TRPV1 Gates Tissue Access and Sustains Pathogenicity in Autoimmune Encephalitis
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Multiple sclerosis (MS) is a chronic progressive, demyelinating condition whose therapeutic needs are unmet, and whose pathoetiology is elusive. We report that transient receptor potential vanilloid-1 (TRPV1) expressed in a major sensory neuron subset, controls severity and progression of experimental autoimmune encephalomyelitis (EAE) in mice and likely in primary progressive MS. TRPV1−/− B6 congenics are protected from EAE. Increased survival reflects reduced central nervous systems (CNS) infiltration, despite indistinguishable T cell autoreactivity and pathogenicity in the periphery of TRPV1-sufficient and -deficient mice. The TRPV1+ neurovascular complex defining the blood-CNS barriers promoted invasion of pathogenic lymphocytes without the contribution of TRPV1-dependent neuropeptides such as substance P In MS patients, we found a selective risk-association of the missense rs877610 TRPV1 single nucleotide polymorphism (SNP) in primary progressive disease. Our findings indicate that TRPV1 is a critical disease modifier in EAE, and we identify a predictor of severe disease course and a novel target for MS therapy.
Multiple sclerosis (MS) is a debilitating inflammatory disease of the central nervous system (CNS) characterized by progressive demyelination and axonal damage (1). Its etiology remains unclear, though tissue damage is autoimmune in nature, with several genetic disease risk loci mapped to T cell function (2, 3, 4). A key step in disease is thought to be impairment of the blood-brain barrier (BBB), a logical, but elusive target for therapeutic intervention (5). This physical barrier is established early during embryogenesis and involves a neurovascular unit of pericytes, astrocytes, neurons, endothelial cells, and microglia (PANEM) (6). Normally, tight junctions and low vascular adhesiveness greatly inhibit extravasation into CNS tissue (5). Disruption of the BBB triggers a complex cascade of events in which the interactive PANEM tissue elements respond to secreted mediators of immune or neuronal derivation (7, 8, 9).
A blood-tissue barrier exists in peripheral tissues, although it is not as restrictive as the BBB. Extravasation into peripheral tissues can be induced by activating transient receptor potential vanilloid-1 (TRPV1), a prominent member of the TRP ion channel superfamily (10). TRPV1 is expressed on the major subset of sensory afferent neurons that integrate noxious stimuli including heat and acidity (11, 12, 13). The outcome of peripheral TRPV1 activation and consequent cation influx in TRPV1+ sensory neuron is two-fold; (i) electric afferent signal transduction to the CNS, generating pain perception and (ii), local efferent release of neuropeptides such as substance P (sP) and calcitonin gene-related peptide (CGRP). Long perceived as largely a heat sensor, TRPV1 is emerging as a major controller of pleiotropic functions in different tissue milieus. Abnormal TRPV1 activity can alter the severity and progression of peripheral inflammatory conditions dramatically, including type 1 diabetes (T1D), colitis and arthritis, through mechanisms largely mediated by its local efferent secretory function: too little in T1D, too much in colitis and arthritis (14, 15, 16).
TRPV1 also is expressed within CNS regions, including the spinal cord dorsal horn and areas of the brain (17, 18, 19, 20, 21). TRPV1 function in these locations remains a topic of debate. Broad expression patterns have fostered the view that central TRPV1 plays a role in pain processing and thermoregulation, and independent studies have suggested that PANEM cells may all express TRPV1, characterizing the BBB as a TRPV1-rich microenvironment (18,20,22). Activation of TRPV1 by application of the agonist capsaicin directly to the brain disrupts BBB integrity (23), and peripheral TRPV1 stimulation also opens the BBB and blood spinal cord barriers (BSCB) (24), supporting recent evidence that TRPV1 may modify cytokine signaling during CNS neuroinflammation and implying a disease-modifying role (25).
We investigated this possibility with a focus on EAE-autoimmunity. TRPV1−/−mice are dramatically protected from disease, developing minimal and delayed disease onset, reduced clinical scores and reduced demyelination. TRPV1 signaling effectively increases BSCB and BBB permeability, actively promoting inflammatory cell extravasation, which is minimal in the absence of TRPV1. A model is provided to explain how TRPV1 signaling in the PANEM complex may be sustained at body temperature. We conducted parallel studies of patients from the Canadian Collaborative Project on Genetic Susceptibility to Multiple Sclerosis (CCPGSMS), a large and lengthy population-based study (26, 27, 28), focusing on the extremely polymorphic TRPV1 locus (29). We found a significant missense single nucleotide polymorphism (SNP) selection bias in patients with a very progressive disease course, indicating that the unexpected core role of TRPV1 may extend to patients with MS.
