TLR2-expressing macrophages are required and sufficient for rhinovirus-induced airway inflammation

Abstract

Background

We have shown that rhinovirus (RV), a cause of asthma exacerbations, colocalizes with CD68- and CD11b-positive airway macrophages following experimental infection in humans. We have also shown that RV-induced cytokine expression is abolished in TLR2−/− bone marrow-derived macrophages.

Objective

We hypothesize that TLR2+ macrophages are required and sufficient for RV-induced airway inflammation in vivo.

Methods

To determine the requirement and sufficiency of TLR2 for RV-induced airway responses, naïve and ovalbumin-sensitized and challenged C57BL/6 wild-type and TLR2−/− mice were infected with RV1B followed by IgG or anti-TLR2. Bone marrow chimera experiments using OVA-treated C57BL/6 and TLR2−/− mice were also performed. Finally, naïve TLR2−/− mice underwent intranasal transfer of bone marrow-derived wild type macrophages.

Results

RV1B infection of naïve wild-type mice induced an influx of airway neutrophils and CD11b+ exudative macrophages which was reduced in TLR2−/− mice. In allergen-exposed mice, RV-induced neutrophilic and eosinophilic airway inflammation and hyperresponsiveness were reduced in TLR2−/− and anti-TLR2-treated mice. Transfer of TLR2−/− bone marrow into wild type ovalbumin-treated, C57BL/6 mice blocked RV-induced airway responses, whereas transfer of wild type marrow to TLR2−/− mice restored them. Finally, transfer of wild-type macrophages to naïve TLR2−/− mice was sufficient for neutrophilic inflammation after RV infection, whereas macrophages treated with IL-4 (to induce M2 polarization) were sufficient for eosinophilic inflammation, mucous metaplasia and airways hyperresponsiveness.

Conclusions

TLR2 is required for early inflammatory responses induced by RV, and TLR2+ macrophages are sufficient to confer airway inflammation to TLR2−/− mice, with the pattern of inflammation depending on macrophage activation state.

Introduction

Rhinovirus (RV) is the most common cause of asthma exacerbations in children and adults. However, the precise mechanisms by which RV induces disease exacerbation are not completely known. Although airway epithelial cells are thought to be a major target of RV, infection of epithelial cells is spotty after experimental infection ¹. Cytokine-producing cells of the monocyte/macrophage lineage may also interact with RV. We have noted colocalization of RV with CD68, a sialoadhesin associated with macrophage lineage cells, in the lungs of ovalbumin (OVA)-sensitized and -challenged, RV-infected mice ² and human asthmatics experimentally infected with RV ³. In OVA-treated mice, depletion of macrophages decreased RV-induced airway inflammation and hyperresponsiveness, suggesting that macrophages play a direct role in the response to RV ². In this model, RV infection induces an influx of CD45+, CD68+, F4/80+, Ly6c+, CD11b^high inflammatory monocytes and exudative macrophages into the lung which, if preceded by OVA treatment, express the M2 alternative-activation markers CD206 and CD301 and produce IL-13 and other type 2 cytokines ⁴.

Pattern-recognition receptors, including members of the Toll-like receptor (TLR) family, appear to play a key role in sensing RV infection. Inhibition of TLR3, an endosomal receptor that interacts with double-stranded RNA, decreases RV-induced interferon expression in cultured airway epithelial cells ⁵. TLR3 null mice infected with RV1B show normal IFN responses and unchanged viral titers, but reduced lung inflammatory responses and airways responsiveness ⁶. On the other hand, RV-induced cytokine expression and viral attachment were abolished in bone marrow-derived macrophages from TLR2 but not TLR3 knockout mice ⁷, suggesting a specific requirement of TLR2 in macrophage-mediated responses. TLR2-dependent cytokine expression did not depend on viral endocytosis or replication. TLR2, a membrane surface receptor which recognizes microbe membrane constituents such as lipoteichoic acids, peptidoglycan and lipopeptides, was recently shown to be required and sufficient for RV-induced NF-κB activation in cultured airway epithelial cells and HEK cells, respectively ⁸. TLR2 inhibition also blocked responses to replication-deficient UV-irradiated virus. In addition, we have shown that TLR2 is required for RV-induced IRAK-1 degradation in airway epithelial cells ⁹. Together, these data suggest that TLR2 recognizes some component of the RV viral capsid.

