Neuroendocrine Modulation of IL-27 in Julian Roewe, Maximilian Higer, Dennis R. Riehl, Adrian Gericke, Markus P. Radsak and Markus Bosmann This information is current as of September 26, 2021. J Immunol published online 23 August 2017 http://www.jimmunol.org/content/early/2017/08/23/jimmun ol.1700687 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 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 23, 2017, doi:10.4049/jimmunol.1700687 The Journal of Immunology

Neuroendocrine Modulation of IL-27 in Macrophages

Julian Roewe,* Maximilian Higer,* Dennis R. Riehl,* Adrian Gericke,† Markus P. Radsak,‡ and Markus Bosmann*

Heterodimeric IL-27 (p28/EBV-induced gene 3) is an important member of the IL-6/IL-12 family. IL-27 is predominantly synthesized by mononuclear phagocytes and exerts immunoregulatory functional activities on lymphocytic and nonlymphocytic cells during infection, autoimmunity or neoplasms. There is a great body of evidence on the bidirectional interplay between the autonomic nervous system and immune responses during inflammatory disorders, but so far IL-27 has not been defined as a part of these multifaceted neuroendocrine networks. In this study, we describe the role of catecholamines (as mediators of the sympathetic nervous system) related to IL-27 production in primary mouse macrophages. Noradrenaline and adrenaline

dose-dependently suppressed the release of IL-27p28 in LPS/TLR4-activated macrophages, which was independent of a1 adre- noceptors. Instead, b2 adrenoceptor activation was responsible for mediating gene silencing of IL-27p28 and EBV-induced gene 3. Downloaded from The b2 adrenoceptor agonists formoterol and salbutamol mediated suppression of IL-27p28 production, when triggered by zymosan/TLR2, LPS/TLR4, or R848/TLR7/8 activation, but selectively spared the polyinosinic-polycytidylic acid/TLR3 pathway.

Mechanistically, b2 adrenergic signaling reinforced an autocrine feedback loop of -derived IL-10 and this synergized with inhibition of the JNK pathway for limiting IL-27p28. The JNK inhibitors SP600125 and AEG3482 strongly decreased + + intracellular IL-27p28 in F4/80 CD11b macrophages. In endotoxic shock of C57BL/6J mice, pharmacologic activation of b2 adrenoceptors improved the severity of shock, including hypothermia and decreased circulating IL-27p28. Conversely, IL-27p28 was 2.7-fold increased by removal of the catecholamine-producing adrenal glands prior to endotoxic shock. These data suggest a http://www.jimmunol.org/ novel role of the sympathetic neuroendocrine system for the modulation of IL-27–dependent acute inflammation. The Journal of Immunology, 2017, 199: 000–000.

nterleukin-27 is a heterodimeric glycosylated protein of the (7, 8). The central role of mononuclear phagocytes for IL-27 IL-6/IL-12 family of (1–3). It consists of the sig- production is underscored by our previous observation that deple- I nature subunit p28 (also known as IL-27p28), IL-27A, or IL- tion of these cells resulted in .85% reduction of circulating IL-27 30 (2). The second subunit of IL-27, EBV-induced gene 3 (EBI3), concentrations during acute inflammation (9). IL-27 is released

is shared with heterodimeric IL-35 (EBI3/p35) (4). The p28 and by mononuclear phagocytes during viral, bacterial, protozoan, and by guest on September 26, 2021 EBI3 subunits interact with noncovalent bonds for the formation fungal infections (10–13). For example, living or inactivated of IL-27 to bind to the specific, high-affinity IL-27RA receptor Gram-negative bacteria, including Escherichia coli or Salmonella also known as WSX-1 (5, 6). enteritidis, provoke the production of IL-27 (14, 15). On the The gene expression and release of IL-27 mainly occurs in molecular level, the agonistic activation of pathogen recognition mononuclear phagocytes such as macrophages and dendritic cells receptors such as TLR2, TLR3, TLR4, TLR7, and TLR9 is suf- in direct response to microbial patterns or other immune signals ficient to initiate abundant gene expression of IL-27 (10, 16, 17). Both Toll/IL-1R domain–containing adaptor inducing IFN-b and *Center for Thrombosis and Hemostasis, University Medical Center, Johannes MyD88 adaptor proteins are required for IL-27p28 production in Gutenberg University Mainz, 55131 Mainz, Germany; †Department of Ophthalmol- LPS-activated macrophages (9, 18, 19). spe- ogy, University Medical Center, Johannes Gutenberg University Mainz, 55131 cifically promotes the gene expression of IL-27p28 rather than Mainz, Germany; and ‡Third Department of Medicine, University Medical Center, Johannes Gutenberg University Mainz, 55131 Mainz, Germany EBI3 following TLR4 activation (20). The downstream signaling ORCIDs: 0000-0002-0503-1274 (M.H.); 0000-0002-3991-5721 (M.P.R.). pathways involve recruitment of IFN regulatory factors 1, 3, and 9 Received for publication May 9, 2017. Accepted for publication July 31, 2017. and NF-kB (c-Rel) transcription factors to the promoter region of This work was supported by the B. Braun Foundation, Federal Ministry of Education the IL-27p28 gene (18, 19, 21). Additionally, IL-27 production is and Research Grant 01EO1503 (to M.B.), Deutsche Forschungsgemeinschaft Grants greatly amplified by type I and type II IFNs based on autocrine/ BO3482/3-1, BO3482/3-3, and BO3482/4-1 (to M.B.), European Union Marie Curie paracrine-positive feedback loops (20, 21). Alternatively, PI3K/ Career Integration Grant 334486 (to M.B.), and by a Clinical Research Fellowship of the European Hematology Association (to M.B.). The authors are responsible for the Akt and STAT3 represent inhibitory signaling pathways for an- contents of this publication. tagonizing IL-27p28 gene expression in LPS/TLR4-activated Parts of the data in this study appeared in the thesis of M.H. macrophages (9, 16). Address correspondence and reprint requests to Prof. Markus Bosmann, Center for The prominent functions of IL-27 following ligation to the Thrombosis and Hemostasis, University Medical Center, Johannes Gutenberg Uni- IL-27RA/gp130 receptor complex on various subsets include versity Mainz, Langenbeckstrasse 1, 55131 Mainz, Germany. E-mail address: [email protected] activation of JAK1/2, asymmetric STAT1/STAT3, and Egr-2 sig- The online version of this article contains supplemental material. naling pathways culminating in the release of IL-10 (5, 22–25). Abbreviations used in this article: BMDM, bone marrow–derived macrophage; CSS, IL-27 signaling also stimulates dendritic cells for induction of the clinical severity score; EBI3, EBV-induced gene 3; NOS2, NO synthase 2; PEM, immunoregulatory molecule CD39 (26). These properties situate peritoneal elicited macrophage; poly(I:C), polyinosinic-polycytidylic acid; RT-PCR, IL-27 as a major T cell–suppressive cytokine required for regaining real-time PCR; WT, wild-type. immune homeostasis following clearance of infections and for Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 preventing T cell–mediated lethal inflammation (12). IL-27 antag-

