ACE2-independent SARS-CoV-2 virus entry through cell surface GRP78 on monocytes - evidence from a translational clinical and experimental approach.

BACKGROUND: SARS-CoV-2 infects host cells via an ACE2/TMPRSS2 entry mechanism. Monocytes and macrophages, which play a key role during severe COVID-19 express only low or no ACE2, suggesting alternative entry mechanisms in these cells. In silico analyses predicted GRP78, which is constitutively expressed on monocytes and macrophages, to be a potential candidate receptor for SARS-CoV-2 virus entry. METHODS: Hospitalized COVID-19 patients were characterized regarding their pro-inflammatory state and cell surface GRP78 (csGRP78) expression in comparison to healthy controls. RNA from CD14+ monocytes of patients and controls were subjected to transcriptome analysis that was specifically complemented by bioinformatic re-analyses of bronchoalveolar lavage fluid (BALF) datasets of COVID-19 patients with a focus on monocyte/macrophage subsets, SARS-CoV-2 infection state as well as GRP78 gene expression. Monocyte and macrophage immunohistocytochemistry on GRP78 was conducted in post-mortem lung tissues. SARS-CoV-2 spike and GRP78 protein interaction was analyzed by surface plasmon resonance, GST Pull-down and Co-Immunoprecipitation. SARS-CoV-2 pseudovirus or single spike protein uptake was quantified in csGRP78high THP-1 cells. FINDINGS: Cytokine patterns, monocyte activation markers and transcriptomic changes indicated typical COVID-19 associated inflammation accompanied by upregulated csGRP78 expression on peripheral blood and lung monocytes/macrophages. Subsequent cell culture experiments confirmed an association between elevated pro-inflammatory cytokine levels and upregulation of csGRP78. Interaction of csGRP78 and SARS-CoV-2 spike protein with a dissociation constant of KD = 55.2 nM was validated in vitro. Infection rate analyses in ACE2low and GRP78high THP-1 cells showed increased uptake of pseudovirus expressing SARS-CoV-2 spike protein. INTERPRETATION: Our results demonstrate that csGRP78 acts as a receptor for SARS-CoV-2 spike protein to mediate ACE2-independent virus entry into monocytes. FUNDING: Funded by the Sino-German-Center for Science Promotion (C-0040) and the Germany Ministry BMWi/K [DLR-grant 50WB1931 and RP1920 to AC, DM, TW].

Nanoparticle-Exposure-Triggered Virus Reactivation Induces Lung Emphysema in Mice.

Nanoparticles (NPs) released from engineered materials or combustion processes as well as persistent herpesvirus infection are omnipresent and are associated with chronic lung diseases. Previously, we showed that pulmonary exposure of a single dose of soot-like carbonaceous NPs (CNPs) or fiber-shaped double-walled carbon nanotubes (DWCNTs) induced an increase of lytic virus protein expression in mouse lungs latently infected with murine γ-herpesvirus 68 (MHV-68), with a similar pattern to acute infection suggesting virus reactivation. Here we investigate the effects of a more relevant repeated NP exposure on lung disease development as well as herpesvirus reactivation mechanistically and suggest an avenue for therapeutic prevention. In the MHV-68 mouse model, progressive lung inflammation and emphysema-like injury were detected 1 week after repetitive CNP and DWCNT exposure. NPs reactivated the latent herpesvirus mainly in CD11b+ macrophages in the lungs. In vitro, in persistently MHV-68 infected bone marrow-derived macrophages, ERK1/2, JNK, and p38 MAPK were rapidly activated after CNP and DWCNT exposure, followed by viral gene expression and increased viral titer but without generating a pro-inflammatory signature. Pharmacological inhibition of p38 activation abrogated CNP- but not DWCNT-triggered virus reactivation in vitro, and inhibitor pretreatment of latently infected mice attenuated CNP-exposure-induced pulmonary MHV-68 reactivation. Our findings suggest a crucial contribution of particle-exposure-triggered herpesvirus reactivation for nanomaterial exposure or air pollution related lung emphysema development, and pharmacological p38 inhibition might serve as a protective target to alleviate air pollution related chronic lung disease exacerbations. Because of the required precondition of latent infection described here, the use of single hit models might have severe limitations when assessing the respiratory toxicity of nanoparticle exposure.

Cytokine signaling converging on IL11 in ILD fibroblasts provokes aberrant epithelial differentiation signatures.

