Sepsis is an abnormal pathological state and consists of an initial acute hyper-inflammatory phase, to fight the infection followed by an immune-tolerant, hibernating phase to aid with recovery. Different metabolic processes and reprogramming unique to each phase have been described and play a role in the different, varied clinical manifestations in the two phases. Hyper-inflammatory phase is characterized by increased aerobic glycolysis (Warburg effect). This phase occurs as the host tissues reacts, with the aim of ramping up energy production, activating the innate immune cells to increase its pathogen killing capacity and control the spread of the infection and defend against the invading microbes [5, 10,11,12]. The late hypo-inflammatory, immune-tolerant phase is characterized by increased fatty acid oxidation in immune cells [10, 11]. A recent analysis of differential gene expression in the SARS-CoV-2 infected lung cell lines demonstrates upregulation of genes involved in mitochondrial cytokine/inflammatory signaling and downregulation in the mitochondrial organization, respiration, and autophagy genes . These findings provide evidence that mitochondrial disruption hinders an effective immune response, increases inflammation, and severity in the Covid-19-related sepsis.
Metabolic and immune consequences, and release of DAMP (Fig. 1)
A key feature of early sepsis is hypoxia. At the cellular level, hypoxia correlates with a switch to aerobic (glycolytic) respiration with alteration of normal oxidative phosphorylation and mitochondrial function due to reduced activity of complex I–IV and complex V of electron transport chain. There is increased oxygen consumption, elevated ATP production, and hyperglycemia. In addition, due to increased cell turnover, there is increased requirement for nucleotides and increase in pentose phosphate pathway [4, 5, 10, 12]. These altered metabolic pathways result in production of excessive cytoplasmic and mitochondrial ROS. Failure to clear the ROS by senescent mitochondria may impair and damage mitochondrial structure, lipid membranes, biogenesis units, and alter the intact mitochondrial DNA [5, 14]. Other mitochondrial factors exacerbating and causing elevated ROS include altered lipid peroxidation, glutathione (GSH) depletion, and altered iron metabolism . Loss of mitochondrial membrane integrity may release mtDNA in the systemic circulation. These circulating mtDNA fragments are also known as danger-associated molecular patterns (DAMP) and are similar to the pathogen associated molecular patterns (PAMP) which are released by microbes and identified by pathogen recognition receptors (PRR) on the immune cells. Nakahira et al. in a prospective study of 200 patients were first to demonstrate the association of elevated cell-free, plasma mtDNA levels with increased ICU-related mortality. They suggested mtDNA as a DAMP capable of propagating the inflammatory response seen in sepsis and other inflammatory conditions and a predictive biomarker for ICU-related mortality . Other DAMP proteins associated with mitochondrial derangement include mtROS, mt formyl peptides, Cytochrome C, cardiolipin, carbamoyl phosphate synthetase, heat shock proteins (HSP), and high-mobility group box proteins-1 (HMBG-1) [5, 6, 17, 18] (Fig. 1).
These endogenously derived DAMP molecules from senescent aged, disrupted mitochondria can activate the innate immune system similar to the PAMP molecules obtained from virus particles. The activation of the PRR (family of Toll-like receptors) results in the activation of immune cells consisting of neutrophils, macrophages, dendritic cells, and natural killer cells. Activated innate immunity initiates the transcription of genes which lead to the production of signal molecules such as prostaglandins, pro-inflammatory cytokines, chemokines, and interleukins, and also activates the complement system, thus evoking the inflammatory cascade [16, 19].
DAMP have also shown to activate the (Nuclear factor kappa-light chain enhancer of B cell) NF-Kβ pathway, activation of inflammasommes, and (stimulator of interferon genes) STING pathways. These mechanisms exacerbate and compound tissue dysfunction involving cardiovascular, immune, metabolic, endocrine, coagulation, and thermoregulatory pathways, and are responsible for the systemic effects and multi-organ failure [5, 16, 19,20,21]. Previous reports from septic patients and septic animal models indicate decrease in intact mtDNA and an increase in circulating free mtDNA fragments [16, 18].
