Response of the immune system in infection by SARS-CoV-2
The innate immunological system functions as the first line of defense of the host against infection by SARS-CoV-2; it is crucial for identifying and eliminating the infected cells, and at the same time, for coordinating an adaptive immunity response. The immune response of the host in cases of COVID-19 can be described as an early local immune response (antiviral defense) and a later local/systemic response phase, followed by uncontrolled inflammatory responses and cytokine storm syndromes (Figure 4). Because SARS-CoV-2 initially affects the upper respiratory tract, its first interactions with the immunological system during the inductive and effector phases, should take place predominantly on the surfaces of the respiratory and oral mucosae[57,59]. Exposure to the virus antigens causes immunoglobulin (Ig)A-mediated responses in the mucosa[60,61] that can be accompanied by the systemic production of IgA, but the systemic production can be absent, transitory, or delayed. The response involving IgA antibodies of the mucosa that maintains an essentially noninflammatory medium and can be particularly prevalent in young people with a mild infection by SARS-CoV-2 without evidence of pneumonia.
Figure 4 Immune response and oxidative stress in severe acute respiratory syndrome-coronavirus-2 infection
. A: Oxidative stress; B: Adaptive immune response; C: Innate immune response; D: Cytokine storm. Created with BioRender.com.
If the immunological system of the individual does not counteract the virus during the initial phase of exposure through a rapid early response, the virus advances to the lower respiratory tract (LRT)[62,63]. Once the virus reaches terminal respiratory pathways and the alveoli, B and T cells are activated, which results in the production of specific anti-SARS-CoV-2 antibodies, with the predominance of an inflammatory environment dominated by IgG. The S and N proteins are the two principal antigens of the coronavirus that induce the production of Ig[64,65]. The IgA, IgM, and IgG antibodies against the N and S proteins, and the IgM and IgG antibodies against the protein receptor-binding domain, as well as the presence of neutralizing antibodies (nAbs) against SARS-CoV-2 are positive from day 1 after the appearance of symptoms. The antibody levels, especially IgG, increase during the disease course, while a limited increase of IgA and IgM is observed. The antibodies against the S1 and N antigens persist for at least 3 mo after the infection[60,65]. In addition, higher levels of nAbs as well as IgG and IgM antibodies and anti-S1 and -N antibodies have been observed in patients with very severe symptoms. In addition, depletion of memory B cells of IgM was associated with worse outcomes, including a higher mortality rate and a greater risk of developing superimposed infections.
In response to viral invasion, the innate immunological system recognizes viral nucleic acids by host recognition-pattern (HRP) receptors, which are expressed in innate immune cells (e.g., neutrophils, dendritic cells, epithelial cells, and macrophages), and by toll-like receptors (TLRs) and retinoic acid-inducible gene I-like (NOD-like) receptors (NLRs) (Figure 4C). For the production of cytokines and the induction of an antiviral state[57,58], as a response to specific pathogen-associated molecular patterns and damage-associated molecular patterns (DAMPs)[57,68]. HRP receptors can activate antiviral responses in neighboring cells and recruit innate and adaptive immune cells and the participation of phagocytes such as macrophages and neutrophils, as well as natural killer (NK) cells[59,68].
Once the virus was detected by an HRP receptor, intracellular signaling pathways are triggered that activate interferon regulatory factor 3 (IRF3) and the nuclear factor kappa beta (NF-κB) signaling cascade. The initial phase of the production of interferon (IFN) mediated by IRF3 performs the initial phase of the innate immune response to detect and brake viral replication. Viral detection stimulates the production of type-1 and type-3 IFN, which results in the expression of interferon-simulated genes ISG (IRF1, IFI44L, and IFIT3) and antiviral genes (OAS3 and ADAR), and the release of large amounts of inflammatory procytokines like interferon gamma (IFN-γ), interleukin (IL)-1RA, IL-6, IL-8, IL-10, and IL-19, monocyte chemoattractant protein 1 (MCP-1), MCP-2, and MCP-3, bonding with C-X-C motif chemokines, including CXCL9, CXCL10, and CXCL5, and tumor necrosis factor alpha (TNF-α). Stimulation of TLRs activates NF-κΒ, giving rise to the production of inflammatory markers deriving from monocytes (IL-1, TNF-α, and IL-6) to control the infection by means of direct antiviral pathways and the recruitment of other leukocytes. Signaling is elevated 24 h after infection by SARS-CoV-2, leading to the progressive loss of the pulmonary alveolar epithelial function. The activation of complement, and especially of the C5a/C5aR1 axis, was also implicated in the pulmonary pathology of COVID-19.