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700687 2 NEUROENDOCRINE REGULATION OF IL-27 onizes Th17 cell development and Th17 cell functions via STAT1 culture dishes and subsequent segregation from nonadherent cells as de- (26–29). The differentiation of type 1 regulatory T cells is induced scribed by us before (9). Peritoneal elicited macrophages (PEM) were by IL-27 (30). Alternatively, IL-27 mediates the survival of both collected 4 d after i.p. injection of thioglycollate (1.5 ml, 2.4% w/v; Becton + + Dickinson, Franklin Lakes, NJ). Macrophages were cultured in RPMI 1640 activated CD4 and CD8 T cells (31, 32). The clonal expansion of (Thermo Fisher Scientific, Waltham, MA) containing 0.1% BSA (Sigma- naive CD4+ T cells and Th1-mediated responses including IFN-g Aldrich, St. Louis, MO) and 100 U/ml penicillin-streptomycin (Thermo production can be activated by IL-27 (2, 33, 34). Additionally, IL- Fisher Scientific) at 37˚C, 5% CO2. At the end of experiments, macrophage 27 synergizes with type I and type II IFNs for limiting innate supernatants were collected, cleared from cellular debris by centrifugation (500 3 g, 5 min, 4˚C), and cryopreserved at 280˚C until further protein lymphoid cell type 2–like innate immune responses (35, 36). Hence, analysis. Adherent macrophages were either lysed for later mRNA ex- the functional properties of IL-27 can encompass immunosuppres- traction or carefully detached from plates for flow cytometry analysis. sion or promotion of inflammation, and its overall effects are most likely dependent on the specific disease context, other inflammatory Endotoxic shock signals, and the total amounts of IL-27 generated (1). LPS (10 mg/kg body weight) from E. coli serotype 0111:B4 (Sigma-Aldrich) The autonomic nervous system with its sympathetic and para- with and without formoterol (200 nmol/kg body weight; Sigma-Aldrich) was sympathetic transmitters orchestrates immune responses, inflam- injected into the peritoneum of C57BL/6J mice. At 3–12 h time points, small volumes of EDTA-anticoagulated blood samples were collected by retro- mation, and immunity (37, 38). Catecholamines are the primary orbital puncture under deep anesthesia with isoflurane. Plasma samples mediators of the sympathetic arm of the autonomic nervous sys- were obtained by centrifugation (2000 3 g, 10 min, 4˚C) and stored at 280˚C tem (39). Noradrenaline is released by nerves innervating the until further analysis. At the end of experiments, mice were euthanized by primary and secondary lymphoid organs such as thymus, bone CO2 inhalation and spleens were isolated for mRNA studies. For survival studies, LPS (10 mg/kg body weight; i.p.) with and without Downloaded from marrow, lymph nodes, and spleen (40, 41). Additionally, nor- formoterol (200 nmol/kg body weight; i.p.) was administered followed by a adrenaline is also produced in the medulla of adrenal glands, with second injection of formoterol (200 nmol/kg body weight; i.p.) or an the latter representing the predominant source of adrenaline re- equivalent volume of vehicle (PBS) after 24 h. Body surface temperature lease into the bloodstream (40). Additionally, leukocytes can was measured with an infrared thermometer over the abdomen (153 IRB; transform into a relevant source of catecholamines during in- Bioseb, Vitrolles, France). The clinical severity score (CSS; ranging from 1 [no symptoms] to 5 [dead]) was assessed as described elsewhere (57). flammation (42). Noradrenaline and adrenaline act with varying To study the role of endogenous glucocorticoid release, C57BL/6J mice affinity through nine distinct subtypes of adrenoceptors (a1A, a1B, were treated with mifepristone (30 mg/kg body weight i.p.) (58), whereas http://www.jimmunol.org/ a1D, a2A, a2B, a2C, b1, b2, b3) (43). Mononuclear phagocytes the control group received the same volume of 3.49% DMSO (v/v) in PBS, were shown to express the a and the b adrenoceptors (44). and both groups of mice received LPS (10 mg/kg body weight; i.p.) 30 min 1 2 later. EDTA-anticoagulated blood samples were collected after 8 h by These receptor subtypes transmit neuroendocrine stress signals for retro-orbital puncture as described above. orchestrating the functional participation of mononuclear phago- cytes in inflammation. A variety of mononuclear phagocyte func- ELISA tions such as chemotaxis (45, 46), phagocytic efficiency (47), Ag ELISA kits for mouse IL-27p28, IL-10, MCP1, and KC were purchased presentation (48), cell proliferation (49), and most notably cyto- from R&D Systems (Minneapolis, MN). The mouse IL-6 ELISA was from kine release (50–52) are affected by adrenoceptor activation. BioLegend (San Diego, CA). Cell-free supernatants or plasma were ana- In the present study, we examined the hypothesis that adrenergic lyzed according to the manufacturer’s protocol. Briefly, the samples were by guest on September 26, 2021 diluted in PBS supplemented with 0.1% BSA to fit into the standard range mediators are important for the release of IL-27 by selectively LPS/ and the optical densities of oxidized tetramethylbenzidine were measured TLR4-activated macrophages. We show that catecholamines with an Opsys MR Dynex microplate reader (Dynex Technologies, Chantilly, potently suppress the production of IL-27 via activation of b2 VA). The IL-27p28 ELISA is specified by the manufacturer to display adrenoceptors by mechanisms involving IL-10 and the JNK sig- 4.6% cross-reactivity with recombinant mouse IL-27 (p28/EBI3 fusion). naling pathway. These data expand our current view on the en- Reverse transcription and real-time PCR tanglements of the sympathetic nervous system via catecholamines during acute inflammatory responses. Macrophages or spleen homogenates (TissueLyser II; Qiagen, Valencia, CA) were processed and total RNA was isolated with help of the RNeasy mini kit (Qiagen). Next, cDNA was generated via reverse transcription Materials and Methods (high-capacity cDNA reverse transcription kit; Applied Biosystems, Foster Animals City, CA). For real-time PCR (RT-PCR), 1–2 ng of cDNA was complemented with iQ SYBR Green master mix (Bio-Rad Laboratories, Hercules, CA) as Mouse studies were performed in accordance with the Animal Protection well as specific forward and reverse primers at a concentration of 0.5 mM each. Act of Germany, the State Investigation Office of Rhineland-Palatinate, the Reactions were performed with a C1000 thermal cycler (Bio-Rad Laborato- 2DDCt Federation of European Laboratory Animal Science Associations guide- ries) and results were normalized to GAPDH using the 2 method. Primer lines, and directive 2010/63/EU of the European Parliament and of the sequences were as follows: mouse GAPDH, 59-TACCCCCAATGTGTCC- Council of the European Union. C57BL/6J mice and IL-102/2 mice GTCGTG-39 (forward), 59-CCTTCAGTGGGCCCTCAGATGC-39 (reverse); (B6.129P2-Il10tm1Cgn/J) were originally obtained from The Jackson Lab- mouse IL-27p28, 59-GGCCATGAGGCTGGATCTC-39 (forward), 59-AACATTT- 2/2 2/2 oratory (Bar Harbor, ME) (53). The generation of a1A-AR and a1B-AR GAATCCTGCAGCCA-39 (reverse); mouse EBI3, 59-GGCTGAGCGAATCAT- mice has been previously described (54–56). These strains were backcrossed CAA-39 (forward), 59-GAGAGAGAAGATGTCCGGGAA-39 (reverse); mouse for nine generations on a C57BL/6Slc background and used along with the IL-10, 59-AGACACCTTGGTCTTGGAGC-39 (forward) and 59-TTTGAAT- corresponding wild-type (WT) control strain. All mouse strains were bred TCCCTGGGTGAGA-39 (reverse);mousearginase1,59-CAGAAGAATGG- and genotyped at the animal facilities of the University Medical Center of the AAGAGTCAG-39 (forward) and 59-CAGATATGCAGGGAGTCACC- Johannes Gutenberg University Mainz in a specific pathogen-free environ- 39 (reverse). ment under standardized conditions with a 12-h light/dark cycle, temperature of 22 6 2˚C, humidity of 55 6 10%, and with free access to food and tap Flow cytometry water. Adrenalectomized C57BL/6 mice and sham operated C57BL/6 mice Intracellular IL-27p28 was trapped in the macrophages by incubation for were purchased from Taconic Biosciences (Hudson, NY) and were housed as 12 h with monensin (2 mM; Becton Dickinson). Following gentle de- described above. The adrenalectomized C57BL/6 mice received normal tachment of macrophages, Fc receptors were blocked by incubating with saline (NaCl 0.9%) as drinking water to maintain their fluid and electrolyte anti-CD16/CD32 Ab (TruStain FcX; BioLegend). After surface marker balances in the absence of adrenal hormones. staining for CD11b–Pacific Blue (clone M1/70; BioLegend) and F4/80- Macrophage preparation and incubation allophycocyanin (clone BM8; BioLegend), cells were fixed and per- meabilized (Cytofix/Cytoperm Plus kit; Becton Dickinson). Subsequently, Bone marrow–derived macrophages (BMDM) were generated by incu- the cells were stained with IL-27p28–PE (clone MM27-7B1; eBioscience, bating isolated bone marrow for 7 d with L929 cell–conditioned medium in San Diego, CA), NO synthase 2 (NOS2)–Alexa Fluor 488 (clone CXNFT; The Journal of Immunology 3 eBioscience), or CD206-PE/Cy7 (clone C068C2; BioLegend). Matched in LPS/TLR4-activated macrophages derived from mice with fluorochrome-labeled isotype Abs were used as controls. At least 50,000 genetic deficiency of the a1 adrenoceptors (a1A, a1B) to mac- cells were acquired with a FACSCanto II (Becton Dickinson), and data rophages from WT mice (C57BL/6Slc) after LPS treatment analyses were performed using WinList for Win32 3.0 software (Verity Software, Topsham, ME) and FlowJo VX for Windows (FlowJo, Ashland, (Fig. 1D). Importantly, catecholamines were equally active to OR) with pregating on mononuclear phagocytes according to forward and suppress IL-27p28 release by macrophages derived from a1A- 2/2 2/2 side scatter plots. AR or a1B-AR mice as compared with macrophages from Bead-based phosphoprotein assay WT mice (Fig. 1D). For comparison, the concentrations of IL-27p28 are depicted as relative values normalized to LPS alone The phosphorylation status of JNK was assessed using the phospho-JNK for each individual mouse strain and pooled from several inde- (Thr183/Tyr185) single-plex set from Bio-Rad Laboratories following the manufacturer’s protocol. Briefly, 2 3 106 macrophages were lysed in the pendent experiments. There was an inconsistent trend of lower 2/2 presence of inhibitors for proteases and phosphatases (Bio-Plex cell lysis absolute concentrations of IL-27p28 in LPS-activated a1B-AR kit; Bio-Rad Laboratories). All magnetic bead washing steps were per- macrophages (data not shown). We also used prazosin as a non– formed with a Bio-Plex Pro II wash station (Bio-Rad Laboratories). Fluo- subtype selective a adrenoceptor antagonist. Indeed, adrenaline rescence intensities corresponding to phospho-JNK content were measured 1 in a Luminex 200/Bio-Plex 200 system with Bio-Plex Manager software and noradrenaline retained their activities as suppressors of LPS- 6.1 (Bio-Rad Laboratories). induced IL-27p28 release during a1 adrenoceptor blockade with prazosin (Fig. 1E). Collectively, the data presented in Fig. 1D and Reagents 1E pointed against the involvement of a1 adrenoceptors for LPS (E. coli serotype O111:B4), salbutamol (a-[(tert-butylamino)methyl]- modulating the production of IL-27 in macrophages.

