IL-4 enhances interferon production by virus-infected human mast cells
Authors: Liliana Portales-Cervantes, Owen M. Crump, Sarah Dada, Christopher R. Liwski, Jelena Gotovina, Ian D. Haidl, Jean S. Marshall
Journal: Journal of Allergy and Clinical Immunology (2020)

It is well recognized that individuals with allergic diseases such as asthma and atopic dermatitis clear some viral infections poorly compared with healthy subjects.1Cheung D.S. Grayson M.H. Role of viruses in the development of atopic disease in pediatric patients.Curr Allergy Asthma Rep. 2012; 12: 613-620Crossref PubMed Scopus (14) Google Scholar,2Wollenberg A. Wetzel S. Burgdorf W.H. Haas J. Viral infections in atopic dermatitis: pathogenic aspects and clinical management.J Allergy Clin Immunol. 2003; 112: 667-674Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar Respiratory viral infections, including respiratory syncytial virus (RSV), influenza A virus (IAV), and rhinovirus, can promote asthma exacerbations and are associated with increased morbidity and mortality. Type I (α/β) and type III (λ) interferons are critical for antiviral defense, inhibit viral replication, and mobilize host defense mechanisms. Deficient IFN production in individuals with allergies has been associated with impaired antiviral responses.3Wark P.A. Johnston S.L. Bucchieri F. Powell R. Puddicombe S. Laza-Stanca V. et al.Asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus.J Exp Med. 2005; 201: 937-947Crossref PubMed Scopus (945) Google Scholar,4Contoli M. Message S.D. Laza-Stanca V. Edwards M.R. Wark P.A. Bartlett N.W. et al.Role of deficient type III interferon-lambda production in asthma exacerbations.Nat Med. 2006; 12: 1023-1026Crossref PubMed Scopus (816) Google Scholar Some authors have reported that TH2-associated mediators, such as IL-4, IL-13, or IgE binding to FcεRI, critically downregulate antiviral mechanisms and IFN production in virus-infected plasmacytoid dendritic cells and epithelial cells.5Contoli M. Ito K. Padovani A. Poletti D. Marku B. Edwards M.R. et al.Th2 cytokines impair innate immune responses to rhinovirus in respiratory epithelial cells.Allergy. 2015; 70: 910-920Crossref PubMed Scopus (94) Google Scholar Mast cells are both key players in allergic disease and critical sentinel cells for host defense against infection. They have been demonstrated to respond to a variety of viral infections, including common respiratory viruses, through the production of mediators that include substantial amounts of a wide range of IFNs, usually without degranulation.6Portales-Cervantes L. Haidl I.D. Lee P.W. Marshall J.S. Virus-infected human mast cells enhance natural killer cell functions.J Innate Immun. 2017; 9: 94-108Crossref PubMed Scopus (6) Google Scholar, 7Al-Afif A. Alyazidi R. Oldford S.A. Huang Y.Y. King C.A. Marr N. et al.Respiratory syncytial virus infection of primary human mast cells induces the selective production of type I interferons, CXCL10, and CCL4.J Allergy Clin Immunol. 2015; 136: 1346-1354.e1Abstract Full Text Full Text PDF PubMed Google Scholar, 8Akoto C. Davies D.E. Swindle E.J. Mast cells are permissive for rhinovirus replication: potential implications for asthma exacerbations.Clin Exp Allergy. 2017; 47: 351-360Crossref PubMed Scopus (16) Google Scholar These responses can be evaluated in vitro by using primary cultured human mast cells such as cord blood–derived human mast cells. We hypothesized that mast cells may modify their IFN expression in the context of an IL-4– or IL-13–rich microenvironment. The impact of IL-4 or IL-13 treatment on human mast cell IFN responses to viral infection was therefore examined. In initial studies, human mast cells were pretreated for 48 hours with IL-4 and then infected with the double-strand RNA virus reovirus. The levels of production of IFN-α2 and IFN-λ1 in response to these infections were assessed. Notably, IL-4 pretreatment significantly enhanced the IFN responses of mast cells following viral infection (Fig 1, A). These responses were dose dependent (Fig 1, B) and consistent across multiple human donors, although there was some variation in the magnitude of effect between individuals. The ability of IL-4 to modify the IFN responses to the ssRNA viruses RSV and IAV was also examined, focusing on IFN-α2 because IFN-λ1 was not substantially induced in mast cells by these infections. The IFN-α2 response to both viruses was significantly enhanced by IL-4 pretreatment of mast cells (Fig 1, C and D) or by treatment of mast cells with IL-4 at the time of infection (Fig 1, E). Notably, for both RSV and reovirus infection, the observed increase in IFN responses took place in the absence