Dimethyl Sulfoxide Modulates T Cell–Mediated Immunity to Attenuate Influenza A Virus Infection

Po-Hsuan Jeng,1,2 Meng-Wei Tsai,3 Shing-Hwa Huang,4 Tien-Ru Huang,1 Mei-Fei Chen,3 Gu-Jiun Lin,3,* Shao-Cheng Liu1,*1Department of Otolaryngology–Head and Neck Surgery, Tri-Service General Hospital, National Defense Medical University, Taipei, Taiwan; 2Graduate Institute of Medical Sciences, National Defense Medical University, Taipei, Taiwan; 3Graduate Institute of Biology and Anatomy, College of Medicine, National Defense Medical University, Taipei, Taiwan; 4Department of General Surgery, En Chu Kong Hospital, New Taipei City, Taiwan*These authors contributed equally to this workCorrespondence: Gu-Jiun Lin, Graduate Institute of Biology and Anatomy, College of Medicine, National Defense Medical University, No. 161, Section 6, Minquan East Road, Neihu District, Taipei, 114, Taiwan, Email [email protected] Shao-Cheng Liu, Department of Otolaryngology–Head and Neck Surgery, Tri-Service General Hospital, National Defense Medical University, No. 325, Section 2, Cheng-Gong Road, Neihu District, Taipei, 114, Taiwan, Email [email protected]: Influenza A virus (IAV) remains a major threat because vaccines provide limited cross-protection and antiviral resistance is rising. Dimethyl sulfoxide (DMSO) is immunomodulatory, but its effect on IAV has not been tested in vivo. We evaluated its efficacy and mechanism in a murine H1N1 model.Methods: Male C57BL/6 mice were intranasally challenged with an LD50 of A/Puerto Rico/8/1934 (H1N1) and given daily subcutaneous DMSO (0.22 g/kg/day) or phosphate-buffered saline (PBS) on days 1– 7. Survival was monitored for 21 days; lung and bronchoalveolar lavage fluid (BALF) were collected on day 8. Lung histology (hematoxylin and eosin), BALF cytokines (sandwich ELISA), lung and splenic IFN-γ–producing CD4+ and CD8+ T cells (flow cytometry), and whole-lung RNA sequencing (differential expression, KEGG enrichment) were assessed. BALF viral burden was measured by neuraminidase activity assay and TCID50. In vitro, splenic CD8+ T cells exposed to 0%, 0.5%, or 1.0% DMSO were immunoblotted for lymphocyte-activation gene 3 (LAG3).Results: DMSO significantly improved 21-day survival and attenuated alveolar inflammatory infiltration. Day-8 BALF viral titers were significantly lower with DMSO (mean 2.2× 103 vs 4.3× 104 TCID50/mL; p = 0.034), as was neuraminidase activity. BALF IFN-γ and TGF-β were elevated, whereas TNF-α, IL-6, and IL-1β were not. IFN-γ+ CD8+ T cells increased in the lung but not the spleen; CD4+ populations were unchanged. RNA sequencing showed enrichment of cytokine–cytokine receptor interaction and PI3K/Akt signaling and upregulation of T-box factors, PPAR-γ, and Nek kinases. In vitro, DMSO accelerated LAG3 induction in CD8+ T cells within 12– 48 h.Conclusion: DMSO is associated with attenuated IAV severity, a lower day-8 airway viral burden, and a localized expansion of IFN-γ–producing CD8+ T cells, without a concomitant rise in the pro-inflammatory cytokines TNF-α, IL-6, or IL-1β. These associations are correlative and support further investigation of DMSO as a host-directed adjunct for influenza.Keywords: dimethyl sulfoxide, influenza A virus, CD8+ T cell, interferon-gamma, PI3K/Akt signaling, immunomodulation