Tissue Proteomic Analysis Identifies Mechanisms and Stages of Immunopathology in Fatal COVID-19.

Immunopathology occurs in the lung and spleen in fatal coronavirus disease (COVID-19), involving monocytes/macrophages and plasma cells. Antiinflammatory therapy reduces mortality, but additional therapeutic targets are required. We aimed to gain mechanistic insight into COVID-19 immunopathology by targeted proteomic analysis of pulmonary and splenic tissues. Lung parenchymal and splenic tissue was obtained from 13 postmortem examinations of patients with fatal COVID-19. Control tissue was obtained from cancer resection samples (lung) and deceased organ donors (spleen). Protein was extracted from tissue by phenol extraction. Olink multiplex immunoassay panels were used for protein detection and quantification. Proteins with increased abundance in the lung included MCP-3, antiviral TRIM21, and prothrombotic TYMP. OSM and EN-RAGE/S100A12 abundance was correlated and associated with inflammation severity. Unsupervised clustering identified "early viral" and "late inflammatory" clusters with distinct protein abundance profiles, and differences in illness duration before death and presence of viral RNA. In the spleen, lymphocyte chemotactic factors and CD8A were decreased in abundance, and proapoptotic factors were increased. B-cell receptor signaling pathway components and macrophage colony stimulating factor (CSF-1) were also increased. Additional evidence for a subset of host factors (including DDX58, OSM, TYMP, IL-18, MCP-3, and CSF-1) was provided by overlap between 1) differential abundance in spleen and lung tissue; 2) meta-analysis of existing datasets; and 3) plasma proteomic data. This proteomic analysis of lung parenchymal and splenic tissue from fatal COVID-19 provides mechanistic insight into tissue antiviral responses, inflammation and disease stages, macrophage involvement, pulmonary thrombosis, splenic B-cell activation, and lymphocyte depletion.

Tissue-Specific Immunopathology in Fatal COVID-19.

Rationale: In life-threatening coronavirus disease (COVID-19), corticosteroids reduce mortality, suggesting that immune responses have a causal role in death. Whether this deleterious inflammation is primarily a direct reaction to the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or an independent immunopathologic process is unknown.Objectives: To determine SARS-CoV-2 organotropism and organ-specific inflammatory responses and the relationships among viral presence, inflammation, and organ injury.Methods: Tissue was acquired from 11 detailed postmortem examinations. SARS-CoV-2 organotropism was mapped by using multiplex PCR and sequencing, with cellular resolution achieved by in situ viral S (spike) protein detection. Histologic evidence of inflammation was quantified from 37 anatomic sites, and the pulmonary immune response was characterized by using multiplex immunofluorescence.Measurements and Main Results: Multiple aberrant immune responses in fatal COVID-19 were found, principally involving the lung and reticuloendothelial system, and these were not clearly topologically associated with the virus. Inflammation and organ dysfunction did not map to the tissue and cellular distribution of SARS-CoV-2 RNA and protein between or within tissues. An arteritis was identified in the lung, which was further characterized as a monocyte/myeloid-rich vasculitis, and occurred together with an influx of macrophage/monocyte-lineage cells into the pulmonary parenchyma. In addition, stereotyped abnormal reticuloendothelial responses, including excessive reactive plasmacytosis and iron-laden macrophages, were present and dissociated from viral presence in lymphoid tissues.Conclusions: Tissue-specific immunopathology occurs in COVID-19, implicating a significant component of the immune-mediated, virus-independent immunopathologic process as a primary mechanism in severe disease. Our data highlight novel immunopathologic mechanisms and validate ongoing and future efforts to therapeutically target aberrant macrophage and plasma-cell responses as well as promote pathogen tolerance in COVID-19.

The Secretome Profiling of a Pediatric Airway Epithelium Infected with hRSV Identified Aberrant Apical/Basolateral Trafficking and Novel Immune Modulating (CXCL6, CXCL16, CSF3) and Antiviral (CEACAM1) Proteins.