Materials and Methods
All mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and maintained in our vivarium in a pathogen-free, temperature-controlled environment on a 12 h light:dark cycle. Experiments were performed using age- and gender-matched mice under approved protocols and in agreement with animal ethics guidelines.
Induction and Transfer of EAE
EAE was induced in mice 6–8 weeks old. For active immunization, mice were immunized in each flank with 50 ng of MOG35–55 peptide (Alpha Diagnostic, San Antonio, TX, USA) emulsified in CFA (1:1) (Sigma-Aldrich, St. Louis, MO, USA). Where indicated, 200 ng pertussis toxin (Sigma-Aldrich) was given intraperitoneally (IP) on the day of EAE induction and 48 h later. Passive immunization was performed as described (30). Briefly, donor mice were immunized as described above. Draining lymph node cells were removed 8 d after immunization and cultured in the presence of 5 µg/mL MOG35–55 peptide for 72 h; 1 × 107 cells were then injected intravenously (IV) into recipient mice along with 200 ng of pertussis toxin (Sigma-Aldrich) IP on the day of injection and 48 h later. For TPRV1 antagonist administration, mice were given 30 mg/kg capsazepine dissolved in 10% Tween-80 (Sigma-Aldrich), 10% ethanol, and 80% saline on 7 consecutive days. Animals were followed for a minimum of 24 d and disease was scored using the following scale: 0, asymptomatic; 1, limp tail; 2, abnormal righting reflex and/or hind limb weakness; 3, unilateral hind limb paralysis; 4, bilateral hind limb paralysis; 5, moribund or death.
Histology and Immunofluorescence
Spinal cord and brain tissue was dissected at peak disease, 16–18 d after active immunization and 24 d after passive immunization. Tissue was fixed for up to 72 h in 10% buffered formalin before staining with hematoxylin and eosin (H&E) or Luxol fast blue (LFB). Infiltration was scored by a blinded certified pathologist using the following scoring system: 1, normal, with intravascular nonadherent leukocytes; 2, intravascular adherent leukocytes; 3, perivascular infiltration. Demyelination was quantified using ImageJ software analysis (NIH, Bethesda, MD, USA).
Mouse spinal cord and brain tissue was dissected and snap frozen. Astrocytes were isolated from neonatal mouse brains as described previously (31) and from C8-D1A cell line (ATCC, Manassas, VA, USA) in vitro cultures. Extracted total protein was electrofractionated on 10% acrylamide gels (Invitrogen [Life Technologies, Carlsbad, CA, USA]), blotted, and probed overnight at 4°C with rabbit anti-TRPV1 antibody (1:200, Calbiochem [EMD Millipore, Billerica, MA, USA]) and rabbit anti-β-actin antibody (1:1000, Cell Signaling, Danvers, MA, USA). The following day, bands were visualized using the Western Lightning-ECL kit (PerkinElmer, Waltham, MA, USA).
Proliferation and Cytokine Secretion
Splenocytes or inguinal lymph node cells (4 × 105) were incubated in 96-well plates with HL-1 media (Lonza, Basel, Switzerland) and stimulated with MOG35–55 for 72 h. For proliferation experiments, 1 mCi of [3H]thymidine was added for 18 h, prior to harvest and liquid scintillation counting. T cell and B cell specific proliferation was measured as described above following stimulation with 1 µg/mL α-CD3 plus 0.25 µg/mL α-CD28, or 1 µg/mL α-CD40 (BD Pharmingen [BD Biosciences, San Jose, CA, USA]). For cytokine analysis, supernatants were collected after 72 h of culture. IFN-γ, IL-10, and IL–4 (BD Biosciences), IL-12 and TNF-α (eBioscience, San Diego, CA, USA), and IL-17 (R&D Systems, Minneapolis, MN, USA) were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocols.
CNS cells were isolated as described previously (32). Splenocytes or CNS cells (1.5 × 106) were incubated at 4°C for 15 min in 100 µL 2% fetal bovine serum (FBS)/phosphate-buffered saline (PBS) with 10 µg/mL Fc-blocker (eBioscience). Cells were incubated for 30 min with the following fluorescent antibodies: CD11b (1:100), CD3 (1:100), CD4 (1:75), CD8 (1:100), B220 (1:100), Igmol/L (1:100) (eBioscience), CD11c (1:100), CD62L (1:150), CD44 (1:100) (BD Biosciences), or CD45.2 (1:75) (Invitrogen [Life Technologies]). Samples were run on an LSR-II flow cytometer (BD Biosciences) and FACS plots were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).
