PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Clin Immunol. Author manuscript; available in PMC May 1, 2011.
Published in final edited form as:
PMCID: PMC2875067
NIHMSID: NIHMS198594
Influenza-Induced Production of Interferon-Alpha is Defective in Geriatric Individuals
David H. Canaday, Naa Ayele Amponsah, Leola Jones, Daniel J. Tisch, Thomas R. Hornick, and Lakshmi Ramachandracorresponding author
David H. Canaday, Geriatric Research, Education and Clinical Center (GRECC), Cleveland VA Medical Center, Cleveland, OH 44106, USA; Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA;
corresponding authorCorresponding author.
Lakshmi Ramachandra: lxr2/at/case.edu
Background
The majority of deaths (90%) attributed to influenza are in person’s age 65 or older. Little is known about whether defects in innate immune responses in geriatric individuals contribute to their susceptibility to influenza.
Objective
Our aim was to analyze interferon-alpha (IFN-alpha) production in peripheral blood mononuclear cells (PBMCs) isolated from young and geriatric adult donors, stimulated with influenza A or Toll-like receptor (TLR) ligands. IFN-alpha is a signature anti-viral cytokine that also shapes humoral and cell-mediated immune responses.
Results
Geriatric PBMCs produced significantly less IFN-alpha in response to live or inactivated influenza (a TLR7 ligand) but responded normally to CpG ODN (TLR9 ligand) and Guardiquimod (TLR7 ligand). All three ligands activate plasmacytoid dendritic cells (pDCs). While there was a modest decline in pDC frequency in older individuals, there was no defect in uptake of influenza by geriatric pDCs.
Discussion and Conclusion
Influenza-induced production of IFN-alpha was defective in geriatric PBMCs by a mechanism that was independent of reduced pDC frequency or viability, defects in uptake of influenza, inability to secrete IFN-alpha, or defects in TLR7 signaling.
Keywords: Aging, influenza, interferon-alpha, plasmacytoid dendritic cells
Over 32,000 deaths per year occur in a typical influenza season with over 90% of deaths in persons over 65 years of age [1]. With the current H1N1 global pandemic, the number of influenza cases is expected to dramatically increase resulting in greater numbers of deaths than usual even in individuals over age 65. They are less susceptible than younger individuals to 2009 H1N1 but continue to have greater risk of complications if they develop active influenza infection. Unfortunately, the seasonal influenza vaccine has poor efficacy in this older group. The rates of protection against hospitalization for influenza and pneumonia are estimated to be only 33% in individuals over age 65 [2]. A better understanding of the pathogenesis of influenza infection and disease in older persons is required to develop more effective vaccines or immunomodulatory strategies to reduce morbidity and mortality in this group.
There are likely multiple mechanisms for this increased morbidity and mortality with aging. While decline in T cell function has been extensively documented in the elderly [311], potential changes in immune function that impact innate anti-viral responses have been largely unexplored. Type I interferons (IFNs) were first identified for their antiviral properties against influenza [12, 13] and have been subsequently shown to induce the transcription of several genes that help degrade viral RNA and block viral replication [1416]. In addition to their anti-viral properties, type I IFNs have multiple effects on human mononuclear populations including T cells and B cells, and reduced type I IFN production could result in decreased induction of cell-mediated immunity [17].
Plasmacytoid dendritic cells (pDCs) are the main producers of type I IFNs after influenza activation [18]. Influenza virus ssRNA induces expression of type I IFNs in pDCs via activation of Toll-like receptor 7 (TLR7) [1921]. IFNs induce expression of MxA, a protein with anti-viral activity [2224] that renders pDCs resistant to virus-induced apoptosis [25]. pDCs have been found in all of the relevant respiratory mucosal sites in humans including nasal mucosa, lung, and bronchalveolar lavage fluid [2629]. In the influenza challenge mouse model, pDCs in the respiratory tract were found to make two-thirds of the IFN-alpha generated supporting the idea that pDCs in spite of their low numbers in respiratory mucosal sites produce the majority of IFN-alpha [30].
Several animal models suggest a clear role for IFN-alpha in protection against influenza. Ferrets and Guinea pigs have been found to be representative models of human disease as they are susceptible to human influenza strains, are able to transmit infection [3133], and express the MxA gene [34]. Recent studies in ferrets and Guinea pigs show that exogenous IFN-alpha reduces viral shedding and morbidity to influenza A virus [35, 36]. The mouse model has shown variable results regarding the importance of IFN-alpha as many of the studies use BALB and B6 mice that lack the MxA gene [34].
In the current study, we have assessed response of peripheral blood mononuclear cells (PBMCs) isolated from control (<35 years) and geriatric (>65 years) individuals to live influenza. IFN-alpha production was significantly reduced in pDCs from geriatric individuals in response to influenza but not to other TLR ligands that also activate pDCs.
Donors
Subjects were recruited from the Cleveland, VA, Case Western Reserve University and University Hospitals of Cleveland according to IRB approved protocols.
Media
Unless otherwise stated, cells were cultured in 96-well round-bottom plates at 37°C in 5% CO2 in X-Vivo 15 serum-free media (Lonza-BioWhittaker, Walkersville, MD, USA) supplemented with Penicillin and Streptomycin (Lonza-BioWhittaker).
Cell Isolation
Blood was obtained in heparin containing green top Vacutainer tubes (BD Biosciences) and processed within 4 h. PBMCs were isolated from blood samples (geriatric and control) by Ficoll Plaque Plus (GE Healthcare/Amersham Biosciences) density gradient centrifugation according to the manufacturer’s instructions. PBMCs were resuspended in X-Vivo-15 serum-free media and counted. In some experiments, pDCs were depleted from PBMCs using a CD304 (BDCA-4/Neuropilin-1) Microbead Kit from Miltenyi Biotec (Germany) according to the manufacturer’s instructions. Multiple cell types were also purified from PBMCs using positive selection Microbead Kits from Miltenyi Biotec. The cell types isolated included: monocytes (CD14 beads), pDCs (BDCA-4/Neuropilin-1 beads), B cells (CD19 beads), myeloid DCs (mDCs, BDCA-1 beads), and T/NK cells (CD2 beads).
