Autophagy is also known as type II programmed cell death. Moderate autophagy can degrade damaged organelles, denaturated macromolecules and intracellular pathogens, and then provide raw materials for cell metabolism (Levine and Kroemer, 2019; Zhu et al., 2019). Excessive autophagy and insufficient autophagy lead to disease (Lavandero et al., 2015). It was confirmed that autophagy is closely associated with proliferation of pathogenic microorganisms (Wang et al., 2020b). The stage and site of pathogenic infection, type of infected cell, and physiological state of the host body all affect the activity and outcome of autophagy. Therefore, identifying the initiation process of autophagy can help us understand its multiple functions.

Mammalian target of rapamycin (mTOR) is a key factor of autophagy initiation. It is widely known that mTOR is composed of two protein complexes mTORC1 and mTORC2, which have different structures and functions and are involved in many physiological processes. Unc-51-like kinase 1 (ULK1) is an indispensable component in autophagy vesicles, which can form the ULK1 complex with autophagy related 13 (Atg13), 200-kDa FAK family kinase interaction protein (FIP200), and Atg101 to induce autophagy (Chen et al., 2014; Hurley and Young, 2017). Under normal circumstances, mTORC1 suppresses autophagy by directly inhibiting the ULK1 complex activity. However, under stress conditions, mTORC1 is phosphorylated leading to the rapid disinhibition of ULK1 and Atg13, which triggers autophagy. In addition, mTORC1 also negatively regulates autophagy by phosphorylating autophagy/Beclin-1 regulator 1 (Ambra1) at Ser52 and 4’,6-diamidino-2-phenylindole (DAP1) at Ser3 and Ser51 (Koren et al., 2010; Cianfanelli and Cecconi, 2015). Conversely, mTORC2 indirectly inhibits autophagy through activation of the AKT/mTORC1 signaling axis. The mechanism involves the AKT-dependent phosphorylation inhibition of tuberous sclerosis complex 1/2 (TSC1/2) which induces Rheb activity that promotes mTORC1 activation (Bernard et al., 2020). Another major mTOR related signaling pathway is the AMP-activated protein kinase (AMPK) -mTOR pathway. The regulation of AMPK is extremely complex. Activation of AMPK phosphorylates TSC2 at Ser792 of mTORC1. Furthermore, PIM2 directly phosphorylates TSC2 at Ser1798 to activate mTORC1 (Lee et al., 2010; Lu et al., 2013). Moreover, AMPK initiates autophagy by directly phosphorylating ULK1 at Ser317 and Ser777 in response to stress (Pagano et al., 2014). In addition, a novel signaling axis AMPK- E3 ligase S-phase kinase-associated protein 2 (SKP2)-co-activator-associated arginine methyltransferase 1 (CARM1), was found to regulate autophagy activation by nutrient starvation (Shin et al., 2016). A recent study indicated that PKCα phosphorylated ULK1 at Ser423, which prevented autosomal formation and inhibited autophagy (Wang et al., 2018a). Atg13, ULK1, and TFEB can be negatively regulated by mTORC1. The secretion of TFEB during stress helped regulate the expression of genes involved in lysosomal biogenesis and lipid catabolism (Settembre et al., 2013).

The processes of autophagy initiation induced by different pathogenic microorganisms are also different. It was shown that Salmonella typhimurium invasion breaks through vesicles and enters the cytoplasm to selectively activate mTOR and degrade AMPK to escape autophagy (Liu et al., 2018). Streptococcus pneumoniae PavA activates the AMPK signaling pathway to induce autophagy in alveolar epithelial cells by inhibiting the mTOR pathway (Kim et al., 2017). In H. pylori infected gastric epithelial cells, inactivated transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) inhibits AMPK phosphorylation, as well as autophagy and cell survival (Lv et al., 2014).

