How Toll-like receptors influence Parkinson's disease in the microbiome-gut-brain axis.

Recently, a large number of experimenters have found that the pathogenesis of Parkinson's disease may be related to the gut microbiome and proposed the microbiome-gut-brain axis. Studies have shown that Toll-like receptors, especially Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4), are key mediators of gut homeostasis. In addition to their established role in innate immunity throughout the body, research is increasingly showing that the Toll-like receptor 2 and Toll-like receptor 4 signaling pathways shape the development and function of the gut and enteric nervous system. Notably, Toll-like receptor 2 and Toll-like receptor 4 are dysregulated in Parkinson's disease patients and may therefore be identified as the core of early gut dysfunction in Parkinson's disease. To better understand the contribution of Toll-like receptor 2 and Toll-like receptor 4 dysfunction in the gut to early α-synuclein aggregation, we discussed the structural function of Toll-like receptor 2 and Toll-like receptor 4 and signal transduction of Toll-like receptor 2 and Toll-like receptor 4 in Parkinson's disease by reviewing clinical, animal models, and in vitro studies. We also present a conceptual model of the pathogenesis of Parkinson's disease, in which microbial dysbiosis alters the gut barrier as well as the Toll-like receptor 2 and Toll-like receptor 4 signaling pathways, ultimately leading to a positive feedback loop for chronic gut dysfunction, promoting α-synuclein aggregation in the gut and vagus nerve.

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

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.

SARS-CoV-2 envelope protein triggers depression-like behaviors and dysosmia via TLR2-mediated neuroinflammation in mice.

BACKGROUND: Depression and dysosmia have been regarded as primary neurological symptoms in COVID-19 patients, the mechanism of which remains unclear. Current studies have demonstrated that the SARS-CoV-2 envelope (E) protein is a pro-inflammatory factor sensed by Toll-like receptor 2 (TLR2), suggesting the pathological feature of E protein is independent of viral infection. In this study, we aim to ascertain the role of E protein in depression, dysosmia and associated neuroinflammation in the central nervous system (CNS). METHODS: Depression-like behaviors and olfactory function were observed in both female and male mice receiving intracisternal injection of E protein. Immunohistochemistry was applied in conjunction with RT-PCR to evaluate glial activation, blood-brain barrier status and mediators synthesis in the cortex, hippocampus and olfactory bulb. TLR2 was pharmacologically blocked to determine its role in E protein-related depression-like behaviors and dysosmia in mice. RESULTS: Intracisternal injection of E protein evoked depression-like behaviors and dysosmia in both female and male mice. Immunohistochemistry suggested that the E protein upregulated IBA1 and GFAP in the cortex, hippocampus and olfactory bulb, while ZO-1 was downregulated. Moreover, IL-1ß, TNF-α, IL-6, CCL2, MMP2 and CSF1 were upregulated in both cortex and hippocampus, whereas IL-1ß, IL-6 and CCL2 were upregulated in the olfactory bulb. Furtherly, inhibiting microglia, rather than astrocytes, alleviated depression-like behaviors and dysosmia induced by E protein. Finally, RT-PCR and immunohistochemistry suggested that TLR2 was upregulated in the cortex, hippocampus and olfactory bulb, the blocking of which mitigated depression-like behaviors and dysosmia induced by E protein. CONCLUSIONS: Our study demonstrates that envelope protein could directly induce depression-like behaviors, dysosmia, and obvious neuroinflammation in CNS. TLR2 mediated depression-like behaviors and dysosmia induced by envelope protein, which could serve as a promising therapeutic target for neurological manifestation in COVID-19 patients.

Effects of 5α-dihydrotestosterone on the modulation of monocyte/macrophage response to Staphylococcus aureus: an in vitro study.

