Pannexin1 Channels Are Required for Chemokine-Mediated Migration of CD4+ T Lymphocytes: Role in Inflammation and Experimental Autoimmune Encephalomyelitis.

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J Immunol. Author manuscript; available in PMC 2017 May 15. Published in final edited form as: J Immunol. 2016 May 15; 196(10): 4338–4347. doi:10.4049/jimmunol.1502440.

Pannexin1 Channels are required for chemokine-mediated migration of CD4+ T Lymphocytes: Role in Inflammation and Experimental Autoimmune Encephalomyelitis Stephani Velasquez*,†, Shaily Malik*,†, Sarah E. Lutz‡, Eliana Scemes§, and Eliseo A. Eugenin*,†,*

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*Public

Health Research Institute (PHRI), Rutgers New Jersey Medical School, Rutgers the State University of NJ, Newark, NJ, USA

†Department

of Microbiology, Biochemistry and Molecular Genetics, Rutgers New Jersey Medical School, Rutgers the State University of NJ, Newark, NJ, USA ‡Department

of Neurobiology and Behavior, University of California, Irvine CA, USA

§Department

of Neuroscience, Albert Einstein College of Medicine, Bronx, New York, USA

Abstract

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Pannexin1 (Panx1) channels are large high conductance channels found in all vertebrates that can be activated under several physiological and pathological conditions. Our published data indicates that HIV infection results in the extended opening of Panx1 channels (5 min – 60 min), allowing for the secretion of ATP through the channel pore with subsequent activation of purinergic receptors, which facilitates HIV entry and replication. In the current report we demonstrate that chemokines, which bind CCR5 and CXCR4, especially SDF-1α/CXCL12, result in a transient opening (peak at 5 min) of Panx1 channels found on CD4+ T lymphocytes which induces ATP secretion, focal adhesion kinase phosphorylation, cell polarization and subsequent migration. Increased migration of immune cells is key for the pathogenesis of several inflammatory diseases including multiple sclerosis (MS). Here we show that genetic deletion of Panx1 reduces the number of the CD4+ T lymphocytes migrating into the spinal cord of mice subjected to experimental autoimmune encephalomyelitis, an animal model of MS. Our results indicate that opening of Panx1 channels in response to chemokines is required for CD4+ T lymphocyte migration, and we propose that targeting Panx1 channels could provide new potential therapeutic approaches to decrease the devastating effects of MS and other inflammatory diseases.

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Keywords Connexin; Gap Junctions; Neuroimmunology; Neuroinflammation; multiple sclerosis

*

Corresponding author: Eliseo Eugenin Ph.D., Public Health Research Institute (PHRI) and Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers New Jersey Medical School, Rutgers The State University of New Jersey, Newark, NJ. [email protected].

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Introduction Pannexins (Panx) constitute a protein family of large conductance, plasma membrane channels (1). Three members have been identified, Panx1, 2, and 3. Panx1 is expressed in all tissues including T lymphocytes (2, 3). Panx 2 expression was initially identified to be restricted to the central nervous system (CNS), however recent work has identified that Panx2 is readily expressed in several organs including eyes, lungs and colon (4, 5). Panx3 is localized in osteoblasts, synovial fibroblasts and chondrocytes (6). Structurally, all Panx(s) consist of a cytosolic N-terminal domain, four transmembrane domains with two extracellular loops and a cytosolic C-terminal domain (7). Although their structure is similar to connexins (Cxs), Panxs and Cxs do not share sequence homology (8).

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Normally Panx channels are in a closed state due to their high conductance. However, several laboratory conditions such as metabolic inhibition using antimycin A and iodoacetic acid, and chemotherapeutic drugs such as staurosporine, doxorubicin and etoposide, result in Panx1 channel opening (9, 10). Nevertheless physiological and infectious conditions that result in activation of Panx1 channels are poorly explored. Our previous work has demonstrated that pathogens such as HIV results in biphasic activity of Panx1 channels, first during early infection due to the binding of the virus to its host receptors (5 min – 60 min), and the second late after infection (10 h – 24 h), probably due to the release of new virions (11). We showed that Panx1 channel activity in response to HIV binding to CD4 and the chemokine receptors CCR5 or CXCR4 allowed for ATP release and subsequent activation of purinergic receptors, thereby facilitating entry of the virus into uninfected macrophages (12). Although our data indicates that HIV binding to CD4 and CCR5 or CXCR4, resulted in activation of Panx1 channels, it remains unknown whether physiological ligands of these chemokine receptors also trigger opening of the channel.

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Several publications indicate that ATP released through Panx1 channels is essential as a “find me” signal in several inflammatory conditions, including spinal cord injury, epileptic seizures and in experimental autoimmune encephalitis (EAE), a murine model of multiple sclerosis (MS) (13–19). In agreement with these studies, we found that SDF-1α/CXCL12, a ligand for CXCR4, induces a well-controlled opening of the Panx1 channels by a G protein coupled receptor mechanism. This resulted in release of the intracellular inflammatory factor ATP through the Panx1 pore, activation of purinergic receptors, and subsequent phosphorylation of focal adhesion kinase (FAK) leading to the initiation of a migratory phenotype. In the current study, we provide evidence that pharmacological blockade or genetic deletion of Panx1 channels inhibits CD4+ T lymphocyte polarization and migration induced by the chemokine SDF-1α/CXCL12. Furthermore, using a Panx1 deficient mouse subjected to EAE, we here show that Panx1 deficiency delayed clinical symptoms due to the decreased infiltration of CD4+ T lymphocytes into the CNS. In conclusion, our data provides evidence that opening of Panx1 channels is necessary for migration of immune cells into areas of inflammation in response to chemokines.

J Immunol. Author manuscript; available in PMC 2017 May 15.