To assess pDC frequency and effectiveness of pDC depletion, 5×105 PBMCs were stained in X-Vivo-15 media using the following antibodies: anti-HLA-DR-Pacific Blue (Biolegend, San Diego, CA, USA), Lineage Cocktail 2 (Lin2)-fluorescein isothiocyanate (FITC, Becton Dickinson, San Jose, CA, USA), and anti-CD123-PE (eBioscience, San Diego, CA, USA). Cells were incubated in the dark on ice for 30 min, washed with PBS, and resuspended in 2% paraformaldehyde (Electron Microscopy Sciences). pDC frequency was determined by gating on HLA-DR+, Lin−, and CD123+ cells on a clinically certified LSRII flow cytometer (Becton Dickinson).
Viruses
Influenza A/PR/8/34 (H1N1, Charles River, Wilmington, MA, USA), influenza A/Hong Kong/8/68 (H3N2, BEI Resources, Manassas, VA, USA), and influenza A/Denver/1/57 (H1N1, BEI Resources) in allontoic fluid were stored as aliquots at −80°C.
A/PR/8/34 was UV-inactivated with 18,000 J using a Stratagene UV Crosslinker 1800. UV inactivation of virus was assessed by infecting Vero E6 cells (ATCC, Manassas, VA, USA). Vero E6 cells (3×104/well) were plated overnight in 24-well plates in DMEM (Hyclone, Thermo Fisher Scientific, Waltham, VA, USA) containing 10% fetal calf serum (Hyclone). Media was aspirated and replaced with fresh media containing trypsin (20 µg/ml, Thermo Fisher Scientific). Cells were infected with either live or UV-inactivated A/PR/8/34 at 1.6×105 EID50/well and 0.32×105 EID50/ml for 48 h at 37°C in 5% CO2. Cells were washed with warm PBS, fixed with freshly made 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) for 15 min at room temperature, washed again, and permeabilized with ice-cold methanol for 20 min at −20°C. Cells were incubated for 1 h at room temperature with blocking buffer (PBS containing 10% goat serum (Invitrogen, Carlsbad, CA, USA) and 5% BSA (Sigma-Aldrich, St. Louis, MO, USA)) and then stained with anti-influenza A monoclonal-FITC antibody (Chemicon-Millipore, Billerica, MA, USA) or isotype control mouse IgG1 (BD Pharmingen, San Diego, CA, USA). Cells were incubated overnight at 4°C, washed three times with PBS, and observed with a Leica DMI 6000 B inverted microscope.
To generate FITC-labeled virus, purified A/PR/8/34 (200 µl, Charles River) was dialyzed with PBS pH 8.2 for 2 h, incubated with 10 µl FITC (FLUOS 20 mg/ml stock in DMSO, Roche, Mannheim, Germany) for 25 min at room temperature and dialyzed twice in 200 ml PBS pH 7.3 for 1 h for each exchange. FITC-A/PR/8/34 was frozen at −80°C in aliquots.
Incubation of Cells with Influenza and Ligands
PBMCs (1×106 cells/well) in 200 µl of X-Vivo media were incubated with or without A/PR/8/34 (low = 0.32×105 EID50/ml, high = 1.6×105 EID50/ml), A/Hong Kong/8/68 (1.8×105 EID50/ml), A/Denver/1/57 (1.1×106 EID50/ml), type “A” CpG ODN 2216 (3 µg/ml, InvivoGen, San Diego, CA, USA), ODN 2216 control that lacks CpG motif (3 µg/ml, InvivoGen), Guardiquimod (1 µg/ml, InvivoGen), or Poly(I:C)-LMW/LyoVec (4 µg/ml, InvivoGen) for 20 h at 37°C. Concentration of reagents was determined in initial experiments by incubating control PBMCs with various dilutions of reagents (virus, TLR ligand) for 20 h, analyzing IFN-alpha in supernatants and selecting concentrations for the subsequent studies that were on the linear part of the IFN-alpha response (data not shown). For inhibitor studies using chloroquine (10 µM, Sigma-Aldrich) or control and IRS 661 ODNs (1.4 µM, manufactured as phosphorothioate ODNs by Invitrogen [19]), inhibitors were added 15 min prior to the addition of A/PR/8/34 or ligands and remained in the culture throughout. Supernatants were stored in microfuge tubes or 96-well plates at −80°C for further analyses.
For experiments that compared A/PR/8/34-induced IFN-alpha production by various cell types, the following number of cells were added per well to reflect their anticipated proportion in 5×105 PBMCs: pDCs (2×104 cells/well), B cells (5×104 cells/well), mDCs (2×104 cells/well), monocytes (1×105 cells/well), and T cell/NK cells (2×105 cells/well).
ELISAs
IFN-alpha and IFN-beta levels in supernatants were assessed with the Verikine™ Human IFN-Alpha Multi-subtype ELISA kit (detection limit 12.5 pg/ml), and Verikine™ Human IFN-Beta ELISA kit (detection limit 25 pg/ml, PBL Interferon Source, Piscataway, NJ, USA), respectively. The human IFN-alpha kit from eBioscience was used for the pDC depletion, cell type specific, and inhibitor experiments. Kits were used according to the manufacturer’s instructions. All samples and standards were inactivated with a final concentration of 0.5% Triton X-100 (Bio-Rad, Richmond, CA, USA).
Analysis of Uptake of Influenza by pDCs
PBMCs (1×106) were pulsed with or without FITC-A/PR/8/34 for 1 h at 37°C. Cells were washed once with media and chased for 30 min at 37°C. Cells were then washed twice with media, resuspended in MACS buffer (Miltenyi Biotec), and stained with the following antibodies: anti-HLA-DR-Pacific Blue (Biolegend), BDCA-4-APC (Miltenyi Biotec), and CD123-PE-Cy7 (eBioscience). Cells were incubated in the dark on ice for 30 min, washed with PBS, resuspended in 2% paraformaldehyde (Electron Microscopy Sciences), and analyzed by flow cytometry.
Annexin Assay
PBMC (2×106) were placed in 400 µl X-Vivo medium±1.6×105 EID50/ml A/PR/8/34 at 37°C for 8 h in microfuge tubes. Cells were stained with LIVE/DEAD Violet Fixable Dead Cell Stain Kit (Molecular Probes, Invitrogen), Lin-FITC, CD123-PE, HLA-DR-PECY5 (BD), and Annexin V-APC (eBioscience) in high calcium concentration annexin buffer according to the manufacturer’s instructions. Cells were fixed in 2% paraformaldehyde (Electron Microscopy Sciences) in annexin buffer and pDCs analyzed for Annexin V staining by flow cytometry.