HRP receptors, particularly the nucleotide-binding oligomerization domain-like (NOD)-like receptor (NLR) family, subsequent to the SARS-CoV-2 infection, assemble a multiprotein complex called inflammasome NLRP3, which results in the activation of caspase-1. Activated caspase-1 splits from the pro-interleukin IL-1β, pro-IL-18, and gasdermin D (GSDMD) and releases the GSDMD N-terminal fragment that can be oligomerized within membranes to form membrane pores and “pyroptosis”. The excision of caspase-1-dependent GSDMD leads to the release of IL-1β and IL-18, which are key mediators of the inflammatory response, and their increase in the plasma have been correlated with COVID-19 mortality or severity. Apoptosis, another type of cell death, also occurs during SARS-CoV-2 infection and is driven by the excision of the caspase-8, -9, -10 initiators of the executioner caspase-3 and -7. Apoptotic caspase-3 activates gasdermin E (GSDME) to induce the lytic form of cell death; the protein ORF3a of the SARS-CoV-2 virus also induces the excision of caspase-8 and -9 and causes apoptosis. The activation associated with the death pathways of the inflammatory cells can give rise to critical tissue damage, severe inflammation, and lactate dehydrogenase (LDH), which is a marker of cell death that has high concentrations in COVID-19 patients. The LDH concentration is considered a predictive factor for the early recognition of pulmonary lesions in severe cases.
Macrophages are a key respiratory system. They produce chemokines and IFN-β. Infected dendritic cells (DCs) produce antiviral cytokines, like IFN-α and IFN-β; the proinflammatory cytokine TNF, IL-6, and high levels of the inflammatory chemokines CCL3, CCL5, CCL2, and CXCL10. The cytokines/chemokines are key factors for the chemotaxis of neutrophils, monocytes, and activated T cells. Activated neutrophils, whose main function is the elimination of pathogens and dendrites by means of phagocytosis, release leukotrienes and reactive oxygen species (ROS) that induce local damage to pneumocytes and the endothelium, which in turn leads directly to acute lung lesions.
Neutrophils can also develop DNA networks called neutrophilic extracellular traps (NETs) through the process of NETosis, or the release of nucleic acids enveloped in histones, which retain viral particles and promote the inactivation of the viral infection and cytokine production to restrict replication of the virus. NETosis is conditioned to the production of ROS by means of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In addition to the physical containment promoted by NETosis, NETs contain proteases and cytotoxic enzymes that permit the neutrophils to centralize lethal proteins at the sites of infection. A variety of stimuli, including toxic factors, viruses, and proinflammatory cytokines such as TNF-α and IL-8, can drive neutrophils to release NETs (Figure 4D). The uncontrolled production of NETs is correlated with the severity of the disease and the extension of the pulmonary lesion, with acute respiratory insufficiency and multiple organic dysfunction syndromes. NETs also contribute to the formation of thrombi, or immunothrombosis, which can amplify the production of cytokines. The inflammatory process comprises a triggering of thrombotic complications that are usually observed in patients with COVID-19, and immunothrombotic dysregulation appears to be an important marker of the severity of the disease[77,78]. In SARS-CoV-2, elevation of the neutrophil/ lymphocyte ratio (NLR), a marker of infection and systemic inflammation, suggests a poor disease prognosis. In addition, patients with COVID-19 have the lowest lymphocyte count and the highest neutrophil count and NLR during severe disease[67,79,80].
