The dynamic interplay of M1/M2-like macrophages and adenosine deaminase activity modulates COVID-19 severity

The COVID-19 pandemic has highlighted the importance of understanding immune regulation underlying disease severity. Adenosine deaminase (ADA), an enzyme with ADA1 and ADA2 isoforms, modulates immune responses and is mainly expressed in lymphoid tissues. This study examined ADA activity and its relationship with macrophage polarization in COVID-19.

A total of 120 COVID-19 patients stratified by disease severity and 40 healthy controls were included. Total ADA (tADA), ADA1, and ADA2 activities were measured using the Giusti-Galanti colorimetric method in both patient sera and culture supernatants. Cytokine and chemokine profiles associated with M1- and M2-like macrophages were quantified by ELISA. To mechanistically evaluate macrophage polarization, naïve monocytes were co-cultured with SARS-CoV-2-responsive lymphocytes under a COVID-19 microenvironment (pCOV) generated using viral peptide pools in the presence of COVID-19 patient sera. Macrophage subsets were characterized by flow cytometry using canonical surface markers. The relationship between ADA2 activity and macrophage polarization was functionally assessed using pentostatin, whereas EHNA was used exclusively for the differential measurement of ADA isoenzyme activities.

Elevated total ADA activity was observed in critical patients, correlating with disease severity. Cytokine profiling revealed a hybrid M1/M2-like phenotype in severe cases, characterized by simultaneous pro- and anti-inflammatory mediator release. ADA2 activity showed a strong positive correlation with M2-associated factors, and flow cytometry confirmed the presence of CD206⁺CD86⁺ hybrid macrophages.

Overall, our findings suggest that ADA2 regulates macrophage plasticity in COVID-19, promoting M1/M2 hybrid polarization and contributing to immune dysregulation. ADA2 may serve as a biomarker for disease progression and a therapeutic target to modulate macrophage-driven inflammation in severe COVID-19.