Role of HIF-α/Sirtuins in sepsis (Fig. 1)
Recent investigations have highlighted mechanisms in acute inflammation and sepsis cascade by HIF-α (hypoxia-inducible factor-Iα)–Sirtuins (silent mating type information regulator) signaling pathway [10, 11, 22]. Tissue hypoxia is a central feature of sepsis. There is upregulation of HIF-α signaling in tissue hypoxia. These signaling molecules and transcription factors are involved in both the hyper-inflammatory phase (elevated HIF-α/low Sirtuins) and the second immune-tolerant, recovery phase of sepsis (elevated Sirtuins/low HIF-α).
Sirtuins (types 1–7) are a highly conserved family of proteins known for their anti-inflammatory and anti-oxidant properties. These proteins belong to the (nicotinamide adenine dinucleotide) NAD+ dependent class III histone deacetylase (HDAC) family of enzymes and different types are present in different compartments of cells such as cytoplasm, mitochondria, and nucleus. Sirtuins are (NAD+) energy sensors of a cell and are considered as guardians of homeostasis. Sirtuins type 3, 4, 5 are localized to the mitochondria [10, 11, 22] (Fig. 1). NAD+ levels decline with aging and are further reduced in chronically stressed cells such as patients with diabetes, hypertension, smokers, and obesity thereby reducing the activity of Sirtuin. These groups of patients have the highest risk for severe sepsis and mortality as currently observed in the Covid-19 pandemic . Recent experiments on SARS-CoV-2 infected murine, human lung cell lines, and autopsy specimens suggest upregulation of poly (ADP) ribose polymerase (PARP) family of genes which are involved in the consumption of NAD+. Overexpression of these PARP genes consume and depress the levels of NAD+ and are overexpressed in these cells . Reduced activity and depletion of NAD+ further attenuate protective function of Sirtuin [23, 24].
Hypoxic cells upregulate the transcription factor, HIFα [12, 20]. In general, stabilization of HIFα and an increase in its hyper-inflammatory activity occur with loss or dysfunctional protective action of the cellular Sirtuins. This has been demonstrated in the hyper-inflammatory stage of animal models of sepsis [10,11,12, 22].
During this initial hyper-inflammatory phase with associated tissue hypoxia, the PRR (Toll-like receptors) upregulate a number of proliferative pathways such as NF-ĸB, hyper activation of NLRP3 inflammasommes, mitogen-activated protein kinase (MAPK), and mammalian target of rapamycin (mTOR) that trigger the cytokine storm [23,24,25].
Along with increased proliferative activity and increased cell turn over, the accompanying aerobic glycolytic pathway and dysfunctional oxidative phosphorylation release ROS which results in downregulation of Sirtuins. Low Sirtuin activity itself promotes elevated ROS due to inefficient clearance. Elevated ROS also downregulates prolyl hydroxylase (PHD). These enzymes inactivate HIF-α. Taken together, these factors stabilize HIF-α and prevent it from undergoing ubiquitination by E3 ligases (von Hippel Lindau factor, VHL) for proteasomal degradation. Stabilized HIF-α translocates to the nucleus and activates key genes upregulating glycolysis and production of pro-inflammatory cytokines and maintains the vicious cycle of a hyper-inflammatory state [10,11,12, 22]. Key HIF-α targets include glucose transporter 1 (Glut1), hexokinase (HK), lactic dehydrogenase (LDH), pyruvate dehydrogenase kinase (PDHK), and cyclo-oxygenase (COX-2). These enzymes shift metabolism away from oxidative phosphorylation and promoting glycolytic signature and further fueling anabolic pathways [10,11,12, 22] (Fig. 1).