The inflammatory responses in the respiratory system intended to eliminate SARS-CoV-2 result in the generation of metabolic-acid waste material that, together with an increase in respiratory muscular work, lead to the development of metabolic acidosis. Metabolic acidosis compromises adaptive cellular immunity and the efficient eradication of SARS-CoV-2. Activated neutrophils and the T lymphocytes depend mainly on glycolytic metabolism for their proliferation, differentiation, and function, which results in the accumulation of lactic acid. A low pH induces anergy in CD8+ T cells, suppresses NK cells, and inhibits the function of CD4+ T cells. Acidosis also increases the levels of circulating glucocorticoids; thus, their anti-inflammatory and immunosuppressor properties compromise immunity against viruses to an even greater degree.
Infection by SARS-CoV-2 promotes mechanisms that antagonize proinflammatory signals, particularly the signaling of IFN-I and IFN-III, but increases the expression of chemokines and proinflammatory cytokines in order to counteract the host’s innate immune response[57,58,63]. Thus, the expansion and early differentiation of T cells depend on the direct action of IFN-I. The descending production of interferons promotes intracellular antiviral defenses in neighboring epithelial cells that can limit viral dissemination, while the release of IL-6 and IL-1β from other immune cells promotes neutrophil recruitment and immune cell activation.
The three most critical components of the adaptive immune responses are viral protein-specific CD4+ T cells, CD8+ T cells, and nAbs. The nAbs produced by B cells can bind to and neutralize the extracellular SARS-CoV-2 proteins. If the Abs cannot prevent the virus from entering cells, cytotoxic CD8+ T cells are called upon to destroy the cells directly infected with their granules[83,84]. Pulmonary cytotoxic CD8+ T cells recognize and induce apoptosis in cells infected through direct mechanisms (i.e. cell–cell contact) and indirect mechanisms with the participation of the perforin and granzyme-secreted cytolytic enzymes, as well as with the cytokines IFN-γ and TNF-α. However, cytotoxic cells by nature do not prevent the infection, they destroy already infected cells, thus reducing propagation of the infection (Figure 4B). Transitory increases of the CD8 effector T and memory T cells constitute an effective and efficient response during early viral infection. High counts of CD8+ T cells in the lungs are correlated with better control of SARS-CoV-2[80,85].
CD4+ T cells, the third arm, are auxiliaries and coordinators of the production of Abs and of the activation of the cytotoxic CD8+ T cells[83,84]. After being infected with SARS-CoV-2, CD4+ T cells are activated and differentiate to Th1 cells or circulating T follicular helper T cells (Tfh)[68,80] that secrete proinflammatory cytokines, such as IL-2, IL-6, IFN-γ, and granulocyte-macrophage colony-stimulating factor (GM-CSF) that participate in the activation, proliferation, and differentiation of cytotoxic T lymphocytes. In addition, elevated levels of cytokines secreted by Th2 cells, such as IL-4 and IL-10), which inhibit Th1 inflammatory responses have been reported. The severity of SARS-CoV-2 infection has been related to diminished adaptive immunity responses, mainly because of depletion of T cells and lymphopenia, alteration of the differentiation of T follicular helper (Tfh) cells[63,87], low levels of CD8+ NK cells, CD4+ auxiliary T cells, and memory T cells. However, Abs by themselves do not correlate with the severity of the disease[64,83]. It is probable that the level of inflammation and the amount of proinflammatory cytokines are associated with the activation and depletion of T cells, but it has not yet been determined whether the early response reaches a state of depletion in individuals with severe hyperinflammation[63,70].
