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Rhinovirus Infection Promotes Eosinophilic Airway after Prior Exposure to House Mite Allergen Downloaded from Amit K. Mehta and Michael Croft

ImmunoHorizons 2020, 4 (8) 498-507 doi: https://doi.org/10.4049/immunohorizons.2000052

http://www.immunohorizons.org/content/4/8/498 http://www.immunohorizons.org/ This information is current as of September 26, 2021.

Supplementary http://www.immunohorizons.org/content/suppl/2020/08/13/4.8.498.DCSupp Material lemental References This article cites 35 articles, 12 of which you can access for free at: http://www.immunohorizons.org/content/4/8/498.full#ref-list-1 by guest on September 26, 2021 Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://www.immunohorizons.org/alerts

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Infectious Disease

Rhinovirus Infection Promotes Eosinophilic Airway Inflammation after Prior Exposure to House Dust Mite Allergen

Amit K. Mehta,*,1 and Michael Croft*† *Center for and Inflammation, La Jolla Institute for , La Jolla, CA 92037; and †Department of Medicine, University of California, San Diego, La Jolla, CA 92093 Downloaded from

ABSTRACT Respiratory virus infection normally drives -dominated airway inflammation, yet some viral infections result in an http://www.immunohorizons.org/ -dominated response in individuals such as allergic asthmatics. One idea is that viral infection simply exacerbates an ongoing type 2 response to allergen. However, prior exposure to allergen might alter the virus-induced innate response such that type 2–like eosinophilic inflammation can be induced. To test this, mice were sensitized intranasally with house dust mite allergen and then at later times exposed to rhinovirus RV1B via the airways. RV1B infection of naive mice led to the expected neutrophilic lung inflammatory response with no or production. In contrast, if mice were exposed to RV1B 1–4 wk after house dust mite inhalation, when the allergen response had subsided, infection led to eosinophilia and mucus production and a much stronger response that were partially or fully steroid resistant. In accordance, RV1B infection resulted in elevated expression of several inflammatory factors in allergen–pre-exposed mice, specifically those associated with type 2 , namely CCL17, CXCL1, CCL2, IL-33, and IL-13. In vitro studies further showed that RV infection led to greater production of chemokines and in human bronchial epithelial cells that were previously stimulated with allergen, reinforcing the notion of an altered virus by guest on September 26, 2021 response after allergen exposure. In conclusion, we report that prior allergen exposure can modify responsiveness of cells in the lungs such that a qualitatively and quantitatively different inflammatory activity results following virus infection that is biased toward type 2–like airway disease. ImmunoHorizons, 2020, 4: 498–507.

INTRODUCTION Cold viruses might drive this type of response, including rhinovirus (RV) (1–5). Typical of most viruses, RVs largely result There is considerable interest in the inflammatory response to in a neutrophil-dominated innate inflammatory response post- viruses that infect the respiratory tract. Most viruses are thought infection, with little or no signs of type 2 immunity, such as mucus to drive a type 1/17–like , resulting in lung production or infiltration of eosinophils into the lungs. Thus, what neutrophilia; however, a number of viral respiratory tract dictates the induction of a neutrophilic versus eosinophilic infections have been linked to type 2–like inflammation typical response is not clear. A number of studies have tried to address of . It has been suggested that viral infections may cause up this in mouse models with coexposure to RV and allergen at the to 90% of asthma-like symptoms in children and 60% in adults. same time. In some studies, with house dust mite (HDM) allergen,

Received for publication June 16, 2020. Accepted for publication July 28, 2020. Address correspondence and reprint requests to: Prof. Michael Croft, Division of Immune Regulation, La Jolla Institute for Immunology, 9420 Athena Circle, La Jolla, CA 92037. E-mail address: [email protected] ORCID: 0000-0002-6062-3635 (M.C.). 1Current address: Fate Therapeutics, La Jolla, CA. This work was supported by National Institutes of Health Grant AI070535 (to M.C.). A.K.M. performed experiments. M.C. and A.K.M. wrote the manuscript. Abbreviations used in this article: BAL, bronchoalveolar lavage; DC, ; HDM, house dust mite; i.n., intranasal, intranasally; PAS, periodic acid–Schiff; RV, rhinovirus. The online version of this article contains supplemental material. This article is distributed under the terms of the CC BY-NC-ND 4.0 Unported license. Copyright © 2020 The Authors

