The depletion of mtDNA copy number induced by transfection with TFAM siRNA every 48 hours for 8 days resulted in a significant impairment of both mitochondrial oxygen consumption and immune functions in THP-1 cells. These results confirm the findings from Chapter 6 in which ρ0 THP-1 cells, lacking mtDNA due to treatment with ethidium bromide, also had a significantly impaired ability to release TNFα in response to LPS and a complete loss of mtDNA-dependent OXPHOS complex activity. A concern with the interpretation of the results from ρ0 cells is that ethidium bromide is a non-specific DNA intercalating agent that has been shown to significantly alter nuclear gene
expression in addition to depleting mtDNA (Magda et al., 2008). However, given the consistency of the findings between different techniques and the more targeted action of TFAM siRNA, the reduced LPS-induced TNFα release by THP-1 cells lacking mtDNA does not appear to occur as a result of ‘off target’ effects of the method of mtDNA depletion.
In Chapter 6 it was also noted that the ρ0 THP-1 cells generated by incubation with ethidium bromide had an immune phenotype consistent with differentiation into alternatively-activated macrophage-like cells, with reduced pro-inflammatory cytokine release but increased phagocytosis of Staphylococcus aureus (Tiemessen et al., 2007; Murray and Wynn, 2011). However, unlike alternatively-activated macrophages, THP-1 cells with TFAM siRNA-induced mtDNA depletion did not release more anti-
inflammatory cytokines and actually had an inhibition of Escherichia coli phagocytosis. The differences in the phagocytic ability of ρ0 THP-1 cells and those depleted of mtDNA after transfection with TFAM siRNA may reflect effects of the different methods of depleting mtDNA. Alternatively, it is possible that there are specific differences in the ability of the THP-1 cells to phagocytose Gram positive and Gram negative bacteria (Skovbjerg et al., 2010). This theory is supported by the significantly lower median proportion of control THP-1 cells internalising bacteria when exposed to
Staphyloccocus aureus compared to Escherichia coli (14.7% (IQR 14-15) vs 24.8% (IQR
23.8-27.5), p=0.03). It may, therefore, be valuable to measure the effects of mtDNA depletion on the ability of THP-1 cells to phagocytose a variety of bacterial species.
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In addition to these key differences in immunological phenotype, the alterations in THP-1 cell metabolism in response to changes in mtDNA levels also refute the theory that differentiation into macrophage-like cells is occurring. Firstly, after transfection with TFAM siRNA THP-1 cells had a significant impairment of mitochondrial respiration, which contrasts with the up-regulation of OXPHOS activity seen in alternatively-
activated macrophages (Rodríguez-Prados et al., 2010). Furthermore, after removal of
TFAM siRNA a recovery in the ability of THP-1 cells to release TNFα occurred in parallel
with the restoration of mtDNA copy number and mitochondrial oxygen consumption. Although there is a degree of plasticity in macrophage differentiation, an increase in the ability to release pro-inflammatory cytokines happens during the shift towards a classically-activated macrophage phenotype, a process that involves a switch to glycolytic respiration rather than the recovery of OXPHOS activity that is seen in THP-1 cells after removal of TFAM siRNA (Murray and Wynn, 2011; O'Neill and Grahame Hardie, 2013).
The impairment of TNFα production by THP-1 cells after transfection with TFAM siRNA or treatment with ethidium bromide, therefore, appears to be due to the
consequences of mtDNA depletion. A reduction in TNFα release in response to an ex
vivo treatment with LPS is a key phenotypic feature of deactivated septic monocytes
that is linked to an increase risk of developing secondary infections and dying
(Cavaillon and Adib-Conquy, 2007; Hall et al., 2011). Small pilot studies have suggested that immunotherapies that restore LPS-induced TNFα release by septic monocytes may improve clinical outcomes (Döcke et al., 1997; Meisel et al., 2009). In this context the recovery of TNFα release in association with restoration of mtDNA copy number in THP-1 cells following removal of TFAM siRNA is potentially interesting. If this finding is confirmed in primary human monocytes, it is possible that critically ill patients with evidence of monocyte deactivation and mtDNA depletion could benefit from
interventions to stimulate mitochondrial biogenesis. Such an approach would provide a novel, biomarker-targeted therapy aimed at stimulating a specific aspect of the innate immune response that is dysfunctional in sepsis (Dare et al., 2009; Hotchkiss et
al., 2013b). However, stimulating mitochondrial biogenesis may not provide a panacea
for sepsis-induced monocyte dysfunction, as phagocytosis did not fully recover after removal of TFAM siRNA, despite restoration of mtDNA copy number.
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7.4.4 Conclusion
Inhibiting POLG expression by transfection of THP-1 cells with 30nM POLG siRNA for 8 days did not result in significant changes in mtDNA copy number or mitochondrial respiration. In contrast, transfection with siRNA targeted against TFAM produced a decrease in mtDNA copy number that occurred in parallel with an impairment of THP-1 cell immune functions, including a reduced ability to release TNFα in response to LPS, a key phenotypic feature of deactivated septic monocytes. Furthermore, the restoration of THP-1 cell mtDNA copy number after removal of TFAM siRNA was associated with the recovery of LPS-induced TNFα release. These results support the suggestion in Chapter 6 that immune dysfunction in THP-1 cells may be caused by depletion of mtDNA, a finding of potential importance to understanding sepsis-induced monocyte deactivation.
The underlying mechanisms behind the association between impaired immunity and mtDNA depletion will be explored in more detail in Chapter 8 . It remains possible that the effects on the ability of THP-1 cells to release TNFα are due to the metabolic consequences of the loss of mitochondrial respiration or that they occur due to a direct impact of the loss of mtDNA on immune and inflammatory signalling pathways (Dietl et al., 2010; West et al., 2011b).
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