Lymphocytes gradually decrease as the disease advances, which results in immunosuppression manifested as atrophy of the organs of the immune system, secondary infection, and multiple organ dysfunction syndrome. Lymphopenia is consistent with overrepresentation of nonfunctional T lymphocytes, with increased percentages of virgin Th lymphocytes (i.e. CD45RA+, CXCR3−, CCR4−, CCR6−, and CCR10−) and a persistent low frequency of markers associated with effector memory T cells, TFH cells, and regulatory T cells (Tregs). Lymphocytopenia is negatively correlated with inflammatory biochemical parameters (ferritin, fibrinogen, PCR, D-dimer, LDH) and the percentage of lymphocytes and positively correlated with the neutrophil count. From an immunological point of view, lymphopenia could depend on the possibly dysfunctional deactivation of dendritic cells and on the increased concentration of cytokines such as TNF-α, IL-6, and IL-10, which act as negative regulators of the proliferation and survival of the T lymphocytes. The production of acute-phase proteins such as ferritin and CRP, in addition to affecting the equilibrium of pro- and anticoagulant pathways (i.e. increasing D-dimer), can induce lymphocyte apoptosis.
The host capacity to generate efficient T cell responses after infection by SARS-CoV-2 probably depends on the directed epitopes, the presence or absence of pre-existing cross-reactive T cells, and genetic factors such as the human lymphocyte antigen (HLA) type, and the repertory of T cell receptors (TCRs). Given that activated T cells in elderly persons and in those with chronic disease present reduced responses to IFN-I, a longer time is needed to generate effective adaptive immune responses because of the deterioration of the immune functions such as the production of virgin T cells and memory T cells, which diminish with aging, and present asynchronous immune responses with high Ab levels and weak T cell responses. Delayed activation of SARS-CoV-2-specific T cells and a reduction of the clarification of the virus increase the risk of cytokine storm, the earlier appearance of severe disease, and increased mortality.
In contrast with innate immune responses, which are produced before the infection and participate fully in the elimination of the virus, the adaptive immune responses begin 4-7 d after infection. If the body does not generate effective adaptive antiviral responses in time to eliminate the virus, the innate immune responses will be maintained, but without eliminating the virus in an effective manner and even leading to systemic inflammatory responses and the uncontrolled release of inflammatory cytokines.
The inflammatory cytokine storm, also known as the cytokine release syndrome, is a severe excessive immune response caused by positive biofeedback circuit damage of immune cells by the cytokines[67,75,87]. The formation of a cytokine storm leads to a “suicide attack” that not only limits additional propagation of the virus, but also induces secondary tissue damage. The marked release of proinflammatory cytokines causes lymphopenia, lymphocyte dysfunction, granulocyte and monocyte anomalies, coagulation disorders such as capillary extravasation syndrome, formation of thrombi, and even the combined immunodeficiency syndrome[76,81]. A series of destructive effects on tissues, including destabilization of the interactions among endothelial cells, damage to the vascular barrier, diffuse alveolar damage characterized by the formation of hyaline membranes, ARDS[57,68], tissue toxicities that affect the respiratory, hematological, gastrointestinal, cardiovascular, renal, hepatic, and neurological systems, multiorgan failure and, ultimately death may occur[58,68,75,76]. Despite the large number of studies much of the physiology of the immune response in COVID-19 has yet to be described.
Oxidative stress and infection by SARS-CoV-2
Oxidative stress is the result of disequilibrium between the oxidant system, which consists principally of free radicals, ROS), and reactive nitrogen species (RNS), and the antioxidant systems that neutralize the free radicals. Reactive oxygen and nitrogen species (RONS) are characterized by unpaired valance electrons, obliging them to react with diverse biological molecules[90,91]. ROS comprise the hydroxyl (OH) radicals, superoxide anion (O2−), singlet oxygen (¹O2), hydrogen peroxide (H2O2), and ozone (O3). RNS include nitric oxide (NO), peroxynitrite (ONOO−), nitrosyl cation (NO+), the nitrosyl anion (NO−), and nitrose acid (NH2O2). Under physiological conditions, the reactive species play an important role in cellular signaling (redox signaling) and the regulation of cytokines, and growth factors such as immunomodulators, cellular differentiation, and others. However, when the equilibrium of oxidant agents and antioxidant systems is disturbed, harmful effects are generated[90,91]. The damage caused by free radicals affects cellular membranes by lipid peroxidation, oxidation, protein denaturalization, DNA damage that can induce inflammatory immune responses and increase the risk of mutations and tumorigenesis, and apoptosis. In general, hydroxyl radicals are highly reactive and are responsible for the greatest cellular damage modification of biomolecules induced by ROS. H2O2 is considered the least harmful and can travel to and penetrate cell membranes, and the superoxide is intermediately harmful.
