Blockade of endolysosomal acidification suppresses TLR3-mediated pro-inflammatory signaling in airway epithelial cells

Pejler et al., Journal of Allergy and Clinical Immunology, doi:10.1016/j.jaci.2024.05.031

Jun 2024

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In Vitro study showing that blocking endolysosomal acidification with bafilomycin A1, monensin, or niclosamide suppresses TLR3-mediated pro-inflammatory signaling in human small airway epithelial cells (HSAECs) stimulated with TLR3 agonists mimicking viral RNA. The inhibitors reduced expression of pro-inflammatory cytokines IL-6, TNF, IL-8 and IL-1β at both mRNA and protein levels. The findings suggest a potential therapeutic strategy to reduce excessive cytokine production in response to respiratory virus infection. HCQ is not used in this study but also blocks endolysosomal acidification and may have similar potential.

9 preclinical studies support the efficacy of niclosamide for COVID-19:

1 In Silico study1

8 In Vitro studies2-9

In Silico studies predict inhibition of SARS-CoV-2 with niclosamide or metabolites via binding to the spikeA,1, M^proB,1, RNA-dependent RNA polymeraseC,1, PLproD,1, nucleocapsidE,1, and helicaseF,1 proteins. Niclosamide inhibits endolysosomal acidification and suppresses TLR3-mediated pro-inflammatory signaling in human small airway epithelial cells stimulated with TLR3 agonists mimicking viral RNA2, modulates host lipid metabolism and reduces infectious SARS-CoV-2 virion production in Vero E6 cells4, reduces CD147 protein levels and inhibits SARS-CoV-2-induced upregulation of CD147 in A549-ACE2 cells, including the highly glycosylated form of CD147 which has been implicated in COVID-19 disease progression and post-COVID-19 cardiac complications5, blocked the formation of syncytia mediated by SARS-CoV-2 spike protein pseudovirus-producing cells6, may reduce inflammation, NLRP3 formation, and caspase-1 activity9, may inhibit viral uncoating, replication, and assembly via disruption of pH gradients and reduced ATP production in host cells8, and shows strong synergy when combined with ivermectin7.

Important missing comments or errors? Let us know.

Study covers niclosamide and HCQ.

1. Haque et al., Exploring potential therapeutic candidates against COVID-19: a molecular docking study, Discover Molecules, doi:10.1007/s44345-024-00005-5.springer.com, doi.org.

2. Pejler et al., Blockade of endolysosomal acidification suppresses TLR3-mediated pro-inflammatory signaling in airway epithelial cells, Journal of Allergy and Clinical Immunology, doi:10.1016/j.jaci.2024.05.031.sciencedirect.com, doi.org.

3. Walia et al., SARS-CoV-2 virulence factor ORF3a blocks lysosome function by modulating TBC1D5-dependent Rab7 GTPase cycle, Nature Communications, doi:10.1038/s41467-024-46417-2.nature.com, doi.org.

4. Garrett et al., Niclosamide as a chemical probe for analyzing SARS-CoV-2 modulation of host cell lipid metabolism, Frontiers in Microbiology, doi:10.3389/fmicb.2023.1251065.frontiersin.org, doi.org.

5. Yang et al., Repurposing Niclosamide as a Novel Anti-SARS-CoV-2 Drug by Restricting Entry Protein CD147, Biomedicines, doi:10.3390/biomedicines11072019.mdpi.com, doi.org.

6. Sheng et al., A pseudovirus-based method to dynamically mimic SARS-CoV-2-associated cell-to-cell fusion and transmission, Acta Biochimica et Biophysica Sinica, doi:10.3724/abbs.2023129.scichina.com, doi.org.

7. Jitobaom et al., Synergistic anti-SARS-CoV-2 activity of repurposed anti-parasitic drug combinations, BMC Pharmacology and Toxicology, doi:10.1186/s40360-022-00580-8.biomedcentral.com, doi.org.

8. Needham, D., The pH Dependence of Niclosamide Solubility, Dissolution, and Morphology: Motivation for Potentially Universal Mucin-Penetrating Nasal and Throat Sprays for COVID19, its Variants and other Viral Infections, Pharmaceutical Research, doi:10.1007/s11095-021-03112-x.springer.com, doi.org.

