CCR2 Plays a Critical Role in Dendritic Cell Maturation: Possible Role of CCL2 and NF- κB

This information is current as Fabio Jimenez, Marlon P. Quinones, Hernan G. Martinez, of September 27, 2021. Carlos A. Estrada, Kassandra Clark, Edgar Garavito, Jessica Ibarra, Peter C. Melby and Seema S. Ahuja J Immunol published online 19 April 2010 http://www.jimmunol.org/content/early/2010/04/19/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 19, 2010, doi:10.4049/jimmunol.0803494 The Journal of Immunology

CCR2 Plays a Critical Role in Dendritic Cell Maturation: Possible Role of CCL2 and NF-kB

Fabio Jimenez,*,†,1 Marlon P. Quinones,*,†,1 Hernan G. Martinez,*,† Carlos A. Estrada,† Kassandra Clark,*,† Edgar Garavito,† Jessica Ibarra,† Peter C. Melby,*,† and Seema S. Ahuja*,†

We postulated that CCR2-driven activation of the transcription factor NF-kB plays a critical role in dendritic cell (DC) maturation (e.g., migration, costimulation, and IL-12p70 production), necessary for the generation of protective immune responses against the intracellular pathogen Leishmania major. Supporting this notion, we found that CCR2, its ligand CCL2, and NF-kB were required for CCL19 production and adequate Langerhans cell (LC) migration both ex vivo and in vivo. Furthermore, a role for CCR2 in upregulating costimulatory molecules was indicated by the reduced expression of CD80, CD86, and CD40 in Ccr22/2 bone marrow-derived dendritic cells (BMDCs) compared with wild-type (WT) BMDCs. Four lines of evidence suggested that CCR2 Downloaded from plays a critical role in the induction of protective immunity against L. major by regulating IL-12p70 production and migration of DC populations such as LCs. First, compared with WT, Ccr22/2 lymph node cells, splenocytes, BMDCs, and LCs produced lower levels of IL-12p70 following stimulation with LPS/IFN-g or L. major. Second, a reduced number of LCs carried L. major from the skin to the draining lymph nodes in Ccr22/2 mice compared with WT mice. Third, early treatment with exogenous IL-12 reversed the susceptibility to L. major infection in Ccr22/2 mice. Finally, disruption of IL-12p70 in radioresistant cells, such as LCs, but not in BMDCs resulted in the inability to mount a fully protective immune response in bone marrow chimeric mice. Collectively, our http://www.jimmunol.org/ data point to an important role for CCR2-driven activation of NF-kB in the regulation of DC/LC maturation processes that regulate protective immunity against intracellular pathogens. The Journal of Immunology, 2010, 184: 000–000.