of evidence of enhanced levels of viral infection of mast cells. In contrast, elevated IAV gene expression was observed following IL-4 treatment of mast cells (see Fig E1, A-D in this article's Online Repository at www.jacionline.org). In view of these findings, the ability of both human epithelial cells and peripheral blood monocytes to modify their IFN response to viral infection following IL-4 treatment was examined. Peripheral blood monocyte production of IFN-α2 and IFN-λ1 or Calu-3 cell production of IFN- λ1 in response to viral infection was neither enhanced nor inhibited by prior IL-4 treatment (see Fig E2, A and B in this article's Online Repository at www.jacionline.org). Because decreased IFN production could be mediated by alterations in RNA sensor expression, the impact of IL-4 treatment of mast cells on selected RNA sensors was also examined (see Table E1 in this article's Online Repository at www.jacionline.org). The expression levels of most of these were unchanged, with only a modest increase in TLR3 mRNA observed. However, this increase was not accompanied by increased responsiveness to transfection with the TLR3 ligand high-molecular-weight polyinosinic-polycyitidylic acid sodium salt (see Fig E2, C and D). To examine the requirement for extended IL-4 pretreatment and potentially for IL-4–induced maturation, mast cells were exposed to IL-4 at the time of viral infection. Such acute IL-4 treatment had an enhancing impact on IFN production similar to that at 48 hours before IL-4 exposure (Fig 1, A). IL-4 exerts its activity by interacting with either type I (IL-4Rα and the common γ chain [γC] subunits) or type II (IL-4Rα and IL-13Rα1) IL-4 receptor complexes. Because the latter receptor complex is shared with IL-13, these cytokines often induce similar biologic effects. We therefore examined the receptor complex mediating IL-4 enhanced IFN responses to viral infection. Antibody blockade of either IL-4Rα or γC, or pharmacologic inhibition of STAT6 with AS1517499, resulted in loss of IL-4–enhanced IFN responses to viral infection (Fig 2, A and B). In contrast, inhibition of IRS-2 with NT157 did not significantly modify the IFN-λ1 response to IL-4 (n = 6; 1337 ± 378.6 pg/mL for reovirus vs 1998 ± 518.1 pg/mL for reovirus + IL-4 and 1181 ± 363.5 pg/mL for reovirus + NT-157 vs 1672 ± 466.4 pg/mL for reovirus + NT-157 + IL-4). Taken together, these data suggest that the action of IL-4 in enhancing mast cell IFN-α2 and IFN-λ1 responses to viral infection occurs through the type I IL-4 receptor. To further define the potential role of the type II IL-4 receptor, mast cells were pretreated with IL-13 in place of IL-4. Although enhanced STAT6 phosphorylation was detected in response to IL-13, no significant enhancement of IFN responses to viral infection was observed (see Fig E3, A and B in this article's Online Repository at www.jacionline.org). To further consider the impact of allergic disease on mast cell responses to viral infection, we also evaluated the impact of IgE-mediated activation of IL-4–primed mast cells on IFN-α2 and IFN-λ1 responses to viral infection. Notably, concurrent IgE-mediated activation increased the IFN-α2 and IFN-λ1 responses of mast cells to viral infection by 1.5- to 53-fold (Fig 2, C). Overall, these data demonstrate that unlike its established impact on bronchial epithelial cells5Contoli M. Ito K. Padovani A. Poletti D. Marku B. Edwards M.R. et al.Th2 cytokines impair innate immune responses to rhinovirus in respiratory epithelial cells.Allergy. 2015; 70: 910-920Crossref PubMed Scopus (94) Google Scholar and plasmacytoid dendritic cells,9Tel J. Torensma R. Figdor C.G. de Vries I.J. IL-4 and IL-13 alter plasmacytoid dendritic cell responsiveness to CpG DNA and herpes simplex virus-1.J Invest Dermatol. 2011; 131: 900-906Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar IL-4 treatment of mast cells induces an increased type I and type III IFN response to mucosal viral infection. This response is mediated by the type I IL-4 receptor and does not occur in response to IL-13. These findings suggest that care should be taken when considering potential local impacts of IL-4 or IgE blockade on viral host responses at mast cell–rich sites. The optimal size, nature, and longevity of the local IFN responses necessary to combat mucosal viral infection while avoiding damaging inflammatory responses remains poorly defined, especially in the context of asthma. Our data demonstrate novel mast cell and IL-4–dependent processes that could substantially modify such responses to viral infection in allergic individuals. We thank Nong Xu, Maria Vaci, and Alexander Edgar for their excellent