Introduction: Interstitial lung disease (ILD) is a heterogenous group of lung disorders where destruction and incomplete regeneration of the lung parenchyma often results in persistent architectural distortion of the pulmonary scaffold. Continuous mesenchyme-centered, disease-relevant signaling likely initiates and perpetuates the fibrotic remodeling process, specifically targeting the epithelial cell compartment, thereby destroying the gas exchange area. Methods: With the aim of identifying functional mediators of the lung mesenchymal-epithelial crosstalk with potential as new targets for therapeutic strategies, we developed a 3D organoid co-culture model based on human induced pluripotent stem cell-derived alveolar epithelial type 2 cells that form alveolar organoids in presence of lung fibroblasts from fibrotic-ILD patients, in our study referring to cases of pulmonary fibrosis, as well as control cell line (IMR-90). Results: While organoid formation capacity and size was comparable in the presence of fibrotic-ILD or control lung fibroblasts, metabolic activity was significantly increased in fibrotic-ILD co-cultures. Alveolar organoids cultured with fibrotic-ILD fibroblasts further demonstrated reduced stem cell function as reflected by reduced Surfactant Protein C gene expression together with an aberrant basaloid-prone differentiation program indicated by elevated Cadherin 2, Bone Morphogenic Protein 4 and Vimentin transcription. To screen for key mediators of the misguided mesenchymal-to-epithelial crosstalk with a focus on disease-relevant inflammatory processes, we used mass spectrometry and characterized the secretome of end stage fibrotic-ILD lung fibroblasts in comparison to non-chronic lung disease (CLD) patient fibroblasts. Out of the over 2000 proteins detected by this experimental approach, 47 proteins were differentially abundant comparing fibrotic-ILD and non-CLD fibroblast secretome. The fibrotic-ILD secretome profile was dominated by chemokines, including CXCL1, CXCL3, and CXCL8, interfering with growth factor signaling orchestrated by Interleukin 11 (IL11), steering fibrogenic cell-cell communication, and proteins regulating extracellular matrix remodeling including epithelial-to-mesenchymal transition. When in turn treating alveolar organoids with IL11, we recapitulated the co-culture results obtained with primary fibrotic-ILD fibroblasts including changes in metabolic activity. Conclusion: We identified mediators likely contributing to the disease-perpetuating mesenchymal-to-epithelial crosstalk in ILD. In our alveolar organoid co-cultures, we were able to highlight the importance of fibroblast-initiated aberrant epithelial differentiation and confirmed IL11 as a key player in fibrotic-ILD pathogenesis by unbiased fibroblast secretome analysis.

Sirtuin 6 inhibits MWCNTs-induced epithelial-mesenchymal transition in human bronchial epithelial cells via inactivating TGF-ß1/Smad2 signaling pathway.

Multi-walled carbon nanotubes (MWCNTs) have been developed with numerous beneficial applications. However, rodent models demonstrate that exposure to MWCNTs via respiratory pathways results in pulmonary fibrosis. Therefore, they could elicit a potential risk of pulmonary fibrosis in humans due to occupational or consumer exposure. Sirtuin 6 (SIRT6), a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, has been proved to prevent fibrosis in the liver, renal and myocardial tissues. In this present study, we aimed to explore the role of SIRT6 in MWCNTs-induced epithelial-mesenchymal transition (EMT), one of the major contributor of lung fibrogenesis in human bronchial epithelial BEAS-2B cells. We found that the protein level of SIRT6 was elevated after exposure to MWCNTs in BEAS-2B cells. Overexpression of SIRT6 significantly inhibited MWCNTs-induced EMT and EMT-like cell behaviors in BEAS-2B cells. Moreover, wild-type SIRT6 was found to decrease MWCNTs-induced phosphorylation of Smad2, but not mutant SIRT6 (H133Y) without histone deacetylase activity. In conclusion, our study demonstrated that SIRT6 inhibited MWCNTs-induced EMT in BEAS-2B cells through TGF-ß1/Smad2 signaling pathway, which depended on its deacetylase activity, and provided evidences that targeting SIRT6 could be a potential novel therapeutic strategy for MWCNTs-induced pulmonary fibrosis.

Sirtuin 6 inhibits myofibroblast differentiation via inactivating transforming growth factor-ß1/Smad2 and nuclear factor-κB signaling pathways in human fetal lung fibroblasts.

