Respiratory -Induced TLR7 Activation Controls IL-17 −Associated Increased Mucus via IL-23 Regulation

This information is current as Nicholas W. Lukacs, Joost J. Smit, Sumanta Mukherjee, of September 27, 2021. Susan B. Morris, Gabriel Nunez and Dennis M. Lindell J Immunol published online 12 July 2010 http://www.jimmunol.org/content/early/2010/07/16/jimmun ol.1000733 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 16, 2010, doi:10.4049/jimmunol.1000733 The Journal of Immunology

Respiratory Virus-Induced TLR7 Activation Controls IL-17–Associated Increased Mucus via IL-23 Regulation

Nicholas W. Lukacs,* Joost J. Smit,† Sumanta Mukherjee,* Susan B. Morris,* Gabriel Nunez,* and Dennis M. Lindell‡

The response to respiratory syncytial virus (RSV), negative strand ssRNA virus, depends upon the ability to recognize specific path- ogen-associated targets. In the current study, the role of TLR7 that recognizes ssRNA was examined. Using TLR72/2 mice, we found that the response to RSV infection in the lung was more pathogenic as assessed by significant increases in inflammation and mucus production. Although there appeared to be no effect of TLR7 deficiency on type I IFN, the pathology was associated with an alteration in responses with increases in mucogenic IL-4, IL-13, and IL-17. Examination of dendritic cells from TLR72/2 animals indicated a preferential activation of IL-23 (a Th17-promoting ) and a decrease in IL-12 production. 2 2 Neutralization of IL-17 in the TLR7 / mice resulted in a significant decrease in the mucogenic response in the lungs of the RSV- Downloaded from infected mice. Thus, without TLR7-mediated responses, an altered immune environment ensued with a significant effect on airway epithelial cell remodeling and goblet cell hyper/metaplasia, leading to increased mucus production. The Journal of Immunology, 2010, 185: 000–000.

he role ofinnate immune responses for establishing the most ssRNA species, respectively, and initiate important cytokine medi-

appropriate and least pathologic responses for destruction ator pathways for the initiation of the immune responses. These http://www.jimmunol.org/ T and clearance of the infectious or deleterious agents is the earliest responses are likely critical to establishing the proper im- first line of defense and is responsible for recognizing the vast array mune response. of pathogens with a limited set of fixed germline-encoded receptors. TheRSVFproteinhasbeenreportedtobindandactivateTLR4,the To deal with this challenge with infectious agents, innate immunity receptor for LPS (6), and distinct polymorphisms to TLR4 have been relies on the detection of patterns or conserved molecular motifs associated with disease severity in patient populations (7–11). Ac- unique to various classes of pathogens (1–3). The heterogeneity of tivation of specific during RSV infection of epithelial viral glycoproteins along with their ability to genetically drift from cells can be upregulated via TLR3. Our laboratory identified that season to season creates even greater challenges for innate immune although CCL5 and CXCL10 can be upregulated via a TLR3- recognition of . To circumvent these obstacles, the innate dependent pathway, CXCL8 is dependent upon a MyD88-dependent by guest on September 27, 2021 immune system has evolved mechanisms to detect characteristics pathway (12). Thus, multiple TLR pathways likely contribute to the of viral nucleic acids that are either distinct in structure (dsRNA) or generation of an effective anti-RSV response. Interestingly, our find- subcellular location (ssRNA). Recognition of viral nucleic acids ings suggest that RSV infection of TLR32/2 mice (a MyD88- triggers the induction of type I IFNs that induce an antiviral state independent mechanism) leads to altered immune environment and in virally infected cells and activate immunoregulatory functions in promotes increased IL-13 that is associated with increases in mucus nearby cells. Because respiratory syncytial virus (RSV) is an production (13). Thus, alteration of the dsRNA recognition pathway ssRNA-negative sense virus, both ssRNA and dsRNA species are leads to a pathogenic phenotype. Likewise, when MyD88 pathways formed and provide targets for the . A subset are deleted in mice, an even more pathogenic environment is induced of pattern recognition receptors includes the TLRs, which recognize with increased eosinophilia, mucus overexpression, and an overall different pathogen-associated molecular patterns and activate induction of a Th2 cytokine environment (14). In the current study, NF-kB and other innate signaling pathways (1, 4, 5). In particular, we have examined one of the important MyD88-dependent path- TLR3 and TLR7/8 recognize pathogen-associated dsRNA and ways associated with viral RNA detection, TLR7. Although the present perception is that plasmacytoid dendritic cells (pDCs) are the primary cell population that expresses TLR7 (15, 16), several *Department of Pathology, University of Michigan Medical School, Ann Arbor, MI other cell populations appear to be able to upregulate its expression, 48109; ‡Center for Immunity and Immunotherapies, Seattle Children’s Research In- stitute, Seattle, WA 98105; and †Institute for Risk Assessment Studies, Utrecht Uni- including myeloid DCs (mDCs), B cells, and (17–19). versity, Utrecht, The Netherlands In the present studies, the deletion of TLR7 results in the generation Received for publication March 9, 2010. Accepted for publication June 12, 2010. of pathogenic responses during RSV infection and initiates an in- This work was supported by National Institutes of Health Grants R01AI073876 (to crease in a number of pathogenic cytokines, especially IL-17, which N.W.L.), R01AI036302 (to N.W.L.), and T32HL07749 (to D.M.L.). appears to be responsible for the increased mucus responses observed 2 2 Address correspondence and reprint requests to Dr. Nicholas W. Lukacs, University in TLR7 / mice. of Michigan Medical School, 1302 Catherine Street, 5214 Med. Sciences I, Ann Arbor, MI 48109-0602. E-mail address: [email protected] Abbreviations used in this paper: BMDC, bone marrow-derived dendritic cell; DC, den- Materials and Methods dritic cell; LALN, lung-associated lymph node; mDC, myeloid dendritic cell; MOI, mul- Mice tiplicity of infection; PAS, periodic acid-Schiff; pDC, plasmacytoid dendritic cell; PMN, polymorphonuclear cell; poly I:C, polyinosinic:polycytidylic acid; QPCR, quantitative B6 wild-type (WT) mice were purchased from The Jackson Laboratory (Bar 2/2 PCR; RSV, respiratory syncytial virus; UC, uninfected control; WT, wild-type. Harbor, ME). TLR7 mice on a B6 background were originally provided by Dr. S. Akira (Osaka, Japan) and a breeding colony subsequently estab- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 lished at the University of Michigan (Ann Arbor, MI). All animal work