498 https://doi.org/10.4049/immunohorizons.2000052

ImmunoHorizons is published by The American Association of Immunologists, Inc. ImmunoHorizons RHINOVIRUS INDUCES ALLERGIC LUNG INFLAMMATION 499 the investigators failed to demonstrate enhancement of type 2 Institute for Immunology Animal Care Committee in accordance inflammation upon concomitant airway exposure to RV; however, with guidelines of the Association for the Assessment and they did find an increased neutrophil response characteristic of Accreditation of Laboratory Animal Care. that which can be induced by RV infection alone (6–8). Alternatively, other studies found that RV infection given at the RV generation and experimental protocols same time as the model allergen OVA or HDM, or 1 d later, led to RV1B stocks were generated as described elsewhere (16). For enhanced lung infiltration of eosinophils, augmented mucus allergen experiments, mice were sensitized intranasally (i.n.) with production, and an overall greater type 2 response than driven 50 mg of HDM extract (Greer Labs, Lenoir, NC) given on days 0, 1, by the allergen alone (9–14). The disparity between these results is 7, and 8 and then challenged acutely with 25 mg of HDM given on not clear but could be due to the dose of allergen and strength of days 21, 22, and 23. Twelve or fourteen days or 4 wk later, mice response to the allergen, the content of LPS in the allergen that, were either exposed to PBS or infected with 50 mlofRV1B(;5 3 normally, at high levels promotes type 1/17 immunity or the 107 PFU/ml) given i.n. Mice were sacrificed 24 h after the infection amount of virus used. Regardless, in either case, the data implied for all analyses. For studies with steroid treatment, mice were that infection acted through amplifying the production of type 1/17 sensitized and challenged with HDM as above and either received or type 2 inflammatory mediators in the lungs induced by the PBS or (Sigma-Aldrich) i.n. at 0.5 mg/kg of body Downloaded from allergen. weight in a volume of 50 ml, 30–60 min before the RV1B challenge. An alternative mechanism is also possible to explain a link For some experiments, RV1B was inactivated by exposure to UV between type 2 lung inflammation and virus infection, although at light for 20 min immediately before infection. In other experi- present underexplored. In this scenario, prior allergen exposure ments, mice were sensitized and challenged with HDM as above, could transiently or permanently alter the infectivity of lung with coexposure to PBS or RV1B given i.n. on the same day as the http://www.immunohorizons.org/ epithelialcellsorotherlungcellsbythevirusorcouldaltertheir last challenge with HDM. responsiveness to viral danger signals such that the virus infection alone triggers a response that resembles that normally induced by Bronchoalveolar lavage analysis and lung histology allergen. Along these lines, one publication found that mice that Bronchoalveolar lavage (BAL) was performed 24 h after the RV1B were repeatedly challenged with pneumonia virus and cockroach challenge. BAL fluid was examined for cellular content. BAL cells allergen subsequently displayed both lung eosinophilia and were counted, and BAL cell differentials were determined using neutrophilia and epithelial cell mucus secretion when infected flow cytometry based on cell surface markers. BAL cells were with RV 1 mo later (15). In this study, we present experimental preincubated with mouse Fc-blocking Ab (2.4G2) and then stained evidence in the mouse that supports these data and furthers the with fluorochrome-conjugated Abs (all from BD Biosciences, La idea that prior allergen exposure can modify the airway response Jolla, CA) to Siglec F (PE), Ly-6C (FITC), Gr-1 (APC), CD11b (PE- by guest on September 26, 2021 to virus. Whereas RV infection of naive mice resulted in airway Cy7), CD11c (eFluor 450), and CD45 (PerCp-Cy5.5) for 20 min at neutrophilia without significant eosinophilia or mucus production, 4°C.SampleswereanalyzedonanLSRIIflow cytometer and infection of mice .1wkandupto4wkaftertheywereexposedto FlowJo software. Part of the lung tissue was kept in RNAlater HDM allergen resulted in strong eosinophilia and mucus pro- solution (Invitrogen, San Diego, CA) for RNA isolation. For lung duction together with neutrophilia and an overall larger lung histology, 5–6-mm sections were cut and stained with H&E for inflammatory response. These data correlated with greater examining cell infiltration and periodic acid–Schiff (PAS) staining induction of type 2 immune-related inflammatory factors in vivo for mucus scoring. Magnification 3200 was used for histologic shortly after RV infection, including chemokines, IL-33, and IL-13. scoring, and at least five fields werescored to obtain the average for Supporting an altered and exacerbated epithelial response, in vitro each mouse, as described elsewhere (16). studies with human bronchial epithelial cells demonstrated that HDM exposure several days prior to virus infection led to an RV infection of epithelial cells intensified response to the virus. Collectively, these results suggest BEAS-2B cells were seeded in 6- or 12-well culture plates as that prior allergen exposure may skew the viral infection toward previously described (16) and 24 h later stimulated with HDM type 2 immunity and allergic-like symptoms and lead to overall (100 mg/ml) for 72 h. Cells were then infected with RV1B at a exaggerated lung inflammation. multiplicity of infection of 0.1 for 6 h and samples extracted for further analyses.