In the pathology of COVID-19, the cytokine storm is an important source of endogenous oxidative stress, and excessive production of ROS that in turn stimulates the increased release of cytokines, causing an exaggeration of the already initiated inflammatory responses (Figure 4A)[93-97]. The interaction of ROS and cytokines generates a self-sustaining cycle involving the cytokine storm and the production of oxidative stress that eventually leads to a high pulmonary protein exudate with a low hemoglobin carrier, the generation of free radicals and proteases, and an increase in the permeability and entry of edematous fluid into the alveoli. The results in deficient gas exchange in the lungs, pulmonary hypoxia, cytopathic hypoxia, damage to the epithelium, acute pulmonary lesions, disseminated coagulation, multiorgan failure and death in patients with COVID-19[95-98].
The cytokine storm with hyperinflammation accompanied by cytopenia and hyperferritinemia is known to generate ROS, by means of the Fenton reaction (Fe²+ + H2O2→ Fe³+ + HO- + HO-). Additionally, the cytokines and endotoxins stimulate an isoform of nitric oxide synthase (iNOS), the inducible isoform NO, which stimulates the production of NO that in turn reacts with the superoxide to yield peroxynitrite (ONOO−)[90,96]. Both peroxynitrite and NO are toxic to mitochondria, producing dysfunctional mitochondria that, in turn, result in cytopathic hypoxia[96,99]. In addition, they cause a possible oxidative storm with all of the harmful effects of RONS, in particular the peroxidation of lipids and oxidation of membrane proteins that contribute to the transformation and hyalinization of the pulmonary alveolar membranes, with lethal respiratory difficulty.
SARS-CoV-2 activates oxidant-sensitive pathways through inflammatory responses following activation of the NF-κΒ pathway. The reduction of oxygen saturation leads to the generation of superoxide radicals and H2O2 by the mitochondria. Hydrogen peroxide triggers the expression of genes that positively regulate proinflammatory cytokines, such as IL-1, IL-6, and TNF-α, and inducible nitric oxide synthase (iNOS) by means of the activation of the (iNOS) NF-κΒ pathway[94,96,98,100]. That, together with the ROS, activates NLRP3 inflammasomes. IFN-γ, IL-1β, IL-2, IL-6, and TNF-α stimulate the generation of NO. IL-6 and TNF-α give rise to superoxide generation in neutrophils, and hydrogen peroxide stimulates the generation of IL-6[98,102]. In cyclical fashion, the proinflammatory cytokines activate macrophages, neutrophils, and endothelial cells through NADPH oxidase (NOx) to produce more superoxide and H2O2. Patients with COVID-19 exhibit an overactivation of NOx2, a mechanism that favors ischemic events related to thrombotic events and associated with severe disease.
At the same time, the IFN-γ pathways are activated by oxidative stress induced by the inflammation intended to combat the infection by the virus. Circulation of the inflammatory cytokines and ROS damage erythrocytes, leading to the generation of heme and free iron and diminish the circulating nitric oxide (NO), which worsens the existing ischemia of the organs. Deterioration of the mitochondria leads to cytopathic hypoxia, which results in a partial reduction of oxygen with the generation of ROS and the reduced energy production. In addition, macrophages and activated neutrophils produce respiratory bursts that generate superoxide radicals and H2O2[94,98] that maintain the oxidative stress.