technical assistance. Umbilical cord blood samples and peripheral blood samples were obtained following approval from the research ethics board of the Izaak Walton Killam Health Centre, Halifax, Nova Scotia, Canada. Adult blood donors provided written informed consent. The viruses used in this study were kindly provided by Dr Shashi Gujar (type 3 Dearing reovirus; Dalhousie University, Halifax, Nova Scotia, Canada), Dr Robert Anderson (RSV long strain; Dalhousie University), Dr Mark Peeples (recombinant GFP-expressing RSV; Center for Vaccines and Immunity, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio), and Dr Craig McCormick (mouse-adapted and human pandemic IAV, H1N1 A/CA/07/2009; Dalhousie University). Cord blood–derived human mast cells were differentiated by long-term culture of cord blood–derived mononuclear cells according to Enoksson et alE1Enoksson M. Ejendal K.F.K. McAlpine S. Nilsson G. Lunderius-Andersson C. Human cord blood-derived mast cells are activated by the Nod1 agonist M-TriDAP to release pro-inflammatory cytokines and chemokines.J Innate Immun. 2011; 3: 142-149Crossref PubMed Scopus (41) Google Scholar following the adaptation described by Portales-Cervantes et al.E2Portales-Cervantes L. Haidl I.D. Lee P.W. Marshall J.S. Virus-infected human mast cells enhance natural killer cell functions.J Innate Immun. 2017; 9: 94-108Crossref PubMed Scopus (15) Google Scholar Mast cell purity was at least 95% according to c-Kit (anti-CD117 antibody) expression. Typically, more than 99% of mast cell cultures were positive for tryptase and devoid of dendritic cells.E3Al-Afif A. Alyazidi R. Oldford S.A. Huang Y.Y. King C.A. Marr N. et al.Respiratory syncytial virus infection of primary human mast cells induces the selective production of type I interferons, CXCL10, and CCL4.J Allergy Clin Immunol. 2015; 136: 1346-1354. e1Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Human monocytes were isolated from healthy donors. Briefly, PBMCs were separated by Lympholyte-H cell separation media (Cedarlane, Burlington, Ontario, Canada) density gradient centrifugation. Monocytes were isolated by positive selection using an EasySep human monocyte enrichment kit (STEMCELL Technologies, Vancouver, British Columbia, Canada) and cultured in RPMI 1640 (Thermo Fisher Scientific, Mississauga, Ontario, Canada) supplemented with 10% FBS (Gibco, Gaithersburg, Md) and 10 ng/mL of GM-CSF (BioLegend, San Diego, Calif) before virus infection. Calu-3 cells were purchased from ATCC (Manassas, Va) and cultured in a Dulbecco modified Eagle medium/Ham's F-12 nutrient mixture (Thermo Fisher Scientific) supplemented with 10% FBS. Mast cells, monocytes, and Calu-3 cells were activated with 10 ng/mL of IL-4 (BioLegend) at 37°C for up to 48 hours in their corresponding culture medium. Control cells were left nonstimulated. The culture medium was removed, and the cells were resuspended in plain RPMI, counted. and infected with 5 multiplicities of infection (MOI) of reovirus at 37°C for 1 hour. Once the reovirus was washed off, the cells were resuspended in activation medium (culture medium with FCS reduced to 1% for monocytes and Calu-3 cells and RPMI supplemented with 1% FCS, 10 ng/mL of stem cell factor, and 100 μg/mL of soybean trypsin inhibitor for mast cells) at 1 × 106 cells per mL for 24 hours. IL-4 was added to cultures of IL-4–primed mast cells following infection. In addition to reovirus, IL-4–primed mast cells were exposed to 5 MOI of IAV for 1 hour or 4 MOI of RSV for 2 hours. Where indicated, mast cells were primed with 10 or 50 ng/mL of IL-13 following infection with reovirus as already mentioned. For TLR3 activation, IL-4–primed or control mast cells were transfected with high-molecular-weight polyinosinic-polycyitidylic acid sodium salt (poly(I:C)) (Invivogen, San Diego, Calif) following the manufacturer's instructions. Briefly, both Lipofectamine 2000 (Thermo Fisher Scientific) and 10 μg/mL of poly(I:C) were mixed at a 1:1 ratio. Mast cells (1 × 106 cells/mL) were cultured in activation medium in the presence of poly(I:C) or Lipofectamine alone (control) at 37°C for 24 hours. For FcεRI crosslinking experiments, mast cells were sensitized with 2.5 μg/mL of human IgE (MilliporeSigma, Burlington, Mass) in the presence of 10 ng/mL of IL-4 for 96 hours at 37°C. The culture medium was removed, and the cells were washed and subsequently infected with reovirus. Following infection, the mast cells were resuspended at 1 × 106 cells/mL in activation medium and treated with 2.5 μg/mL of anti-human IgE (SouthernBiotech, Birmingham, Ala) or IgG1 (BioLegend) antibodies at 37°C for up to 24 hours after infection. Culture