4-hydroxy-m-xylene-a,a9-diol), formoterol ((R*,R*)-N-[2-hydroxy-5-[1- Downloaded from b hydroxy-2-[[2-(4-methoxyphenyl)-1-ethylethyl]amino]ethyl]phenyl]formamide IL-27 is silenced by 2 adrenoceptor agonists fumarate), and mifepristone (RU-486) were purchased from Sigma- To further characterize the role of adrenoceptors for regulating the Aldrich. Prazosin hydrochloride and salmeterol xinafoate were pur- chased from Tocris Bioscience (Bristol, U.K.). Orciprenaline (Alupent) gene expression of IL-27, we designed experiments using b2 was from Boehringer Ingelheim (Ingelheim, Germany). The TLR agonists adrenoceptor agonists. Macrophages from C57BL/6J mice were polyinosinic-polycytidylic acid [poly(I:C)], zymosan (Saccharomyces activated with LPS (100 ng/ml) alone or combined with the cerevisiae) and R848 were from InvivoGen (San Diego, CA). Adrenaline

highly b2 selective adrenoceptor agonist, (R,R)-formoterol http://www.jimmunol.org/ was obtained from Jenapharm (Jena, Germany) and noradrenaline was 26 from Sanofi Aventis (Gentilly, France). Recombinant mouse IL-10 and (10 M). At different time points (0–24 h), the mRNA was IL-4 were purchased from PeproTech (Rocky Hill, NJ). JNK inhibitors extracted and expression levels of IL-27p28 were analyzed by AEG3482 and SP600125 were obtained from Tocris Bioscience. Neu- RT-PCR and normalized to GAPDH mRNA (Fig. 2A). The LPS- tralizing anti-mouse IL-10 receptor Abs (clone 1B1.3a) were affinity pu- induced accumulation of mRNA for IL-27p28 peaked around 6 h rified using protein G–Sepharose (GE Healthcare, Munich, Germany), and was profoundly diminished by the addition of formoterol. according to a standard protocol, and used together with corresponding azide-free control rat IgG1k Abs (BioLegend). The treatment of macrophages with formoterol alone did not result in altered gene expression of IL-27p28 as compared with Statistical analysis resting untreated macrophages (Ctrl; Fig. 2A). Next, we used