Nitroxidative stress is a state of physiological imbalance mainly caused by excessive ROS and RNS. Under normal conditions, low levels of ROS/RNS trigger the hosts protective immune response, which is of great significance for anti-infection, anti-inflammatory, and tumor inhibition. However, excessive ROS/RNS directly leads to membrane rupture, DNA peroxidation damage, and cell dysfunction, such as loss of energy metabolism, changes in cell signal transduction, and gene mutation. During some pathogenic microorganisms infection, the host’s antioxidant system and metabolic balance of free radicals becomes disordered, resulting in nitroxidative stress.

ROS components include O₂, H₂O₂ and –OH, and RNS components includes NO, NO₂, and ONOO− (Stamler et al., 1992). ROS functions as a signaling molecule in a variety of intracellular processes, which lead to cell proliferation, apoptosis, and defense against microorganisms (Fratelli et al., 2005; Scherz-Shouval et al., 2007). Endogenous ROS is mainly induced by the mitochondrial respiratory chain (Zou et al., 2017). Specific enzymes of the NADPH oxidase (NOX) family and double oxidase family are involved in ROS production. These enzymes catalyze the conversion of intracellular O₂ to O₂ ^-, and then O₂ ^- is converted to H₂O₂ in the presence of superoxide dismutase. Subsequently, H₂O₂ reacts with metal ions to form –OH (Si et al., 2015). For example, sodion, a second messenger, interacts with phospholipids and controls oxidase in mitochondria. Furthermore, sodion helps transfer electrons from the substrate to oxygen ions, and subsequently promotes ROS formation and causes oxidative stress (Hernansanz-Agustin et al., 2020). Studies reported that SoxRS and OxyR are the main redox response transcription factors involved in oxidative stress in bacteria. OxyR senses H₂O₂ and organic peroxide, and SoxRS regulates O₂ ⁻-mediated oxidative stress. In addition, other stress factors including RpoS and PerR are also involved in regulating oxidative stress responses (Fei et al., 2020). ROS and RNS are produced through different processes including ultraviolet irradiation, metal-catalyzed reactions, electron transport reactions, and inflammation (Valko et al., 2006). Many studies have shown that a variety of pathogens induce nitroxidative stress (Li et al., 2019). The increase of ROS/RNS production was found during microbial infection, or infection with hepatitis C virus, Herpes simplex virus type 1, E. coli, and Salmonella (Molteni et al., 2014; Rhen, 2019). When pathogenic microorganisms infect a host, ROS and RNS can be beneficial and detrimental via anti-inflammatory, anti-pathogen, protective immunity, or cytotoxicity functions (Umezawa et al., 1997). Moderate insulin-like growth factor 1 promoted mitochondrial ROS synthesis and NO synthase gene expression which participated in the elimination of P. falciparum (Drexler et al., 2014). Activation of TLR4 helped clear invading pathogens through the production of NO which was induced by increasing the activity of iNOS via guanosine triphosphate cyclohydrolase (Deng et al., 2015). Inhibition of mitochondrial ROS production impaired the ability of immune cells to kill Salmonella typhimurium in mice (West et al., 2011). In addition, excessive ROS/RNS was closely associated with tissue and organ damage (Deng et al., 2012). For example, excessive ROS triggered apoptosis by inducing the expression of NLRP3, caspase-1, and ASC (Dai et al., 2019).

