Granulocytes and monocytes/macrophages of the myeloid lineage are the chief cellular agents of innate immunity. Here, we have examined the inflammatory response in mice with conditional knockouts of the hypoxia responsive transcription factor HIF-1alpha, its negative regulator VHL, and a known downstream target, VEGF. We find that activation of HIF-1alpha is essential for myeloid cell infiltration and activation in vivo through a mechanism independent of VEGF. Loss of VHL leads to a large increase in acute inflammatory responses. Our results show that HIF-1alpha is essential for the regulation of glycolytic capacity in myeloid cells: when HIF-1alpha is absent, the cellular ATP pool is drastically reduced. The metabolic defect results in profound impairment of myeloid cell aggregation, motility, invasiveness, and bacterial killing. This role for HIF-1alpha demonstrates its direct regulation of survival and function in the inflammatory microenvironment.

IntroductionThe major outer membrane component of gram-negative bacteria, lipopolysaccharide (LPS) or endotoxin, is a potent activator of monocyte or macrophage function leading to responses that are both protective and injurious to the host. 1 LPS induces the expression of the procoagulant molecular tissue factor (TF) and inflammatory cytokines such as tumor necrosis factor alpha (TNF-␣). 2 LPS binds to LPS-binding protein (LBP) in serum, followed by the binding of the LBP/LPS complex to CD14, which activates signal transduction pathways and transcription factors and induces gene expression. 1 However, the requirement for CD14 can be bypassed by treating cells with high doses of LPS, 3 suggesting that CD14 serves to present the LBP/LPS complex to a less abundant receptor. Recently, it was shown that the Lps gene responsible for LPS hyporesponsiveness in C3H/HeJ mice is a mutant form of the Toll-like receptor (Tlr)4 gene. 4 These studies and others demonstrated that TLR4 is the long-sought LPS signaling receptor. 5 LPS has been shown to activate members of the mitogenactivated protein kinase (MAPK) family, including extacellular signal-regulated kinases (ERK)1/2, 6,7 c-Jun amino terminal kinases (JNKs), 8 and p38. 9 Inhibition of MAPK kinase (MEK) in monocytes by a specific inhibitor U0126 reduced LPS induction of several inflammatory cytokines, including interleukin-1, interleukin-8, and TNF-␣ as well as prostaglandin E 2 , indicating a role for the ERK1/2 pathway in LPS signaling independent of the JNK and p38 pathways. 10 LPS stimulation of monocytes activates many transcription factors, including the nuclear factor NF-B/Rel family, which induce genes encoding various inflammatory mediators. 2 In unstimulated monocytes the NF-B/Rel proteins are retained in the cytoplasm by their interaction with the inhibitors IBs. 11 LPS stimulation of monocytes leads to the phosphorylation of IBs by IB kinases (IKKs), leading to the rapid translocation of NF-B/ Rel proteins to the nucleus. 12 We and others have demonstrated that LPS activation of IKK␤ is required for B-dependent transcription and TNF-␣ expression in human monocytes and THP-1 cells. [13][14][15] The transcription factor Egr-1 is an 80-kd nuclear phosphoprotein containing 3 zinc-finger DNA-binding domains. 16 It is rapidly activated in various cell types in response to a variety of stimuli. [17][18][19] For instance, LPS stimulation of murine macrophages induced a rapid induction of Egr-1 transcription, messenger RNA (mRNA), and protein. 20 The Egr-1 promoter contains several serum response elements (SREs) and Ets binding sites that mediate induction. [21][22][23] Serum response factor (SRF) and ternary complex factors (TCFs) form a ternary complex at these SRE and Ets sites. The TCF The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 U.S.C. section 1734. For personal use only. on May 9, 2018. by guest www.bloodjou...

Sepsis is associated with a systemic activation of coagulation and an excessive inflammatory response. Anticoagulants have been shown to inhibit both coagulation and inflammation in sepsis. In this study, we used both genetic and pharmacologic approaches to analyze the role of tissue factor and protease-activated receptors in coagulation and inflammation in a mouse endotoxemia model. We used mice expressing low levels of the procoagulant molecule, tissue factor (TF), to analyze the effects of TF deficiency either in all tissues or selectively in hematopoietic cells. Low TF mice had reduced coagulation, inflammation, and mortality compared with control mice. Similarly, a deficiency of TF expression by hematopoietic cells reduced lipopolysaccharide (LPS)-induced coagulation, inflammation, and mortality. Inhibition of the downstream coagulation protease, thrombin, reduced fibrin deposition and prolonged survival without affecting inflammation. Deficiency of either protease activated receptor-1 (PAR-1) or protease activated receptor-2 (PAR-2) alone did not affect inflammation or survival. However, a combination of thrombin inhibition and PAR-2 deficiency reduced inflammation and mortality. These data demonstrate that hematopoietic cells are the major pathologic site of TF expression during endotoxemia and suggest that multiple protease-activated receptors mediate crosstalk between coagulation and inflammation.