**2.2 Innate immune system**

Unlike adaptive immunity, innate immunity is not specific to individual pathogens, but is instead a host system which allows for conserved responses to broad classes of pathogens. A key facet of the innate immune response is inflammation, which allows for the destruction and removal of infected or damaged cells, as well as tissue cleanup and infiltration of additional immune effector cells to the site of infection. A key component of innate immunity is inflammation. Chemokines and pro-inflammatory cytokines respectively attract innate immune cells to the site of infection and activate them, thereby promoting their various effector functions. Neutrophils are early responder cells which perform phagocytosis of pathogens and kill microorganisms via the release of soluble anti-microbial molecules and neutrophil extracellular traps. Dendritic cells take up pathogens and damaged cell components via phagocytosis and migrate to the lymph nodes, where they present antigen on class II MHC to activate CD4+ T cells. Likewise, macrophages can perform similar antigen presenting functions, and are also key orchestrators of the immune response via the release of cytokines and chemokines. A variety of other cell types are active during an innate immune response to pathogens such as SARS-CoV-2, and we point readers to a recent review on this topic [23].

#### **2.3 Monocytes**

The remainder of this chapter will principally focus on the contributions of monocytes and macrophages to COVID-19. These are innate immune cells of myeloid lineage. Monocytes arise in the bone marrow from common myeloid progenitor cells, which are also the precursor to erythrocytes, mast cells, and neutrophils among other cell types [24]. Monocytes circulate in the bloodstream and perform important innate immune effector functions, including antigen presentation, phagocytosis, and immune signaling through cytokine production [25]. Monocytes recognize broad classes of pathogen (e.g., gram-negative bacteria, dsRNA viruses, etc.) via pathogen binding to cell surface and intracellular pattern recognition receptors (PRRs) and respond by phagocytosis and/or cytokine production to further orchestrate the immune response [25].

In humans, three traditional monocyte phenotypes are widely recognized: classical (CD14<sup>+</sup> CD16<sup>−</sup> ), intermediate (CD14<sup>+</sup> CD16<sup>+</sup> ), and non-classical (CD14dimCD16+ ) [26]. Classical monocytes make up the bulk of circulating monocytes (>80%) and are highly phagocytic and potent cytokine producers. Intermediate monocytes are enriched for antigen presenting MHC molecules and produce cytokines under PRR binding, and non-classical monocytes have patrolling and wound healing functions and are less responsive to PRR binding compared to classical monocytes [27]. However, intermediate and non-classical monocytes are also associated with increased basal inflammation and are increased in circulation in a number of chronic diseases [27], thereby suggesting that proportional increases in CD16<sup>+</sup> monocyte populations may be reflective of detrimental innate immune responses.

#### **2.4 Macrophages**

Macrophages are tissue mononuclear phagocytes that participate in innate immune defense and stimulation of the adaptive immune system. During an inflammatory event, peripheral monocytes invade tissue and differentiate to macrophages to carry out host defense, tissue remodeling, and cellular signaling activities. Tissue enrichment of monocyte-derived macrophages is therefore a hallmark of many infections and contributes to immunopathology during acute disease [28–30]. Additionally, many long-lived tissue resident macrophages are not monocytic in origin and instead arise during embryonic development [31]. Tissue resident macrophages often have distinct nomenclature (e.g. Kupffer cells – liver; microglia – brain; osteoclasts – bone) and can perform highly diverse functions depending on tissue environment.

Macrophages are phenotypically heterogeneous and can be polarized along the inflammatory spectrum to mediate diverse functions which are pro-inflammatory (e.g., T cell stimulation) or anti-inflammatory (e.g., wound healing) in nature [32]. Macrophages are often classified as M1/pro-inflammatory or M2/anti-inflammatory depending on their polarization signals and their expression of cell surface or intracellular markers, which is recognized to be an oversimplification but persists due to the utility of studying these phenotypes, especially *in vitro*.

Within the tissue, macrophages play principal roles in phagocytosis of dead cells, cell debris, and extracellular pathogens, and additionally present antigens to CD4+ T cells to activate adaptive immune responses [33]. Macrophages also recognize pathogens through PRR binding and produce cytokines and chemokines to orchestrate the immune response, including both initiation and resolution of inflammation, the latter of which is key to successful tissue repair.

### **3. Monocytes and macrophages in COVID-19**

A substantial body of evidence now demonstrates the importance of immune function during COVID-19 [22, 23]. Here we will focus on the potential contributions of monocytes and macrophages to severe COVID-19, especially as it pertains to the hyperinflammatory environment characteristic of severe disease [34–36]. A large number of cytokines have been shown to be upregulated during COVID-19 and associated with poor outcomes, including interleukin (IL)-1α, IL-1β, IL-2, IL-6, IL-8, IL-10, IL-17A, IL-33, monocyte chemoattractant protein-1 (MCP-1), granulocyte colony stimulating factor (G-CSF), interferon (IFN)-γ, IFNγ-inducible protein (IP10), and tumor necrosis factor-α (TNFα) among others [37–43].

