such as CD8+ Treg cells which have been extensively studied recently,[3] may exist. Altogether, these results pave the way for our future investigation on the mechanism employed by HQGZWW to protect against MS/EAE.

Acknowledgments

The authors would like to thank for Dr. Huang Yang from Xiangya Hospital, Central South University, China giving us the critical comments on current study.

Financial support and sponsorship

This work was supported by Key Plans of Hunan Administration Traditional Chinese Medicine (No. 201915 to YP), the grants from the Natural Science Foundation of Hunan Province, China (No. 2018JJ6043 to YP), and the Health and Family Plans commission of Hunan Province, China (No. B20180815 to YP).

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Thompson AJ, Banwell BL, Barkhof F, Carroll WM, Coetzee T, Comi G, et al. Diagnosis of multiple sclerosis: 2017 revisions of the McDonald criteria. Lancet Neurol 2018;17:162-73.  Back to cited text no. 1
    
2.
Doshi A, Chataway J. Multiple sclerosis, a treatable disease. Clin Med 2016;16 Suppl 6:s53-9.  Back to cited text no. 2
    
3.
Reich DS, Lucchinetti CF, Calabresi PA. Multiple sclerosis. N Eng J Med 2018;378:169-80.  Back to cited text no. 3
    
4.
Wollebo HS, White MK, Gordon J, Berger JR, Khalili K. Persistence and pathogenesis of the neurotropic polyomavirus JC. Ann Neurol 2015;77:560-70.  Back to cited text no. 4
    
5.
Valenzuela RM, Pula JH, Garwacki D, Cotter J, Kattah JC. Cryptococcal meningitis in a multiple sclerosis patient taking natalizumab. J Neurol Sci 2014;340:109-11.  Back to cited text no. 5
    
6.
Song L, Zhou QH, Wang HL, Liao FJ, Hua L, Zhang HF, et al. Chinese herbal medicine adjunct therapy in patients with acute relapse of multiple sclerosis: A systematic review and meta-analysis. Complement Ther Med 2017;31:71-81.  Back to cited text no. 6
    
7.
Wang Y, Lin XM, Zheng GQ. Traditional Chinese medicine for Parkinson's disease in China and beyond. J Altern Complement Med 2011;17:385-8.  Back to cited text no. 7
    
8.
Wang Xiaoqing TQ, Yanhua L. Effect of Buyang Huanwu decoction on immune Regulation of EAE mice. Chin J Immunol 2017;1:52-5.  Back to cited text no. 8
    
9.
Tian Q, Li Y, Yu J. Immune regulation of Buyang Huanwu decoction on mononuclear macrophages in EAE. Chin J Pathophysiol 2017;33:200-7.  Back to cited text no. 9
    
10.
Huang XP, Ding H, Wang B, Qiu YY, Tang YH, Zeng R, et al. Effects of the main active components combinations of Astragalus and Panax notoginseng on energy metabolism in brain tissues after cerebral ischemia-reperfusion in mice. Pharmacogn Mag 2015;11:732-9.  Back to cited text no. 10
    
11.
Yuan CX, Chu T, Liu L, Li HW, Wang YJ, Guo AC, et al. Catalpol induces oligodendrocyte precursor cell-mediated remyelination in vitro. Am J Transl Res 2015;7:2474-81.  Back to cited text no. 11
    
12.
Yang T, Zheng Q, Wang S, Fang L, Liu L, Zhao H, et al. Effect of catalpol on remyelination through experimental autoimmune encephalomyelitis acting to promote Olig1 and Olig2 expressions in mice. BMC Complement Altern Med 2017;17:240.  Back to cited text no. 12
    
13.
Choi JH, Lee MJ, Jang M, Kim EJ, Shim I, Kim HJ, et al. An oriental medicine, hyungbangpaedok-san attenuates motor paralysis in an experimental model of multiple sclerosis by regulating the T Cell response. PloS one 2015;10:E0138592.  Back to cited text no. 13
    
14.
Nie WP, Zhao L, Deng CQ. The role of Huangqiguizhiwuwu Decoction on autoimmune disease. J Hunan Chin Med 2019;35:171-3.  Back to cited text no. 14
    
15.
Turobov VI, Danilkovich AV, Shevelev AB, Biryukova YK, Pozdniakova NV, Azev VN, et al. Efficacy of Synthetic Peptide Corresponding to the ACTH-Like Sequence of Human Immunoglobulin G1 in Experimental Autoimmune Encephalomyelitis. Front Pharmacol 2018;9:113.  Back to cited text no. 15
    
