Sciatic-Vagal Nerve Stimulation by Electroacupuncture Alleviates Inflammatory Arthritis in Lyme Disease-Susceptible C3H Mice

Abstract

Lyme disease is caused by Borrelia burgdorferi, and the pathogenesis of the disease is complex with both bacterial and host factors contributing to inflammatory responses. Lyme disease affects different organs including joints and results in arthritis. Immune responses stimulated by B. burgdorferi through toll-like receptors cause infiltration of leukocytes, which produce inflammatory cytokines and facilitate spirochete clearance. However, arthritic manifestations and chronic fatigue syndrome-like symptoms persist long after completion of antibiotic treatment regimens in a significant number of patients. To counter the effects of inflammation, treatment by non-steroidal anti-inflammatory drugs, hydroxychloroquine, or synovectomy to eradicate inflammatory arthritis in the involved joint could be employed; however, they often have long-term consequences. Acupuncture has been used for a long time in Asian medicine to diminish pain during various ailments, but the effects and its mechanism are just beginning to be explored. Control of inflammation by neuronal stimulation has been exploited as a systemic therapeutic intervention to arrest inflammatory processes. Our objective was to determine whether activation of the sciatic-vagal network by electroacupuncture on ST36 acupoint, which is used to control systemic inflammation in experimental models of infectious disorders such as endotoxemia, can also alleviate Lyme arthritis symptoms in mice. This aim was further strengthened by the reports that sciatic-vagal neuronal network stimulation can lead to dopamine production in the adrenal medulla and moderate the production of inflammatory factors. We first assessed whether electroacupuncture affects spirochete colonization to attenuate Lyme arthritis. Interestingly, bioluminescent B. burgdorferi burden detected by live imaging and qPCR were similar in electroacupuncture- and mock-treated mice, while electroacupuncture induced a lasting anti-inflammatory effect on mice. Despite the discontinuation of treatment at 2 weeks, the simultaneous decrease in neutrophils in the joints and inflammatory cytokine levels throughout the body at 4 weeks suggests a systemic and persistent effect of electroacupuncture that attenuates Lyme arthritis. Our results suggest that electroacupuncture-mediated anti-inflammatory responses could offer promising healthcare benefits in patients suffering from long-term Lyme disease manifestations.

Keywords: Borrelia burgdorferi; Lyme arthritis; electroacupuncture; inflammation; sciatic nerve; vagus nerve.

Figure 1

Colonization of organs of 4-week-old C3H/HeJ mice by Borrelia burgdorferi N40 strain did not change after EA treatment. (A, B) Real-time images of a representative female C3H/HeJ infected mouse (10 mice for each treatment) using IVIS-200 displayed bioluminescence as a semi-quantitative indicator of B. burgdorferi colonization in different organs of mice at 2 (A) and 4 weeks (B) post-infection. Spirochete burden was comparable in mock- and EA-treated mice. (C) Overall, light emission in representative male C3H/HeJ mouse, representing colonization of organs and spirochetal burden at 2 weeks post-infection, was not different from that observed in female mice (A) and was comparable in mock- and EA-treated mice. (D) Determination of burden of B. burgdorferi by real-time qPCR confirmed results obtained by live imaging (C), such that mock- and EA-treated mice showed similar levels of heart and joint colonization of 5 mice each. Bars indicate median values. (E) Increasing infection dose to 10⁵ B. burgdorferi/mouse did not appreciably increase over colonization levels of organs in mice infected by 10⁴ spirochetes/mouse. (F) SCID mice showed higher colonization of organs by B. burgdorferi (higher bioluminescence radiance) than immunocompetent mice (A–E); however, spirochete burden was comparable in mock- versus EA-treated mice. EA, electroacupuncture; SCID, severe combined immunodeficient.

Figure 2

Attenuation of joint inflammation and arthritis in EA-treated mice. (A, B) Mediolateral and not craniocaudal measurements of tibiotarsus joints of 10 mice showed a significant reduction in swelling after EA treatment. Bars indicate median values. (C–F) Severe arthritis in tibiotarsus joint manifested by an increase in synovial space (arrow 1), higher leukocyte infiltration (blue stain, arrow 2), and synovial hyperplasia and erosion of cartilage (arrow 3) in composite tibiotarsus image (C) were observed in mock-treated Borrelia burgdorferi-infected mice. Infiltration of leukocytes as depicted by blue-stained dots reduced in EA-treated mice consistently. Bar in panel F represents a size of 100 µm. EA, electroacupuncture.