Statistical Analysis
The magnitude of IFN-alpha production and pDC frequency was compared between geriatric and control patients. The distribution of these data were evaluated using scatter plots and were shown to be non-normally distributed. Therefore, Mann–Whitney, nonparametric two-tailed t tests were used for statistical comparisons and graphics. Results in the text present both median and mean. All analyses were performed using GraphPad Prism version 5.02 for Windows, GraphPad Software, San Diego, CA, USA, (www.graphpad.com).
PBMCs from Geriatric Individuals Produce Less IFN-Alpha in Response to Influenza than PBMCs from Younger Controls
Subjects are described in Table 1. The control population included 38 individuals in the age range of 21–35 with an average age of 27. The geriatric population consisted of 43 individuals in the age range of 66–96 with an average age of 84. The younger individuals were healthy and not on medications.
Table 1
Table 1
Clinical Information of Study Subjects
PBMCs isolated from control or geriatric individuals were incubated with or without low (0.32×105 EID50/ml) or high (1.6×105 EID50/ml) concentrations of A/PR/8/34 (H1N1) for 6 or 20 h and supernatants were assayed for IFN-alpha by ELISA (Fig. 1a,b). IFN-alpha was barely detectable in the 6 h supernatants (data not shown). In 20 h supernatants, at low-dose A/PR/8/34, geriatric PBMCs (n=22, mean=76 pg/ml, median=4.8 pg/ml) produced 78.2% (p=0.012) less IFN-alpha than control PBMCs (n=21, mean=348 pg/ml, median=156 pg/ml). At high-dose A/PR/8/34, geriatric PBMCs (n=24, mean=2115 pg/ml, median=1,326 pg/ml) produced 66.5% (p=0.0006) less IFN-alpha than control PBMCs (n=20, mean=6,203 pg/ml, median=4,325 pg/ml). No response was seen with media or allantoic fluid alone. No IFN-beta was detected in the supernatants.
Fig. 1
Fig. 1
PBMCs from geriatric individuals produce less IFN-alpha in response to A/PR/8/24 than PBMCs from younger controls. Control and geriatric PBMCs (1 × 106 cells/ cellswell.well) in 200 µl of X-Vivo media were incubated with or without a A/PR/8/34 (more ...)
In the geriatric subjects there were several subgroups based on medications and comorbidities. For several of the subgroups we had enough subjects to do a subgroup analysis. These included subjects on statins or aspirin (ASA). We found that their influenza-induced IFN-alpha levels were not different than those not on these medications (p=0.53 statins, p=0.28 ASA). Also, diabetes mellitus was the most common comorbid condition in the geriatric population, and those geriatric subjects with diabetes mellitus were not different in their induction of IFN-alpha (p=0.45). Based on these subgroup analyses, we feel fairly confident that the comparisons between the young control and older geriatric populations are yielding valid results across a general geriatric group.
Reduced response to live influenza was not due to increased apoptosis of geriatric cells making IFN-alpha, since geriatric PBMCs stimulated with UV-inactivated A/PR/8/34 (high dose) for 20 h also made significantly less IFN-alpha (p=0.0001, n=12, mean=391 pg/ml, median=312 pg/ml) than control PBMCs (n=15, mean=2,566 pg/ml, median=2,114 pg/ml; Fig. 1c). This also suggests that IFN-alpha production to A/PR/8/34 is at least partially replication dependent and is consistent with that seen by others to influenza and other ssRNA viruses [37, 38]. A lack of differences in apoptosis was confirmed by flow cytometric analysis of Annexin V and LIVE/DEAD violet staining of PBMCs after live A/PR/8/34 infection and gating on pDCs. We found no difference (p=0.83) in Annexin V and LIVE/DEAD staining in pDC from nine controls (15.9% SD 10.7) and seven geriatric individuals (15.5% SD 11.4). This further supports that apoptosis of pDC is not the cause of the reduction in IFN-alpha observed in older individuals.
To determine if the reduced response was seen with other strains of influenza A, control and geriatric PBMCs were incubated with A/Hong Kong/8/68 (H3N2, 1.8×105 EID50/ml) and A/Denver/1/57 (H1N1, 1.1×106 EID50/ml) for 20 h and IFN-alpha concentrations was assayed in supernatants (Fig. 2). Geriatric PBMCs (n=14, mean=12,904 pg/ml, median=10,408 pg/ml) made 54% less IFN-alpha (p=0.0095) than control PBMCs (n=11, mean=28,105 pg/ml, median=22,241 pg/ml) in response to A/Denver/1/57. Geriatric PBMCs (n=13, mean=5,365 pg/ml, median=3,976 pg/ml) also made 58% less IFN-alpha (p=0.0065) than control PBMCs (n=11, mean 12,842 pg/ml, 9,034 pg/ml) in response to A/Hong Kong/8/68.
Fig. 2
Fig. 2
PBMCs from geriatric individuals make less IFN-alpha in response to clinical isolates A/Hong Kong/8/68 and A/Denver/1/57.Control and geriatric PBMCs (1 × 106 cells/well) in 200 µl of X-Vivo media were incubated with or without a A/Hong (more ...)
In conclusion, geriatric PBMCs made significantly less IFN-alpha in response to influenza A. This reduced response was not dependent on the viability of the virus, suggesting that viral-mediated apoptosis of geriatric PBMCs was not responsible for the diminished response.
Influenza Activates pDCs Via TLR7
Influenza has been shown to activate pDCs via TLR7 [1921]. While influenza may additionally signal via the retinoic acid-inducible gene (RIG-I) [39], a cytoplasmic RNA helicase that recognizes 5’triphosphate RNA [40, 41], studies have also shown that NS1 protein of influenza inhibits RIG-1 [41, 42]. Additionally, pDCs, which produce large amounts of IFN-alpha, use the TLR system rather than RIG-I for viral detection [43]. Since our studies used total PBMC cultures, experiments were performed to confirm that pDCs were the primary producers of IFN-alpha in response to influenza and to determine whether influenza signaled via TLR7 or RIG-I. PBMCs and PBMCs lacking pDCs were activated with A/PR/8/34 and with other TLR ligands (TLR9 ligand, CpG ODN and TLR7 ligand, and Guardiquimod) known to activate pDCs. Depletion of pDCs from PBMCs completely abrogated the response to all three ligands (Fig. 3a). We further confirmed that pDCs were the primary source of IFN-alpha production in PBMCs by examining responses of purified cell types found in PBMCs, including purified pDCs, mDCs, monocytes, B cells, and mixed T cell/NK populations, to influenza. Only purified pDCs made IFN-alpha in response to A/PR/8/34 stimulation (Fig. 3b).