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Materials and Methods Materials RPMI medium was purchased from Life Technologies (Carlsbad, CA). Fetal bovine serum was purchased from Lonza (Walkersville, MD). Human and mouse SDF-1α/CXCL12 was purchased from R&D Systems (Minneapolis, MN). The Panx1 mimetic blocking peptide 10Panx1 (WRQAAFVDSY) and the scramble peptide (FADRYWAQVS) were synthesized by PeproTech (Rocky Hill, NJ). All other reagents were purchased from Sigma unless otherwise indicated in the text. Isolation of human PBMCs and CD4+ T lymphocytes

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Anti-coagulated human blood was obtained from leukopacks from the New Jersey/New York Blood Center. PBMCs were isolated by over-layering with Ficoll-Paque (Amersham Bioscience, Uppsala, Sweden) according to the procedure described by the manufacturer. To obtain a pure population of human CD4+ T lymphocytes (92–99% CD4+ T lymphocytes), monocytes were removed from PBMCs by using CD14-coupled magnetic beads, and subsequently, CD4+ T lymphocytes were isolated with CD4-coupled magnetic beads (Miltenyi Biotec, Germany). Isolation of CD4+ T lymphocytes from animal spleens For CD4+ T lymphocytes, normal or Panx1 KO mice were sacrificed. The spleen was removed and prepared into a cell suspension. CD14 positive cells were isolated using MACS cell separation beads (Miltenyi Biotech GmbH, Germany). T cells were grown in RPMI medium containing IL-2 (20 U/ml) and PHA (1 µg/ml) for 24 h and subjected to treatment.

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Dye uptake and time-lapse microscopy to determine pannexin1 channel activity

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To characterize the functional state of Panx1 channels, dye-uptake experiments using ethidium (Etd) bromide were performed. Cells were washed twice in HBSS and then exposed to Locke's solution (containing 154 mM NaCl, 5.4 mM KCl, 2.3 mM CaCl2, 5 mM HEPES, pH 7.4) with 5 μM Etd, and time-lapse microscopy was performed. Phase-contrast and fluorescence microscopy with time-lapse imaging were used to record cell appearance and fluorescence-intensity changes in each experimental condition. Fluorescence was recorded every 30 s. The NIH ImageJ program was used for offline image analysis and fluorescence quantification. For data representation and calculation of Etd uptake slopes, the average of two independent background fluorescence (FB, expressed in arbitrary unitsA.U.) was subtracted from mean fluorescent intensity (F1). Results of this calculation (F1−FB), from at least 20 cells, were averaged and plotted against time (min). Slopes were calculated using Microsoft Excel software and expressed as A.U./min. Due to the fact that we are calculating the slope of the curve between two time points, dead cells are unable to maintain an increasing Etd uptake resulting in a slope close to 0. Thus, these cells are eliminated from the analysis. Microscope and camera settings remained the same in all experiments. Dead cells or cells with a damaged plasma membrane were clearly identified during the time-lapse microscopy as a result of their nonspecific Etd uptake, determined by


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lack of time dependency and stability in dye uptake (not inhibited by blockers), and were not quantified. Evaluation of the migratory phenotype induced by chemokines using confocal microscopy

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CD4+ T lymphocytes were cultured at 2×106 cells in 1 mL of RPMI 1640 with 10% FBS in the presence or absence of SDF-1α/CXCL12 (100 ng/mL). SDF-1α/CXCL12 was used to examine cellular polarization and process formation as a measure of the migratory phenotype. Pretreatment for 15 min with probenecid (500 μM), pertussis toxin (100 ng/ml), apyrase (50 U/ml), BzATP (1 µM), oATP (100 μM) or mimetic peptides to Panx1 (200μM: PeproTech, NJ) was used to examine the mechanism by which Panx-1 channels mediate cell migration. CD4+ T lymphocytes were fixed with 2 % paraformaldehyde after treatment and staining for tubulin and actin was performed at different time points to quantify the migratory phenotype of T cells using confocal microscope. Images were obtained using a Nikon A1 confocal microscope and analyses were performed using NIS Elements (Nikon, Japan). Immunostaining of CD4+ T lymphocytes and mice spinal cords

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Human CD4+ T lymphocytes and mouse spinal cord sections (20 µm) were permeabilized with 0.01 % Triton-×100. Cells and tissue were washed with PBS and blocked using 0.9 % fish gelatin, 50 μM EDTA, 1 % horse serum, and 1 % globulin-free albumin. CD4+ T lymphocytes and spinal cord sections were incubated with mouse monoclonal α-tubulin antibody (1:2000, Sigma), Panx1 rabbit polyclonal antibody (1:500; Life Technologies, CA), and CD4 rat monoclonal antibody conjugated to biotin (1:200; Abcam, United Kingdom). Immunoreactivity was visualized with goat anti-mouse FITC, streptavidin Cy3, goat antirabbit FITC; actin was stained using Texas Red-X conjugated to phalloidin (Life Technologies, CA). Samples were mounted with ProLong Gold antifade containing DAPI (Life Technologies, CA). Images were obtained using a Nikon A1 confocal microscope and analyses were performed using NIS Elements (Nikon, Japan). ATP Release Assay

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Primary human CD4+ T lymphocytes were treated with SDF-1α/CXCL12, probenecid, mimetic peptide and/or scramble mimetic peptide for 1, 5, 10, 15, 30, 45, and 60 minutes. Supernatants were collected after each time point and ATP concentration was determined using the ATPlite luminescence assay system (PerkinElmer, MA) by combining 100μL of sample with 100μL of ATPlite reagent. Luminescence was measured using a PerkinElmer’s EnVision Multilabel Plate Reader. The extracellular concentration of ATP was determined by comparing sample luminescence to a standard curve of 0.39 to 100nM ATP using the ATP standard provided by the manufacturer. Transmigration Assay CD4+ T lymphocytes (3×105 cells) isolated from leukopaks were added to the top of a 3 μM pore tissue culture insert to assay transmigration. Media alone or SDF-1α/CXCL12 (100ng/ml, R&D Systems) were added to the bottom chamber to generate a chemotactic


