IL-33 Signaling Regulates Innate IL-17A and IL-22 Production via Suppression of Prostaglandin E2 during Fungal Infection This information is current as of September 26, 2021. Jaleesa M. Garth, Kristen M. Reeder, Matthew S. Godwin, Joseph J. Mackel, Chad W. Dunaway, Jonathan P. Blackburn and Chad Steele J Immunol published online 7 August 2017 http://www.jimmunol.org/content/early/2017/08/04/jimmun Downloaded from ol.1602186

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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 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 7, 2017, doi:10.4049/jimmunol.1602186 The Journal of Immunology

IL-33 Signaling Regulates Innate IL-17A and IL-22 Production via Suppression of Prostaglandin E2 during Lung Fungal Infection

Jaleesa M. Garth, Kristen M. Reeder, Matthew S. Godwin, Joseph J. Mackel, Chad W. Dunaway, Jonathan P. Blackburn, and Chad Steele

Members of the IL-1 family play protective and regulatory roles in immune defense against the opportunistic mold Aspergillus fumigatus. In this study, we investigated the IL-1 family member IL-33 in lung defense against A. fumigatus. IL-33 was detected in the naive lung, which further increased after exposure to A. fumigatus in a dectin-1–independent manner. Mice deficient in the for IL-33 (Il1rl12/2) unexpectedly demonstrated enhanced lung clearance of A. fumigatus. IL-33 functioned as a negative

regulator of multiple inflammatory , as IL-1a, IL-1b, IL-6, IL-17A, and IL-22 were significantly elevated in fungal- Downloaded from exposed Il1rl12/2 mice. Subsequently, IL-33 administration to normal mice attenuated fungal-induced IL-17A and IL-22, but not IL-1a, IL-1b, or IL-6, production. IL-33–mediated regulation of IL-17A and IL-22 did not involve the modulation of IL-23 but 2/2 rather PGE2; PGE2 was significantly increased in fungal-exposed Il1rl1 mice, and normal mice produced less PGE2 after fungal exposure when administered IL-33, suggesting that IL-33–mediated regulation of IL-17A and IL-22 occurred at the level of

PGE2. This was confirmed by in vivo cyclooxygenase 2 inhibition, which attenuated fungal-induced IL-17A and IL-22, as well as 2/2 IL-1a, IL-1b, and IL-6, production in Il1rl1 mice, resulting in impaired fungal clearance. We also show that a PGE2 receptor http://www.jimmunol.org/ agonist increased, whereas a PGE2 synthase inhibitor decreased, the levels of IL-17A and IL-22 but not IL-1a, IL-1b, or IL-6. This study establishes novel mechanisms of innate IL-17A/IL-22 production via PGE2 and regulation of the PGE2/IL-17A/IL-22 axis via IL-33 signaling during lung fungal exposure. The Journal of Immunology, 2017, 199: 000–000.

oncerns over the rise in invasive fungal infections (IFIs) further shown that the fungal b-glucan receptor dectin-1 drives IL-1a over the last several decades as the result of modern and IL-1b production in vivo during IA (10). A recent study has C medical interventions (new, more effective immunosup- demonstrated that IL-1R was essential for survival, with IL-1a pressive drugs/regimens) are mounting (1). IFIs caused by Aspergillus promoting leukocyte recruitment and IL-1b promoting antifungal by guest on September 26, 2021 fumigatus remain one of the most lethal human infectious diseases. activity (11). Studies in humans have shown that A. fumigatus Invasive aspergillosis (IA) is particularly insidious as the result of strongly induces IL-36g and IL-36Ra, but not IL-36a, which were a combination of increasing antifungal drug resistance, extremely dependent on dectin-1 and TLR4 (12). Inhibiting IL-36 signaling high mortality, and an inherent difficulty in diagnosis (2, 3). Al- was found to abrogate the induction of protective Th17 and Th1 though IA is the most devastating aspect of A. fumigatus exposure responses. With respect to immunopathogenic effects of IL-1 and accounts for 20–40% of IFIs in solid organ and hemato- family members during IA, IL-1a and IL-1b are thought to be poietic transplantation (4, 5), colonization with, or sensitization the primary drivers of inflammation in chronic granulomatous dis- to, A. fumigatus has dramatic effects on lung function in asth- ease via decreased autophagy and increased inflammasome activa- matics (6) and individuals with cystic fibrosis (7). tion (13). Finally, analysis of polymorphisms within the IL-1 family The IL-1 family of cytokines is composed of 11 members: 7 with revealed that a single nucleotide polymorphism in IL-1Ra VNTR2 proinflammatory characteristics (IL-1a,IL-1b,IL-18,IL-33, (rs380092), IL-1a-889C (rs1800587), and IL-1b (rs1143627) cor- IL-36a, IL-36b, and IL-36g) and 4 with anti-inflammatory char- relates with the incidence or risk for developing IA in hematological acteristics (IL-1Ra, IL-36Ra, IL-37, and IL-38). Early studies in patients (14). experimental models of A. fumigatus infection documented the induction of IL-1 family members, such as IL-1b (8, 9). We have IL-33 is recognized as 1 of 11 members of the IL-1 family because of its cluster, structure, and processing (reviewed in Ref. 15). IL-33 has been studied in a variety of in- Department of Medicine, University of Alabama at Birmingham, Birmingham, AL flammatory conditions, including allergic asthma, colitis, rheu- 35294 matoid arthritis, and cardiovascular diseases (reviewed in Ref. 16). Received for publication January 3, 2017. Accepted for publication July 20, 2017. Likewise, IL-33 has been investigated in a variety of viral (influ- This work was supported by Public Health Service Grants HL096702, HL122426, and HL136211. enza, respiratory syncytial virus), bacterial (sepsis, Gram-negative keratitis, Gram-positive infection), and fungal (Candida Address correspondence and reprint requests to Dr. Chad Steele, School of Medicine, University of Alabama at Birmingham, 1900 University Boulevard, THT 437A, albicans peritonitis, fungal asthma) infection models (17). By Birmingham, AL 35294. E-mail address: [email protected] and large, IL-33 mediates the induction of type 2/Th2 responses The online version of this article contains supplemental material. and suppression of Th1 and inflammatory responses. However, Abbreviations used in this article: BL/6, C57BL/6; COX-2, cyclooxygenase 2; IA, IL-33 has the potential to mediate a diverse array of functions, such invasive aspergillosis; IFI, invasive fungal infection; mPGES, microsomal PGE syn- as recruitment and reactive oxygen species/reactive ni- thase; NSAID, nonsteroidal anti-inflammatory drug. trogen intermediates induction, depending on the site and type of Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 insult (17). Although IL-33 has yet to be investigated in IA, blockade