Fig. 3
Fig. 3
pDC are the source of IFN-alpha in PBMC stimulated with influenza. PBMCs, PBMCs lacking pDCs, or purified cell types were incubated in 200 µl of X-Vivo media with or without A/PR/8/34 (high = 1.6 × 105 EID50/ml), CpG ODN 2216 (3 µg/ml) (more ...)
All endosomal TLRs (TLR7, TLR9, and TLR3) signal within the acidic environment of the endosome and can be inhibited by reagents (e.g., chloroquine) that prevent endosomal acidification [4446]. RIG-I is a cyotosolic RNA helicase that cannot be inhibited by chloroquine. To determine whether A/PR/8/34 signaled via a TLR or RIG-I, PBMCs were incubated with A/PR/8/34, Guardiquimod, CpG ODN, and a known RIG-I ligand, poly(I;C):LyoVec, in the presence or absence of chloroquine (Fig. 4a). While chloroquine inhibited IFN-alpha production in response to A/PR/8/34, Guardiquimod, and CpG ODN, it had little effect on IFN-alpha production in response to Poly(I;C): LMW LyoVec supporting observations made by others, that influenza signals via a TLR. TLR7 signaling can be inhibited in cells incubated with the inhibitory ODN IRS 661 [19]. ODN IRS 661 inhibited IFN-alpha production in response to both A/PR/8/34 (80% inhibition) as well as Guardiquimod (82% inhibition) but had no effect on responses to CpG ODN (Fig. 4b) that signals via TLR9. Together, these results support the observation that influenza activates pDCs in a TLR7-dependent manner.
Fig. 4
Fig. 4
Influenza-induced IFN-alpha is acidification and TLR7 dependent. a PBMCs were stimulated with RIG-I ligand Poly(I:C) LyoVec (4 µg/ml), CpG ODN 2216 (3 µg/ml), Guardiquimod (1 µg/ml), or A/PR/8/34 (high = 1.6 × 105 EID50 (more ...)
Geriatric PBMCs Have Reduced Frequency of pDCs
Since influenza activates pDCs via TLR7, reduced pDC numbers in the elderly may account for the decrease in response to influenza. It has been reported that the number of circulating pDCs declines during aging [4749]. Shodell and Siegal reported a decline in pDC numbers as a percentage of all mononuclear cells gated. pDC were 0.29% in individuals 18–29 years of age to 0.17% in individuals 60–91 years of age [49]. We analyzed pDC frequency in control (n=22) and geriatric (n=25) PBMCs by flow cytometry (Fig. 5). pDCs were identified as HLA-DR+, Lin–, and CD123+. We have similarly observed that pDC frequency significantly declined 38% (p=0.038) from a median of 0.27 (mean=0.27) in young controls to a median of 0.14 (mean=0.17) in geriatric PBMCs. Reduced pDC frequency in geriatric PBMCs may partially account for the reduced response to influenza A.
Fig. 5
Fig. 5
pDC frequency is lower in geriatric individuals. Control and geriatric PBMCs (5×105) were stained using anti-HLA-DR, anti-Lin2, and anti-CD123 and pDC frequency was quantified by gating on HLA-DR+, Lin2−, and CD123+ cells on a clinically (more ...)
Geriatric PBMCs are not Defective in Production of IFN-Alpha in Response to TLR9 Ligand CpG ODN or Other TLR7 Ligand Guardiquimod
In addition to a decrease in pDC frequency, reduced IFN-alpha responses of geriatric pDCs to influenza maybe due to defects in production of IFN-alpha or in expression and/or signaling via TLR7. Therefore, response of control and geriatric pDCs to type “A” CpG ODN (a TLR9 ligand, [50, 51]) and Guardiquimod (a TLR7 ligand, [52]) were analyzed. Control and geriatric PBMCs were stimulated with or without CpG ODN 2216 (3 µg/ml) or Guardiquimod (1 µg/ml) for 20 h and supernatants were assayed for IFN-alpha by ELISA (Fig. 6a,b). No IFN-alpha was detected in medium controls or when ODN 2216 control was used (data not shown). While geriatric pDCs (n=24) made slightly less IFN-alpha (mean=21,392 pg/ml, median=15,286 pg/ml) than control pDCs (n=22, mean=25,316 pg/ml, median=21,192 pg/ml) in response to CpG ODN 2216 the 15.5% decrease was not statistically significant (p=0.22). Response of geriatric PBMCs (n=24, mean=411 pg/ml, median=263 pg/ml) to Guardiquimod was equivalent (p=0.91) to that of control PBMCs (mean=390 pg/ml, median=275 pg/ml).
Fig. 6
Fig. 6
Geriatric PBMCs are not defective in production of IFN-alpha in response to TLR9 ligand CpG ODN or other TLR7 ligand Guardiquimod. PBMCs (1 × 106 cells/well) were incubated in 200 µl of X-Vivo media with a CpG ODN 2216 (3 µg/ml) (more ...)
Geriatric pDCs were therefore not defective in production of IFN-alpha following activation with TLR9 or TLR7. Since pDC frequency is lower in geriatric individuals, geriatric pDCs should produce more IFN-alpha on a per cell basis than control pDCs, to account for the levels of IFN-alpha made in response to Guardiquimod as well as CpG ODN. Alternatively, a higher percentage of geriatric pDCs responded to both these ligands to produce IFN-alpha.
Geriatric pDCs are Not Defective in Uptake of Influenza A
Since geriatric pDCs were not defective in IFN-alpha production or TLR signaling, reduced uptake of influenza by geriatric pDCs could additionally contribute to the defective response. To assay uptake of influenza, control and geriatric PBMCs were pulsed with FITC-A/PR/8/34 for 1 h, washed, and then chased an additional 30 min at 37°C to maximize uptake. pDCs were identified by staining with anti-HLA-DR, anti-BDCA-4, and anti-CD123, and the mean fluorescence intensity (MFI) of FITC label in pDCs was analyzed by flow cytometry (Table 2). Mean MFI for control pDCs was 13,109 while the mean MFI for geriatric pDCs was slightly higher at 13,523 (p=0.56). Although little difference in uptake of FITC-A/PR/8/34 was observed between controls and geriatrics, differences in IFN-alpha production in response to A/PR/8/34 between controls and geriatrics persisted (Table 2).