BACKGROUND: Staphylococcus aureus (S. aureus) is a pathogen responsible for a wide range of clinical manifestations and potentially fatal conditions. There is a paucity of information on the influence of androgens in the immune response to S. aureus infection. In this study, we evaluated the influence of the hormone 5α-dihydrotestosterone (DHT) on mouse peritoneal macrophages (MPMs) and human peripheral blood monocytes (HPBMs) induced by S. aureus. METHODS: An in vitro model of MPMs from BALB/c sham males, orchiectomised (OQX) males, and females was used. Cells were inoculated with 10 µL of S. aureus, phage-type 80 or sterile saline (control) for 6 h. The MPMs of OQX males and females were pre-treated with 100 µL of 10-2 M DHT for 24 h before inoculation with S. aureus. The concentration of the cytokines TNF-α, IL-1α, IL-6, IL-8, and IL-10; total nitrites (NO-2); and hydrogen peroxide (H2O2) were measured in the supernatant of MPM cultures. In addition, the toll-like receptor 2 (TLR2) and nuclear factor kappa B (NF-kB) genes that are involved in immune responses were analysed. For the in vitro model of HPBMs, nine men and nine women of childbearing age were selected and HPBMs were isolated from samples of the volunteers' peripheral blood. In women, blood was collected during the periovulatory period. The HPBMs were inoculated with S. aureus for 6 h and the supernatant was collected for the analysis of cytokines TNF-α, IL-6, IL-12; and GM-CSF, NO-2, and H2O2. The HPBMs were then removed for the analysis of 84 genes involved in the host's response to bacterial infections by RT-PCR array. GraphPad was used for statistical analysis with a p value < 0.05. RESULTS: Our data demonstrated that MPMs from sham males inoculated with S. aureus displayed higher concentrations of inflammatory cytokines and lower concentrations of IL-10, NO-2, and H2O2 when compared with MPMs from OQX males and females. A similar result was observed in the HPBMs of men when compared with those of women. Previous treatment with DHT in women HPBMs increased the production of pro-inflammatory cytokines and decreased the levels of IL-10, NO-2, and H2O2. The analysis of gene expression showed that DHT increased the activity of the TLR2 and NF-kB pathways in both MPMs and HPBMs. CONCLUSIONS: We found that DHT acts as an inflammatory modulator in the monocyte/macrophage response induced by S. aureus and females exhibit a better immune defence response against this pathogen.

The SARS-CoV-2 E protein induces Toll-like receptor 2-mediated neonatal lung injury in a model of COVID-19 viremia that is rescued by the glucocorticoid ciclesonide.

SARS-CoV-2 viremia is associated with increased acute lung injury (ALI) and mortality in children and adults. The mechanisms by which viral components in the circulation mediate ALI in COVID-19 remain unclear. We tested the hypothesis that the SARS-CoV-2 envelope (E) protein induces Toll-like receptor (TLR)-mediated ALI and lung remodeling in a model of neonatal COVID-19. Neonatal C57BL6 mice given intraperitoneal E protein injections revealed a dose-dependent increase in lung cytokines [interleukin 6 (Il6), tumor necrosis factor (Tnfα), and interleukin 1 beta (Il1ß)] and canonical proinflammatory TLR signaling. Systemic E protein induced endothelial immune activation, immune cell influx, and TGFß signaling and lung matrix remodeling inhibited alveolarization in the developing lung. E protein-mediated ALI and transforming growth factor beta (TGFß) signaling was repressed in Tlr2-/-, but not Tlr4-/- mice. A single dose of intraperitoneal E protein injection induced chronic alveolar remodeling as evidenced by a decrease in radial alveolar counts and increase in mean linear intercepts. Ciclesonide, a synthetic glucocorticoid, inhibited E protein-induced proinflammatory TLR signaling and ALI. In vitro, E protein-mediated inflammation and cell death were TLR2-dependent in human primary neonatal lung endothelial cells and were rescued by ciclesonide. This study provides insight into the pathogenesis of ALI and alveolar remodeling with SARS-CoV-2 viremia in children, whereas revealing the efficacy of steroids.NEW & NOTEWORTHY We reveal that the envelope protein of SARS-CoV-2 mediates acute lung injury (ALI) and alveolar remodeling through Toll-like receptor activation, which is rescued by the glucocorticoid, ciclesonide.

