Intestinal barrier interactions with specialized CD8 T Cells

Copyright: © 2017 Konjar, Ferreira, Blankenhaus and Veldhoen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.The trillions of microorganisms that reside in the gastrointestinal tract, essential for nutrient absorption, are kept under control by a single cell barrier and large amounts of immune cells. Intestinal epithelial cells (IECs) are critical in establishing an environment supporting microbial colonization and immunological tolerance. A large population of CD8+ T cells is in direct and constant contact with the IECs and the intraepithelial lymphocytes (IELs). Due to their location, at the interphase of the intestinal lumen and external environment and the host tissues, they seem ideally positioned to balance immune tolerance and protection to preserve the fragile intestinal barrier from invasion as well as immunopathology. IELs are a heterogeneous population, with a large innate-like contribution of unknown specificity, intercalated with antigen-specific tissue-resident memory T cells. In this review, we provide a comprehensive overview of IEL physiology and how they interact with the IECs and contribute to immune surveillance to preserve intestinal homeostasis and host-microbial relationships.Members of the Veldhoen laboratory are supported by European Union H2020 ERA project (N°667824—EXCELLtoINNOV), publication costs were provided by LISBOA-01-0145-FEDER-007391, projeto cofinanciado pelo FEDER através POR Lisboa 2020—Programa Operacional Regional de Lisboa, do PORTUGAL 2020, e pela Fundação para a Ciência e a Tecnologia.info:eu-repo/semantics/publishedVersio

Identification of continuous human B-cell epitopes in the VP35, VP40, nucleoprotein and glycoprotein of Ebola virus

Ebola virus (EBOV) is a highly virulent human pathogen. Recovery of infected patients is associated with efficient EBOV-specific immunoglobulin G (IgG) responses, whereas fatal outcome is associated with defective humoral immunity. As B-cell epitopes on EBOV are poorly defined, we sought to identify specific epitopes in four EBOV proteins (Glycoprotein (GP), Nucleoprotein (NP), and matrix Viral Protein (VP)40 and VP35). For the first time, we tested EBOV IgG+ sera from asymptomatic individuals and symptomatic Gabonese survivors, collected during the early humoral response (seven days after the end of symptoms) and the late memory phase (7-12 years post-infection). We also tested sera from EBOV-seropositive patients who had never had clinical signs of hemorrhagic fever or who lived in non-epidemic areas (asymptomatic subjects). We found that serum from asymptomatic individuals was more strongly reactive to VP40 peptides than to GP, NP or VP35. Interestingly, anti-EBOV IgG from asymptomatic patients targeted three immunodominant regions of VP40 reported to play a crucial role in virus assembly and budding. In contrast, serum from most survivors of the three outbreaks, collected a few days after the end of symptoms, reacted mainly with GP peptides. However, in asymptomatic subjects the longest immunodominant domains were identified in GP, and analysis of the GP crystal structure revealed that these domains covered a larger surface area of the chalice bowl formed by three GP(1) subunits. The B-cell epitopes we identified in the EBOV VP35, VP40, NP and GP proteins may represent important tools for understanding the humoral response to this virus and for developing new antibody-based therapeutics or detection methods

Identification of continuous human B-cell epitopes in the VP35, VP40, nucleoprotein and glycoprotein of Ebola virus

Ebola virus (EBOV) is a highly virulent human pathogen. Recovery of infected patients is associated with efficient EBOV-specific immunoglobulin G (IgG) responses, whereas fatal outcome is associated with defective humoral immunity. As B-cell epitopes on EBOV are poorly defined, we sought to identify specific epitopes in four EBOV proteins (Glycoprotein (GP), Nucleoprotein (NP), and matrix Viral Protein (VP)40 and VP35). For the first time, we tested EBOV IgG+ sera from asymptomatic individuals and symptomatic Gabonese survivors, collected during the early humoral response (seven days after the end of symptoms) and the late memory phase (7-12 years post-infection). We also tested sera from EBOV-seropositive patients who had never had clinical signs of hemorrhagic fever or who lived in non-epidemic areas (asymptomatic subjects). We found that serum from asymptomatic individuals was more strongly reactive to VP40 peptides than to GP, NP or VP35. Interestingly, anti-EBOV IgG from asymptomatic patients targeted three immunodominant regions of VP40 reported to play a crucial role in virus assembly and budding. In contrast, serum from most survivors of the three outbreaks, collected a few days after the end of symptoms, reacted mainly with GP peptides. However, in asymptomatic subjects the longest immunodominant domains were identified in GP, and analysis of the GP crystal structure revealed that these domains covered a larger surface area of the chalice bowl formed by three GP(1) subunits. The B-cell epitopes we identified in the EBOV VP35, VP40, NP and GP proteins may represent important tools for understanding the humoral response to this virus and for developing new antibody-based therapeutics or detection methods

Type I and type III interferons drive redundant amplification loops to induce a transcriptional signature in influenza-infected airway epithelia

