Distinct Responses of Lung and Spleen Dendritic Cells to the TLR9 Agonist CpG Oligodeoxynucleotide This information is current as Li Chen, Meenakshi Arora, Manohar Yarlagadda, Timothy of September 23, 2021. B. Oriss, Nandini Krishnamoorthy, Anuradha Ray and Prabir Ray J Immunol 2006; 177:2373-2383; ; doi: 10.4049/jimmunol.177.4.2373 http://www.jimmunol.org/content/177/4/2373 Downloaded from References This article cites 62 articles, 32 of which you can access for free at: http://www.jimmunol.org/content/177/4/2373.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 23, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Distinct Responses of Lung and Spleen Dendritic Cells to the TLR9 Agonist CpG Oligodeoxynucleotide1 Li Chen,* Meenakshi Arora,* Manohar Yarlagadda,* Timothy B. Oriss,* Nandini Krishnamoorthy,* Anuradha Ray,*† and Prabir Ray2*† Dendritic cells (DCs) sense various components of invading pathogens via pattern recognition receptors such as TLRs. CpG oligodeoxynucleotides (ODNs), which mimic bacterial DNA, inhibit allergic airways disease and promote responses in the spleen to bacterial components. Because many TLR agonists are currently being tested for potential therapeutic effects, it is important to characterize the expression and function of TLRs in different tissues. We show that both myeloid and plasmacytoid DCs in the spleen express TLR9, the receptor for CpG ODNs, but lung DCs show no detectable expression in either subset. TLR4 expression in contrast was detected on both lung and spleen DCs. LPS was superior to CpG ODN in increasing the allostimulatory potential of lung DCs and their expression of CD40. However, both agonists efficiently stimulated spleen DCs. CpG ODNs administered to Downloaded from mice efficiently inhibited Th2 cytokine production both in the lung draining lymph node and in the spleen. Surprisingly, inhibition of Th2 cytokine production was evident despite high levels of expression of GATA-3 and additional transcription factors that regulate Th2 responses. Although in the spleen CpG ODNs induced IL-6, a key cytokine induced via TLR9-MyD88 signaling, no IL-6 was detectable in lung LN cells. These studies show for the first time that lung DCs lack TLR9 expression, but, despite this deficiency, CpG ODNs induce potent inhibitory effects on Th2 cytokine production in the lung without inducing expression of the proinflammatory cytokine, IL-6, which has been linked to chronic diseases in the lung and the gut. The Journal of Immunology, http://www.jimmunol.org/ 2006, 177: 2373–2383. endritic cells (DCs)3 play an important role in Ag pre- tegic distribution, and their ability to capture and process Ag, and sentation in different tissues (1, 2), including the lung (3, present them to T cells in the LNs make DCs the key APCs in the D 4). Research since the late 1980s has shown an impor- lung and in other mucosal surfaces. tant role for DCs in pulmonary immune responses (3, 5–13). DCs Lung DCs are constantly exposed to a myriad of inhaled agents. are present in high density in the proximal airways and are well However, the lung maintains homeostasis in the face of constant positioned to capture Ags. Overall, pulmonary DCs are immature provocation by these multiple stimuli. Interestingly, although LPS by guest on September 23, 2021 in that they express low levels of costimulatory molecules (CD80, and CpG oligodeoxynucleotides (ODNs) are both bacterial com- CD86, and CD40). During inflammation, increased numbers of ponents, LPS induces severe inflammation in the lung precipitating DCs are rapidly recruited from bone marrow progenitors to the sepsis (20, 21), while CpG ODNs suppress allergic airway inflam- airway epithelium, where these immature DCs efficiently capture mation (22–25) and also growth of Mycobacterium tuberculosis in Ags due to their high phagocytic ability (6, 12, 14). Subsequent to the lungs (25). Like the lung, the spleen also encounters pathogens Ag capture, the DCs undergo maturation, which is influenced by being the principal filter unit for pathogens that enter the blood- the microenvironment, and the mature DCs traffic to local draining stream. Administration of CpG ODNs to mice has been shown to lymph nodes (LNs). The transition from immature to mature state cause transient splenomegaly (26), and CpG ODNs potentiate ef- is accompanied by the production of various cytokines and che- fects of TLR agonists in the spleen (27, 28). Bacterial CpG motifs mokines by the DCs that regulate their ability to interact with naive have been