The Spike protein of SARS-CoV-2 signals via Tlr2 in zebrafish.

One of the most studied defense mechanisms against invading pathogens, including viruses, are Toll-like receptors (TLRs). Among them, TLR3, TLR7, TLR8 and TLR9 detect different forms of viral nucleic acids in endosomal compartments, whereas TLR2 and TLR4 recognize viral structural and nonstructural proteins outside the cell. Although many different TLRs have been shown to be involved in SARS-CoV-2 infection and detection of different structural proteins, most studies have been performed in vitro and the results obtained are rather contradictory. In this study, we report using the unique advantages of the zebrafish model for in vivo imaging and gene editing that the S1 domain of the Spike protein from the Wuhan strain (S1WT) induced hyperinflammation in zebrafish larvae via a Tlr2/Myd88 signaling pathway and independently of interleukin-1ß production. In addition, S1WT also triggered emergency myelopoiesis, but in this case through a Tlr2/Myd88-independent signaling pathway. These results shed light on the mechanisms involved in the fish host responses to viral proteins.

Nasopharyngeal epithelial cells from patients with coronavirus disease 2019 express abnormal levels of Toll-like receptors.

Aberrant activation of the immune system has been attributed with etiology and pathogenesis of coronavirus disease 2019 (COVID-19). Here, the transcript levels of toll-like receptors (TLRs) were measured in the nasopharyngeal epithelial cells obtained from COVID-19 patients to assess the involvement of these molecules in the clinical outcome of COVID-19 patients. Nasopharyngeal swab samples were used to obtain epithelial cells from 120 COVID-19 patients and 100 healthy controls. COVID-19 cases were classified into those having clinical symptoms/needing for hospitalization, having clinical symptoms/not needing for hospitalization, and those without clinical symptoms|. The mRNA expression levels of TLRs were measured in the nasopharyngeal epithelial cells. Overall, mRNA expression of TLR1, TLR2, TLR4, and TLR6 was significantly higher in COVID-19 cases compared to controls. The mRNA expression of TLRs were all higher significantly in the samples from COVID-19 patients having clinical symptoms and needing hospitalization as well as in those with clinical symptoms/not needing for hospitalization in comparison to controls. TLR expression was significantly higher in those with clinical symptoms/needing for hospitalization and those with clinical symptoms/not needing for hospitalization compared to COVID-19 cases without clinical symptoms. In cases with clinical symptoms/needing for hospitalization and those with clinical symptoms/not needing for hospitalization, there was a correlation between TLR expression and clinicopathological findings. In conclusion, aberrant expression of TLRs in the nasopharyngeal epithelial cells from COVID-19 cases may predict the severity of the diseases and necessity for supportive cares in the hospital.

Pam2CSK4-adjuvanted SARS-CoV-2 RBD nanoparticle vaccine induces robust humoral and cellular immune responses.

As the global COVID-19 pandemic continues and new SARS-CoV-2 variants of concern emerge, vaccines remain an important tool for preventing the pandemic. The inactivated or subunit vaccines themselves generally exhibit low immunogenicity, which needs adjuvants to improve the immune response. We previously developed a receptor binding domain (RBD)-targeted and self-assembled nanoparticle to elicit a potent immune response in both mice and rhesus macaques. Herein, we further improved the RBD production in the eukaryote system by in situ Crispr/Cas9-engineered CHO cells. By comparing the immune effects of various Toll-like receptor-targeted adjuvants to enhance nanoparticle vaccine immunization, we found that Pam2CSK4, a TLR2/6 agonist, could mostly increase the titers of antigen-specific neutralizing antibodies and durability in humoral immunity. Remarkably, together with Pam2CSK4, the RBD-based nanoparticle vaccine induced a significant Th1-biased immune response and enhanced the differentiation of both memory T cells and follicular helper T cells. We further found that Pam2CSK4 upregulated migration genes and many genes involved in the activation and proliferation of leukocytes. Our data indicate that Pam2CSK4 targeting TLR2, which has been shown to be effective in tuberculosis vaccines, is the optimal adjuvant for the SARS-CoV-2 nanoparticle vaccine, paving the way for an immediate clinical trial.