During the second phase of sepsis, a hypo-metabolic state will ensue with decreased oxygen consumption, resumption of mitochondrial respiration, and ATP production. Majority of the mortality associated with sepsis occurs during this phase. This hypo-metabolic state allows cells to enter an anti-inflammatory hibernating state allowing for slow recovery of cellular function. However, this cyto-protective, immunosuppressive state may also be detrimental to clear infections . Moreover, secondary nosocomial infections may occur during this phase. For example: clostridium difficile infections, and secondary bacterial and candida infections. Often these infections are multidrug resistant and increase sepsis-related morality in the setting of the hypo-inflammatory phase . Biochemically, this phase is associated with reduced activity of HIF-α and upregulation of Sirtuin activity. Sirtuins with their deacetylase function inactivate NF-ĸB, HIF-α, high-mobility group box proteins-1 (HMBG-1), and other DAMP molecules, thus decreasing the overall cytokine load [23,24,25]. Sirtuins also coordinate switch from glucose to fatty acid oxidation during this phase. The molecular mechanisms for this remain unclear [10, 11, 14, 22, 25]. With cell recovery, mitochondrial biogenesis is initiated and there are upregulation of markers of mitochondrial biogenesis such as PAR gamma coactivator-1α (PGC-1α), Transcription factor A for mitochondria (TFAM), and nuclear respiratory factors (NRF-1) [9, 15, 23].
Mitochondria localization may be necessary for SARS-CoV-2 replication
SARS-COV-2 virus uses ACE2 receptor for cell entry and TMPRSS2 for spike protein (S protein) priming . Variations of ACE 2 receptors in different ethnic populations may play a role in the virulence and transmissibility of this virus .
SARS-CoV-2 protein and human–protein interactions that connect multiple complex biological processes such as replication, protein trafficking, and ubiquitination have been observed. As a group SARS-CoV viruses produce accessory proteins called open reading frames (ORF) which interact with mitochondrial outer membrane receptors. One particular interaction involves ORF-9 interaction with mitochondrial antiviral signaling systems (MAVS, TOMM.70) . MAVS is a mitochondrial import receptor and also functions as cytoplasmic viral recognition receptor. ORF has shown to suppress MAVS activity, thus limiting the initial host cell, innate immune, interferon, and antiviral response [28,29,30]. Similar other SARS-CoV-2 virus non-structural protein (NSP4, NSP5, NSP7, and NSP8) interaction with human mitochondrial membranes and matrix functions has been demonstrated. These viral proteins have been demonstrated to associate with RNA processing, electron transport and mitochondrial signaling, and trafficking proteins. Thus, viral–mitochondrial interactions may disrupt both the membrane integrity and functional aspects of the mitochondria .
Once inside the cytoplasm, the SARS-CoV-2 virus has to replicate from a single-stranded RNA (ss RNA) through an intermediate double-stranded RNA (ds RNA). This predisposes the virus to the antagonism through their recognition by Toll-like receptors, RIG-1 receptors, and mitochondrial antiviral signaling systems (MAVS) and, thus, activation of innate immunity as described earlier. SARS-CoV-2 virus evades this detection by forming double-membrane vesicles (DMV) around its dsRNA, thus shielding it from detection. Viruses need intracellular organelles such as mitochondria and endoplasmic reticulum for their replication and dissemination [31, 32].
Based on computational machine learning models, it has recently been demonstrated by Wu et al. that the SARS-CoV-2 RNA genome and all sub-genomic RNAs are enriched in the host mitochondrial matrix and nucleolus . Open reading frame (ORF) from corona viral genome has been identified in the mitochondria. Based on these models, they predicted that mitochondrial residency may be required to the formation of double-membrane vesicles which is a critical in corona virus being able to replicate un-abated by evading cellular defenses. In other words, the virus simply may hijack the mitochondria and use its machinery for its own replication and sustenance . In doing so, it may damage the mt DNA and this may cause leakage of the mt DNA into the cytoplasm with then acts as a trigger for activation of innate immunity . Previously, an association between high mitochondrial viral RNA and decrease in mitochondrial functional integrity has been demonstrated with HIV RNA virus . This hypothesis needs further validation with SARS-CoV-2. These findings suggest that mitochondrial involvement by the SARS-CoV-2 RNA virus may actually assist with virulence and transmissibility. Mechanisms by which the virus gains access into the mitochondria and cause dysfunction remain to be investigated.