supernatants were harvested for cytokine analysis following centrifugation, and the cells were lysed in RLT buffer (Qiagen, Hilden, Germany) for mRNA analysis. Total RNA was isolated with an RNeasy Mini kit (Qiagen). cDNA was generated with a QuantiTect Reverse Transcription Kit (Qiagen). Gene expression was analyzed in a CFX96 Real-Time Detection system (Bio-Rad, Hercules, Calif) by using primers from Bio-Rad (IFNA2, STING) or Qiagen (IFNL1, TLR3, TLR7, TLR9, PKR, IFIH1, and DDX58) and SsoAdvanced Universal SYBR green supermix (Bio-Rad). Gene expression was normalized to at least 2 reference genes, HPRT and TBP (Qiagen). The level of reovirus and IAV infection in human mast cells was analyzed by droplet digital PCR by using primer pairs (Sigma, Oakville, Ontario, Canada) for the genes S4 and NS1,E4Slaine P.D. MacRae C. Kleer M. Lamoureaux E. McAlpine S. Warhuus M. et al.Adaptive mutations in influenza A/California/07/2009 enhance polymerase activity and infectious virion production.Viruses. 2018; 10Crossref Scopus (9) Google Scholar respectively, in a C1000 touch thermal cycler (Bio-Rad). The data were analyzed in a QX200 droplet reader (Bio-Rad) by using Quantasoft software (Bio-Rad). IFN-α2 and IFN-λ1 were measured by using the Bio-Plex Pro-human cytokine assay (Thermo Fisher Scientific) or an ELISA kit from R&D Systems (Minneapolis, Minn), respectively, following the manufacturers instructions. Mast cells were pretreated with 4 μg/mL of mouse IgG2a (BioLegend), anti-human IL-4Rα (R&D Systems), or anti-human γC (R&D Systems) antibodies for 1.5 hours at 37°C and 5% CO2. In separate experiments, mast cells were treated with the STAT6 inhibitor AS1517499 (Axon Medchem, Reston, Va) or the IRS 1/2 inhibitor NT-157 (Axon) for 1 hour at 37°C. Following mast cell pretreatment with either antibodies or inhibitors, mast cells were set up for reovirus infection as already mentioned. Mast cells were resuspended in activation medium in the presence or absence of 10 ng/mL of IL-4 when indicated. Antibodies and chemical inhibitors were readded to the cell culture following infection. Cytokine analysis was performed 24 hours after infection, with the result represented in terms of the percent increase in IFN-λ1 production as follows: (Mast cell IFN production under treatment – Mast cell basal IFN production)/Mast cell basal IFN production ×100, with "treatment" corresponding to 10 ng/mL of IL-4), isotype control plus IL-4, anti-IL-4Rα antibody plus IL-4, or anti-γC antibody plus IL-4 and "basal" indicating the absence of IL-4 or antibodies in mast cell cultures before or after infection. Alternatively, IFN-λ1 production was represented as IFN-λ1 production by IL-4–activated reovirus-infected mast cells minus IFN-λ1 production by reovirus-infected mast cells. Human mast cells infected with the recombinant GFP expressing RSVE5Hallak L.K. Spillmann D. Collins P.L. Peebles M. Glycosaminoglycan sulfation requirements for respiratory syncytial virus infection.J Virol. 2000; 74: 10508-10513Crossref PubMed Scopus (222) Google Scholar were fixed in 1% paraformaldehyde. For intracellular staining of non-GFP RSV, cells were manipulated as described by Al-Afif et al.E3Al-Afif A. Alyazidi R. Oldford S.A. Huang Y.Y. King C.A. Marr N. et al.Respiratory syncytial virus infection of primary human mast cells induces the selective production of type I interferons, CXCL10, and CCL4.J Allergy Clin Immunol. 2015; 136: 1346-1354. e1Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar STAT6 phosphorylation was analyzed in mast cells stimulated in culture medium with 10 ng/mL of IL-4 or 10 and 50 ng/mL of IL-13 for 30 minutes at 37°C. Cells were fixed with 4% paraformaldehyde following permeabilization in 100% methanol. Cells were resuspended in 1% BSA/PBS and stained with anti–phospho-STAT6 monoclonal antibody (Thermo Fisher Scientific) or a relevant isotype control. Mast cells were analyzed in a FACSCanto II system (BD Biosciences, San Jose, Calif). Data was analyzed with FlowJo X software (FlowJo, LLC, Ashland, Ore). Distribution of data was examined by the Shapiro-Wilk and Kolmogorov-Smirnov tests. Depending on data distribution, repeated-measures ANOVA or the Friedman test were carried out for multiple comparisons with the Bonferroni or Dunn test on selected pairs using post hoc testing, respectively. Differences were considered significant at P < .05. Statistical analyses were performed by using GraphPad Prism software (version 5.0, GraphPad Software Inc, San Diego, Calif) and Instat software (GraphPad Software Inc). ND, Not detected. Data are shown as means plus or minus SEMs. Gene expression is normalized to that of HPRT (n = 7).