CD49d⁺ neutrophils induce FcεRI expression on lung dendritic cells in a mouse model of postviral asthma

Abstract

The increasing prevalence of atopy and asthma remains unexplained but may be due to infection with respiratory viruses. In support of this hypothesis, we showed that experimental asthma after viral infection in mice depended on type I IFN-driven up-regulation of the high-affinity receptor for IgE (FcεRI) on conventional dendritic cells (cDCs) in the lung. Here we demonstrate that FcεRI expression on lung cDCs depends on an unexpected activity of a CD49d⁺ subset of polymorphonuclear neutrophils (PMNs) that are found in the lungs of wild-type C57BL6 but not IFNAR^−/− mice. Expression of FcεRI depends in part on a CD11b-dependent interaction between PMNs and cDCs. This study demonstrates a novel PMN-cDC interaction in the lung that is necessary for the ability of viral infection to induce atopic disease.

INTRODUCTION

The increasing prevalence of asthma and atopic disease is a major public health problem (1). Many hypotheses have been proposed to explain this epidemic of allergic disease (2, 3). One hypothesis is based on epidemiological studies that have correlated severe viral infections early in life to the subsequent development of asthma and allergic disease (4–6). However, until recently mechanistic studies were lacking to indicate how viral infection could lead to atopic disease.

Recently, using a mouse model of asthma triggered by a transient viral infection, we identified a potential mechanism by which a Th1 anti-viral response induces Th2 atopic disease. We demonstrated that mice who survive a severe Sendai virus (SeV) infection develop chronic airway hyperreactivity and mucous cell metaplasia, similar to human infants infected with Respiratory Syncytial Virus (7). We also showed that acute development of airway hyperreactivity was dependent on the expression of the high affinity receptor for IgE (FcεRI) on conventional dendritic cells (cDCs) in the lung. The increased cDC FcεRI expression in the lung during SeV infection required intact type I IFN receptor signaling. Importantly, crosslinking of FcεRI led to production of CCL28 and recruitment of IL-13 producing Th2 cells, which in turn drove the subsequent development of chronic asthma (8). Therefore, blocking induction of FcεRI on the cDC has clear therapeutic implications in preventing post-viral atopic disease.

The present study sought to extend our prior observations and identify the specific cells involved in the type I IFN-dependent induction of FcεRI on lung cDC following SeV infection.

MATERIALS AND METHODS

Mouse generation and handling

C57BL6 mice were from The Jackson Laboratory. Mice deficient in type I IFN receptor (IFNAR^−/−; C57BL6 background) were a kind gift of J. Sprent (Garvan Institute, Sydney, Australia). Mice 6–20 wks old were used for all experiments. Mice were housed, handled, and experiments performed according to protocols approved by the Institutional Animal Care and Use Committee at the Medical College of Wisconsin.

Mice were inoculated intranasally (i.n.) with 2×10⁵ pfu of SeV (Fushimi strain; American Type Culture Collection) and monitored daily for weight and activity. 100 μg of anti-Gr-1, anti-NK1.1, anti-mPDCA-1 or control IgG mAb were given s.c. on days -1, 1, 3, and 5 post-inoculation (PI) SeV. In some experiments, mice were inoculated i.n. with 10 μg LPS (from E. coli O55:B5, Sigma) and sacrificed 1 day PI.

Cell purification and culture

Lung cDC were obtained from lung digest as previously described (9). Briefly, mice were euthanized, the inferior vena cava severed, and the right cardiac ventricle injected with PBS, before bronchoalveolar lavage (BAL) performed with 1ml PBS. Lungs were removed, minced, and incubated in digest media for 1 h at 37°C, EDTA was added to the media (2mM final concentration) for the last 15 min. The single cell suspension was filtered through 40-μm pore cell strainers before removing erythrocytes by NH₄Cl hypotonic lysis (Sigma-Aldrich). cDC were purified using positive immunomagnetic selection with CD11c MACS beads (Miltenyi Biotec), with >95% purity achieved after two serial purifications (8).

Neutrophils (PMN; ≥ 85% pure) were isolated from BAL of mice 1 or 3 days PI. Subsets of PMN were obtained by sorting for CD49d (FACSAria, BD). PMN from BAL of SeV infected mice and lung cDC from uninfected mice were cultured together for 48 h in complete RPMI (Sigma-Aldrich) supplemented with 10% fetal calf serum and penicillin/streptomycin (Invitrogen) media at 37°C with 5% CO₂. PMN viability was ≥ 95% at the start of culture and decreased to 38 ± 2.6% by 48 hours.