For cDNA gene arrays, RNA from brain and spinal cord was isolated from mice 8 d after immunization using TRIzol Reagent (Invitrogen [Life Technologies]), followed by PureLink RNA Mini Kit (Invitrogen [Life Technologies]). Gene arrays used the Mouse Gene 1.0 ST array (Affymetrix, Santa Clara, CA, USA) and analysis by Expression Console (Affymetrix). Predicted and unknown genes were excluded from analysis.
Blood Spinal Cord/Brain Barrier Analysis
Evans Blue dye flux into brain and spinal cord tissue was determined as described (24). Briefly, 4 mL/kg of 2% Evans Blue dye (Sigma-Aldrich) was injected IV and after 2 h animals were perfused with PBS. The spinal cord and supraspinal tissues were immediately dissected, removing the dura mater. The lumbar spinal cord, thoracic spinal cord, brain stem, cerebellum and frontal cortex were further dissected and weighed. Tissue was incubated in formamide (Sigma-Aldrich) at 60° C for 72 h, and dye concentration in extracts was determined by spectrophotometry (620 nm) in a 96-well plate reader.
To measure permeability of large molecule (infrared-labeled IgG), 5 µg/g of donkey anti-goat IgG, IRDye 800CW labeled (LI-COR Biosciences, Lincoln, NE, USA) was injected IV and, after 30 min, animals were perfused with PBS. Spinal cord and supraspinal tissues were immediately dissected and scanned with an Odyssey Infrared Imager (LI-COR Biosciences).
For magnetic resonance imaging, a multichannel 7.0 T magnet was used to acquire in vivo 2D T1-weighted MR images (100 nm in-plane resolution) of the thoracic and lumbar spinal cord. Mice were injected with a bolus of Gd-DTPA (1.5 mmol/kg) IP and imaged at 1% isoflurane according to established protocols. Quantitative measurements of signal intensity were determined by normalizing the spinal cord intensity to the average intensity of two phantoms included in each coil (microcapillary tubes containing 1% Gd-DTPA in agar).
All subjects used in the study were ascertained through the ongoing Canadian Collaborative Project on the Genetic Susceptibility to MS (CCPGSMS), as described (26, 27, 28). DNA and anonymized clinical charts from a total of 163 sporadic MS patients were identified for analysis. The disability of the sporadic MS patients was assessed carefully and recorded at study entry with the Expanded Disability Status Scale (EDSS) determined by neurologists involved in the CCPGSMS. Here, patients were classified as having either “benign” or “malignant” MS based on EDSS scores sustained or achieved over designed time intervals. The benign MS cases (n = 112) fell into the relapsing-remitting clinical subtype where minimal disability (that is, EDSS ≤3) was sustained over a period >20 years from disease onset. By contrast, the malignant MS cases (n = 51), a subgroup of MS patients acquiring significant disability (that is, EDSS >6; the need for a walking aid or worse) within 5 years of disease onset, had the primary progressive form of the disease.
Total genomic DNA, extracted from whole blood as part of the CCPGSMS (33), was used to type SNPs. Genotyping of SNPs was performed using the Sequenom MassEXTEND protocol (https://doi.org/www.sequenom.com). Only conservative and moderate genotyping calls were accepted in this study. Samples having aggressive or low probability quality genotypes were reanalyzed.
Statistical significance between two means was assessed by Mann-Whitney and unpaired t tests, and Welch correction was employed where appropriate. Analysis of curves was performed using two-way analysis of variance (ANOVA). Human data was analyzed using the PLINK analysis package (34). Statistical significance was two-tailed and set at 5%, with error bars showing a single standard deviation.
All supplementary materials are available online at https://doi.org/www.molmed.org .
TRPV1 is Pathogenic in EAE
We also induced disease via active immunization with MOG35–55 peptide, without the classical injections of pertussis toxin, generating mild to moderate disease scores in B6 mice over a 40-d period after immunization, while TRPV1−/− mice were essentially asymptomatic. TRPV1+/− heterozygotes developed intermediate disease scores (Supplementary Figure 1B), suggestive of a gene dose effect.
