Abstract
IMT504 is a non-CPG, non-coding synthetic oligodeoxinucleotide (ODN) with immunomodulatory properties and a novel inhibitory role in pain transmission, exerting long-lasting analgesic effects upon multiple systemic administrations. However, its mechanisms of anti-nociceptive action are still poorly understood. In the present study in male adult rats undergoing complete Freund’s adjuvant-induced hindpaw inflammation, we focused in the analysis of the immunomodulatory role of IMT504 over the cellular infiltrate, the impact on the inflammatory milieu, and the correlation with its anti-allodynic role. By means of behavioral analysis, we determined that a single subcutaneous administration of 6 mg/kg of IMT504 is sufficient to exert a 6-week-long full reversal of mechanical and cold allodynia, compromising neither acute pain perception nor locomotor activity. Importantly, we found that the anti-nociceptive effects of systemic IMT504, plus quick reductions in hindpaw edema, were associated with a modulatory action upon cellular infiltrate of B-cells, macrophages and CD8+ T-cells populations. Accordingly, we observed a profound downregulation of several inflammatory leukocyte adhesion proteins, chemokines and cytokines, as well as of β-endorphin and an increase in the anti-inflammatory cytokine, interleukin-10. Altogether, we demonstrate that at least part of the anti-nociceptive actions of IMT504 relate to the modulation of the peripheral immune system at the site of injury, favoring a switch from pro- to anti-inflammatory conditions, and provide further support to its use against chronic inflammatory pain.
Similar content being viewed by others
Data Availability
Available if requested.
References
Baddack-Werncke U, Busch-Dienstfertig M, González-Rodríguez S, Maddila SC, Grobe J, Lipp M, Stein C, Müller G (2017) Cytotoxic T cells modulate inflammation and endogenous opioid analgesia in chronic arthritis. J Neuroinflammation 14:1–11. https://doi.org/10.1186/s12974-017-0804-y
Bannon AW, Malmberg AB (2007) Models of nociception: hot-plate, tail-flick, and formalin tests in rodents. In: current protocols in neuroscience. John Wiley & Sons, Inc, New York City, p unit 8.9
Bas DB, Su J, Wigerblad G, Svensson CI (2016) Pain in rheumatoid arthritis: models and mechanisms. Pain Manag 6:265–284. https://doi.org/10.2217/pmt.16.4
Basso L, Boué J, Mahiddine K, Blanpied C, Robiou-du-Pont S, Vergnolle N, Deraison C, Dietrich G (2016) Endogenous analgesia mediated by CD4+ T lymphocytes is dependent on enkephalins in mice. J Neuroinflammation 13:1–10. https://doi.org/10.1186/s12974-016-0591-x
Bauer S, Kirschning CJ, Häcker H et al (2001) Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci U S A 98:9237–9242. https://doi.org/10.1073/pnas.161293498
Boué J, Blanpied C, Brousset P, Vergnolle N, Dietrich G (2011) Endogenous opioid-mediated analgesia is dependent on adaptive T cell response in mice. J Immunol 153:485–493. https://doi.org/10.4049/jimmunol.1003335
Bui TM, Wiesolek HL, Sumagin R (2020) ICAM-1: a master regulator of cellular responses in inflammation, injury resolution, and tumorigenesis. J Leukoc Biol 108:787–799. https://doi.org/10.1002/JLB.2MR0220-549R
Chahin A, Opal SM, Zorzopulos J, Jobes DV, Migdady Y, Yamamoto M, Parejo N, Palardy JE, Horn DL (2015) The novel immunotherapeutic oligodeoxynucleotide imt504 protects neutropenic animals from fatal pseudomonas aeruginosa bacteremia and sepsis. Antimicrob Agents Chemother 59:1225–1229. https://doi.org/10.1128/AAC.03923-14
Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL (1994) Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 53:55–63. https://doi.org/10.1016/0165-0270(94)90144-9
