Induction and Role of Indoleamine 2,3 Dioxygenase in Mouse Models of Influenza A Virus Infection

<div><p>Influenza infection stimulates protective host immune responses but paradoxically enhances lung indoleamine 2,3 dioxygenase (IDO) activity, an enzyme that suppresses helper/effector T cells and activates Foxp3-lineage regulatory CD4 T cells (Tregs). Influenza A/PR/8/34 (PR8) infection stimulated rapid elevation of IDO activity in lungs and lung-draining mediastinal lymph nodes (msLNs). Mice lacking intact IDO1 genes (IDO1-KO mice) exhibited significantly lower morbidity after sub-lethal PR8 infection, and genetic or pharmacologic IDO ablation led to much faster recovery after virus clearance. More robust influenza-specific effector CD8 T cell responses manifested in lungs of PR8-infected IDO1-KO mice, though virus clearance rates were unaffected by IDO ablation. Similar outcomes manifested in mice infected with a less virulent influenza A strain (X31). IDO induction in X31-infected lungs was dependent on IFN type II (IFNγ) signaling and was restricted to non-hematopoietic cells, while redundant IFN type 1 or type II signaling induced IDO exclusively in hematopoietic cells from msLNs. Memory T cells generated in X31-primed IDO1-KO mice protected mice from subsequent challenge with lethal doses of PR8 (100×LD₅₀). However recall T cell responses were less robust in lung interstitial tissues, and classic dominance of TCR Vβ8.3 chain usage amongst memory CD8⁺ T cells specific for influenza nucleoprotein (NP₃₆₆) did not manifest in IDO1-KO mice. Thus, influenza induced IDO activity in lungs enhanced morbidity, slowed recovery, restrained effector T cell responses in lungs and shaped memory T cell repertoire generation, but did not attenuate virus clearance during primary influenza A infection.</p></div

Influenza A infection stimulates IDO in lungs and draining msLNs.

<p>Mice were infected with 750 PFU of X31. IDO expression and activity in lungs and msLNs were measured at the times indicated after infection. <b>ABE.</b> Tissue kynurenine (A) or IDO enzyme activity (B, E) were assessed in lungs and msLNs of X31-infected WT (B6), IDO1-KO (B6) and B6→IDO1-KO chimeric mice. <b>CD.</b> IDO protein was assessed in lungs of X31-infected WT mice by Western blot (C) and immunohistochemical analyses (D). Influenza infected lung cells were identified by detecting NP in adjacent sections (D). Data are the means (+/−1sd) of >3 mice/group from at least two independent experiments. Original magnifications, 200×. **p<0.01, ***p<0.0001.</p

IDO shapes influenza-specific memory CD8 T cell generation.

<p>X31-primed WT and IDO1-KO (B6) mice were challenged with PR8 and influenza-specific T cell responses in BAL and lung parenchyma were assessed at 5 dpi. <b>A–C.</b> Numbers of total cells (A), NP₃₆₆-specific T cells (B), PB1-specific T cells (C). <b>D.</b> Repertoires of NP₃₆₆-specific T cells in BAL and lung parenchyma were analyzed by staining cells for TCR Vβ8.3 at 5 dpi. <b>E.</b> Repertoires of long-term NP₃₆₆-specific memory T cells in spleen were analyzed using a panel of antibodies against various TCR Vβ subtypes. Data were compiled from 2 experiments and statistical analyses were performed using Student's unpaired <i>t</i> test. *p<0.05, ****p<0.0001.</p

Dose response of PIV5-H7.