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gradient. For Panx1 blocking experiments, CD4+ T lymphocytes were pretreated with probenecid (500 μM) or mimetic peptides to Panx1 (200μM) for 30 minutes before the transmigration assay, and then the pretreated cells were added to the top of the tissue culture insert and allowed to transmigrate for 2 hours. Each transmigration condition was performed in 4 replicate tissue culture inserts. After 2 hours the cells that transmigrated across the filter were collected from the bottom chamber, and counted using trypan blue dye and by Flow Cytometry. Flow Cytometry

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CD4+ T lymphocyte cell surface markers were analyzed using fluorochrome-coupled mAb specific for human CD4 (clone OKT4; Biolegend, CA), CD3 (clone BW264/56; Miltenyi Biotec, Germany), as well as the corresponding isotype control APC mouse IgG2bk (BD Pharmagin, CA) and mouse IgG2a FITC negative control (DakoCytomation, Denmark). Starting and transmigrated populations of CD4+ T lymphocytes were analyzed for CD3 and CD4 using FACS. Antibodies were titrated to determine optimal concentrations for staining. CD4+ T lymphocytes were incubated in the dark on ice for 30 min with the appropriate antibodies. Following staining, cells were washed with FACS buffer (calcium and magnesium free PBS supplemented with 1% BSA) and fixed with 4% paraformaldehyde. Events were acquired with a BD Accuri C6 flow cytometer and analyzed by Flowjo software (v10.0.6; TreeStar, OR). Western Blot Analysis

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Control, SDF-1α/CXCL12, probenecid, and mimetic peptide treated cells were lysed with RIPA buffer (Cell Signaling, Beverly, MA) containing protease inhibitors, and 80 µg of protein were electrophoresed on a 4–20% polyacrylamide gel (Bio-Rad, CA), and transferred onto nitrocellulose membranes. Membranes were probed with rabbit monoclonal antibodies to FAK (Tyr576/577), FAK (Tyr 397), FAK (Tyr 925) or GAPDH (Cell Signaling) after SDF-1α/CXCL12 (100ng/ml, R&D Systems) treatment in the presence and absence of Panx1 blockers. Densitometric analysis was performed using NIH ImageJ software. Panx1 knockout mice and EAE model

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Female C57Bl/6 mice were obtained from IFFA CREDO and Charles River. Panx1 deficient mice were obtained from U.C. Davis KOMP (allele: Panx1tm1a (KOMP) Wtsi) as heterozygous (HT) mice on C57Bl/6 background and bred to homozygosity (Panx1 KO and Panx1 WT) and maintained in an SPF animal facility at The Albert Einstein College of Medicine. EAE was induced as previously described (15, 20). Clinical signs of disease were scored on a 0–8 scale where, 0: No signs; 1: Loss of tail tone; 2: Paralyzed tail; 3: Hind limb weakness; 4: Hind limb hemi-paralysis; 5: Complete hind limb paralysis; 6: Complete hind limb paralysis with forelimb weakness; 7: Tetraplegia; 8: Moribund. Histopathology Non-EAE and EAE mice paraffin embedded sections from thoracic, lumbar and sacral spinal cords were processed. Twenty μm thick sections were stained with hematoxylin and eosin


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(H&E). Images were obtained using Pannoramic desk whole slide imaging system (PerkinElmer, MA). Numbers of infiltrating leukocytes were quantified using NIH ImageJ software. Statistical analysis Analysis of variance was used to compare the different groups; *P≤ 0.01 as compared to controls and #P≤ 0.01 as compared to SDF-1α/CXCl12 treatment for all statistical analyses performed in this study. For the experiments using the control and knockout animals, confident interval for the differences between two means was applied, and a difference of P≤0.05 was considered significant.

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SDF-1α/CXCL12 results in transient activation of Panx1 channels on CD4+ T lymphocytes We have previously shown that HIV infection of CD4+ T lymphocytes activates Panx1 channels in a biphasic manner and dependent on the binding of the virus with CD4 and the corresponding chemokine receptor (CXCR4 and/or CCR5) (11). To assess whether endogenous chemokine binding to CXCR4 or CCR5 resulted in activation of Panx1 channels, Etd uptake experiments were performed.

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As observed in figure 1A, control cells showed minimal Etd staining during the time course analyzed indicating that the channel is in a closed state (30 sec-168 h or 10080 min; Figure 1B; black circles). In contrast treatment of human CD4+ T lymphocytes with SDF-1α/ CXCL12 (100 ng/mL), the ligand for CXCR4 induced a transient increase of Etd uptake (only detected up to 5 min; *P≤0.01, Figure 1B; red circles). No Etd uptake was observed when CD4+ T lymphocytes were pre-incubated with probenecid (Prob), siRNA to Panx1 (siRNA) or the blocking extracellular peptide (PEP) to Panx1 channels in response to SDF-1α/CXCL12 (Figure 1C). Negative controls such as scrambled peptide (SCR) did not alter Etd uptake induced by SDF-1α/CXCL12 (Figure 1C).

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Similar results of transient Etd influx were observed for chemokines that bind CCR5 such as RANTES/CCL5, MIP-1α/CCL3, and MIP-1β/CCL4 (data not shown). In all these studies, dead cells were not considered, because when the plasma membrane is compromised Etd enters the cells in an unspecific manner abolishing the time dependent entry of the dye. Thus, these cells were easily identified and eliminated from the study. In our study only 2– 3 % of cell death was observed (data not represented). Chemokine mediated Etd influx was transient and only detected up to 5 min as compared to the extended opening (5–60 min) observed with several HIV isolates (11). Our Etd uptake data indicates that chemokine treatment induced the opening of a large channel, that is sensitive to probenecid and the extracellular Panx1 channel blocking peptide, but not lanthanum or the Cx43 hemichannel blocking peptide (Figure 1C and data not shown), supporting our hypothesis that chemokines open Panx1 channels. Furthermore, Etd uptake induced by SDF-1α/CXCL12 treatment was sensitive to pertussis toxin (P.T.), suggesting that G protein coupled receptors,


