Innate And Adaptive Immunity During SARS-CoV-2 Infection: Biomolecular Cellular Markers And Mechanisms

Abstract

The coronavirus 2019 (COVID-19) pandemic was caused by a positive sense single-stranded RNA (ssRNA) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, other human coronaviruses (hCoVs) exist. Historical pandemics include smallpox and influenza, with efficacious therapeutics utilized to reduce overall disease burden through effectively targeting a competent host immune system response. The immune system is composed of primary/secondary lymphoid structures with initially eight types of immune cell types, and many other subtypes, traversing cell membranes utilizing cell signaling cascades that contribute towards clearance of pathogenic proteins. Other proteins discussed include cluster of differentiation (CD) markers, major histocompatibility complexes (MHC), pleiotropic interleukins (IL), and chemokines (CXC). The historical concepts of host immunity are the innate and adaptive immune systems. The adaptive immune system is represented by T cells, B cells, and antibodies. The innate immune system is represented by macrophages, neutrophils, dendritic cells, and the complement system. Other viruses can affect and regulate cell cycle progression for example, in cancers that include human papillomavirus (HPV: cervical carcinoma), Epstein–Barr virus (EBV: lymphoma), Hepatitis B and C (HB/HC: hepatocellular carcinoma), and human T cell Leukemia Virus-1 (T cell leukemia). Bacterial infections also increase the risk of developing cancer (e.g., Helicobacter pylori). Viral and bacterial factors can cause both morbidity and mortality alongside being transmitted within clinical and community settings by affecting a host's immune response. Therefore, it is appropriate to contextualize advances in single-cell sequencing in conjunction with other laboratory techniques allowing insights into immune cell characterization. These developments offer improved clarity and understanding that overlap with autoimmune conditions that could be affected by innate B cells (B1+ or marginal zone cells) or adaptive T cell responses to SARS-CoV-2 infection and other pathologies. Thus, this review starts with an introduction to host respiratory infection before examining invaluable cellular messenger proteins and then individual immune cell markers.

Keywords: COVID-19; B-cells; neutrophils; dendritic cells; T-cells; NK-cells; monocytes; macrophages; innate; adaptive; cytokines; chemokines; adhesion molecules; antibody; cluster of differentiation; receptors; proteins; SARS-CoV-2; serology

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1. Introduction 

1.1. Overview

The causal virion SARS-CoV-2 of the COVID-19 pandemic contains more than four immunogenic proteins composed of spike (S protein), nucleocapsid (N protein), envelope (E protein), and membrane (M protein) and associated subunits with accessory proteins [1,2]. Current therapeutic development occurred before/after March 2020 when the World Health Organization (WHO) declared a pandemic; SARS-CoV-2 is known to spread between animals and between generations [3]. It is thought that around 15% of COVID-19 disease mortality could be due to pneumonia or acquired respiratory distress syndrome (ARDS) [4]. Current vaccine immunogens were largely developed as pre-fusion S protein derivatives with newer candidates progressing through clinical trials with a goal to reduce chronic COVID-19 disease burden within populations as research continues. The SARS-CoV-2 genome is approximately 30 kilobases encoding 9860 amino acids and is defined by open reading frames (ORF) and non-structural proteins (NSP) required for viral propagation within all animal hosts [5]. The SARS-CoV-2 genome hosts 16 ORFs that encode 29 proteins required for viral propagation and host immune response inhibition. For example, ORF1a and ORF1ab encode polypeptides cleaved into 16 NSPs. Molecular testing methods (e.g., PCR) are commonly used diagnostic tools that allow the analysis and detection of specific RNA sequences within samples. SARS-CoV-2 infects cells via respiratory pathways and type II pneumocytes (ATII) using angiotensin-converting enzyme 2 (ACE-2) as the predominant receptor for entry [6]. Disruption and infection of ATII cells expressing ACE2 occur through phospholipid membranes. Other receptors expressed on all leukocytes, platelets, and endothelial cells include varying clusters of differentiation markers (CD), for example, CD3, CD4, and CD19, among others. Some are also currently implicated in initial SARS-CoV-2 cellular entry that includes type II transmembrane protease (TMPRSS2), asialoglycoprotein receptor-1 (ASGR1) and kringle containing transmembrane protein 1 (KREMEN1), dipeptidyl peptidase 4 (DPP4), neuropilin (NRP1), CD147 and vimentin [7–12]. Therefore, because of cellular infection, immune cells regulate and develop within primary lymph organs (e.g., bone marrow and thymus), but also through a network of secondary lymph organs (e.g., tonsils and others) utilizing lymph nodes (LNs) and cellular membranes that allow cellular permeability and lymphocyte migration to process infectious pathogenic proteins at all barriers through the nervous, digestive, endocrine, respiratory, circulatory, muscular, and skeletal systems. Technological advancement since 2017 has also allowed greater phenotypic analysis and, therefore, it is clearer now that SARS-CoV-2 proteins have different host roles. Analyses have confirmed that M protein is vital for assembling, S protein is for cellular receptor entry, and N and E proteins appear to be potential pore-forming proteins [13,14]. Single-cell RNA sequencing (scRNA-Seq), spectral flow cytometry (FACS), and mass cytometry (CyTOF) can detect markers enabling phenotypic analysis of all immune cell subsets [15–18]. It is noteworthy that antibody proteins involved in testing for SARS-CoV-2 infections have variable factors and human antibody concentrations. These are measured by predominant monoclonal antibody diagnostics for which many scales exist that undergo validation regardless of manufacturer. For example, institutions measure concentrations in sera utilizing several assays that measure binding antibodies (BAU/mL), others measure neutralizing antibodies (nAb in IU/mL), and others measure concentration (ng/mL) as standardization occurs to ensure consistency on a global scale (Supplementary Materials and Data S1–S5) [19,20]. Therefore, in this paper, we review current existing laboratory and clinical studies that have measured and illustrated statistical significance to illustrate the changing immune cellular maturation environment, to hypothesize that many of the inflammatory effects seen with SARS-CoV-2 infection can be dysregulated adaptive immune responses. Multiple bacterial, viral, and fungal pathologies have both immunological and genetic variability factors affected by cell cycle regulation and antigen presentation factors. These affect multiple pathologies, and risk is therefore affected by genetic susceptibility factors. This includes genes that encode proteins expressed on immune cell membranes, such as human leukocyte antigens (HLA), referred to as the major histocompatibility complex (MHC). These present short protein (peptide) fragments to the immune system cells and localized cellular markers affected by other mediator proteins, as we discuss below. The immune cells discussed in this paper provide overall context with further cell characterization through the interaction of B cells, T cells, and each of the other four cell subtypes discussed below, classified specifically by cluster of differentiation (CD) proteins expressed on cell surfaces reliant on cytokine and chemokines acting as immune cell signals affected by external pathogens.

1.2. Current SARS-CoV-2 Vaccine Immunogen Responses

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Current vaccine antigens or viral antigens prime the immune system to recognize pathogenic proteins via epitopes that can mutate, thereby affecting immune cell recognition through B cell and T cell receptors (BCR/TCR). Prevention of chronic COVID-19 disease to pre-Omicron variants was estimated in these ratios within current vaccine immunogens, developed by Pfizer/BioNTech, Astra Zeneca, Sinopharm, and Novavax: BNT162b2: 95.3%, AZD1222: 70.4%, BBIBP-CorV: 79%. Immunogen development indicates NVX-CoV2373 at 72% when screened against Omicron BA.1 and BA4/BA5 [21]. Additional risk reduction of COVID-19 disease was estimated at 86%. Population studies show variable SARS-CoV-2 protein antibody responses (76%:24% response/non-response). The estimated production of functional antibodies to S protein immunogens currently spans 6 months to 1.5 years. SARS-CoV-2 S protein mutations are now well documented in other studies to ascertain potential epitopes that affect immune response [22]. Functional cellular T cell responses, either helper (TH) or cytotoxic (TC), are also suggestive of CD4+: CD8+ activity occurrence in the ratio 96%:54% in COVID-19 disease [21]. Compared to other respiratory viruses such as influenza, the SARS-CoV-2 S protein possesses higher mutational rates within the spike/ACE2 interface, and the emergence of Omicron variants support this, denoted by BA1, BA2, BA2.75, BA4, BA5, BQ1, and XBB [23]. Fortunately, technology and laboratory techniques exist that facilitate accurate cell profiling and allow comparisons of relevant immune cells. Therefore, in this paper, we will discuss the route of infection, predominant cytokine and protein markers, and, finally, individual cellular responses of predominant immune cell lineages in order of B cells, neutrophils, monocytes/macrophages/dendritic cells (DC), natural killer (NK cells), and T cell subtypes.