In the present study, we hypothesized that TLR2+ macrophages are responsible for RV-induced airway inflammation in vitro and in vivo. To test this, we examined the requirement of TLR2 for RV-induced responses in human peripheral blood monocyte-derived macrophages. Next, we infected naïve and allergen-challenged TLR2−/− and anti-TLR2-treated mice with RV1B, a minor group virus which replicates in mouse airways ¹⁰. Bone marrow chimera experiments using OVA-treated C57BL/6 and TLR2−/− mice were also performed. Finally, to specifically examine the role of TLR2 expressed on airway macrophages, we transferred wild-type bone-marrow derived macrophages to TLR2 knockout mice and examined their effects on RV-induced airway responses.

Materials and Methods

Macrophages

To obtain mouse macrophages, bone marrow was isolated and cultured using L929 mouse fibroblast supernatants as a source of macrophage colony-stimulating factor (M-CSF), as described ^{7, 11}. To obtain human macrophages, CD14+ peripheral blood monocytes (Precision Bioservices, Frederick, MD) were incubated with 50 ng/ml M-CSF (Peprotech Rocky Hill, NJ) for seven days. Selected cultures were also incubated with 50 ng/ml IL-4 (Peprotech), 30 μg/ml monoclonal anti-TLR2 neutralizing antibody (clone T2.5, eBioscience, San Diego, CA) or isotype control antibody

Animals

C57BL/6 mice WT and TLR2−/− (B6.129-Tlr2^tm1Kir/J TLR2 KO) were purchased from Jackson Laboratory (Bar Harbor, ME). All animal usage was approved by the Institutional Animal Care and Use Committee and followed guidelines set forth in the Principles of Laboratory Animal Care from the National Society for Medical Research. Mice were housed in the pathogen-free facility within the Unit for Laboratory Animal Medicine at the University of Michigan.

RV infection and allergen sensitization

RV1B (ATCC, Manassas, VA), a minor group virus that infects mouse cells ¹², was partially purified from infected HeLa cell lysates by ultrafiltration using a 100 kD cut-off filter and titered by plaque assay ^{10, 13}. Intact virus fails to go through the filter and is concentrated. Retentates from uninfected HeLa cells were used for sham infections. Viral preparations tested negative for Mycoplasma (Mycoprobe Mycoplasma Detection Kit, R&D Systems, Minneapolis, MN) or LPS (Pierce Endotoxin Quantitation Kit, Thermoscientific, Rockford, IL). For infection of cultured mouse macrophages, RV1B was purified by sucrose gradient centrifugation ¹³. Finally, human macrophages were infected with either sucrose gradient-purified RV1B or RV39 (ATCC), a major group virus. Viral proteins were detected by silver staining and anti-RV16 antibody (QED Biosciences, San Diego, CA).

Six-to-eight week-old wild-type and TLR2−/− mice, each with a C57BL/6 genetic background, were inoculated with 50 μl of 1×10⁸ PFU RV1B or an equivalent volume of sham HeLa cell lysate. In certain experiments, mice were sensitized intraperitoneally with 50 μl of 2 mg/ml endotoxin-free OVA in phosphate-buffered saline (PBS) mixed with alum or PBS control on days 0 and 7, followed by intranasal challenge with 50 μl of 2 mg/ml OVA or PBS on days 7, 12 and 13. These mice were inoculated with RV1B or sham on day 14 of the protocol. Lungs were harvested for analysis 1–3 days after infection. Finally, selected mice were treated intranasally with anti-TLR2 (100 μg in 50 μl) following the last OVA challenge.

Bone marrow chimeras

Chimeric mice were used to determine the source of TLR2 critical to RV-induced airways inflammation and hyperresponsiveness. Adoptive transfer of bone marrow from wild type to TLR2 null and wild type mice, and conversely the transfer of TLR2−/− bone marrow into TLR2−/− and wild-type mice, were performed as described^{14, 15}. Briefly, mice underwent ¹³⁷Cs-gamma-irradiation (13 Gy) from a Gammacell 40 Exactor Irradiator (Best Theratronics, Ottawa, ON) located in the Unit for Laboratory Animal Medicine-managed Experimental Irradiation Core facility. After a stabilization period of 6–8 weeks to allow clonal repopulation of the lung and periphery by transferred stem cells, mice were exposed to OVA with or without RV.