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probably CXCR4, the receptor for SDF-1α/CXCL12 is required for Panx1 opening (data not shown). To further demonstrate that Panx1 channels are opened in response to SDF-1α/CXCL12, CD4+ T lymphocytes were isolated from the Panx1 knockout and control animals (Figures 1 D and E). CD4+ T lymphocytes obtained from control animals replicated our data obtained using human cells. SDF-1α/CXCL12 treatment induced a transient opening of the channel (Figure 1D). In contrast, no uptake was detected in CD4+ T lymphocytes isolated from the Panx1 knockout mice at any time analyzed in response to SDF-1α/CXCL12 (Figure 1E). SDF-1α/CXCL12 induced cell polarization of human and mice CD4+ T lymphocytes by a mechanism dependent on opening of Panx1 channels


The major function of all chemokines is to regulate physiological and pathological cellular trafficking of particular cell populations into different tissues (21). Here we tested the hypothesis that opening of Panx1 channels in response to chemokine stimulus is required for cellular polarization and migration. Human control CD4+ T lymphocytes with and without probenecid treatment remained circular, and minimal polarization or formation of processes was detected at all time points analyzed (13.88 ± 1.6% of cells with processes; Figure 2A and E). Human CD4+ T lymphocytes treated with SDF-1α /CXCL12 (100ng/mL) resulted in cell polarization and process formation in a time dependent manner (69.9 ± 6.1% of cells with processes; Figure 2B see arrow). Cells pre-treated with the Panx1 channel blocker, probenecid (Prob, 500 μM) or the mimetic peptide 10Panx1 (200 μM) to the extracellular loop of Panx1 prevented cell polarization induced by SDF-1α /CXCL12 (11.9 ± 4.7% of cells with processes; Figure 2C and D). Treatment of CD4+ T lymphocytes with scrambled peptide did not prevent cell polarization induced by SDF-1α /CXCL12 (63.9 ± 7.7% of cells with processes).

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Since the study was conducted using human primary cells, donor variability was expected. All donors had differences in the percentage of cells with processes in response to SDF-1α/ CXCL12, the time course of migration, and baseline control cell polarization. Despite the inherent variability of human cells, donor variability, SDF-1α/CXCL12 always increased polarization over control/untreated conditions and Panx1 channels blockers prevented the chemokine induced cell polarization (supplemental Figure 1, seven representative donors are shown). Figure 2E represents the summary of all donors. In the presence of a chemoattractant, cells become polarized and rearranged their cellular components developing a leading and a trailing edge (22, 23). In all donors SDF-1α /CXCL12 increased process formation at all-time points analyzed (*P≤0.01 as compared to control cells, Figure 2E). Blockade of Panx1 channels by either Prob or mimetic peptide always prevented cell polarization (#P≤0.01 as compared to cells treated only with SDF-1α /CXCL12; Figure 2E). Furthermore, cell polarization induced by SDF-1α /CXCL12 was also dependent on G protein coupled receptor activation, secreted ATP, and activation of purinergic receptors. As described in our model (Figure 3D), we hypothesize that binding of SDF-1α/CXCL12 to its receptor CXCR4 opens the Panx1 channel. Open Panx1 channels release intracellular messengers such as ATP that subsequently activates purinergic receptors to trigger cell polarization and migration. Thus, to examine the role of G protein coupled receptors, J Immunol. Author manuscript; available in PMC 2017 May 15.

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extracellular ATP and purinergic receptors, we pretreated the CD4+ T lymphocytes with pertussis toxin (P.T.), apyrase (Apy), and oxidized ATP (oATP) before treatment with SDF-1α/CXCL12 (Figure 2F). All blockers reduced the numbers of cells with processes, despite treatment with SDF-1α/CXCL12, supporting our hypothesis (Figure 2F). In agreement with our model, addition of BzATP (1–10 µM) to directly activate purinergic receptors also results in cell polarization in a P.T. and Panx1 channel independent manner (data not shown).

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To further determine whether Panx1 channels are necessary to mediate a migratory phenotype in response to SDF-1α /CXCL12. Panx1 knockout animals were used. CD4+ T lymphocytes were isolated from spleen, cultured and exposed to different treatments including SDF-1α /CXCL12 in the presence of probenecid, P.T., apyrase, BzATP or oATP. CD4+ T lymphocytes obtained from the control animals without treatment remained circular with minimal polarization detected (Figure 2G). CD4+ T lymphocytes obtained from control animals and treated with SDF-1α /CXCL12 (100 ng/mL) resulted in cell polarization and process formation in a time dependent manner (Figure 2G, 5 min). CD4+ T lymphocytes obtained from Panx-1 knockout animals did not shown any polarization in control and SDF-1α/CXCL12 treated conditions (Figure 2G). In addition, polarization of CD4+ T lymphocytes obtained from control animals and treated with SDF-1α/CXCL12 requires extracellular ATP, activation of G protein coupled receptors, and purinergic receptors, because preincubation of the cells with Apyrase (Apy), Pertussis toxin (P.T.) and oATP abolished cell polarization (Figure 2G). In conclusion, opening of Panx1 channels in response to SDF-1α/CXCL12 treatment is essential for cell polarization induced by chemokine stimuli.

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Panx1 is localized on the leading edge during cell polarization induced by SDF-1α / CXCL12 and Panx1 channel activity is required for FAK phosphorylation Our data indicates that opening of Panx1 channels is essential for cell polarization, a critical step in cellular migration. However, the cellular location of this protein during migration and the downstream signaling activated by this channel are not defined. Confocal analysis of CD4+ T lymphocytes under untreated conditions shows a uniform distribution of Panx1 channels on the plasma membrane (Figure 3A; control). SDF-1α /CXCL12 treatment results in cell polarization and relocalization of Panx1 channels to the leading edge of the cells (Figure 3A; SDF-1α /CXCL12; see arrow).

