Influenza vaccination reveals sex dimorphic imprints of prior mild COVID-19.

Acute viral infections can have durable functional impacts on the immune system long after recovery, but how they affect homeostatic immune states and responses to future perturbations remain poorly understood1-4. Here we use systems immunology approaches, including longitudinal multimodal single-cell analysis (surface proteins, transcriptome and V(D)J sequences) to comparatively assess baseline immune statuses and responses to influenza vaccination in 33 healthy individuals after recovery from mild, non-hospitalized COVID-19 (mean, 151 days after diagnosis) and 40 age- and sex-matched control individuals who had never had COVID-19. At the baseline and independent of time after COVID-19, recoverees had elevated T cell activation signatures and lower expression of innate immune genes including Toll-like receptors in monocytes. Male individuals who had recovered from COVID-19 had coordinately higher innate, influenza-specific plasmablast, and antibody responses after vaccination compared with healthy male individuals and female individuals who had recovered from COVID-19, in part because male recoverees had monocytes with higher IL-15 responses early after vaccination coupled with elevated prevaccination frequencies of 'virtual memory'-like CD8+ T cells poised to produce more IFNγ after IL-15 stimulation. Moreover, the expression of the repressed innate immune genes in monocytes increased by day 1 to day 28 after vaccination in recoverees, therefore moving towards the prevaccination baseline of the healthy control individuals. By contrast, these genes decreased on day 1 and returned to the baseline by day 28 in the control individuals. Our study reveals sex-dimorphic effects of previous mild COVID-19 and suggests that viral infections in humans can establish new immunological set-points that affect future immune responses in an antigen-agnostic manner.

The differential expression of toll like receptors and RIG-1 correlates to the severity of infectious diseases.

The toll like receptors (TLRs) and RIG-1 are proteins involved in the initial reaction of the innate immune system to infectious diseases and, thus, can provide much information to the surgical pathologist in terms of the molecular dynamics of the infection. The TLRs (TLR1, 2, 3, 4, 7, 8) and RIG-1 distribution as determined by immunohistochemistry was examined in the following diseases: human papillomavirus (n = 30 including 15 squamous intraepithelial lesions (SIL), 5 cancers, and 10 controls); molluscum contagiosum (n = 8 including 4 controls), SARS-CoV2 (n = 52 including 20 mild, 5 fatal, and 27 controls) and reovirus infection as oncolytic therapy. Mild, regressing infection (molluscum contagiosum, mild SARS-CoV2 and low grade SIL) each showed the same pattern: marked up regulation of at least three of the TLRs/RIG-1 with decreased expression of none compared to the controls. Severe infection (fatal SARS-CoV2, and cervical cancer) each showed marked decrease expression in at least three of the TLRs/RIG-1. We recently documented an equivalent marked decrease expression of the TLRs/RIG-1 in the placenta in fatal in utero infections. The reoviral infected tissues showed an overall pattern of marked increase expression of TLRs/RIG-1, consistent with a strong anti-viral response. Thus, the in situ testing of infectious diseases by a panel of these early infectious disease recognition proteins may allow the surgical pathologist to predict the outcome of the disease which, in turn, may assist in the understanding of the role of the TLRs/RIG-1 in determining the fate of a given infectious process.

Human genetic basis of severe or critical illness in COVID-19.

Coronavirus Disease 2019 (COVID-19) caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to considerable morbidity and mortality worldwide. The clinical manifestation of COVID-19 ranges from asymptomatic or mild infection to severe or critical illness, such as respiratory failure, multi-organ dysfunction or even death. Large-scale genetic association studies have indicated that genetic variations affecting SARS-CoV-2 receptors (angiotensin-converting enzymes, transmembrane serine protease-2) and immune components (Interferons, Interleukins, Toll-like receptors and Human leukocyte antigen) are critical host determinants related to the severity of COVID-19. Genetic background, such as 3p21.31 and 9q34.2 loci were also identified to influence outcomes of COVID-19. In this review, we aimed to summarize the current literature focusing on human genetic factors that may contribute to the observed diversified severity of COVID-19. Enhanced understanding of host genetic factors and viral interactions of SARS-CoV-2 could provide scientific bases for personalized preventive measures and precision medicine strategies.

Nucleic acid-sensing toll-like receptors: Important players in Sjögren's syndrome.