Because persistent and excessive nitroxidative stress can lead to host tissue damage, organisms have an antioxidant system that maintains redox homeostasis. There are two types of antioxidant systems: antioxidant enzymes and non-enzymatic antioxidants (Halliwell, 1996). Antioxidants help to mediate peroxidation by catalyzing the formation of active oxygen intermediates which lead to remove free radicals of oxygen and nitrogen. For example, the massive accumulation of ROS promotes the production of superoxide dismutase (SOD), which catalyzes the conversion of superoxide into O₂ and H₂O₂ (Lipinski et al., 2010). Then, catalase scavenges peroxide by catalyzing the conversion of H₂O₂ into O₂ and H₂O (He et al., 2017). In addition, the GPx family and Trx system, endogenous antioxidants, also participate in the elimination of nitroxidative stress (Papp et al., 2007; Liu et al., 2014a). Moreover, several signaling pathways, of which nuclear factor erythroid 2-related factor 2 (NRF2) signaling is the most important, are involved in regulating nitroxidative stress. ROS directly oxidizes the cysteine residues on Kelch-like ECH-associated protein 1 (KEAP1) freeing NRF2 from the KEAP1-NRF2 complex. After free NRF2 translocates into the nucleus, it promotes the expression of multiple antioxidant genes by binding to their regulatory regions (Schieber and Chandel, 2014). Furthermore, NO^- triggers NRF2 transcription and the expression of ferroportin which activate the antioxidant system and nutrition immunity (Nairz et al., 2013). In turns, inhibiting NRF2 signaling increased ROS levels by suppressing the expression of HO-1 during H. pylori infection (Ko et al., 2016).

TLR4 is known for recognizes a broad variety of substances including bacterial lipopolysaccharide (LPS), viral structural protein, fungal mannan, and parasitic glycoinositolphospholipids and has a key role in activating innate immunity (Kawai and Akira, 2011; Mu et al., 2011). For example, when Gram-negative bacteria infect a host, bacterial LPS is directly recognized by TLR4 in the presence of LPS binding protein, CD14, and myeloid differentiation factor 2 (MD-2). Subsequently, TLR4 undergoes oligomerization and recruits downstream adaptor proteins through its Toll-interleukin-1 receptor domains, which initiates signal transduction (Lu et al., 2008). Similar to other TLRs, TLR4 also has two types of signal transduction. After TLR4 is combined with MyD88, phosphorylated IRAK-4 activates the TRAF6-TAK1-NF-κB/MAPK signaling axis which promotes the release of inflammatory factors, such as IL-1β, IL-6, and TNF-α (Monlish et al., 2016; Dajon et al., 2017). Theses cytokines are responsible for inflammation, autophagy and nitroxidative stress. In the MyD88-independent pathways, TRAM is selectively recruited to TLR4 to link TLR4 and TRIF, which induces the production of type I IFN and other proinflammatory cytokines through the activation of IRF3, NF-κB, and MAPK signaling (Kawai and Akira, 2011).

However, when TLR4 is overactivated or its negative regulatory system is obstructed, TLR4 can induce endotoxic shock, autoimmune disease, and even cytokine storm where the immune system attacks the host. The SARS-CoV-2 virus has spread worldwide since 2020, infecting billions of people and killing millions of people (Harrison et al., 2020). Cytokine storm is thought to be an important cause of death in patients with severe and critical COVID-19, which is caused by the SARS-CoV-2 virus (Chen et al., 2020). Compared with a severe influenza group, PBMCs from COVID-19 patients showed a high inflammatory response, and a marked TNF/IL-1β driven inflammatory response. IFN, TNF-α, and IL-1β co-drive inflammatory responses in the monocytes of patients with severe COVID-19, but not in those of patients with mild COVID-19 (Lee et al., 2020). Further research showed that TLR4 is probably involved in recognizing the spike protein of SARS-CoV-2 and then inducing inflammatory responses (Bhattacharya et al., 2020; Choudhury and Mukherjee, 2020). After SARS-CoV-2 virus infects a host, the SARS-CoV-2 spike trimer directly binds to TLR4 and the trigger section of IL-1β. Inhibiting TLR4 completely blocks SARS-CoV-2-induced IL-1β. In contrast, ACE2-deficient or TMPRSS2-inhibition did not affect SARS-CoV-2-induced IL-1β (Zhao et al., 2021). This indicates that if TLR4 signals are not effectively controlled, they can seriously threaten health. Several tightly-regulated mechanisms regulate TLR4 signal transduction to avoid excessive immune response ( Table 1 ) (Liu et al., 2016a).