The respiratory epithelium comprises polarized cells at the interface between the environment and airway tissues. Polarized apical and basolateral protein secretions are a feature of airway epithelium homeostasis. Human respiratory syncytial virus (hRSV) is a major human pathogen that primarily targets the respiratory epithelium. However, the consequences of hRSV infection on epithelium secretome polarity and content remain poorly understood. To investigate the hRSV-associated apical and basolateral secretomes, a proteomics approach was combined with an ex vivo pediatric human airway epithelial (HAE) model of hRSV infection (data are available via ProteomeXchange and can be accessed at https://www.ebi.ac.uk/pride/ with identifier PXD013661). Following infection, a skewing of apical/basolateral abundance ratios was identified for several individual proteins. Novel modulators of neutrophil and lymphocyte activation (CXCL6, CSF3, SECTM1 or CXCL16), and antiviral proteins (BST2 or CEACAM1) were detected in infected, but not in uninfected cultures. Importantly, CXCL6, CXCL16, CSF3 were also detected in nasopharyngeal aspirates (NPA) from hRSV-infected infants but not healthy controls. Furthermore, the antiviral activity of CEACAM1 against RSV was confirmed in vitro using BEAS-2B cells. hRSV infection disrupted the polarity of the pediatric respiratory epithelial secretome and was associated with immune modulating proteins (CXCL6, CXCL16, CSF3) never linked with this virus before. In addition, the antiviral activity of CEACAM1 against hRSV had also never been previously characterized. This study, therefore, provides novel insights into RSV pathogenesis and endogenous antiviral responses in pediatric airway epithelium.

An Open-Format Enteroid Culture System for Interrogation of Interactions Between Toxoplasma gondii and the Intestinal Epithelium.

When transmitted through the oral route, Toxoplasma gondii first interacts with its host at the small intestinal epithelium. This interaction is crucial to controlling initial invasion and replication, as well as shaping the quality of the systemic immune response. It is therefore an attractive target for the design of novel vaccines and adjuvants. However, due to a lack of tractable infection models, we understand surprisingly little about the molecular pathways that govern this interaction. The in vitro culture of small intestinal epithelium as 3D enteroids shows great promise for modeling the epithelial response to infection. However, the enclosed luminal space makes the application of infectious agents to the apical epithelial surface challenging. Here, we have developed three novel enteroid-based techniques for modeling T. gondii infection. In particular, we have adapted enteroid culture protocols to generate collagen-supported epithelial sheets with an exposed apical surface. These cultures retain epithelial polarization, and the presence of fully differentiated epithelial cell populations. They are susceptible to infection with, and support replication of, T. gondii. Using quantitative label-free mass spectrometry, we show that T. gondii infection of the enteroid epithelium is associated with up-regulation of proteins associated with cholesterol metabolism, extracellular exosomes, intermicrovillar adhesion, and cell junctions. Inhibition of host cholesterol and isoprenoid biosynthesis with Atorvastatin resulted in a reduction in parasite load only at higher doses, indicating that de novo synthesis may support, but is not required for, parasite replication. These novel models therefore offer tractable tools for investigating how interactions between T. gondii and the host intestinal epithelium influence the course of infection.

Integrative analysis of Paneth cell proteomic and transcriptomic data from intestinal organoids reveals functional processes dependent on autophagy.

Paneth cells are key epithelial cells that provide an antimicrobial barrier and maintain integrity of the small-intestinal stem cell niche. Paneth cell abnormalities are unfortunately detrimental to gut health and are often associated with digestive pathologies such as Crohn's disease or infections. Similar alterations are observed in individuals with impaired autophagy, a process that recycles cellular components. The direct effect of autophagy impairment on Paneth cells has not been analysed. To investigate this, we generated a mouse model lacking Atg16l1 specifically in intestinal epithelial cells, making these cells impaired in autophagy. Using three-dimensional intestinal organoids enriched for Paneth cells, we compared the proteomic profiles of wild-type and autophagy-impaired organoids. We used an integrated computational approach combining protein-protein interaction networks, autophagy-targeted proteins and functional information to identify the mechanistic link between autophagy impairment and disrupted pathways. Of the 284 altered proteins, 198 (70%) were more abundant in autophagy-impaired organoids, suggesting reduced protein degradation. Interestingly, these differentially abundant proteins comprised 116 proteins (41%) that are predicted targets of the selective autophagy proteins p62, LC3 and ATG16L1. Our integrative analysis revealed autophagy-mediated mechanisms that degrade key proteins in Paneth cell functions, such as exocytosis, apoptosis and DNA damage repair. Transcriptomic profiling of additional organoids confirmed that 90% of the observed changes upon autophagy alteration have effects at the protein level, not on gene expression. We performed further validation experiments showing differential lysozyme secretion, confirming our computationally inferred downregulation of exocytosis. Our observations could explain how protein-level alterations affect Paneth cell homeostatic functions upon autophagy impairment.This article has an associated First Person interview with the joint first authors of the paper.

Proteomic Profiling of Enteroid Cultures Skewed toward Development of Specific Epithelial Lineages.

