Profiling of lung SARS-CoV-2 and influenza virus infection dissects virus-specific host responses and gene signatures

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which emerged in late 2019 has spread globally, causing a pandemic of respiratory illness designated coronavirus disease 2019 (COVID-19). A better definition of the pulmonary host response to SARS-CoV-2 infection is required to understand viral pathogenesis and to validate putative COVID-19 biomarkers that have been proposed in clinical studies. METHODS: Here, we use targeted transcriptomics of formalin-fixed paraffin-embedded tissue using the NanoString GeoMX platform to generate an in-depth picture of the pulmonary transcriptional landscape of COVID-19, pandemic H1N1 influenza and uninfected control patients. RESULTS: Host transcriptomics showed a significant upregulation of genes associated with inflammation, type I interferon production, coagulation and angiogenesis in the lungs of COVID-19 patients compared to non-infected controls. SARS-CoV-2 was non-uniformly distributed in lungs (emphasising the advantages of spatial transcriptomics) with the areas of high viral load associated with an increased type I interferon response. Once the dominant cell type present in the sample, within patient correlations and patient-patient variation, had been controlled for, only a very limited number of genes were differentially expressed between the lungs of fatal influenza and COVID-19 patients. Strikingly, the interferon-associated gene IFI27, previously identified as a useful blood biomarker to differentiate bacterial and viral lung infections, was significantly upregulated in the lungs of COVID-19 patients compared to patients with influenza. CONCLUSION: Collectively, these data demonstrate that spatial transcriptomics is a powerful tool to identify novel gene signatures within tissues, offering new insights into the pathogenesis of SARS-COV-2 to aid in patient triage and treatment.Arutha Kulasinghe, Chin Wee Tan, Anna Flavia Ribeiro dos Santos Miggiolaro, James Monkman, Habib SadeghiRad, Dharmesh D. Bhuva, Jarbas da Silva Motta Junior, Caroline Busatta Vaz de Paula, Seigo Nagashima, Cristina Pellegrino Baena, Paulo Souza-Fonseca-Guimaraes, Lucia de Noronha, Timothy McCulloch, Gustavo Rodrigues Rossi, Caroline Cooper, Benjamin Tang, Kirsty R. Short, Melissa J. Davis, Fernando Souza-Fonseca-Guimaraes, Gabrielle T. Belz, and Ken O, Byrn

The epithelium of canine palatine tonsils

The palatine tonsil is positioned to play a key role in protecting the body against ingested microorganisms. These microorganisms must traverse the tonsillar epithelium to initiate immune reactions, but no information is available from dogs on the structure of this epithelium. In this study, the morphology and ultrastracture of the epithelium of the palatine tonsil of dogs was examined using light and electron microscopy. The epithelium is of two types: reticular and non-reticular. Reticular epithelium, which is invaded by lymphoid cells, is located over the apices of nodules, separated indistinctly by islands of non-reticular squamous epithelium. The reticular epithelium contains M cells which are more abundant towards the periphery of lymphoid nodules. The apical membrane of the M cells forms folds from which microvilli extend into the lumen of the oropharynx. Abluminally, cytoplasmic processes enfold clusters of lymphocytes. Numerous desmosomes secure the marginal plasma membrane and lateral membranous interdigitations to adjacent epithelial cells, thereby maintaining the integrity of the tonsillar epithelium. Epithelial discontinuities, which are few, occupy similar positions to M cells and appear to contain migrating lymphocytes. A fenestrated basement membrane allows cell transport between the intraepithelial passageways of the reticular epithelium and the subepithelial lymphoid tissues

An investigation of the use of chromium, platinum and gold coating for scanning electron-microscopy of casts of lymphoid-tissues

Resin casts replicate the internal structure of organs and provide a three-dimensional representation of the arrangement of vessels and intercellular spaces. Casting media are insulators and must be coated with a conductor to prevent sample charging and to allow the adequate production of secondary electrons from the specimen to generate sufficient signal to form a clear image

Contemporary analysis of MHC-related immunodominance hierarchies in the CD8(+) T cell response to influenza A viruses