Table 2
Table 2
Analysis of Uptake of FITC-Labeled A/PR/8/34 by Control and Geriatric pDCs
In conclusion, geriatric pDCs were defective in the production of IFN-alpha in response to influenza A by a mechanism that was independent of reduced pDC viability, defects in uptake of influenza, inability to secrete IFN-alpha, or defects in TLR7 signaling.
Susceptibility of older individuals to influenza has often been ascribed to defects in T cell function. The contribution of the innate immune response to this defect remains unclear. Innate immune responses to influenza consist importantly of vigorous production of type I IFNs. They are potent anti-viral cytokines that also regulate other aspects of innate and adaptive immunity. We observed a significant decrease in the levels of IFN-alpha in supernatants from geriatric PBMCs activated with influenza. Surprisingly, no defect in IFN-alpha production was observed in geriatric PBMCs activated with CpG ODN 2216 or Guardiquimod. While influenza activates pDCs via TLR7, CpG ODN 2216 and Guardiquimod activate pDCs via TLR9 and TLR7, respectively. Our observations clearly indicate no defect in TLR7 or TLR9 signaling or IFN-alpha production in the pDC population in the elderly. Consistent with our findings, Jing et al. recently reported that the frequency of IFN-alpha secreting pDCs was reduced in PBMCs from healthy elderly subjects activated with influenza but not CpG 2216 [47]. We also observed a decline in pDC frequency in the elderly similar to that reported by other groups [4749]. Less IFN-alpha secreting pDCs coupled with decline in pDC frequency in geriatric PBMCs may account for the decreased response to influenza in geriatric PBMCs. But geriatric pDCs would have to make more IFN-alpha on a per pDC basis in response to Guardiquimod and CpG ODN 2216 (Fig. 6) to account for their normal response to these ligands despite lower pDC frequency.
Defect in IFN-alpha production in geriatric pDCs was only observed with influenza but not with other ligands that also target intracellular TLRs in pDCs. Interaction of ligands with TLR7 and TLR9 occurs in acidic endocytic vesicles, is pH dependent and is abrogated by agents like chloroquine and bafilomycin A that increase endosomal pH [4446]. Interaction of influenza viral ssRNA with TLR7 requires virus fusion and uncoating from endocytic vacuoles by a process that is pH dependent and occurs in late endosomes through a type I fusion process [53, 54]. Wang et al. demonstrated that increasing intraendosomal pH from approximately 4.5 to 5.2 with chloroquine significantly decreased IFN-alpha production in human pDCs in response to influenza virus but not to TLR7 ligand R848 that is an imidazoquinoline compound like Guardiquimod [55]. Increasing intraendosomal pH to 5.8 abrogated IFN-alpha production in response to both R848 and influenza. Therefore, subtle variations in late endosomal pH may impact IFN-alpha production in response to influenza but not other TLR7 ligands. We speculate that a slight increase in pH in late endosomes (and maybe all endosomal compartments) in the elderly may impact influenza virus fusion and uncoating and lead to inhibition of IFN-alpha production in response to influenza (which is very pH sensitive) but have little impact on IFN-alpha production to other TLR7 and TLR9 ligands (which may not be as pH sensitive).
The intracellular location of a TLR ligand may also determine the resulting biological response [56]. In human pDCs localization of CpG ODNs to transferrin-receptor-positive early endosomes led exclusively to IFN-alpha production while localization of CpG ODNs to LAMP-1 positive late endosomes promoted maturation of pDCs [56]. Similarly, in murine pDCs retention of Type “A” CpG ODN in endosomal compartments promoted IFN-alpha induction [57] while rapid transfer to lysosomal vesicles, as seen with conventional DCs, led to little IFN-alpha production. When Type “A” CpG ODN was manipulated for endosomal retention, robust production of IFN-alpha was observed [57]. In geriatric pDCs, viral ssRNA may be rapidly transferred to lysosomes, resulting in reduced IFN-alpha production.
Entry of influenza virus into cells has been studied extensively and involves binding of the virus to sialic acid-containing receptors on the cell surface followed by internalization by receptor-mediated endocytosis [58]. Since decreased uptake of influenza virus by geriatric pDCs may also lead to decreased IFN-alpha production, we compared uptake of FITC-labeled virus in geriatric and control pDC by flow cytometry. No defect in uptake of influenza by geriatric pDCs was observed. Although the experiment was designed with an incubation period to allow virus to bind cells, followed by a chase period to maximize uptake of virus, the possibility that virus remained on the cell surface cannot be ruled out.
In addition to their anti-viral properties, type I IFNs mediate both innate and adaptive immune responses. Therefore, reduced IFN-alpha levels in geriatric individuals after influenza stimulation could have a number of deleterious effects on both innate and adaptive immunity in older adults. Type I IFNs can induce the production of multiple cytokines, chemokines, and other molecules like IL-15 [59], CCXCL10, CCL4, CCL2, and IL-1RA [60]. Type I IFNs have been shown to play a modulatory role in differentiation of human T cells to Th1 development [61, 62] and in the development of CD8+ T central memory cells [63, 64]. Presence of IFN-alpha was shown to have mixed effects on proliferation in human memory CD4+ T cells depending on the antigen stimulation [65]. In murine systems, type I IFNs act directly on both CD4+ and CD8+ T cells to allow clonal expansion in response to viral infection [66, 67]. An in vitro study by Jego et al. [68] using human DCs and B cells showed that IFN induces activation and IL-6 secretion by DCs, which was required for differentiation of B cells into antibody-secreting cells. Isotype switching of B cells is also enhanced by type I IFNs [69]. In murine systems, following influenza virus infection, type I IFN receptor signals directly activated local B cells [70] and type I IFN directly modulated respiratory tract B cell responses [71]. Type I IFN has also been shown to enhance B cell receptor-dependent B cell responses [72]. A number of DC functions are enhanced by type I IFNs including MHC-I cross priming and DC maturation [7375]. Therefore, innate immune defects in IFN-alpha production by older individuals could lead to multiple defects in their adaptive immune responses.