SARS-CoV-2 modulates inflammatory responses of alveolar epithelial type II cells via PI3K/AKT pathway.

Background: SARS-CoV-2 infects through the respiratory route and triggers inflammatory response by affecting multiple cell types including type II alveolar epithelial cells. SARS-CoV-2 triggers signals via its Spike (S) protein, which have been shown to participate in the pathogenesis of COVID19. Aim: Aim of the present study was to investigate the effect of SARS-CoV2 on type II alveolar epithelial cells, focusing on signals initiated by its S protein and their impact on the expression of inflammatory mediators. Results: For this purpose A549 alveolar type II epithelial cells were exposed to SARS CoV2 S recombinant protein and the expression of inflammatory mediators was measured. The results showed that SARS-CoV-2 S protein decreased the expression and secretion of IL8, IL6 and TNFα, 6 hours following stimulation, while it had no effect on IFNα, CXCL5 and PAI-1 expression. We further examined whether SARS-CoV-2 S protein, when combined with TLR2 signals, which are also triggered by SARS-CoV2 and its envelope protein, exerts a different effect in type II alveolar epithelial cells. Simultaneous treatment of A549 cells with SARS-CoV-2 S protein and the TLR2 ligand PAM3csk4 decreased secretion of IL8, IL6 and TNFα, while it significantly increased IFNα, CXCL5 and PAI-1 mRNA expression. To investigate the molecular pathway through which SARS-CoV-2 S protein exerted this immunomodulatory action in alveolar epithelial cells, we measured the induction of MAPK/ERK and PI3K/AKT pathways and found that SARS-CoV-2 S protein induced the activation of the serine threonine kinase AKT. Treatment with the Akt inhibitor MK-2206, abolished the inhibitory effect of SARS-CoV-2 S protein on IL8, IL6 and TNFα expression, suggesting that SARS-CoV-2 S protein mediated its action via AKT kinases. Conclusion: The findings of our study, showed that SARS-CoV-2 S protein suppressed inflammatory responses in alveolar epithelial type II cells at early stages of infection through activation of the PI3K/AKT pathway. Thus, our results suggest that at early stages SARS-CoV-2 S protein signals inhibit immune responses to the virus allowing it to propagate the infection while in combination with TLR2 signals enhances PAI-1 expression, potentially affecting the local coagulation cascade.

Mucosal TLR2-activating protein-based vaccination induces potent pulmonary immunity and protection against SARS-CoV-2 in mice.

Current vaccines against SARS-CoV-2 substantially reduce mortality, but protection against infection is less effective. Enhancing immunity in the respiratory tract, via mucosal vaccination, may provide protection against infection and minimise viral spread. Here, we report testing of a subunit vaccine in mice, consisting of SARS-CoV-2 Spike protein with a TLR2-stimulating adjuvant (Pam2Cys), delivered to mice parenterally or mucosally. Both routes of vaccination induce substantial neutralising antibody (nAb) titres, however, mucosal vaccination uniquely generates anti-Spike IgA, increases nAb in the serum and airways, and increases lung CD4+ T-cell responses. TLR2 is expressed by respiratory epithelia and immune cells. Using TLR2 deficient chimeric mice, we determine that TLR2 expression in either compartment facilitates early innate responses to mucosal vaccination. By contrast, TLR2 on hematopoietic cells is essential for optimal lung-localised, antigen-specific responses. In K18-hACE2 mice, vaccination provides complete protection against disease and sterilising lung immunity against SARS-CoV-2, with a short-term non-specific protective effect from mucosal Pam2Cys alone. These data support mucosal vaccination as a strategy to improve protection in the respiratory tract against SARS-CoV-2 and other respiratory viruses.