GraphPad Prism v6.00 software was used for statistical analyses. In vitro salbutamol (also known as albuterol) as another Food and Drug by guest on September 26, 2021 experiments were repeated three times (biological replicates), and each Administration–approved drug with b2 adrenoceptor agonistic independent experiment was performed in duplicate wells (technical rep- activities. Likewise, salbutamol was a potent inhibitor of IL-27p28 licates) unless stated otherwise in the figure legends. In vivo data were generated with the numbers of mice as indicated in the figure legends. All mRNA (Fig. 2B). The gene expression of EBI3 was also studied, values are depicted as mean with error bars representing SEM. Statistical because it is the second subunit of heterodimeric IL-27 (2). Indeed, significance was analyzed using the unpaired two-tailed Student t test, one- LPS promoted an accumulation of the mRNA for EBI3, but the way ANOVA with Dunnett multiple comparisons test, two-way ANOVA, relative increase (5- to 25-fold) appeared to be less pronounced or log-rank Mantel–Cox tests for survival studies. We considered differ- ∼ ences significant at p , 0.05. as compared with IL-27p28 mRNA ( 1000-fold). This obser- vation may be related to the fact that unstimulated resting macrophages already contained higher baseline mRNA for Results EBI3 as compared with IL-27p28 mRNA (data not shown). Most Catecholamines antagonize the IL-27–dependent immune importantly, the induction of EBI3 was significantly reduced by response of macrophages the selective b2 adrenoceptor agonists formoterol (Fig. 2C) To study the role of adrenergic mediators for the IL-27–dependent and salbutamol (Fig. 2D) in cultures of LPS/TLR4-activated immune response, we used cultures of LPS/TLR4-activated macrophages. macrophages (Fig. 1A). LPS (100 ng/ml) induced a time- Time course studies revealed that the release of IL-27p28 (as dependent appearance of IL-27p28 in supernatants of mac- detected by ELISA) was constantly suppressed by the presence of rophages derived from C57BL/6J mice. The addition of either formoterol in cultures of LPS/TLR4-activated macrophages de- adrenaline (1026 M) or noradrenaline (1026 M) potently pre- rived from C57BL/6J mice (Fig. 3A). Similar effects were noted vented the release of IL-27p28 to supernatants of macrophage when salbutamol was used as a selective b2 adrenoceptor agonist cultures at all the time points studied (Fig. 1A). When the mRNA (Fig. 3B). Comparable results were obtained in peritoneal- levels for IL-27p28 were assessed by RT-PCR, it was observed elicited and bone marrow–derived C57BL/6J macrophage prep- that both adrenaline and noradrenaline blunted the gene expression arations (BMDM; data not shown). In dose-response studies, 210 of IL-27p28 (Fig. 1B). In dose-response experiments, the IC50 of formoterol used in concentrations as low as 10 Mdisplayeda adrenaline regarding suppression of IL-27p28 protein release in high potency for inhibition of IL-27p28 (Fig. 3C). Alternatively, LPS/TLR4-activated macrophages was ∼1028 M, whereas nor- the inhibitory effects of the short-acting salbutamol were less 2 adrenaline appeared to be somewhat less potent at concentrations pronounced with concentrations ,10 7 M(Fig.3D).Wecon- 26 ,10 M (Fig. 1C). firmed that the b2 adrenoceptor agonist formoterol was equally To approach the question whether the observed effects of cat- effective to suppress IL-27p28 in macrophages with genetic echolamines were mediated through a-orb-adrenoceptors, we deficiency of the a1 adrenoceptors (a1A, a1B) as compared with compared the suppressive effects of adrenaline and noradrenaline WT macrophages (Fig. 3E). This was in accordance with the 4 NEUROENDOCRINE REGULATION OF IL-27 Downloaded from FIGURE 1. Release of IL-27p28 in macrophages is suppressed by catecholamines. (A) Macrophages (BMDM) from C57BL/6J mice were incubated with either LPS (100 ng/ml) alone or in combination with adrenaline (1026 M) or noradrenaline (1026 M). At several time points, the supernatants were collected followed by detection of IL-27p28 by ELISA. (B) RT-PCR analysis of mRNA for IL-27p28 in LPS/TLR4-activated macrophages either with or without addition of adrenaline (1026 M) or noradrenaline (1026 M), normalized to GAPDH expression. (C) Dose-response curves of the effects of adrenaline (1029–1026 M) and noradrenaline (1029–1026 M) on the release of IL-27p28 by LPS/TLR4-activated macrophages. IL-27p28 concentrations with LPS alone were used as 100% value (ELISA). (D) IL-27p28 release from macrophages derived from mice with genetic deletion of the a1A-adre-

2/2 2/2 http://www.jimmunol.org/ noceptor (a1A-AR ), a1B-adrenoceptor (a1B-AR ), or macrophages of the corresponding WT control strain (C57BL/6Slc). Macrophages were stimulated with LPS alone or coincubated with either adrenaline (1026 M) or noradrenaline (1026 M) for 24 h. For normalization the concentrations of IL- 27p28 after LPS alone treatment for each mouse strain were used as 100% values. (E) Inhibitory effects of catecholamines on LPS-induced IL27p28 release 26 after 30 min preincubation with the a1 blocker, prazosin (10 M), versus DMSO vehicle (0.004% v/v) (24 h, ELISA). Ctrl denotes resting control macrophages that received either DMSO vehicle or prazosin alone. Statistical significance was tested versus LPS stimulation of DMSO vehicle (*) or LPS stimulation of prazosin-treated cells (#). All data are representative of three independent experiments each performed in duplicate wells. Error bars represent SEM. *p , 0.05, ****p , 0.0001, ####p , 0.0001. ns, not significant. subsequent observation that incubation of macrophages with the (IL-6) in LPS-activated macrophage cultures (Supplemental Fig. by guest on September 26, 2021 a1 antagonist, prazosin, did not alter the inhibitory effects of 1A–C) (59). formoterol (Fig. 3F). The nonselective b1/b2 adrenoceptor ago- Selectivity of b2 adrenoceptor agonists for different pattern nist orciprenaline and the long-acting b2 adrenoceptor agonist salmeterol resulted in a similar blockade of IL-27p28 release by recognition receptor pathways LPS/TLR4-activated macrophages (Fig. 1G). Finally, it was We and others have reported before that several different TLR/ confirmed that formoterol and salbutamol also downregulated pathogen recognition receptor pathways can induce the produc- other proinflammatory (KC, MCP1) and cytokines tion of IL-27p28 in mononuclear phagocytes (9, 16, 18, 21). To

FIGURE 2. Gene expression of IL-27p28 and EBI3 is antagonized by b2 adrenoceptor agonists in macro- phages. (A) Time course (0–24 h) of mRNA for IL- 27p28 from macrophages (C57BL/6J, BMDM) acti- vated by LPS (100 ng/ml) alone or in combination with 26 the selective b2 adrenoceptor agonist formoterol (10 M). Formoterol alone did not alter the gene expression levels of IL-27p28 (RT-PCR). (B) Time course of IL- 27p28 mRNA in LPS/TLR4-activated macrophages when combined with the selective b2 adrenoceptor agonist salbutamol (1026 M) (RT-PCR). (C) RT-PCR for mRNA of EBI3 in macrophages at several time points after LPS and formoterol (1026 M), when used alone or in combination. (D) RT-PCR for EBI3 mRNA in macrophages stimulated with LPS with and without salbutamol or salbutamol (1026 M) alone at different time points (0–24 h). All data are representative of three independent experiments each performed in du- plicate wells. Error bars represent SEM. *p , 0.05,

**p , 0.01, ***p , 0.001 for LPS versus LPS plus b2 adrenoceptor agonists. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. Dose-dependent and time-dependent modulation of IL-27 release by b2 adrenoceptor agonists in macrophages. (A) Time course of IL-27p28 release in supernatants of macrophage cultures (C57BL/6J, PEM) when incubated with LPS alone, LPS in combination with formoterol (1026 M), or formoterol (1026 M) alone (ELISA). (B) Time course of macrophage-derived IL-27p28 release in cultures incubated with LPS alone, LPS in combination with salbutamol (1026 M), or salbutamol (1026 M) alone (ELISA). (C) Dose-response studies of the suppressive effects of formoterol (10212–1026 M) in LPS/TLR4-activated macrophages (24 h, ELISA). (D) Dose-response studies of the inhibitory effects of salbutamol (1029–1026 M) in LPS/TLR4-activated macrophages (24 h, ELISA). (E) Comparison of the suppressive effects of formoterol in LPS/TLR4-activated macrophages derived from either WT mice 2/2 2/2 (C57BL/6Slc), a1A adrenoceptor–deficient mice (a1A-AR ), or a1B adrenoceptor–deficient mice (a1B-AR ). For each genotype the concentrations of IL-27p28 in supernatants were normalized to LPS alone (=100% value). Formoterol was equally effective to suppress IL-27p28 in all strains. (F) Inhibitory 26 effect of formoterol on LPS-induced IL-27p28 release by BMDM following 30 min preincubation with the a1 blocker prazosin (10 M) or DMSO vehicle 26 (0.004% v/v) (24 h, ELISA). (G) Comparison of the inhibitory effects of the b2 adrenoceptor agonists formoterol, orciprenalin, and salmeterol (10 M each) on IL-27p28 release in LPS/TLR4-activated macrophages (24 h, ELISA). Ctrl denotes resting control macrophages. All data are representative of three independent experiments each performed in duplicate wells. Error bars represent SEM. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001, LPS versus LPS plus b2 adrenoceptor agonist. ns, not significant.

