TLRs: Innate Immune Sentries against SARS-CoV-2 Infection

Abstract

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been responsible for a devastating pandemic since March 2020. Toll-like receptors (TLRs), crucial components in the initiation of innate immune responses to different pathogens, trigger the downstream production of pro-inflammatory cytokines, interferons, and other mediators. It has been demonstrated that they contribute to the dysregulated immune response observed in patients with severe COVID-19. TLR2, TLR3, TLR4 and TLR7 have been associated with COVID-19 severity. Here, we review the role of TLRs in the etiology and pathogenesis of COVID-19, including TLR7 and TLR3 rare variants, the L412F polymorphism in TLR3 that negatively regulates anti-SARS-CoV-2 immune responses, the TLR3-related cellular senescence, the interaction of TLR2 and TLR4 with SARS-CoV-2 proteins and implication of TLR2 in NET formation by SARS-CoV-2. The activation of TLRs contributes to viral clearance and disease resolution. However, TLRs may represent a double-edged sword which may elicit dysregulated immune signaling, leading to the production of proinflammatory mediators, resulting in severe disease. TLR-dependent excessive inflammation and TLR-dependent antiviral response may tip the balance towards the former or the latter, altering the equilibrium that drives the severity of disease.

Keywords: COVID-19; SARS-CoV-2; Toll-like receptor; host genetics; innate immunity.

Figure 3

Several TLRs, such as TLR2, TLR3, TLR4 and TLR7, have been associated with the pathogenesis of COVID-19. The interaction of TLR2 and 4 with SARS-CoV-2 proteins triggers downstream pathways leading to the production of pro-inflammatory cytokines, via the activation of multiple adaptor proteins, as shown in Figure 2 [119,122,140,141,142,143]. LOF TLR3 and TLR7 variants negatively regulate anti-SARS-CoV-2 immune responses, inducing an impaired IFN response that could delay viral clearance in the initial disease phase and promote a severe COVID-19 clinical course [39,48,49,50,51,107]. The L412F polymorphism reduces TNFα production and inhibits TLR3-dependent autophagy, promoting viral persistence [115]. SARS-CoV-2 induces senescence in human non-senescent cells and exacerbates the tissue-destructive SAPS, increasing IL-1α, IL-1β, IL-6, IL-8 and GM-CSF mRNA levels in human senescent cells through TLR3 [117]. SAPS, senescence-associated secretory phenotype. Created with BioRender.com.

Figure 1

Pattern recognition receptors (PRRs) and their ligands. PRRs can be classified into two main classes: membrane-bound receptors, such as TLRs and CLRs, and cytoplasmic sensors, including NLRs, ALRs and RLRs. In humans, 10 TLRs respond to a variety of Pathogen Associated Molecular Patterns, including lipopolysaccharide (TLR4), lipopeptides (TLR1, 2, 6 and 10), bacterial flagellin (TLR5), viral dsRNA (TLR3 and 10), viral or bacterial ssRNA (TLR7 and 8) and CpG-rich unmethylated DNA (TLR9). TLR1, 2, 4, 5, 6 and 10 are plasma membrane TLRs, whereas TLR3, 7, 8 and 9 are located on endosomes. TLRs can be homodimers, in the cases of TLRs 3, 4, 5, 7, 8 and 9, or heterodimers as in TLRs 1 and 2 or TLR2 and 6. TLR2 and 4 sense the SARS-CoV-2 Envelope protein (E) and the Spike protein (S), respectively, whereas TLR3 and 7 sense SARS-CoV-2 nucleic acid. CLRs are a family of receptors that recognize carbohydrates, such as β-Glucan and α-Mannan, on the surface of pathogenic microorganisms. NLRs are intracellular PRRs that mainly recognize the diaminopimelic acid iE-DAP of the cell wall of Gram-negative bacteria and MDP in all bacterial cell walls. ALRs recognize the dsDNA of bacteria. RLRs recognize short dsRNA (<1000 bp) and long-chain dsRNA (>1000 bp) of different viruses through ligand-recognition domains. TLRs, Toll-like receptors; CLRs, C-type lectin receptors; NLRs, NOD-like receptors; ALRs, absent in melanoma-2-like receptors; RLRs, RIG-I-like receptors; dsRNA, double-stranded RNA; ssRNA, single-stranded RNA; dsDNA, double-stranded DNA; LPS, lipopolysaccharides; CpG DNA, cytosine-phosphate-guanine DNA; iE-DAP, γ-D-glu-meso-diaminopimelic acid; and MDP, muramyl dipeptide. Created with BioRender.comTLRs are crucial components in the initiation of innate immune responses to a variety of pathogens, triggering the downstream production of pro-inflammatory cytokines, interferons (IFNs), and other mediators [10]. Several findings have highlighted the potential contribution of TLRs to the dysregulated immune response observed in patients with severe COVID-19.

Figure 2

Downstream signaling pathway of TLRs. TLRs are type I integral membrane glycoproteins characterized by the extracellular (or the extraendosomal) domains and a cytoplasmic signaling domain. TLRs share a common structural framework in their extracellular ligand-binding domains. These domains, formed by varying numbers of leucine-rich-repeat motifs, all adopt horseshoe-shaped structures. After ligand binding, two extracellular domains form an “m”-shaped dimer sandwiching the ligand molecule, bringing the transmembrane and cytoplasmic domains into close proximity and triggering a downstream signaling cascade. After ligand-induced dimerization, the cytoplasmic signaling domain associates with adaptor molecules to transmit signaling. Six adaptor proteins have been identified: MyD88, MAL, TRIF and TRAM are recruited to TLR cytoplasmic domains to initiate signaling, whereas SARM and BCAP inhibit TLR responses. All TLRs, except for TLR3, associate with MyD88 and MAL proteins. Otherwise, TLR3 and TLR4 use a TRIF-dependent pathway. Both the MyD88-dependent and TRIF-dependent pathways lead to the activation of downstream molecules: NF-κB, AP-1, and members of the IRF family. These pathways are responsible for the TLR-mediated expression of inflammatory genes, type I, type II and type III IFN genes and interferon stimulated genes (ISGs). MyD-88, myeloid differentiation primary-response gene 88; MAL, MyD88-adaptor-like protein; TRIF, TIR-domain-containing adaptor protein inducing interferon-β (IFNβ); TRAM, TRIF-related adaptor molecule; SARM, sterile α- and armadillo-motif-containing protein; BCAP, B-cell adaptor for PI3K; NF-κB, nuclear factor-κB; AP-1, activating protein-1; and IRF, IFN-regulatory factor. Created with BioRender.com.All nine members of the IRF family have a conserved amino-terminal DNA-binding domain (DBD) [30,31,32,33,34,35] that recognizes the consensus DNA sequence element ISRE [36] in the gene promoters of IFNs and interferon-stimulated gene (ISG) genes [37]. These cytokines, in turn, activate antimicrobial and proinflammatory activities, as well as the maturation of antigen-specific adaptive immune responses. IRF3, IRF5 and IRF7 are primarily responsible for the activation of type I IFN genes downstream of TLR activation, although IRF1 and IRF8 can also contribute [37]. IRF1, IRF3, IRF5 and IRF8 also induce the expression of proinflammatory cytokines such as IL-6, TNF-α, CCL5/RANTES, CXCL10 or CCL2, and other genes in response to TLR activation [38].