IFITM3 restricts virus-induced inflammatory cytokine production by limiting Nogo-B mediated TLR responses.

Interferon-induced transmembrane protein 3 (IFITM3) is a restriction factor that limits viral pathogenesis and exerts poorly understood immunoregulatory functions. Here, using human and mouse models, we demonstrate that IFITM3 promotes MyD88-dependent, TLR-mediated IL-6 production following exposure to cytomegalovirus (CMV). IFITM3 also restricts IL-6 production in response to influenza and SARS-CoV-2. In dendritic cells, IFITM3 binds to the reticulon 4 isoform Nogo-B and promotes its proteasomal degradation. We reveal that Nogo-B mediates TLR-dependent pro-inflammatory cytokine production and promotes viral pathogenesis in vivo, and in the case of TLR2 responses, this process involves alteration of TLR2 cellular localization. Nogo-B deletion abrogates inflammatory cytokine responses and associated disease in virus-infected IFITM3-deficient mice. Thus, we uncover Nogo-B as a driver of viral pathogenesis and highlight an immunoregulatory pathway in which IFITM3 fine-tunes the responsiveness of myeloid cells to viral stimulation.

In silico designing of a novel epitope-based candidate vaccine against Streptococcus pneumoniae with introduction of a new domain of PepO as adjuvant.

BACKGROUND: Streptococcus pneumoniae is the leading reason for invasive diseases including pneumonia and meningitis, and also secondary infections following viral respiratory diseases such as flu and COVID-19. Currently, serotype-dependent vaccines, which have several insufficiency and limitations, are the only way to prevent pneumococcal infections. Hence, it is plain to need an alternative effective strategy for prevention of this organism. Protein-based vaccine involving conserved pneumococcal protein antigens with different roles in virulence could provide an eligible alternative to existing vaccines. METHODS: In this study, PspC, PhtD and PsaA antigens from pneumococcus were taken to account to predict B-cell and helper T-cell epitopes, and epitope-rich regions were chosen to build the construct. To enhance the immunogenicity of the epitope-based vaccine, a truncated N-terminal fragment of pneumococcal endopeptidase O (PepO) was used as a potential TLR2/4 agonist which was identified by molecular docking studies. The ultimate construct was consisted of the chosen epitope-rich regions, along with the adjuvant role (truncated N-PepO) and suitable linkers. RESULTS: The epitope-based vaccine was assessed as regards physicochemical properties, allergenicity, antigenicity, and toxicity. The 3D structure of the engineered construct was modeled, refined, and validated. Molecular docking and simulation of molecular dynamics (MD) indicated the proper and stable interactions between the vaccine and TLR2/4 throughout the simulation periods. CONCLUSIONS: For the first time this work presents a novel vaccine consisting of epitopes of PspC, PhtD, and PsaA antigens which is adjuvanted with a new truncated domain of PepO. The computational outcomes revealed that the suggested vaccine could be deemed an efficient therapeutic vaccine for S. pneumoniae; nevertheless, in vitro and in vivo examinations should be performed to prove the potency of the candidate vaccine.

CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation.