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1602186 2 INNATE REGULATION DURING INVASIVE ASPERGILLOSIS of IL-33R signaling has been shown to improve fungal asthma se- Block (BD Biosciences, San Diego, CA) at 4˚C for 20 min. Thereafter, verity, putatively through the modulation of proallergic responses cells were stained with a single-color LIVE/DEAD Fixable Dead Cell (18). In the current study, we defined the role of IL-33 in lung de- Stain (Invitrogen), followed by labeling with cell-specific markers (Abs were from BD Biosciences, BioLegend, and eBioscience) (24). fense against IA. We show that IL-33 is detrimental for lung anti- fungal defense as a result of its function as a negative regulator of In vivo treatments (IL-33, celecoxib, IL-17A/IL-22 neutralization) IL-17A and IL-22 via suppression of cyclooxygenase 2 (COX-2)/PGE2. In specific experiments, WT BL/6 mice were treated with recombinant murine IL-33 intratracheally. Specifically, mice were challenged with Materials and Methods A. fumigatus and administered IL-33 (1 mgin50ml; R&D Systems) or PBS intratracheally 6 and 24 h thereafter. In other experiments, WT BL/6 and Mice Ilrl12/2 mice were administered celecoxib (100 mg/kg in 250 ml i.p.; Wild-type (WT) C57BL/6 (BL/6) mice, 6–8 wk of age, were obtained Cayman Chemical) or vehicle (ethanol). Mice received celecoxib or ve- from The Jackson Laboratory (Bar Harbor, ME) or Taconic (Hudson, NY). hicle 24 h prior to A. fumigatus challenge and then again at 6 and 24 h Il1rl12/2 (ST2/IL-33R) mice were a kind gift from Dr. A. McKenzie postchallenge. For both IL-33 and celecoxib treatments, 48 h after (Cambridge University). Il1r12/2 mice were obtained from The Jackson A. fumigatus challenge, mice were sacrificed and the were processed Laboratory. All animals were housed in a specific pathogen–free, Asso- for lung digest cell isolation followed by overnight culture and assessment of inflammatory cytokine, IL-22, and PGE2 levels. For in vivo IL-17A and ciation for Assessment and Accreditation of Laboratory Animal Care– 2/2 certified facility and handled according to Public Health Service Office of IL-22 neutralization, Ilrl1 mice were challenged intratracheally with 3 7 Laboratory Animal Welfare policies after review by the University of 7 10 A. fumigatus conidia in 50 ml; 24 h later, mice were administered Alabama at Birmingham Institutional Animal Care and Use Committee. 50 mg of rat anti-mouse IL-17A (cat. no. MAB421) or IL-22 (cat. no. MAB5821) or rat IgG isotype-control Ab or polyclonal goat anti-mouse Preparation of A. fumigatus, in vivo challenge, and lung IL-17A (cat. no. AF-421-NA) or IL-22 (cat. no. AF582) or goat IgG Downloaded from fungal burden assessment isotype-control Ab (cat. no. AB-108-C) (all Abs from R&D Systems) intratracheally. Forty-eight hours after challenge, mice were sacrificed, the A. fumigatus isolate 13073 (American Type Culture Collection, Manassas, left lungs were collected, and fungal burden was assessed as described VA) was maintained on potato dextrose agar for 5–7 d at 37˚C. Conidia above. were harvested by washing the culture flask with 50 ml of sterile PBS supplemented with 0.1% Tween 20. The conidia were passed through a In vitro treatments (PGE2 inhibition, EP receptor agonist) sterile 40-mm nylon membrane to remove hyphal fragments and enumer- In specific experiments, WT BL/6 mice were challenged with A. fumigatus, ated on a hemocytometer. For challenge, mice were lightly anesthetized http://www.jimmunol.org/ and lungs were collected and enzymatically digested 48 h later. Cells were with isoflurane and administered 7 3 107 A. fumigatus conidia in a volume enumerated and plated at 1 3 106 cells in a volume of 0.2 ml in the of 50 ml intratracheally, as previously described (10, 19). Briefly, mice are presence of vehicle, an inhibitor of microsomal PGE synthase (mPGES)-1 held in a vertical upright position, and the tongue is withdrawn from the (Cayman Chemical), or misoprostol, a global EP receptor (PGE receptor) mouth using forceps. A pipette is used to deliver the 50-ml inoculum to 2 agonist (Sigma). Supernatants were collected after 24 h and clarified by the caudal oropharynx, and normal breathing results in fluid aspiration into centrifugation, and inflammatory cytokine and IL-22 levels were quantified the lungs (20). For lung fungal burden analysis, the left lungs were col- by Bio-Plex and ELISA (R&D Systems), respectively (10). lected at 48 h postexposure and homogenized in 1 ml of PBS. Total RNA was extracted from 0.1 ml of unclarified lung homogenate using the Statistics MasterPure Yeast RNA Purification Kit (Epicentre Biotechnologies, Madison, WI), which includes a DNase treatment step to eliminate ge- Data were analyzed using GraphPad Prism Version 5.0 statistical soft- nomic DNA, as previously reported (21). Total RNA was also extracted ware (GraphPad, San Diego, CA). Comparisons between groups were by guest on September 26, 2021 from serial 1:10 dilutions of live A. fumigatus conidia (101–109) and made with the two-tailed unpaired Student t test when data were nor- treated with DNase to form a standard curve. Lung A. fumigatus burden mally distributed. Significance was accepted at a p value ,0.05. was analyzed with real-time PCR measurement of A. fumigatus 18S rRNA [GenBank accession number AB008401 (22)] and quantified using a Results standard curve of A. fumigatus conidia, as previously described (21). As a validation of the real-time PCR method, heat-killed A. fumigatus did not The lack of IL-33 signaling results in enhanced A. fumigatus yield a signal by real-time PCR and were unable to grow on potato dex- lung clearance trose agar plates (21). In addition, no-amplification controls (i.e., no re- verse transcriptase included in the cDNA reaction) yielded a signal , IL-33 is detrimental for lung infection with the Th1-requiring 0.001% by real-time PCR, indicating that the DNase treatment step effi- fungal pathogen Cryptococcus neoformans as a result of its ciently eliminated contaminating A. fumigatus DNA [because DNA is not known propensity to induce type 2/Th2 responses (25). IL-33 is predicative of organism viability (23)]. protective in a model of C. albicans peritoneal sepsis via the in- Lung cell isolation and culture, inflammatory cytokine duction of heightened neutrophil responses (26). Elimination of A. fumigatus from the lung is highly dependent on analysis, and PGE2 analysis (27). Moreover, there is evidence to suggest that innate type 2 Mice were anesthetized by i.p. ketamine/xylazine and sacrificed by ex- responses may also be beneficial in A. fumigatus lung clearance sanguination 48 h postinfection. Both lungs were collected and minced in IMDM (Sigma, St. Louis, MO) supplemented with 1% Penicillin– (28). Collectively, host immune responses initiated by IL-33 Streptomycin–L-glutamine (Mediatech, Herndon, VA), 10% heat-inactivated would appear to favor A. fumigatus lung clearance. In initial FBS (Invitrogen, Carlsbad, CA), and 0.4 mg/ml polymyxin B (Thermo studies, we demonstrate that IL-33 is increased in response to Fisher), followed by incubation for 60 min with tissue culture–grade type acute A. fumigatus challenge (Fig. 1A); however, unlike our pre- IV collagenase (1 mg/ml; Sigma) in a 37˚C orbital shaker at 100 rpm. The cell suspension was filtered through sterile 70- and 40-mm nylon filters, vious report on fungal asthma (29), IL-33 did not require dectin- and RBCs were lysed with ACK buffer (Lonza, Walkersville, MD) to 1–mediated b-glucan recognition for induction (Fig. 1A). To our 2 2 create lung cell preparations. For lung cell cultures, cells were enumerated surprise, mice deficient in IL-33R (ST2/T1/IL1-RL1; Il1rl1 / ) 6 on a hemocytometer and plated at 1 3 10 cells in a volume of 0.2 ml. demonstrated lower A. fumigatus burden in the lung 48 h post- Supernatants were collected after 24 h, clarified by centrifugation, and challenge (Fig. 1B), suggesting that IL-33 induction functions as a stored at 280˚C. Supernatants were analyzed for levels of 32 cy- tokines and using a Bio-Plex Multiplex suspension cytokine negative regulator of fungal clearance. We did not observe a dif- array (Bio-Rad), according to the manufacturer’s instructions (20, 21). The ference in fungal burden prior to 48 h postchallenge (data not data were analyzed using Bio-Plex Manager software (Bio-Rad). IL-22 shown). Intriguingly, augmented fungal clearance in Il1rl12/2 levels were quantified by ELISA (R&D Systems) (10). PGE2 levels were mice at 48 h postchallenge was not a consequence of differential quantified using a PGE Parameter Assay Kit (cat. no. KGE004B; R&D 2 lung cellularity (Fig. 1C), because we observed no significant Systems). In some experiments, neutrophil, inflammatory , and + levels in lung digest cells were analyzed by flow cytometry. changes in neutrophils and (gated on CD11b cells, Cells were washed, and Fc receptors were blocked with Mouse BD Fc followed by Ly6G+ cells as neutrophils and Siglec-F+ cells as The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. The lack of IL-33 signaling results in enhanced A. fumigatus lung clearance. (A) BL/6 WT and dectin-1–deficient (Clec7a2/2) mice were challenged intratracheally with A. fumigatus conidia; at 48 h postexposure, whole lungs were collected, and IL-33 levels were quantified in clarified lung homogenates by ELISA. The figure illustrates cumulative data from two or three independent studies (n = 3–5 mice per group, per study). (B) BL/6 WT and Il1rl12/2 (IL-1RL1; ST2-deficient) mice were challenged intratracheally with A. fumigatus conidia; at 48 h after exposure, lung fungal burden was assessed by real-time PCR analysis of A. fumigatus 18S rRNA levels. The figure illustrates cumulative data from two independent studies (n = 4 or 5 mice per group, per study). Data are expressed as mean A. fumigatus 18S rRNA + SEM. (C) Cumulative flow cytometric data from two independent studies (n = 2–4 mice per group, per study). Data are expressed as the absolute number of live cells in lung digests. (D and E) Lung cells were isolated via enzymatic digestion, Fc blocked, stained with a LIVE/DEAD staining kit, and stained with fluorochrome-conjugated CD11c, CD11b, Ly6G, Ly6C, and Siglec F. Representative flow cytometric plots are included for neutrophils and eosinophils (gated on CD11b+ cells, followed by Ly6G+ cells as neutrophils and Siglec F+ cells as eosinophils) (D) and inflammatory (gated on CD11b+ Ly6C+ cells, followed by gating on CCR2+ cells) (E). (F) BL/6 WT and Il1rl12/2 (IL-1RL1; ST2-deficient) mice were challenged intratracheally with A. fumigatus conidia; at 48 h after exposure, the right lungs were collected and en- zymatically digested, and unfractionated lung cells were cultured in triplicate for 24 h. CCL11/eotaxin, CXCL1/KC, and CCL3/MIP-1a levels were quantified in clarified coculture supernatants by Bio-Plex. The figure shows cumulative data from four independent studies (n = 1 or 2 mice per group, per study). (G) Representative H&E-stained lung sections from WT (left panel) and Il1rl12/2 (right panel) mice challenged intratracheally with A. fumigatus conidia for 48 h (original magnification 3200). (H) Representative Grocott’s methenamine silver–stained lung sections from WT (left panel) and Il1rl12/2 (right panel) mice challenged intratracheally with A. fumigatus conidia for 48 h (original magnification 3200). **p , 0.01, unpaired two-tailed Student t test. eosinophils) (Fig. 1D) or inflammatory monocytes (gated on organism from the lung is enhanced in the absence of IL-33 CD11b+ Ly6C+ cells, followed by gating on CCR2+ cells) (Fig. 1E) signaling. (all of which have been implicated in innate lung clearance of A. fumigatus) (10, 30, 31). Similar data were observed at 12 and IL-33 signaling negatively affects IL-17A and IL-22 after A. 24 h postinfection (Supplemental Fig. 1). This observation is fumigatus exposure supported by no differences in the levels of various chemokines Because we did not observe major cellular changes in the lung produced by lung digest cells (Fig. 1F) and similar levels of in- between A. fumigatus–exposed WT and Il1rl12/2 mice, we ex- flammatory cells and inflammation in H&E-stained lung tissue amined whether there was a difference in inflammatory factors sections (Fig. 1G). Sequential Grocott’s methenamine silver– that could explain improved fungal clearance in the absence of stained lung tissue sections also demonstrated lower levels of IL-33 signaling. We observed significantly higher levels of IL-1a, A. fumigatus organisms in Il1rl12/2 mice (right panel) compared IL-1b, and IL-6, a profile that favors the development of IL-17A with WT mice (left panel) (Fig. 1H). Thus, despite the induction and IL-22, which were also produced at higher levels by lung of IL-33 after acute A. fumigatus exposure, clearance of the digest cells from Il1rl12/2 mice (Fig. 2A). Higher inflammatory 4 INNATE REGULATION DURING INVASIVE ASPERGILLOSIS cytokine levels in the absence of IL-33 signaling suggested that a negative effect on the clearance of A. fumigatus from the lung, IL-33 functioned as a negative regulator of these mediators. To we did not observe an effect of IL-33 treatment on fungal burden confirm this, we challenged mice with A. fumigatus, followed by (Fig. 2D). Thus, the absence of IL-1RL1/ST2 signaling results in treatment with IL-33, which resulted in an ∼65% reduction in augmented lung clearance of A. fumigatus in the presence of IL-17A (Fig. 2B) and an ∼50% reduction in IL-22 production heightened IL-17A and IL-22 production, indicating that signaling (Fig. 2C). Although this suggests that IL-33 treatment could have through IL-1RL1 serves as a negative regulator of these mediators.