Chiu IM, Heesters BA, Ghasemlou N, von Hehn CA, Zhao F, Tran J, Wainger B, Strominger A, Muralidharan S, Horswill AR, Wardenburg JB, Hwang SW, Carroll MC, Woolf CJ (2013) Bacteria activate sensory neurons that modulate pain and inflammation. Nature 502:52–57. https://doi.org/10.1038/nature12479
Choi Y, Young Wook Y, Heung Sik N et al (1994) Behavioral signs of ongoing pain and cold allodynia in a rat model of neuropathic pain. Pain 59:369–376. https://doi.org/10.1016/0304-3959(94)90023-x
Cook AD, Christensen AD, Tewari D, McMahon SB, Hamilton JA (2018) Immune cytokines and their receptors in inflammatory pain. Trends Immunol 39:240–255. https://doi.org/10.1016/j.it.2017.12.003
Coronel MF, Hernando-Insúa A, Rodriguez JM, Elias F, Chasseing NA, Montaner AD, Villar MJ (2008) Oligonucleotide IMT504 reduces neuropathic pain after peripheral nerve injury. Neurosci Lett 444:69–73. https://doi.org/10.1016/j.neulet.2008.07.045
Cunha JM, Cunha FQ, Poole S, Ferreira SH (2000) Cytokine-mediated inflammatory hyperalgesia limited by interleukin-1 receptor antagonist. Br J Pharmacol 130:1418–1424. https://doi.org/10.1038/sj.bjp.0703434
Cunha TM, Verri WA, Silva JS, Poole S, Cunha FQ, Ferreira SH (2005) A cascade of cytokines mediates mechanical inflammatory hypernociception in mice. Proc Natl Acad Sci U S A 102:1755–1760. https://doi.org/10.1073/pnas.0409225102
Dawes JM, Calvo M, Perkins JR, Paterson KJ, Kiesewetter H, Hobbs C, Kaan TKY, Orengo C, Bennett DLH, McMahon SB (2011) CXCL5 mediates UVB irradiation-induced pain. Sci Transl Med 3:90–60. https://doi.org/10.1126/scitranslmed.3002193
Elias F, Flo J, Lopez RA, Zorzopulos J, Montaner A, Rodriguez JM (2003) Strong cytosine-Guanosine-independent Immunostimulation in humans and other Primates by synthetic Oligodeoxynucleotides with PyNTTTTGT motifs. J Immunol 171:3697–3704. https://doi.org/10.4049/jimmunol.171.7.3697
Elias F, Flo J, Rodriguez JM, Nichilo AD, Lopez RA, Zorzopulos J, Nagle C, Lahoz M, Montaner A (2005) PyNTTTTGT prototype oligonucleotide IMT504 is a potent adjuvant for the recombinant hepatitis B vaccine that enhances the Th1 response. Vaccine 23:3597–3603. https://doi.org/10.1016/j.vaccine.2004.12.030
Ercan N, Uludag MO, Agis ER, Demirel-Yilmaz E (2013) The anti-inflammatory effect of diclofenac is considerably augmented by topical capsaicinoids-containing patch in carrageenan-induced paw oedema of rat. Inflammopharmacology 21:413–419. https://doi.org/10.1007/s10787-013-0175-7
Fehrenbacher JC, Vasko MR, Duarte DB (2012) Models of inflammation: carrageenan- or unit 5.4 complete freund’s adjuvant (CFA)-induced edema and hypersensitivity in the rat. Curr Protoc Pharmacol 0 5:Unit5.4. https://doi.org/10.1002/0471141755.ph0504s56
Ghasemlou N, Chiu IM, Julien JP, Woolf CJ (2015) CD11b+Ly6G- myeloid cells mediate mechanical inflammatory pain hypersensitivity. Proc Natl Acad Sci U S A 112:808–817. https://doi.org/10.1073/pnas.1501372112
Gonçalves dos Santos G, Delay L, Yaksh TL, Corr M (2020) Neuraxial cytokines in pain states. Front Immunol 10:1–17. https://doi.org/10.3389/fimmu.2019.03061
Hernando-Insúa A, Montaner AD, Rodriguez JM et al (2007) IMT504, the prototype of the immunostimulatory oligonucleotides of the PyNTTTTGT class, increases the number of progenitors of mesenchymal stem cells both in vitro and in vivo: potential use in tissue repair therapy. Stem Cells 25:1047–1054. https://doi.org/10.1634/stemcells.2006-0479
Hu Z, Deng N, Liu K, Zhou N, Sun Y, Zeng W (2020) CNTF-STAT3-IL-6 Axis mediates neuroinflammatory cascade across Schwann cell-neuron-microglia. Cell Rep 31:1–16. https://doi.org/10.1016/j.celrep.2020.107657
Ivetic A (2018) A head-to-tail view of L-selectin and its impact on neutrophil behaviour. Cell Tissue Res 371:437–453. https://doi.org/10.1007/s00441-017-2774-x
Jain A, Hakim S, Woolf CJ (2020) Unraveling the plastic peripheral Neuroimmune Interactome. J Immunol 204:257–263. https://doi.org/10.4049/jimmunol.1900818