investigate whether the suppressive effects of b2 adrenoceptor ag- hibitory effects of adrenaline, noradrenaline, formoterol, and onists were specific for the TLR4 pathway or a rather global phe- salbutamol, when macrophages were coincubated with either LPS nomenon, we incubated macrophages with receptor agonists for or poly(I:C) (Fig. 4B). In fact, neither of these adrenoceptor ag- TLR2, TLR3, TLR4, and TLR7/8 (Fig. 4A). The receptor agonists onists displayed any significant activity regarding the reduction of for TLR2 (zymosan), TLR3 [poly(I:C)], and TLR7/8 (R848) all poly(I:C)-induced IL-27p28 in macrophages, whereas the release promoted the appearance of IL-27p28, as detected by ELISA after of LPS-induced IL-27p28 was strongly suppressed (Fig. 4B). 24 h, in a similar fashion as observed for the TLR4 (LPS) (Fig. 4A). b Whereas the activation of the pathways for TLR2, TLR4, and 2 adrenoceptor agonists reinforce an IL-10 feedback loop to limit IL-27 release TLR7/8 were significantly inhibited by formoterol (b2), no such effects were noted when poly(I:C) was used to stimulate TLR3- To shed light on the molecular mechanisms mediating the sup- dependent IL-27p28 release (Fig. 4A). Next, we compared the in- pression of IL-27 by b2 adrenoceptor agonists, we first turned our 6 NEUROENDOCRINE REGULATION OF IL-27

chronic enterocolitis and colon carcinoma in situ, which is fre- quently observed in this mouse strain (53). We compared the suppressive effects of formoterol in LPS/TLR4-activated macro- phages from either C57BL/6J (WT) mice to macrophages from IL-102/2 mice (Fig. 5E). Relative values for IL-27p28 were cal- culated for each strain and each individual time point by setting LPS alone to 100%. Although coincubation with formoterol nul- lified the release of IL-27p28 in LPS/TLR4-activated WT mac- rophages, the release of IL-27p28 by IL-102/2 macrophages partly remained intact (Fig. 5E). In detail, in IL-102/2 macro- phages undergoing the LPS plus formoterol treatment the remaining release of IL-27p28 was 41% at the 6 h time point, 58% at the 9 h time point, and 78% at the 24 h time point as compared with LPS alone in IL-102/2 macrophages (Fig. 5E). Hence, IL-10 appeared to be especially important to mediate the b2 adrenergic effects at later time points. This idea is supported by the fact that several hours are required for the generation and build-up of substantial amounts of IL-10 in response to LPS and formoterol.

The peak of mRNA for IL-10 after LPS and formoterol was ob- Downloaded from served after 6 h (Fig. 5A). Fig. 5F shows the distinct inhibitory effects of formoterol (b2) in LPS-activated macrophages from either C57BL/6J (WT) mice or IL-102/2 mice. The absolute concentrations of IL-27p28 after LPS alone were higher in IL-102/2 macrophages as compared with WT macrophages (Fig. 5F).

Finally, recombinant mouse IL-10 greatly suppressed the release http://www.jimmunol.org/ of IL-27p28 from LPS-activated macrophages (Fig. 5G). No such activities of recombinant mouse IL-10 were observed, when IL- FIGURE 4. Selective suppression of IL-27 by b2 adrenoceptor agonists 27p28 was induced by poly(I:C) activation (Fig. 5G). This ob- in dependency of activated pattern recognition receptor pathways. (A) servation may provide an explanation for the data presented in Macrophages (C57BL/6J, PEM) were activated with LPS (TLR4, 100 ng/ Fig. 4A and 4B, where a selective sparing of IL-27p28 inhibition ml), zymosan (TLR2, 10 mg/ml), poly(I:C) (TLR3, 1 mg/ml), or R848 by neuroendocrine sympathomimetic agonists was observed in (TLR7/8, 5 mg/ml) alone or in combination with the b2 adrenoceptor 2 poly(I:C)-activated macrophages. agonist formoterol (10 6 M). IL-27p28 was detected in supernatants by ELISA after 24 h. (B) IL-27p28 release from LPS/TLR4-activated mac- Because IL-10 is a determinant factor for the polarization of by guest on September 26, 2021 rophages was compared with poly(I:C)/TLR3-activated macrophages, when macrophages into an M2 phenotype (60, 61), we studied the role of 2 2 coincubated with adrenaline (10 6 M), noradrenaline (10 6 M), formoterol IL-10–inducing b2 adrenoceptor agonists related to M1/M2 differ- (1026 M), or salbutamol (1026 M). All data are representative of three (A)or entiation. Formoterol and salbutamol increased the amounts of two (B) independent experiments each performed in duplicate wells. Error mRNA for arginase 1, an established M2 marker, when combined bars represent SEM. *p , 0.05, **p , 0.01, ***p , 0.001, LPS versus LPS with LPS (Supplemental Fig. 1D). Recombinant mouse IL-4 induced plus adrenoceptor agonists. ns, not significant. M2 polarization as studied by CD206 expression on F4/80+CD11b+ macrophages by flow cytometry, and the presence of CD206 was further augmented by b2 adrenoceptor agonists (Supplemental Fig. attention to IL-10 (59). The presence of formoterol in cultures of 1E, 1F). The intracellular content of LPS/TLR4-induced NOS2, a LPS/TLR4-activated macrophages (C57BL/6J) significantly in- known M1 marker, was profoundly reduced by formoterol or sal- creased the amounts of mRNA for IL-10 at all the time points butamol (74.7% versus 38.5% or 42.3% F4/80+CD11b+NOS2+ studied (3–24 h; Fig. 5A). Likewise, formoterol promoted the macrophages; Supplemental Fig. 1G, 1H). These findings are in release of IL-10 protein as detected by ELISA in supernatants of accordance with a recent report describing the M2 macrophage activated macrophages, when formoterol was used in a concen- phenotype promoting activities of b2 adrenoceptor agonists (62). tration range of 10210–1026 M (Fig. 5B). Salbutamol significantly b augmented the release of IL-10 by LPS/TLR4-activated macro- 2 adrenoceptor agonists mediate IL-27 suppression through 28 26 the JNK signaling pathway phages in concentrations of 10 –10 M (Fig. 5C). When b2 adrenoceptor agonists were used alone (i.e., in the absence of To investigate the intracellular mechanisms responsible for the LPS), no induction of IL-10 release was detectable (ELISA; data suppression of IL-27 by catecholamines, we turned our attention to not shown). To further characterize the relationship between IL-10 the JNK signaling pathway. We have reported before that b2 and IL-27p28 in LPS/TLR4-activated macrophages, we used adrenoceptor agonists selectively affect phosphorylation of JNK monoclonal neutralizing anti–IL-10R Abs. When anti–IL-10R (59). When macrophages derived from C57BL/6J mice were in- Abs were added together with LPS and the b2 adrenoceptor ag- cubated with LPS (100 ng/ml) alone or in combination with for- 26 onist formoterol (b2), the suppressive effects of formoterol re- moterol (10 M), a significant reduction in intracellular phospho- garding the release of IL-27p28 were almost completely JNK levels (Thr183/Tyr185) was observed with formoterol (Fig. 6A). intercepted (Fig. 5D). Matched isotype control Abs (IgG1) were Similarly, treatment with recombinant mouse IL-10 (10 ng/ml) used as a negative control and did not interfere with the inhibition resulted in a confinement of JNK activation patterns in LPS/ of LPS-induced IL-27p28 formation by formoterol (Fig. 5D). TLR4-activated macrophage cultures (Fig. 6B). Next, the role of IL-10 was studied by using macrophages derived To define the role of JNK activation for the release of IL-27 by from IL-10–deficient mice (IL-102/2). The IL-102/2 mice were macrophages, we used specific pharmacologic JNK inhibitors. The sacrificed #6 wk of age before the spontaneous development of small molecule inhibitor, AEG3482, dose-dependently suppressed The Journal of Immunology 7 Downloaded from