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A critical signaling event in the leading edge during cell polarization and migration is the phosphorylation of focal adhesion kinase (FAK). FAK plays a central role in migration, specifically in cytoskeletal rearrangement in response to external stimuli mediated by Gprotein coupled receptors and calcium (24, 25). To examine whether opening of Panx1 channels participates in FAK phosphorylation in response to SDF-1α /CXCL12, Western blots were performed to analyze 3 different phospho-FAK sites (Tyrosine 397, 925, and 576/577) (26). There was no significant difference in the phosphorylation of FAK in control cells with and without probenecid (Figure 3B, a representative experiment is shown). However, after a 15-minute treatment with SDF-1α/CXCL12 there was a significant increase (SDF, *P≤0.01; n=4) in the phosphorylation of FAK at Tyrosine 397, 925, and 576/577 as


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compared to control conditions in human and mice CD4+ T lymphocytes (Figure 3B and C, the numbers on the bottom of each line represents the experiment shown and Fig. 3C correspond to the data of all 4 donors). Blockade of Panx1 channels prevented phosphorylation of FAK induced by SDF-1α/CXCL12 treatment in human and mouse CD4+ T lymphocytes (Figure 3B and C). Treatment of human and mouse CD4+ T lymphocytes with scrambled mimetic peptide resulted in similar phosphorylation of FAK as is seen in the SDF-1α /CXCL12 treated cells (Figure 3B and C, hCD4). To assure the key role of panx-1 channels in activation of FAK, we performed experiments using CD4+ T lymphocytes isolated from control and Panx1 knockout animals (Figure 3B and C, Panx-1 KO mice or mouse CD4, mCD4). As indicated in Fig.1, SDF-1α/CXCL12 induced opening of Panx1 channels in mouse CD4+ T lymphocytes. Here we demonstrated that Panx1 channel opening is required for FAK phosphorylation (Figure 3B and C, mCD4). CD4+ T lymphocytes isolated from the Panx1 knockout mice were unable to induce changes in cell shape, polarization as well as activation of FAK (Figure 3B and C, mKOCD4). Thus, our hypothesis is that opening of Panx1 channels in response to SDF-1α /CXCL12 treatment is required to phosphorylate FAK, a critical kinase involved in actin rearrangement in the leading edge of the cell (see model; Figure 3D). SDF-1 α/CXCL12 induces the release of ATP through Panx1 channels

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Upon opening of Panx1 channels several intracellular factors can be released including the highly chemotactic and inflammatory factor ATP. Therefore, we evaluated whether opening of Panx1 channels in response to SDF-1α /CXCL12 induced ATP release. Our data demonstrated that SDF-1α /CXCL12 produced an increase in ATP release (*P≤ 0.01; Figure 4). ATP release in response to SDF-1α /CXCL12 was completely prevented by pretreating the cells with probenecid or Panx1 mimetic peptides (Figure 4). Scramble mimetic peptide was used as a control with no specific effects. These results indicate that opening of Panx1 channels in response to SDF-1α /CXCL12 results in the release of ATP into the extracellular space. Opening of Panx1 channel in response to SDF-1α/CXCL12 is essential for chemotaxis of CD4+ T lymphocytes Our previous data demonstrated that opening of Panx1 channels is required for FAK phosphorylation and cell polarization in response to several chemokines. To characterize whether opening of Panx1 channel is required for cellular migration we performed transwell migration assays in response to SDF-1α/CXCL12.

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CD4+ T lymphocytes were seeded on the top chamber and SDF-1α/CXCL12 was placed in the bottom chamber to generate a chemotactic gradient across the 3 μm filter. After 2 hours, cells that transmigrated across the filter and into the bottom chamber were collected, and counted by FACS. In control conditions low number of CD4+ T lymphocytes migrated across the 3μm filter into the bottom chamber (Control and Control + Prob; Figure 5A and B). The addition of SDF-1α/CXCL12 into the lower chamber resulted in a significant increase in the number of cells transmigrating across the filter (SDF-1; Figure 5A and B). Pretreatment of the CD4+ T lymphocytes with Panx1 blockers probenecid or mimetic peptide prevented transmigration induced by SDF-1 α/CXCL12 (SDF-1 + Prob and SDF-1


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+Peptide; Figure 5A and B). Pretreatment of CD4+ T lymphocytes with scrambled mimetic peptide resulted in similar results as CD4+ T lymphocytes induced to migrate in response to SDF-1 α/CXCL12 (SDF-1 + Scr Peptide; Figure 5A and B). Our results demonstrate that opening of Panx1 channels is essential for transmigration of CD4+ T lymphocytes in response to SDF-1α/CXCL12. Thus Panx1 channels are not only important in cell polarization but are also involved in active cell movement. Panx1 channels are required for efficient transmigration of leukocytes into the spinal cords of mice subjected to EAE

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A key feature of MS is the excessive infiltration of autoreactive T lymphocytes into the CNS as well as elevated levels of SDF-1α/CXCL12 in the cerebrospinal fluid and tissue (27–31). Recent work from our collaborators has identified that genetic deletion of Panx1 channels results in a delayed onset of clinical and pathological symptoms of EAE, an animal model for MS (15). However it is unknown whether this delayed onset of clinical symptoms was due to decreased or delayed migration of CD4+ T lymphocytes into the spinal cord. To assess the number of transmigrating CD4+ T lymphocytes, staining for CD4 and Panx1 was performed and analyzed using confocal microscopy (Figure 6A and B). Control WT non-EAE mice had no infiltrating CD4+ T lymphocytes. We demonstrated that spinal cords from acute EAE Panx1 KO mice had fewer CD4+ T lymphocytes present when compared to the spinal cords of Panx1 WT mice. No significant difference in the number of CD4+ T lymphocytes was observed in the spinal cords of chronic EAE Panx1 KO mice and chronic EAE Panx1 WT mice (data not shown). Thus, Panx1 is required for efficient transmigration of CD4+ T lymphocytes into compromised spinal cords in the pathogenesis of EAE.

