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Hypoxia is a common feature of critical illness and may arise as a consequence of a range of mechanisms. Failure to adequately oxygenate the blood as it traverses the lungs, impaired delivery due to vascular obstruction and the cell’s inability to utilise it effectively are all ways in which cells may be exposed to a hypoxic environment. In addition to this, in inflamed or infected tissue, hypoxia may be severe and arise as a result of reduced perfusion, microvascular injury and increased interstitial pressure all of which may be coupled with increased oxygen utilisation by immune cells[253-256].

Under normal conditions the oxygen tension to which cells are exposed lies in the range of 2- 9kPa. This equates to around 2.5-9% oxygen. However, in the context of infection, markedly lower levels of oxygen may be available with nadir values of less than 1% reported[253]. There is burgeoning evidence to suggest that hypoxia is not simply an epiphenomenon associated with infection, but that it does in fact regulate a range of immune processes and contributes towards the activation of the innate response.

These studies explore the hypothesis that as a regulator of the immune response to inflammation, DDAH2 may itself play an important role in modulating NO production in hypoxia.

4.1.1.1 Hypoxia inducible factor regulates the hypoxic response

Hypoxia inducible factors (HIF) were originally discovered in the early 1990s as one of the mechanisms by which erythropoietin was synthesised in hypoxic conditions[257, 258]. Regulated by both oxygen and iron levels, HIF is found in all mammalian cells and has been shown to regulate more than 100 genes in response to hypoxic stress. HIF regulated genes modulate metabolism, vascular tone, new vessel development and apoptosis, with implications in both healthy and disease states[259-262].

The HIF complex is comprised of the constitutive HIF1β which binds to one of two inducible components, HIF1α and HIF 2α. Under normal conditions, the inducible subunits are unstable and as a consequence are readily turned over via the ubiquitin-proteasome pathway[263] and by asparaginyl hydroxylase[264]. In hypoxia, these hydroxylase pathways are inhibited and the HIF proteins are stabilised. As a consequence, HIF1α and HIF 2α accumulate, translocate to the cell nucleus and form a heterodimer with HIF1β. This heterodimer then binds directly to regions of the promoter sequence of its target genes (Hypoxia response elements, HREs) to initiate transcription.

4.1.1.2 Hypoxia inducible factor and innate immune cells

Global knockout of HIF 1α is not compatible with life in murine models, however using a similar technique to that employed in this study, a mouse has been developed that is HIF1α- deficient only in macrophages, granulocytes and microglial cells. Whilst this mouse is phenotypically normal under control conditions, when exposed to an inflammatory stress, it displays significantly impaired macrophage activation and induction of the local inflammatory response[265].

The impact of the reduced macrophage function observed in the HIF1α knockout mouse is an impaired capacity to kill both Gram-positive and Gram-negative bacteria[265, 266]. By contrast, a hypoxic environment appears to improve bactericidal activity of normal macrophages and neutrophils[266, 267]. This process may be mediated by a number of mechanisms including, in part, by the HIF1α-induced upregulation of iNOS [266] and increased cytokine production[267]. It is interesting to note that in contrast to these hypoxia- mediated processes, HIF does not appear to modulate reactive oxygen species (ROS) synthesis by macrophages which appears to be independent of the presence of HIF1α[268] This hypoxia-mediated augmentation of the innate response appears to be synergistically regulated by nuclear factor κB (NF-κB)[269] whereby hypoxia stimulates the activation of NF-κB by inhibiting metabolising hydroxylases. NF-κB can in turn provoke the upregulation of HIF1α, thus HIF synthesis is a major regulator of innate immune response[270].

In animal models of sepsis, HIF1α deletion in macrophages and granulocytes is protective against a normally fatal dose of LPS and significantly reduces the systemic inflammatory state [268].

4.1.1.3 The impact of hypoxia on the inflammatory response

Hypoxia has been shown to induce the synthesis of a number of pro and anti-inflammatory mediators of inflammation by innate immune cells. The list is extensive and includes IL-1, TNF-α, PGE2, IFN-γ and IL-10. This has been demonstrated in both human and murine macrophages with a significant number shown to be HIF-mediated[271, 272].

Studies also report a number of mechanisms by which hypoxia can regulate NO production by immune cells. It is well established that oxygen is essential for the oxidation of NADPH by NOS in the synthesis of NO. Two moles of oxygen are required for the production of 1 mole of NO[273]. For this reason, it is well established that in low oxygen conditions, isolated macrophages, particularly in murine cell lines and primary culture, produce only minimal NO when exposed to a pro-inflammatory stimulus in a hypoxic environment[274]. However, in murine and human cells it has been demonstrated that hypoxia, via the HIF1α- mediated stimulation of iNOS HRE elements, is able to upregulate iNOS synthesis and thus protein expression[272, 275]. As a consequence, murine cells display increased iNOS expression, but no apparent elevation of NO synthesis when cultured in hypoxic conditions. If however, they are subsequently returned to a normoxic environment, this upregulation of