Early studies of influenza virus-specific CD8 T cell-mediated immunity indicated that the level of CTL activity associated with H2D is greatly diminished in mice that also express H2K(k). Such MHC-related immunodominance hierarchies are of some interest, as they could lead to variable outcomes for peptide-based vaccination protocols in human populations. The influence of H2K(k) on the H2D-restricted response profile has thus been looked at again using a contemporary, quantitative, IFN-γ-based flow cytometric assay. The depressive effect of H2K(k) was very apparent for the influenza DPA epitope and was also reproduced when CTL activity was measured for H2D-expressing targets pulsed with the immunodominant NP peptide. The secondary CD8IFN-γ DNP-specific response was much greater in parental H2 than in H2(kx) F mice, but the sizes of the CD8 sets specific for K(k)NP and DNP were essentially equivalent in the F animals. Thus, although the immunodominance profile associated with DNP is lost when H2K(k) is also present, the response is still substantial. A further, MHC-related effect was also identified for the K(k)NS1 epitope, which was consistently associated with a greater CD8IFN-γ response in H2K(k)D recombinant than in (H2K(k)D(k) x H2KD)F mice. The diminished DPA response in H2(kxb)F mice was characterized by loss of a prominent Vβ7 TCR responder phenotype, supporting the idea that TCR deletion during ontogeny shapes the available repertoire. The overall conclusion is that these MHC-related immunodominance hierarchies are more subtle than the early CTL assays suggested and, although inherently unpredictable, are unlikely to cause a problem for peptide-based vaccine strategies

Changing patterns of dominance in the CD8(+) T cell response during acute and persistent murine gamma-herpesvirus infection

The murine γ-herpesvirus MHV-68 causes an acute, transient pneumonitis, followed by an infectious mononucleosis (IM)-like illness with splenomegaly, widespread latent infection of B lymphocytes and an expansion of Vβ4 CD8 T cells. CD8 T cells specific for an H-2D-restricted epitope were prominent during the acute respiratory infection, but their prevalence declined rapidly during the mononucleosis. In contrast, CD8 T cells specific for an H-2K-restricted epitope, apparently expressed by virus-infected B lymphocytes, were most numerous during the mononucleosis illness and were maintained at relatively high frequencies thereafter. The prevalence of all peptide-specific CD8 T cells decreased during the expansion of the Vβ4 CD8 population, which did not recognize any peptide epitopes identified and was apparent also in an MHC class I-deficient environment. The CD8 T cell population recognizing productively infected epithelial cells thus differed substantially from that responding during the IM illness

A Murid gamma-herpesviruses exploits normal splenic immune communication routes for systemic spread

Gamma-herpesviruses (γHVs) are widespread oncogenic pathogens that chronically infect circulating lymphocytes. How they subvert the immune check-point function of the spleen to promote persistent infection is not clear. We show that Murid Herpesvirus-4 (MuHV-4) enters the spleen by infecting marginal zone (MZ) macrophages, which provided a conduit to MZ B cells. Relocation of MZ B cells to the white pulp allowed virus transfer to follicular dendritic cells. From here the virus reached germinal center B cells to establish persistent infection. Mice lacking MZ B cells, or treated with a sphingosine-1-phosphate receptor agonist to dislocate them, were protected against MuHV-4 colonization. MuHV-4 lacking ORF27, which encodes a glycoprotein necessary for efficient intercellular spread, could infect MZ macrophages but was impaired in long-term infection. Thus, MuHV-4, a γHV, exploits normal immune communication routes to spread by serial lymphoid/myeloid exchange

DEA-like Models for Efficiency Evaluation of Hierarchically Structured Units

The knowledge of the internal structure of decision making units (DMUs) gives further insights with respect to the "black box" perspective when considering data envelopment analysis models. We present one-level and two-level hierarchical structures of the DMUs under evaluation. Each unit is composed of consecutive stages of parallel subunits all with constant returns to scale. In particular, the maximization of the relative efficiency of a DMU is studied. For the two-stage situation, different degrees of coordination among the subunits of the hierarchical levels are discussed. When some form of coordination has to be guaranteed, we introduce balancing constraints and we compare two different models. In both cases, we prove that the maximum relative efficiency of a DMU is assessed by comparing it with all the existing subunits