Modulation of human beta-defensin-1 (hBD-1) in plasmacytoid dendritic cells (PDC), monocytes, and epithelial cells by influenza virus, Herpes simplex virus, and Sendai virus and its possible role in innate immunity

Abstract

hBD comprise a family of antimicrobial peptides that plays a role in bridging the innate and adaptive immune responses to infection. The expression of hBD-2 increases upon stimulation of numerous cell types with LPS and proinflammatory cytokines. In contrast, hBD-1 remains constitutively expressed in most cells in spite of cytokine or LPS stimulation; however, its presence in human PDC suggests it plays a role in viral host defense. To examine this, we characterized the expression of hBD-1 in innate immune cells in response to viral challenge. PDC and monocytes increased production of hBD-1 peptide and mRNA as early as 2 h following infection of purified cells and PBMCs with PR8, HSV-1, and Sendai virus. However, treatment of primary NHBE cells with influenza resulted in a 50% decrease in hBD-1 mRNA levels, as measured by qRT-PCR at 3 h following infection. A similar inhibition occurred with HSV-1 challenge of human gingival epithelial cells. Studies with HSV-1 showed that replication occurred in epithelial cells but not in PDC. Together, these results suggest that hBD-1 may play a role in preventing viral replication in immune cells. To test this, we infected C57BL/6 WT mice and mBD-1((-/-)) mice with mouse-adapted HK18 (300 PFU/mouse). mBD-1((-/-)) mice lost weight earlier and died sooner than WT mice (P=0.0276), suggesting that BD-1 plays a role in early innate immune responses against influenza in vivo. However, lung virus titers were equal between the two mouse strains. Histopathology showed a greater inflammatory influx in the lungs of mBD-1((-/-)) mice at Day 3 postinfection compared with WT C57BL/6 mice. The results suggest that BD-1 protects mice from influenza pathogenesis with a mechanism other than inhibition of viral replication.

Figure 1.. Expression of hBD-1 mRNA (A) in fresh human PBMCs using RT-PCR and (B) in mock-stimulated and virus-stimulated PBMCs using real-time qRT-PCR.

(A) BD gene expression in fresh PBMCs without virus stimulation (from Donor #2 in B). Total RNA was extracted using an RNeasy kit (Qiagen) and reverse-transcribed with the specific primers for hBD-1, hBD-2, hBD-3, and hBD-4 (Lanes 1–4, respectively). RNA from NHBE cells was also assayed for hBD-1–4, and gene expression of each BD is shown below. The oligonucleotide sequences are shown in Table 1. The lane labeled with “M” used a 123-bp MW marker to assess size. Only hBD-1 was expressed at the basal level in PBMCs (Lane 1). Below is the basal expression of hBD-1, hBD-2, hBD-3, and hBD-4 in unstimulated NHBE in liquid culture. (B) Individual variation of increased hBD-1 mRNA by viruses. qRT-PCR detected variable stimulation of hBD-1 gene expression in total mRNA from PBMCs of four donors stimulated with PR8 influenza, HSV-1, or Sendai virus for 3, 6, or 18 h. Each sample was assayed in triplicate using the oligonucleotide primer sequences in Table 2. All viruses increased gene expression of hBD-1. Error bars indicate sd of the mean indicated by the height of the bar. Levels were compared with those of fresh PBMCs and seemed to be donor-dependent.

Figure 2.. qRT-PCR detecting hBD-1 and IFN-α1/13 and IFN-α8 gene expression in PDC.

(A) hBD-1 mRNA following a 3- and 6-h stimulation of 96% pure PDC with PR8 influenza virus in two donors (different donors from the semi-quantitative experiments). The increase is significant in both samples, as measured by one-way ANOVA followed by Tukey's test (P<0.001). (B) IFN-α1/13 and IFN-α8 mRNA increases following a 2-h incubation with PR8 influenza and HSV-1 in the same two donors. (A and B) Error bars indicate the sd of the mean value of the qRT-PCR. (C) qRT-PCR of HSV-stimulated, 96%-purified PDC from different donors after 6 h. (D) UV inactivation of HSV-1 prevented the virus-induced increase of hBD-1 mRNA. In all figures, medium-stimulated PDC was given a value of 1.0, and all bars of each donor are relative to their medium-stimulated control.

Figure 3.. qRT-PCR detecting hBD-1 and IFN-α1/13 and IFN-α8 gene expression in monocytes.