MATERIALS AND METHODS Quantitative RT-PCR TRIzol reagent (Invitrogen, Carlsbad, CA) was used for RNA Mice isolation. An aliquot of total RNA (1 mg) was reverse transcribed to C57BL/6 (8–10-wk-old) mice were purchased from The Jackson cDNA by using Transcriptor First Strand cDNA Synthesis Kit Laboratory (Bar Harbor, ME). All RV experiments, including (Roche Diagnostics, Indianapolis, IN). Quantitative PCRs were infection in mice, subsequent animal housing, and all experimental performed on cDNA generated from mouse lung or epithelial cell processes for sample analyses, were conducted in biosafety level 2 lines with SYBR Green (Roche Diagnostics) using primers rooms. All experiments followed the regulations of the La Jolla described elsewhere (16, 17). RV1B viral RNA expression in the

https://doi.org/10.4049/immunohorizons.2000052 500 RHINOVIRUS INDUCES ALLERGIC LUNG INFLAMMATION ImmunoHorizons Downloaded from http://www.immunohorizons.org/ by guest on September 26, 2021

FIGURE 1. Prior exposure to HDM results in an exacerbated and altered lung inflammatory response to RV. Mice were sensitized i.n. with HDM, and after 3 wk, acutely challenged i.n. with HDM over a 3-d period from days 21–23. (A–C) Twelve or fourteen days later on days 34 or 36, mice were exposed i.n to either PBS or RV1B. Separately, naive mice were also exposed i.n. to RV1B. Twenty- four hours after exposure to PBS or RV1B, BAL was assessed for (A) eosinophils, , and . Lung sections were scored for (B) bronchial inflammation with H&E stain and (C) percentage of mucus-producing bronchial epithelial cells with PAS stain. Results are mean 6 SEM from four to six mice per group and representative of three experiments. Original magnifications 320 (B), 3200 (C). (Continued)