Sjögren's syndrome (SS) is a chronic systemic autoimmune disease that affects the salivary and lacrimal glands, as well as other organ systems like the lungs, kidneys and nervous system. SS can occur alone or in combination with another autoimmune disease, such as systemic lupus erythematosus (SLE) or rheumatoid arthritis. The etiology of SS is unknown but recent studies have revealed the implication of the activation of innate immune receptors, including Toll-like receptors (TLRs), mainly through the detection of endogenous nucleic acids, in the pathogenesis of systemic autoimmune diseases. Studies on SS mouse models suggest that TLRs and especially TLR7 that detects single-stranded RNA of microbial or endogenous origin can drive the development of SS and findings in SS patients corroborate those in mouse models. In this review, we will give an overview of the function and signaling of nucleic acid-sensing TLRs, the interplay of TLR7 with TLR8 and TLR9 in the context of autoimmunity, summarize the evidence for the critical role of TLR7 in the pathogenesis of SS and present a possible connection between SARS-CoV-2 and SS.

Contribution of T- and B-cell intrinsic toll-like receptors to the adaptive immune response in viral infectious diseases.

Toll-like receptors (TLRs) comprise a class of highly conserved molecules that recognize pathogen-associated molecular patterns and play a vital role in host defense against multiple viral infectious diseases. Although TLRs are highly expressed on innate immune cells and play indirect roles in regulating antiviral adaptive immune responses, intrinsic expression of TLRs in adaptive immune cells, including T cells and B cells, cannot be ignored. TLRs expressed in CD4 + and CD8 + T cells play roles in enhancing TCR signal-induced T-cell activation, proliferation, function, and survival, serving as costimulatory molecules. Gene knockout of TLR signaling molecules has been shown to diminish antiviral adaptive immune responses and affect viral clearance in multiple viral infectious animal models. These results have highlighted the critical role of TLRs in the long-term immunological control of viral infection. This review summarizes the expression and function of TLR signaling pathways in T and B cells, focusing on the in vitro and vivo mechanisms and effects of intrinsic TLR signaling in regulating T- and B-cell responses during viral infection. The potential clinical use of TLR-based immune regulatory drugs for viral infectious diseases is also explored.

Unique Evolution of Antiviral Tetherin in Bats.

Bats are recognized as important reservoirs of viruses deadly to other mammals, including humans. These infections are typically nonpathogenic in bats, raising questions about host response differences that might exist between bats and other mammals. Tetherin is a restriction factor which inhibits the release of a diverse range of viruses from host cells, including retroviruses, coronaviruses, filoviruses, and paramyxoviruses, some of which are deadly to humans and transmitted by bats. Here, we characterize the tetherin genes from 27 bat species, revealing that they have evolved under strong selective pressure, and that fruit bats and vesper bats express unique structural variants of the tetherin protein. Tetherin was widely and variably expressed across fruit bat tissue types and upregulated in spleen tissue when stimulated with Toll-like receptor agonists. The expression of two computationally predicted splice isoforms of fruit bat tetherin was verified. We identified an additional third unique splice isoform which includes a C-terminal region that is not homologous to known mammalian tetherin variants but was functionally capable of restricting the release of filoviral virus-like particles. We also report that vesper bats possess and express at least five tetherin genes, including structural variants, more than any other mammal reported to date. These findings support the hypothesis of differential antiviral gene evolution in bats relative to other mammals. IMPORTANCE Bats are an important host of various viruses which are deadly to humans and other mammals but do not cause outward signs of illness in bats. Furthering our understanding of the unique features of the immune system of bats will shed light on how they tolerate viral infections, potentially informing novel antiviral strategies in humans and other animals. This study examines the antiviral protein tetherin, which prevents viral particles from escaping their host cell. Analysis of tetherin from 27 bat species reveals that it is under strong evolutionary pressure, and we show that multiple bat species have evolved to possess more tetherin genes than other mammals, some of which encode structurally unique tetherins capable of activity against different viral particles. These data suggest that bat tetherin plays a potentially broad and important role in the management of viral infections in bats.

Toll-like Receptor Mediation in SARS-CoV-2: A Therapeutic Approach.