Jiang BC, Liu T, Gao YJ (2020) Chemokines in chronic pain: cellular and molecular mechanisms and therapeutic potential. Pharmacol Ther 212:1–25. https://doi.org/10.1016/j.pharmthera.2020.107581
Kawai T, Akira S (2011) Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity 34:637–650. https://doi.org/10.1016/j.immuni.2011.05.006
Keating A (2012) Mesenchymal stromal cells: new directions. Cell Stem Cell 10:709–716. https://doi.org/10.1016/j.stem.2012.05.015
Kiguchi N, Maeda T, Kobayashi Y, Fukazawa Y, Kishioka S (2010) Macrophage inflammatory protein-1α mediates the development of neuropathic pain following peripheral nerve injury through interleukin-1β up-regulation. Pain 149:305–315. https://doi.org/10.1016/j.pain.2010.02.025
Knight BE, Kozlowski N, Havelin J, King T, Crocker SJ, Young EE, Baumbauer KM (2019) TIMP-1 attenuates the development of inflammatory pain through MMP-dependent and receptor-mediated cell signaling mechanisms. Front Mol Neurosci 12:1–16. https://doi.org/10.3389/fnmol.2019.00220
Krieg AM, Yi AK, Matson S, Waldschmidt TJ, Bishop GA, Teasdale R, Koretzky GA, Klinman DM (1995) CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374:546–549. https://doi.org/10.1038/374546a0
Krug A, Towarowski A, Britsch S, Rothenfusser S, Hornung V, Bals R, Giese T, Engelmann H, Endres S, Krieg AM, Hartmann G (2001) Toll-like receptor expression reveals CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with Cd40 ligand to induce high amounts of IL-12. Eur J Immunol 31:3026–3037. https://doi.org/10.1002/1521-4141(2001010)31:10<3026::AID-IMMU3026>3.0.CO;2-H
Labuz D, Berger S, Mousa SA, Zöllner C, Rittner HL, Shaqura MA, Segovia-Silvestre T, Przewlocka B, Stein C, Machelska H (2006) Peripheral antinociceptive effects of exogenous and immune cell-derived endomorphins in prolonged inflammatory pain. J Neurosci 26:4350–4358. https://doi.org/10.1523/JNEUROSCI.4349-05.2006
Laumet G, Ma J, Robison AJ, Kumari S, Heijnen CJ, Kavelaars A (2019) T cells as an emerging target for chronic pain therapy. Front Mol Neurosci 12:1–17. https://doi.org/10.3389/fnmol.2019.00216
Laumet G, Edralin JD, Dantzer R, Heijnen CJ, Kavelaars A (2020) CD3+ T cells are critical for the resolution of comorbid inflammatory pain and depression-like behavior. Neurobiol Pain 21:1–8. https://doi.org/10.1016/j.ynpai.2020.100043
Le Blanc K, Mougiakakos D (2012) Multipotent mesenchymal stromal cells and the innate immune system. Nat Rev Immunol 12:383–396. https://doi.org/10.1038/nri3209
Leiguarda C, Coronel MF, Montaner AD, Villar MJ, Brumovsky PR (2018) Long-lasting ameliorating effects of the oligodeoxynucleotide IMT504 on mechanical allodynia and hindpaw edema in rats with chronic hindpaw inflammation. Neurosci Lett 666:17–23. https://doi.org/10.1016/j.neulet.2017.12.032
Leussis MP, Bolivar VJ (2006) Habituation in rodents: a review of behavior, neurobiology, and genetics. Neurosci Biobehav Rev 30:1045–1064. https://doi.org/10.1016/j.neubiorev.2006.03.006
Machelska H, Mousa SA, Brack A, Schopohl JK, Rittner HL, Schäfer M, Stein C (2002) Opioid control of inflammatory pain regulated by intercellular adhesion molecule-1. J Neurosci 22:5588–5596. https://doi.org/10.1523/jneurosci.22-13-05588.2002
Maddila SC, Busch-Dienstfertig M, Stein C (2017) B lymphocytes express Pomc mRNA, processing enzymes and β-endorphin in painful inflammation. J NeuroImmune Pharmacol 12:180–186. https://doi.org/10.1007/s11481-016-9715-4
Monasterio G, Castillo F, Rojas L, Cafferata EA, Alvarez C, Carvajal P, Núñez C, Flores G, Díaz W, Vernal R (2018) Th1/Th17/Th22 immune response and their association with joint pain, imagenological bone loss, RANKL expression and osteoclast activity in temporomandibular joint osteoarthritis: a preliminary report. J Oral Rehabil 45:589–597. https://doi.org/10.1111/joor.12649