<p>BALB/c mice in groups of 15 were infected IN with PIV5-H7 at a dose of 10⁴, 10⁵ and 10⁶ PFU, or PBS or intramuscularly injected with 256 HAU of iH7N9. Mice were rested for 3 weeks then challenged with 10 LD₅₀ of A/Anhui/1/2013 (H7N9) and monitored for (A) survival and (B) weight loss. (<i>P</i><0.05, ANOVA, *compared to PBS on days 2–8; †compared to PBS on days 2, 6, and 8; ^‡compared to PBS on day 8 (10⁴ only) (C) Titers of H7N9 in the lungs of mice Day 3 post H7N9 challenge. (<i>P</i><0.05, Kruskal-Wallis)</p

Identification of Virulence Determinants in Influenza Viruses

To date there is no rapid method to screen for highly pathogenic avian influenza strains that may be indicators of future pandemics. We report here the first development of an oligonucleotide-based spectroscopic assay to rapidly and sensitively detect a N66S mutation in the gene coding for the PB1-F2 protein associated with increased virulence in highly pathogenic pandemic influenza viruses. 5′-Thiolated ssDNA oligonucleotides were employed as probes to capture RNA isolated from six influenza viruses, three having N66S mutations, two without the N66S mutation, and one deletion mutant not encoding the PB1-F2 protein. Hybridization was detected without amplification or labeling using the intrinsic surfaced-enhanced Raman spectrum of the DNA-RNA complex. Multivariate analysis identified target RNA binding from noncomplementary sequences with 100% sensitivity, 100% selectivity, and 100% correct classification in the test data set. These results establish that optical-based diagnostic methods are able to directly identify diagnostic indicators of virulence linked to highly pathogenic pandemic influenza viruses without amplification or labeling

IDO attenuates influenza-specific CD8 T cell responses in X31-infected mice.

<p><b>A–C.</b> B6 and IDO1-KO (B6) mice were infected with X31 virus and total numbers of cells and virus-specific CD8 T cell numbers were evaluated in msLNs at 10 dpi using tetramers specific for NP₃₆₆ (B) and PA (C). Data were compiled from 5 experiments, and statistical analyses were performed using Student's unpaired <i>t</i> test; **p<0.01, ***p<0.001.</p

IDO ablation enhances influenza-specific CD8 T cell responses to PR8 infection.

<p>B6 (WT) or IDO1-KO mice with B6 backgrounds were infected with PR8 virus (50 PFU, i/n) at sub-lethal doses, and 10 days after infection bronchial alveolar lavage (BAL) cells and T cells were counted. <b>A.</b> Representative FACS gating and analysis strategy to detect NP₃₆₆-specific CD8 T cells using tetramers (upper panel) and intracellular cytokine (IFNγ, IL-10) expression after peptide specific stimulation for 2 hrs <i>ex vivo</i> (lower panel). <b>B–F.</b> Numbers of total BAL (B) and NP₃₆₆ (C), PA (D), PB1 (E) CD8 and NP₃₁₁ (F) CD4 specific T cells. Data were analyzed using Student's unpaired <i>t</i> test; *p<0.05, **p<0.01, ***p<0.001, ns, not significant.</p

IFN gene expression following dsRNA treatment of NHBE and NSBE cells.

<p>A) Undifferentiated NHBE and NSBE cells were left untreated or treated exogenously with 50 µg/ml poly I:C. RNA was harvested 24 hours later and analyzed by qRT-PCR for IFN-α -β, or -λ expression. Results are expressed as fold over mock infected cells and normalized to a housekeeping gene. Significant differences in gene expression between NHBE and NSBE cells are indicated by **** where p<0.0001 and were determined as described in the Material and Methods. B) Cells were treated as described in (A) and 24 hours following poly I:C treatment cell lysates were harvested and subjected to Western blotting using an anti-RIG-I or anti-GAPDH antibodies. C) Quantification of band intensity shown in (B) where intensity of the RIG-I band is normalized to that of GAPDH.</p

IFN and ISG expression in undifferentiated NHBE and NSBE cells.

<p>NHBE (A) or NSBE (B) cells remaining in an undifferentiated state were infected at a MOI of 0.01 with the indicated viruses. At 8 and 24 HPI RNA was harvested and levels of type I and III IFN genes and ISGs were quantified by qRT-PCR. Results are expressed as fold over mock infected cells and normalized to a housekeeping gene.</p

Multi-step viral growth in NHBE and NSBE cells.

<p>Undifferentiated NHBE (A) and NSBE (B) cells were infected with the indicated viruses at a MOI of 0.01. At 0, 8, 24, 48, 72, and 96 HPI supernatant was collected and viral titers were determined by plaque assay on MDCK cells.</p