The innate immune system facilitates defense mechanisms against pathogen invasion and cell damage. Toll-like receptors (TLRs) assist in the activation of the innate immune system by binding to pathogenic ligands. This leads to the generation of intracellular signaling cascades including the biosynthesis of molecular mediators. TLRs on cell membranes are adept at recognizing viral components. Viruses can modulate the innate immune response with the help of proteins and RNAs that downregulate or upregulate the expression of various TLRs. In the case of COVID-19, molecular modulators such as type 1 interferons interfere with signaling pathways in the host cells, leading to an inflammatory response. Coronaviruses are responsible for an enhanced immune signature of inflammatory chemokines and cytokines. TLRs have been employed as therapeutic agents in viral infections as numerous antiviral Food and Drug Administration-approved drugs are TLR agonists. This review highlights the therapeutic approaches associated with SARS-CoV-2 and the TLRs involved in COVID-19 infection.

Polymorphisms in the Genes Coding for TLRs, NLRs and RLRs Are Associated with Clinical Parameters of Patients with Acute Myeloid Leukemia.

Toll-like receptors (TLRs), NOD-like receptors (NLRs), and RIG-I-like receptors (RLRs) are major elements of the innate immune system that recognize pathogen-associated molecular patterns. Single-nucleotide polymorphisms (SNPs) in the TLR, NLR, and RLR genes may lead to an imbalance in the production of pro- and anti-inflammatory cytokines, changes in susceptibility to infections, the development of diseases, and carcinogenesis. Acute myeloid leukemia (AML) is a bone marrow malignancy characterized by uncontrolled proliferation of transformed myeloid precursors. We retrospectively analyzed 90 AML patients. We investigated the effect of fifteen SNPs located in the genes coding for RLR1 (rs9695310, rs10738889, rs10813831), NOD1 (rs2075820, rs6958571), NOD2 (rs2066845, rs2066847, rs2066844), TLR3 (rs5743305, rs3775296, 3775291), TLR4 (rs4986791, rs4986790), and TLR9 (rs187084, rs5743836). We observed that TLR4 rs4986791, TLR9 rs5743836, and NOD2 rs2066847 were associated with CRP levels, while RLR-1 rs10738889 was associated with LDH level. Furthermore, we found TLR3 rs5743305 AA to be more common in patients with infections. We also found TLR9 rs187084 C to be associated with more favorable risk, and RLR-1 rs9695310 GG with higher age at diagnosis. In conclusion, the current study showed that SNPs in the genes encoding TLRs, NLRs, and RLRs may be potential biomarkers in patients with AML.

Porcine epidemic diarrhea virus strain FJzz1 infection induces type I/III IFNs production through RLRs and TLRs-mediated signaling.

Interferons (IFNs) including type I/III IFNs are the major components of the host innate immune response against porcine epidemic diarrhea virus (PEDV) infection, and several viral proteins have been identified to antagonize type I/III IFNs productions through diverse strategies. However, the modulation of PEDV infection upon the activation of the host's innate immune response has not been fully characterized. In this study, we observed that various IFN-stimulated genes (ISGs) were upregulated significantly in a time- and dose-dependent manner in LLC-PK1 cells infected with the PEDV G2 strain FJzz1. The transcriptions of IRF9 and STAT1 were increased markedly in the late stage of FJzz1 infection and the promotion of the phosphorylation and nuclear translocation of STAT1, implicating the activation of the JAK-STAT signaling pathway during FJzz1 infection. In addition, abundant type I/III IFNs were produced after FJzz1 infection. However, type I/III IFNs and ISGs decreased greatly in FJzz1-infected LLC-PK1 cells following the silencing of the RIG-I-like receptors (RLRs), including RIG-I and MDA5, and the Toll-like receptors (TLRs) adaptors, MyD88 and TRIF. Altogether, FJzz1 infection induces the production of type-I/III IFNs in LLC-PK1 cells, in which RLRs and TLRs signaling pathways are involved, followed by the activation of the JAK-STAT signaling cascade, triggering the production of numerous ISGs to exert antiviral effects of innate immunity.

Viral proteins recognized by different TLRs.

Virus invasion activates the host's innate immune response, inducing the production of numerous cytokines and interferons to eliminate pathogens. Except for viral DNA/RNA, viral proteins are also targets of pattern recognition receptors. Membrane-bound receptors such as Toll-like receptor (TLR)1, TLR2, TLR4, TLR6, and TLR10 relate to the recognition of viral proteins. Distinct TLRs perform both protective and detrimental roles for a specific virus. Here, we review viral proteins serving as pathogen-associated molecular patterns and their corresponding TLRs. These viruses are all enveloped, including respiratory syncytial virus, hepatitis C virus, measles virus, herpesvirus human immunodeficiency virus, and coronavirus, and can encode proteins to activate innate immunity in a TLR-dependent way. The TLR-viral protein relationship plays an important role in innate immunity activation. A detailed understanding of their pathways contributes to a novel direction for vaccine development.

