f275fa40120a4b77b61a4f8ccb6e0ca4 293372.pdf 95407339948d17084ecdcd6415fe2166b2626831 293372.pdf ab2e6adff7c1e73cac4afedc8649a5a027788ac9c1ccdd587d0203078aba0fac 293372.pdf Title: Nucleotide-Binding Oligomerization Domain 1 (NOD1) Agonists Prevent SARS-CoV-2 Infection in Human Lung Epithelial Cells through Harnessing the Innate Immune Response Subject: The lung is prone to infections from respiratory viruses such as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). A challenge in combating these infections is the difficulty in targeting antiviral activity directly at the lung mucosal tract. Boosting the capability of the respiratory mucosa to trigger a potent immune response at the onset of infection could serve as a potential strategy for managing respiratory infections. This study focused on screening immunomodulators to enhance innate immune response in lung epithelial and immune cell models. Through testing various subfamilies and pathways of pattern recognition receptors (PRRs), the nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) family was found to selectively activate innate immunity in lung epithelial cells. Activation of NOD1 and dual NOD1/2 by the agonists TriDAP and M-TriDAP, respectively, increased the number of IL-8+ cells by engaging the NF-B and interferon response pathways. Lung epithelial cells showed a stronger response to NOD1 and dual NOD1/2 agonists compared to control. Interestingly, a less-pronounced response to NOD1 agonists was noted in PBMCs, indicating a tissue-specific effect of NOD1 in lung epithelial cells without inducing widespread systemic activation. The specificity of the NOD agonist pathway was confirmed through gene silencing of NOD1 (siRNA) and selective NOD1 and dual NOD1/2 inhibitors in lung epithelial cells. Ultimately, activation induced by NOD1 and dual NOD1/2 agonists created an antiviral environment that hindered SARS-CoV-2 replication in vitro in lung epithelial cells. Keywords: NOD-like receptor; innate immunity; respiratory mucosa; viral respiratory infections; SARS-CoV-2 Author: Edurne Garcia-Vidal, Ignasi Calba, Eva Riveira-Muñoz, Elisabet García, Bonaventura Clotet, Pere Serra-Mitjà, Cecilia Cabrera, Ester Ballana and Roger Badia Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Tue May 14 08:22:01 2024 CEST ModDate: Tue May 14 08:26:13 2024 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 17 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2867235 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- PYPZKK+VnURWPalladioL-Bold Type 1 Custom yes yes yes 10 0 KESBIZ+EURM10 Type 1 Builtin yes yes yes 16 0 MMQHMD+URWPalladioL-Bold Type 1 Custom yes yes yes 21 0 OYWPJY+URWPalladioL-Roma Type 1 Custom yes yes yes 27 0 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Immune System Destination: subsection.2.1 Item: Title: Identification of NLRs as Putative Innate Immune Agonists Suitable for the Activation of the Immune Response in the Respiratory Tract Destination: subsection.2.2 Item: Title: NOD1 Agonists Selectively Activate the NF-B and ISRE Signaling Pathways in Human Lung Epithelial Cells Destination: subsection.2.3 Item: Title: Selective Immune Activation by NOD1 Agonist Is Specific for Lung Epithelial Cells Destination: subsection.2.4 Item: Title: NOD1 Agonists Do Not Elicit Immune Innate Activation in PBMCs Destination: subsection.2.5 Item: Title: NOD1 Agonists Inhibit SARS-CoV-2 Infection in Lung Epithelial Cells Destination: subsection.2.6 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Cells Destination: subsection.4.1 Item: Title: Compounds Destination: subsection.4.2 Item: Title: Luminescence and Spectrophotometry Assays Destination: subsection.4.3 Item: Title: Evaluation of Cytotoxicity Destination: subsection.4.4 Item: Title: Flow Cytometry Destination: subsection.4.5 Item: Title: Quantitative RT-Polymerase Chain Reaction (qRT-PCR) Destination: subsection.4.6 Item: Title: Western Blot Destination: subsection.4.7 Item: Title: Transfection and RNA Interference Destination: subsection.4.8 Item: Title: Virus and Infections Destination: subsection.4.9 Item: Title: Statistical Methods Destination: subsection.4.10 Item: Title: References Destination: appendix.A. Info: Title: Nucleotide-Binding Oligomerization Domain 1 (NOD1) Agonists Prevent SARS-CoV-2 Infection in Human Lung Epithelial Cells through Harnessing the Innate Immune Response Author: Edurne Garcia-Vidal, Ignasi Calba, Eva Riveira-Muñoz, Elisabet García, Bonaventura Clotet, Pere Serra-Mitjà, Cecilia Cabrera, Ester Ballana and Roger Badia Subject: The lung is prone to infections from respiratory viruses such as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). A challenge in combating these infections is the difficulty in targeting antiviral activity directly at the lung mucosal tract. Boosting the capability of the respiratory mucosa to trigger a potent immune response at the onset of infection could serve as a potential strategy for managing respiratory infections. This study focused on screening immunomodulators to enhance innate immune response in lung epithelial and immune cell models. Through testing various subfamilies and pathways of pattern recognition receptors (PRRs), the nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) family was found to selectively activate innate immunity in lung epithelial cells. Activation of NOD1 and dual NOD1/2 by the agonists TriDAP and M-TriDAP, respectively, increased the number of IL-8+ cells by engaging the NF-B and interferon response pathways. Lung epithelial cells showed a stronger response to NOD1 and dual NOD1/2 agonists compared to control. Interestingly, a less-pronounced response to NOD1 agonists was noted in PBMCs, indicating a tissue-specific effect of NOD1 in lung epithelial cells without inducing widespread systemic activation. The specificity of the NOD agonist pathway was confirmed through gene silencing of NOD1 (siRNA) and selective NOD1 and dual NOD1/2 inhibitors in lung epithelial cells. Ultimately, activation induced by NOD1 and dual NOD1/2 agonists created an antiviral environment that hindered SARS-CoV-2 replication in vitro in lung epithelial cells. 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