For IFN treatment of PMN, 1000 U/ml mouse IFNβ (R&D) was added to PMN from BAL of mice 1 day PI LPS or to PMN purified from naïve bone marrow and co-cultured with cDC.

Purification of PMN from bone marrow

PMN were isolated from bone marrow as previously described (10). Marrow was flushed from long bones with HBSS/0.1% BSA, pelleted, and resuspended in 3 ml of 45% Percoll (GE Healthcare Bio-Sciences). Solutions of 66, 60, 55, and 50% were prepared by diluting the 100% stock Percoll with HBSS. 3 ml of the 66% solution and 2ml aliquots of each decreasing concentration of Percoll solution were layered over one another in a 15-ml conical tube. The bone marrow single-cell suspension in 45% Percoll was subsequently layered over the prepared Percoll density gradient, followed by centrifugation at 1800g for 30 minutes at room temperature. Cells were collected from the 66–60% interface and washed with HBSS/0.1%BSA. PMN purity was consistently >95% as assessed by flow cytometry. Contaminating cells found at this interface included a small percentage of nucleated erythrocytes and B cells.

Antibodies and flow cytometry analyses

Phytoerythrin-, allophycocyanin-, FITC, or Alexa Fluor 647- labeled antibodies against mouse CD11c (clone N418), FcεRIα (clone MAR-1), CD49d (clone R1-2), Gr1 (clone RB6-8C5), and isotype control IgGs were obtained from eBioscience, BD Pharmingen, and/or Biolegend. Anti-Gr1 (clone RB6-8C5), anti-NK1.1 (clone PK136), anti-mPDCA-1 (clone eBio927), anti-CD11b (clone M1/70), and anti-CEACAM-1 (clone CC1) mAbs were obtained from eBioscience or BD Pharmingen. Stained cells were analyzed with a FACSCalibur flow cytometer (BD) and data analyzed with FlowJo software (Tree Star, Inc).

Morphologic examination

Cells were mounted onto slides using the Shandon Cytospin 4 (Thermo Fisher Scientific) at 300 rpm for 5 minutes, and slides were stained with the Diff- Quick Stain Kit as per manufacturer’s instructions (Fisher).

Immunohistochemistry

Whole lung from SeV infected mice frozen in Tissue-Tek O.C.T. compound (Sakura) and 10 μm sections were obtained with a cryostat. Sections were fixed with acetone, blocked with goat serum, and stained with FITC labeled anti-VCAM-1 (clone 429) mAb or IgG isotype control (Biolegend). Stained lung sections were then examined for fluorescence by a blinded observer.

Real-time PCR assay

mRNA was isolated from whole lung using TRIzol (Sigma-Aldrich). cDNA was then generated with the QuantiTect reverse transcription kit (Qiagen) per manufacturer’s instructions. qPCR assays were performed using StepOnePlus PCR system (Applied Biosystems) and TaqMan Fast Universal PCR Master Mix. TaqMan primer and probes for rodent GAPDH control (4352339E), VCAM-1 (Mm01320970_m1), and TNFα (Mm00443258_m1) were obtained from Applied Biosystems.

Statistical analyses

Unless otherwise stated, all data are presented as mean ± SEM. Student’s t test was used to assess statistical significance between means. Mann-Whitney U test was used for comparison of medians of nonparametric data. For comparison of ratios, Wilcoxin Signed Rank was used. In all cases, significance was set at p < 0.05.

RESULTS AND DISCUSSION

PMNs are required for SeV mediated FcεRI expression on cDCs

We reported previously that lung cDCs express FcεRI by SeV PI day 3, which suggested that an early effector cell was involved. PMN are important effector cells in SeV induced lung disease and constitute >90% of the cells in the BAL of SeV infected mice at day 3 PI (11, 12). Therefore, we depleted PMN and examined FcεRI expression on cDC (11, 12). Treating C57BL6 mice with anti-Gr1 mAb every other day from one day prior to SeV inoculation blocked the upregulation of FcεRI expression on cDC (Fig. 1a,b). Since anti-Gr1 mAb has been reported to cross-react with Ly-6C expressing cells, we examined cDC FcεRI expression when mice were treated with depleting mAbs against NK cells and plasmacytoid dendritic cells (pDC), the 2 major Ly Ly-6C bearing cell types present during the early time points of the infection (13–15). NK cell or pDC depleting mAbs anti-NK1.1 or anti-mPDCA-1, respectively, failed to significantly reduce FcεRI expression on cDCs (Fig. 1a,b). These findings suggested that PMN were required for SeV mediated induction of FcεRI on lung cDC.

Figure 1. PMNs from IFNAR sufficient mice drive expression of FcεRI on lung cDC.