Montaner AD, Denichilo A, Rodríguez JM et al (2011) Addition of the immunostimulatory oligonucleotide IMT504 to a seasonal flu vaccine increases hemagglutinin antibody titers in young adult and elder rats, and expands the anti-hemagglutinin antibody repertoire. Nucleic Acid Ther 21:265–274. https://doi.org/10.1089/nat.2011.0284
Mousa SA, Machelska H, Schäfer M, Stein C (2000) Co-expression of β-endorphin with adhesion molecules in a model of inflammatory pain. J Neuroimmunol 108:160–170. https://doi.org/10.1016/S0165-5728(00)00284-8
Mousa SA, Shakibaei M, Sitte N, Schäfer M, Stein C (2004) Subcellular pathways of β-endorphin synthesis, processing, and release from Immunocytes in inflammatory pain. Endocrinology 145:1331–1341. https://doi.org/10.1210/en.2003-1287
Mousa SA, Shaqura M, Brendl U, al-Khrasani M, Fürst S, Schäfer M (2010) Involvement of the peripheral sensory and sympathetic nervous system in the vascular endothelial expression of ICAM-1 and the recruitment of opioid-containing immune cells to inhibit inflammatory pain. Brain Behav Immun 24:1310–1323. https://doi.org/10.1016/j.bbi.2010.06.008
Nwidu LL, Airhihen B, Ahmadu A (2016) Anti-inflammatory and anti-nociceptive activities of stem-bark extracts and fractions of Carpolobia Lutea (Polygalaceae). J Basic Clin Pharm 8:25–32. https://doi.org/10.4103/0976-0105.195097
Oh SB, Tran PB, Gillard SE, Hurley RW, Hammond DL, Miller RJ (2001) Chemokines and glycoprotein 120 produce pain hypersensitivity by directly exciting primary nociceptive neurons. J Neurosci 21:5027–5035. https://doi.org/10.1523/jneurosci.21-14-05027.2001
Pinho-Ribeiro FA, Verri WA, Chiu IM (2017) Nociceptor sensory neuron–immune interactions in pain and inflammation. Trends Immunol 38:5–19. https://doi.org/10.1016/j.it.2016.10.001
Poole S, Cunha FQ, Selkirk S, Lorenzetti BB, Ferreira SH (1995) Cytokine-mediated inflammatory hyperalgesia limited by interleukin-10. Br J Pharmacol 115:684–688. https://doi.org/10.1111/j.1476-5381.1995.tb14987.x
Prockop DJ, Oh JY (2012) Medical therapies with adult stem/progenitor cells (MSCs): a backward journey from dramatic results in vivo to the cellular and molecular explanations. J Cell Biochem 113:1460–1469. https://doi.org/10.1002/jcb.24046
Przewlocki R, Hassan AHS, Lason W, Epplen C, Herz A, Stein C (1992) Gene expression and localization of opioid peptides in immune cells of inflamed tissue: functional role in antinociception. Neuroscience 48:491–500. https://doi.org/10.1016/0306-4522(92)90509-Z
Rittner HL, Brack A, Machelska H, Mousa SA, Bauer M, Schäfer M, Stein C (2001) Opioid peptide - expressing leukocytes: identification, recruitment, and simultaneously increasing inhibition of inflammatory pain. Anesthesiology 95:500–508. https://doi.org/10.1097/00000542-200108000-00036
Rodriguez JM, Elias F, Montaner AD, Flo J, Lopez RA, Zorzopulos J, Franco RJ, Lenial SP, Lopez Salón M, Pirpignani ML, Solimano J, Garay G, Riveros D, Fernandez J, Cacchione R, Dupont J (2006) Oligonucleotide IMT504 induces an immunogenic phenotype and apoptosis in chronic lymphocytic leukemia cells. Medicina (B Aires) 66:9–16
Rodriguez JM, Marchicio J, López M, Ziblat A, Elias F, Fló J, López RA, Horn D, Zorzopulos J, Montaner AD (2015) PyNTTTTGT and CpG immunostimulatory oligonucleotides: effect on granulocyte/monocyte Colony-stimulating factor (GM-CSF) secretion by human CD56+ (NK and NKT) cells. PLoS One 10:1–20. https://doi.org/10.1371/journal.pone.0117484
Safieh Garabedian B, Poole S, Allchorne A et al (1995) Contribution of interleukin-1β to the inflammation-induced increase in nerve growth factor levels and inflammatory hyperalgesia. Br J Pharmacol 115:1265–1275. https://doi.org/10.1111/j.1476-5381.1995.tb15035.x