(A) hBD-1 mRNA increases relative to mock-stimulated monocytes following a 3- and 6-h stimulation of 100%-purified monocytes stimulated with PR8 influenza, HSV-1, and Sendai viruses in the same two donors as Fig. 2A, plus a new third donor. LPS stimulation (100 ng/ml) was used as a negative control. (B) IFN-α subtypes 8 and 1/3 mRNA increases above that of fresh monocytes from Donors #1 and #2 stimulated for 3 h and 6 h with the same agents as in A. (C) Monocytes stimulated with PR8 influenza and (D) HSV-1 with their UV-inactivated (10 min) counterparts (bars indicate mean and sd of n=3 donors). UV-inactivated influenza stimulated increases in hBD-1 mRNA in monocytes but did not inactivate this effect by HSV-1. In all graphs, medium controls are normalized to 1.0.

Figure 4.. Intracellular hBD-1 in PDC and monocytes in PBMCs stimulated for 2 h with no virus (mock), influenza virus, HSV, or Sendai virus.

Representative data from different donors (#7, #2, and #4 in D) are shown in A (influenza), B (Sendai virus), and C (HSV-1). Filled peaks represent anti-hBD-1 (1:250) antiserum compared with solid lines representing preimmune rabbit serum (1:250). Secondary staining was performed using goat anti-rabbit IgG conjugated to FITC. The ΔMFI between the hBD-1 antiserum and preimmune antiserum is shown in each histogram. PDC stimulated with influenza and Sendai viruses showed the greatest amount of hBD-1 induction. Monocytes stimulated with influenza and HSV showed a much smaller induction of hBD-1. (D) Variation between donors regarding virus-induced hBD-1 in PDC. PBMCs were stimulated for 2 h with virus in the presence of brefeldin A (added 1.5 h after 30-min stimulation), and hBD-1 was analyzed in PDC by flow cytometry as described above. ΔMFI was determined by comparing the geometric MFI of anti-hBD-1 antiserum with the MFI of normal rabbit serum in virus-stimulated cells. The ΔMFI was also determined in unstimulated cells. Δ(ΔMFI) was then determined. Lines indicate the mean Δ(ΔMFI) for each group. Most normal donor PDC responded to each virus, but there was variability in response by one individual to different viruses and also between different donors to a single virus (Donors #6 and #8 did not respond to PR8 influenza). Not all viruses were tested with each donor.

Figure 5.. Effect of virus challenge on hBD-1 gene expression in target epithelial cells.

(A) NHBE cells were treated with influenza virus, and OKF6/TERT cells were treated with HSV-1 at a MOI of 1:1 for 3 h. Total mRNA was isolated, and hBD-1 mRNA levels were quantified by qRT-PCR relative to β-actin. The qRT-PCR was carried out in triplicate, and data are shown as mean ± sd of qRT-PCR values for five cultures/infected cells and two cultures for mock-infected cells. Reduction in hBD-1 mRNA levels is significant by t test (P<0.01). (B) OKF6/TERT cells were treated with HSV-1 at a MOI of 1:1 for 0–8 h. Total mRNA was isolated, and hBD-1 mRNA levels were quantified by qRT-PCR as described in Materials and Methods. Reduction in hBD-1 mRNA levels is significant by one-way ANOVA (*P=0.03). (C) OKF6/TERT cells were challenged for 18 h with live HSV-1 at a MOI of 1:1 or the equivalent viral dose after viral inactivation by UV treatment or at a MOI of 10:1 of live HSV-1 for 18 h. Cells were also treated with 1 μg/ml CpG and 40 μg/ml poly I:C. Results were compared by ANOVA followed by Tukey's testing for multiple comparisons. Each bar represents the mean among three cultures, and error bars indicate sd. Only live HSV suppressed hBD-1 (*P<0.01). Incubation with poly I:C increased hBD-1 levels (†P<0.04). (D) Visualization of GFP in OKF6/TERT cells infected with HSV-1 (n=3; only one represented). Upon replication, when enough GFP is expressed, cells glow fluorescent green (beginning at 6 h).

Figure 6.. Survival and weight loss of WT and mBD-1^(−/−) mice following infection of mice with mouse HK18.

(A) Kaplan-Meyer plot of survival of WT C57BL/6 male mice and mice lacking mBD-1 (KO), the homologue to hBD-1, following intranasal infection with 300 PFU. The survival plots were different at P = 0.027. (B) Comparison of percent body-weight loss in WT mice versus KO following infection; *P ≤ 0.05. (C) Representative micrographs (Table 3) of WT and mBD-1^(−/−) mice infected with mouse-adapted HK18. Mice were infected with 300 PFU and killed 3 days after infection. Each panel represents a lung section from one mouse stained with H&E. A more severe inflammatory leukocytic infiltrate (neutrophils and mononuclear cells) and perivascular edema occurred at 3 h in the KO mice. Objective magnification is 10×. Total magnification is 100×.