Histology and immunofluorescence microscopy

Lungs were fixed with 4% formaldehyde overnight. Five μm-thick paraffin sections and processed for histology or fluorescence microscopy as described ². Lung sections were stained with hematoxylin and eosin or Periodic acid-Schiff (PAS). Anti-TLR2 was labeled with AlexaFluor 555 succinimidyl ester (Life Technologies, Waltham, MA). Eosinophil major basic protein (MBP) was visualized using rat monoclonal anti-mouse MBP (MT-12.7). This antibody was obtained from the Dr. James J. Lee ¹⁶ and directly conjugated to AlexaFluor 555

Determination of viral copy number

Lungs were homogenized and RNA extracted with Trizol (Sigma-Aldrich, St. Louis, MO). Positive strand RNA was measured by quantitative onestep PCR. Copy numbers of positive strand viral RNA were normalized to 18S RNA, which was similarly amplified using gene-specific primers and probes. Primer sequences are shown in the Table.

Table.

Primer sequences for real-time quantitative PCR.

mRNA	Forward primer (5′ to 3′)	Reverse primer (5′ to 3′)
RV	GCT GCC TGC ACA CCC TGA GGG GTGT	GAA ACA CGG ACA CCC AAA GTA GT
TNF-α	ATG CAC CAC CAT CAA GGA CTC AA	ACC ACT CTC CCT TTG CAG AAC TC
CXCL1	TGC ACC CAA ACC GAA GAA GTC AT	CAA GGG AGC TTC AGG GTC AAG
IFN-α	CCA TCC CTG TCC TGA GTG	CCA TGC AGC AGA TGA GTC CTT
IFN-β	GAC GGA GAT GAT GGA GAA GAG TTA G	CCA CCC AGT GCT GGA GAA
IL-13	TCC CTG ACC AAC ATC TCC AAT	ACA GAG GCC ATG CAA TAT CC
CCL24	ACC TCC AGA ACA TGG CGG GC	AGA TGC AAC ACG CGC AGG CT
TLR2	TCG TTC ATC TCT GGA GCA TC	TTG ACG CTT TGT CTG AGG TT
GAPDH	GTC GGT GTG AAC GGA TTT G	GTC GTT GAT GGC AAC AAT CTC

Measurement of cytokine mRNA expression

Lung RNA was extracted with Trizol (Sigma-Aldrich) and analyzed for cytokine gene expression by quantitative real-time PCR using specific primers and probes (Table). Signals were normalized to GADPH using the comparative ΔΔCT method.

Flow cytometry

Mouse lungs were perfused with 5 mM ethylene diaminetetracetic acid (EDTA) in PBS, minced and digested in collagenase. Lung cell suspensions were processed and stained with fluorescent-labeled antibodies against CD45, F4/80, CD11b, CD11c, Ly6c, Gr1, Siglec F, as described previously ⁴. All antibodies were purchased from Biolegend (San Diego, CA) and flow data was analyzed using FlowJo software (TreeStar, Ashland, OR).

Measurement of airway responsiveness

Mice were anesthesized, intubated and ventilated with a Buxco Finepoint system (Wilmington, NC). To assess airways responsiveness, mice were administered increasing doses of nebulized methacholine, as described ¹⁰.

Adoptive transfer of macrophages to the airways

Mouse macrophages were cultured in L929 media for five days, incubated in the presence or absence of IL-4 overnight and then pulsed with carboxyfluorescein diacetate succinimidyl ester (CFSE, Life Technologies) for 10 minutes. Macrophages were transferred to mice intratracheally at 10⁶ cells/mouse 24 hours prior to RV infection.

Results

TLR2 is required for RV-induced cytokine expression in cultured mouse macrophages

We have shown that RV1B-induced cytokine expression is attenuated in TLR2−/− bone marrow-derived macrophages ⁷. We repeated this experiment, substituting virus purified by sucrose gradient centrifugation for virus partially purified by ultrafiltration. RV-induced expression of TNF-α and CXCL1 expression was blocked in TLR2 null cells (Figure 1A).

Figure 1. Infection of C57BL/6 and TLR2 null mice.