Conclusions
Geriatric PBMCs made significantly less IFN-alpha in response to influenza than PBMCs from younger controls. This could have a deleterious effect on anti-influenza responses in geriatric individuals and contribute significantly to the susceptibility of older individuals to influenza.
Acknowledgments
This work was supported by AI077056 (to L.R. and D.C), McGregor Fund grant (to D.C) and Veterans Affairs GRECC and CSR&D Merit grant (D.C. and T.H.). Flow cytometry was performed at the CWRU/UH Center for AIDS Research (NIH Grant AI36219). We thank Megan Ermler for assistance with infecting Vero E6 cells, Scott Howell at Visual Sciences Research Center (NIH Grant P30-EY11373) for assistance with microscopy, Gareth Hardy for IFN-beta analysis and Lucy Jury at the Cleveland VA for help procuring blood samples.
Abbreviations
ASAAspirin
CADCoronary artery disease
COPDChronic obstructive pulmonary disease
CVACerebrovascular accident
DCDendritic cell
IFNInterferon
PBMCsPeripheral blood mononuclear cells
pDCPlasmacytoid DC
TLRToll-like receptor

Contributor Information
David H. Canaday, Geriatric Research, Education and Clinical Center (GRECC), Cleveland VA Medical Center, Cleveland, OH 44106, USA. Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
Naa Ayele Amponsah, Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
Leola Jones, Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
Daniel J. Tisch, Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, OH 44106, USA.
Thomas R. Hornick, Geriatric Research, Education and Clinical Center (GRECC), Cleveland VA Medical Center, Cleveland, OH 44106, USA.
Lakshmi Ramachandra, Department of Pathology Case Western Reserve University, Wolstein 6530, 2103 Cornell Rd., Cleveland, OH 44106, USA.
1. Thompson WW, Shay DK, Weintraub E, Brammer L, Cox N, Anderson LJ, et al. Mortality associated with influenza and respiratory syncytial virus in the United States. JAMA. 2003;289:179–186. [PubMed]
2. Vu T, Farish S, Jenkins M, Kelly H. A meta-analysis of effectiveness of influenza vaccine in persons aged 65 years and over living in the community. Vaccine. 2002;20:1831–1836. [PubMed]
3. Effros RB. Replicative senescence of CD8 T cells: effect on human ageing. Exp Gerontol. 2004;39:517–524. [PubMed]
4. Goronzy JJ, Lee WW, Weyand CM. Aging and T-cell diversity. Exp Gerontol. 2007;42:400–406. [PMC free article] [PubMed]
5. Gupta S, Bi R, Su K, Yel L, Chiplunkar S, Gollapudi S. Characterization of naive, memory and effector CD8+ T cells: effect of age. Exp Gerontol. 2004;39:545–550. [PubMed]
6. Haynes L, Maue AC. Effects of aging on T cell function. Curr Opin Immunol. 2009;21:414–417. [PubMed]
7. Haynes L, Swain SL. Why aging T cells fail: implicatins for vaccination. Immunity. 2006;24(6):663–666. [PubMed]
8. Kovaiou RD, Grubeck-Loebenstein B. Age-associated changes within CD4+ T cells. Immunol Lett. 2006;107:8–14. [PubMed]
9. Miller RA. The aging immune system: primer and prospectus. Science. 1996;273:70–74. [PubMed]
10. Miller RA. Effect of aging in T lymphocyte activation. Vaccine. 2000;18:1654–1660. [PubMed]
11. Nikolich-Zugich J. Ageing and life-long maintenance of T-cell subsets in the face of latent persistent infections. Nat Rev Immunol. 2008;8:512–522. [PubMed]
12. Isaacs A, Lindenmann J. Virus interference. I. The interferon. Proc R Soc Lond B Biol Sci. 1957;147:258–267. [PubMed]
13. Isaacs A, Lindenmann J, Valentine RC. Virus Interference. II. Some properties of interferon. Proc R Soc Lond B Biol Sci. 1957;147:268–273. [PubMed]
14. Hovanessian AG. Interferon-induced and double-standed RNA-acivated enzymes: a specific protein kinase and 2′, 5′ oligoade-nylate synthetases. J Interferon Res. 1991;11:199–205. [PubMed]
15. Kerr IM, Brown RE, Ball LA. Increased sensitivity if cell-free protein synthesis to double-stranded RNA after interferon treatment. Nature. 1974;250:57–59. [PubMed]
16. Silverman RH. Viral encounters with 2′, 5′-oligoadenylate synthetase and RNase L during the interferon antiviral response. J Virol. 2007;81:12720–12729. [PMC free article] [PubMed]
17. Randall RE, Goodbourn S. Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures. J Gen Virol. 2008;89:1–47. [PubMed]
18. Fonteneau JF, Gilliet M, Larsson M, Dasilva I, Munz C, Liu YJ, et al. Activation of influenza virus-specific CD4+ and CD8+ T cells: a new role for plasmacytoid dendritic cells in adaptive immunity. Blood. 2003;101:3620–3626. [PubMed]
19. Barrat FJ, Meeker T, Gregorio J, Chan JH, Uematsu S, Akira S, et al. Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus. J Exp Med. 2005;202:1131–1139. [PMC free article] [PubMed]
20. Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science. 2004;303:1529–1531. [PubMed]
21. Lund JM, Alexopoulou L, Sato A, Karow M, Adams NC, Gale NW, et al. Recognition of single-stranded RNA viruses by Tolllike receptor 7. Proc Natl Acad Sci USA. 2004;101:5598–5603. [PubMed]
22. Krug RM, Shaw M, Broni B, Shapiro G, Haller O. Inhibition of influenza viral mRNA synthesis in cells expressing the interferon-induced Mx gene product. J Virol. 1985;56:201–206. [PMC free article] [PubMed]
23. Pavlovic J, Zurcher T, Haller O, Staeheli P. Resistance to influenza virus and vesicular stomatitis virus conferred by expression of human MxA protein. J Virol. 1990;64:3370–3375. [PMC free article] [PubMed]
24. Samuel CE. Antiviral actions of interderon. Interferon-regulated cellular proteins and their surprisingly selective antiviral activities. Virology. 1991;183:1–11. [PubMed]
25. Cella M, Salio M, Sakakibara Y, Langen H, Julkunen I, Lanzavecchia A. Maturation, activation, and protection of dendritic cells induced by double-stranded RNA. J Exp Med. 1999;189:821–829. [PMC free article] [PubMed]
26. Masten BJ, Olson GK, Tarleton CA, Rund C, Schuyler M, Mehran R, et al. Characterization of myeloid and plasmacytoid dendritic cells in human lung. J Immunol. 2006;177:7784–7793. [PubMed]