Recently, 3D small intestinal organoids (enteroids) have been developed from cultures of intestinal stem cells which differentiate in vitro to generate all the differentiated epithelial cell types associated with the intestine and mimic the structural properties of the intestine observed in vivo. Small-molecule drug treatment can skew organoid epithelial cell differentiation toward particular lineages, and these skewed enteroids may provide useful tools to study specific epithelial cell populations, such as goblet and Paneth cells. However, the extent to which differentiated epithelial cell populations in these skewed enteroids represent their in vivo counterparts is not fully understood. This study utilises label-free quantitative proteomics to determine whether skewing murine enteroid cultures toward the goblet or Paneth cell lineages results in changes in abundance of proteins associated with these cell lineages in vivo. Here, proteomics data confirms that skewed enteroids recapitulate important features of the in vivo gut environment, demonstrating that they can serve as useful models for the investigation of normal and disease processes in the intestine. Furthermore, comparison of mass spectrometry data with histology data contained within the Human Protein Atlas identifies putative novel markers for goblet and Paneth cells.

Kinetoplastid Phylogenomics Reveals the Evolutionary Innovations Associated with the Origins of Parasitism.

The evolution of parasitism is a recurrent event in the history of life and a core problem in evolutionary biology. Trypanosomatids are important parasites and include the human pathogens Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp., which in humans cause African trypanosomiasis, Chagas disease, and leishmaniasis, respectively. Genome comparison between trypanosomatids reveals that these parasites have evolved specialized cell-surface protein families, overlaid on a well-conserved cell template. Understanding how these features evolved and which ones are specifically associated with parasitism requires comparison with related non-parasites. We have produced genome sequences for Bodo saltans, the closest known non-parasitic relative of trypanosomatids, and a second bodonid, Trypanoplasma borreli. Here we show how genomic reduction and innovation contributed to the character of trypanosomatid genomes. We show that gene loss has "streamlined" trypanosomatid genomes, particularly with respect to macromolecular degradation and ion transport, but consistent with a widespread loss of functional redundancy, while adaptive radiations of gene families involved in membrane function provide the principal innovations in trypanosomatid evolution. Gene gain and loss continued during trypanosomatid diversification, resulting in the asymmetric assortment of ancestral characters such as peptidases between Trypanosoma and Leishmania, genomic differences that were subsequently amplified by lineage-specific innovations after divergence. Finally, we show how species-specific, cell-surface gene families (DGF-1 and PSA) with no apparent structural similarity are independent derivations of a common ancestral form, which we call "bodonin." This new evidence defines the parasitic innovations of trypanosomatid genomes, revealing how a free-living phagotroph became adapted to exploiting hostile host environments.

Integrated transcriptomic and proteomic analysis of the global response of Wolbachia to doxycycline-induced stress.

The bacterium Wolbachia (order Rickettsiales), representing perhaps the most abundant vertically transmitted microbe worldwide, infects arthropods and filarial nematodes. In arthropods, Wolbachia can induce reproductive alterations and interfere with the transmission of several arthropod-borne pathogens. In addition, Wolbachia is an obligate mutualist of the filarial parasites that cause lymphatic filariasis and onchocerciasis in the tropics. Targeting Wolbachia with tetracycline antibiotics leads to sterilisation and ultimately death of adult filariae. However, several weeks of treatment are required, restricting the implementation of this control strategy. To date, the response of Wolbachia to stress has not been investigated, and almost nothing is known about global regulation of gene expression in this organism. We exposed an arthropod Wolbachia strain to doxycycline in vitro, and analysed differential expression by directional RNA-seq and label-free, quantitative proteomics. We found that Wolbachia responded not only by modulating expression of the translation machinery, but also by upregulating nucleotide synthesis and energy metabolism, while downregulating outer membrane proteins. Moreover, Wolbachia increased the expression of a key component of the twin-arginine translocase (tatA) and a phosphate ABC transporter ATPase (PstB); the latter is associated with decreased susceptibility to antimicrobials in free-living bacteria. Finally, the downregulation of 6S RNA during translational inhibition suggests that this small RNA is involved in growth rate control. Despite its highly reduced genome, Wolbachia shows a surprising ability to regulate gene expression during exposure to a potent stressor. Our findings have general relevance for the chemotherapy of obligate intracellular bacteria and the mechanistic basis of persistence in the Rickettsiales.

Positional proteomics at the N-terminus as a means of proteome simplification.

One strategy to reduce complexity in proteome analysis is through rational reduction of the proteolytic peptides that constitute the analyte for mass spectrometric analysis. Methods for selective isolation of C- and N-terminal peptides have been developed. In this chapter, we outline the context and variety of methods for selective isolation of N-terminal peptides and detail one method based on negative selection through differential removal of internal peptides.