Audray Fortin

3D-printed alternatives to standard flocked swabs were rapidly developed to provide a response to the unprecedented and sudden need for an exponentially growing amount of diagnostic tools to fight the COVID-19 pandemic. In light of the anticipated shortage, a hospital-based 3D-printing platform was implemented in our institution for the production of swabs for nasopharyngeal and oropharyngeal sampling based on the freely available, open-source design provided to the community by University of South Florida’s Health Radiology and Northwell Health System teams as a replacement for locally used commercial swabs. Validation of our 3D-printed swabs was performed with a head-to-head diagnostic accuracy study of the 3D-printed “Northwell model” with the cobas PCR Media^® swab sample kit. We observed an excellent concordance (total agreement 96.8%, Kappa 0.936) in results obtained with the 3D-printed and flocked swabs, indicating that the in-house 3D-printed swab could be used reliably in the context of a shortage of flocked swabs. To our knowledge, this is the first study to report on autonomous hospital-based production and clinical validation of 3D-printed swabs.

Protein function is regulated by post-translational modifications, among which reversible oxidation of Cys (Cys ox-PTM) emerged as a key regulatory mechanism of cellular responses. The redox regulation of virus-host interactions is well documented, but in most cases, proteins subjected to Cys ox-PTM remain unknown. The identification of Cys ox-PTM sites in vivo is essential to underpin our understanding of the mechanisms of the redox regulation. In this study, we present a proteome-wide identification of reversible Cys ox-PTM sites in vivo during stimulation by oxidants using a maleimide-based bioswitch method coupled to mass spectrometry. We identified 2720 unique Cys ox-PTM sites encompassing 1473 proteins with distinct abundance, location and functions. Label-free quantification (LFQ)-based analysis revealed the enrichment of ox-PTM in numerous pathways, many relevant to virus-host interaction. Here, we focused on the oxidation of STING, the central adaptor of the innate immune type I interferon pathway induced upon detection of cytosolic DNA. We provide the first in vivo demonstration of reversible oxidation of Cys ¹⁴⁸ and Cys ²⁰⁶ of STING. Molecular analyses led us to establish a new model in which Cys ¹⁴⁸ oxidation is constitutive, while Cys ²⁰⁶ oxidation is inducible by oxidative stress or by the natural ligand 2’3’-cGAMP. We show that oxidation of Cys ²⁰⁶ has an inhibitory function to prevent STING hyperactivation through the constraint of a conformational change associated with the formation of inactive polymers containing intermolecular disulfide bonds. This provides new ground for the design of therapies targeting STING relevant to autoinflammatory disorders, immunotherapies and vaccines. Brief summary of the main results: The function of proteins is regulated by post-translational modifications, among which reversible oxidation of Cys recently emerged as a key component. Comprehension of redox regulation of cellular responses requires identification of specific oxidation sites in vivo. Using a bioswitch method to specifically label Cys subjected to reversible oxidation coupled to mass spectrometry, we identified thousands of novel oxidation sites. Many are relevant to virus-host interaction pathways. Here, we focused on the oxidation of STING, an adaptor critical for activating the innate immune type I interferon pathway engaged upon cytosolic DNA sensing. Molecular studies led us to establish a new model in which STING Cys ¹⁴⁸ is oxidized at basal levels, while Cys ²⁰⁶ oxidation is induced by oxidative stress and ligand binding. We show that oxidation of Cys ²⁰⁶ has an inhibitory function to prevent STING hyperactivation. This study provides ground for novel research avenues aimed at designing therapeutics that target this pathway.

Interferon (IFN) β and Tumor Necrosis Factor (TNF) are key players in immunity against viruses. Compelling evidence has shown that the antiviral and inflammatory transcriptional response induced by IFNβ is reprogrammed by crosstalk with TNF. IFNβ mainly induces interferon-stimulated genes by the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway involving the canonical ISGF3 transcriptional complex, composed of STAT1, STAT2, and IRF9. The signaling pathways engaged downstream of the combination of IFNβ and TNF remain elusive, but previous observations suggested the existence of a response independent of STAT1. Here, using genome-wide transcriptional analysis by RNASeq, we observed a broad antiviral and immunoregulatory response initiated in the absence of STAT1 upon IFNβ and TNF costimulation. Additional stratification of this transcriptional response revealed that STAT2 and IRF9 mediate the expression of a wide spectrum of genes. While a subset of genes was regulated by the concerted action of STAT2 and IRF9, other gene sets were independently regulated by STAT2 or IRF9. Collectively, our data supports a model in which STAT2 and IRF9 act through non-canonical parallel pathways to regulate distinct pool of antiviral and immunoregulatory genes in conditions with elevated levels of both IFNβ and TNF.

IFNβ typically induces an antiviral and immunoregulatory transcriptional program through the activation of ISGF3 (STAT1, STAT2 and IRF9) transcriptional complexes. The response to IFNβ is context-dependent and is prone to crosstalk with other cytokines, such as TNFα IFNβ and TNFα synergize to drive a specific delayed transcriptional program. Previous observation led to the hypothesis that an alternative STAT1-independent pathway involving STAT2 and IRF9 might be involved in gene induction by the combination of IFNβ and TNFα. Using genome wide transcriptional profiling by RNASeq, we found that the costimulation with IFNβ and TNFα induces a broad antiviral and immunoregulatory transcriptional program independently of STAT1. Additionally, STAT2 and IRF9 are involved in the regulation of only a subset of these STAT1-independent genes. Consistent with the growing literature, STAT2 and IRF9 act in concert to regulate a subgroup of these genes. Unexpectedly, STAT2 and IRF9 were also engaged in specific independent pathways to regulate distinct sets of IFNβ and TNFα-induced genes. Altogether these observations highlight the existence of distinct previously unrecognized non-canonical STAT1-independent, but STAT2 and/or IRF9-dependent pathways in the establishment of a delayed antiviral and immunoregulatory transcriptional program in conditions where elevated levels of both IFNβ and TNFα are present.