Poorly coordinated iron, especially in the presence of high concentrations of oxygen and reducers have the potential to generate hydrogen peroxide, superoxide, and hydroxyl radicals in the lung. The radical superoxide anion reduces Fe (III) to Fe (II) that, in the presence of H2O2, produces hydroxyl radicals (•OH), which are extremely toxic and promote the formation of lipid peroxidases in the cell membrane and the oxidation of proteins, causing cell death by apoptosis. Hydroxyl radicals plus free iron convert soluble plasma fibrinogen into abnormal fibrin clots in the form of enzymatic degradation-resistant dense and entangled deposits, leading to microthrombosis in the vascular system and in the microcirculation[95,96].
Oxidative damage resulting from SARS-CoV-2 infection can produce viral mutations that affect the immunological response. In addition, overproduction of ROS suppresses T-lymphocyte responses and results in weakened adaptive immunity, altering the structure and function of the circulating lymphocytes, principally TCD4+, with selective depletion reduced antiviral activity of CD8+ T cells. In general, the host response to stress and to combat an inflammatory condition is marked by a strong increase of the cortisol level. Cortisol supports the mechanisms of the host immunological defense in a permissive manner, and high levels of cortisol suppress inflammation and prevent tissue damage. However, in the case of severe COVID-19, patients can develop a corticosteroid insufficiency related to a critical disease. It is known that overproduction of ROS and the weakening of antioxidants are needed for viral replication and the subsequent disease associated with the virus. Viral infections alter antioxidant mechanisms leading to an unbalanced oxidative-antioxidant state and consequent oxidative cellular damage. Exposure to various pro-oxidants generally leads to activation of nuclear factor erythroid 2-related factor 2 (Nrf2) and to an increase of the expression of components of the antioxidant response. However, respiratory virus infections have also been associated with inhibition of the Nrf2 pathways that leads to inflammation and oxidative damage. Nrf2 is a transcription factor responsible for the adaptation of cells and tissues including alveolar epithelium, endothelium, and macrophages to electrophilic or oxidative stress. Under normal conditions, Nrf2 is found in the cytoplasm bound to its inhibitor Keap1, which is directed to Nrf2 for ubiquination and later degradation. In the presence of electrophiles or ROS, the Keap1–Nrf2 complex dissociates and Nrf2 migrates to the nucleus where it stimulates the transcription of target genes with sequences of antioxidant response elements in their promoters. Nrf2 controls the expression of the genes that participate in the antioxidant response, redox homeostasis, and the biogenesis of the mitochondria, etc. In addition, Nrf2 functions as a transcription repressor that inhibits the expression of inflammatory cytokines in the macrophages (e.g., IL-1β, IL-6, and TNF-α). SARS-CoV-2 can interfere with the equilibrium between the transcription factor NF-κB involved in the expression of cytokines and in the activation of Nrf2, responsible for the expression of antioxidant enzymes, including hemoxygenase 1 (HO-1), superoxide dismutase 1 (SOD1), superoxide dismutase 3 (SOD3), glutathione S-transferase (GST), catalase (CAT), and glutathione peroxidase (GPx). Nrf2 also regulates the increase in the production of the antioxidant enzymes NAD(P)H and quinone oxidoreductase (NQO1), and enzymes needed for the biosynthesis of glutathione, which functions as the main cellular antioxidant. In patients with SARS-CoV-2 infection, deficiencies in systems protection against free radicals, as well as deficits in superoxide dismutases (SODs), CAT, and reduced glutathione (GSH) have been described.
Oxidative stress is already increased in the elderly and people with diabetes and chronic cardiovascular diseases[90,91]. Increase in the stress level in response to viral infection affords a possible explanation of the severity of COVID-19 in such patients. In addition, elderly individuals may particularly vulnerable to infection by SARS-CoV-2 because the level and the activity of Nrf2 diminish with age. Therefore, aging is not only associated with alterations in the response to adaptive immunity, but also to a proinflammatory state in the host.