https://doi.org/10.4049/immunohorizons.2000052 ImmunoHorizons RHINOVIRUS INDUCES ALLERGIC LUNG INFLAMMATION 501 lung was assessed using RV1B specificprimers.Dataarepresented there was a small residual number of eosinophils and lymphocytes as normalized to ribosomal protein housekeeping gene L32 for in the airways resulting from the allergen challenges, which was lungsamplesorGAPDHforBEAS-2Bcells. markedly lower than that seen at the peak of the response 1 d after the last challenge (;3 3 104 versus ;240 3 104 eosinophils; Statistical analyses ;7 3104 versus ;120 3104 lymphocytes; compare Fig. 1A, HDM + All results are presented as mean 6 SEM. Statistical analyses were PBS group, with Supplemental Fig. 1A, 1C, PBS group). Neutrophils performed with GraphPad Prism software (GraphPad Software, were not detected compared with a small but easily visualized La Jolla, CA); for comparison of two groups, a Student t test in number at the peak of the allergen response (;8 3 104;seeFig.1A, case of normally distributed data or a Mann–Whitney U test was HDM + PBS group, versus Supplemental Fig. 1B, PBS group). Most performed. For multiple group data, ANOVA or Kruskal–Wallis interestingly, RV infection of these mice, which were previously with parametric (Bonferroni) or nonparametric (Dunn) posttest, exposedtoallergen12dto2wkbeforehand,resultednotonlyina ff respectively, was performed for comparison of groups. Di erences pronounced BAL lymphocyte response but also a strong eosinophil fi between the groups were considered as statistically signi cant response when measured at 24 h postinfection (Fig. 1A, HDM + RV , with p 0.05. group).A24-htimepointwaschosen,asthisisthepeakofthe Downloaded from virus-induced lung neutrophilic inflammatory response in naive (unsensitized) mice. The response in allergen-exposed mice RESULTS greatly contrasted with that induced by RV infection of naive mice, which resulted in BAL neutrophilia as expected, whereas Prior allergen exposure modifies the airway response to RV only a small number of lymphocytes and a negligible number of infection toward eosinophilia and mucus production eosinophils were detected in the airways (Fig. 1A, RV group). http://www.immunohorizons.org/ Varying results have been reported with RV infection of mice There was a trend to an increased neutrophil response in mice together with allergen challenge. In some studies, RV infection at pre-exposed to allergen, although this was not statistically signif- the same time as inhalation of OVA or HDM led to enhanced icant and not augmented to the same extent as the eosinophil allergic type 2 lung inflammation that developed several days after and lymphocyte response (Fig. 1A, HDM + RV group). theinfection(9,10,13,14).Inalternativemodels,whenHDM The results with analysis of BAL also corresponded to the allergen was given together with RV, only an increase in overall lung inflammatory phenotype as measured by tissue neutrophilia was observed but no change in eosinophilia or other fi aspects of Th2-type inflammation (6–8). In our facility, we histology. Scoring of cellular in ltrates around the bronchioles initially tested the effect of RV1B infection in mice that were showed that RV infection of HDM-sensitized mice resulted in an sensitized with HDM for 20 d and then acutely challenged with almost 5-fold increase compared with mice only exposed to HDM fi by guest on September 26, 2021 i.n. HDM over 3 d on days 21, 22, and 23 (Supplemental Fig. 1). In or naive mice receiving RV (Fig. 1B). Signi cantly, RV infection of contrast to the aforementioned reports, RV infection at the time naive mice alsodidnotresult in anyappreciablemucus production, of the final allergen challenge, on day 23, did not change the as visualized by quantitating the proportion of PAS positive – immediate acute lung inflammatory response measured 1 d later bronchial epithelial cells, whereas infection of HDM pre-exposed when the allergen-induced response is at its peak. This included mice led to a doubling of the baseline mucus response that was no significant effect on BAL neutrophilia or eosinophilia, a residual in the allergen-challenged animals (Fig. 1C). Collectively, result possibly explained by such a strong response to the these results show that prior exposure of the lungs to allergen not allergen that any effect of the virus was masked. Thus, the only primes for a greater inflammatory response following general concept of exacerbated airway inflammation after virus infection with RV, but importantly biases the initial immediate infection when there is concomitant allergen exposure may RV response toward an allergic phenotype that includes eosino- depend on the strength of the allergen challenge and time of philia and mucus production. analyses or other factors not examined. We also tested whether the effect of allergen on the RV- To pursue an alternate scenario, we examined the RV-induced induced response was transient or long lasting. Rather than acute response at times after the allergen-driven inflammatory performing the infection at 12–14 d after the last allergen exposure, response had subsided, which usually occurs within 1 wk after mice were rested for 4 wk and then infected with RV (Fig. 1D, exposure. Mice were sensitized and challenged with HDM as Supplemental Fig. 2). RV infection again drove eosinophilia in the before over 23 d and then rested for 12 or 14 d before i.n. infection allergen–pre-exposed animals (Fig. 1D). However, this was less with RV1B on day 34 or 36 (Fig. 1, Supplemental Fig. 2). At this time, than seen when infection was performed after 2 wk (compare with

(D) Four weeks later on day 50, mice were exposed i.n to either PBS or RV1B. Separately, naive mice were also exposed i.n. to RV1B. Twenty-four hours after exposure to PBS or RV1B, BAL was assessed for eosinophils, neutrophils, and lymphocytes. Results are mean 6 SEM from three to four mice per group and representative of two experiments. *p , 0.05, **p , 0.01, ***p , 0.001.

https://doi.org/10.4049/immunohorizons.2000052 502 RHINOVIRUS INDUCES ALLERGIC LUNG INFLAMMATION ImmunoHorizons