FIGURE 5. b2 adrenoceptor agonists activate an autocrine/paracrine IL-10 loop to limit IL-27p28. (A) RT-PCR of mRNA for IL-10 in macrophages (C57BL/6J, PEM) incubated with LPS (100 ng/ml) with and without formoterol (1026 M) at different time points (0–24 h). Ctrl denotes untreated resting macrophages. (B) IL-10 release by macrophages treated with LPS with and without formoterol in different concentrations (10212–1026 M) (24 h, ELISA). (C) IL-10 release by macrophages treated with LPS with and without salbutamol in different concentrations (1029–1026 M) (24 h, ELISA). (D) IL-27p28 release from LPS/TLR4-activated macrophages with addition of a monoclonal blocking anti–IL-10R Ab (aIL-10R; 10 mg/ml) using isotype-matched IgG1 26 Ab (10 mg/ml) as control. Interception of IL-10R signaling nullified the suppressive effects of formoterol (b2,10 M) (24 h, ELISA). (E) Relative release of IL-27p28 at different time points (6–24 h) from macrophages derived from C57BL/6J (WT) mice as compared with macrophages from IL-102/2 mice, http://www.jimmunol.org/ when incubated with the combination of LPS plus formoterol (1026 M) (ELISA). For each time point and mouse strain the IL-27p28 concentration with LPS alone was calculated as 100% value. (F) IL-27p28 in supernatants from WT macrophages and IL-102/2 macrophages incubated with LPS or LPS plus 26 b2 adrenoceptor agonist (formoterol, 10 M) (24 h, ELISA). (G) Comparison of the effects of recombinant mouse IL-10 (10 ng/ml) on the release of IL- 27p28 from macrophages (WT) either induced by a TLR4 agonist (LPS 100 ng/ml) or a TLR3 agonist [poly(I:C) 1 mg/ml]. All data are representative of three independent experiments each performed in duplicate wells. Error bars represent SEM. *p , 0.05, **p , 0.01, ***p , 0.001. For (A)–(C), statistical significance was tested for LPS versus LPS plus b2 adrenoceptor agonist. n.s., not significant.

IL-27p28 release from LPS/TLR4-activated macrophages (Fig. 6C). either AEG3482 or SP600125 resulted in profoundly reduced Likewise, the competitive and reversible JNK inhibitor, SP600125, numbers of IL-27p28–producing macrophages as indicated by the by guest on September 26, 2021 antagonized the appearance of IL-27p28 in cell culture supernatants lower frequencies of IL-27p28+F4/80+ and IL-27p28+CD11b+ of macrophages (Fig. 6D). In flow cytometry experiments, F4/80+ cells, with the latter induced by the presence of LPS (Fig. 6E). In + CD11b double-positive macrophages were costained for intracel- summary, these studies suggested that b2 adrenoceptor activation lular IL-27p28 in the presence of a Golgi transport inhibitor. These antagonized JNK signaling to blunt the production of IL-27p28 in experiments confirmed that the blockade of JNK signaling with macrophages.

FIGURE 6. Role of the JNK signaling pathway in the regulation of IL-27. (A) Phospho-JNK (Thr183/Tyr185) in untreated resting macrophages (Ctrl, C57BL/6J, PEM) as compared with 1 h in- cubation with LPS (100 ng/ml) or LPS plus for- moterol (1026 M); bead-based assay (Bio-Plex 200/ Luminex 200). (B) Phospho-JNK in macrophages after 1 h incubation with LPS or LPS plus recombi- nant mouse IL-10 (10 ng/ml; preincubated over- night). (C) IL-27p28 release from LPS-activated macrophages together with the JNK inhibitor, AEG3482, in different concentrations (5–20 mM; 90 min preincubation) (12 h, ELISA). (D) Dose- response curve of the effects of the JNK inhibitor, SP600125, on the release of IL-27p28 by macro- phages (12 h, ELISA). (E) Flow cytometry of in- tracellular IL-27p28 in F4/80+CD11b+ macrophages incubated with LPS alone or in combination with either AEG3482 or SP600125 (12 h). Regions were defined according to isotype Ab controls (data not shown). All data are representative of three inde- pendent experiments each performed in duplicate wells. Error bars represent SEM. *p , 0.05, **p , 0.01, ***p , 0.001. 8 NEUROENDOCRINE REGULATION OF IL-27

Evidence for the regulation of IL-27 and IL-10 by the b2 the amounts of mRNA for IL-27p28 were quantified in spleen adrenergic system in vivo homogenates of C57BL/6J mice with endotoxemia with and To investigate whether the neuroendocrine regulatory circuits without formoterol (Fig. 7F). In fact, formoterol significantly di- defined in macrophage cultures are also present in vivo, we minished the gene expression of IL-27p28 during endotoxemia employed a model of endotoxic shock in C57BL/6J mice during after 12 h (Fig. 7F). No reciprocal effect was observed for mRNA which splenic macrophages are the major cellular source of IL- of IL-10 after 12 h (data not shown), which may be explained by 27p28 (9, 20). the concept that IL-10 gene expression already occurs earlier Because adrenaline and noradrenaline have a short plasmatic during endotoxemia (Fig. 7E). To study the influence of endogenous catecholamines for the half-live in vivo (t1/2 of ∼2 min), we used formoterol, as a long- LPS/TLR4-induced release of IL-27p28 during endotoxic shock, lasting b2 adrenoceptor agonist in endotoxic shock. One group of mice received injections of LPS alone, whereas the other group we used C57BL/6J mice after surgical removal of the adrenal received formoterol i.p. together with LPS and again formoterol glands. Mice were allowed to recover from the surgery for several i.p. after 24 h. Formoterol treatment significantly reduced the se- weeks before they were subjected to endotoxemia. As a control verity of LPS-induced hypothermia at early time points (Fig. 7A). group, mice after sham surgery were treated in the same fashion. Hypothermia is a predictor of septic shock mortality (63). Addi- Adrenalectomized mice displayed 2.7-fold higher concentrations tionally, all mice were monitored and assessed using a CSS. of IL-27p28 in plasma 8 h after injection of LPS (Fig. 7G). Of note, Formoterol reduced the CSS values, indicating an improvement of in one outlier mouse within the group of adrenalectomy the con- the adverse clinical signs during the course of endotoxic shock centrations of IL-27p28 were even substantially lower as in the (Fig. 7B). There was a nearly significant trend (p = 0.067) for a sham operated control group. We speculate that in this mouse the Downloaded from better survival of the formoterol-treated mice (n = 10 per group; intra-abdominal injection of LPS may not have been i.p. but rather Fig. 7C). accidental intraintestinal or retro-abdominal. Alternatively, in this Subsequently, plasma was collected by repetitive bleeding from outlier animal the surgical removal of the adrenal glands may have the retro-orbital sinus at 3, 6, and 12 h time points (Fig. 7D). At all been unintentionally incomplete. In any case, the concentrations the time points studied, formoterol significantly reduced the of IL-27p28 were significantly higher (p , 0.01) in adrenalecto- plasma concentrations of IL-27p28 in mice with endotoxic shock. mized mice after endotoxemia. To evaluate a potential contribu- http://www.jimmunol.org/ IL-10 was quantified in the plasma samples of the same experi- tion of endogenous glucocorticoids in adrenalectomized mice, we ment (Fig. 7E). Formoterol promoted the appearance of IL-10 in used the glucocorticoid receptor antagonist, mifepristone, according plasma during endotoxemia at 3 and 6 h (Fig. 7E). Additionally, to a published dosing regimen (58). The appearance of IL-27p28 in