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000733 2 VIRAL ACTIVATION OF TLR7 REGULATES IL-23 was performed in accordance with the University of Michigan Committee tage protein A affinity chromotography (Millipore, Bedford, MA). Mice were on Use and Care of Animals policy. treated with ∼3 mg anti–IL-17A Ig or control Ig every other day beginning at day 4 postinfection to allow our studies to address whether blocking IL-17 Respiratory syncytial virus might have therapeutic potential. Our laboratory utilizes the antigenic subgroup A strain of RSV,referred to as Statistics Line 19. This isolate was obtained from a sick infant at the University of Michigan (20) and has been demonstrated in animal models to mimic Data was analyzed using Prism software (GraphPad, San Diego, CA). Unless human infection by stimulating mucus production using an inoculum of otherwise specified, data shown are representative of two or more experi- 1 3 105 PFU/mouse by intratracheal administration (21). ments. Statistical significance in all experiments was determined by one- way ANOVA, followed by a Newman-Keuls post test. Significant differ- Real-time TaqMan PCR ences were regarded as p , 0.05. Statistical comparisions of quantitative The smallest lobe was removed and homogenized in 1 ml Trizol reagent PCR (QPCR) data were determined from normalized cycle threshold values, (Invitrogen, Carlsbad, CA). RNA was isolated as described (Invitrogen), and preconversion to fold increases. 5 mg was reverse-transcribed to assess expression. Detection of cyto- kine mRNA in lung samples was determined using predeveloped primer/ Results probe sets (Applied Biosystems, Foster City, CA) and analyzed using an Increased expression of TLR7 during RSV infection ABI Prism 7500 Sequence Detection System (Applied Biosystems). Tran- script levels of Muc5ac, Gob5, Ifna4,andIfnb were determined using cus- The expression of TLR7 has been identified on multiple immune tom primers, as previously described (22, 23). Gapdh was analyzed as an cell populations but is thought to be most closely associated with internal control and was normalized to Gapdh. Fold changes APCs, such as DCs and B cells. To determine if RSV infection in gene expression levels were calculated by comparison with the gene ex- upregulates TLR7 expression, infected animals were assessed for pression in uninfected mice, which were assigned an arbitrary value of 1. Downloaded from TLR7 expression byQPCR.Thelungs(Fig.1A)demonstrateda time- Lung and lymph node leukocyte isolation dependent increase in TLR7 expression that first appeared on day 4 Lung leukocyteswere isolated from enzyme-dispersed lung tissue. Right lungs and increased and plateaued on days 8 and 12, respectively. When from each mouse were excised, washed in PBS, minced, and digested enzy- cells from the RSV-infected mice (day 8 of infection) were assessed matically for 45 min in 15 ml/lung digestion buffer (RPMI, 5% FCS, 1 mg/ml by intracellular staining for TLR7 by flow cytometry, the primary collagenase [Roche Applied Science, Indianapolis, IN], and 30 mg/ml DNase [Sigma-Aldrich, St. Louis, MO]). Lung-associated lymph nodes (LALNs) cells that expressed TLR7 were B cells, pDCs, and the strongest were dispersed similar to lungs, except that only 5 ml digestion buffer was staining pattern for mDCs (conventional) based upon their median http://www.jimmunol.org/ used. Following erythrocyte lysis using ammonium chloride (NH4Cl) buffer, fluorescence intensity in the lung (Fig. 1B,1C). Thus, it appears that cells were washed and resuspended in media (RPMI, 5% FCS). Total lung numerous immune cells express TLR7, primarily in CD11b+CD11c+ leukocyte numbers were assessed in the presence of trypan blue using a he- mocytometer; viability was .85%. conventional DCs, but there was no increase in TLR7 intensity on a per-cell basis in RSV compared with uninfected animals. Rather, Lymph node restimulation the increase in mRNA expression levels is likely due to an increased LALN cell suspensions were plated in duplicate at 1 3 106 cells/well fol- recruitment of those cells in animals infected with RSV. lowed