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During sepsis there is evidence of mitochondrial OXPHOS dysfunction, excessive mitochondrial ROS production and mtDNA depletion, while the presence of dysfunctional mitochondria can modulate inflammatory responses. In order to compensate for these adverse effects cells must be able to selectively remove dysfunctional organelles and generate new mitochondria to replace them. This compensatory response occurs by the coordinated induction of mitophagy and mitochondrial biogenesis (López-Armada et al., 2013).

1.7.1 Mitochondrial biogenesis

Mitochondrial biogenesis is a highly dynamic process during which pre-existing mitochondria grow and divide. It requires the coordinated expression and interaction of a number of mitochondrial genes encoded by both nuclear DNA and mtDNA (Lee and Wei, 2005). Alterations in physiological conditions or cellular energy requirements trigger a complex network of hormones and signalling pathways that lead to the

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expression of a number of mitochondrial transcription factors (Weitzel and Alexander Iwen, 2011). Peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α) acts as a master regulator of mitochondrial biogenesis through the induction of

nuclear transcription factors such as nuclear respiratory factors 1 and 2 (NRF-1 and -2) (Lee and Wei, 2005; Crouser, 2010). These transcription factors promote the

expression of nuclear genes the products of which form essential mitochondrial constituents or act to influence important mitochondrial functions. In turn, the replication of mtDNA is controlled by mitochondrially-targeted proteins encoded on the nuclear genome, including polymerase-γ (POLG), the DNA polymerase responsible for mtDNA replication, and mitochondrial transcription factor A (TFAM), which directly interacts with mtDNA to promote replication and the transcription of mtDNA-encoded genes (Kang et al., 2007).

There are an increasing number of diverse studies in animal models which indicate that expanding the mitochondrial population is an important process in promoting survival and recovery from sepsis. In one highly innovative study the intra-tracheal instillation of bone marrow derived stromal cells increased alveolar ATP generation and protected against LPS-induced acute lung injury through the direct transfer of mitochondria to alveolar epithelial cells (Islam et al., 2012). Elsewhere, the recovery of metabolic function and mtDNA copy number in hepatocytes was found to be

dependent on the sustained expression of the activators of mitochondrial biogenesis in a murine model of bacterial peritonitis (Haden et al., 2007). In addition, up-regulation of PGC-1α expression has been shown to promote recovery from LPS-induced acute kidney injury in mice and the restoration of mitochondrial and cellular function following oxidant injury in rabbit renal proximal tubular cells (Rasbach and Schnellmann, 2007; Tran et al., 2011).

In animal sepsis models the induction of mitochondrial biogenesis appears to be directly triggered by the binding of ligands to PRRs and the resultant activation of inflammatory signalling pathways (Drabarek et al., 2012). Mice treated with a sub- lethal dose of heat-killed Escherichia coli were found to have an initial loss of mtDNA and mitochondrial proteins, which was rapidly reversed following the activation of mitochondrial biogenesis in a process dependent on the stimulation of toll-like

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Staphylococcus aureus Tlr-2-/- _{and Tlr-4}-/- _{knock-out mice had increased mortality in} association with a diminished induction of PGC-1α expression and persistent mtDNA depletion (Sweeney et al., 2010). In another study pharmacological inhibition of the inflammatory transcription factor NF-κB significantly delayed the increases in NRF-1, TFAM and mtDNA copy number that occurred in response to LPS-induced

inflammation in mice and also in human cell lines (Suliman et al., 2010).

In contrast to these animal studies, the evidence for mitochondrial biogenesis induction in human patients with sepsis is less well established. A study of 16 muscle biopsies from patients with sepsis-induced multi-organ failure showed that the survivors had early increases in expression of PGC-1α and mitochondrial antioxidants (Carré et al., 2010). However, a similar investigation looking at muscle cell

transcriptome revealed that incomplete and uncoordinated expression of

mitochondrial transcription factors and genes during critical illness may lead to a failure to maintain adequate mitochondrial function (Fredriksson et al., 2008). Additional longitudinal studies will, therefore, be important to clarify the precise role of mitochondrial biogenesis, particularly in human monocytes, during the recovery of critically ill patients following an inflammatory insult.

1.7.2 Mitophagy

Through encapsulation in an autophagosome and subsequent lysosomal degradation, damaged and dysfunctional cellular contents are catabolised during autophagy (Lee et

al., 2012). Mitophagy is a specialised form of autophagy which involves the removal of

mitochondria from a cell in response to developmental demands or in order to maintain quality control (Youle and Narendra, 2011). Dysfunctional mitochondria, particularly those generating excessive ROS or with depolarisation of the mitochondrial membrane potential (Δψm, generated by the transport of protons across the inner mitochondrial membrane during OXPHOS), are selectively targeted for mitophagy by the accumulation of phosphatase and tensin homologue-induced putative kinase-1 (PINK1) which leads to the recruitment of the mitophagy activator Parkin (Narendra et

al., 2008; Frank et al., 2012; Gilkerson et al., 2012; Hill et al., 2012).

There is emerging evidence that mitophagy is a critical compensatory response in sepsis. In general there is a lack of clinical data from human patients with sepsis;

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although one study found that PINK1 levels were higher (suggesting increased mitophagy) in PBMCs from 8 septic patients compared to 14 critically ill controls (Mannam et al., 2014). This is consistent with murine models in which a septic insult has been found to lead to the induction of mitophagy in association with the recovery of mitochondrial function (Carchman et al., 2013; Chang et al., 2015). Conversely, in a rabbit model of critical illness non-survivors had evidence of inadequate autophagy alongside increased organ damage and a greater impairment of mitochondrial

respiration (Gunst et al., 2013). Other studies have indicated that inhibiting autophagy leads to the accumulation of damaged mitochondria and the persistence of oxidative stress following an inflammatory stimulus (Nakahira et al., 2011; Zhou et al., 2011; Motori et al., 2013). Furthermore, defective or inhibited mitophagy may also result in a failure to clear the mitochondrial DAMPs arising from dysfunctional mitochondria, which can exacerbate inflammation due to an increase in NLRP3 inflammasome formation (Nakahira et al., 2011; van der Burgh et al., 2014).

Murine sepsis models also suggest that the induction of mitophagy is closely

integrated with the activation of mitochondrial biogenesis following an inflammatory insult. After an intra-abdominal Staphylococcus aureus infection there was concurrent up-regulation of mitophagy and the transcription of the key biogenesis regulators Pgc-

1α and Tfam in the pulmonary tissue of mice (Chang et al., 2015). Elsewhere, after

both caecal ligation and puncture (CLP) and LPS exposure, mice in which the resultant contemporaneous induction of mitophagy and mitochondrial biogenesis was blunted had a higher mortality (Mannam et al., 2014). Separately, the simultaneous activation of mitophagy and mitochondrial biogenesis occurring after either CLP or treatment with LPS has been shown to be abolished by inhibiting TLR-4 signalling (Carchman et

al., 2013). Intriguingly, this study also suggests that the presentation of mtDNA to the

intracellular PRR TLR-9 during mitochondrial degradation may activate mitochondrial biogenesis, as inhibiting either mitophagy or TLR-9 signalling prevented the LPS- induced up-regulation of PGC-1α, NRF-1 and TFAM.

Thus, there is growing evidence from animal models to suggest that mitophagy is an essential part of the adaptive response to sepsis which is closely integrated with mitochondrial biogenesis. However, there is a clear need for clinical studies in order to confirm the relevance of these findings to human sepsis.

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