27. Tsoumakidou M, Tzanakis N, Papadaki HA, Koutala H, Siafakas NM. Isolation of myeloid and plasmacytoid dendritic cells from human bronchoalveolar lavage fluid. Immunol Cell Biol. 2006;84:267–273. [PubMed]
28. Hartmann E, Graefe H, Hopert A, Pries R, Rothenfusser S, Poeck H, et al. Analysis of plasmacytoid and myeloid dendritic cells in nasal epithelium. Clin Vaccine Immunol. 2006;13:1278–1286. [PMC free article] [PubMed]
29. Gill MA, Long K, Kwon T, Muniz L, Mejias A, Connolly J, et al. Differential recruitment of dendritic cells and monocytes to respiratory mucosal sites in children with influenza virus or respiratory syncytial virus infection. J Infect Dis. 2008;198:1667–1676. [PMC free article] [PubMed]
30. Jewell NA, Vaghefi N, Mertz SE, Akter P, Peebles RS, Jr, Bakaletz LO, et al. Differential type I interferon induction by respiratory syncytial virus and influenza a virus in vivo. J Virol. 2007;81:9790–9800. [PMC free article] [PubMed]
31. Lowen AC, Mubareka S, Tumpey TM, Garcia-Sastre A, Palese P. The guinea pig as a transmission model for human influenza viruses. Proc Natl Acad Sci USA. 2006;103:9988–9992. [PubMed]
32. Matsuoka Y, Lamirande EW, Subbarao K. The ferret model for influenza. Curr Protoc Microbiol. 2009 Chapter 15:unit 15G 12.
33. Tumpey TM, Maines TR, Van Hoeven N, Glaser L, Solorzano A, Pappas C, et al. A two-amino acid change in the hemagglutinin of the 1918 influenza virus abolishes transmission. Science. 2007;315:655–659. [PubMed]
34. Staeheli P, Grob R, Meier E, Sutcliffe JG, Haller O. Influenza virus-susceptible mice carry Mx genes with a large deletion or a nonsense mutation. Mol Cell Biol. 1988;8:4518–4523. [PMC free article] [PubMed]
35. Van Hoeven N, Belser JA, Szretter KJ, Zeng H, Staeheli P, Swayne DE, et al. Pathogenesis of 1918 pandemic and H5N1 influenza virus infections in a guinea pig model: antiviral potential of exogenous alpha interferon to reduce virus shedding. J Virol. 2009;83:2851–2861. [PMC free article] [PubMed]
36. Kugel D, Kochs G, Obojes K, Roth J, Kobinger GP, Kobasa D, et al. Intranasal administration of alpha interferon reduces seasonal influenza A virus morbidity in ferrets. J Virol. 2009;83:3843–3851. [PMC free article] [PubMed]
37. Hornung V, Schlender J, Guenthner-Biller M, Rothenfusser S, Endres S, Conzelmann KK, et al. Replication-dependent potent IFN-alpha induction in human plasmacytoid dendritic cells by a single-stranded RNA virus. J Immunol. 2004;173:5935–5943. [PubMed]
38. Sandbulte MR, Boon AC, Webby RJ, Riberdy JM. Analysis of cytokine secretion from human plasmacytoid dendritic cells infected with H5N1 or low-pathogenicity influenza viruses. Virology. 2008;381:22–28. [PMC free article] [PubMed]
39. Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, Matsui K, et al. Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature. 2006;441:101–105. [PubMed]
40. Hornung V, Ellegast J, Kim S, Brzozka K, Jung A, Kato H, et al. 5′-Triphosphate RNA is the ligand for RIG-I. Science. 2006;314:994–997. [PubMed]
41. Pichlmair A, Schulz O, Tan CP, Naslund TI, Liljestrom P, Weber F, et al. RIG-I-mediated antiviral responses to single-stranded RNA bearing 5′-phosphates. Science. 2006;314:997–1001. [PubMed]
42. Guo Z, Chen LM, Zeng H, Gomez JA, Plowden J, Fujita T, et al. NS1 protein of influenza A virus inhibits the function of intracytoplasmic pathogen sensor, RIG-I. Am J Respir Cell Mol Biol. 2007;36:263–269. [PubMed]
43. Kato H, Sato S, Yoneyama M, Yamamoto M, Uematsu S, Matsui K, et al. Cell type-specific involvement of RIG-I in antiviral response. Immunity. 2005;23:19–28. [PubMed]
44. Hacker H, Mischak H, Miethke T, Liptay S, Schmid R, Sparwasser T, et al. CpG-DNA-specific activation of antigen-presenting cells requires stress kinase activity and is preceded by non-specific endocytosis and endosomal maturation. EMBO J. 1998;17:6230–6240. [PubMed]
45. Yi AK, Peckham DW, Ashman RF, Krieg AM. CpG DNA rescues B cells from apoptosis by activating NFkappaB and preventing mitochondrial membrane potential disruption via a chloroquine-sensitive pathway. Int Immunol. 1999;11:2015–2024. [PubMed]
46. Yi AK, Tuetken R, Redford T, Waldschmidt M, Kirsch J, Krieg AM. CpG motifs in bacterial DNA activate leukocytes through the pH-dependent generation of reactive oxygen species. J Immunol. 1998;160:4755–4761. [PubMed]
47. Jing Y, Shaheen E, Drake RR, Chen N, Gravenstein S, Deng Y. Aging is associated with a numerical and functional decline in plasmacytoid dendritic cells, whereas myeloid dendritic cells are relatively unaltered in human peripheral blood. Hum Immunol. 2009;70(10):777–784. [PubMed]
48. Perez-Cabezas B, Naranjo-Gomez M, Fernandez MA, Grifols JR, Pujol-Borrell R, Borras FE. Reduced numbers of plasmacytoid dendritic cells in aged blood donors. Exp Gerontol. 2007;42:1033–1038. [PubMed]
49. Shodell M, Siegal FP. Circulating, interferon-producing plasmacytoid dendritic cells decline during human ageing. Scand J Immunol. 2002;56:518–521. [PubMed]
50. Bauer S, Kirschning CJ, Hacker H, Redecke V, Hausmann S, Akira S, et al. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci USA. 2001;98:9237–9242. [PubMed]
51. Krug A, Rothenfusser S, Hornung V, Jahrsdorfer B, Blackwell S, Ballas ZK, et al. Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells. Eur J Immunol. 2001;31:2154–2163. [PubMed]
52. Morris GE, Parker LC, Ward JR, Jones EC, Whyte MK, Brightling CE, et al. Cooperative molecular and cellular networks regulate Toll-like receptor-dependent inflammatory responses. FASEB J. 2006;20:2153–2155. [PubMed]
53. Mukhopadhyay S, Kuhn RJ, Rossmann MG. A structural perspective of the flavivirus life cycle. Nat Rev Microbiol. 2005;3:13–22. [PubMed]
54. Lamb RA, Krug RM. Orthomyxoviridae: the viruses and their replication. In: Knipe DM, Howley PM, editors. Fields virology. 4th ed. Philadelphia: Lippincott Williams & Wilkins; 2001. pp. 1487–1532.