by restimulation with either media or RSV (multiplicity of infection [MOI] ∼0.5). Cells were incubated at 37˚C for 24 h and supernatants col- Deletion of TLR7 enhances RSV-induced goblet cell lected for analysis on the Bio-Rad Bioplex 200 system according to the hyperplasia by guest on September 27, 2021 manufacturer’s protocol (Bio-Rad, Hercules, CA). Kits (Bio-Rad) contain- The role of TLRs for the recognition of pathogen-associated molec- ing Ab beads to Th cytokines (IL-17, IFN-g, IL-4, IL-5, IL-13) were used to assay for cytokine production in each of the samples. ular patterns, such as virus-associated ssRNA by TLR7, is thought tobecriticalintherecognitionofaninfectiousprocess.TLR7signaling Lung protein analysis is dependent upon MyD88 adaptor-mediated pathways, and our pre- The levels of CCL5 and CXCL10 in lung homogenates were assessed using vious studies have established that deletion of MyD88 results in multiplex bead-based arrays (Invitrogen) and analyzed on the Bioplex 200 a skewed response with heightened levels of Th2 cytokines, eosino- system (Bio-Rad). philia, and increased mucus production. In the present studies, when 2 2 Generation of bone marrow-derived DCs TLR7 / mice were infected with RSV,there was also an increase in 2/2 2/2 pathologic responses similar to that observed in the MyD88 mice, Bone marrow was harvested from B6 or TLR7 mice and seeded in tissue- especially the expression of mucus and increased numbers of goblet culture flasks in RPMI 1640-based complete media with 10 ng GM-CSF/ml 2 2 (R&D Systems, Minneapolis, MN). Cells were fed every 3 d, and loosely cells in the airways (Fig. 2A). Similarly, we observed that TLR7 / adherent cells were collected after 10 d. The majority of the cells, .85%, were mice had increased expression of mucus-associated Muc5ac + CD11c bone marrow-derived DCs (BMDCs) and subsequently were infected and Gob5 compared with RSV-infected WT animals (Fig. 2B). In- with RSV (MOI ∼0.5) or stimulated with specific TLR ligands: CpG (Hycult Biotech, Uden, The Netherlands), LPS (Sigma-Aldrich), or 3M-011 (3M, St. terestingly, there was not a remarkable increase in inflammatory cells 2/2 Paul, MN). The cells were then assessed for gene and protein expression as in the TLR7 mice as evidenced in the lung sections or individual well as used for flow cytometric analysis. populations of cells including T cells and DC subsets in either the Flow cytometric analysis lung or draining lymph nodes (LALNs), as assessed by flow cytom- etry (Fig. 2C). When we examined viral clearance, we found no Except TLR7, all Abs for flow cytometry (BD Pharmingen, San Diego, CA; significant changes as assessed by viral gene expression within Biolegend, San Diego, CA; or eBioscience, San Diego, CA) were used to the lungs of infected mice as well as assessing viral titer at day 3 of stain intact cells per the manufacturer’s instructions and analyzed on an LSR II cytometer (BD Biosciences, San Jose, CA). For TLR7 staining, cells were infection by viral plaque assays (data not shown). Thus, in the absence permeabilized using fixation/permeabilization solution (BD Biosciences) of TLR7, there was primarily a pathogenic alteration of the RSV- and stained with anti-mouse TLR7 Abs (Imgenex, San Diego, CA). Isotype induced responses that was primarily centered on airway pathology. control Abs were used to demonstrate specificity of our staining and to 2 2 establish the criteria for our flow cytometry populations. Alteration in cytokine profiles in TLR7 / mice with RSV- induced disease Anti–IL-17A Abs Several studies have identified that type I IFN is most often as- Anti-mouse IL-17Awas generated by immunization of rabbits with recombi- nant murine IL-17A. The polyclonal sera was tested for cross-reactivity to sociated with an appropriate and that TLR7 can a panel of other cytokines, including other IL-17 family members, and no mediate these effects (24–28). Given the above data indicating that cross-reactivity was observed. Abs were isolated from total sera using Mon- mDCs appeared to have the highest expression of TLR7, we chose The Journal of Immunology 3