PGE2, but not IL-23, is increased in the absence of IL-33 signaling IL-23 is the most recognized positive regulator of IL-17A and IL- 22 production and we have previously reported that IL-23 drives IL-17A and IL-22 production in the lung after A. fumigatus ex- posure (19, 24). Therefore, we speculated that the increased pro- duction of IL-17A and IL-22 in the absence of IL-33 signaling was most likely the result of increased IL-23. However, Il1rl12/2 mice had similar levels of IL-23 in the lung as WT mice (Fig. 3A). There also was no difference in IL-23 production by A. fumigatus– stimulated bone marrow–derived dendritic cells between WT and 2 2 Il1rl1 / mice (Supplemental Fig. 2). These data suggested that Downloaded from IL-17A and IL-22 were elevated in Il1rl12/2 mice via an IL-23– independent mechanism. PGE2 has recently been described as a potentiator of innate IL-22 production (32); therefore, we ques- tioned whether PGE2 was modulated in the absence of IL-33 signaling. Results show that, at baseline, lung cells from naive 2/2 WT and Il1rl1 mice produced similar levels of PGE2 (Fig. 3B); http://www.jimmunol.org/ however, upon exposure to A. fumigatus, lung cells from Il1rl12/2 mice demonstrated significantly higher PGE2 production (Fig. 3B). These data suggest that IL-33 signaling regulates PGE2 production during A. fumigatus exposure. We next examined mice treated with IL-33 after A. fumigatus exposure and found that, like IL-17A and IL-22, PGE2 levels were significantly lower (Fig. 3C). Thus, IL-33 is a negative regulator of PGE2, which may function as a positive regulator of IL-17A and IL-22 production during lung fungal exposure. by guest on September 26, 2021 COX-2 inhibition results in attenuated inflammatory cytokine production in Il1rl12/2 mice after A. fumigatus exposure PG-endoperoxide synthase 2 (i.e., COX-2) initiates the conversion of arachidonic acid to multiple PGs, including PGE2. A recent study in humans reported that individuals with various systemic inflammatory syndromes demonstrated decreased COX-2 and PGE2 expression coupled with lower IL-22 levels (32). Using an experimental murine model of LPS-induced systemic inflamma- tion, this study demonstrated that PGE2–EP4 signaling promoted FIGURE 2. IL-33 signaling negatively affects IL-17A and IL-22 pro- homeostasis of type 3 innate lymphoid cells and drove them to 2 2 duction after A. fumigatus exposure. BL/6 WT and Il1rl1 / (IL-1RL1; produce IL-22 (32). To determine whether this axis also functioned ST2-deficient) mice were challenged intratracheally with A. fumigatus in the induction of lung-derived IL-22, as well as other inflamma- conidia; at 48 h after exposure, the right lungs were collected and enzy- tory mediators, we examined the effects of COX-2 inhibition. matically digested and unfractionated lung cells were cultured in triplicate 2 2 Treating A. fumigatus–exposed Il1rl1 / mice with celecoxib/ for 24 h. (A) IL-1a, IL-1b, IL-6, IL-17A, and IL-22 levels were quantified in clarified coculture supernatants by Bio-Plex or ELISA. The figure il- Celebrex, a COX-2–selective nonsteroidal anti-inflammatory drug lustrates cumulative data from four independent studies (n = 1 or 2 mice (NSAID), significantly lowered IL-1a (Fig. 4A), IL-1b (Fig. 4B), IL-6 per group, per study). (B and C) BL/6 WT mice were challenged with (Fig. 4C), IL-17A (Fig. 4D), and IL-22 (Fig. 4E) production. A. fumigatus and were administered IL-33 (1 mgin50ml) or PBS intra- Consequently, the attenuation of these mediators resulted in less 2 2 tracheally 6 and 24 h later. Forty-eight hours after exposure, the right lungs efficient fungal clearance in Il1rl1 / mice (Fig. 4F). Despite our were collected and enzymatically digested, and unfractionated lung cells previous reports showing a role for IL-17A and IL-22 in lung were cultured in triplicate for 24 h. IL-17A (B) and IL-22 (C) levels in defense against A. fumigatus (19, 24), neutralization of both me- coculture supernatants were quantified by Bio-Plex or ELISA. The figure diators in Il1rl12/2 mice unexpectedly did not affect their ability illustrates cumulative data from three independent studies (n = 1 or 2 mice to clear A. fumigatus from the lung (Fig. 4G). The effects of per group, per study). (D) BL/6 WT mice were challenged with A. fumigatus COX-2 inhibition were also observed in WT mice (e.g., IL-22 and were administered IL-33 (1 mgin50ml) or PBS intratracheally 6 and 6 6 24 h later. Forty-eight hours after exposure, lung fungal burden was assessed levels were 504 67 and 66 61 pg/ml in vehicle-treated (n =4) by real-time PCR analysis of A. fumigatus 18S rRNA levels. The figure il- and Celebrex-treated mice (n = 4), respectively; p = 0.001). Thus, lustrates cumulative data from four independent studies (n =3or4miceper inhibition of COX-2 signaling in the absence of IL-1RL1 during group, per study). Data are expressed as mean A. fumigatus 18S rRNA + A. fumigatus exposure compromises lung production of multiple SEM. **p , 0.01, ***p , 0.001, unpaired two-tailed Student t test. inflammatory mediators, leading to impaired fungal clearance. The Journal of Immunology 5