TRPV1−/− Mice Generate Systemic Pathogenic Autoreactivity but Fail to Infiltrate the CNS
Adoptive Transfer of EAE Fails in TRPV−/− Mice
BSCB and BBB Collapse in EAE Is TRPV1-Dependent and Requires Lymphocytes
Given the intimate proximity of TRPV1+ cellular PANEM elements at the BBB and BSCB, we monitored barrier integrity during EAE progression. We detected no increase in the permeability of Evans Blue prior to d 3 after immunization (Figures 6A, B). Evans Blue extravasation following injection of MOG35–55 or of pertussis toxin alone was similar in brain and spinal cord regions of B6 and TRPV1−/− mice, rarely, if at all, exceeding baseline d 0 levels (Figures 6A, B). However, after immunization with MOG plus pertussis toxin, permeability was massive by d 11, particularly in the spine of B6 mice (Figures 6A–C) and perhaps reminiscent of the recently discovered lumbar BSCB tissue entry pathway (35). Significantly lower leakage levels were seen in TRPV1−/− mice, closely resembling lymphocyte-free RAG1−/− B6 congenics (Supplementary Figure 3). These observations extended to large molecule leakage, demonstrated by infrared-labeled IgG, with results comparable to Evans Blue extravasation (Figures 6D, E).
We demonstrate TRPV1 expression within astrocytes isolated from the brains of B6 mice and within the astrocyte cell line C8-D1A (Supplementary Figure 4A), similar to previous reports (18,20). This PANEM cell type plays a fundamental role in BSCB and BBB permeability, and further investigation into astrocyte functions mediated by TRPV1 is warranted. We also noted that TRPV1 expression levels within the CNS remain unchanged during the course of EAE (Supplementary Figure 4B), despite previous reports of altered expression in other disease states (37). Conversely, the expression of numerous hematopoetic cell lineage genes is upregulated significantly compared with TRPV1−/− mice, serving as an independent corollary of our data where B6 mice developed severe disease (Supplementary Figures 5A-C). B6 mice also showed increased transcription of numerous genes typically restricted to neuronal or nonhematopoietic tissue (Supplementary Figure 5D), providing a number of opportunities for future research investigating BSCB and BBB integrity. Collectively, TRPV1-dependent BSCB and BBB compromise per se requires antigen, adjuvant and lymphocytes, hallmarks of a cognate immune response.
Given the role of TRPV1 in promoting the severity of encephalitis, we questioned if a TRPV1 antagonist could elicit disease protection. We administered capsazepine subcutaneously and intranasally from d 0 to d 6 after immunization at a dose effective in other models (38). Despite our attempts, we found no significant difference between B6 mice treated with the antagonist and those, which received vehicle alone (Supplementary Figure 6), though we believe that adjustments to the dose, timing, and possibly the antagonist itself, may be prudent future approaches.
A Role for TRPV1, but Not Tac1, in EAE and MS
Tac1 and TRPV1 SNP frequencies in benign and malignant MS patients.
Sample size (n)
Sex ratio (F:M)
Mean age of onset (y)
Mean duration of disease (y)
Frequency (benign MS)
Frequency (malignant MS)
p Value (nominal)
Odds ratio (95% CI)
Our inability to accurately predict the clinical course of MS, and subsequently tailor patient therapy, reflects our failure to fully understand disease etiology and pathogenesis. Here we identify TRPV1 as a major progression element in murine EAE and as a likely participant in MS.
We went to considerable efforts to determine if, as in T1D, the pathogenic role of TRPV1 had no detectable impact on disease-promoting autoimmune profiles, including numeric, functional and subset distributions of autoreactive lymphocyte lineages. TRPV1 may be expressed on antigen presenting cells (39,40), but we conclude that it has negligible effects during the initiation and progression phases of EAE autoimmunity. Conversely, the numeric size of T cell and B cell infiltrates in the CNS remains minuscule in the absence of TRPV1, despite equal pool sizes of disease-associated lymphocytes systemically. Similar results were observed following adoptive transfer of highly activated autoreactive cells, where B6 recipients developed acute disease and TRPV1−/− recipients were almost entirely protected with sparse cellular infiltration while their lymphocytes were highly pathogenic in B6 hosts.
Our attention therefore shifted to the barriers that control lymphocyte egress into the CNS. Dramatic breakdown of BSCB and BBB integrity preceding peak disease was absent in TRPV1−/− mice, suggesting that TRPV1 itself is a participant in the PANEM cluster that grants passage to effector cells. This effect on permeability was not all encompassing, as small molecule permeability was still intact irrespective of TRPV1 expression. We provide data suggesting that one of the cell types mediating this effect may be astrocytes, and the functional consequences of this expression during EAE should be examined further. Interestingly, a relationship between TRPV1 and CNS access also is seen in nonobese diabetic (NOD) mice (41), a mouse strain found to carry a hypofunctional TRPV1 mutant gene (14).