1.3. Respiratory Microenvironment

Respiratory tract organs affected are the nose, throat, larynx, trachea, bronchi, and lungs exposed to external antigens composed of surface epithelial cell layers. An adult human lung surface area contains approximately 700 million alveoli, with a surface area of 70 m2 and a diameter of between 200 µm and 500 µm, covered by capillaries. Within this defined alveolar layer are ciliated type I pneumocytes (ATI) cells, as well as type II pneumocyte (ATII) cells and alveolar Mφ (AMφ) that regulate respiration, secretion of surfactant, and immune cell regulation, respectively, alongside goblet cells, basal cells, and other cell types [24]. Early studies (n = 7) in chronic SARS-CoV-2-induced disease show direct infection of ATII cells through the glycocalyx and surfactant layer, thereby compromising homeostatic barriers and valve functions through increased pressure of inhaled O2 or exhaled CO2 within nanobubbles across cell membranes where CO2 is produced through the tricarboxylic acid (TCA) cycle [25]. The glycocalyx layer is known to contain an abundance of proteins that affect vascular function (e.g., syndecans) that can be degraded and affect the vasculature, such as matrix metalloproteinases (MMP), heparanase, and hyaluronidase, through the action of cytokines (IL-1β and others) [26,27]. This process of respiration is dependent on membrane thickness and gas solubility of O2, N2, and CO2 nanobubbles [28] (see Figure 1).

Figure 1. Overview of SARS-CoV-2 immune cell interactions.

1.4. Cytokine and Serum Proteins during SARS-CoV-2 Infection 

Serum protein elevation, documented as a “cytokine storm” that is elevated or dysfunctional in SARS-CoV-2-induced chronic COVID-19 disease, occurs in many other pathologies [32]. Cytokines are a group of short-lived proteins released by various cells acting as intercellular messengers. Cytokine synthesis and secretory mechanisms include release from lysosomes, shedding of vesicles from plasma membranes, and release from plasma membranes. Many studies document these, which are not the main topic of this review. In comparison, in influenza (genus Influenza A/B/C/D) infection, the cytokines IL-1β, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, TNF-α, and IFN-γ are relevant to immune cell interaction, as outlined below in figures. During SARS-CoV-2 infection-induced COVID-19 disease, other proteins have been considered, which include transforming and vascular endothelial cellular growth factors (TGF-β/VEGF) in conjunction with specific MMPs (MMP2, MMP3, MMP9) [33–36]. These represent tissue re-modeling proteins with specific chemotactic factors also required to direct leukocyte chemotaxis between germinal center lymph systems (GC) and throughout the body [34–36]. Relevant chemokines considered below include CXCL10 (IP-10), CCL2 (MCP-1), CCL3 (MIP1-α), and CCL11 [33–37]. However, prior to the 2020 pandemic, in a related coronavirus (MERS-CoV) causing Middle East Respiratory Syndrome (MERS), the cytokine proteins IL-1β, IL-6 and IL-8 were highlighted as key to host response, whereas, in infection, CXCL10 and other pleiotropic chemokines are further investigated that utilize CXCR3 expressed on Mφ, T cells, DCs, and both NK/B cells [38–40]. The above cytokines and chemokines are, therefore, all innate/adaptive regulators that contribute towards infection control and regulation within blood serum. Studies indicate that COVID-19-associated coagulopathy (CAC) is a causal factor in chronic disease with complexes formed between innate immune cells affecting coagulation and fibrinolytic processes through unknown mechanisms. Categorization of COVID-19 has, therefore, occurred in vascular endothelial cell dysfunction, hyper-inflammatory response, and hypercoagulability, documenting this aspect of SARS-CoV-2 induced pathology with resulting serum elevation in plasma levels of D-dimer, C-reactive protein, P-selectin, and fibrinogen [41]. More recently, in a yet-to-be-reviewed pre-print, 7315 proteins were investigated specifically in chronic COVID-19 disease, relating to complement protein as key in the coagulation pathways; complement C1q subcomponent subunits A, B, and C (C1QA, C1QB, and C1QC) were mainly enriched in lungs and LNs [42]. Complement factors C3, C5, C7, and C9, in contrast, were commonly upregulated in LNs and aorta/vessel walls with downregulated SP-C in ATII cells [42]. Investigations indicate two other proteins, the receptor for advanced glycation end-products (RAGE/AGER) and chloride intracellular channel (CLIC5), also associated with ATI cells, but that an SP-C-related protein was downregulated specific to ATII cells, as above [42]. Interestingly, the authors noted a significant reduction in IL-12 production in LN that can affect DC maturation, as discussed below. Many cell cycle regulatory proteins were measured, such as a cyclin-dependent kinase (CDK2), but also origin replication complex (ORC) and nucleoporins (NUC), which were observed to be upregulated in LNs. Furthermore, many protein changes were noted as corresponding with tissue cellular changes within the glycocalyx. In a similar case-control study looking at biomarkers in seropositive individuals (n = 400), significant changes also occurred to E-selectin (CD62) and cathepsin B, and persistent symptoms may be associated with iron-sulfur cluster co-chaperone protein (HSCB), heat shock protein HSP 90-beta (HSP90AB1), amyloid-beta precursor protein (APP), phospholipase D Family Member 3 (PLD3), cystatin-C (CST3), and calprotectin (S100-A9) [43].

1.5. Pre-2022 Laboratory Research Context

Since 2015, research studies have clarified that SP could modulate host immune responses in lung inflammation and, therefore, may be a therapeutic target during increased dysregulation seen in chronic COVID-19 disease [44]. There is a discordance in the literature, and this may have been overlooked during the influenza 2009 H1N1 pandemic [44]. As numerous reports (n = 10) clarify, with SARS-CoV-2 infection, there is extensive alveolar damage with endothelial injury of cell membranes, vascular thrombosis, occlusion of alveolar capillaries, edema with angiogenic vessel growth, and lymphocyte migration [44]. The resulting mechanisms controlling cytokine regulation occur between all leukocytes with resulting questions over immune cells and respective interleukins (IL), growth factors (GF), chemokines (CXC), and respective receptors or ligands (e.g., CXCR3 and/or CXCR4) that require further clarification below [45]. SARS-CoV-2 pathogenesis begins with disrupted membrane homeostasis with resulting syncytia formation, cell fusion, and multinucleate cells accompanied by immune system dysregulation [46–48]. This formation of syncytia could be initiated by transmembrane proteins (e.g., TMEM16) that regulate phospholipid-rich cell membranes including phosphatidylserine (PS) [49–51]. Braga et al. utilized cell fusion inhibition assays (CFIA) and in situ measurement of viral RNA assays (n = 41) studies in affected individuals to clarify that SARS-CoV-2 infected individuals had fused cell syncytia dominant that contained napkin, which processes SP-B common to ATII cells [50]. They noted that, by regulating an ion channel dependent on calcium and one scramblase enzyme that regulates PS, the S protein clearly appears to activate transmembrane proteins (TMEM) at the cell membrane surface or within organelle membranes [49]. For example, one of these TMEM16 is part of a protein family consisting of calcium-dependent ion channels responsible for PS regulation in a normal calcium- and arginine-rich layer [49]. Concurrently, it is now known that SARS-CoV-2 ORF3a may affect a calcium-regulated ion channel, TMEM16F, regulated by PS, that can augment procoagulant activity through tenase and prothrombinase complexes, which are key regulators of the coagulation pathway [50–52]. Therefore, such localized changes infer routes of SARS-CoV-2 entry within the epithelial microenvironment. Indeed, the carbohydrate-rich glycocalyx layer covering mucosal epithelial cells also contains a mixture of mucin (MUC) glycoproteins, glycosaminoglycans, and other glycoproteins, which extend and surround cilia and normally function to clear larger bacteria. Expansive research recently revealed that cilia, microvilli, and mucus function remain key for SARS-CoV-2 adhesion and receptor-mediated entry into epithelial cells, which appear to act as adhesives. MUC proteins are high-molecular-weight proteins that form mucus clusters. In COVID-19 disease, initially (n = 16) two types of mucins were extensively investigated, of which membrane-tethered MUC1 and the gel-forming MUC5AC appeared at significantly elevated levels. Therefore, normal pathogenic clearance via mucin proteins could be disrupted, facilitating SARS-CoV-2 entry to allow viral persistence [53–56]. Importantly, other research indicates that, in addition to ORF3a, other SARS-CoV-2 proteins including E and ORF8 can act to assemble and form toxic ion channels [57,58].