9. Munson et al., Niclosamide and ivermectin modulate caspase-1 activity and proinflammatory cytokine secretion in a monocytic cell line, British Society For Nanomedicine Early Career Researcher Summer Meeting, 2021, web.archive.org/web/20230401070026/https://michealmunson.github.io/COVID.pdf.archive.org.

a. The trimeric spike (S) protein is a glycoprotein that mediates viral entry by binding to the host ACE2 receptor, is critical for SARS-CoV-2's ability to infect host cells, and is a target of neutralizing antibodies. Inhibition of the spike protein prevents viral attachment, halting infection at the earliest stage.

b. The main protease or M^pro, also known as 3CL^pro or nsp5, is a cysteine protease that cleaves viral polyproteins into functional units needed for replication. Inhibiting M^pro disrupts the SARS-CoV-2 lifecycle within the host cell, preventing the creation of new copies.

c. RNA-dependent RNA polymerase (RdRp), also called nsp12, is the core enzyme of the viral replicase-transcriptase complex that copies the positive-sense viral RNA genome into negative-sense templates for progeny RNA synthesis. Inhibiting RdRp blocks viral genome replication and transcription.

d. The papain-like protease (PLpro) has multiple functions including cleaving viral polyproteins and suppressing the host immune response by deubiquitination and deISGylation of host proteins. Inhibiting PLpro may block viral replication and help restore normal immune responses.

e. The nucleocapsid (N) protein binds and encapsulates the viral genome by coating the viral RNA. N enables formation and release of infectious virions and plays additional roles in viral replication and pathogenesis. N is also an immunodominant antigen used in diagnostic assays.

f. The helicase, or nsp13, protein unwinds the double-stranded viral RNA, a crucial step in replication and transcription. Inhibition may prevent viral genome replication and the creation of new virus components.

Pejler et al., 19 Jun 2024, multiple countries, peer-reviewed, 4 authors. Contact: aida.paivandy@uu.se.

In Vitro studies are an important part of preclinical research, however results may be very different in vivo.

This Paper Niclosamide All

Blockade of endolysosomal acidification suppresses TLR3-mediated pro-inflammatory signaling in airway epithelial cells

PhD # Gunnar Pejler, MSc # Xinran O Zhao, BSc Ella Fagerström, PhD Aida Paivandy

Journal of Allergy and Clinical Immunology, doi:10.1016/j.jaci.2024.05.031

Background: Endolysosomal compartments are acidic and contain low pH-dependent proteases, and these conditions are exploited by respiratory viruses, such as SARS-CoV-2 and influenza virus, for escaping into the cytosol. Moreover, endolysosomes contain various pattern recognition receptors (PRRs), which respond to virus-derived pathogen-associated molecular patterns (PAMPs) by production of pro-inflammatory cytokines/chemokines. However, excessive pro-inflammatory responses can lead to a potentially lethal cytokine storm. Objectives: Here we investigated the endosomal PRR expression profile in primary human small airway epithelial cells (HSAECs), and whether blockade of endolysosomal acidification affects their cytokine/chemokine production after challenge with virus-derived stimulants. Methods: HSAECs were exposed to stimulants mimicking virus-derived PAMPs, either in the absence or presence of compounds causing blockade of endolysosomal acidification, followed by measurement of cytokine expression and release. Results: We show that toll-like receptor 3 (TLR3) is the major endosomal PRR expressed by HSAECs, and that TLR3 expression is strongly induced by TLR3 agonists, but not by a range of other PRR agonists. We also demonstrate that TLR3 engagement with its agonists elicits a robust pro-inflammatory cytokine/chemokine response, which is profoundly suppressed through blockade of endolysosomal acidification, by bafilomycin A1, monensin, or niclosamide. Using TLR3 reporter cells, it was confirmed that TLR3 signaling is strongly induced by Poly(I:C) and that blockade of endolysosomal acidification efficiently blocked TLR3 signaling. Finally, we show that blockade of endolysosomal acidification causes a reduction in the levels of TLR3 mRNA and protein. Conclusion: These findings show that blockade of endolysosomal acidification suppresses TLR3-dependent cytokine and chemokine production in HSAECs. Clinical implication: These findings may be exploited for therapeutic strategies aiming to ameliorate the cytokine storm in response to respiratory virus infection. Capsule summary. This study shows that blockade of endolysosomal acidification in human airway epithelial cells causes reduced TLR3 signaling and reduced output of proinflammatory cytokines and chemokines.

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