FIGURE 1. TLR7 expression during RSV in- fection. A, Expression of TLR7 in the lungs and lung draining lymph nodes at various times post RSV infection. Expression of TLR was assessed Downloaded from via QPCR (TaqMan) from whole-lung/lymph node RNA. Each time point represents the mean of four to five mice, and error bars represent the SEM. B, Histograms depict expression of TLR7 by various cell types (B cells, pDCs, and mDCs) determined via flow cytometric analysis. Leuko- cytes from the lungs of control and RSV-infected http://www.jimmunol.org/ mice (day 8 postinfection) were isolated via en- zymatic digests. The resulting cell suspensions were stained with Abs to surface markers, then permeabilized to stain TLR7, with the gray area representing the control Ab stain and black lines the TLR7 Ab. C, Columns represent the medi- an fluorescence intensity of TLR-specific Ab staining in the various cell populations from the

collagenase-dispersed lungs of mice. pp , 0.05. by guest on September 27, 2021

to focus on BMDCs as an in vitro model to assess the role of TLR7 dependent upon type I IFN. The TLR72/2 DCs were able to ma- in the innate immune response to RSV. When we examined type I ture properly as demonstrated by examination of CD80 expression IFN in DCs after RSV infection, there was no alteration of either (Fig. 3D). These data suggest that a defect in type I IFN-associated IFNa4 or IFNb in the TLR72/2 compared with WT DC pop- responses are unlikely to be the proximate cause for the dysregu- ulations (Fig. 3A). Furthermore, examining chemokines that are lated response to RSV observed in TLR72/2 mice. associated with type I IFN induction, CCL5 and CXCL10, dem- To begin to understand the changes in immune responses in the onstrated a similar induction with not only RSV but also a TLR3/ TLR72/2 animals that correspond to the pathologic alterations polyinosinic:polycytidylic acid (poly I:C) stimulus (Fig. 3B). In observed, lungs from RSV-infected mice were isolated and dis- addition, when we examined the type I IFN-dependent transcrip- persed into single-cell suspensions. The single-cell suspensions tion factor IFN regulatory factor-1 in RSV-infected or poly I:C- were then given RSV (MOI 0.5) to initiate a recall response to stimulated DCs, it was similarly upregulated in WT and TLR72/2 RSV, and cytokine levels in supernatants were assessed using DCs (Fig. 3C). Finally, several studies have demonstrated that DC bioplex proteomics analysis after 24 h (Fig. 4A). The data indicate maturation in general and specifically during RSV infection is that although there was no alteration in IFN-g production in the 4 VIRAL ACTIVATION OF TLR7 REGULATES IL-23