16.
Wagner CA, Roqué PJ, Mileur TR, Liggitt D, Goverman JM. Myelin-specific CD8+ T cells exacerbate brain inflammation in CNS autoimmunity. J Clin Invest 2020;130:203-13.  Back to cited text no. 16
    
17.
Peng Y, Zhu FZ, Chen ZX, Zhou JX, Gan L, Yang SS, et al. Characterization of myelin oligodendrocyte glycoprotein (MOG) 35-55-specific CD8+ T cells in experimental autoimmune encephalomyelitis. Chin Med J (Engl) 2019;132:2934-40.  Back to cited text no. 17
    
18.
Zhang J, Markovic-Plese S, Lacet B, Raus J, Weiner HL, Hafler DA. Increased frequency of interleukin 2-responsive T cells specific for myelin basic protein and proteolipid protein in peripheral blood and cerebrospinal fluid of patients with multiple sclerosis. J Exp Med 1994;179:973-84.  Back to cited text no. 18
    
19.
Chitnis T, Khoury SJ. Cytokine shifts and tolerance in experimental autoimmune encephalomyelitis. Immunol Res 2003;28:223-39.  Back to cited text no. 19
    
20.
Sinha S, Boyden AW, Itani FR, Crawford MP, Karandikar NJ. CD8(+) T-Cells as Immune Regulators of Multiple Sclerosis. Front Immunol 2015;6:619.  Back to cited text no. 20
    
21.
Sasaki K, Bean A, Shah S, Schutten E, Huseby PG, Peters B, et al. Relapsing-remitting central nervous system autoimmunity mediated by GFAP-specific CD8 T cells. J Immunol 2014;192:3029-42.  Back to cited text no. 21
    
22.
Ford ML, Evavold BD. Modulation of MOG 37-50-specific CD8+T cell activation and expansion by CD43. Cell Immunol 2006;240:53-61.  Back to cited text no. 22
    
23.
Crawford MP, Yan SX, Ortega SB, Mehta RS, Hewitt RE, Price DA, et al. High prevalence of autoreactive, neuroantigen-specific CD8+ T cells in multiple sclerosis revealed by novel flow cytometric assay. Blood 2004;103:4222-31.  Back to cited text no. 23
    
24.
Lolli FM, Martini H, Citro A, Franceschini D, Portaccio E, Amato MP, et al. Increased CD8+ T cell responses to apoptotic T cell-associated antigens in multiple sclerosis. J Neuroinflammation 2013;10:94.  Back to cited text no. 24
    
25.
Trandem K, Zhao J, Fleming E, Perlman S. Highly activated cytotoxic CD8 T cells express protective IL-10 at the peak of coronavirus-induced encephalitis. J Immunol 2011;186:3642-52.  Back to cited text no. 25
    
26.
Peng Y, Shao H, Ke Y, Zhang P, Xiang J, Kaplan HJ, et al. In vitro activation of CD8 interphotoreceptor retinoid-binding protein-specific T cells requires not only antigenic stimulation but also exogenous growth factors. J Immunol 2006;176:5006-14.  Back to cited text no. 26
    
27.
Peng Y, Liu CP. Characterization of proteolipid protein-peptide-specific CD (4)(+) T cell of experimental allergic encephalomyelitis in Biozzi AB/H mice. Chin Med J (Engl) 2002;115:521-4.  Back to cited text no. 27
    
28.
Huang Sujie LX, Zequan Z. Effect of Huangqi Guizhi Wuwu decoction on immune inflammatory factors IL-33 and IL-35 in EAE mice. Hebei Chengde, Specialized Committee of Chinese Society for the combination of traditional. Chin West Med 2015:226-30.  Back to cited text no. 28
    
29.
Sun Y, Wang S, Huang M, Jing Y, Xie K, Cheng X. Therapeutic effect and immunomodulatory mechanism of radix astragali seu hedysari on experimental autoimmune encephalomyelitis mouse. ACTA Univ Tradit Med Sinensts Pharmocol Shanghai 2017;27:58-62.  Back to cited text no. 29
    
30.
Xing YX, Liu BY, Zhao YJ, Zhang LH, Rodolfo Thome, Xue HQ, et al. Immunomodulatory and neuroprotective mechanisms of Huangqi glycoprotein treatment in experimental autoimmune encephalomyelitis. Folia Neuropathol. 2019;57:117-28. doi: 10.5114/fn.2019.85843.  Back to cited text no. 30
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]



 

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