A. RV1B infection of bone marrow-derived macrophages from wild type C57BL/6 and TLR2−/− mice. B, C. RV infection of human macrophages derived from CD14+ peripheral blood monocytes. Macrophages were cultured the presence or absence of IL-4 and infected with sucrose gradient-purified RV1B (B) or RV39 (C). Whole lung mRNA expression was measured by quantitative PCR. Signals were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the comparative CT (2−ΔΔCT) method and expressed as fold-change versus GAPDH. (N=3 samples per condition, mean±SEM, *different from wild type or IgG control, p<0.05, ANOVA.)

TLR2 is required for RV-induced cytokine expression in cultured human macrophages

In this set of experiments, macrophages were generated from human CD14+ peripheral blood monocytes and infected with sucrose gradient-purified RV1B or RV39. Selected cultures were treated with IL-4 or anti-TLR2. RV1B- and RV39-infected human macrophages each produced CXCL2 and CXCL8 (Figures 1B, 1C). Incubation with IL-4 shifted the macrophage response to RV towards an M2 polarization pattern, with infected cells producing the eosinophil chemoattractant CXCL11 and IL-13 (Figures 1B, 1C). With both minor and major group RV, cytokine responses were blocked by anti-TLR2.

TLR2 knockout does not affect RV copy number

We sought to determine whether TLR2 is required for RV-induced responses in vivo. First, to assess whether TLR2 plays a role in RV replication or clearance in the lung, wild type C57BL/6 and TLR2−/− mice were infected with RV for up to three days. Lungs were harvested at different time points post-infection and processed for positive strand viral RNA. Measurement of RV copy number between wild-type and TLR2−/− mice showed no difference at each of the indicated time points (Figure 2A).

Figure 2. RV-induced airway inflammation is TLR2-dependent.

Wild-type and TLR2−/− mice infected with intranasally with RV or sham HeLa cell lysate control, sacrificed and the lungs harvested at different time points. A. RV positive strand RNA was assessed 24–72 h after infection and normalized by total μg of RNA. B. Transcript levels of TLR2 from lungs of mice infected with RV. C. BAL neutrophils. D–G. Transcript levels of pro-inflammatory cytokines CXCL1 and TNF-α (D, E) and the type I interferons IFN-α and IFN-β (F, G) were assessed by quantitative PCR and results expressed as fold change over GADPH. (For A–G, N=3 mice per group, mean±SEM, *different from wild type, p<0.05, ANOVA.) H. Hematoxylin and eosin staining of lung sections from sham and RV-infected mice harvested 24 h post-inoculation. Magnification, 200X.

RV induces TLR2 mRNA expression

It has been shown previously that RV increases TLR2, TLR7 and TLR8 expression in cultured airway epithelial cells ⁸. In cells inoculated with replication-deficient UV-irradiated virus, only TLR2 was induced. We tested whether TLR2 mRNA expression was increased in the lungs of RV-infected mice (Figure 2B). TLR2 transcripts were increased 24 hours after infection and persisted to the 48 hour time point.

TLR2 knockout attenuates RV-induced airway inflammation

Following RV infection of C57BL/6 mice, CXCL1 and TNF-α mRNA expression were increased significantly beginning at four hours post-infection and remained so 48 hr post-infection (Figures 2D, 2E). Pro-inflammatory cytokine levels were significantly reduced in TLR2−/− mice. The type I interferons IFN-α and IFN-β were induced following RV infection (Figures 2F, 2G). IFN levels were significantly reduced in TLR2−/− mice.

To further examine differences in RV response between wild type and TLR2−/− mice, histological sections were stained with H&E. Lungs of wild-type animals revealed peribronchial inflammatory and intraluminal infiltrates (Figure 2H). In contrast, RV-infected TLR2−/− mice showed very little inflammation. Consistent with our previous findings, lung digests from infected wild-type mice showed a significant influx of CD11b+Ly6c+ exudative macrophages, whereas influx was absent from RV-infected TLR2−/− mice (Figure 3).

Figure 3. RV-induced infiltration of CD11b+ Ly6c+ exudative macrophages into the airiways is TLR2-dependent.

A. Lung single-cell suspensions were gated for cell viability, surface expression of CD45 and F4/80, and then CD11b and Ly6c. B. Group mean data for total CD45+, F4/80, CD11b+ and Ly6c+ cells per lung (n=3).