(a) Representative histograms of FcεRIα expression on purified lung cDC 5 days post inoculation (PI) with SeV in mice given 100 μg of control IgG, anti-Gr1, anti-NK1.1, or anti-mPDCA-1 mAb s.c. on days -1, 1, 3, and 5 PI (b) Only anti-Gr1 treatment significantly inhibited expression of FcεRIα on lung cDC. Data from 3 mice per group in 2 separate experiments (total of n = 6 mice/group). * p< 0.05 versus control IgG group. (c) Representative histograms of FcεRIα expression on purified lung cDC after 48 h culture with PMN from the BAL of day 3 PI SeV WT or IFNAR^−/−mice. (d) MFI of FcεRIα on cDC cultured as in (c), and percent of cDC expressing FcεRIα after culture as in (c). Data from a total of 12 BAL donor and 4 cDC donor mice per group used for 2 separate experiments (n=6 BAL and 2 cDC donors/experiment). * p< 0.05 versus WT.

Next, we tested whether PMN could induce FcεRI on the cDC in vitro. By performing transwell studies, we found that purified naïve lung cDC were induced to express FcεRI after 48 hours of culture with PMN isolated from the BAL of day 3 PI SeV infected WT mice. This effect was seen only when cDC were cultured in direct contact with PMN and did not occur when the cells were cultured for only 24 hours (data not shown). Moreover, consistent with previous reports on crosstalk between PMN and DC, a 10:1 excess of PMN provided the strongest signal for cDC FcεRIα induction (data not shown) (16).

PMN from mice deficient in type I IFN receptor (IFNAR^−/−) fail to induce FcεRI expression on cDC

We previously demonstrated that type I IFN acted on a cell type other than the cDC to induce FcεRI expression on lung cDC during SeV infection (8). Therefore, we next determined if type I IFN signaling was necessary for the PMN to induce FcεRI on cDC. PMN were isolated from the BAL of day 3 PI SeV infected WT or IFNAR^−/− mice and co-cultured with lung cDC purified from naïve WT mice. In contrast to PMN isolated from IFNAR^−/− mice, PMN from WT mice induced the expression FcεRI on lung cDC after 48h (Fig. 1c,d).

CD49d expression defines a subset of PMN required for FcεRIα induction

PMN have been grouped into two distinct functional subsets based in part on the surface expression of CD49d (17). In contrast to PMN isolated from WT mice, very few PMN isolated from the BAL on day 3 PI from IFNAR^−/− mice expressed CD49d (Fig. 2a). This result suggested that the CD49d expressing subset of PMN might be important for induction of FcεRI on lung cDC. Therefore, PMN from WT BAL were purified by cell sorting into the CD49d⁺ or CD49⁻ populations. As shown in figure 2b, both of these populations appeared morphologically similar with typical features of PMN. These subsets of PMN were separately cultured with naïve cDC for 48h. Only CD49d⁺ PMN were capable of inducing FcεRIα expression on lung cDC (Fig 2c, d).

Figure 2. SeV infection leads to accumulation of CD49d⁺ PMN, which induce FcεRIα on lung cDC.

(a) Representative contour plots for Gr-1 versus CD49d expression on BAL cells from day 3 PI SeV infected WT and IFNAR^−/− mice, and quantification of these data (mean ± sem, n = 5 per group, * p< 0.05). (b) CD49d⁺ and CD49d⁻ PMN have similar morphology. (c) CD49d⁺ PMN are sufficient to drive FcεRI expression on cDC. Representative histograms showing expression of FcεRIα on lung cDC after 48 h culture with CD49d⁺ PMN or CD49d⁻ PMN from BAL of day3 PI SeV infected WT mice. (d) Quantification of data in (c), showing mean ± sem fold MFI and percent of cDC expressing FcεRI after culture with CD49d⁺ and CD49d⁻ PMNs. Data from a total of 20 BAL donor mice and 4 cDC donor mice split into 2 separate experiments (10 BAL and 2 cDC donors/experiment); * p < 0.05 versus CD49d⁺. (e) SeV but not LPS inoculation specifically induces the CD49d⁺ PMN. WT mice were treated with LPS or SeV i.n. and 1 day later sacrificed and BAL and blood removed. Expression of CD49d versus Gr1 was determined by flow cytometry, with mean ± sem percent of CD49d⁺ PMN in the BAL and blood expressing CD49d shown (n = 4 per group, * p< 0.05).