shown to promote chronic intestinal inflammation via T cells to direct T cell differentiation (1, 2, 6, 15–19). Their stra- secretion of proinflammatory cytokines such as IL-6 and IFN-␥ (29). CpG ODNs are increasingly being used in various clinical trials (30). Therefore, it is important to better understand the effects *Department of Medicine, Pulmonary, Allergy, and Critical Care Division, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213; and †Department of Immu- of CpG ODNs in different tissues and the expression characteris- nology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213 tics of TLR9, which is the best-studied receptor for CpG ODNs. Received for publication November 17, 2005. Accepted for publication May Because little is known about TLR expression in lung DCs, which 26, 2006. are the key APCs in the lung, we investigated whether CpG ODNs The costs of publication of this article were defrayed in part by the payment of page exert similar or differential effects on DCs from the lung vs the charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. spleen, which serve distinct functions in the body. 1 This work was supported by Grants HL 60207 (to P.R.) and HL 77430 (to A.R.) We show that while LPS had similar effects on lung and spleen from the National Institutes of Health. DCs and the DCs in turn stimulated CD4ϩ T cells in a comparable 2 Address correspondence and reprint requests to Dr. Prabir Ray, Department of Med- fashion, CpG ODNs exerted differential effects on lung and spleen icine, Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh DCs both in in vitro assays as well as when administered to mice. School of Medicine, 3459 Fifth Avenue, MUH 628 NW, Pittsburgh, PA 15213. E- mail address: [email protected] Studies of the expression profile of the two TLRs at the two lo- 3 Abbreviations used in this paper: DC, dendritic cell; alum, aluminum hydroxide; cations showed that while TLR4 expression is comparable be- cRPMI, complete RPMI; CT, cholera toxin; GUS, ␤-glucuronidase; IDO, indoleam- tween lung and spleen DCs, only the latter express TLR9 with no ine 2,3-dioxygenese; i.n., intranasal; LN, lymph node; pDC, plasmacytoid DC; mDC, myeloid DC; ODN, oligodeoxynucleotide; TRIF, Toll/IL-1 receptor domain-contain- detectable expression in lung DCs. Despite the lack of TLR9 ex- ing adapter-inducing IFN-␤; wt, wild type. pression in lung DCs, CpG ODN administration caused potent Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 2374 DISTINCT RESPONSES OF LUNG AND SPLEEN DCs TO CpG ODN inhibition of allergen-induced Th2 cytokine production in both the Flow cytometry spleen and the lung LN. However, only in the spleen, production Cells were first incubated for 5 min with Fc block (10 ␮g/ml; BD Bio- of IL-6, a key cytokine downstream of TLR9 (31–33) and MyD88 sciences) to minimize nonspecific Ab binding and were then incubated (34), was noted. Interestingly, this inhibition of cytokine produc- with saturating concentrations of appropriate Abs for 5 min on ice in 2% tion was evident in the presence of high levels of expression of FBS/PBS, after which the cells were washed in the same buffer. Before GATA-3, which we and others (35, 36) previously demonstrated to be intracellular TLR9 staining, DCs were stained with dye-conjugated Abs against the cell surface molecules CD11c, MHC class II, and B220. The the master regulator of Th2 differentiation. With the recent identifi- cells were washed, fixed for 30 min using freshly prepared Fix/Perm so- cation of TLR9-independent mechanisms of CpG action (37, 38), our lution (eBioscience), and were washed in PBS and 1ϫ permeabilization data suggest that CpG ODNs can exert both TLR9-dependent (IL-6) buffer (eBioscience) successively to permeabilize the cells. The cells were and -independent effects to regulate immune responses. incubated with biotinylated mAb against murine TLR9 (clone 5G5) or with isotype control (mouse IgG2a), as described (41). Streptavidin-PerCP (BD Biosciences) was used for detection. TLR9 staining was also investigated Materials and Methods using another anti-TLR9 Ab IMG-431 (Imgenex). All samples were ana- Mice lyzed using a FACSCalibur flow cytometer and CellQuest software (BD Biosciences). Dead cells were excluded using forward and side light scatter Ϫ/Ϫ BALB/cByJ, CD40 mice (backcrossed to BALB/c background), and properties. Cell sorting was conducted using a FACSAria cell sorter and C57BL/6 male mice, obtained from The Jackson Laboratory, and DO11.10 FACS Diva software (BD Biosciences).