Could a Lower Toll-like Receptor (TLR) and NF-κB Activation Due to a Changed Charge Distribution in the Spike Protein Be the Reason for the Lower Pathogenicity of Omicron?

The novel SARS-CoV-2 Omicron variant B.1.1.529, which emerged in late 2021, is currently active worldwide, replacing other variants, including the Delta variant, due to an enormously increased infectivity. Multiple substitutions and deletions in the N-terminal domain (NTD) and the receptor binding domain (RBD) in the spike protein collaborate with the observed increased infectivity and evasion from therapeutic monoclonal antibodies and vaccine-induced neutralizing antibodies after primary/secondary immunization. In contrast, although three mutations near the S1/S2 furin cleavage site were predicted to favor cleavage, observed cleavage efficacy is substantially lower than in the Delta variant and also lower compared to the wild-type virus correlating with significantly lower TMPRSS2-dependent replication in the lungs, and lower cellular syncytium formation. In contrast, the Omicron variant shows high TMPRSS2-independent replication in the upper airway organs, but lower pathogenicity in animal studies and clinics. Based on recent data, we present here a hypothesis proposing that the changed charge distribution in the Omicron's spike protein could lead to lower activation of Toll-like receptors (TLRs) in innate immune cells, resulting in lower NF-κB activation, furin expression, and viral replication in the lungs, and lower immune hyper-activation.

Intracellular TLRs of Mast Cells in Innate and Acquired Immunity.

Mast cells (MCs) distribute to interface tissues with environment, such as skin, airway, and gut mucosa, thereby functioning as the sentinel against invading allergens and pathogens. To respond to and exclude these external substances promptly, MCs possess granules containing inflammatory mediators, including heparin, proteases, tumor necrosis factor, and histamine, and produce these mediators as a consequence of degranulation within minutes of activation. As a delayed response to external substances, MCs de novo synthesize inflammatory mediators, such as cytokines and chemokines, by sensing pathogen- and damage-associated molecular patterns through their pattern recognition receptors, including Toll-like receptors (TLRs). A substantial number of studies have reported immune responses by MCs through surface TLR signaling, particularly TLR2 and TLR4. However, less attention has been paid to immune responses through nucleic acid-recognizing intracellular TLRs. Among intracellular TLRs, human and rodent MCs express TLR3, TLR7, and TLR9, but not TLR8. Some virus infections modulate intracellular TLR expression in MCs. MC-derived mediators, such as histamine, cysteinyl leukotrienes, LL-37, and the granulocyte-macrophage colony-stimulating factor, have also been reported to modulate intracellular TLR expression in an autocrine and/or paracrine fashion. Synthetic ligands for intracellular TLRs and some viruses are sensed by intracellular TLRs of MCs, leading to the production of inflammatory cytokines and chemokines including type I interferons. These MC responses initiate and facilitate innate responses and the subsequent recruitment of additional innate effector cells. MCs also associate with the regulation of adaptive immunity. In this overview, the expression of intracellular TLRs in MCs and the recognition of pathogens, including viruses, by intracellular TLRs in MCs were critically evaluated.

Elevated expression of TLR2 and its correlation with disease activity and clinical manifestations in adult-onset Still's disease.

This study investigated the role of Toll-like receptor 1 (TLR1), TLR2, TLR4, TLR7, and TLR9 in patients with adult-onset Still's disease (AOSD). This study included 20 patients with AOSD and 15 healthy controls (HCs). TLR expression in the peripheral blood was quantified using flow cytometry; TLR expression pattern, in the skin lesions and lymph nodes (LNs) of patients with AOSD, was evaluated immunohistochemically. Significantly higher mean intensities of cells presenting TLR2 and TLR7 from whole blood were observed in patients with AOSD than in HCs. TLR2 expression in whole cells correlated with systemic scores, levels of lactate dehydrogenase and ferritin and serum levels of interleukin-1ß (IL-1ß), IL-6, and IL-18. The percentage of TLR2-positive inflammatory cells was higher in skin biopsy samples from patients with AOSD than those in HCs. TLR9-expressing positive inflammatory cell counts were higher in skin lesions from patients with AOSD than those in the HC, eczema, and psoriasis groups. The expression levels of TLR1, TLR4, TLR7, and TLR9 were higher in LNs of patients with AOSD than in those with T cell lymphoma and reactive lymphadenopathy. Circulating TLR2- and TLR7-positive cells may contribute to the pathogenesis of AOSD. Furthermore, immunohistochemical staining for TLRs in skin lesions and LNs may aid in differentiating AOSD from similar conditions.

