147: Mechanistic insights into the anti-inflammatory actions of IMT504 � Focus on Macrophages

Topic: Basic Science
Subtopic: Pain category (ICD-11), Chronic neuropathic pain, , , , , , , , , , Neuroimmunology, Pharmacological interventions, Drug development

Background and Aims: Neuropathic pain, caused by a lesion or disease of the somatosensory system, is a debilitating condition with very limited therapeutic options. Macrophages are central to neuroinflammation driving the pathophysiology of neuropathic pain. IMT504, a non-CpG oligodeoxynucleotide, effectively reduces neuropathic pain-like behavior, through modulation of the immune microenvironment. However, if IMT504 directly modulates macrophages and through what types of signaling pathways, remains unknown. In this study, we employed bone marrow-derived macrophages (BMDMs) to evaluate potentially relevant events induced by the ODN. We also address the relevance of IL-10 in the mechanisms of action of IMT504 by use of knockout mice.

Methods: To determine IMT504-BMDM interactions, we employed a cell internalization assay on C57BL/6 mice BMDMs, after incubation with IMT504-AF488 10 µg/ml±0.1% Trypan Blue, followed by flow-cytometry analysis. ELISA analysis of TNFα, IL-6, IFN-β, IL-10, TGF-β, CXCL1, CCL2 in supernatant was evaluated for the following conditions in BMDMs: naïve control, IMT504-treated (3, 10, 30 ug/ml), LPS-stimulated (100 ng/ml for 6, 12 or 24h) and IMT504 pre-treated, LPS-stimulated. Finally, evaluation of mechanical and cold allodynia was performed in WT and IL-10 KO mice with spared nerve injury (SNI), 7 days after injury, and following 4 consecutive 20 µg intrathecal IMT504 administrations.

Results: More than 96% BMDMs showed IMT504 internalization across various time-points (P<0.0001); in addition, signs of accumulation were observed upon continuous exposure to ODN. IMT504 pre-treatment of BMDMs, followed by LPS stimulation, significantly enhanced IL-10 (P = 0.0009) and IFN-β (P = 0.0049) release compared to LPS alone at 12 and 24h; notably, IMT504 alone did not induce cytokine secretion. Intrathecal IMT504 successfully blocked mechanical and cold allodynia for up to eight days post-treatment. Analysis of pain-like behavior in IL-10 KO mice receiving IMT504 revealed a significant anti-allodynic effect, except at 24 to h post-administration.

Conclusions: IMT504 is rapidly and abundantly internalized by BMDMs. Its cytokine release modulation occurs only during inflammatory challenges, indicating that IMT504 acts as an immunomodulatory primer rather than a direct activator. Our results also confirm that intrathecal IMT504 is equally effective in reducing pain-like behavior in rats and mice. And while studies in knockout mice show that IL-10 is a required mediator, the remaining early and late-stage anti-allodynic effects suggest the involvement of IL-10-dependent and independent pathways. Finally, based on IMT504’s molecular structure and induction in IFN-β release, these effects are likely to involve DNA-sensing pathways, which will require further investigation.

References: • Ye, Y., H. Cheng, Y. Wang, Y. Sun, L. Dong Zhang, and J. Tang. "Macrophage: A Key Player in Neuropathic Pain." International Reviews of Immunology 43, no. 5 (2024): 326–39.
• Coronel, María Florencia, Marcelo José Villar, and Pablo Rodolfo Brumovsky. "Multitargeting Neuroimmune Pathways: Novel Candidates for Chronic Pain Relief." Trends in Pharmacological Sciences 46, no. 10 (2025): 940–45.
• Casadei, M., E. Fiore, J. Rubione, L. M. Domínguez, M. F. Coronel, C. Leiguarda, M. García, et al. "IMT504 Blocks Allodynia in Rats with Spared Nerve Injury by Promoting the Migration of Mesenchymal Stem Cells and by Favoring an Anti-inflammatory Milieu at the Injured Nerve." Pain 163, no. 6 (2022): 1114–29.
• Rubione, J., S. M. Sbrascini, B. Miguel, et al. "Modulation of the Inflammatory Response by Pre-emptive Administration of IMT504 Reduces Postoperative Pain in Rats and Has Opioid-Sparing Effects." The Journal of Pain 24, no. 6 (2023): 991–1008.
• Fiore, N. T., S. R. Debs, J. P. Hayes, S. S. Duffy, and G. Moalem-Taylor. "Pain-Resolving Immune Mechanisms in Neuropathic Pain." Nature Reviews Neurology 19, no. 4 (2023): 199–220.
• Yu, X., H. Liu, K. A. Hamel, et al. "Dorsal Root Ganglion Macrophages Contribute to Both the Initiation and Persistence of Neuropathic Pain." Nature Communications 11, no. 1 (2020): 264.

Presenting Author: Bernardo Miguel
Submitting Author: Bernardo Miguel

Senior Author: Thiago Mattar Cunha

IASP 2026 Global Year theme of Neuropathic Pain: Yes

• Conceicao E. Anibal Silva: PhD - Department of Pharmacology, Ribeirao Preto Medical School, University of Sao Paulo, Brasil
• Pablo R. Brumovsky: MD,PhD - Institute for Research in Translational Medicine (CONICET/Austral University), Argentina
• Thiago Mattar Cunha: PhD - Department of Pharmacology, Ribeirao Preto Medical School, University of Sao Paulo, Brasil