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Discussion We have previously shown that HIV binding to CD4 and CCR5/CXCR4 stimulated a biphasic (early, 5–60 min and late 10–24 h) opening of Panx1 channels. However, the mechanism by which these channels are activated by HIV or other physiological stimulus is unknown. Here we show that physiological concentrations of chemokines, which bind CXCR4 and CCR5, induce a transient (5 min) activation of Panx1 channels on CD4+ T Lymphocytes which results in ATP release, FAK phosphorylation, cell polarization and transmigration. We propose that blocking Panx1 channels could be a potential therapeutic target to reduce inflammation and transmigration of immune cells into compromised tissues.

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Panx1 channels are usually closed in order to prevent degradation of the electrochemical gradient across the plasma membrane (32). Currently the mechanisms of Panx1 opening are poorly explored but it has been suggested that one mechanism involves mechanical stimulation. An increase in fluid shear force such as the application of suction to a patch of membrane or physical stretching of the entire cell increases the permeability of the channel (8, 33–36). Panx1 channels have also been shown to open in response to increasing extracellular potassium concentration (18, 19, 37). Recent work has identified that proteolytic cleavage of Panx1 C-terminal tail by caspases 3 or caspases 7 results in ATP secretion, cellular recruitment and inflammation in association with apoptosis (13, 38). As was previously described opening of Panx1 channels results in changes in ionic gradients, as


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well as the release of several factors, including ATP (38–42). ATP is released from intact apoptotic cells, serving as a danger signal that recruits phagocytic cells (13). Here we identified that SDF-1α/CXCL12 stimulation induces activation of Panx1 channels which results in the release of ATP through the channel since blockade of Panx1 channels prevented ATP release.


Our data indicates that, in contrast to HIV infection which results in a prolonged activation of this channel, chemokines that bind to CCR5 and CXCR4 induce a transient activation of Panx1 channels. In this regard, it has been shown that gp120 activates a different signaling pathway than the one induced by chemokines (43–50). Once HIV or specific chemokines bind to the chemokine receptor, activation of GPCR occurs which results in the recruitment of the G protein complex inducing the exchange of GTP for GDP and causing the disassociation of the βγ dimer from the α unit resulting in downstream signaling (51–53). In agreement, our data demonstrates that pertussis toxin blocks the opening of Panx1 channels induced by SDF-1α/CXCL12 treatment. Evidence in the literature suggests that HIV-1 gp120 triggers Pyk2 phosphorylation in CD4+ cells. However, HIV-1 gp120 binding to CXCR4 fails to induce phosphorylation of ERK1/2 MAPK even though SDF-1α/CXCL12 binding to CXCR4 clearly induces phosphorylation of ERK1/2 MAPK in CD4+ cells (47, 54). Chemokines are thought to bind to a pocket formed by the transmembrane helices found in G-protein coupled receptors which allow a conformational change that promotes guanine nucleotide exchange in G-proteins (55). Chemokines bind this pocket at their N termini; HIV envelope does not induce an activated conformation of CCR5 to enter and can bind away from the pocket. This suggests that some of the elements of the binding site are distinct between HIV and chemokines, which bind to the same receptors (45). Taking this evidence into consideration, the differential temporal activation of Panx1 channels seen in HIV infection and chemokine stimulation could be a result of differential downstream signaling cascade induced by different binding sites used by the virus and the chemokines. Here we demonstrated that FAK is phosphorylated at Tyrosine576/577, Tyrosine925 and Tyrosine397 residues during SDF-1α/CXCL12 stimulation and this phosphorylation is prevented when Panx1 channels are blocked as well as in CD4+ T lymphocytes from Panx1 knockout mice. Phosphorylation and activation of FAK has been described to play an integral role in cytoskeletal rearrangement induced by G-protein coupled receptor stimuli such as CXCL12 (24, 25). Based on the present results and above considerations, it is proposed that Panx1 channels in concert with purinergic receptors induce the phosphorylation of FAK located at the leading edge of polarizing cell resulting in cell migration.

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MS is an autoimmune inflammatory demyelinating disease characterized by destruction of myelin and migration of autoreactive T cells into the nervous system (56). In MS, SDF-1α/ CXCL12 contributes to the transmigration of autoreactive T lymphocytes into the CNS and is elevated in inflamed endothelial cells, astrocytes, and cerebro spinal fluid (CSF) of MS patients (29–31). The recent work of our collaborators has demonstrated that inhibition or deletion of Panx1 improved the neurological and pathological symptoms of EAE (15). Our present work demonstrates that this delayed onset of clinical symptoms was due to a decreased migration of CD4+ T lymphocytes into the spinal cord. CD4+ Th1 and Th17 cells


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that migrate into the CNS induce EAE and CD4+ cells are significantly increased in lesions, serum and CSF of MS patients (57–59). Therefore, it is likely that clinical symptoms may be delayed by specifically targeting the T cell subset involved in the damage by controlling Panx1 channel activity to reduce transmigration of activated leukocytes into the CNS. In conclusion, Panx1 channels are transiently opened in response to chemokine gradients and opening is required for the pathogenesis of several diseases such as HIV and MS. We propose that blocking these channels can be exploited as a therapeutic target to reduce the devastating consequences of several inflammatory diseases.

Supplementary Material Refer to Web version on PubMed Central for supplementary material.

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Acknowledgments We would like to thank the New Jersey/New York Blood Center, the Alfred P. Sloan Foundation Minority fellowship (to S.V.) and Mount Sinai NeuroAIDS Disparities Summer Institute, R25 MH080663 (to S.V.). This work was partially supported by NIH-NINDS training grant T32 NS07439 (to S.E.L.), National Multiple Sclerosis Society fellowship FG2035A1 (To S.E.L.), RO1-NS052245 (to E.S.), the National Institute of Mental Health grant, MH096625, and PHRI funding (to E.A.E).