55. Wang JP, Liu P, Latz E, Golenbock DT, Finberg RW, Libraty DH. Flavivirus activation of plasmacytoid dendritic cells delineates key elements of TLR7 signaling beyond endosomal recognition. J Immunol. 2006;177:7114–7121. [PubMed]
56. Guiducci C, Ghirelli C, Marloie-Provost MA, Matray T, Coffman RL, Liu YJ, et al. PI3K is critical for the nuclear translocation of IRF-7 and type I IFN production by human plasmacytoid predendritic cells in response to TLR activation. J Exp Med. 2008;205:315–322. [PMC free article] [PubMed]
57. Honda K, Ohba Y, Yanai H, Negishi H, Mizutani T, Takaoka A, et al. Spatiotemporal regulation of MyD88-IRF-7 signalling for robust type-I interferon induction. Nature. 2005;434:1035–1040. [PubMed]
58. Matlin KS, Reggio H, Helenius A, Simons K. Infectious entry pathway of influenza virus in a canine kidney cell line. J Cell Biol. 1981;91:601–613. [PMC free article] [PubMed]
59. Surh CD, Boyman O, Purton JF, Sprent J. Homeostasis of memory T cells. Immunol Rev. 2006;211:154–163. [PubMed]
60. Decalf J, Fernandes S, Longman R, Ahloulay M, Audat F, Lefrerre F, et al. Plasmacytoid dentritic cells initiate a complex chemokine and cyokine network and are a viable drug target in chronic HCV patients. J Exp Med. 2007;204:2423–2437. [PMC free article] [PubMed]
61. Farrar JD, Smith JD, Murphy TL, Murphy KM. Recruitment of Stat4 to the human interferon-alph/beta receptor requires activated Stat2. J Biol Chem. 2000;275:2693–2697. [PubMed]
62. Rogge L, D'Ambrosio D, Biffi M, Penna G, Minetti LJ, Presky DH, et al. The role of Stat4 in species-specific regulation of Th cell development by type I IFNs. J Immunol. 1998;161:6567–6574. [PubMed]
63. Davis AM, Ramos HJ, Davis LS, Farrar JD. Cutting edge: a T-bet-independent role for IFN-alpha/beta in regulating IL-2 secretion in human CD4+ central memory T cells. J Immunol. 2008;181:8204–8208. [PMC free article] [PubMed]
64. Ramos HJ, Davis AM, Cole AG, Schatzle JD, Forman J, Farrar JD. Reciprocal responsiveness to interleukin-12 and interferon-alpha specifies human CD8+ effector versus central memory T-cell fates. Blood. 2009;113:5516–5525. [PubMed]
65. Gallagher KM, Lauder S, Rees IW, Gallimore AM, Godkin AJ. Type I interferon (IFN alpha) acts directly on human memory CD4+ T cells altering their response to antigen. J Immunol. 2009;183:2915–2920. [PubMed]
66. Kolumam GA, Thomas S, Thompson LJ, Sprent J, Murali-Krishna K. Type I interferons act directly on CD8 T cells to allow clonal expansion and memory formation in response to viral infection. J Exp Med. 2005;202:637–650. [PMC free article] [PubMed]
67. Havenar-Daughton C, Kolumam GA, Murali-Krishna K. Cutting Edge: the direct action of type I IFN on CD4 T cells is critical for sustaining clonal expansion in response to a viral but not a bacterial infection. J Immunol. 2006;176:3315–3319. [PubMed]
68. Jego G, Palucka AK, Blanck JP, Chalouni C, Pascual V, Banchereau J. Plasmacytoid dendritic cells induce plasma cell differentiation through type I interferon and interleukin 6. Immunity. 2003;19:225–234. [PubMed]
69. Le Bon A, Schiavoni G, D'Agostino G, Gresser I, Belardelli F, Tough DF. Type i interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo. Immunity. 2001;14:461–470. [PubMed]
70. Coro ES, Chang WL, Baumgarth N. Type I IFN receptor signals directly stimulate local B cells early following influenza virus infection. J Immunol. 2006;176:4343–4351. [PubMed]
71. Chang WL, Coro ES, Rau FC, Xiao Y, Erie DJ, Baumgarth N. Influenza virus infection causes global respiratory tract B cell response modulation via innate immune signals. J Immunol. 2007;178:1457–1467. [PubMed]
72. Braun D, Caramalho I, Demengeot J. IFN-a/b enhances BCR-dependent B cell responses. Int Immunol. 2002;14:411–419. [PubMed]
73. Ito T, Amakawa R, UInaba M, Ikehara S, Inaba K, Fukuhara S. Differential regulation of human blood dendritic cell subsets by IFNs. J Immunol. 2001;166:2961–2969. [PubMed]
74. Le Bon A, Etchart N, Rossmann C, Ashton M, Hou S, Gewert D, et al. Cross-priming of CD8+ T cells stimulated by virus-induced type I interferon. Nat Immunol. 2003;4:1009–1015. [PubMed]
75. Tough DF. Type I interferon as a link between innate and adaptive immunity between through dendritic cell stimulation. Leuk Lymphoma. 2004;45:257–264. [PubMed]