BACKGROUND: Coronavirus-induced disease 19 (COVID-19) infects more than three hundred and sixty million patients worldwide, and people with severe symptoms frequently die of acute respiratory distress syndrome (ARDS). Recent studies indicated that excessive neutrophil extracellular traps (NETs) contributed to immunothrombosis, thereby leading to extensive intravascular coagulopathy and multiple organ dysfunction. Thus, understanding the mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced NET formation would be helpful to reduce thrombosis and prevent ARDS in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: We incubated SARS-CoV-2 with neutrophils in the presence or absence of platelets to observe NET formation. We further isolated extracellular vesicles from COVID-19 patients' sera (COVID-19-EVs) to examine their ability to induce NET formation. RESULTS: We demonstrated that antagonistic mAbs against anti-CLEC5A mAb and anti-TLR2 mAb can inhibit COVID-19-EVs-induced NET formation, and generated clec5a-/-/tlr2-/- mice to confirm the critical roles of CLEC5A and TLR2 in SARS-CoV-2-induced lung inflammation in vivo. We found that virus-free extracellular COVID-19 EVs induced robust NET formation via Syk-coupled C-type lectin member 5A (CLEC5A) and TLR2. Blockade of CLEC5A inhibited COVID-19 EVs-induced NETosis, and simultaneous blockade of CLEC5A and TLR2 further suppressed SARS-CoV-2-induced NETosis in vitro. Moreover, thromboinflammation was attenuated dramatically in clec5a-/-/tlr2-/- mice. CONCLUSIONS: This study demonstrates that SARS-CoV-2-activated platelets produce EVs to enhance thromboinflammation via CLEC5A and TLR2, and highlight the importance of CLEC5A and TLR2 as therapeutic targets to reduce the risk of ARDS in COVID-19 patients.

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.

Inhibition of IRAK4 dysregulates SARS-CoV-2 spike protein-induced macrophage inflammatory and glycolytic reprogramming.

Escalated innate immunity plays a critical role in SARS-CoV-2 pathology; however, the molecular mechanism is incompletely understood. Thus, we aim to characterize the molecular mechanism by which SARS-CoV-2 Spike protein advances human macrophage (MÏ´) inflammatory and glycolytic phenotypes and uncover novel therapeutic strategies. We found that human MÏ´s exposed to Spike protein activate IRAK4 phosphorylation. Blockade of IRAK4 in Spike protein-stimulated MÏ´s nullifies signaling of IRAK4, AKT, and baseline p38 without affecting ERK and NF-κB activation. Intriguingly, IRAK4 inhibitor (IRAK4i) rescues the SARS-CoV-2-induced cytotoxic effect in ACE2+HEK 293 cells. Moreover, the inflammatory reprogramming of MÏ´s by Spike protein was blunted by IRAK4i through IRF5 and IRF7, along with the reduction of monokines, IL-6, IL-8, TNFα, and CCL2. Notably, in Spike protein-stimulated MÏ´s, suppression of the inflammatory markers by IRAK4i was coupled with the rebalancing of oxidative phosphorylation over metabolic activity. This metabolic adaptation promoted by IRAK4i in Spike protein-activated MÏ´s was shown to be in part through constraining PFKBF3, HIF1α, cMYC, LDHA, lactate expression, and reversal of citrate and succinate buildup. IRAK4 knockdown could comparably impair Spike protein-enhanced inflammatory and metabolic imprints in human MÏ´s as those treated with ACE2, TLR2, and TLR7 siRNA. Extending these results, in murine models, where human SARS-CoV-2 Spike protein was not recognized by mouse ACE2, TLRs were responsible for the inflammatory and glycolytic responses instigated by Spike protein and were dysregulated by IRAK4i therapy. In conclusion, IRAK4i may be a promising strategy for severe COVID-19 patients by counter-regulating ACE2 and TLR-mediated MÏ´ hyperactivation. IRAK4i therapy counteracts MÏ´ inflammatory and glycolytic reprogramming triggered by Spike protein. This study illustrates that SARS-CoV-2 Spike protein activates IRAK4 signaling via ACE2 as well as TLR2 and TLR7 sensing in human MÏ´s. Remarkably, IRAK4i treatment can dysregulate both ACE-dependent and independent (via TLR sensing) SARS-CoV-2 Spike protein-activated inflammatory and metabolic imprints.

SARS-CoV-2 Envelope (E) Protein Binds and Activates TLR2 Pathway: A Novel Molecular Target for COVID-19 Interventions.

