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Background: Increased levels of CO2 reduce macrophage function during inflammatory processes and wound healing. However, little is known how macrophages sense CO2 and adapt upon high levels of CO2 during differentiation and activation. We, therefore, elucidated the effects of CO2 on gene and protein expression during basic inflammatory processes, such as monocyte differentiation and macrophage activation. Methods: Monocyte differentiation was induced by phorbol 12-myristate 13-acetate (PMA). Primary macrophages (BMDMs) were polarized using different cytokines to induce proinflammatory (M1) macrophages (lipopolysaccharides) or immunomodulatory (M2) macrophages (interleukin-4). Cells were simultaneously subjected to different levels of CO2. Morphological changes, mRNA and protein expression of markers for cell differentiation and macrophage polarization were determined. Cell culture medium was buffered and intracellular pH (pHi) was monitored to determine pH-dependent effects of CO2. Direct effects of CO2 on PKC protein function were analyzed. Results: High levels of CO2 attenuated PMA-induced cell differentiation of human monocytes. In BMDMs, CO2 significantly reduced transcript levels of M1-marker. Mechanistically, CO2 mitigated PMA-stimulated PKC activity and function. Experiments with buffered medium revealed that changes in pH were responsible for most, but not all, of the CO2-mediated effects on monocyte differentiation and macrophage polarization. Pharmacological and genetic inhibition of carbonic anhydrases (CA) uncoupled the CO2-elicited intracellular pH-response, thereby preventing CO2-sensitivity during monocyte differentiation, activation, and migration, which could be reversed by CRISPR-mediated upregulation of CA2. Conclusion: Carbon dioxide is highly immunomodulatory and might thus affect wound healing properties in vivo. In M1 (but not M2) macrophages, CO2 is sensed by CA2-driven changes in pHi. MJS received funding from the German Research Foundation (DFG; STR 1570/1-1) and the Braun Foundation (Braun®; BBST-D-18-00018). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.