The Effect of Low Oxygen With and Without Steady-state Hydrogen Peroxide on Cytokine Gene and Protein Expression of Monocyte-Derived Macrophages Henry Owegi 1 , Hongtao Li 2 , Mark Bouwens 3 , Stephane Egot-Lemaire 4 , Sebastian Mueller 5 , Roy W. Geib 2 , Gabi N. Waite 4 1 Department of Chemistry and Physics, Indiana State University; 2 Department of Microbiology and Immunology, Indiana University School of Medicine, Terre Haute, IN, USA; 3 Cell Biology and Immunology Group, Wageningen University, Wageningen, The Netherlands; 4 Department of Cellular and Integrative Physiology, Indiana University School of Medicine; Terre Haute, IN, USA; 5 Department of Internal Medicine, Salem Medical Center, University of Heidelberg, Heidelberg, Germany. ABSTRACT An early event during inflammation and infection is the migration of monocytes into tissues where they differentiate into macrophages. Such monocyte-derived macrophages face an unfavorable environment characterized by extremely low oxygen tension and accumulation of reactive oxygen species such as hydrogen peroxide. Previous experiments showed that a macrophage cell line cultured under these conditions responded to inflammatory stimulants with an increased respiratory burst compared to cells kept at regular ambient oxygen conditions. The objective of the present study was to test if this effect was accompanied by an increased cytokine production. Using cytokine protein arrays and real time gene expression analysis, we indeed found that low oxygen exposure increased expression of characteristic macrophage inflammatory cytokines such as IL-1, IL-6, and TNF-alpha. Interestingly, cells exposed to low oxygen and steady-state hydrogen peroxide levels did not show this increase, indicating different cell mechanisms might be involved in increasing macrophage hydrogen peroxide and cytokine release. Keywords: cytokines, chemokines, transcription factors, THP-1, hypoxia, respiratory burst, inflammation, HIF-1α, Ο 2 , Η 2 Ο 2 . INTRODUCTION Macrophages are highly versatile immune cells. They are derived from monocytes which are produced in bone marrow and released into blood. Monocytes turn into macrophages when they leave the blood and enter areas of infection [1]. There, they produce reactive oxygen species to kill engulfed pathogens or to attack larger pathogens outside of the cells. This marks the onset of inflammation and wound healing. There are different types of macrophages. Some, classically activated macrophages, called M1, specialize in producing reactive oxygen species, while others alternatively activated macrophages, called M2, are more active during healing by clearing up debris via phagocytosis [1]. The different actions of macrophages, from recruitment towards specialization, are mainly controlled by cytokines and chemokines that are produced by other immune cells and by the