2/2 FIGURE 3. PGE2, but not IL-23, is increased in the absence of IL-33 signaling. (A) BL/6 WT and Il1rl1 (IL-1RL1; ST2-deficient) mice were challenged intratracheally with A. fumigatus conidia; whole lungs were collected, and IL-23 levels were quantified in clarified lung homogenates by ELISA 48 h after exposure. The figure illustrates cumulative data from two independent studies (n = 4 or 5 mice per group, per study). (B) BL/6 WT and Il1rl12/2 (IL-1RL1; ST2-deficient) mice were challenged intratracheally with A. fumigatus conidia; at 48 h after exposure, the right lungs were collected and en- zymatically digested, and unfractionated lung cells were cultured in triplicate for 24 h. PGE2 levels were quantified in clarified coculture supernatants by enzyme immunoassay. The figures illustrates cumulative data from three independent studies (n = 1 or 2 mice per group, per study). (C) BL/6 WT mice were challenged with A. fumigatus and were administered IL-33 (1 mgin50ml) or PBS intratracheally 6 and 24 h later. Forty-eight hours after exposure, the right lungs were collected and enzymatically digested, and unfractionated lung cells were cultured in triplicate for 24 h. PGE2 levels were quantified in clarified coculture supernatants by enzyme immunoassay. The figure illustrates cumulative data from three independent studies (n = 1 or 2 mice per group, Downloaded from per study). *p , 0.05, ***p , 0.001, unpaired two-tailed Student t test.