This paper presents a molecular characterization of the interaction between the SARS-CoV-2 envelope (E) protein and TLR2. We demonstrated that the E protein, both as a recombinant soluble protein and as a native membrane protein associated with SARS-CoV-2 viral particles, interacts physically with the TLR2 receptor in a specific and dose-dependent manner. Furthermore, we showed that the specific interaction with the TLR2 pathway activates the NF-κB transcription factor and stimulates the production of the CXCL8 inflammatory chemokine. In agreement with the importance of NF-κB in the TLR signaling pathway, we showed that the chemical inhibition of this transcription factor leads to significant inhibition of CXCL8 production, while the blockade of the P38 and ERK1/2 MAP kinases only results in partial CXCL8 inhibition. Overall, our findings propose the envelope (E) protein as a novel molecular target for COVID-19 interventions: either (i) by exploring the therapeutic effect of anti-E blocking/neutralizing antibodies in symptomatic COVID-19 patients, or (ii) as a promising non-spike SARS-CoV-2 antigen candidate for inclusion in the development of next-generation prophylactic vaccines against COVID-19 infection and disease.

Does SARS-CoV-2 affect neurodegenerative disorders? TLR2, a potential receptor for SARS-CoV-2 in the CNS.

The coronavirus (COVID-19) pandemic, caused by severe acute respiratory system coronavirus 2 (SARS-CoV-2), has created significant challenges for scientists seeking to understand the pathogenic mechanisms of SARS-CoV-2 infection and to identify the best therapies for infected patients. Although ACE2 is a known receptor for the virus and has been shown to mediate viral entry into the lungs, accumulating reports highlight the presence of neurological symptoms resulting from infection. As ACE2 expression is low in the central nervous system (CNS), these neurological symptoms are unlikely to be caused by ACE2-virus binding. In this review, we will discuss a proposed interaction between SARS-CoV-2 and Toll-like receptor 2 (TLR2) in the CNS. TLR2 is an innate immune receptor that recognizes exogenous microbial components but has also been shown to interact with multiple viral components, including the envelope (E) protein of SARS-CoV-2. In addition, TLR2 plays an important role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Based on these observations, we hypothesize that TLR2 may play a critical role in the response to SARS-CoV-2 infiltration in the CNS, thereby resulting in the induction or acceleration of AD and PD pathologies in patients.

Novel Synthetic Lipopeptides as Potential Mucosal Adjuvants Enhanced SARS-CoV-2 rRBD-Induced Immune Response.

As TLR2 agonists, several lipopeptides had been proved to be candidate vaccine adjuvants. In our previous study, lipopeptides mimicking N-terminal structures of the bacterial lipoproteins were also able to promote antigen-specific immune response. However, the structure-activity relationship of lipopeptides as TLR2 agonists is still unclear. Here, 23 synthetic lipopeptides with the same lipid moiety but different peptide sequences were synthesized, and their TLR2 activities in vitro and mucosal adjuvant effects to OVA were evaluated. LP1-14, LP1-30, LP1-34 and LP2-2 exhibited significantly lower cytotoxicity and stronger TLR2 activity compared with Pam2CSK4, the latter being one of the most potent TLR2 agonists. LP1-34 and LP2-2 assisted OVA to induce more profound specific IgG in sera or sIgA in BALF than Pam2CSK4. Furthermore, the possibility of LP1-34, LP2-2 and Pam2CSK4 as the mucosal adjuvant for the SARS-CoV-2 recombinant RBD (rRBD) was investigated. Intranasally immunized with rRBD plus either the novel lipopeptide or Pam2CSK4 significantly increased the levels of specific serum and respiratory mucosal IgG and IgA, while rRBD alone failed to induce specific immune response due to its low immunogenicity. The novel lipopeptides, especially LP2-2, significantly increased levels of rRBD-induced SARS-CoV-2 neutralizing antibody in sera, BALF and nasal wash. Finally, Support vector machine (SVM) results suggested that charged residues in lipopeptides might be beneficial to the agonist activity, while lipophilic residues might adversely affect the agonistic activity. Figuring out the relationship between peptide sequence in the lipopeptide and its TLR2 activity may lay the foundation for the rational design of novel lipopeptide adjuvant for COVID-19 vaccine.

