Orchestration of the immune response by dendritic cells

International audienc

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

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

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