References


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Figure 1. SDF-1α/CXCL12 results in activation of Panx1 channels in CD4+ T Lymphocytes

(A) Representative images of phase (bright field), Fluorescence (Etd) and the merge (merge) of both images after 5 minutes of fluorescent recording under control or SDF-1α treated conditions. In control cells no Etd uptake (top row of images) was observed during the time course analyzed. SDF-1α/CXCL12 (100ng/mL) treatment resulted in an increase in fluorescence in a time dependent manner (bottom row of images). (B) Quantification of Etd uptake in human CD4+ T lymphocytes. No Etd uptake was recorded from control cells during all the time points analyzed (black circles). SDF-1α/CXCL12 treatment resulted in a


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transient increase in Etd fluorescence (peak at 5 min; P≤0.01) remaining at a steady control level until 168 h or 10080 min, the last time point analyzed (red circles). (C) SDF-1α/ CXCL12 induced Etd uptake is sensitive to pretreatment with Probenecid (Prob), Panx-1 siRNA (siRNA) and Panx1 blocking peptide (PEP). Scrambled peptide did not alter Etd uptake induced by SDF-1α/CXCL12. (D) Murine CD4+ T lymphocytes have a similar behavior to human cells. CD4+ T lymphocytes isolated from control animals replicate the results described in panel B. (E) In contrast, CD4+ T lymphocytes obtained from the Panx1 knockout mice and treated with SDF-1α/CXCL12 were unable to uptake Etd. *P≤0.01 denote significance as compared with control conditions (n=6) and # P≤0.01 as compared to SDF-1α/CXCL12 conditions.

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Figure 2. Opening of Panx1 channels is required for cellular polarization in response to SDF-1α / CXCL12

(A–D) Representative 3D images of human CD4+ T lymphocytes stained for DAPI (Nucleus, blue staining), tubulin (FITC, green staining), actin (Texas-red, red staining) and merge (all the colors). (A) Control conditions show a round cell with peripheral actin and a tubulin scaffold with no processes. (B) Stimulation of CD4+ T lymphocytes with SDF-1α/ CXCL12 (100 ng/ml) induced cell polarization and formation of long processes (arrow denote formation of the process). (C and D) Pretreatment of CD4+ T lymphocytes with Panx1 channel blocker, probenecid (SDF1 + Prob, 500 μM) or the mimetic peptide 10Panx1 J Immunol. Author manuscript; available in PMC 2017 May 15.

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(SDF-1 + Peptide, 200 μM) prevent polarization and the formation of processes induced by SDF-1α/CXCL12. Scramble mimetic peptide was used as a control (SDF-1 + Scr, 200 μM). (E) Mean percentage of cells with processes was analyzed for all donors (n=7), time points (1, 5, 10, 15, 30, 45 and 60 minutes) and treatments. (F) Correspond to the pre-incubation of human CD4+ T lymphocytes with apyrase to degrade extracellular nucleotides such as ATP (Apy), pertussis toxin (P.T.) to block activation of G protein coupled receptors such as CXCR4, or oATP to block purinergic receptors (oATP). Treatment of cells with SDF-1α/ CXCL12 in the presence of these blockers abolished cell polarization. (G) Experiments using murine CD4+ T lymphocytes indicate that Apy, P.T. and oATP are necessary for cell polarization in response to SDF-1α/CXCL12. In addition, murine CD4+ T lymphocytes isolated from panx-1 knockout mice do not polarize in response to SDF-1α/CXCL12. *P≤0.01 denote significance as compared to control conditions and #P≤0.01 denote significance as compared to SDF-1α/CXCL12 treated conditions. Error bars represent standard error (n=7).

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Figure 3. Panx1 is localized on the leading edge of the polarized cell in response to SDF-1α/ CXCL12 treatment and opening of Panx1 channel is required for phosphorylation of focal adhesion kinase in response to SDF-1α/CXCL12

(A) Human CD4+ T lymphocytes were stained nucleus (DAPI; blue), Panx1 (FITC; green), and actin (Texas-red; red) in untreated and SDF-1α/CXCL12 treated conditions. Confocal analysis indicates that most of the Panx1 protein is localized at the processes of the CD4+ T lymphocytes treated with SDF-1α/CXCL12 (SDF-1) or the leading edge of the cell (Arrow). (B) Analysis of FAK using human and murine CD4+ T lymphocytes (obtained from control and panx-1 knockout animals). Western blot showing the FAK phosphorylation under


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untreated conditions (Control), control with probenecid (Control+Prob), after a 30-minute treatment with SDF-1α/CXCL12 (100ng/ml; SDF-1), and after pretreatment with, Probenecid (500 μM; SDF-1+Prob), mimetic peptide (200 μM; SDF-1+ Pep), and scrambled mimetic peptide (200 μM; SDF-1+ Scr) followed by a 30-minute treatment with SDF-1α/ CXCL12. Total GAPDH was used as a loading control. The numbers indicated in the bottom of each line correspond to the quantification of the mean fold change of the phosphorylation bands as compared to control conditions (n=4, a representative experiment is shown). (C) Quantification of western blot analysis normalized to Total FAK. The bars represent antibodies used to detect phosphorylation of FAK antibody against FAK Tyr397 (black bars), Tyr925 (white bars) and Tyr567/577 (red bars). This graph correspond to all results obtained using human CD4 cells (hCD4), mouse CD4 cells (mCD4), and CD4 isolated from the panx-1 knockout mouse (mKOCD4). (D) A schematic representation of proposed events taking place at the leading edge of a migratory cell after the initial binding of SDF-1α/ CXCL12 to CXCR4. SDF-1α binding to its chemokine receptor CXCR4 is shown to induce activation of Panx1 channels and the release of ATP through the channel pore. Extracellular ATP binding to purinergic receptors are shown to activate focal adhesion kinase (FAK) as well as other migratory proteins to facilitate cytoskeletal rearrangement and cell polarization. *P≤0.01 denotes significance as compared to control conditions and #P≤0.01 denote significance as compared to SDF-1α/CXCL12 treated conditions.