FIGURE 7. Influence of adrenergic activation on the by guest on September 26, 2021 severity and cytokine release of endotoxic shock. (A) Body surface temperature during the early phase of endotoxic shock (LPS 10 mg/kg body weight i.p.; n =10) with and without formoterol (200 nmol/kg body weight i.p.; n = 10). (B) CSS during endotoxic shock with and without formoterol (n = 10 per group). (C) Survival of endotoxic shock in the presence or absence of formoterol (200 nmol/kg body weight i.p.; two in- jections at 0 and 24 h; the control group received PBS vehicle in addition to LPS; n = 10 per group). (D) Serial measurements of IL-27p28 in plasma at 3–12 h after administration of LPS with and without formoterol in two groups of C57BL/6J mice (ELISA). (E) Plasma concentrations of IL-10 from the same experiment as shown in (D) (ELISA). (F) RT-PCR of mRNA for IL- 27p28 in spleen homogenates of endotoxemic mice (n = 7) or endotoxemic mice with cotreatment of for- moterol (n = 4) (12 h). (G) IL-27p28 in plasma 8 h following endotoxemia of C57BL/6 mice with prior sham surgery (Sham-OP) or adrenalectomy surgery (ADR-X) several weeks before LPS injection. (H) IL-27p28 in plasma 8 h following endotoxemia of C57BL/6J mice with the selective glucocorticoid receptor antagonist mifepristone (30 mg/kg body weight i.p., 30 min before LPS challenge) or DMSO vehicle in- jection (n = 5 per group). Experiments were performed with cohoused n = 4–10 male mice per group, and each symbol (circles, squares, triangles) represents one ani- mal. Error bars represent SEM. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. ns, not significant. The Journal of Immunology 9 plasma was not significantly influenced by mifepristone treatment IL-27p28 as compared with noradrenaline (Fig. 1C). Indeed, during endotoxic shock (Fig. 7H), suggesting that endogenous subsequent experiments revealed that the inhibition of IL-27 in catecholamines rather than endogenous glucocorticoids are major macrophages appeared as mainly mediated by b2 adrenoceptor regulators of IL-27p28 release in endotoxic shock (Figs. 7G, 8). agonists (Figs. 1D, 1E, 2, 3). The single doses of catecholamines added to our macrophage cultures were typically higher as com- Discussion pared with continuous physiologic concentrations (1–20 3 1029 M), In this study, we have investigated the influence of neuroendocrine although plasma catecholamine levels can increase up to 20-fold 2 sympathetic mediators on the gene expression and production of (2–8 3 10 8 M) during severe stress and sepsis (66, 67). IL-27 by macrophages. Our findings suggest that adrenaline and We have focused our studies on macrophages because mono- noradrenaline interact with b2 adrenoceptors to mediate a context- nuclear phagocytes are considered an exclusive cellular source of dependent suppression of IL-27 release by LPS/TLR4-activated IL-27 (7, 9), although this view was recently challenged by 2 macrophages rather than poly(I:C)/TLR3-activated macrophages. uncovering a unique population of IL-27–producing Foxp3 The underlying mechanisms of these phenomena appear to rely CD11a+CD49d+ Ag-specific CD4+ T cells during malaria infec- on a feedback loop with IL-10, wherein poly(I:C)/TLR3-induced tion (68). IL-27 is insensitive to the counterbalancing effects of IL-10. Our data suggest that catecholamines suppress IL-27p28 release Additionally, b2 adrenoceptor agonists synergistically with IL-10 by inhibition of LPS/TLR4-induced phosphorylation of the JNK downmodulate the IL-27–stimulating intracellular JNK signaling signaling pathway in primary mouse macrophages (Figs. 6, 8). pathway. The proposed molecular mechanisms are summarized in JNK proteins belong to the MAPK family of stress-induced in- Fig. 8. tracellular signals. In fact, we have demonstrated before that Downloaded from The data of our current study extend the knowledge in the catecholamines via their b2 adrenergic activities negatively reg- neuroimmune field by to our knowledge, first describing that IL-27 ulate the phosphorylation of JNK (59, 69). The IFN-g–mediated is regulated by catecholamines, the principal hormones of multi- IL-27 protein content was positively regulated by JNK in human faceted acute stress responses (64). Adrenaline and noradrenaline monocytic cells (70). Additionally, using Theiler’s murine en- are well known to affect a multitude of immune functions such as cephalomyelitis virus–infected RAW264.7 cells the pharmaco-