RV infection of OVA-sensitized and -challenged mice induces a synergistic increase in airway eosinophils which is dependent on TLR2

Since TLR2 knockout resulted in a significant reduction in RV-mediated airway inflammation in naïve mice, we next examined whether TLR2 plays a role in RV-mediated exacerbation of allergic airway disease. Wild type and TLR2−/− C57BL/6 mice were sensitized and challenged with OVA for two weeks and then infected with RV overnight. As shown previously^{2, 4}, wild type mice sensitized with OVA showed an influx of neutrophils and eosinophils into the bronchoalveolar lavage fluid (Figure 4A, 4B). RV infection of allergen-sensitized and -challenged mice showed a synergistic increase in airway eosinophils; TLR2−/− knockout significantly reduced RV-induced neutrophils and eosinophils, but had no effect on airway inflammation induced by OVA alone. TLR2−/− mice showed fewer eosinophils in the airway tissue (Figure 4C). The TLR2−/− knockout also significantly attenuated RV-induced airways hyperresponsiveness in OVA-treated mice (Figure 4D).

Figure 4. TLR2 is required for RV-induced airway inflammation and responsiveness in OVA-sensitized and -challenged mice.

A–D. Female C57BL/6 wild-type or TLR2 null mice were exposed to either PBS or OVA before infection with RV or sham control for 24 h. BAL neutrophils (A) and eosinophils (B). (N=3 mice per group, mean±SEM, *different from wild type, †different from OVA/sham, p<0.05, ANOVA.) C. Lung sections stained with hematoxylin and eosin are shown. (Original magnification, 200X; arrows in inset indicate eosinophils.) D. Airways responsiveness was measured in tracheotomized animals exposed to increasing doses of methacholine (N=3 mice per group, mean±SEM, *different from wild type OVA/RV mice, p<0.05, two-way ANOVA.) E–G. C57BL/6 wild-type mice were exposed to either PBS or OVA before treatment with IgG or anti-TLR2 and infection with RV or sham control. E. Lung neutrophils and eosinophils were assessed by flow cytometry, gating on high-complexity, CD45+, Gr1+, Siglec F+ cells. Whole lung mRNA expression of CXCL1 (G) and CCL24 (H) was measured by qPCR. (N=3 mice per group), mean±SEM, *different from IgG, †different from OVA/sham, p<0.05, ANOVA.)

We also administered neutralizing antibody against TLR2 to determine the requirement of TLR2 for RV-induced responses. Antibody was administered just prior to RV infection. Lung neutrophils and eosinophils were identified by flow cytometry using the markers CD45, CD11b, Gr1 and Siglec F ¹⁷. Anti-TLR2 significantly decreased RV-induced neutrophilic and eosinophilic airway inflammation (Figure 4E). Consistent with this, OVA-treated, RV-infected mice showed additive or synergistic increases in TNF-α and CXCL1 mRNA expression which were decreased by treatment with anti-TLR2 neutralizing antibody given prior to RV infection (Figure 4F, 4G).

Bone marrow chimeras

To determine the relative contributions of TLR2+ bone marrow-derived and structural cells to RV-induced airways inflammation and hyperresponsiveness, we performed adoptive transfer of wild type bone marrow into TLR2−/− and wild type mice and, conversely, the transfer of TLR2−/− bone marrow into TLR2−/− and wild-type mice. Six weeks after transfer, mice were sensitized and challenged with OVA as described above, and exposed to sham or RV. BAL fluid was harvested for cell counts. All mice showed neutrophilic inflammation in response to OVA exposure and sham infection (Figure 5A). However, only wild type and TLR2−/− mice reconstituted with wild type bone marrow showed a significant increase in BAL neutrophils after RV infection. A similar pattern was observed with BAL eosinophils. Only wild type and TLR2−/− mice reconstituted with wild type bone marrow showed a significant increase in BAL eosinophils after RV infection. However, wild type mice reconstituted with TLR2−/− bone marrow showed a trend towards increased airway eosinophils after RV infection. Also, TLR2−/− mice reconstituted with TLR2−/− bone marrow showed few eosinophils after OVA treatment either before or after RV infection.

Figure 5. Bone marrow chimeras.