Because we had previously shown a type I IFN dependence in this response, we wanted to determine if type I IFN induced expression of CD49d on PMN. Bone marrow derived PMN were cultured with murine IFNβ; however CD49d expression was not induced (data not shown). Further, co-culturing naïve WT bone marrow PMN with naïve lung cDC in the presence of IFNβ did not result in expression of FcεRI on the cDC (data not shown). Therefore, it appears that the effect of type I IFN in this response is indirect, acting on a cell other than the PMN.

Because CD49d is an adhesion molecule that binds VCAM, we explored the possibility that differential expression of VCAM-1 or TNF (which induces VCAM-1 expression) in the lungs of WT and IFNAR^−/− mice with SeV infection could explain the difference in accumulation of CD49d⁺ PMN (18). However, using histology, we did not find any difference in VCAM-1 protein on the lung endothelium of WT or IFNAR^−/− mice. In addition, we did not find any difference in whole lung TNF or VCAM-1 message (data not shown). Thus, differential VCAM-1 expression is not the mechanism through which CD49d⁺ PMN accumulate in the lungs of WT mice.

Viral specificity of the response

LPS treatment of WT mice failed to induce FcεRI expression on lung cDC (data not shown). Therefore, we compared the frequency of CD49d⁺ PMN in the BAL or blood of mice following LPS treatment or infection with SeV. Because LPS causes a rapid influx of PMN in the first 24 h after administration, we examined the PMN isolated from the BAL or blood 1 d following administration of LPS or SeV. CD49d⁺ PMNs were found only in the BAL or blood of mice infected with SeV (Fig. 2e). Thus, the ability of SeV but not LPS to induce FcεRI expression was related to the preferential accumulation of CD49d⁺ PMN in the lungs of SeV infected mice.

PMN CD11b mediates induction of FcεRI on lung cDC

We next wanted to determine what proteins were involved in the cognate interaction between lung cDC and PMN. One likely target was CD49d, however, the CD49d mAb used for cell sorting PMN is a blocking antibody (19). Therefore, since flow-sorted CD49d⁺ PMN induced FcεRIα expression on lung cDC, CD49d must not be directly involved in the induction of FcεRIα expression on cDC. We next focused on two ligands for DC-SIGN that have been implicated in interactions between PMN and cDC: CD11b and CEACAM-1 (20, 21). Culturing PMN with a blocking mAb to CD11b before addition to cDC significantly inhibited induction of FcεRI by 80% (Fig. 3). Blocking CEACAM-1 on PMN did not affect FcεRI expression, nor did addition of an anti-DC-SIGN mAb to the cDC (data not shown). Since we demonstrated previously that naïve lung cDC do not express CD11b, we believe the effects of anti-CD11b were mediated through blockade of CD11b on the PMN (9). CD11b expression is not restricted to the CD49d⁺ PMN subset, and in fact the CD49d⁻ PMN have greater expression of CD11b than CD49d⁺ PMNs (fold MFI of 202 ± 30 versus 72 ± 3, respectively; p < 0.003, n = 5 mice per group); therefore, even the modest level of CD11b expression is sufficient to mediate the PMN:cDC interaction, although additional mechanisms must be involved in induction of FcεRI on lung cDC. Moreover, it is not surprising that the induction of FcεRI on the cDC should involve novel mechanisms outside the reported interactions between CD11b, CEACAM-1, and DC-SIGN, as those have been reported to lead to a Th1 mediated response, not a Th2 directed one (20–22). Our current studies are focused on understanding these other factors involved in this unique PMN – cDC interaction.

Figure 3. PMN induce FcεRI on cDC through a partial CD11b dependent process.

(a) CD49d⁺ PMN sorted from BAL of day 3 PI SeV infected WT mice were treated with an IgG control mAb or a blocking anti-CD11b mAb prior to culture with lung cDC. Representative histograms showing FcεRIα expression on lung cDC after 48 h culture. (b) Quantification of data in (a), showing mean ± sem fold MFI and percent of cDC expressing FcεRI after culture. * p < 0.05. Data from a total of 20 BAL donor and 4 cDC donor mice split into 2 separate experiments (10 BAL and 2 cDC donors/experiment).

In summary, our data are the first to show SeV infection specifically increased the accumulation of CD49d⁺ PMN in the lung, which in turn induced cDC to express FcεRI. Furthermore, the increased expression of FcεRI on cDC required the cognate interaction of CD49d⁺ PMN with cDC, a process dependent primarily on the expression of CD11b on the PMN. These unexpected results implicate CD49d⁺ PMN as an essential effector cell in the induction of chronic asthma induced by viral infection. These novel results suggest that one focus of future therapeutic attempts to block translation of viral illness into atopic disease should be the CD49d⁺ PMN population.