Ag presentation, leukocyte proliferation and traffic, secretion of logic blockage of JNK was associated with a reduction of mRNA http://www.jimmunol.org/ cytokines and Abs, and balancing Th1/Th2 lymphocyte responses for IL-27p28 (10). Theiler’s murine encephalomyelitis virus in- (64). The cellular effects of catecholamines are mediated by fection of macrophages is thought to activate the TLR3 and TLR7 several classes of adrenoceptors, which all belong to the 7-trans- pattern recognition pathways (10). Interestingly, there was a membrane domain spanning G protein–coupled receptor family negligible potency of catecholamines regarding the selective in- (41). These adrenoceptors activate cAMP and several kinase sig- hibition of poly(I:C)/TLR3-induced IL-27p28 in our experiments. naling pathways (e.g., PKA, CREB, p38 MAPK), but mediate This is partly explained by the insensitivity of poly(I:C)/TLR3- inhibition of NF-kB (41, 65). In general, adrenaline exerts a induced IL-27p28 for autocrine/paracrine IL-10 (Fig. 5G). In higher affinity for b2 adrenoceptors than does noradrenaline. This conclusion, catecholamines appear to modulate IL-27p28 release is reflected by our initial observation that equimolar concentra- in a context-dependent manner rather during bacterial than viral by guest on September 26, 2021 tions of adrenaline were more potent for the inhibition of infections. A traditional concept of stress hormones such as catecholamines (and glucocorticoids) has been that these mediators act as unilateral suppressors of immune responses and inflammation. The rationale for this view is that reducing the severity of inflammation (e.g., joint swelling, pain) is vitally important to preserve organ function during a “fight or flight” type stress response. Indeed, the idea of catecholamines acting as profound antagonists of the acute in- flammatory response is supported by our findings that b2 adre- noceptor agonists increase the release of anti-inflammatory IL-10 by macrophages (Fig. 5). These data are in accordance with the clinical observations that catecholamines induce IL-10 release in patients suffering from acute myocardial infarction or accidental brain injury (71, 72). In LPS-activated human whole blood, both noradrenaline and adrenaline increase the release of IL-10 but decrease IL-12 con- centrations (51). These effects are reversible by administration of the nonselective b1/b2 adrenoceptor blocker, propranolol (51). In human monocytes, adrenaline downregulates TNF-a but increases TNF receptor expression (52). The production of the CCL3 (MIP1a) is antagonized by exogenous and endogenous FIGURE 8. Schematic of the proposed mechanisms of IL-27 regulation catecholamines in murine macrophages and mice after endotoxic by neuroendocrine mediators in macrophages. Activation of the TLR4 shock (73). Catecholamines suppress the intracellular concentra- pathway by LPS promotes phospho-JNK (and other signaling cascades; tions of IL-6 but do not affect IL-8 in LPS-activated human data not shown) for facilitating the gene expression and release of IL-27 by monocytes (74). All these findings would point toward a uniform macrophages. Catecholamines via b adrenoceptor activation antagonize 2 immunosuppression by catecholamines. phospho-JNK and suppress the release of IL-27. b2 adrenoceptor agonists (e.g., formoterol, salbutamol) promote the release of IL-10 in an autocrine/ Alternatively, the conceptual data interpretation is more complex paracrine feedback loop that via IL-10R limits phospho-JNK and reduces for the suppression of IL-27 by catecholamines via b2 adreno- the release of IL-27. Activation of IL-27 production by the TLR3 pathway ceptors. It is well established that IL-27 exerts both proin- is unresponsive to the effects of b2 adrenoceptors and/or IL-10. flammatory and anti-inflammatory properties (1). IL-27 can drive 10 NEUROENDOCRINE REGULATION OF IL-27 a rapid clonal expansion of IFN-g–producing CD4+ T cells (2). The This correlates with our findings that formoterol reduced plasma chemotaxis of activated T cells to the site of local inflammation may IL-27p28 together with the severity of endotoxic shock (Fig. 7). be stimulated by IL-27 via the induction of CXCL10 release in lung In human patients with sepsis and septic shock, continuous fibroblasts (75). Additionally, IL-27 is required for the IL-21–de- infusions of noradrenaline and (in severe cases) adrenaline are pendent production of high-affinity Abs in the germinal centers of routinely used as powerful vasopressors to treat hypotension and lymph nodes (76). In contrast, the anti-inflammatory actions of IL- concomitant organ hypoperfusion. We speculate that the extent of 27 include the promotion of T cell–derived release of IL-10 and immune-modulatory IL-27 production is most likely affected by antagonizing Th17 cells (22, 27, 28). In skin fibroblasts, IL-27 catecholamine therapy and this may determine the overall func- stimulates the expression of IL-18 binding protein, which is a sol- tional effects of this immune-modulatory cytokine in human sepsis. uble receptor analog and natural antagonist of the T cell activating Moreover, IL-27 has been suggested as a biomarker for the di- cytokine, IL-18 (77). IL-27 induces the expression of immune- agnosis of pediatric and adult sepsis patients (88, 89), but we now regulatory CD39 on dendritic cells to limit T cell responses (26). caution that catecholamine therapy as a confounding factor may In consequence of these findings, it is not surprising that genetic distort IL-27 concentrations. A similar situation is imagined for disruption of IL-27RA signaling results in lethal inflammation be- chronic pulmonary obstructive disease, which is associated with cause of the inability to normalize T cell reactivity after clearance of elevated serum concentrations of IL-27 during disease exacerba- an infectious pathogen (12). Thus, fine-tuning of IL-27–dependent tions that are frequently treated with inhalations of b2 adrenoceptor immune responses by catecholamines presumably relays multilay- agonists and glucocorticoids (90). Low doses of glucocorticoids ered context-dependent influences on the prevailing inflammatory are also included in supportive pharmacotherapy regimens of sep- states and not by any means transmits an obstinate adrenergic im- sis. Despite our results with the glucocorticoid antagonist mife- Downloaded from munosuppression. In fact, emerging studies have recently substan- pristone (Fig. 7H), we cannot exclude the possibility that persistent tiated the idea about proinflammatory properties of catecholamines. and abnormal concentrations of endogenous or exogenous glu- For instance, a b2 adrenoceptor agonist promoted neutrophil infil- cocorticoids may modulate IL-27. In light of the importance of an tration and damage of colon tissue in a DSS-induced colitis model, adequate stress-hormone response for critically ill patients, it is whereas opposite effects were noted by administration of a nonse- not surprising that experimental blockade of b1/b2 adrenergic sig- lective b1/b2 adrenergic blocker (78). In another study, noradrena- naling is a disadvantage for sepsis survival (67, 91). http://www.jimmunol.org/ line induced elevated microarray gene expression profiles of In summary, our present study adds new information to the inflammatory cytokines and chemokines in resting and activated diverse interactions of neuroendocrine pathways with the immune memory CD8+ T cells from humans (79). Lymphocyte trafficking is system. To our knowledge, we provide the first evidence that IL- controlled by b2 adrenoceptor activation (80). Finally, blockade of 27–dependent immune functions are under the control of b2 a2 adrenoceptors or catecholamine-synthesizing enzymes greatly adrenoceptor signaling in mononuclear phagocytes. In the future, suppressed inflammation in two models of lung injury (42). Some of it will be interesting to continue the research on the functional the apparently discrepant findings on the inflammatory properties of consequences of these observations, especially related to IL-27RA– catecholamines are apparently reconciled by data suggesting dif- expressing target cells of immune-modulatory IL-27 during in- by guest on September 26, 2021 ferential roles of b2 adrenoceptors (anti-inflammatory) as compared flammatory milieus. with a adrenoceptors (proinflammatory) (81, 82). Nonetheless, for IL-27p28 release we did not observe any roles of a1 adrenoceptor Acknowledgments signaling relaying the effects of catecholamines in macrophages We cordially thank Fabien Meta and Foruzandeh Samangan for technical (Fig. 1D, 1E), although radioligand binding studies have shown such assistance and Lisa Kubacki for secretarial assistance. M.B. thanks Peter cells to express a1 adrenoceptors (44). A. Ward, Ulrich Walter, and Heiko Muhl€ for constant support and men- In the past, the adrenal glands and sympathetic nerves were torship. considered as the major sources of catecholamines. However, accumulating data suggest that catecholamines are also produced Disclosures by immune cells. For example, human neutrophils contain cat- The authors have no financial conflicts of interest. echolamines and their metabolites, suggesting autoregulatory ad- renergic mechanisms (83). Activation of macrophages with LPS has been reported to induce tyrosine hydroxylase, the rate-limiting References 1. Bosmann, M., and P. A. Ward. 2013. Modulation of inflammation by - enzyme responsible for catecholamine generation, and conse- 27. J. Leukoc. Biol. 94: 1159–1165. quently the synthesis of noradrenaline and dopamine (49). Cat- 2. Pflanz, S., J. C. Timans, J. Cheung, R. Rosales, H. Kanzler, J. Gilbert, L. Hibbert, echolamines derived from mononuclear phagocytes enhance acute T. Churakova, M. Travis, E. 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