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1.6. Role of Toll-like Receptors (TLR) or TLR-Induced IFN Dysregulation

In order to mount an anti-viral response, usually type I IFN is produced [59]. Current research contradicts this, as type I IFN production presents as beneficial and detrimental in COVID-19 disease; however, studies examining Middle Eastern respiratory syndrome (MERS) and respiratory syncytial virus (RSV) indicate that the timing of type I IFN production affects the cellular response [59,60]. Additional considerations are surface and cytosol pattern recognition receptors (PRRs) that initiate downstream signaling cascades utilizing NF-kB, type I IFN, and inflammasome pathways [43–46,61]. These include damage-associated molecular proteins (DAMP) that encompass a myriad of proteins surrounding and within nuclear and extracellular spaces that include ten conserved Toll-like receptors (TLRs), retinoic acid-inducible gene-I-(RIG-I)-like receptors, Nod-like receptors (NLRs), AIM2-like receptors, and intracellular DNA and RNA sensors, that lead to production of pro-inflammatory or anti-viral cytokines necessary for antigen-specific adaptive responses [61,62]. For example, IL-1RA is a DAMP receptor, that, once released intracellularly, binds to and initiates IL-1α release, which is supported by case studies (n = 71) that showed this was the case in chronic COVID-19 disease concurrently with IL-10, which is largely immunosuppressive [63,64]. It is known that SARS-CoV-2 proteins are recognized by cellular sensors and, therefore, the roles of TLR3/4/7 are of interest in terms of which immune cells express these. TLR3 is more abundant in NK cells, whereas TLR4 is more common in Mφ. Toll-like receptors (TLRs) transduce signals via MyD88 and TRIF. Most TLRs use MyD88 to trigger inflammatory cytokine production; TLR3 is the exception and signals exclusively through TRIF, whilst TLR4 is unique in that it can bind and signal through either MyD88 or TRIF to nuclear transcription factors. Previous in vitro studies indicate that TLR3/7 can be associated with IL-1α, IL-1β, IL-4, and IL-6 release [65]. Therefore, other studies investigated the nature of TLR7 as a risk factor in severe COVID-19 disease [66]. The role of TLRs in immune cell signaling is largely unclear, and will undoubtedly need further research, but is implicated in T-cell signaling [67]. TLR4 presents on monocytes, Mφ, and DCs, and in some non-immune cells, such as endothelial cells, and has a role in both LPS-induced Gram-negative bacterial CD14 immune cell trafficking, and, interestingly, may regulate RORγt + regulatory T cell responses in colitis [68–70]. Clinical trials regarding newer therapeutics affecting TLR are ongoing (NCT05089110, NCT04526977, and NCT05293236) in both COVID and HIV pathologies that will clarify this further (see Supplementary Materials). The role of SP-A, as discussed above, is under research, and it is plausible that TLR4 expression has differential effects within select organ systems depending on activation, as seen in neonates, where TLR2/4 activation was shown to stimulate downstream extracellular signal regulated kinase (ERK) and protein kinase B (AKT) with IL-6 pathways unchanged between children and adults [71]. Expression on both platelets and alveolar Mφ could affect thrombotic and immune pathways simultaneously with reduced expression on ATII cells, and confirmation in animal studies, which have recently been shown to link TLR4 to intestinal cytokine mRNA expression [72–74]. TLR4 clearly has an influence on platelets through aggregation and P-selectin expression, and the formation of mixed aggregates between platelets and neutrophils, and in microbes with LPS triggers synthesis and/or secretion of von Willebrand factor (vWF), platelet factor 4 (CXCR4), and thromboxaneA2 (TXA2), alongside NETosis with CD11b upregulation and other adhesion molecules (Supplementary Data S1) [75,76]. Originally identified in 1957 by Isaacs and Lindemann, IFNs were found in secretions to inhibit viral and tumor growth. They are currently classified into three groups and individual subtypes: Type I, II, and III. Type I IFNs consist of IFN-α and IFN-β (also IFN-δ, IFN-ε, IFN-κ, IFN-τ, IFN-ω, and IFN-ζ) but also within Type II is IFN-γ, whilst type III IFNs encompasses IFN-λ [77]. With regard to SARS-CoV-2 sensitivity to IFN, early clinical case studies indicate SARS-CoV-2 sensitivity to IFN-α and IFN-β in vitro, however, more recent tissue studies indicate that IFN-α and IFN-β response could paradoxically facilitate the viral propagation from the respiratory epithelium to the vasculature through direct endothelial cell infection [78,79]. Recently, type III IFN-λ has been investigated and is under clinical research following earlier studies (n = 257) that document reduced IFN- λ2 during chronic COVID-19 disease [80]. The cellular source of SARS-CoV-2 infection-induced IFN production is largely unknown presently, as IFN receptors are located within B cells, monocytes, Mφ, T lymphocytes, glial cells, neurons, and plasmacytoid dendritic cells (pDCs), among others [60,61]. Interestingly, epithelial response in vitro studies shows that IFN-γ can promote SARS-CoV-2 infection in cell culture to enhance cell differentiation within enterocytes in vitro [81]. IFN-λ has been seen to be activated by bacteria, including Staphylococcus aureus [82]. It is of note that type II IFN and type III IFN can be secreted by NK and T cells, and few studies document whether type III IFN affects antibody classs witching. Therefore, as a key mediator of anti-viral responses within the respiratory tract, it is now being seen that IFNA2 and IFNG gene expression in the respiratory tract is accompanied by increases in IFNB1, and also with reduced early IFNA2, but that this IFN response seems to occur in sera rather than tissues [83–85].

2. Innate Immune Systems and SARS-CoV-2 Research

2.1. B Cell Development Dependency on T cell Activation

B lymphocytes represent 10% of white blood cells (leukocytes). Central to innate immune responses as pathogen sensors, these develop in germinal centers (GC) and then are distributed throughout the lymphatic system network by secretion of immunoglobulins (Ig), determining detection and neutralization of antigens through cellular development processes [86]. B cells respond to non-host antigens dependent on receptors that include antibodies shed from the cell surface (e.g., IgM, CD79a, and CD79b) (see Figure 2). 

Figure 2. B cell and T cell interactions

B cell development from hematopoietic precursor cells (HPSC) occurs in stages from pro-B cells, pre-B cells, immature B cells, and growing into mature B cells in the fetal liver and then in bone marrow. B cell responses are defined by CD markers evolving into mature B cell subpopulations, such as B-1, B-2, and regulatory B cells [87,88]. Research on newer B cell subtypes defined by other phenotypic CD markers with single-cell sequencing has occurred since 2017. Remarkably, B lymphocytes synthesize up to 1011 antibodies, or B cell receptors (BCR), within a host that undergoes clonal selection and somatic hypermutation (SHM) leading to the specificity of antigenic epitope protein recognition. BCR consists of a transmembrane section extending through the cytoplasm with protein sequences that depend on co-activation or stimulation from other proteins to activate B cells. Other CD molecules define B cell development or lineage (e.g., CD19, CD21). These are relevant to cell-residing locations, developmental stages, maturation, and activation states. CD10 expression occurs on first-stage B cell lineage cells (e.g., pro-B, pre-B cell, and GC) and can change throughout maturation with others shown (see Figure 3) [89]. Moreover, CD27 exclusively resides within memory B plasma cells, whilst CD5 characterizes B-1 cells and DCs (see Figure 3). B cell receptor (BCR) complexes with other T cell markers (TCR) influencing maturation and antigen presentation result in pre-GC memory B cells (pre-GC MBCs) and short-lived plasma cells (SLPCs) that produce low-affinity early antibodies. Other B cells reach the GC, where antibody affinity and selection can occur by clonal selection/SHM, modifying protein structure via class-switching recombination (CSR), resulting in long-lived plasma cells (LLPCs) and memory B cells (MBCs) with specific antibody isotypes, but also plasmablasts (PB) that produce Ig of the five main isotypes that occur as multimeric proteins (IgM, IgG, IgA, IgE, and IgD) in normal host-specific immune responses. These are indicated within these ranges in sera IgG: 80%, IgA:15%, IgM:5%, and IgD:0.2%, with trace amounts of IgE (see Table 1) [90].

Figure 3. B cell phenotypes during maturation.

Table 1. Antibody isotypes concentrations in sera and complement activation ability [83].

2.3. Role of B Cell Markers in Current Research

CD19 has long been used as a B cell biomarker [104]. In recent years, this has expanded into characterization, as below, of B cells by receptors expressed at different maturation stages throughout B cell lineages, which include naïve B cells, unswitched memory B cells, switched memory B cells, and double-negative (DN) B cells, but also, alongside this, chemokine receptors responsible for systemic lymphatic direction. Some studies suggest there is no consensus on DN B cells; however, these recently characterized DN B cell's cellular markers are clearer (see Figure 3 or Supplementary Data S2). Further DN B cell analysis of CD11c has refined these into subsets expressing CXCR5 hypothesized to emerge from naïve B cell activation outside of the GC [105]. Researchers expanded this recently to two other subtypes, DN3 and DN4 B cells (see Figure 3). Enrichment within certain DN B cell subsets has been discussed to play a key role in other comparatively well-characterized autoimmune diseases (multiple sclerosis (MS), systemic lupus erythematosus (SLE), myasthenia gravis (MG), and rheumatoid arthritis (RA) [105–107]. For example, CD27+ IgD+ has been suggested to have impaired gene signaling in RA through VH3-23D to VH1-8, affecting production or, rather, reduced BCR diversity during selection [108]. Thus, recent subtypes have examined the DN CD11c B cell phenotype (n = 18) to show these in autoimmune pathologies compared to healthy controls (SLE, Sjøgren’s syndrome). Furthermore, B cells (CD19) expressing CD11c+ together with elevated levels of CD69, Ki-67, CD45RO, and CD45RA, as metabolic markers and B cell memory phenotype markers, along with a lack of DN cell markers CD21, could well escape normal immune cell regulation [109,110]. Therefore, the depletion of B cell subsets has also been examined in age, referred to as age-associated B cells (ABCs), with regard to affecting the production of autoantibodies [111–118].