Disengaging the COVID-19 Clutch as a Discerning Eye Over the Inflammatory Circuit During SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the cytokine release syndrome (CRS) and leads to multiorgan dysfunction. Mitochondrial dynamics are fundamental to protect against environmental insults, but they are highly susceptible to viral infections. Defective mitochondria are potential sources of reactive oxygen species (ROS). Infection with SARS-CoV-2 damages mitochondria, alters autophagy, reduces nitric oxide (NO), and increases both nicotinamide adenine dinucleotide phosphate oxidases (NOX) and ROS. Patients with coronavirus disease 2019 (COVID-19) exhibited activated toll-like receptors (TLRs) and the Nucleotide-binding and oligomerization domain (NOD-), leucine-rich repeat (LRR-), pyrin domain-containing protein 3 (NLRP3) inflammasome. The activation of TLRs and NLRP3 by SARS-CoV-2 induces interleukin 6 (IL-6), IL-1ß, IL-18, and lactate dehydrogenase (LDH). Herein, we outline the inflammatory circuit of COVID-19 and what occurs behind the scene, the interplay of NOX/ROS and their role in hypoxia and thrombosis, and the important role of ROS scavengers to reduce COVID-19-related inflammation.

Individual genetic variability mainly of Proinflammatory cytokines, cytokine receptors, and toll-like receptors dictates pathophysiology of COVID-19 disease.

Innate and acquired immunity responses are crucial for viral infection elimination. However, genetic variations in coding genes may exacerbate the inflammation or initiate devastating cytokine storms which poses severe respiratory conditions in coronavirus disease-19 (COVID-19). Host genetic variations in particular those related to the immune responses determine the patients' susceptibility and COVID-19 severity and pathophysiology. Gene polymorphisms such as single nucleotide polymorphisms (SNPs) of interferons, TNF, IL1, IL4, IL6, IL7, IL10, and IL17 predispose patients to the severe form of COVID-19 or severe acute respiratory syndrome coronavirus-2 (SARS-COV-2). These variations mainly alter the gene expression and cause a severe response by B cells, T cells, monocytes, neutrophils, and natural killer cells participating in a cytokine storm. Moreover, cytokines and chemokines SNPs are associated with the severity of COVID-19 and clinical outcomes depending on the corresponding effect. Additionally, genetic variations in genes encoding toll-like receptors (TLRs) mainly TLR3, TLR7, and TLR9 have been related to the COVID-19 severe respiratory symptoms. The specific relation of these mutations with the novel variants of concern (VOCs) infection remains to be elucidated. Genetic variations mainly within genes encoding proinflammatory cytokines, cytokine receptors, and TLRs predispose patients to COVID-19 disease severity. Understanding host immune gene variations associated with the SARS-COV-2 infection opens insights to control the pathophysiology of emerging viral infections.

mRNA expression of toll-like receptors 3, 7, 8, and 9 in the nasopharyngeal epithelial cells of coronavirus disease 2019 patients.

BACKGROUND: The etiopathogenesis of coronavirus disease 2019 (COVID-19) stem partially from the abnormal activation of the innate and adaptive immune systems. Here in the current investigation, the mRNA expression levels of toll-like receptors (TLRs) were evaluated in the nasopharyngeal epithelial cells from COVID-19 patients. METHODS: Epithelial cells were obtained using nasopharyngeal swab samples from 90 COVID-19 patients and 50 controls. COVID-19 cases were classified into those without symptoms, with symptoms but not hospitalized, and with symptoms and hospitalized. To determine the mRNA expression levels of TLRs, first RNA was extracted and cDNA was synthesized, and finally Real-time PCR was exerted. RESULTS: It was seen that the transcript levels of TLR3, TLR7, TLR8, and TLR9 were overexpressed in the COVID-19 patients with clinical symptoms needing hospitalization as well as in those with clinical symptoms without needing for hospitalization compared to controls. Upregulation of TLRs was associated with clinical presentations of the patients. CONCLUSIONS: Modulation of TLR3, TLR7, TLR8, TLR9 in the epithelial cells of COVID-19 cases may estimate the disease severity and requirement for hospitalization.