Fibroblast-to-myofibroblast differentiation, which is characterized by increased expression of α-smooth muscle actin, is known to be involved in the pathogenesis of idiopathic pulmonary fibrosis. Sirtuin 6 (SIRT6), a member of the sirtuin family, has been proved to inhibit epithelial-to-mesenchymal transition during idiopathic pulmonary fibrosis. However, the function of SIRT6 in lung myofibroblast differentiation is still obscure. Transforming growth factor-ß1 (TGF-ß1) is one of the main factors that can powerfully promote myofibroblast differentiation. In the current study, we aimed to explore the role of SIRT6 in the cellular model of fibroblast-to-myofibroblast differentiation induced by TGF-ß1 using human fetal lung fibroblasts (HFL1). We demonstrated that the SIRT6 protein level is upregulated by TGF-ß1 in HFL1 cells. Overexpression of SIRT6 significantly suppresses TGF-ß1-induced myofibroblast differentiation in HFL1 cells. Mechanistically, SIRT6 decreases phosphorylation and nuclear translocation of Smad2 under TGF-ß1 stimulation. Nevertheless, mutant SIRT6 (H133Y) without histone deacetylase activity fails to inhibit phosphorylation and nuclear translocation of Smad2. Meanwhile, SIRT6 interacts with the nuclear factor-κB (NF-κB) subunit p65 and represses TGF-ß1-induced NF-κB-dependent transcriptional activity, which is also dependent on its deacetylase activity. Overexpression of wild-type SIRT6 but not the H133Y mutant inhibits the expression of NF-κB-dependent genes including interleukin (IL)-1ß, IL-6 and matrix metalloproteinase-9 (MMP-9) induced by TGF-ß1, all of which have been demonstrated to promote myofibroblast differentiation. Collectively, our study reveals that SIRT6 prevents TGF-ß1-induced lung myofibroblast differentiation through inhibiting TGF-ß1/Smad2 and NF-κB signaling pathways.

Fluoride induces apoptosis via inhibiting SIRT1 activity to activate mitochondrial p53 pathway in human neuroblastoma SH-SY5Y cells.

There has been a great concern about the neurotoxicity of fluoride since it can pass through the blood-brain barrier and accumulate in the brain. It has been suggested that apoptosis plays a vital role in neurotoxicity of fluoride. However, whether p53-mediated apoptotic pathway is involved is still unclear. Our results showed that apoptosis was induced after treatment with 40 and 60 mg/L of NaF for 24 h in human neuroblastoma SH-SY5Y cells. Exposure to 60 mg/L of NaF for 24 h significantly upregulated the levels of p53 and apoptosis-related proteins including PUMA, cytochrome c (cyto c), cleaved caspase-3 and cleaved PARP, whereas downregulated Bcl-2 in SH-SY5Y cells. Meanwhile, fluoride increased p53 nuclear translocation, cyto c release from mitochondria to cytoplasm and mitochondrial translocation of Bax in SH-SY5Y cells. Fluoride-induced increases of apoptotic rates and apoptosis-related protein levels were significantly attenuated by inhibiting p53 transcriptional activity with pifithrin-α. In addition, fluoride inhibited the deacetylase activity of SIRT1 and increased p53 (acetyl K382) level in SH-SY5Y cells. Apoptosis and upregulation of cleaved caspase-3, cleaved PARP and p53 (acetyl K382) induced by fluoride could be ameliorated by SIRT1 overexpression or its activator resveratrol in SH-SY5Y cells. Taken together, our study demonstrates that fluoride induces apoptosis by inhibiting the deacetylase activity of SIRT1 to activate mitochondrial p53 pathway in SH-SY5Y cells, which depends on p53 transcriptional activity. Thus, SIRT1 may be a promising target to protect against neurotoxicity induced by fluoride.

Sirtuin 6 inhibits epithelial to mesenchymal transition during idiopathic pulmonary fibrosis via inactivating TGF-ß1/Smad3 signaling.

Sirt6 which is implicated in the control of aging, cancer, and metabolism, has been shown to have anti-fibrosis function in heart and liver. However, whether Sirt6 inhibits idiopathic pulmonary fibrosis remains elusive. Epithelial to mesenchymal transition has been found to be involved in the pathogenesis of idiopathic pulmonary fibrosis. In the present study, forced expression of Sirt6 significantly abrogated TGF-ß1-induced epithelial to mesenchymal transition-like phenotype and cell behaviors in A549 cells. Additionally, activation of TGF-ß1/Smad3 signaling pathway and binding of Smad3-Snail1 were ameliorated by overexpression of wild-type Sirt6 but not mutant Sirt6 (H133Y) without histone deacetylase activity. Meanwhile, upregulation of epithelial to mesenchymal transition-related transcription factors by TGF-ß1 were also restored by overexpression of wild-type Sirt6 but not mutant Sirt6. Furthermore, in vivo study showed that lung targeted delivery of Sirt6 using adeno-associated virus injection blunted bleomycin-induced pulmonary epithelial to mesenchymal transition and fibrosis. Overall, our findings unravel that Sirt6 acts as a key modulator in epithelial to mesenchymal transition process, suggesting Sirt6 may be an attractive potential therapeutic target for idiopathic pulmonary fibrosis.