PGE2 supports production of IL-17A and IL-22, but not IL-6, the mechanism behind elevated PGE2 in the absence of IL-33 IL-1a, or IL-1b, by lung cells from A. fumigatus–exposed mice signaling is not a result of enhanced IL-1a and IL-1b production.

Although PGE2 is a product of COX-2 signaling, it is unclear http://www.jimmunol.org/ whether PGE2 itself affects inflammatory mediator production. To Discussion address this, we cultured lung cells from infected mice with an In this article, we demonstrate that, despite the induction of IL-33 inhibitor of mPGES-1, which converts the COX-2 product PGH2 in the lung in response to A. fumigatus, IL-33 signaling negatively into the biologically active PGE2. This demonstrated that specific regulated immune responsiveness to A. fumigatus, as evidenced by 2/2 inhibition of PGE2 attenuated IL-17A and IL-22 production mice deficient in IL-33R signaling (ST2; Il1rl1 mice) dis- (Fig. 5A) but not that of IL-6, IL-1a, or IL-1b (Fig. 5B). Con- playing lower fungal burden postchallenge. Assessment of po- versely, treating lung cells from infected mice with the global EP tential mechanisms to explain enhanced fungal clearance in the receptor (PGE2 receptor) agonist misoprostol increased IL-6, absence of IL-33 signaling revealed a specific profile of elevated IL-17A, and IL-22 production (Fig. 5C) over vehicle-treated cytokines: IL-1a, IL-1b, IL-6, IL-17A, and IL-22. Despite ob- by guest on September 26, 2021 lung cells, but it did not increase production of IL-1a and IL-1b serving elevated levels of these mediators, they did not correlate (Fig. 5D). Misoprostol was not effective at inducing these medi- with any changes in lung cellularity, an observation itself sup- ators in lung cells from naive mice (Supplemental Fig. 3). Col- ported by the lack of changes in the levels of multiple chemokines. lectively, our data reveal that PGE2 specifically promotes the As a result, we focused on the IL-17A/IL-22 axis, because of production of IL-17A and IL-22 during lung fungal exposure. their known abilities to induce soluble antimicrobial responses from the lung (34) and our previous work documenting a b Increased IL-1 and IL-1 signaling is not the mechanism a protective role for IL-17A and IL-22 in host defense against associated with enhanced PGE2 and IL-22 production in 2/2 A. fumigatus (19, 24). An intriguing finding from the current study Il1rl1 mice after A. fumigatus exposure was that IL-33 functioned as a negative regulator of IL-17A and Thus far, data indicate that the level of PGE2 directly correlates IL-22. Relatively few cytokines have been identified as negative with the level of IL-17A and IL-22 after A. fumigatus exposure; regulators of IL-17A and IL-22. The most well-recognized include 2/2 however, it is not clear why PGE2 is elevated in Il1rl1 mice TGF-b, which promotes IRF4-mediated suppression of IL-17A after A. fumigatus exposure. In addition to IL-17A and IL-22, data and IL-22 (35). Others include IL-27, which promotes c-Maf– in Fig. 2A showed that IL-1a and IL-1b were significantly ele- mediated inhibition of IL-17A and IL-22, and IL-25, which favors vated in Il1rl12/2 mice after A. fumigatus exposure. IL-1b has type 2 cytokine induction and, consequently, IL-17A and IL-22 previously been implicated in PGE2 induction (33). In turn, mice suppression. Overall, our findings suggest the addition of IL-33 to deficient in the receptor for IL-1a and IL-1b demonstrated an this list, because the absence of IL-1RL1/ST2 signaling results in ∼60% reduction in PGE2 levels (Fig. 6A), as well as a profound heightened IL-17A and IL-22 production, whereas increasing reduction in the production of IL-22 (Fig. 6B), suggesting that the IL-33 levels results in decreased IL-17A and IL-22 production, increase in IL-1a and/or IL-1b in the absence of IL-33 signaling indicating that IL-33 signaling through IL-1RL1 serves as a was potentially responsible for the increase in PGE2 and, thus, negative regulator of IL-17A and IL-22. IL-22 production. To confirm this, we determined whether IL-1a Based on our previous work (19, 24), our hypothesis behind and IL-1b levels were modulated in mice that received IL-33 after elevated IL-17A and IL-22 levels in the absence of IL-33 signaling A. fumigatus challenge. Despite the fact that IL-33 signaling im- was that IL-33 negatively regulated IL-23, which we have shown paired PGE2 (Fig. 3C) and IL-22 (Fig. 2C) production, IL-33 to drive IL-17A and IL-22 production in the lung (19, 24). administration had no effect on the levels of IL-1a or IL-1b However, this was found not to be the case, resulting in a search (Fig. 6C). There was also no synergy between IL-1b and PGE2 for an IL-23–independent mechanism of enhanced IL-17A and IL- receptor stimulation, because the addition of IL-1b, in conjunction 22 production. A role for PGE2 in the generation of IL-17A and with misoprostol, to lung cells from A. fumigatus–infected mice IL-22 has been controversial. It has been reported that PGE2 may did not augment production of IL-22 (Supplemental Fig. 3). Thus, enhance IL-17A production from previously polarized Th17 cells 6 INNATE REGULATION DURING INVASIVE ASPERGILLOSIS Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. COX-2 inhibition results in attenuated inflammatory cytokine production in Il1rl12/2 mice after A. fumigatus exposure. (A–E) BL/6 WT and Il1rl12/2 (IL-1RL1; ST2-deficient) mice were administered celecoxib i.p. (100 mg/kg in 250 ml) or vehicle (V; [ethanol]) 24 h prior to A. fumigatus challenge and then again at 6 and 24 h postchallenge. Thirty-six to forty-eight hours after A. fumigatus challenge, mice were sacrificed, the right lungs were collected and enzymatically digested, and unfractionated lung cells were cultured for 24 h. IL-1a (A), IL-1b (B), IL-6 (C), IL-17A (D), and IL-22 (E) levels in coculture supernatants were quantified by Bio-Plex or ELISA. The figure illustrates cumulative data from four independent studies (n = 3 or 4 mice per group, per study). (F) Mice were treated as in (A); 48 h after exposure, lung fungal burden was assessed by real-time PCR analysis of A. fumigatus 18S rRNA levels. The figure illustrates cumulative data from two independent studies (n = 4 mice per group, per study). (G) Il1rl12/2 (IL-1RL1; ST2-deficient) mice were challenged intratracheally with A. fumigatus conidia, and 24 h thereafter, mice were administered 50 mg of rat anti-mouse IL-17A or IL-22 (R&D Systems), rat IgG isotype control Ab or goat anti-mouse IL-17A, or IL-22 (R&D Systems) or goat IgG isotype control Ab. Forty-eight hours after challenge, mice were sacrificed, the left lungs were collected, and fungal burden was assessed. The figure illustrates cumulative data from two or three