Macrophage specific DDAH2 knockout in sepsis

The demonstration that excess mortality in DDAH2 deficient animals with sepsis arose during the first 24 hours of infection and was not mediated by vascular dysfunction, led to the suggestion that an impaired innate immune response was responsible. We therefore used the LoxP Cre recombinase mediated macrophage DDAH2 deficient mice in our sepsis model to explore this hypothesis.

6.3.3.1 Mortality

The knockout of DDAH2 in macrophages and granulocytes resulted in a near identical pattern of exaggerated early mortality to that seen in the global knockout mice. The degree of illness severity was similar in the Ddah2MΦ- mice to that seen in the Ddah2-/- mice when temperature was measured at 18 hours after the onset of sepsis, consistent with a similar pattern of early deterioration in the knockout mice of both groups. At termination, temperatures were similar suggesting consistent illness severity at the threshold for experimental cessation. The similarities in response across the two transgenic models suggest that it is innate immune cell macrophage DDAH2 that plays a critical role in the regulation of the innate immune response and that this has a significant impact on the systemic response to infection.

6.3.3.2 Cardiovascular function in sepsis

Assessing the cardiovascular impact of macrophage specific knockout of DDAH2 in septic mice proved challenging. The attempt to undertake in vivo radiotelemetry in Ddah2MΦ- and Ddah2flox/flox controls proved impossible when both knockout and control groups developed features of proximal gastrointestinal tract ischaemia making continuing with the experiment impossible. The mechanism for this apparent ischaemic complication was not immediately clear. Given that the finding arose in both groups of mice, it did not appear likely that this was DDAH2 mediated, instead it was suggested that a LoxP mediated phenomenon common to both groups might be responsible. The Cre LoxP model has been associated with a number of off target genetic and developmental effects [366, 367], and whilst unreported in the literature, a vascular malformation impairing mesenteric blood supply which only became apparent when the great vessel circulation was disrupted is a possible mechanism for this and may arise as a consequence of unidentified genetic variability introduced following breeding between floxed and Cre mice.

Instead of in vivo monitoring, measurement of anaesthetised blood pressure using the Millar catheter was considered the best alternative approach. These experiments revealed no significant differences at six hours after the onset of sepsis in systolic or diastolic blood pressure between the knockout and control groups. This method is limited by the single time point employed and the requirement for anaesthesia to facilitate catheter insertion. The use of volatile anaesthesia in sepsis mandated an early time point for assessment of haemodynamics as the volatile anaesthetic agents used in maintaining the sedation cause systemic vasodilatation. This both increases the risk of death causes by catastrophic hypotension and also reduces the sensitivity of the system to detect subtle differences in blood pressure between knockout and control groups. This contributes to the significant hypotension and also the similarities between the groups when tested.

6.3.3.3 Methylarginine handling

Plasma methylarginines in the macrophage specific knockout mice and their floxed controls were similarly elevated to the level seen in the global knockout models. No significant differences were observed in systemic concentrations of NO or methylarginines between Ddah2MΦ- _{and the relevant controls. The absence of differences between these two models in} the systemic concentrations does not necessarily reflect changes concentrations of MAs and local NO synthesis in macrophages. This is because whilst effects of DDAH2 knockout in macrophages at a cellular level may have a significant impact, the contribution that they make to the ‘pool’ of MAs and NO found in the plasma is relatively modest. This is in contrast to the impact of global knockout where multiple tissues are contributing additional MAs to the plasma.

6.3.4 Strengths and limitations

These studies explore, for the first time, the impact of DDAH2 knockout on the whole organism response to severe infection. The strengths of this study include the use of two different transgenic approaches. Both of which are associated with their own challenges, however the consistency of the response to sepsis across them makes the finding that DDAH2 is critical in regulating the systemic innate response to sepsis robust.

Corroborating this finding with the observations that bacterial load is significantly elevated in the abdominal cavity and also the blood in both knockout models suggests that the impacts of DDAH2 knockout on monocyte function observed previously lead to impaired bactericidal activity in vivo.

This, coupled with the observation that both at rest and in both models of sepsis, there are minor differences only between vascular function in knockout mice and their controls suggests that the hypotension seen in the knockout models is likely to be mediated by an exaggerated inflammatory state caused by an inability to eradicate bacteria rather than intrinsic dysfunction of vascular activity.

The inability to undertake in vivo monitoring of haemodynamics in the macrophage specific knockout mice and their controls is a limitation of this study. Had this been possible, it would have been valuable to observe whether the hypotension seen in the global knockout mice was also seen in the Ddah2MΦ- mice. Had it been present, this would have confirmed the observation that intrinsic vascular dysfunction did not play a role in the sepsis induced hypotension seen in DDAH2 global knockout mice.

An additional limitation includes the use of only male mice in this study. It has been reported that female mice display quantitatively different responses to the septic insult[368]. It is not clear how this would affect the findings presented here but is the subject of further exploration.

6.3.5 Future work

In addition to understanding the role of sex hormones in DDAH2 knockout models of sepsis, it would also be interesting to study the impact of sepsis on the only other tissue that expresses DDAH2 exclusively – the heart. The development of a cardiac specific DDAH2 knockout mouse would provide valuable insights in to the role of DDAH2 in regulating the cardiac stress response.

Whilst understanding the mechanisms of mortality provides potential diagnostic, risk stratification and therapeutic insights, none of the studies undertaken here explore the impact of knockout on survivors from sepsis. With increasing interest in the survivor syndrome of sepsis and the long term consequences of the disease, a ‘sepsis survivor’ model, developed for use in the DDAH1 and DDAH2 knockout rats that the group is currently developing will provide great insights into the mechanisms of recovery from severe infections.

6.3.6 Summary statement

 Global knockout of DDAH2 in mice results in a developmentally normal mouse with a subtle hypertensive phenotype associated with exertion

 Global DDAH2 knockout results in systemic and organ specific dysregulation of methylarginine concentrations, with increased clearance of both L-NMMA and ADMA in the urine

 Global knockout of DDAH2 causes hypotension, impaired bactericidal activity and excess early mortality in a caecal ligation and puncture model of septic shock.

 In sepsis, global DDAH2 knockout is associated with systemic derangement of methylarginines

 Macrophage specific knockout of DDAH2 recapitulates the impaired bactericidal activity and early mortality from sepsis of global knockout suggesting the macrophage DDAH2 is a critical player in the innate immune response.

7 Endogenous inhibitors of nitric oxide synthesis and their

regulators in human sepsis

7.1 Introduction

The work reported here, in conjunction with that undertaken previously presents strong evidence from animal experimentation that DDAH1 and DDAH2 both play important roles in the response to sepsis. It appears that in rodent models, DDAH1 inhibition or knockout leads to a protective effect based upon improvement in vascular tone and preservation of organ perfusion[47, 213, 223]. In contrast, knockout of DDAH2 results in significant impairment of the immune response which results in reduced ability to eradicate bacteria. This in turn leads to excess mortality in rodent models of sepsis[30].

These observations provide mechanistic insight into the role of each DDAH isoform in regulating NO synthesis in their respective tissue distributions. Understanding the relevance of these observations in human disease requires an alternative approach. Building upon small studies in the area to date, this chapter focuses on the largest observational study of methylarginines and their regulators ever undertaken in patients with septic shock.