Anti-inflammatory and anti-viral actions of anionic pulmonary surfactant phospholipids.

Pulmonary surfactant is a mixture of lipids and proteins, consisting of 90% phospholipid, and 10% protein by weight, found predominantly in pulmonary alveoli of vertebrate lungs. Two minor components of pulmonary surfactant phospholipids, phosphatidylglycerol (PG) and phosphatidylinositol (PI), are present within the alveoli at very high concentrations, and exert anti-inflammatory effects by regulating multiple Toll like receptors (TLR2/1, TLR4, and TLR2/6) by antagonizing cognate ligand-dependent activation. POPG also attenuates LPS-induced lung injury in vivo. In addition, these lipids bind directly to RSV and influenza A viruses (IAVs) and block interaction between host cells and virions, and thereby prevent viral replication in vitro. POPG and PI also inhibit RSV and IAV infection in vivo, in mice and ferrets. The lipids markedly inhibit SARS-CoV-2 infection in vitro. These findings suggest that both POPG and PI have strong potential to be applied as both prophylaxis and post-infection treatments for problematic respiratory viral infections.

Differential Effect of SARS-CoV-2 Spike Glycoprotein 1 on Human Bronchial and Alveolar Lung Mucosa Models: Implications for Pathogenicity.

BACKGROUND: The SARS-CoV-2 spike protein mediates attachment of the virus to the host cell receptor and fusion between the virus and the cell membrane. The S1 subunit of the spike glycoprotein (S1 protein) contains the angiotensin converting enzyme 2 (ACE2) receptor binding domain. The SARS-CoV-2 variants of concern contain mutations in the S1 subunit. The spike protein is the primary target of neutralizing antibodies generated following infection, and constitutes the viral component of mRNA-based COVID-19 vaccines. METHODS: Therefore, in this work we assessed the effect of exposure (24 h) to 10 nM SARS-CoV-2 recombinant S1 protein on physiologically relevant human bronchial (bro) and alveolar (alv) lung mucosa models cultured at air-liquid interface (ALI) (n = 6 per exposure condition). Corresponding sham exposed samples served as a control. The bro-ALI model was developed using primary bronchial epithelial cells and the alv-ALI model using representative type II pneumocytes (NCI-H441). RESULTS: Exposure to S1 protein induced the surface expression of ACE2, toll like receptor (TLR) 2, and TLR4 in both bro-ALI and alv-ALI models. Transcript expression analysis identified 117 (bro-ALI) and 97 (alv-ALI) differentially regulated genes (p ≤ 0.01). Pathway analysis revealed enrichment of canonical pathways such as interferon (IFN) signaling, influenza, coronavirus, and anti-viral response in the bro-ALI. Secreted levels of interleukin (IL) 4 and IL12 were significantly (p < 0.05) increased, whereas IL6 decreased in the bro-ALI. In the case of alv-ALI, enriched terms involving p53, APRIL (a proliferation-inducing ligand) tight junction, integrin kinase, and IL1 signaling were identified. These terms are associated with lung fibrosis. Further, significantly (p < 0.05) increased levels of secreted pro-inflammatory cytokines IFNγ, IL1ꞵ, IL2, IL4, IL6, IL8, IL10, IL13, and tumor necrosis factor alpha were detected in alv-ALI, whereas IL12 was decreased. Altered levels of these cytokines are also associated with lung fibrotic response. CONCLUSIONS: In conclusion, we observed a typical anti-viral response in the bronchial model and a pro-fibrotic response in the alveolar model. The bro-ALI and alv-ALI models may serve as an easy and robust platform for assessing the pathogenicity of SARS-CoV-2 variants of concern at different lung regions.