Fig. 1A). Infection in this case additionally resulted in a much Prior allergen exposure leads to selective upregulation of stronger neutrophil response than seen in unsensitized mice, lung inflammatory gene expression upon RV infection whereas the lymphocyte response was not enhanced (Fig. 1D). To further explore the effect of prior allergen exposure on the RV- These data suggest that the modifying effect of prior allergen induced response, we assessed the expression of transcripts for a exposure on the magnitude and quality of the lung inflammatory range of inflammatory factors in the lungs, some of which were response to virus can persist but that it is most pronounced in previously associated with virus infection (11, 18). Rather than an terms of resulting in an allergic phenotype when infection occurs overall greater response induced by RV in HDM-presensitized within a few weeks of allergen exposure. mice, we saw differential effects depending on the gene examined Downloaded from http://www.immunohorizons.org/ by guest on September 26, 2021

FIGURE 2. RV drives greater lung inflammatory gene expression in mice previously exposed to HDM. Mice were i.n. sensitized and challenged with HDM as in Fig. 1 and subsequently exposed i.n. to RV1B or PBS 12–14 d later. Separately, naive mice were also exposed i.n. to RV1B. After 24 h, lung tissue was analyzed for expression of mRNA for (A) CXCL1, (B) CXCL2, (C) CCL17, (D) CCL5, (E) CCL11, (F) CCL2, (G) IL-6, (H) GM-CSF, (I) IL-33, (J) IL-13, (K) RV1B, and (L) IFN-l. Results are mean 6 SEM from four to six mice per group and a representative of two experiments. *p , 0.05, **p , 0.01.

https://doi.org/10.4049/immunohorizons.2000052 ImmunoHorizons RHINOVIRUS INDUCES ALLERGIC LUNG INFLAMMATION 503

(Fig. 2). Most notably, expression of CXCL1/KC (the murine inactivated virus to prevent replication. Showing UV treatment homolog of IL-8), CCL17 (TARC), CCL2 (MCP-1), IL-33, and IL-13 was effective, lung neutrophilia induced by RV was strongly were significantly upregulated in these mice above levels seen in suppressed in naive mice, as well as those pre-exposed to HDM naive unsensitized mice infected with RV. Upward trends were (Supplemental Fig. 3). In contrast, the enhanced eosinophil seen in expression of CXCL2 (MIP-2a), CCL5 (RANTES), and and lymphocyte response that resulted from RV infection of IL-6 induced by RV in mice previously exposed to allergen com- HDM-sensitized mice was comparable in mice receiving UV- pared with naive mice, although these were not statistically signif- treated virus. icant. No obvious differences in CCL11 (eotaxin-1) or GM-CSF were found, even though mRNA for these genes was upregulated by RV. Allergen exposure leads to enhanced induction of We did not detect IL-5 mRNA induced by RV in either naive mice inflammatory genes in bronchial epithelial cells or mice previously exposed to HDM (data not shown). postinfection with RV We also assessed the expression of IFN-l as a surrogate for the The preceding data suggested that prior allergen exposure level of infection achieved by RV, and this was reduced in allergen- modified the early response to RV infection independent of viral sensitized mice. Moreover, transcripts for RV1B were detected at replication. Because RV is thought to primarily infect epithelial lower levels in these mice compared with unsensitized mice (Fig. cells, the results also implied that altered responsiveness of these Downloaded from 2). It is not clear why lower levels were seen. As similar or cells in the airways might be one of several potential explanations enhanced levels of or chemokine transcripts were for the different activity of the virus in allergen–pre-exposed present, it is unlikely that differing virus levels were responsible animals. To further investigate this possibility, we stimulated the forthe induced ; however,the reducedexpression human bronchial epithelial cell line BEAS2-B in vitro with HDM of IFN-l and viral RNA could have been related to the induced and tested the expression of a range of inflammatory genes http://www.immunohorizons.org/ allergic response. In this regard, prior studies have found an postinfection with RV1B several days later (Fig. 3). HDM extract inverse correlation between IgE and production of IFN-a by contains several entities that might modify epithelial cell re- plasmacytoid dendritic cells (DC) in asthma patients and that IgE sponsiveness, including PAR-2–acting proteases and TLR4 ligands signaling to these cells limits IFN secretion induced by influenza (20, 21). Pre-exposure to HDM led to increased expression of all virus (19). We additionally performed an experiment with UV- molecules assayed. In some cases, this was additive, being the sum by guest on September 26, 2021

FIGURE 3. Prior exposure of bronchial epithelial cells to HDM leads to enhanced expression of inflammatory genes upon infection with RV. BEAS-2B cells were treated with HDM for 3 d and then subsequently exposed to RV1B or mock media. Separate cells were untreated (UN) or exposed to RV1B alone. Cells were examined for mRNA expression of (A) CXCL1, (B) CXCL2, (C) CCL2, (D) CCL5, (E) IL-8, and (F) RV1B. Results are mean 6 SEM from duplicate cultures and a representative of at least two experiments each. *p , 0.05, **p , 0.01.