independent studies (n = 3 or 4 mice per group). Data are expressed as mean A. fumigatus 18S rRNA + SEM. *p , 0.05, **p , 0.01, ***p , 0.001, unpaired two-tailed Student t test. T, treated. or memory Th17 cells (36, 37); however, recent data show that were not determined. However, as described earlier, a recent report PGE2 has the complete opposite (i.e., negative) effect on Th17 demonstrated that the COX2/PGE2 axis functioned in the generation generation from naive T cells (38). In this latter study, the fungal of innate IL-22 production in humans and mice (32). In this study, pathogen C. neoformans was shown to produce its own PGE2, using an experimental model of LPS-induced systemic inflammation, which negatively affected IL-17A production but had no effect on PGE2–EP4 signaling promoted homeostasis of type 3 innate lym- IL-22 (38). Other studies showed that anti-CD3 activation of phoid cells and drove them to produce IL-22. We found that, similar 2/2 human PBMCs in the presence of exogenous PGE2 promoted to IL-17A and IL-22, PGE2 levels were elevated in Il1rl1 mice; IL-17A production but significantly dampened IL-22 production treating mice with IL-33 resulted in the lowering of IL-17A, IL-22, (39). Human PBMCs stimulated with the fungus C. albicans and PGE2 levels in the lung. Although IL-17A and IL-22 levels were exhibited increased IL-17A and IL-22 production that was slightly reduced after IL-33 treatment (approximately two thirds and one half, affected by the NSAID diclofenac (p , 0.05 for IL-17A; IL-22 respectively), they were not completely eliminated. Moreover, IL-33 was slightly lower but not statistically significant) (40). In this treatment had no effect on the levels of IL-1a,IL-1b,orIL-6. report, EP receptor agonists were moderately effective at restoring Consequently, we did not observe an effect of IL-33 treatment on NSAID-inhibited IL-17A production, although the effects on IL-22 A. fumigatus lung clearance. Based on these results, we hypothesize The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 5. PGE2 supports IL-17A and IL-22, but not IL-6, IL-1a, or IL-1b, production by lung cells from A. fumigatus–exposed mice. (A and B) BL/6 WT and Il1rl12/2 (IL-1RL1; ST2-deficient) mice were challenged with A. fumigatus conidia; at 48 h after exposure, mice were sacrificed, the right lungs were collected and enzymatically digested, and unfractionated lung cells were cultured in the presence of the mPGES inhibitor (I) or vehicle (V; DMSO)in triplicate for 24 h. IL-17A and IL-22 levels (A) and IL-6, IL-1a, and IL-1b levels (B) in coculture supernatants were quantified by Bio-Plex or ELISA. The figure illustrates cumulative data from three independent studies (n = 1 or 2 mice per group, per study). (C and D) BL/6 WT mice were exposed, and cells A C were isolated as in ( ). Cells were cultured in the presence of misoprostol or vehicle in triplicate for 24 h. IL-6, IL-17A, and IL-22 levels ( ) and IL-1a and by guest on September 26, 2021 IL-1b levels (D) in coculture supernatants were quantified by Bio-Plex or ELISA. The figure illustrates cumulative data from two independent studies (n =1 or 2 mice per group, per study). *p , 0.05, **p , 0.01, ***p , 0.001, unpaired two-tailed Student t test. that IL-33 treatment does not facilitate a level of immunosuppres- increased IL-6, IL-17A, and IL-22 production but not IL-1a or sion that compromises fungal clearance. In contrast, when targeting IL-1b. Conversely, specific inhibition of PGE2 had a negative PGE2 via COX-2 inhibition, IL-17A and IL-22 levels, as well as effect on IL-17A and IL-22 production from lung cells, but not IL-1a, IL-1b and IL-6 levels, in Il1rl12/2 mice were significantly IL-1a,IL-1b, or IL-6 production. The novelty in our findings is reduced, which correlated with reduced A. fumigatus lung clear- not only that the COX-2/PGE2 axis drives innate IL-17A and ance. An unexpected finding from our work was that neutral- IL-22 production in a mucosal tissue (i.e., the lung), suggesting ization of IL-17A and IL-22 in Il1rl12/2 mice did not impair a conserved mechanism of IL-17A and IL-22 induction, it also A. fumigatus lung clearance. Based on observations with IL-33 identifies putative upstream regulators of the PGE2/IL-17A/IL-22 treatment (reduced IL-17A and IL-22 levels; intact IL-1a, axis (i.e., IL-33 signaling). A question arising from our findings is IL-1b, and IL-6 levels; and no effect on fungal clearance) and how does IL-33 signaling regulate PGE2 induction and, thus, celecoxib/Celebrex treatment (reduced IL-17A, IL-22, IL-1a, control the level of inflammatory cytokines during fungal expo- IL-1b, and IL-6 levels and a negative effect on fungal clearance), sure? IL-1 signaling was a prime candidate responsible for increased 2/2 2/2 the mechanism of resistance in Il1rl1 mice appears to be quite PGE2 in Il1rl1 mice, because IL-1a and IL-1b were elevated in complex. Specifically, intact (IL-33 treatment) or elevated (Il1rl12/2 these mice after fungal exposure, and IL-1 signaling is known to mice) IL-1a, IL-1b, and/or IL-6 may compensate in some manner induce PGE2 (41, 42). However, despite a profound reduction in when IL-17A and IL-22 levels are manipulated. It would be of IL-17A and IL-22 levels, IL-1R deficiency resulted in only a 2/2 2/2 interest in future studies to cross Il1rl1 mice with Il1r moderate reduction in PGE2 production. Moreover, treating mice mice, which would eliminate IL-1a and IL-1b signaling in tandem with IL-33 did not attenuate the levels of IL-1a and IL-1b, despite with IL-33 signaling and may result in mice deficient in IL-17A significantly lower PGE2 levels. Collectively, these results suggest and IL-22 induction, as well as IL-1a and IL-1b signaling. that other IL-1–independent factors were responsible for elevated 2/2 When targeting PGE2 via COX-2 inhibition, IL-17A and IL-22 PGE2 levels in Il1rl1 mice. To this end, we assessed additional levels, as well as IL-1a, IL-1b, and IL-6 levels, in Il1rl12/2 mice factors that were increased in A. fumigatus–exposed Il1rl12/2 were significantly reduced, further suggesting that COX-2–medi- mice, yet were decreased in mice that were treated with IL-33. ated PGE2 contributed in some way to the heighted IL-17A and This analysis identified the cytokines IL-3, IL-17A, and IFN-g. IL-22 production observed in these mice. To confirm that PGE2 Although published reports suggest that each of these may induce had the specific ability to induce IL-17A and IL-22, addition of a PGE2 (43–47), neutralization of IL-3, IL-17A, or IFN-g in lung 2/2 PGE2 agonist to lung cells from A. fumigatus–exposed mice cell cultures from Il1rl1 mice did not result in attenuated PGE2 8 INNATE REGULATION DURING INVASIVE ASPERGILLOSIS