Following 13 Gy ¹³⁷Cs-gamma-irradiation, wild type bone marrow was transferred into TLR2−/− and wild type mice and, conversely, TLR2−/− bone marrow was transferred into TLR2−/− and wild-type mice. Six-to-eight weeks after transfer, mice were sensitized and challenged with OVA and exposed to sham or RV. A. BAL fluid was harvested for cell counts. B. Airways responsiveness was assessed by exposure to increasing doses of inhaled methacholine. Total respiratory resistance was measured with a Buxco plethysmograph. (N=6, mean±SEM, *different from sham, p<0.05, one-way or two-way ANOVA, as applicable.)

Next, we assessed airways cholinergic responsiveness (Figure 5B). Mice undergoing irradiation and bone marrow transfer followed by OVA exposure and RV infection tended to have lower airway responses than non-irradiated mice (compare to Figure 4), leading us to increase the maximal concentration of methacholine. Only wild type and TLR2−/− mice reconstituted with wild type bone marrow showed a significant increase in airways responsiveness after RV infection. Similar to the BAL cell counts, wild type mice reconstituted with TLR2−/− bone marrow showed a trend towards increased responsiveness after RV infection.

Adaptive transfer of wild-type macrophages to the airways of TLR2−/− mice

Since we have previously shown that macrophages are required for RV-induced airway inflammation ^{2, 4} and that TLR2 is essential for RV-induced cytokine production by macrophages ⁷, we tested whether transferring TLR2-positive wild type bone marrow-derived macrophages would be sufficient to increase airway inflammation in TLR2−/− mice infected with RV. Bone marrow-derived macrophages from wild-type or TLR2−/− mice were transferred to TLR2−/− mice intranasally. Twenty-four hours later, animals were infected with RV. Transferred, CFSE-stained macrophages were detectable in recipient animals (Figure 6A). TLR2−/− mice receiving wild-type macrophages showed a significant increase in neutrophil influx in response to RV compared to mice receiving TLR2−/− macrophages (Figures 6B, 6C). There was no change in airway eosinophils. Next, to simulate prior antigen sensitization and challenge, bone marrow macrophages were treated with 20 μg/ml IL-4 and then transferred to TLR2−/− hosts. We have previously shown that IL-4 treatment is sufficient to polarize bone marrow-derived macrophages to an alternatively-activated M2 phenotype which is characterized in part by type 2 cytokine production ⁷. TLR2−/− mice receiving IL-4-polarized wild-type macrophages showed significant increases in both neutrophils and eosinophils in response to subsequent RV infection. The presence of eosinophils was verified by staining with myelin basic protein (Figure 6C). Transfer of IL-4-treated TLR2−/− macrophages had no effect. It should be noted that the level of inflammation was lower than that of wild-type mice infected with RV (Figure 4). Transfer of untreated macrophages increased whole lung mRNA expression of the neutrophil chemoattractant CXCL1, whereas transfer of IL-4-treated macrophages increased expression of both CXCL1 and the eosinophil chemoattractant CCL24 (Figure 6B). Transfer of IL-4-treated wild type macrophages to TLR2−/− mice induced mucous metaplasia, as evidenced by PAS staining (Figure 6D). Transfer of untreated wild type macrophages or IL-4-treated TLR2−/− macrophages had no effect (not shown). Finally, we examined the effect of IL-4-treated macrophages on RV-induced airways responsiveness (Figure 6E). Compared to IL-4-treated TLR2−/− macrophages, transfer of IL-4-treated wild-type macrophages caused a small but significant increase in methacholine response. Together, these data suggest that the TLR2+ M2-polarized macrophage is sufficient for the type 2 cytokine response to RV.

Figure 6. Adoptive transfer of bone marrow-derived macrophages from wild-type C57BL/6 mice to TLR2−/− mice partially restores the inflammatory response to RV.