Saloman JL, Cohen JA, Kaplan DH (2020) Intimate neuro-immune interactions: breaking barriers between systems to make meaningful progress. Curr Opin Neurobiol 62:60–67. https://doi.org/10.1016/j.conb.2019.11.021
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682. https://doi.org/10.1038/nmeth.2019
Schreiter A, Gore C, Labuz D, Fournie-Zaluski MC, Roques BP, Stein C, Machelska H (2012) Pain inhibition by blocking leukocytic and neuronal opioid peptidases in peripheral inflamed tissue. FASEB J 26:5161–5171. https://doi.org/10.1096/fj.12-208678
Sitte N, Busch M, Mousa SA, Labuz D, Rittner H, Gore C, Krause H, Stein C, Schäfer M (2007) Lymphocytes upregulate signal sequence-encoding proopiomelanocortin mRNA and beta-endorphin during painful inflammation in vivo. J Neuroimmunol 183:133–145. https://doi.org/10.1016/j.jneuroim.2006.11.033
Stein C, Machelska H (2011) Modulation of peripheral sensory neurons by the immune system: implications for pain therapy. Pharmacol Rev 63:860–881. https://doi.org/10.1124/pr.110.003145
Taskinen HS, Röyttä M (2000) Increased expression of chemokines (MCP-1, MIP-1α, RANTES) after peripheral nerve transection. J Peripher Nerv Syst 5:75–81. https://doi.org/10.1046/j.1529-8027.2000.00009.x
Tisoncik JR, Korth MJ, Simmons CP, Farrar J, Martin TR, Katze MG (2012) Into the eye of the cytokine storm. Microbiol Mol Biol Rev 76:16–32. https://doi.org/10.1128/mmbr.05015-11
Vanderwall AG, Milligan ED (2019) Cytokines in pain: harnessing endogenous anti-inflammatory signaling for improved pain management. Front Immunol 10:1–15. https://doi.org/10.3389/fimmu.2019.03009
Wang Z, Chu X, Li N, Fu L, Gu H, Zhang N (2020) Engineered DNA nanodrugs alleviate inflammation in inflammatory arthritis. Int J Pharm 577:1–35. https://doi.org/10.1016/j.ijpharm.2020.119047
Wiedemann F, Link R, Pumpe K, Jacobshagen U, Schaefer HE, Wiesmüller KH, Hummel RP, Jung G, Bessler W, Böltz T (1991) Histopathological studies on the local reactions induced by complete fReund’s adjuvant (CFA), bacterial lipopolysaccharide (LPS), and synthetic lipopeptide (P3C) conjugates. J Pathol 164:265–271. https://doi.org/10.1002/path.1711640313
Woolf CJ (2020) Capturing novel non-opioid pain targets. Biol Psychiatry 87:74–81. https://doi.org/10.1016/j.biopsych.2019.06.017
Woolf CJ, Allchorne A, Safieh Garabedian B, Poole S (1997) Cytokines, nerve growth factor and inflammatory hyperalgesia: the contribution of tumour necrosis factor α. Br J Pharmacol 121:417–424. https://doi.org/10.1038/sj.bjp.0701148
Xanthos DN, Sandkühler J (2014) Neurogenic neuroinflammation: inflammatory CNS reactions in response to neuronal activity. Nat Rev Neurosci 15:43–53. https://doi.org/10.1038/nrn3617
Zhang N, Inan S, Cowan A, Sun R, Wang JM, Rogers TJ, Caterina M, Oppenheim JJ (2005) A proinflammatory chemokine, CCL3, sensitizes the heat- and capsaicin-gated ion channel TRPV1. Proc Natl Acad Sci U S A 102:4536–4541. https://doi.org/10.1073/pnas.0406030102
Zhao G, Jin H, Li J, Su B, du X, Kang Y, Wang X, Wang B (2009) PyNTTTTGT prototype oligonucleotide IMT504, a novel effective adjuvant of the FMDV DNA vaccine. Viral Immunol 22:131–138. https://doi.org/10.1089/vim.2008.0073
Zhou Q, Bao Y, Zhang X, Zeng L, Wang L, Wang J, Jiang W (2014) Optimal interval for hot water immersion tail-flick test in rats. Acta Neuropsychiatr 26:218–222. https://doi.org/10.1017/neu.2013.57
Zorzopulos J, Opal SM, Hernando-Insúa A, Rodriguez JM, Elías F, Fló J, López RA, Chasseing NA, Lux-Lantos VA, Coronel MF, Franco R, Montaner AD, Horn DL (2017) Immunomodulatory oligonucleotide IMT504: effects on mesenchymal stem cells as first-in-class immunoprotective/immunoregenerative therapy. World J Stem Cells World Jour 9:45–67. https://doi.org/10.4252/wjsc.v9.i3.45
Acknowledgements
We would like to thank Mrs. Gabriela Periz, Mr. Guillermo Gastón, Mr. Franco Puebla and Mr. Santiago Cabrera, for animal care and manipulation.