#### **3.1 Monocyte infiltration into infected tissue**

The infiltration of monocytes and subsequent increase in monocyte-derived macrophages is a hallmark of infection severity for a number of viruses [28–30]. Several observations have now suggested that this is also the case in COVID-19. A variety of single cell RNA sequencing studies have noted increases in monocytes and macrophages in collected bronchoalveolar lavage fluid that are associated with severe disease [44–46]. Notably, infiltrating monocyte-derived macrophages seem ultimately to be the principal contributors to disease severity, rather than resident alveolar macrophages [45, 47, 48]. Likewise, several postmortem analyses of patients who died due to COVID-19 have found significant numbers of monocytes and monocyte-derived macrophages in tissues, especially lungs [49–52].

*Targeting Mononuclear Phagocytes to Treat COVID-19 DOI: http://dx.doi.org/10.5772/intechopen.98967*

A key drawback to observational human studies is that they cannot empirically determine whether monocyte infiltration and macrophage increases are due to SARS-CoV-2 infection itself, due to the ethical issues with infecting human subjects with the virus itself in a prospective manner. Animal studies are often used in such instances, and this is complicated in COVID-19 research by the general non-susceptibility of traditional laboratory mouse models to infection by SARS-CoV-2. This is due to significant sequence differences between mouse and human angiotensin converting enzyme 2 (ACE2), the principal receptor for SARS-CoV-2 [53].

Nevertheless, while standard mouse models are not susceptible to viral infection, a large number of other laboratory animals have been established as models of COVID-19. Experimental infection in these animals invariably leads to monocyte and macrophage infiltration into pulmonary tissue, as has been shown to date with human ACE2-expressing transgenic mice [54, 55], Syrian hamsters [56], rhesus macaques [57–59], and African green monkeys [60] among others. Given the associations with disease shown in human sequencing and pathology studies, as well as the empirical evidence from animal studies demonstrating monocyte/macrophage infiltration during infection, it appears clear that these cells are responding to localized tissue infection during COVID-19. However, the functions being mediated by monocytes and macrophages during the course of SARS-CoV-2 infection are not yet entirely clear.

#### **3.2 Phenotypic shifts and hyperinflammation**

In addition to infiltration into infected tissues, various observational studies have shown shifts in monocyte and macrophage phenotypes towards hyperinflammatory states in COVID-19. One of the most consistent changes noted in COVID-19 immune profiling studies is a decrease in monocyte expression of human leukocyte antigen (HLA)-DR, an MHC class II protein. HLA-DR downregulation has been seen in other inflammatory conditions such as sepsis and is linked to disease severity [61]. In COVID-19, HLA-DR downregulation is a prominent feature [38, 45, 62–66], and could signify immune exhaustion in virus-stimulated cells that could lead to impaired inflammation and antigen presentation, and therefore to defects in adaptive immune responses.

Likewise, both monocytes [67–70] and lung macrophages [44, 45, 71] produce increased levels of pro-inflammatory cytokines during acute SARS-CoV-2 infection as noted in human observational studies. Pro-inflammatory macrophage responses have also been noted in the lungs of experimentally infected non-human primates [60, 72, 73], thereby linking macrophage inflammation to infection with SARS-CoV-2 itself (and not some other biological factor). Therefore, it appears that SARS-CoV-2 infection causes inflammatory responses in macrophages and monocytes, although the immediate proximal cause of this response cannot be identified solely via immune cell phenotype profiling.

#### **3.3 Responses to direct infection**

Both macrophage infiltration to infected tissues and resulting hyperinflammation in these cells can be explained by indirect mechanisms (e.g., infected cells activating monocytes/macrophages via cytokine signaling). However, several *in vitro* studies have demonstrated that monocytes and macrophages react to infection with SARS-CoV-2 by mounting pronounced inflammatory responses [74–76]. It is likely that infection of these cells is abortive (i.e., does not produce additional infectious virus) [74, 75, 77], so these responses may be mediated by viral protein binding to cellular receptors rather than by recognition of replicating viral RNA.

**Figure 1.**

*Schematic of lung macrophage populations during COVID-19. Uninfected lungs (left) maintain a resident population of alveolar macrophages. During SARS-CoV-2 infection (right), infiltrating monocyte-derived macrophages become activated and produce pro-inflammatory cytokines. Created with BioRender.com.*

Additionally, immunometabolic activation has been demonstrated in infected monocytes, which upregulate glycolytic activation [78] and accumulate intracellular lipid droplets [79] when exposed to SARS-CoV-2. Metabolic reprogramming is a hallmark of innate immune activation [80], and thus may be a mechanism by which myeloid cells mount their initial inflammatory responses to SARS-CoV-2. A schematic of lung macrophage populations with and without SARS-CoV-2 infection is shown in **Figure 1**.