Figure 3

Fluorescence immunohistopathological examination of male mice joints to determine the effect of EA on CD4+ Th17 (CCR6 staining), Th1 (CxCR3 staining), and Tregs (CD39 staining) infiltration. (A–C) Both Th1 and Th17 cell infiltration diminished in whole joint after EA treatment significantly. Treg levels in EA-treated mice appear to be sufficient, albeit reduced, to counterbalance the effect of Th17 and Th1 in causing inflammatory Lyme arthritis. All three fluorescence signal pixels were counted using the microscope and are presented here (B,C). Statistical analysis was conducted using two-tailed unpaired Student’s t-tests for unequal variance to determine significant differences between the paired groups (NS, not significant; *p < 0.05, **p < 0.01). EA, electroacupuncture.

Figure 4

Systemic effect of EA on proinflammatory cytokines levels in serum and extracts from heart and spleen of Borrelia burgdorferi-infected C3H/HeJ male mice. (A) Proinflammatory cytokine TNF-α reduced significantly after EA treatment in serum, and heart and spleen extracts, while IL-2 reduced significantly only in heart and not spleen extracts or serum. (B) IL-17A, IL-17F, and IL-22 cytokines produced mostly by Th17 cells were reduced significantly in heart in EA-treated mice at 4 weeks post-infection (p.i.), while spleen also showed significant reduction in IL-22 levels. Bars indicate median values. Statistical analysis was conducted using two-tailed unpaired Student’s t-tests for unequal variance to determine significant differences between the paired groups (NS, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 5

Effect of EA treatment on cytokines production by CD4+ Th1 and Th2 cells in serum, spleen, and heart extracts of Borrelia burgdorferi-infected C3H/HeJ male mice. IL-4, IL-5, and IL-13 cytokines that are primarily produced by Th2 cells were reduced significantly in heart after EA treatment while proinflammatory Type II IFN-γ cytokines were reduced significantly in heart as well as spleen. Overall lower levels of these cytokines were detected by LEGENDplex in EA-treated mice in this study. Bars indicate median values. Statistical analysis was conducted using two-tailed unpaired Student’s t-tests for unequal variance to determine significant difference between the paired groups (NS-Not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). EA, electroacupuncture.

Figure 6

Comparative analysis of mock- and EA-treated female immunocompetent and SCID C3H female mouse joint sections to determine inflammation. (A–D) Leukocyte infiltration was reduced in EA-treated mice (B and D versus A and C) irrespective of immunological genotype of mice. Leukocyte infiltration (arrows) showed association with joint swelling (not shown) and diminished in the joints of EA- compared to mock-treated mice. Cartilage damage and increase in synovial space resolved in these young mice at this stage. (E–H) Neutrophils (red, anti-Ly6G rabbit antibodies followed by anti-rabbit TRITC-conjugated antibodies) were the predominant leukocytes in the joints of all infected mice, and their levels were reduced in EA-treated mice. Only a few macrophages in panels E–H (purple, anti-F4/80-APC antibodies, arrows) and T cells in panels E and F (green, anti-CD3-Alexa Fluor 488 antibodies, arrowheads) were observed in tibiotarsus joints. (I–L) Higher magnification of sections shown in panels E–H. Green cells in SCID mice joints (G, H, K, L) are likely NKT cells. EA, electroacupuncture; SCID, severe combined immunodeficient.

Figure 7

Model summarizing effect of Borrelia burgdorferi infection and EA treatment on joint inflammatory responses and arthritis attenuation. High concentration of lipoproteins on B. burgdorferi surface signal through TLR2 on antigen-presenting cells to cause naive CD4+ cells to differentiate in different types of T cells in response to various combinations of cytokines produced. These Treg, Th17, or Th1 cells then produce listed cytokines (marked by black arrows). Neuromodulation of brain through sciatic nerve electrostimulation by EA results in signaling of adrenal glands through vagus nerve to produce dopamine (marked by blue arrows). Binding of dopamine neurotransmitter to receptor present on lymphocytes results in activation of Tregs, which then suppress Th1- and Th17-mediated inflammatory Lyme arthritis. EA, electroacupuncture.