2.4. B Cell Antibody Responses during Respiratory Infections

IgG1 and IgG3 were initially associated with severe disease in older adults with COVID-19 (n = 123) disease and accompanying irregularities in neutralizing antibodies (nAbs), chemokines, and T cell responses, which is an anomaly as IgG3 was previously thought to provide enhanced pathogen response [91,119,120]. However, IgG deficiency has been observed to be associated with increased mortality risk in Chronic Obstructive Pulmonary Disease (COPD) patients (n = 489) in these ratios: 56% IgG1: 27%: IgG2: 24% IgG3: 31% IgG4 [120]. A previous study (n = 105) comparing serology of hCoV-229E, hCoV-OC43, hCoV-NL63, and hCoV-HKU1 elucidated that participants show in other hCoV antibody responses with regard to IgG that were 99%:100%:98%:91%, with IgA in nasal wash samples, detected in between 8% and 31% of participants [121]. Lower pathogenic hCoVs represent 15–30% of common cold respiratory tract infections in humans each year, also with seropositivity estimated at 90% in adults, indicating that T cell roles require further clarification [122]. A non-coronavirus antigen comparison would therefore be the influenza virus that expresses haemagglutinin (HA) and neuraminidase (NA) proteins that present seasonally. In these cases, research indicates increases in serum IgM-specific HA antibodies during the 2009 H1N1 pandemic in these ratios: IgM (86–94%), IgG (100%), and IgA (76 to 96%) [123,124].

2.5. B Cells and Antibody Responses to SARS-CoV-2 Infection

In late 2020, Plume et al. carried out a unique study (in brief, see Table 2) examining SARS-CoV-2 protein antigen fragments, analyzing these against antibody isotypes against most of the virus antigens, and they indicated that seroconversion at day 20 may cause issues, as 97.3% reacted against the chosen epitopes of SARS-CoV-2, but E protein was not investigated in this study [125–127]. The overall frequency of individual antibody responders within individuals (n = 103) in this study, which was conducted between April 2020 to January 2021, is indicated (see Table 2). Differential polyclonal antibody responses would normally occur as immune cells mature against different viral protein antigens. Therefore, the frequency of individuals producing polyclonal antibodies against subtypes of SARS-CoV-2 S protein (S1, S2), M protein, and N protein antigens in pre-January 2021 samples was examined. This ranged with IgG dominant against RBD, S1, and M protein domains in 100% of their samples in moderate to severe disease (see Supplementary Data S3). The frequency of IgA responders was seen as 24.7–35.6%, reacting against the whole S protein domain, RBD, S1, S2, M, and N protein domains, with the latter not predominant in severity. It is noteworthy that this study recognized limitations of IgE assay sensitivity at that juncture, and would be worthy of further investigation in comparable studies [126,127].

Table 2. Frequency of Individual Serological Response During SARS-CoV-2 Infection (%) [127]. Copyright permission refers to citations and/or Supplementary Data.

B cell memory and production of S-protein-specific Ig has been measured, and it can now be seen that a putative role for unknown B cell subtypes, such as DN2 B cells downregulating CXCR5, or other direction-specific receptors such as CD62L (L selectin) that are usually expressed, could affect the immune responses [125]. CD19+ CD24+ CD27+ CD38+ B cells indicate that antibody responses to SARS-CoV-2 S protein-specific B cells are increasing, although further details can be observed regarding the two other relevant transitional B cells (see Supplementary Data S2) [107,126]. In contrast with other studies, it was found that a 32 amino acid peptide (V551–L582) in the currently mapped RBD domain could be an immunodominant B cell epitope, equating to 58.7% of IgG samples tested [127]. However, E protein assays were performed in concurrent studies that did not appear, in historical analysis, to have similar viral neutralization properties pre–2020, indicating that prior exposure may not have occurred as epitopes differ in viral antigens. Initial plaque neutralization assays indicate that sufficient concentrations of nAbs may occur to S1, RBD, and potentially to N protein SARS-CoV-2 domains with predominantly IgG response. As above, naïve B cells expressing IgD+CD19+CD27− (see Figure 3, Table 3, and Supplementary Data S2) can be sole predictors of antibody titrations compared to control groups with high significance (p = 0.009) [128].

Table 3. B cell phenotypes (adapted from Li et al.) [107]. Copyright permission refers to citations and/or Supplementary Data S2, also see Figure 3.

Nonetheless, chronic COVID-19 disease patients presenting with DN (IgD− CD27−) B cells are shown to experience worse disease severity and complications. The DN1 subset is noteworthy for holding potential for early activated memory cells, whereas the DN2 cells encompass antibody PB–PB-secreting cells that have been primed beforehand; however, it remains uncertain what impact each DN B cell subtype and mechanistic property has on disease resolution [129]. Interestingly, earlier in the pandemic, the nature of the SARS-CoV-2 S2 domain antibody response was indicative that it was comparatively immunogenic, stimulating both IgA and IgG. Statistically, eighty-six percent (86%) of individuals were seen to have antibodies against this conserved S2 domain, with more concentrations of antibodies produced against the S2 domain than the RBD domain [130]. SARS-CoV-2 can be considered novel by not eliciting a higher level of secretory IgA, such as both influenza and lower pathogenic hCoVs. In fact, chronic COVID-19 disease did stimulate elevated levels of five serum antibody types of IgM, IgG1, IgA1, IgG2, and IgG3 [131–133]. It was demonstrated in chronic COVID-19 disease that significantly higher IgG1, IgG2, and IgG3 occurred at day 3 alongside transient elevation in IgA1, disappearing by day 7; the impact of this is unclear, as are the cellular mechanisms that affect this, but research is ongoing. A concurrent study also confirmed that IgM–IgA1, IgM–IgG1, and IgM–IgG2 were enriched in acute SARS-CoV-2 infection, demonstrating an initial robust immune response [84]. Therefore, along these lines, IgA variability was investigated in sera to determine cellular phenotypes, including FACS analysis (n = 135) of PB B cells to show in acute infection that IgM and IgG were secreted between 10 and 15 days after infection, in these ratios: IgM: 10.5% (range 4.2–54.1), IgG: 27.9% (range 7.4–64.8). Therefore, B plasma cells (PBs) that produce IgA were further quantified by expression of proliferation markers and B cell markers Ki67+CD19loCD27hiCD38hi to produce IgA: 61.4% (range 18.1–87.6) occurring in these antibody subtypes, IgA1:66% (range 26.8–88.5), compared to IgA2: 31.6% (range 3.7–70.8), which are in line with above overall mucosal immune responses [134]. However, individuals with dominant IgA responses were seen to have an associated risk of mortality in severe COVID-19 disease, as below, that experienced dysregulated myelopoietic responses. High IgA to low IgG titrations can cause pathological consequences in the host, involving decreased pathogenic phagocytosis, increased cellular apoptosis, and increased NETosis, as reported in late–stage fatal COVID-19 disease. Individuals possessing a high IgG to IgA ratio experience increased inflammatory dampening through immune response, resulting in better prognoses and early–late–stage disease resolution. Limited data exists to reveal why SARS-CoV-2-induced COVID-19 disease exhibits such a novel antibody profile regarding IgG1/IgA1 responses. It is considered that alterations to the Ig structure can produce complications, either increasing infections or immune complex formation and in other pathological diseases such as dengue antibody–dependent enhancement (ADE), IgG antibody structure was examined. These changes include glycosylation (glycan or carbohydrate adjoining hydroxyl or other functional groups) and fucosylation (transfer of sugar fucose from a GDP–fucose to other proteins or glycans), and, therefore, this could affect leukocyte extravasation and selectin–mediated binding through cellular membranes, which is recognized as a potential factor in cancer therapeutics [135,136]. Therefore, studies during the pandemic (n = 33) examined this in chronic COVID-19 disease to confirm that IgG, against the SARS-CoV-2 RBD protein, could potentially affect Mφ release of IL-1β, IL-6, IL-8, and TNF [137]. However, IgG3 and IgM are inferred to be responsible for 80% of the neutralization of SARS-CoV-2, with suggestions that IgG3 glycosylation affects SARS-CoV-2 binding specificity to the S protein [132,138]. As mentioned earlier, glycosylation can occur where an N–N-linked glycan forms within the IgG-Fc region. In accordance with examining the IgG subtypes, a recent study from Brazil examined the avidity of IgG (n = 47) to SARS-CoV-2 proteins to show an increase in IgG1 and IgG3 levels at day 8, and IgG4 concentration levels were less detectable during the study period. Mortality at 8–21 days showed higher anti–RBD IgG4 levels in comparison with the recovered, which contradicts other studies and is relatively unknown with regard to IgG4 pathology research [139]. Initial screens of N/S/E SARS-CoV-2 proteins in smaller cohorts (n = 320) indicated that anti-N IgG and anti-N IgA were produced in response to SARS-CoV-2, and IgG antibodies were produced to S1 and E proteins, but also that the anti–E protein antibodies evoked were not significantly higher, which is indicative of the current immunogens within clinical trials and those used in lateral flow testing [140]. Currently, in vitro, it has been determined that specific memory B cells are produced for up to 6 months, compared to S protein, with IgG measured at 66% and IgM at 100%, which would be consistent with either natural infection or vaccination [126].