of PGE2 generation impaired, IL-17A and IL-22 production. Overall, the current body of work adds depth to our understanding of the role that specific IL-1 family members play in IL-17A and IL-22 regulation and innate lung defense. Moreover, these find- ings lay the foundation for determining whether IL-33 signaling affects PGE2 and/or IL-17A and IL-22 induction in other IL-17A/ IL-22–dependent models of inflammation, such as colitis and pso- riasis, or infection models requiring IL-17A/IL-22 for defense, such as Gram-negative bacterial pneumonia and influenza.

Disclosures The authors have no financial conflicts of interest.

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Corrections

Garth, J. M., K. M. Reeder, M. S. Godwin, J. J. Mackel, C. W. Dunaway, J. P. Blackburn, and C. Steele. 2017. IL-33 signaling regulates innate IL-17A and IL-22 production via suppression of prostaglandin E2 during lung fungal infection. J. Immunol. 199: 2140–2148.

The authors discovered mislabeled axes in Fig. 6C. Specifically, the y-axis should read “pg/ml (lung digest cells - 48 h)” instead of “IL-22 pg/ml (lung digest cells - 48 h).” In addition, “IL-1a” and “IL-1b” should be included on the x-axis.

The corrected version of Fig. 6 is shown below. The figure legend was correct as published and is shown below for reference. The online version of this article has been corrected and now differs from the print version as originally published.

We apologize for this oversight.

2/2 FIGURE 6. Increased IL-1R signaling is not the mechanism associated with enhanced PGE2 and IL-22 in Il1rl1 mice after A. fumigatus exposure. BL/6 WT and Il1rl12/2 (IL-1R1–deficient) mice were challenged intratracheally with A. fumigatus conidia, and 48 h after exposure, the right lungs were collected and enzymatically digested, and unfractionated lung cells were cultured in triplicate for 24 h. PGE2 levels were quantified in clarified coculture supernatants by enzyme immunoassay (A), and IL-22 levels were quantified in clarified coculture supernatants by ELISA (B). The figures illustrate cu- mulative data from four independent studies (n 5 1 or 2 mice per group, per study). (C) BL/6 WT mice were challenged with A. fumigatus and administered IL-33 (1 mgin50ml) or PBS intratracheally 6 and 24 h later. Forty-eight hours after exposure, the right lungs were collected and enzymatically digested, and unfractionated lung cells were cultured in triplicate for 24 h. IL-1a and IL-1b levels were quantified in clarified coculture supernatants by Bio-Plex. The figure illustrates cumulative data from two independent studies (n 5 1 or 2 mice per group, per study). ***p , 0.001, unpaired two-tailed Student t test. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701301

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