FIGURE 2. Role of TLR7 in RSV-induced mucus production, goblet cell hyperplasia, and pulmonary in- flammation. A, PAS staining of lung sections from con- trol and TLR72/2 mice at day 9 postinfection dem- onstrated increased mucus production as indicated by the pink-red stain (original magnification 3400). In B, the expression of mucus-associated genes Muc5ac and Downloaded from Gob5 was determined by QPCR of whole-lung RNA (day 9 postinfection). Columns represent the mean 6 SEM of five mice per group. The experiment was per- formed three times with similar results. C, Effect of TLR7 deficiency on leukocyte recruitment to the lungs and LALNs at day 9 of infection. Cells were recovered

via enzymatic digestion of tissue, and the numbers of http://www.jimmunol.org/ CD45+, CD4+, CD8+, CD11b+CD11c+ mDCs, and CD11c+B220+ pDCs were determined via flow cytom- etry. pp , 0.05. by guest on September 27, 2021

TLR72/2 mice compared with WT, a very significant increase in of IL-17 observed in the TLR72/2 mice following RSV infection. IL-17A production could be observed, as well as significantly more Based on evidence from the literature regarding the recognition of Th2 cytokines IL-4 and IL-13 (Fig. 4A). Among these, only IL- RSV via TLR4, we hypothesized that TLR4 may mediate the IL- 17A was persistently elevated in lung homogenates at day 9 post- 23p19–promoting signal in BMDCs. To test this hypothesis, infection (27.24 6 5.67 pg/ml versus 8.30 6 1.98 pg/ml in con- BMDCs were stimulated with various TLR agonists, and the trols; p , 0.05). Thus, the increased production of IL-17A, as well induction of IL-23p19 was assessed via QPCR. Compared to other as Th2 cytokines IL-4 and IL-13, corresponded with associated TLR agonists CpG (TLR9) and 3M-011 (TLR7), LPS was the most increases in mucus gene expression and goblet cell hyperplasia. potent inducer of IL-23p19 (Fig. 4C). Taken together, these data In order to further examine if there was an alteration in APC suggest that IL-12/IL-23 signaling in response to RSV may be me- function in the TLR72/2 mice that would alter the subsequent diated by the opposing actions of TLR7 and TLR4, with TLR7 production of T cell-derived cytokines, we again used BMDCs favoring IL-12 and TLR4 favoring IL-23. from WT and TLR72/2 mice. BMDCs were infected with RSV To determine whether TLR7 deficiency would alter the balance (MOI ∼0.5) or other TLR agonists and assessed for expression of of IL-12 versus IL-23 signaling in vivo, we assessed the induction IL-6, IL-12p40 (the common chain), as well as the IL-12–and IL- of IL-12 and IL-23 in the lungs of RSV-infected mice. TLR72/2 23–specific chains p35 and p19, respectively. No significant mice and WT controls were infected with RSV, and the expression increased differences in the mRNA expression of induction of of IL12 p35, IL-12/IL-23p40, and IL-23 p19 were assessed at day IL-6 were found, but rather a trend toward a decrease (WT 15.0 6 3 postinfection via QPCR of lung RNA. No detectable induction 3.0-fold versus TLR7 8.7 6 1.6-fold increase over uninfected). of IL-12p40 was observed in either group (Fig. 4D), and the in- However, the data in Fig. 4B illustrate that although IL-12p40 ex- duction of IL-12p35 was similar between the two groups (Fig. pression was similar between the WT and TLR72/2 BMDCs, there 4D). Similar to the results obtained with BMDCs in vitro, a dra- was a significant decrease in IL-12p35 and concomitant increase in matic increase in IL-23p19 was observed in the lungs of TLR72/2 IL-23p19 in TLR72/2 BMDCs. These data indicate a skewing of mice, relative to WT controls (Fig. 4D). In addition, when we the DC response to RSV, which could explain the increased level examined other IL-17–associated cytokines in lungs of infected The Journal of Immunology 5