Funding
This work was supported by Argentinean National Agency for the Promotion of Science and Technology (PICTO-Startup 2016–0091 and PICT 2017–0969 (PRB)), Austral University (grant n° 80020160200010UA01, CL), International Brain Research Organization (IBRO, CL), Fondecyt (grant n°1181622) and CEDENNA AFB180001 Projects (LC).
Author information
Authors and Affiliations
Contributions
All authors state to have discussed the results and commented on the manuscript. Candelaria Leiguarda and Drs. Luis Constandil, Alejandro Montaner, Marcelo J. Villar and Pablo R. Brumovsky discussed and designed the project. Candelaria Leiguarda performed most experimental and analytical work, in collaboration with Drs. Constanza Potiliski (molecular array), Julia Rubione (flow cytometry), Pablo Tate (histology) and Veronica Bisagno (open-field test). The manuscript was written by Candelaria Leiguarda and Pablo Brumovsky, with assistance from all other authors (through discussion of all results and the strategy for its presentation in manuscript format).
Corresponding author
Ethics declarations
Competing Interests
None.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Fig. S1
Single s.c. IMT504 results in long-lasting reductions in mechanical allodynia. (A) A single s.c. administration of IMT504 (6 mg/kg; n = 6) blocks mechanical allodynia during 6 full weeks before returning to values comparable to CFA-VEH rats. (B) AUC of data in (A) (P = 0.0006, t = 4.95, df = 10; Hedges’g CFA-VEH: vs CFA-IMT504: 2.86). (PPTX 1.66 mb)
Fig. S2
Flow cytometry gating strategy for the isolation of myeloid and lymphoid populations. Cell surface antibodies were used to identify myeloid and lymphoid populations and subpopulations for B and T cells, T cytotoxic cells, monocytes and macrophages. All samples were first gated on a forward scatter (FS)/ side scatter (SS) plot and selected accordingly. (A) Flow cytometry shows myeloid (CD45+CD11b/c+; line outlines) and lymphoid (CD45+CD11b/c−; dotted lines outlines) cells recruitment into the hindpaw after CFA injection and IMT504 treatment. Myeloid cells were selected for their higher complexity and size as compared with lymphocytes at the FS/SS plot. (B) Monocytes were first gated for CD45+CD11b/c+ population and further phenotyped for CD172a+. (C) Macrophages population was selected by gating CD4+CD3− subset and further gating HIS36+ cells. (D) B-cells were gated for CD45+CD45RA+ population while (E) T-cells were then gated for CD45+CD3+ population and further gated for the subset of interest (CD4+CD8− and CD4−CD8+). Data was analyzed using FlowJo software. Myeloid and lymphoid population counts are expressed as percentage values, while subpopulations counts are expressed in absolute values. (PPTX 251 kb)
Rights and permissions
About this article
Cite this article
Leiguarda, C., Potilinski, C., Rubione, J. et al. IMT504 Provides Analgesia by Modulating Cell Infiltrate and Inflammatory Milieu in a Chronic Pain Model. J Neuroimmune Pharmacol 16, 651–666 (2021). https://doi.org/10.1007/s11481-020-09971-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11481-020-09971-2