The response of cells to virus infection depends on Interferons (IFNs), a group of cytokines which activate the expression of hundreds of genes that help control viral replication inside infected cells. While type I IFN was discovered in 1957, type III IFN (IFNλ, IL-28/29) was characterized recently and is known for its role in the response to hepatitis C virus. Airway epithelia are the primary target of influenza virus, and we studied how infection induces IFNs and which IFN is most important for the epithelial anti-influenza response. We found that infected epithelia detect virus through the cytoplasmic RIG-I/MAVS recognition system, leading to activation of the transcription factor IRF7 and subsequent induction of both type I and III IFNs. All ensuing cellular responses to infection are dependent on the production and secretion of IFNs, as responses are lost in epithelia lacking receptors for both type I and III IFNs. Finally, gene induction is indistinguishable in single receptor-deficient and wild-type cells, indicating that the two IFN systems are completely redundant in epithelia. Thus, influenza infection of airway epithelia induces, via a RIG-I/MAVS/IRF7 dependent pathway, both type I and III IFNs which drive two overlapping and redundant amplification loops to upregulate antiviral genes

In primary MTEC, influenza A infection induces an IFN signature.

<p>Total RNA from mock and PR8 infected cells was analysed using Affymetrix Mouse Genome 430 2.0 microarrays at 24(≥4-fold change relative to mock infected; t-test unpaired, p<0.01, Benjamini-Hochberg multiple test correction). (A) K-means clustering of the differentially expressed genes. (B) Heat map of the 234 upregulated-gene, as shown in (A). The range of fold changes is expressed in a log2 scale. (C) Ingenuity Pathway Analysis of the upregulated genes in influenza infected MTEC.</p

Lack of type I and III IFN signalling in the stromal compartment increases susceptibility to IAV infection.

<p>Chimeric mice were infected i.n. with 10⁵ TCID₅₀ PR8, (A) Weight loss and mortality were measured. Graphs show mean ± SEM and are representative of 2 independent experiments (n = 6). (B) Viral replication was assessed by qRT-PCR on total lung RNA at 4 days post infection. *** p<0.001, ** p<0.01 by 2-way ANOVA with Bonferroni post tests (weight loss), Log-rank (Mantel-Cox) Test (survival) or unpaired t test (RT-PCR quantification).</p

Residual IFN production is responsible for ISG induction in MAVS−/− epithelia.

<p>(A) qRT-PCR analysis of Rsad2, Oasl2 and STAT1 in MAVS−/− epithelia infected with PR8 in the presence or absence of blocking anti-IFNAR and neutralizing anti-IL28A/B antibodies. (B) Wild-type epithelia were PR8-infected in the presence or absence of brefeldin A (2.5 µg/ml). Expression of the indicated genes was determined by qRT-PCR at 24 hpi. All transcripts were normalized to HPRT levels and then expressed as fold induction relative to the mean of mock infected controls, +/− SEM. Asterisks indicate differences that are statistically significant (unpaired t test; *, P<0.05; **, P<0.01).</p

In primary MTEC, type I and III IFNs are induced upon influenza virus A infection, in a MAVS and replication dependent way.

<p>(A) Mouse tracheal epithelial cells were costained for ZO-1 (green) and β tubulin IV (red), or Mucin 5A (red) and Clara cell secretory protein (CCSP) (green). Images were acquired at ×20 magnification. (B) Total RNA from mock infected and PR8 infected (moi = 0.3) MTEC was analysed using Affymetrix Mouse Genome 430 2.0 microarrays. The signal intensity of each probe was first normalized on the median intensity of that probe across the control group and then represented as log2 fold change relative to the controls. Asterisks indicated statistically significant differences (unpaired t test; **, p<0.01). (C) qRT-PCR analysis of IL-28A/B and IFNβ1 transcripts of MTEC derived from wild-type, TLR7^−/−, MyD88^−/−, TRIF^−/− and MAVS^−/− mice, mock infected or infected with either PR8 or heat inactivated PR8. Fold induction is relative to mock treated samples at 24 hpi +/− SEM. (D) IL-28A/B level in the supernatants of the indicated cultures were measured by ELISA 24 hpi.</p

Basal expression of signalling intermediates is similar in wild-type and IFNAR1^−/−IL-28Rα^−/− double knock-out MTEC.

<p>(A) Relative expression of IRF3, IRF1, IRF7 and IRF9 was determined in both mock infected and PR8 infected MTEC by qRT-PCR. The transcripts quantities are expressed as ratio to HPRT levels +/− SEM. (B) The same RNA samples were analyzed for STAT1, STAT2, STAT3, STAT4 and STAT6 expression.</p

IRF3 is not required for IFN induction in primary MTEC.

<p>(A) MTEC derived from wild-type or IRF3^−/− mice were mock treated or infected with PR8. RNA was analyzed at the indicated time by qRT-PCR, normalized to the amount of HPRT transcripts and measured as fold induction relative to the level in the corresponding control samples. (B) Expression of IL-28A/B and IFNβ was measured by qRT-PCR at 24 hpi, in epithelial cultures of the indicated genotypes. (C) Protein levels of IL-28A/B in the supernatants of mock and PR8 infected cells were measured by ELISA 24 hpi or, where indicated, at 48 hpi. Error bars indicate the SEM of replicates.</p