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2.6. Role of B Cell Markers during SARS-CoV-2 Infection and Other Conditions

Antibody responses to SARS-CoV-2 immunogens can be attained in individuals on anti-CD20 therapy via the onset of B cell repopulation [150]. In the absence of B cells, a strong T cell response is nevertheless generated, which may help to protect against chronic SARS-CoV-2-induced COVID-19 disease in this high-risk population [150]. Therefore, it is essential to understand the nature of this response. Researchers in 2020 found, earlier in the pandemic, that within chronic COVID-19 disease (n = 52), affected individuals’ overall quantities of B cells did not significantly change. However, compared to healthy individuals, DN1 B cells significantly decrease with severity, with increases in DN2 B cells seen between moderate to chronic disease, but note a significant increase in patients during disease severity of DN3 cells, but with no concurrent changes in the DN4 B cells. Other B cell subsets were therefore investigated to discover a novel subset of B cells named “transitional B cells or TR” that may correlate with clinical outcome as measured by B cells expressing more CD24 than CD21 [129]. This was an interesting finding because CD24 expression is known to affect cell migration, invasion, and proliferation, while expression or lack of CD21 is associated with B cell switching and memory with complement proteins. CD21 is also expressed on follicular dendritic cells and known to associate as a complex with complement proteins (C3dg, C3d, and inactive C3b) on the antigen surface, together with CD19/CD81 [115,129,151]. Interestingly, increased TR cells did correlate with COVID-19 disease routinely used blood protein markers detected, including neutrophil/lymphocyte ratio, acute phase proteins, ferritin levels, D-dimer, and others. The exact nature of DN B cells requires further clarification, as subsets are associated with SLE [152]. As before, the B cell DN1 reduction/DN2 increase in chronic COVID-19 was accompanied by high levels of CD69 and CD89 in DN2 cells, alongside what appears to be DN2 selection of IgG, but also suggests that DN3 cells may produce VH4-34 IgG autoreactive antibodies, some of which could be protective [118]. There were initial indications that germline Ig variable heavy chain VH4-34 showed decreased SHM frequencies, which would affect B cell Ig maturation through the SHM process [153]. Unswitched memory B cells (CD27+ IgD+ ), historically, are part of normal and pathological immune responses with reduced overall IgM-secreting B cells. For example, in RA, unswitched B cells are thought to occur due to gene recombination, contributing to antibody selection by VH3-23D to VH1-8 [108]. Interestingly, the BCR repertoire of these cells was altered in RA, exhibiting some of the same markers as DN2 cells, such as CD11c, FcRL5, and transcription factor (T-bet) [154,155]. While antibodies generated by B cells are historically well-characterized, it is unclear why SARS-CoV-2 generates high antibody responses in chronic severity and not in acute infection. The timing of an antibody response is important in antibody-based therapeutics, as drug application influences patient outcomes [156,157]. Naïve B cells are activated with the assistance of follicular T (TFH) cells [158]. Therefore, this novel antibody expression caused by SARS-CoV-2-infection-induced chronic COVID-19 disease was found to be of three antibody classes and isotypes, including IgM, IgG1, IgA1, IgG2, and IgG3, which require further analysis. Recent analysis of SARS-CoV-2 S protein immunogens indicate Ig expression by spike-specific B cells at six months was produced in these ranges: IgG: 61.33–77.46%, with concurrent IgA: 3.04–7.37%, and IgM: 12.30–24.97%, to note significant reduction in IgG/IgA with significant increase in B cell-specific IgM at six months [159,160]. As discussed earlier, B cells develop in GCs and, through a small cohort study (n = 15), a role for circulating TFH cells was elucidated to show S–protein–specific B cells develop through SHM. At five months, 66% of this cohort had B memory cells to vaccine immunogens, and this research suggested that there was a slight increase in nAb [160–163]. Concurrent with other studies, unsurprisingly, minor differences in memory–switched cells as the main population were represented (median: 59.92%) [163]. Their analysis examined SARS-CoV-2–specific B cell markers, CD27 and CD38, that were accompanied by a significant increase in CD27hiCD38hi PBs. This occurred in recovered individuals compared to uninfected individuals at six months, with IgD+CD27+ and IgD− CD27+ B cells that were significantly reduced in chronic SARS-CoV-2 infection [161,163]. The extra follicular response remains under investigation and Woodruff et al., in a cohort study, suggested that the DN2/DN1 B cell ratio may underpin some of the serology anomalies in severe COVID-19 disease with CXCR5 downregulated and CXCR3 upregulated. CXCR5 is a chemokine constitutively expressed on specifically B cells and TFH cells responsible for directing B cells to GCs, while CXCR3 has multiple ligands, including CXCL8/9/10, but is preferentially expressed on TH1 cells and the majority of the T cell population, DCs, and memory B cells. Evidence is emerging that upregulation or changes in these and other chemokines (CXCR3, CXCR5, CCR7) in acute infection and downregulation in severity would be a further indicator relevant to the maturation of DCs [162,163]. Statistical significance was apparent between antibody-secreting cell (ASC) expansion with high levels of CD21−B cells independent of duration of infection [164,165]. B cells control antibody secretion, and reports indicate that IL-10 and IL-21 are responsible for B cell class-switching to IgG1, IFN-γ class-switching to IgG2, and TGF-β switching from IgA1 to IgA2 responses [133]. Research shows that IgG1 and IgG3 (n = 123) correlate in chronic SARS-CoV-2 severity with a cytokine IL-1β response [119]. IgG2 is thought to be more relevant to bacterial responses to capsular polysaccharide antigens. Concurrent in vitro studies also indicate that SARS-CoV-2 IgA1 and IgG3 may have a protective neutralizing effect in SARS-CoV-2 infection [164,165]. Further research would be required to clarify this claim. Other studies (n = 82) confirm that in chronic SARS-CoV-2 infection, within seven days, the serum antibody response is 60% IgA, 53.3% IgM, and 46.7% IgG, with IgG reaching 100% by day 2 [166,167]. Therefore, a single-cell transcriptomic study analysis of severe COVID-19 disease in detail highlights that DN1 cells express IgA2 genes, and can therefore potentially secrete IgA2, whilst DN3 B cells were seen to express IgM genes absent in DN2 cells, with DN4 cells possessing IgE genes and corresponding Fc receptor genes (see Figure 3) [118]. Interestingly, this raises the possibility that there are distinct T cell-independent and T cell-dependent pathways in B cells during COVID-19 disease, which is now suggested in other concurrent studies. IgE, therefore, can be produced by DN4 B cells, but serology from COVID-19 patients was not considered to be statistically relevant to this cellular subset [118,129,168,169]. IgE is known to cause mast cell degranulation through a higher affinity FcεRI receptor, and assay sensitivities require validation and development for this antibody normally seen in allergic responses with the IgG response predominant during infection. It is notable that the H2 receptor, present in gastric mucosa, brain, and mast cells, was targeted using an antagonist famotidine in trials, and was seen to have some effects in modulating SARS-CoV-2-infection-caused symptoms with synergism with macrophage TH2 cytokines [170–172].