Genetic deletion of TRPV1 produced significant disease protection in B6 MOG-EAE, including day of onset, maximum clinical scores and overall survival. Fittingly, one major genomic interval (eae7) that modulates the severity and duration of clinical signs during EAE (42,43) includes TRPV1. It is likely that the contribution of TRPV1 in this locus is considerable, as precedence has been shown in another T cell-mediated autoimmune disease, T1D (idd4.1) (14). Intermediate disease scores in TRPV1+/− mice support the idea that therapeutic targeting of TRPV1 by antagonists would, on face value, have a rationale, but might have to suppress significantly more than 65% of TRPV1 activity, that is, the levels observed here in TRPV1+/− heterozygotes. This may explain why our attempts to limit disease severity using capsazepine were unsuccessful. The efficacy of additional TRPV1 antagonists, currently approved for clinical use (44), should be investigated further to treat EAE and MS.
On the basis of observations in the EAE model, we analyzed a total of 163 individuals from the CCPGSMS program who were stratified into two groups on opposite ends of the clinical spectrum, thus examining TRPV1 and Tac1 (sP-coding) genes as disease modifiers rather than determinants of overall disease risk, a comparison that generally yields more modest associations (45). Patients with the most severe progression showed an overrepresentation of certain TRPV1 SNPs. The nonsynonymous mutation rs877610 is located within the intracellular C-terminal domain of TRPV1 in proximity to known binding sites, while rs224534, which also results in an amino acid change, lies within an extracellular region between the first two transmembrane domains. It will take considerable sequencing efforts to more fully characterize the allelic heterogeneity of TRPV1 in general and in patient populations such as MS. Furthermore, it will be important to determine if these alleles render TRPV1 activity hypo-functional or hyperfunctional. Even though sP is functionally linked to TRPV1, Tac1 SNPs showed no bias in our patient population. In retrospect, our failure to find relevant Tac1 SNPs should not surprise, given our comparison within a disease population rather than comparing patients with normal controls as before (46,47), and it coincides with the lack of EAE protection observed here in Tac1−/− mice.
We have made several comparisons between encephalitis and T1D, given the relatively high prevalence, in some populations, of MS and T1D comorbidity (48). The brain has the highest energy budget among organs: 20% of the total energy for 2% of body mass (49). To use abundant glucose, the PANEM complex must sustain high volume one-way traffic of insulin that makes the PANEM complex an insulin-rich milieu comparable to some extent to pancreatic islets. Insulin receptor ligation by TRPV1+ terminals raises TRPV1 currents, resetting activation thresholds to room temperature (50,51). Thus, there is potential for TRPV1 activation outside of its normal temperature constraints where specific TRPV1 alleles may confer a risk scenario. Alternatively, lipid TRPV1 agonists also may contribute to this process, as they have been reported to be upregulated in the brain and spinal cord during neuroinflammatory conditions such as EAE (52,53). The presence of enzymes responsible for catalyzing the biosynthesis of these endogenous agonists in the brain has also been reported previously (54). Anandamide and palmitoylethanolamide can either activate or desensitize TRPV1 (55), and their actions on TRPV1 within the CNS in the context of encephalitis should be strongly considered (56).
Collectively, our data make TRPV1 a prominent factor controlling disease progression/severity in EAE. Although numerically limited, our human observations employed a stringently selected subset of a large and well-characterized, population-based patient cohort, linking differences in disease course to polymorphisms in the human TRPV1 locus. While considerable work will be required to characterize the TRPV1 locus in MS patients and their relatives, allele sequences are likely to be identified that can predict severe course and thus rationalize aggressive therapies. TRPV1 continues to be a challenging drug target in humans (57,58), yet TRPV1-targeted treatments might have the potential to replace drug targeting of the immune system with its associated toxicities.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This work was supported by grants from the Canadian Institutes of Health Research (CIHR), the MS Society of the United Kingdom, and the MS Society of Canada Scientific Research Foundation. We thank L Morikawa for excellent assistance with histopathology. G Paltser is a recipient of awards from CIHR and the Banting and Best Diabetes Centre (Toronto). LS Cahill is a recipient of a CIHR postdoctoral fellowship.
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