FIGURE 3. Effect of TLR7 de- ficiency on the induction of type I IFN, the IFN response pathway, and DC maturation. In A, the induction of IFNa4 and IFNb in RSV-infected con- trol and TLR72/2 BMDCs was assessed via QPCR. B, The induction of chemokines CCL5 and CXCL10 in control B6 and TLR72/2 BMDCs. BMDCs were cultured with media alone, poly I:C (20 mg/ml), 3M-011 (10 mM), or RSV, and the levels of chemokines in the supernatant were measured at 24 h via Bioplex multi- plex assay. The expression of IRF-1 (C) was assessed via QPCR was also assessed at 24 h. D, Effect of TLR7 deficiency on DC maturation in re- sponse to RSV. The expression of CD80 by RSV-infected control B6 Downloaded from and TLR72/2 BMDCs was assessed via flow cytometry at 24 h. Represen- tative histograms depict staining with CD80-specific Abs (open histo- grams), with shaded histograms rep- resenting staining with isotype control http://www.jimmunol.org/ Abs. Columns represent the mean of triplicate samples 6 SEM. Similar results were obtained in two indepen- dent experiments.

mice, there were no significant changes in IL-6, a cytokine re- we also examined the cytokine profile (Fig. 5C) and found that quired for IL-17A induction (Fig. 4E). Importantly, no alteration although anti–IL-17 strikingly reduced the expression of IL-17 in in a closely related IL-17F and no changes in IL-17–inducible the lung, it had no significant effect on other cytokines, including by guest on September 27, 2021 chemokines CXCL1 or CXCL2 were observed during RSV IL-4 and IL-13. In addition, when we examined the neutrophil infection at day 3 post RSV infection (Fig. 4E). Together, these accumulation in the TLR72/2, there was a significant increase in in vitro and in vivo data support the conclusion that dysregulation polymorphonuclear cells (PMNs) at day 8 of infection that was in innate cytokine production in BMDCs, away from Th1 reduced, but still present, when animals were treated with anti– promoting IL-12 and in favor of Th17-promoting IL-23, results IL-17 (WT-RSV, 0.5 6 0.5%; TLR7-RSV, 19.3 6 6.8%; TLR7- in dysregulated immune responses in the lungs of TLR72/2 mice, RSV–anti–IL-17, 6.0 6 3.7% PMNs of total BAL leukocytes). leading to increased pathology. Thus, these data overall suggest that, in the absence of TLR7 sig- naling, inappropriate activation of the IL-17A pathway during RSV IL-17 overexpression is a dominant pathogenic signal in infection plays a prominent role in promoting increased mucus pro- 2/2 TLR7 animals duction and alters inflammation. Our data indicated that the predominant cytokine that was upregu- lated in TLR72/2 animals was IL-17. Results have suggested that Discussion IL-17 may directly induce mucus production from epithelial cell The response to viral infections within the lung must proceed in populations in the lung (29, 30). To address the role played by IL-17 a balanced and programmed manner to optimally clear the virus in the immunopathology observed in RSV-infected TLR72/2 mice, without damaging critical pulmonary function or promoting we generated anti–IL-17 Abs and treated TLR72/2 animals using pathologic responses. This is particularly important in individuals passive immunization during RSV infection. Control or anti–IL-17 with underlying disease, such as those with chronic obstructive Abs were administered systemically (i.p.) beginning at day 4 post- pulmonary disease, asthma, or end-stage fibrotic illness. It is clear infection. The levels of IL-17 in lung homogenates and bronchoal- that the determination of the direction of the immune responses veolar lavage were significantly decreased in Ab-treated mice, relies on innate immune cell activation and the mediators that they demonstrating the effectiveness of in vivo neutralization (data not produce. In the current study, the innate response and subsequent shown). Compared to control IgG-treated, RSV-infected TLR72/2 T cell response to RSV, an ssRNA virus, depends upon the pres- mice, IL-17–neutralized mice had decreased mucus production, as ence of ssRNA recognition through TLR7 to promote a relatively assessed via the degree of periodic acid-Schiff (PAS) staining in nonpathogenic response. Original studies described that TLR7 lung sections (Fig. 5A). As a more quantitative measure, we was necessary for recognition of ssRNA and elicited both IL-12 assessed the expression of the mucus-associated genes Muc5ac and type I IFNs necessary for Th1-type immune responses (31– and Gob5 in the lungs of control IgG-treated and anti–IL-17– 33). The response of the TLR72/2 BMDCs in the present studies treated TLR72/2 mice. Compared to control IgG-treated mice, demonstrated that without TLR7, there was a change in the bal- anti–IL-17–treated TLR72/2 mice exhibited significantly reduced ance of innate cytokines with increases in Th17 inducing IL-23 expression of these mucus-associated genes (Fig. 5B). In addition, and a decrease in Th1 inducing IL-12. Interestingly, in the absence 6 VIRAL ACTIVATION OF TLR7 REGULATES IL-23