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Figure 4. SDF-1α/CXCL12 treatment results in the release of ATP through the Panx1 channels pore

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Untreated CD4+ T Lymphocytes (Control) and untreated cells in the presence of probenecid (Control + Probenecid) had minimum release of ATP in the media. In contrast, CD4+ T Lymphocytes stimulated with SDF-1α/CXCL12 had a significant increase (*P≤0.01 as compared to control conditions; n=3) in ATP release at all time points examined (1, 5, 10,15, 30,45, 60 minutes) Pre-treatment of CD4+ T lymphocytes with probenecid (SDF-1+ probenecid) and mimetic peptide (SDF-1 + peptide) prevented the increase in extracellular ATP levels induced by SDF-1α/CXCL12 treatment alone. CD4+ T lymphocytes pre-treated with Scramble mimetic peptide (SDF-1 + Scr Peptide) was used as a control. *P≤0.01 denotes significance as compared to control conditions. Error bars represent standard error (n=3).


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Figure 5. Blocking Panx1 channels significantly prevents migration of CD4+ T lymphocytes in response to SDF-1α/CXCL12

(A) CD4+ T lymphocytes were added to the top of a 3 μm tissue culture insert and cells were allowed to transmigrate for 2 hours in response to SDF-1α/CXCL12 (100ng/ml) in the presence and absence of Panx1 blockers. After transmigration cells were collected from the lower chamber and counted using trypan blue staining and FACS. Dot plots of a representative experiment are shown. In each plot, representative numbers of transmigrating cells are shown on the top right hand corner (B) Untreated CD4+ T Lymphocytes (Control) and untreated cells in the presence of probenecid (Control+ Prob) had few cells J Immunol. Author manuscript; available in PMC 2017 May 15.

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transmigrating. CD4+ T lymphocytes exposed to SDF-1α/CXCL12 (SDF-1) induced a significant number of cells to transmigrate across the tissue culture insert. Pre-treatment of CD4+ T lymphocytes with probenecid (SDF-1+ prob) and mimetic peptide (SDF-1 + peptide) significantly reduced the number of transmigrating CD4+ T Lymphocytes in response to SDF-1α/CXCL12. Pre-treatment of CD4+ T lymphocytes with scramble mimetic peptide (SDF-1 + Scr) was used as a control. *P≤0.01 denotes significance as compared to control conditions and #P≤0.01 denotes significance as compared to SDF-1α/ CXCL12 treated conditions. Error bars represent standard error (n=3).


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Author Manuscript Author Manuscript Author Manuscript Figure 6. Panx1 is required for efficient transmigration of CD4+ T lymphocytes into the spinal cord of EAE mice

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(A) Panx1 knockout and WT spinal cords obtained from the acute (12–13 dpi) phases of EAE were stained with anti-Panx1 (Green) and anti-CD4 (red) antibodies and counterstained with DAPI (blue). WT mice that were not induced to develop EAE were used as a control. (B) Spinal cords of Panx1 knockout mice derived from the acute phase of EAE exhibited fewer number of CD4+ T lymphocyte cells compared to WT mice. In agreement, these Panx1 KO animals have a delayed onset of clinical symptoms. No significant difference in terms of number of infiltrating cells was seen between WT and Panx1 deficient mouse spinal cords obtained from the chronic phase (35 dpi) of EAE (data not shown). Non-EAE controls J Immunol. Author manuscript; available in PMC 2017 May 15.

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WT and Panx1 KO spinal cords displayed limited number of infiltrating CD4+ T lymphocytes. *P≤0.01 and error bars represent standard error. n.s.= not significant


Th40 cells (CD4+CD40+ Tcells) drive a more severe form of Experimental Autoimmune Encephalomyelitis than conventional CD4 T cells.

Role of glycine N-methyltransferase in the regulation of T-cell responses in experimental autoimmune encephalomyelitis.

Suppressor T cells prevent experimental autoimmune encephalomyelitis in mice.

The Role of ERK Signaling in Experimental Autoimmune Encephalomyelitis.

Cytokine-regulated neutrophil recruitment is required for brain but not spinal cord inflammation during experimental autoimmune encephalomyelitis.

Experimental Autoimmune Encephalomyelitis in Marmosets.

IL-15-dependent CD8+ CD122+ T cells ameliorate experimental autoimmune encephalomyelitis by modulating IL-17 production by CD4+ T cells.

Functional genomics analysis of vitamin D effects on CD4+ T cells in vivo in experimental autoimmune encephalomyelitis ‬.

Intraperitoneal injection of CD4+ T cells induces CD5 B cells. Implications for a regulatory role of CD5 B cells in experimental autoimmune encephalomyelitis.

Cutting Edge: Integrin α4 Is Required for Regulatory B Cell Control of Experimental Autoimmune Encephalomyelitis.

B Lymphocytes Are Required during the Early Priming of CD4+ T Cells for Clearance of Pneumocystis Infection in Mice.

Anti-CD48 Monoclonal Antibody Attenuates Experimental Autoimmune Encephalomyelitis by Limiting the Number of Pathogenic CD4+ T Cells.

Loss of Allograft Inflammatory Factor-1 Ameliorates Experimental Autoimmune Encephalomyelitis by Limiting Encephalitogenic CD4 T-Cell Expansion.

Grb2 Is Important for T Cell Development, Th Cell Differentiation, and Induction of Experimental Autoimmune Encephalomyelitis.

Role of CD8+ T cells in murine experimental allergic encephalomyelitis.

Change of paradigm: CD8+ T cells as important helper for CD4+ T cells during asthma and autoimmune encephalomyelitis.

Skeletal muscle findings in experimental autoimmune encephalomyelitis.

Phospholipase D1 mediates lymphocyte adhesion and migration in experimental autoimmune encephalomyelitis.

Increased sleep fragmentation in experimental autoimmune encephalomyelitis.

T cells in the spinal cord in experimental autoimmune encephalomyelitis are matrix adherent and secrete tumor necrosis factor alpha.

Interleukin-7 is required for CD4(+) T cell activation and autoimmune neuroinflammation.

Nigella sativa amliorates inflammation and demyelination in the experimental autoimmune encephalomyelitis-induced Wistar rats.

Suppression of experimental autoimmune encephalomyelitis by sulfasalazine.

Combinatorial Effect of Metformin and Lovastatin Impedes T-cell Autoimmunity and Neurodegeneration in Experimental Autoimmune Encephalomyelitis.