https://doi.org/10.4049/immunohorizons.2000052 504 RHINOVIRUS INDUCES ALLERGIC LUNG INFLAMMATION ImmunoHorizons of the residual HDM activity and the level of activity induced by wasseeninwhichRVinfectionresultedinmRNAexpressionthat RV in cells not previously stimulated with HDM. This was seen for was more than the sum of each separate activity. This was CXCL1, CXCL2, and CCL2. In other cases, a synergistic activity particularly obvious for CCL5 and, to a lesser extent, IL-8. In Downloaded from http://www.immunohorizons.org/ by guest on September 26, 2021

FIGURE 4. The lung inflammatory response to RV in HDM-sensitized mice is partially corticosteroid resistant. Mice were i.n. sensitized and challenged with HDM as in Fig. 1 and subsequently exposed i.n. to RV1B or PBS 12–14 d later. Separately, naive mice were also exposed i.n. to RV1B. Dexamethasone (Dex.) was given i.n. 30–60 min before RV1B or PBS. After 24 h, BAL was analyzed for (A) eosinophils, (B) neutrophils, and (C) lymphocytes. Lung sections stained with PAS [(D), top] and scored for bronchial inflammation in H&E-stained sections (left panel) and percentage of mucus-producing bronchial epithelial cells (right panel). Results are mean 6 SEM from three to four mice per group and a representative of two experiments. Original magnification 3200 (D). *p , 0.05.