FIGURE 4. A, Effect of TLR7 deficiency on early lung cytokine production during RSV infection. Control and TLR72/2 mice were infected with RSV, and the lungs were harvested at day 3 postinfection. Single-cell suspensions were obtained via enzymatic digests, and cells were restimulated with RSV (MOI ∼0.5). Cytokine production was assessed via Bioplex multiplex assay. Simi- lar results were obtained in three indepen- dent experiments. B, Role of TLR7 in mediating IL-12 versus IL-23 signaling in vitro. BMDCs were isolated and cultured from WT and TLR72/2 mice in the presence of media, the TLR7 agonist 3M-011, or RSV (MOI ∼0.5) for 24 h. The induction of IL12p35, IL-12/23p40, and IL-23p19 were Downloaded from determined via QPCR. C, Effect of TLR agonists on the induction of IL-23 p19 in DCs. BMDCs from control B6 mice were treated with media alone, CpG (1 mg/ml), LPS (1 mg/ml), or 3M-011 (10 mM) and the expression of IL-23 p19 was assessed at 4 h via QPCR. D, Effect of TLR7 defi- http://www.jimmunol.org/ ciency on the induction of IL-12 and IL-23 in vivo. Control and TLR72/2 mice were infected with RSV, and the expression of IL-12p35, IL-12/IL-23p40, and IL-23p19 were determined at day 3 postinfection via QPCR of whole-lung RNA. E, Control and TLR72/2 mice were infected with RSV, and the expression of IL-6, IL-17F, CXCL1, and CXCL2 were determined at day 3 postinfec- tion via QPCR of whole-lung RNA. Data by guest on September 27, 2021 represent mean 6 SEM from lungs of five to six mice per group. pp , 0.05.

of TLR7, no alteration in RSV clearance could be detected. How- to the cell surface may provide an early innate activation signal ever, the immune response and subsequent pulmonary phenotype that is important to sensing the infection. Discrete amino acid was greatly affected, with a significant increase in cytokines and changes in F protein appear to regulate the induction of IL-13 and mucus production, demonstrating that a link between viral clear- mucus in different strains of RSV (48) and may be linked to ance and immunopathology was not evident. Although several TLR4 activation. A recent study also examined the role of immune cytokines that were upregulated, IL-4, IL-13, and IL- TLR2/TLR6 in RSV infection and found that they had a role in 17, have clearly been linked to the generation of pathogenic innate cell cytokine and production along with effi- responses, IL-17A was the most noteworthy. Numerous studies cacy of viral clearance (49). A most profound pathogenic effect have suggested that by targeting TLR activation in general and was found when MyD882/2 mice were used with RSV infection TLR7 specifically, a skewing of the immune response away from and included increased Th2 cytokines, enhanced mucus produc- Th2 could be accomplished (27, 34–39). In addition, a recent tion, and profound eosinophilia (14). This latter response was study demonstrated that type I IFN induced by TLR7 regulates likely due to the removal of multiple TLR pathways, including IL-17 production (40). It may be the absence of this pathway that TLR2, TLR4, and TLR7. The response in MyD882/2 mice alters the response, perhaps through regulation of IL-23. Thus, our appeared to be of a similar intensity as that described in findings support these previous findings using a clinically relevant STAT12/2 mice (30, 50), perhaps linking it to a type I IFN re- animal model of pulmonary viral infection. sponse that may be important early in the antiviral immunity. In Several studies have examined the role of TLRs in RSVand other addition, when the only MyD88-independent sensing TLR was respiratory viral infections. TLR4 was first implicated in RSV removed, TLR3, a pathogenic effect was also observed linked to infections (6, 41), and although initially controversial (42), nu- alteration of IL-13 production, leading to increased mucus pro- merous studies have identified a clear connection between TLR4 duction (12). Recent studies have clearly established that TLR4 and RSV clearance (6, 41, 43, 44) as well as a link involving pathways lead to increased expression of IL-23 via TLR4 polymorphisms and severe RSV-induced disease in infants AP-1 activation, which can be especially enhanced by coactiva- (9, 45–47). The ability of RSV F protein to bind and induce tion with IFN-g (51). Interestingly, IL-23 along with IL-1b activation of TLR4 suggests that even the initial binding of RSV induces IL-17 from g/d T cells and can subsequently enhance The Journal of Immunology 7

