Dataset Information

Tissue-Specific Protease Dynamics in the Pig Respiratory Tract During Influenza A Virus Infection

ABSTRACT: Influenza A virus (IAV) is a zoonotic pathogen capable of infecting diverse avian and mammalian hosts, causing seasonal epidemics and occasional global pandemics in humans. Viral entry requires proteolytic activation of hemagglutinin (HA). While serine proteases such as TMPRSS2, and HAT are known HA activators, the respiratory tract harbours additional proteases whose contributions to infection remain unclear. Dysregulation of these proteases can enhance viral replication, tissue damage, and inflammation, highlighting the need for a systems-level view of the proteolytic landscape. Here, we use high-throughput, proteome-wide proteomics and N-terminomics to identify 112 host proteases across the nasal mucosa, trachea, and lung. We monitor and validate 28 proteases with targeted proteomics and microfluidic qPCR, representing a comprehensive degradome analysis in the respiratory tract of the highly translational pig model of influenza infection. We show that protease abundance and activity were highly tissue-specific: while the nasal mucosa showed selective activation of broad- and narrow-specificity proteases alongside robust antiviral responses, the trachea exhibited modest modulation with subtle shifts in protease–inhibitor balance, and the lung maintained predominantly active proteases despite lower viral loads but severe tissue damage, indicative of immune-mediated pathology. Sequence motif analysis revealed distinct cleavage preferences across tissues, indicating differential protease processing across the studied respiratory tissues in antiviral pathways, antigen processing, and tissue remodelling. Several identified proteases, including ST14, KLKB1, PRSS8, and LGMN, were increased and functionally active upon infection, suggesting roles in viral processing and host immune regulation. Collectively, our results define a spatially organised proteolytic network that shapes tissue-specific antiviral host responses and contributes to H1N1 influenza pathogenesis.

INSTRUMENT(S):

ORGANISM(S): Sus Scrofa Domesticus (domestic Pig)

TISSUE(S): Lung, Nasal Mucosa, Trachea

DISEASE(S): Swine Influenza

SUBMITTER: Konstantinos Kalogeropoulos

LAB HEAD: Konstantinos Kalogeropoulos

PROVIDER: PXD074287 | Pride | 2026-03-02

REPOSITORIES: Pride

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			Action	DRS
	20240110_MK_EVO_AMH_FAIMS_1CV_Aurora15_58min_1478_DIA_500ng_NPO127.raw	Raw
	20240110_MK_EVO_AMH_FAIMS_1CV_Aurora15_58min_1478_DIA_500ng_NPO128.raw	Raw
	20240110_MK_EVO_AMH_FAIMS_1CV_Aurora15_58min_1478_DIA_500ng_NPO129.raw	Raw
	20240110_MK_EVO_AMH_FAIMS_1CV_Aurora15_58min_1478_DIA_500ng_NPO130.raw	Raw
	20240110_MK_EVO_AMH_FAIMS_1CV_Aurora15_58min_1478_DIA_500ng_NPO131.raw	Raw

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Project description:Influenza A virus (IAV) infection is a global respiratory disease, which annually leads to 3-5 million cases of severe illness, resulting in 290,000-650,000 deaths. Additionally, during the past century, four global IAV pandemics have claimed millions of human lives. The epithelial lining of the trachea plays a vital role during IAV infection, both as point of viral entry and replication as well as in the antiviral immune response. Tracheal tissue is generally inaccessible from human patients, which makes animal models crucial for the study of the tracheal host immune response. In this study, pigs were inoculated with swine- or human-adapted H1N1 IAV to gain insight into how host adaptation of IAV shapes the innate immune response during infection. In-depth multi-omics analysis (global proteomics and RNA sequencing) of the host response in upper and lower tracheal tissue was conducted, and results were validated by microfluidic qPCR. Additionally, a subset of samples was selected for histopathological examination. A classical innate antiviral immune response was induced in both upper and lower trachea after infection with either swine- or human-adapted IAV with upregulation of genes and higher abundance of proteins associated with viral infection and recognition, accompanied by a significant induction of interferon stimulated genes with corresponding higher proteins concentrations. Infection with the swine-adapted virus induced a much stronger immune response compared to infection with a human-adapted IAV strain in the lower trachea, which could be a consequence of a higher viral load and a higher degree of inflammation. Central components of the JAK-STAT pathway, apoptosis, pyrimidine metabolism, and the cytoskeleton were significantly altered depending on infection with swine- or human-adapted virus and might be relevant mechanisms in relation to antiviral immunity against putative zoonotic IAV. Based on our findings, we hypothesize that during host adaptation, IAV evolve to modulate important host cell elements to favor viral infectivity and replication.

Project description:Influenza A virus (IAV) is a zoonotic pathogen capable of infecting diverse avian and mammalian hosts, causing seasonal epidemics and occasional global pandemics in humans. Viral entry requires proteolytic activation of hemagglutinin (HA). While serine proteases such as TMPRSS2, and HAT are known HA activators, the respiratory tract harbours additional proteases whose contributions to infection remain unclear. Dysregulation of these proteases can enhance viral replication, tissue damage, and inflammation, highlighting the need for a systems-level view of the proteolytic landscape. Here, we use high-throughput, proteome-wide proteomics and N-terminomics to identify 112 host proteases across the nasal mucosa, trachea, and lung. We monitor and validate 28 proteases with targeted proteomics and microfluidic qPCR, representing a comprehensive degradome analysis in the respiratory tract of the highly translational pig model of influenza infection. We show that protease abundance and activity were highly tissue-specific: while the nasal mucosa showed selective activation of broad- and narrow-specificity proteases alongside robust antiviral responses, the trachea exhibited modest modulation with subtle shifts in protease–inhibitor balance, and the lung maintained predominantly active proteases despite lower viral loads but severe tissue damage, indicative of immune-mediated pathology. Sequence motif analysis revealed distinct cleavage preferences across tissues, indicating differential protease processing across the studied respiratory tissues in antiviral pathways, antigen processing, and tissue remodelling. Several identified proteases, including ST14, KLKB1, PRSS8, and LGMN, were increased and functionally active upon infection, suggesting roles in viral processing and host immune regulation. Collectively, our results define a spatially organised proteolytic network that shapes tissue-specific antiviral host responses and contributes to H1N1 influenza pathogenesis.

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Tissue-Specific Protease Dynamics in the Pig Respiratory Tract During Influenza A Virus Infection

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