3. Inflammatory Cells and Phagocytes

3.1. Neutrophil Introduction

Polymorphonuclear neutrophils (PMN) are granular and trilobed, being the most common circulating leukocyte, representing between 40% and 80% of leukocytes in normal adults. Neutrophil infiltration in respiratory tissues is characteristic of many in- inflammatory diseases [173]. Neutrophils are granular, acting against antigens by dispersing azurophilic cytoplasmic granules using the actions of proteolytic enzymes (e.g., myeloperoxidase, elastases, and proteinase–3) but also lactotransferrin, lysozyme, or reactive oxygen species (ROS), which are also anti–microbial, for clearing pathogens [174,175]. Pathogenic stimuli trigger cellular calcium release via endoplasmic reticulum (ER), resulting in activation of protein kinase C (PKC) and assembly of the NADPH oxidase complex generating ROS. Neutrophils form hematopoietic stem cells (HPSCs) in the bone marrow and are short–lived, between 1 and 7 days, and traverse cell membranes by selectin–dependent capture and integrin-mediated adhesion (see Supplementary Data S1), after which migration to tissues occur, and they survive for 1–2 days while circulating and clearing by phagocytosing Mφ. Development of neutrophils occurs in bone marrow from progenitor neutrophils and can be broadly classified according to CD markers as CD81+CD43+CD15+CD63+CD66b+, which differentiate into immature neutrophils expressing CD11b+CD66b+CD101+/−CD10−CD16+/− before maturing in the bone marrow to express CD11b+CD66b+CD101+CD10+CD16 [176]. CD16 is co-expressed on other cells including NK cells, monocytes, Mφ, and certain T cells [176]. CD16 (FcγRIII) has subtypes including CD16a and CD16b (FcγRIIIa/FcγRIIIb), whilst CD11 and, specifically, CD11b are classified as more relevant to migration and lung inflammation [177–179]. It is notable that CXCR2 and CXCR4 appear to be key regulators within this cell subset implicated in lung fibrosis, but these also modulate cellular mitochondrial activity, neutrophil migration, and neutrophil homing utilizing adhesion receptors that include CD62L [180–184]. However, more recently, CD11b and CD18, which are ubiquitously expressed, are now thought to require CD47 in epithelial transmigration [185,186]. Interestingly, Alberca et al. examined a novel cell subtype, defined as myeloid-derived suppressor (MDSC) cells, within laboratory case studies: CD33+CD11b+HLA–DR−CD14−CD66b+ and CD33+CD11b+HLA– DR−CD14+CD66b−cells. In peripheral blood markers of chronic COVID-19 disease, this was seen to correlate possible M–MDSC and polymorphonuclear P–MDSC, which have been linked to chronic inflammation [186]. These MDSC–defined cells have been described initially in cancer, HCV, and HIV to affect T cell proliferation. Recent clarification on putative phenotypes emerging as M–MDSC (CD11bloD14+CD15−HLA–DR−) and P–MDSC as CD11bloCD14−CD15+ HLA–DR− have been suggested to affect TREGS as below through TGF–β affecting the overall balance of both TREGS and self–tolerant DCs [187].

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3.2. Neutrophil Cellular Markers after Host SARS-CoV-2 Infection during COVID-19 Disease

During chronic COVID-19 disease, it is believed that neutrophils form neutrophil extracellular traps (NETs), as we discussed above, where the cell signaling within is disrupted, causing active degranulation or “NETosis/neutrophil apoptosis” [188]. The exact mechanisms of NETosis contribution remain unknown [189]. Therefore, recent case studies (n = 64) focused on identifying neutrophils' cellular markers further. During COVID-19 disease, some appear to suppress stimulation of IFN-γ production with unknown cell subsets that stimulate T cell proliferation but fail to activate T cells [190]. Several authors suggest host driven immune response is causal in the lack of production of type I and III interferons in conjunction with elevated chemokines, and IL-6 is a causal factor in coronavirus pathology [191,192]. Whilst IL-6 could be the predominant cytokine regulator of NETosis, other protein markers are clearer, those being extracellular DNA (cDNA), neutrophil elastase (NE) activity, or myeloperoxidase-DNA (MPO-DNA), and these correlate with disease severity, measured in neutrophils by markers CD33loCD16+CD11b+ [193]. Researchers recently found (n = 155) that NE, histone-DNA, MPO-DNA, and free double-stranded DNA (dsDNA) were increased with concurrent DNase reduction and exacerbation of neutrophil stimulation occurring via IL-8, CXCR2, and DAMPs with impaired degradation of NETs via DNase 1 and DNase 11L3, which are suggested to act as regulators of neutrophil DNA metabolism [194]. Recent abstracts also imply a correlation between these and either membrane-bound or soluble CD13 [195]. A comprehensive neutrophil analysis (n = 384) utilizing single-cell analysis classified six cell states defined by inflammatory gene signature (IGS) to indicate that concordant IgA1:IgG1 ratios are elevated in coronavirus disease mortality, with IgG indicating antibody-dependent neutrophil phagocytosis and IgA2 inducing apoptosis [133]. Interestingly, neutrophils expressed significantly increased levels during maturation of CD32 (FcγRII), CD16b (FcγRIIIb), and CD89 (FcαR), the main Ig receptors which have respective ligands on B cells, above. No known studies currently exist connecting type III IFN with this isotype switching. A similar investigation into IgA2 (n = 97) confirmed that anti-SARS-CoV-2 IgA2 in severe COVID-19 disease correlated with cDNA [131]. Syncytia formation and NETosis are likely in the formation of immune complexes as an imbalance due to or caused by coagulopathy and immunothrombosis [131,193]. Endothelial cells in both animal and human studies revealed endothelial cells could be directly infected; however, studies show colocalization with CD31 within a disrupted inflamed endothelial layer, as clearly seen by upregulation of many adhesion molecules (e.g., P-selectin) and chemotactic factor release of CXCL10 alongside IL-6 (see Supplementary Data S4) [196,197]. Additional factors involved in platelet coagulation are vWF, with elevated P-selectin and E-selectin upregulation, observed in chronic COVID-19 disease patients, that are all involved with endothelial dysfunction [76,198]. Exploring this further, Kuchroo et al. performed a single-cell analysis study (n = 168) of infected SARS-CoV-2 patients that differentiated between neutrophil and monocyte populations with monocyte markers (CD16hiCD66b/CD14−CD16hiHLA–DRlo) to find T helper 17 (TH17) cell response generated IFN-γ and granzyme B [199]. In this key finding, CD14−CD16hi monocytes were enriched in severe infection, and it was confirmed that HLA-DR upregulation correlated with severity. In 2020, it was shown in chronic COVID-19 disease patients that IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ with C-reactive protein (CRP) correlated with IL-10 [34]. IL-1 is a key cytokine involved with neutrophil activation, which shares homology and similar functions with the aforementioned TLR families [64,200]. IL-1α and IL-1β have been implicated in COVID-19 disease in other studies. Therefore, the exact mechanisms of the regulatory enzyme glycosyltransferase, α-1,6-fucosyltransferase (FUT8) were examined, only to find little correlation with disease prognosis, but it was found that receptor expression was upregulated within other myeloid monocyte compartments that express CD16a (FcγRIIIa), also including classical (CD14hi/+, CD16−−) and intermediate (CD14hi/+, CD16lo/+), and non-classical (CD14−−/lo, CD16+ ) markers that also include CD11c DCs that are also HLA-DR+ myeloid cells [146,201–204]. Clinical trials remain ongoing to further clarify cytokines IL-1 β, IL-6, IL-8, TNF-α, TGF-β, IFN-γ, IL-17, IL-21, IL-22, IL-23, and IL-10, and ROS production in COVID pneumonia, with results awaiting (Supplementary Data) (NCT04930757, NCT04434157, and NCT05520918). As above, neutrophil proteins disrupted during NETosis naturally will affect extracellular ions and, specifically, calcium homeostasis required by other cytoskeletal proteins with intracellular enzymes, not necessarily degraded, that include MPO, histones, and other proteases within this cytokine and immune cell environment [205].

3.3. Monocyte Cellular Development

Since the advent of FACS and the discovery of monocytes by Ehrlich and Metchnikoff, currently identified monocyte subsets are broadly defined by classical (CD14hi/+, CD16−), intermediate (CD14hi/+, CD16lo/+), and non-classical (CD14−/lo, CD16+ ) markers [203,206,207]. Monocytes represent around 10% of the leukocyte population, and they are short-lived (1–2 days) while circulating in blood, bone marrow, and spleen (see Figure 4).