Toll-like receptor-agonist-based therapies for respiratory viral diseases: thinking outside the cell.

Respiratory virus infections initiate in the upper respiratory tract (URT). Innate immunity is critical for initial control of infection at this site, particularly in the absence of mucosal virus-neutralising antibodies. If the innate immune response is inadequate, infection can spread to the lower respiratory tract (LRT) causing community-acquired pneumonia (as exemplified by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/coronavirus disease 2019). Vaccines for respiratory viruses (influenza and SARS-CoV-2) leverage systemic adaptive immunity to protect from severe lung disease. However, the URT remains vulnerable to infection, enabling viral transmission and posing an ongoing risk of severe disease in populations that lack effective adaptive immunity.Innate immunity is triggered by host cell recognition of viral pathogen-associated molecular patterns via molecular sensors such as Toll-like receptors (TLRs). Here we review the role of TLRs in respiratory viral infections and the potential of TLR-targeted treatments to enhance airway antiviral immunity to limit progression to severe LRT disease and reduce person-to-person viral transmission. By considering cellular localisation and antiviral mechanisms of action and treatment route/timing, we propose that cell surface TLR agonist therapies are a viable strategy for preventing respiratory viral diseases by providing immediate, durable pan-viral protection within the URT.

Toll-like receptors matter: plasmacytoid dendritic cells in COVID-19.

Plasmacytoid dendritic cells (pDC) have the unique ability to rapidly mount high-level antiviral type I interferon (IFN-I) responses during diverse virus infections. In COVID-19 patients, reduced pDC numbers correlate with diminished IFN-I serum levels and enhanced disease severity. However, the molecular mechanisms underlying SARS-CoV-2-mediated pDC stimulation to induce cytokine responses are still largely unclear. In this issue of the EMBO Journal, van der Sluis and colleagues tackled this question by using an innovative hematopoietic stem and progenitor cells (HSPC)-pDC system that allows gene editing and the detailed analysis of pDC sensing mechanisms.

Molecular dynamics simulations reveal the selectivity mechanism of structurally similar agonists to TLR7 and TLR8.

TLR7 and TLR8 are key members of the Toll-like receptor family, playing crucial roles in the signaling pathways of innate immunity, and thus become attractive therapeutic targets of many diseases including infections and cancer. Although TLR7 and TLR8 show a high degree of sequence homology, their biological response to small molecule binding is very different. Aiming to understand the mechanism of selective profiles of small molecule modulators against TLR7 and TLR8, we carried out molecular dynamic simulations on three imidazoquinoline derivatives bound to the receptors separately. They are Resiquimod (R), Hybrid-2 (H), and Gardiquimod (G), selective agonists of TLR7 and TLR8. Our MD trajectories indicated that in the complex of TLR7-R and TLR7-G, the two chains forming the TLR7 dimer tended to remain "open" conformation, while the rest systems maintained in the closed format. The agonists R, H, and G developed conformational deviation mainly on the aliphatic tail. Furthermore, we attempted to quantify the selectivity between TLR7 and TLR8 by binding free energies via MM-GBSA method. It showed that the three selected modulators were more favorable for TLR7 than TLR8, and the ranking from the strongest to the weakest was H, R and G, aligning well with experimental data. In the TLR7, the flexible and hydrophobic aliphatic side chain of H has stronger van der Waals interactions with V381 and F351 but only pick up interaction with one amino acid residue i.e. Y353 of TLR8. Unsurprisingly, the positively charged side chain of G has less favorable interaction with I585 of TLR7 and V573 of TLR8 explaining G is weak agonist of both TLR7 and TLR8. All three imidazoquinoline derivatives can form stable hydrogen bonds with D555 of TLR7 and the corresponding D543 of TLR8. In brief, the set of total 400ns MD studies sheds light on the potential selectivity mechanisms of agonists towards TLR7 and TLR8, indicating the van der Waals interaction as the driving force for the agonists binding, thus provides us insights for designing more potent and selective modulators to cooperate with the hydrophobic nature of the binding pocket.