https://doi.org/10.4049/immunohorizons.2000052 ImmunoHorizons RHINOVIRUS INDUCES ALLERGIC LUNG INFLAMMATION 505 contrast to the in vivo results, we did not find CCL17, CCL11, or but perhaps the response to HDM in our study was quantitatively IL-33 upregulated by RV1B in unstimulated or stimulated cells greater than that to cockroach allergen, allowing the altered (data not shown). An increase in RV1B mRNA was also detected in responsiveness to RV without a previous virus infection. Compared BEAS-2B cells first stimulated with HDM (Fig. 3), although this with normal subjects, allergic asthmatic patients known to be was not sufficient to explain all of the effects on inflammatory gene infected with RV have been reported to either show enhanced expression. These results suggest that the altered response to RV neutrophilic inflammation (23) or both eosinophilic and neutro- in allergen–pre-exposed animals could in part be related to en- philic inflammation (24, 25) in the lower airways. Our model hanced responsiveness of epithelial cells that are directly infected system, and the aforementioned cockroach system, displayed both with RV in the lungs. of these phenotypes. Collectively, the data suggest that the viral response in the lungs of allergic individuals recently exposed to The allergic-type airway response to RV in allergen–pre- their allergen might differ in both magnitude and quality compared exposed mice is partially steroid resistant with nonallergic individuals or allergic individuals that did not It is of interest to know whether a virus-induced inflammatory recently inhale their allergen. response in the lungs is amenable to conventional anti-inflammatory The explanation for the allergic phenotype of eosinophilia and treatment. We then finally asked if the allergic-like response to RV mucus production induced by respiratory virus infection is not Downloaded from seen in mice previously sensitized with HDM was affected by complete. The virus-induced allergic response could be explained dexamethasone, given only during the time of infection. Importantly, by greater infectivity of allergen-primed lung cells or an altered eosinophil, neutrophil, and lymphocyte accumulation induced in the responsiveness of the cells that become infected. Although we BAL of RV-infected HDM-sensitized mice was largely unaffected by cannot rule out the former, we did not detect greater levels of viral steroid treatment (Fig. 4A–C). When lung tissue inflammation was mRNA in vivo in allergen–pre-exposed mice, and in line with this http://www.immunohorizons.org/ assessed by histologic scoring of H&E sections, we did find a IFN-l, which often correlates with viral load, was not enhanced. statistically significant reduction in mice treated with dexametha- Moreover, UV-inactivated virus also elicited the same allergic sone, although much of the RV-driven response was still intact. This phenotype. Th2 cytokines have been suggested to enhance RV likely reflected the combined eosinophil, neutrophil, and lymphocyte infectivity by upregulating expression of receptors like ICAM-1 accumulation but suggested that some aspect of the cellular response (26), in line with the observation in some studies that severity of was sensitive. Additionally, the RV-driven elevation in number of allergic symptoms in humans correlated with prolonged or mucus-producing bronchial epithelial cells was blocked by steroid persisting virus infection (24). Nevertheless, more studies have treatment down to levels seen in mice exposed to HDM but not RV found that allergic symptoms do not correlate with increased viral (Fig. 4D). Collectively, these data show that prior exposure to load or delayed clearance (23, 27). Therefore, greater overall allergen results in an allergic-likeimmediateresponsetoRVinthe infectivity might not explain the altered response to RV, and by guest on September 26, 2021 lungs that is, in part, steroid resistant. differential responsiveness of infected cells could be important. Given the rapid nature of the response that we measured, taking place within 24 h of infection, it is logical that the cells that were DISCUSSION infected with the virus would control the early lung influx of eosinophils, lymphocytes, and neutrophils that we observed. Many prior publications have highlighted the ability of RV to Lung epithelial cells can produce a number of chemokines and exacerbate an ongoing allergen-driven lung inflammatory re- cytokines when infected with RV (28), and enhanced production sponse when virus infection occurs simultaneously or within a day of chemokines has been linked to respiratory infection in allergic of allergen inhalation (5–10, 12–14, 22). In this report, we show asthmatic subjects (29). Our data with analyses of inflammatory that animals that were sensitized with HDM allergen several molecules upregulated in vivo in part support this notion, with weeks prior to RV infection displayed an altered immediate lung CXCL1/KC, CCL17, and CCL2 being significantly elevated in RV- inflammatory response that resembles the mixed eosinophilic and infected, allergen–pre-exposed mice over levels seen in RV- neutrophilic responses normally induced by allergens, a phenotype infected naive mice, which could contribute, at least in part, to the that was not evident with the same viral infection in a nonallergic enhanced immune infiltrate. Our in vitro studies with the human animal. bronchial epithelium cell line also in part support the notion that Our data directly support a previous study that also asked if chemokine production could explain some of the allergic lung exposure to allergen could modify the subsequent response to phenotype. However, there was not complete concordance RV, with results that were quite similar to the data presented in between these data and the in vivo data. Differences between in this study (15). The model system differed from ours in using vitro and in vivo data might be due to BEAS-2B cells not being cockroach allergen together with a prior infection with mouse representative of the epithelial cells infected in the mouse lung or pneumonia virus, and it was only in mice previously exposed to that some of these chemokines we observed in vivo were derived both virus and allergen that the authors found that RV infection from nonepithelial cells. We did not see significant differences in resulted in eosinophilia and mucus production along with CCL11/eotaxin-1 in vivo or in vitro that has been more specifically neutrophilia. It is not obvious why this study did not observe a linked to eosinophil recruitment. It is not clear if this was due to similar phenomenon with only prior cockroach allergen exposure, sampling at the wrong time or simply that other factors might be

https://doi.org/10.4049/immunohorizons.2000052 506 RHINOVIRUS INDUCES ALLERGIC LUNG INFLAMMATION ImmunoHorizons responsible for the eosinophilia observed. IL-33 is an epithelial In summary, we show that allergic mice that are exposed to cell–derived molecule, although it can also be made by macro- HDM allergen several weeks prior to RV infection display rapid phages. Recent experiments have suggested that IL-33 can be as inflammation in the lungs that is not only of greater magnitude potent as IL-5 and CCL11 in controlling eosinophil migration (30, than that induced in allergen-naive mice but also resembles a 31). We found increased IL-33 production in RV-infected HDM response to allergen. Our studies have implications for viral allergic mice, and the previously mentioned study demonstrated induction of lung inflammation and factors that might modulate enhanced IL-33 expression in lung epithelial cells in cockroach the immune response that results in the lungs after respiratory allergen allergic mice and that blocking IL-33 partially reduced infection. eosinophilia driven by RV (15), supporting a role for this cytokine. These results then suggest that enhanced production of chemo- DISCLOSURES kines and IL-33 by RV-infected epithelial cells in allergen–pre- exposed mice islikelyto contribute in part to the altered eosinophil The authors have no financial conflicts of interest. and lymphocyte response. This may apply to any epithelial cell that was previously stimulated by allergen (via TLR ligands, proteases, and other mechanisms). 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