Figure 4. Antigen-presenting cell roles in SARS-CoV-2 infection

3.4. Monocyte Cellular Markers during Host SARS-CoV-2 Infection

In COVID-19 disease, it was intimated that classical CD14++CD16−− monocytes were a source of upregulated chemokine CCR2, along with a neutrophil chemoattractant IL-8 (CXCL8) and TNF-α with upregulated gene expression and synthesis of IL-1β and IL-18 with fewer confirmed CD14+CD16++ monocytes. Moreover, the downregulation of HLA-DR in severe patients (n = 12) was seen to affect overall viral antigen presentation [201,211,212]. These cellular populations are further characterized by CD195 (CCR5), as well as TNF-α receptors CD120a/CD120b (TNFR1/2). Both of these receptors were found in blood serum and up-regulated along with ADAM17, known to affect L-selectin (CD62) shedding, with ADAM17, a TNF-α convertase, also upregulated in inflammatory bowel disease (IBD) [213,214]. Other soluble immune cell shedding markers were measured in sera (sCD14 and sCD163), and, although unrelated to disease severity, they correlated with standard blood sera proteins (acute phase protein, ferritin, LDH, CRP, and procalcitonin) [215]. In addition, CCR5 inhibition studies, during prolonged SARS-CoV-2 infection and disease, demonstrated that changes to CD14/CD16 subsets occur, affecting pro-inflammatory cytokines alongside CD4+/CD8+ T cell reduction. These researchers showed that IL-2, IL-4, CCL3, IL-6, IL-10, IFN-γ, and VEGF were elevated and, moreover, TREG cells decreased with concurrent GM-CSF reduction, affecting monocyte development [216]. FACS analysis was used for NK cell analysis and in differentiating CD14hi/+, CD16−−monocytes by CD16 marker to find occurrence via inflammasome activation (NLRP3) evidenced by caspase-1 activity in severe COVID-19 disease. This concurred with the dysregulation of mitochondrial superoxide and lipid peroxidation markers of oxidative stress. These findings were later confirmed during gasdermin D cleavage studies [217]. Gasdermin D (GSDMD) is known as a pore-forming protein significantly found to be activated by SARS-CoV-2 infection of neutrophils, as measured by caspase 1/3 activation as a potential NETosis and pyroptosis stimulator [218,219]. In addition, 6% of SARS-CoV-2 infected monocytes were found to have other pyroptotic markers when measuring GSDMD, IL-1β, IL-1RA, IL-18, and LDH, as well as three key chemokines: CCL7, CXCL9, and CXCL10 (see Figure 5) [220]. In vitro studies showed this could be causal in IL-1β secretion by SARS-CoV-2-exposed monocytes [221]. Specifically, the numbers of circulating classical monocytes (CD14hi/+, CD16−) were enriched with downregulation of CCR2 and HLA-DR, but the numbers of intermediate (CD14hi/+, CD16lo/+) and non-classical (CD14−/lo, CD16+ ) monocytes increased [222]. Alternative transcriptomic analysis confirmed that intermediate CD14hi/+CD16lo/+ possessed a temporal interferon-stimulated gene signature (ISG) in acute SARS-CoV-2 infection (IRF7, IFI44L, IFIT1, and IFIT3). The analysis also, remarkably, illustrated that IL-8 (CXCL8) and IL-1β together with CCL3 were substantially upregulated without induction of pro-inflammatory cytokine genes such as TNF, IL-6, IL-1, CCL3, CCL4, or CXCL2 in the cells that possessed reduced HLA-DR expression and reduced antigen presentation capability [223]. On the other hand, more recently, in a SARS-CoV-2 case-control study (n = 37), it has been clarified that there was an initial increase in classical monocytes (CD14hi/+, CD16−−) with a decrease in intermediate (CD14hi/+, CD16lo/+) and a gradual normalization of non-classical monocytes (CD14−/lo, CD16+ ) 6–7 months after follow-up, with changes to other cell subtypes below [203,204,207,224].

3.5. Macrophages Metabolism and Function

In 1950–1970, macrophage (Mφ) metabolic cycles were closely examined in what was then known as the Warburg effect, where Mφ in tumors was seen to change metabolic profiles. Indeed, recent research shows that activation of Mφ or DCs with a range of stimuli (LPS, TLR3 ligand poly (I: C), type I IFN) induces a metabolic switch. Metabolic profiles thus change from oxidative phosphorylation (OXPHOS) to glycolysis with a resultant reduction in the TCA cycle, while lactate production drives Mφ metabolism and fluxes upwardly through the pentose phosphate pathway [225]. Mφ is the most abundant immune cell type within the lung, classified as alveolar φ (AMφ) or interstitial (iMφ). Macrophages (Mφ) originate from blood monocytes that migrate between vascular tissues with morphology recognizing TLRs, pathogen-associated molecular patterns (PAMP), and pathogenic antigens. Conclusions are difficult to draw with reference to Mφ interactions with B/T cells, as explained below (see Figure 6).

Figure 5. Monocyte cell phenotypes. 

3.6. Macrophage Classification

Less data has defined interstitial macrophages (iMφ), in comparison to alveolar macrophages (AMφ) being well-defined as regulators of lung pulmonary immune responses. AMφ and iMφ are both tissue-resident phagocytic cells that also include brain microglia, liver Kupffer cells, and others. Therefore, AMφ are distinct in their ability to induce and inhibit inflammatory responses on exposure to pathogens, and change cell surface markers utilizing complement opsonization receptors and other pattern recognition molecules, as above, that facilitate phagocytosis of either cell debris or pathogens [226]. Mφ characterization subsequently loosely differentiates between varying inflammatory phenotypes, commonly referred to as M0 (non-activated), M1 (pro-inflammatory), and M2 (anti-inflammatory) by polarization and cytokine secreted but are currently not defined by CD nomenclature [227,228]. As M-CSF and GM-CSF induce differentiation, it was suggested that Mφ are subdivided into M1φ secreting cytokines IL1-β, IL-6, IL-12, and TNF-α, with M2-like secreting TGF-β, IL-10, IL-4, and IL-13 (see Figure 7) [229].

 Figure 6. Macrophage process and role in infection.

Figure 7. Macrophage phenotypes during polarization.

3.7. Macrophage Metabolism Role during Polarization and SARS-CoV-2 Infection

Macrophage polarization is the process where Mφ evolves through maturation and adopts different functional and secretory programs in response to signals from the microenvironment in which they are located at a given point in time. This dual innate and adaptive capacity relates to multiple roles in all organisms as effector cells are involved at the center of most biological processes. More specifically, they are involved in the elimination of cellular debris, pathogens, embryonic development, and tissue repair utilizing an array of immune system cells that include B lymphocytes, DCs, TH1, TH2, NK cells, and others, below (see Figures 1–12) [232]. It is noteworthy that IFN-γ is thought to polarize M1φ, causing up-regulation of inflammatory cytokines upon viral infection whilst inhibiting growth and enhancing apoptosis of lung cells in vitro [233]. Dysregulation of AMφ polarity therefore needs to be considered in context with other in vitro or in vivo respiratory research where both fibrosis and inflammation can occur (e.g., silicosis) [234]. M1φ and M2φ, along with gene protein markers within bronchoalveolar lavage fluid (BALF), are one way of ascertaining polarity state alterations associated with the disease. Cellular staining methods such as hematoxylin/eosin and trichrome staining on lung tissue can alternatively be utilized. M1φ/M2φ phenotypes appear to undergo differential phenotype changes affecting T cells and the resulting Ig class switching, as well as differential antigen presentation alongside chemokine and cytokine release within both respiratory and mucosal compartments, which are affected by the factors below. M1φ can produce nitric oxide synthase (iNOS), which uses L-arginine to produce nitric oxide (NO) while M2φ utilizes arginase 1 (ARG1), which hydrolyses L-arginine to L-ornithine for collagen synthesis. Therefore, during infection and/or phagocytosis of Mφ, changes in extracellular metabolites could occur, affecting polarization and changing the oxidative phosphorylation balance dependent on amino acid consumption. Furthermore, it is possible that M1φ metabolizing extracellular arginine into NO and L-citrulline with increased glycolysis, fatty acid synthesis, and ATP metabolism could change the levels of metabolites. In comparison, M2φ shows enhanced OXPHOS and glutamine metabolism therefore representing a metabolic cellular shift that could occur similarly [235–237]. Research is comparatively unclear on whether M2φ activation is glycolysis dependent. Therefore, metabolism in COVID-19-affected individuals is essentially relevant to immune cell function where patients’ decreased tryptophan was seen with elevations in L-kynurenine, which usually increases with age [238]. Tryptophan is an essential amino acid regulated by enzymes indoleamine 2,3-dioxygenase-1 (IDO-1) or indoleamine 2,3–dioxygenase–2 (IDO-2), eventually leading to producing kynurenine. Researchers clarified IDO-2 in a case-control cohort study (n = 21) with a similar pathology to confirm that both IDO-1 and IDO-2 occurred in abundance within and outside AT1, AT2 cells, interstitial, and endothelial cells, with IDO-2 being largely localized to the lungs rather than tissues. Selected immune cells (Mφ, DCs, and neutrophils) migrate within the respiratory compartment in SARS-CoV-2-infection-induced COVID-19 disease [238,239]. Therefore, this apparent confirmation that IDO was expressed in the disease, along with the limited availability of other research, suggests that known selected M2φ markers IL-10/CXCR4 may increase, while T cell homing receptors CCR7 and IL-12A (IL-12p35) have been known to decrease in other fibrotic conditions [240]. M1φ and M2φ phenotypes are clearer with exposure to other in vivo bacterial and viral agents [241,242]. Fibrosis occurs around vascular compartments and within endothelial layers and is understandably linked to COVID-19 disease and long-term sequelae where tissue stiffens, with concurrently decreased oxygenation and lung dysfunction. For example, Galectin-3, as a carbohydrate-binding protein, is produced in the lungs by AMφ and epithelial cells. M2φ secretion of TGF-β or IL-10 can therefore either stimulate the secretion of tissue-modeling proteins or regulate TREG cells in acute lung injury. TGF-β is considered to act synergistically with -AMφ in the secretion of retinal dehydrogenase (RALDH), an enzyme that catalyzes the retinal to retinoic acid conversion within the cell, which is critical to the transcription factor retinoic acid-related orphan receptor gamma t (RORγt) [235,243,244]. Recent literature suggests that M2φ is dependent on higher energy production [235,236,244]. Therefore, as outlined above, the only other relevant immune system cells, mast cells, basophils, and eosinophils, are discussed elsewhere but were investigated in 2021.