Bone marrow cells from either wild type or TLR2−/− mice were cultured in L929 media for five days, incubated in the presence or absence of IL-4 overnight, pulsed with carboxyfluorescein diacetate succinimidyl ester (CFSE) and instilled intratracheally into TLR2−/− mice. A. Immunofluorescence images showing CFSE-pulsed cells in the airways (green=CFSE, red=TLR2, blue=DAPI; magnification, 200x). B, C. BAL cell counts, whole lung mRNA expression and airway histology from mice 24 h after sham or RV infection. Transfer of untreated macrophages from wild type mice to TLR2−/− mice was sufficient for RV-induced neutrophilic airway inflammation, and transfer of IL-4-treated wild-type macrophages was sufficient for RV-induced eosinophilic inflammation. (N=6–9; as pooled value from three separate experiments, each with three mice per group), mean±SEM, *different from transfer of TLR2−/− cells, †different from transfer of untreated wild type cells, p<0.05, ANOVA.) Transfer of untreated macrophages increased whole lung mRNA expression of the neutrophil chemoattractant CXCL1, whereas transfer of IL-4-treated macrophages increased expression of both CXCL1 and the eosinophil chemoattractant CCL24 (N=3, mean±SEM, *different from transfer of TLR2−/− cells, †different from transfer of untreated wild type cells, p<0.05, ANOVA.) C. Airway sections from mice described in (B) were stained with hematoxylin and eosin and anti-major basic protein (MBP). D. Effect of macrophage adoptive transfer on airway mucus, as demonstrated by PAS staining. E. Effect of macrophage transfer on airway cholinergic responsiveness. TLR2−/− mice treated with IL-4-treated wild-type or TLR2−/− macrophages and then infected with RV. (N=6, mean±SEM, *different from transfer of TLR2−/− cells, p<0.05, two-way ANOVA.)

Discussion

We have found that TLR2 is required for RV-induced airway responses in naïve as well as allergen-sensitized mice. TLR2−/− mice showed significantly attenuated airway inflammation and responsiveness in response to RV infection. In addition, transfer of TLR2-expressing bone marrow cells, as well as IL-4-treated bone marrow-derived macrophages, to TLR2−/− mice was sufficient to induce airway inflammation and hyperresponsiveness upon subsequent infection with RV. These data not only demonstrate a significant role for TLR2 in the innate immune response to RV, but also identify the airway macrophage as a critical cell target and effector of RV infection.

Recent studies have uncovered a potential role for TLR2 in the sensing of RV infection. TLR2, a membrane surface receptor which recognizes bacterial lipopeptides and lipoteichoic acid, was recently shown to be required and sufficient for RV-induced NF-κB activation in cultured airway epithelial cells and HEK cells, respectively ⁸. TLR2 inhibition also blocked responses to replication-deficient UV-irradiated virus. Similarly, we previously found that RV-induced cytokine expression and viral attachment were abolished in bone marrow-derived macrophages from TLR2 knockout mice ⁷, suggesting a specific requirement of TLR2 in macrophage-mediated responses. TLR2-dependent cytokine expression did not depend on viral endocytosis or replication. We also colocalized RV and TLR2 on the macrophage cell surface. In the present study, we confirmed that TLR2 is required for RV-induced cytokine expression in macrophages, this time using a highly-purified viral preparation. Finally, we found that TLR2 was required for RV-induced airway responses in vivo. Together, these data suggest that surface TLR2 responds to a molecular pattern on the viral capsid, rather than to double-stranded viral RNA, a product of viral replication. At first blush, such an interaction is surprising, since picornaviruses are non-enveloped and their capsid does not include a lipid component. On the other hand, picornaviruses interact with cellular proteins during entry into the cell and virus particle assembly, interactions which alter the structure of the future viral capsid. For example, RV and other picornavirus particles are specifically modified by having myristic acid covalently bound to the VP4 capsid protein ^{18, 19}. Such a modification might serve as a ligand for TLR2. Although VP4 normally resides on the capsid inner surface, binding of RV to its cellular receptor induces conformational changes in the virus, including translocation of VP4 the exterior viral surface ²⁰. This altered particle interacts directly with membranes without further involvement of the cellular virus receptor ²¹. It is also conceivable that there is no direct interaction between RV and TLR2; instead, viral capsid could bind with one of TLR2’s many coreceptors, including CD14, scavenger receptors, integrins, dectin-1 or CXCR4²². Finally, TLR2 could be activated by RV-mediated release of a damage-associated molecular pattern.

There is increasing evidence that genetic differences in TLR2 expression influence the development of asthma. Peripheral blood monocytes from farmers’ children with reduced risk of atopy express significantly higher amounts of CD14 and TLR2 than those from non-farmers’ children ²³. Later, the TLR2/−16934 polymorphism, a marker for a group of highly linked TLR2 single nucleotide polymorphisms, was associated with reduced risk of asthma in children of farmers ²⁴. While the functional significance of this polymorphism is not known, it was suggested that this variant could result in increased TLR2 protein expression. On the other hand, the −16934 polymorphism has also been associated with wheeze and bronchial hyperresponsiveness in day care attendees ²⁵, consistent with the notion that TLR2 expression heightens the inflammatory response to RV and other respiratory viral infections. We speculate that an increase in TLR2 expression or function could heighten inflammatory responses to RV infection, whereas a loss of TLR2 could protect against RV exacerbations.