FIGURE 5. Neutralizing IL-17 beginning on day4ofRSVinfectionofTLR72/2 mice attenu- ates mucus production. A, PAS staining of lung sections from control Ig or anti–IL-17–treated, RSV-infected TLR72/2 mice at day 8 postinfec- tion (original magnification 3100). Expression of mucus-associated genes Muc5ac and Gob5 (B), Downloaded from IL-4, IL-13, and IL-17 (C) was assessed via QPCR of lung RNA in uninfected control (UC), control Ig RSV-infected mice, and in anti–IL-17–treated animals. Columns represent the mean fold increase over uninfected controls, and error bars represent SEM. pp , 0.05. http://www.jimmunol.org/ by guest on September 27, 2021

CD4 T cell-derived IL-17 (52). The latter mechanism may be immunopathology of the response. Thus, there may be multiple critical in driving IL-17 in the absence of TLR7 signaling by associated mechanisms for induction of severe disease that may shifting the balance away from IL-12 and toward IL-23. Al- result from genetic or environmental alteration of pattern recog- though the present studies did not observe an increase in nition receptor expression. IL-6 levels in TLR72/2 mice nor in isolated DCs, and because Establishing an altered immune environment of the lung early in IL-6 and IL-17 may be critical and synergize to induce mucus development may have subsequent impact on later responses in gene expression (29), the pathologic phenotype in the TLR72/2 specific susceptible individuals. Although it is still controversial may be due to the increase in IL-17 in the presence of sufficient whether severe RSV disease predisposes children to subsequent IL-6 production. Thus, it is clear that the pathogen-sensing sys- pulmonary disease, clear epidemiology studies have linked early tem induced by multiple TLRs is critical at several levels for RSV disease with persistent pulmonary problems (55–60). In- driving an appropriate antiviral response. terestingly, several studies have now linked TLR7 polymorphisms In the context of innate cell activation, TLR2 and TLR4 are with altered responses to HIV and -induced found predominantly on the surface of innate cells and allow the responses, and, most interesting, the TLR7 polymorphisms have early recognition of the virus, subsequently followed by TLR3 and been associated with development of asthma (61–64). Thus, sim- TLR7 that are found primarily in the endosomic compartment and ilar to TLR4 polymorphism studies, a link may exist between recognize ds- and ssRNA. These later TLRs appear to have a prom- TLR7 polymorphisms and RSV disease and associated persistent inent impact on shaping the T cell immune response. In addition, pulmonary problems. An early and consistent observation related because RSV infects predominantly via cytoplasmic entry, other to IL-17 overproduction has been the induction of CXC chemo- nonendosomic sensing systems are also important, such as retinoic kines that leads to increased neutrophil accumulation that can acid-inducible gene-I (53). A recent study that used mice deficient impact local tissue damage and increase mucus production via in the primary adapter molecule for retinoic acid-inducible gene-I, elastase release (65–67). Although we did not observe changes mitochondrial antiviral signaling protein, infected with RSV in CXC chemokines in the TLR72/2 animals, we were able to detect demonstrated a significant abrogation of early cytokine pro- an increase in PMN accumulation in TLR72/2 that was reduced duction, but pathogenesis later in disease was not investigated with neutralization of IL-17. The changes in the ability of particular (54). Alteration of any of these pathogen-sensing pathways may or multiple TLR systems may lead to multiple disease phenotypes lead to an alteration in either the clearance of the virus and/or the in the lung including asthma and chronic obstructive pulmonary 8 VIRAL ACTIVATION OF TLR7 REGULATES IL-23 disease, diseases that most severely manifest themselves through viral 19. Barrat, F. J., and R. L. Coffman. 2008. Development of TLR inhibitors for the treatment of autoimmune diseases. Immunol. 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