Figure 8. Functional diversity of dendritic cells in maturation

Figure 9. Dendritic cell phenotypes

Figure 10. Natural killer cell phenotype diversity and maturation

 Figure 11. T-Cell phenotype diversity and developmental cellular markers

Figure 12. T cell phenotype diversity and developmental cellular markers

3.8. Macrophage Phenotypes, Cytokines and Chemokines during SARS-CoV-2 Infection

SARS-CoV-2-infected Mφ in vitro were seen to colocalize at endothelial cell membranes, expressing CD31 (PECAM-1) alongside endothelial cell endosomes and also displaying activation markers to exosomes expressing mRNA for IL-1β, caspase 1, and NLRP3 from infected individuals [249]. It is notable that complement opsonization receptors include CR1/CR2, but also exosomes CR3 and CR4 (β2 integrin), as well as CD11b/CD18 (αMβ2) expressed on neutrophils that can bind iC3b being an efficient phagocyte receptor, although many integrin subunits are also upregulated (see Supplementary Data S1). Therefore HLA-DR (encoded on chromosome 6p21.31), was found to present S protein antigens and combinational peptide units of S1/S2/RBD, so this represented a key finding that antigen presentation was occurring [233]. Interestingly, both Mφ and MDSC express CD68 and CD163, which were investigated in 2018 in the context of thrombocytopenia (ITP) to try and clarify MDSCs phenotypes further. Initial indications that chemokine receptors and ligands directed leukocyte migration were evident with CCL2/CCL3 and eotaxin. It was also indicated that IL-1β may expand both these cell types within ITP patients prior [250]. Further, single-cell sequencing of SARS-CoV-2 within other inflammatory disorders (RA/CD/UC) clarified that, in BALF samples during COVID-19 disease, preferential expression occurs of CXCL10, CXCL9, CCL2, CCL3, and IL-1β (also GBP1, STAT1 gene proteins). These were also induced by IFN-γ and TNF-α, therefore clarifying that M1φ are pro-inflammatory in COVID-19 disease. However, Mφ subpopulations are further characterized by HLA-DR, CD195 (CCR5), and TNFR1/TNFR2 expression, which is also higher on intermediate monocytes, followed by classical and then non-classical monocytes as well as Mφ [251]. A recent preprint suggests that, within acute SARS-CoV-2 infection, monocytes change IGS from innate immune functions as CD14+ monocytes develop into pro-thrombotic, showing differential upregulation of MHC II alongside MHC I downregulation (HLA-DR/HLA-ABC), with accompanying gene signatures downregulated that would affect IFN production (e.g., IFNA1, IFNA2), but also TLR7 and AIM2, affecting increased expression of pathways involved in hemostasis and immunothrom bosis [207]. In contrast, TNFR2 is expressed at high levels in non-classical monocytes, followed by intermediate, and then the lowest expression was in classical monocytes [252]. Enlarged monocytes with M2φ characteristics also secrete IL-6, IL-10, and TNF-α, and express surface receptors CD11b+ , CD14+ , CD16+ , CD68+ , CD80+ , CD163+ , and CD206+/CD14hi/+ . CD14hiCD16− Mφ were observed to display inflammasome activation, as evidenced by caspase-1/ASC-speck formation in severe COVID-19 disease when compared to mild or healthy controls [221]. It is established that M2φ are TH2-like and can produce allergic cytokines, which are related to tissue remodeling and pathology that includes IL-4/IL-13. However, histamine H1 Mφ receptor and eosinophil H4 also share this role [171,253]. CD68 and CD163 increase in severity alongside CD163 and TREGS. It is possible that M2φ, together with suppressor TREGS, promotes this immunosuppressive environment. However, it is of note that other studies found that both M1φ/M2φ phenotypes could markedly upregulate CD38+ CD23+ in disease, which can prime DCs and naïve T cells [254]. Moreover, it was clarified by gene protein analysis that differential M1φ or M2φ polarization could be induced in vitro with M1φ expressing IL-6, TLR4, CXCL9, CXCL10, and CXCL11, while M2φ expressed CD206, CCL17, and CCL22 (with gene markers STAT6, IRF4) [233,255]. This was an interesting finding, as TLR4 is historically activated by bacterial antigens, while CCL17 and CCL22 seem to be relevant as DC and Mφ chemokines. Therefore, on Mφ, it appears that, aside from M1φ secreting IL-1β, IL-8 and IL-18, additional chemokines are expressed, such as CXCL16 together with CCL2, while anti-inflammatory M2φ expresses transglutaminase 2 (TGM2), apolipoprotein E (APOE), α2-macroglobulin (A2M), CCL13, and CCL26. Interestingly, a role for a triggering receptor expressed on myeloid cells 2 (TREM2) protein in the potential toxicity of M1φ cells that hold an affinity for the CXCR3 receptor seems clearer than before. TREM2 has been shown to be expressed on newly differentiated Mφ, acting as a sensor and activator of T cell responses in SARS-CoV-2 infection. Recent notable preprints confirm the role of TREM2 and iMφ in orchestrating respiratory inflammation [256–258].

4. Dendritic Cells

4.1. Dendritic Cell Overview

Dendritic cells were formally identified in 1873 (Langerhans cells), and by Steinman and Cohn in 1973 in vivo in the spleen, based on unique morphology, with a finite lifespan of days, distinguishing them from Mφ, and they are replenished by hematopoiesis from precursor HPSCs [265]. Initially found to be potent stimulators of the mixed lymphocyte reaction, this elucidated their role as central to antigen presentation by expression of high levels of MHC class II molecules and integrin CD11c [202]. Therefore, in combination with migration ability between non-lymphoid and lymphoid organs, they hold a key with superior capacity to affect T cell development and function. DCs can be defined by migration to secondary lymphoid tissues and priming TN (naïve) cells in combination with Mφ in primary lymphoid organs. In 1994, a key development came in research describing in vitro cell culture methods for developing DC-like cells from monocytes using GM-CSF and IL-4 [266,267]. Compared to other APCs, such as Mφ and B cells, these are considered the most efficient APC priming T cells via both MHC class I/II molecules and delivering antigens to CD4+ and CD8+ T cells. DCs developed from naïve to mature from a combined monocyte/DC pool that was thought to be CD103+ DCs that could process influenza virus in LN networks by cross-presentation and were potent stimulators of CD8+ T cells [268]. DCs are formed from a heterogeneous population of bone-marrow-produced cells classified as plasmacytoid DC (pDCs), type 1 conventional DCs (cDC1), type 2 (cDC2), myeloid DCs (mDCs), and Langerhans cells, but also monocytic DCs (MoDC) with CD14 subtypes above that evolve from hematopoietic stem progenitor cells (HPSC) (see Table 4).

5. Discussion

SARS-CoV-2 and other viruses evolved as zoonotic infections that can cross animal barriers. It is necessary to consider that, regardless of the origins of SARS-CoV-2, there was a genetic homology of 96.5% with Homophilus affinis, and cellular recombination events are necessary for both an immune response and continued viral propagation within animal hosts. Currently, variant surveillance data on SARS-CoV-2 variants in other animals includes monitoring in different animal populations such as mink (1320) and bats (8), with comparatively less monitoring in pet and zoo animals (see Supplementary Data). Records suggest that only two pathogens have largely been eradicated in humans and animals, which are smallpox (an Orthopoxviridae variola) and rinderpest (a Paramyxoviridae morbillivirus), respectively [421]. Therefore, further consideration of current surveillance within and across animal populations occurs in order to monitor other potential pathogenic coronaviruses (e.g., infectious bronchitis in birds, porcine delta coronavirus, and feline coronavirus) to prevent such zoonotic infections recurring in the future. Experimental research between the 16th and 18th centuries throughout the world, begun by Edward Jenner in the late 1700s on cowpox, eventually led to the modern-day interpretation and use of the word “vaccine,” referring to the substance being derived from vaccinia (cowpox). The smallpox pandemic caused by variola virus was eventually declared eradicated in 1980 by the World Health Organization, through research development utilizing what some would consider the pioneering vaccine immunogens by which standards are measured. Many other vaccine immunogens now exist, with research development improvements summarized here that illustrate the goals and research of thousands of people across the world. Estimates of overall smallpox immunity are around 50 years, largely due to pioneers in this field that also include Ehrlich, Medawar, and Edelman, and many others whose research into syphilis, actively acquired tolerance, and the structure of antibody molecules laid the foundations for Kohler and Milstein’s discovery of how to produce monoclonal antibodies.

cistanche tubulosa-improve immune system

6. Conclusions

After extensive research, we have quantified and outlined the nature of current specific receptors and proteins relevant to clinical laboratories and medical research by documenting both innate and adaptive immune system cells within current coronavirus immunology data and other pathologies to date. B cell generation of antibodies, followed by neutrophils in pathophysiology, releases initial cytokines and correlates that are dependent on antigen-presenting cells of monocyte, macrophage, and dendritic cell lineages. However, each of these relates to defined T cell lineages. There are evidential increases in not only S protein immunogen responses but also in N and M proteins. In this article, we considered cellular markers according to current immunological literature and dictations from experts in the field. Many senior scientists between April and September 2020 wrote letters in this regard (see Supplementary Materials) that demonstrated overall infection antibody positivity of 23% (NY), 18% (London), and 11% (Madrid) are dependent on the other 10 or more T cell subtypes performing all the regulatory functions of the immune system, which are arguably more important in all pathologies [427–445].