Concerning the cellular source of TLR2 required and sufficient for RV-induced airway responses, we found that transfer of TLR2+ bone marrow cells to TLR2−/− mice was sufficient to confer airways inflammation and responsiveness to RV. Intranasal transfer of IL-4-treated wild type macrophages to TLR2−/− mice was also sufficient for RV-induced airway inflammation and hyperresponsiveness. Taken together, these data implicate the macrophage to be the primary cell responsible for RV-induced airway responses. However, airway epithelial cells are also likely to play a role. First, inflammatory and functional responses to RV following transfer of IL-4-treated TLR2+ macrophages did not reach the level of wild type mice. Second, wild type mice reconstituted with TLR2−/− bone marrow maintained an intermediate level of airway eosinophils and methacholine responsiveness to RV infection. Airway epithelial cells are a source of chemokines after RV infection ^{2, 26}. Also, since significant viral replication does not occur in macrophages, we speculate that epithelial cells are needed as a reservoir for new macrophage-binding viral particles.

Although transfer of TLR2-expressing macrophages to TLR2−/− mice was sufficient to induce airway inflammation upon subsequent infection with RV, untreated macrophages elicited only neutrophilic inflammation, whereas IL-4-treated macrophages elicited both neutrophilic and eosinophilic inflammation. These data are consistent with the notion that ex vivo IL-4 treatment induces M2 polarization, leading to production of eosinophil-attracting chemokines upon RV infection in vivo. We have shown that allergen sensitization and challenge induces alternative activation of lung macrophages, as evidenced by expression of the M2 markers arginase-1, Ym-1, CD206/Mrc1 and CD301/Mgl-2, as well as the secretion of M2 cytokines CCL11, CCL17, CCL22 and CCL24 ^{2, 4, 7}. In the present study, IL-4 treatment increased RV-induced CCL11 and IL-13 expression. Increasing evidence from animal models ^{9, 27–30} and humans ^{31, 32} have implicated macrophages with features of M2 polarization in the pathogenesis of allergic asthma. Based on previous work showing colocalization of RV with CD68- and CD11b-positive macrophages in the airways of human asthmatics experimentally infected with RV (2), we propose that interactions between RV and polarized macrophages are responsible, at least in part, for the increased airway inflammation observed following RV infection of patients with asthma.

There are limitations to our current study. First, while we determined the requirement of TLR2-expressing bone marrow cells for RV-induced airway responses, we did not determine the specific requirement of TLR2-expressing macrophages, which would require a tissue-specific knockout. Second, the absence of strong viral replication in the RV mouse model ¹⁰ could minimize the apparent role of airway epithelial cells which would ordinarily be needed to sustain viral responses by serving as a reservoir for viral replication.

We conclude that, in mice with allergic airways disease, TLR2 is required for early inflammatory responses induced by RV. Macrophage TLR2 expression was also partially sufficient for RV-induced airway inflammation. Future studies examining the mechanism of TLR2 regulation could provide a new therapeutic target for viral-induced asthma exacerbation.

Key Messages.

• In cultured human macrophages, cytokine responses to either rhinovirus (RV)-1B or RV-39 are blocked by neutralizing antibody to TLR2.

• RV1B-induced airway inflammation is blocked in TLR2−/− and anti-TLR2-treated mice.

• Transfer of TLR2−/− bone marrow into wild type ovalbumin-treated, C57BL/6 mice blocked RV-induced airway responses, whereas transfer of wild type marrow restored them.

• Transfer of wild-type macrophages to TLR2−/− mice is sufficient to confer airway neutrophilic inflammation after RV infection, and transfer of IL-4-treated macrophages (to shift cells to an M2 phenotype) is associated with neutrophilic and eosinophilic inflammation, mucous metaplasia and airways hyperresponsiveness, analogous to RV-infected mice with allergic airways disease.

Abbreviations

CFSE

carboxyfluorescein diacetate succinimidyl ester

MBP

major basic protein

OVA

ovalbumin

RV

rhinovirus