endritic cell (DC) maturation is a highly regulated cellular CCL19, a major ligand for CCR7, is produced by maturing DCs event that includes at least three processes: migration, and by high endothelial venules, creating a gradient from the site D upregulation of costimulatory surface molecules (along of its release to the lymphatics (5, 6, 16, 17). Cognate interactions with Ag presentation), and the release of polarizing , such between this gradient and CCR7, present on the DC’s surface, as IL-12 (1–4), by Th cells. Models describing events occurring in drives the cells into the draining lymph nodes (DLNs) (6, 17, 18). by guest on September 27, 2021 each of the three processes described above have emerged (5, 6), yet Moreover, it is likely that induction of CCL19 production and the the full array of factors coordinating DC maturation remains un- parallel maturation process are initially triggered by known. To date, these models build on disjointed evidence sug- produced early on, which have the ability to provide initial signals gesting that DC maturation processes are driven by programs leading to NF-kB–dependent gene transactivation. triggered by transcription factors, such as NF-kB (7–10). Several models have been widely used in the literature to study The study of the biology of DCs residing in the skin, such as LCs. In this study, we took advantage of an ex vivo model of ear Langerhans cells (LCs) and dermal DCs, has provided significant skin explants and an in vivo model of contact hypersensitivity. (19– insight into the mechanisms underlying DC maturation (11–13), 21). In the former model, skin from mouse ears is separated into particularly as it relates to migration (5, 6). For instance, LC mi- two layers and placed in a petri dish containing culture media. The gration is dependent on selective changes in the levels of chemo- physical separation of the two skin layers induces LCs migration attractant molecules, known as chemokines, and to changes in LC from the epidermis into the dermal lymphatics. LCs are collected responsiveness to these chemoattractants, a consequence in the as they fall off from the lymphatic channels directly into the petri upregulation of receptors such as CCR7 (6, 14, 15). dish (19). The in vitro model involves painting the skin with FITC plus an irritant to induce the migration of LC carrying FITC to the *Audie L. Murphy Division, Veterans Administration Center for Research on AIDS DLNs (20, 21). In a previous study, our laboratory successfully and HIV-1 Infection, South Texas Veterans Health Care System; and †Department of tested the hypothesis that, in addition to CCR7, CCR2 plays a key Medicine, University of Texas Health Science Center, San Antonio, TX 78229 role in LC trafficking during inflammation using these models. 1F.J. and M.P.Q. contributed equally to this work. We showed that mice with genetic inactivation of CCR2 Received for publication October 21, 2008. Accepted for publication March 8, 2010. exhibited impaired LC migration and an inability to control in- This work was supported by National Institutes of Health Grants RO1-AR052755 and fection with the intracellular pathogen Leishmania major (21). RO1-AI48644 and a Veterans Affairs merit grant. Notably, our group (and others) has previously shown that CCR2- Address correspondence and reprint requests to Dr. Seema S. Ahuja, Department of dependent signals result in the induction of NF-kB translocation in Medicine (MC 7870), University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900. E-mail address: ahuja@uthscsa. astrocytes, but it remains unknown whether CCR2-dependent edu activation of NF-kB is also linked to CCR2-dependent modulation Abbreviations used in this paper: BM, bone marrow; BMDC, bone marrow-derived of DC function. This seems plausible considering that DC matu- dendritic cell; DC, dendritic cell; DLN, draining lymph node; Flt3L, Flt3 ligand; ration has been shown associated with changes in the expression KLH, keyhole limpet hemocyanin; KO, knockout; LC, Langerhans cell; LTC4, leu- of CCR2 (22) and that maturation is thought to be modulated by kotriene C4; PB, parasite burden; PDTC, pyrrolidine dithiocarbamate; rm, recombi- nant murine; RPA, RNase protection assay; RR, radioresistant; WT, wild-type. NF-kB, as described above.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803494 2 CCR2 IN DC MIGRATION AND Th1 RESPONSE

We surmised that migration defects in CCR2-deficient mice nine, and antibiotics. The limiting dilution culture method was used to might be a component of the broader disturbances affecting DC quantify parasite burden (21). In brief, relevant organs were weighed and maturation. Hence, using mice with genetic inactivation of CCR2, homogenized between two frosted slides. The homogenate was placed in 96-well plates in triplicate, and 5-fold serial dilutions were performed in we sought to investigate the impact of CCR2 in DC migration and complete medium 199. Parasites were cultured for 2 wk at 27˚C. The plates maturation, aiming to test the hypothesis that CCR2-driven acti- were scored for the presence of motile parasites in individual wells using an vation of NF-kB could play a critical role in DC maturation inverted microscope at 3200 magnification. The parasite scores are the log processes, which in turn might be necessary for the generation of values of the reciprocal from the last positive dilution. PKH26 (Sigma-Aldrich) labeling of L. major was performed as de- protective immune responses against the intracellular pathogen scribed by Quinones et al. (24). A minimum of 2.5 3 107 PKH-labeled L. major. In this paper, we show that CCR2, potentially through parasites were used to infect mice. Injection with a lower amount of the induction of NF-kB translocation, plays a role in DC matu- parasites made the identification of cells carrying the parasite unreliable. ration involving CCL19 production, LC migration; Ag pre- This adjustment in the dose was performed to account for the loss of sentation with upregulation of costimulatory molecules in DCs; parasite viability associated with PKH labeling and to increase our ability to detect fluorescent-labeled parasites in vivo. Parasite viability was pre- and the release of the Th1-inducing IL-12. Furthermore, served following PKH labeling and in vivo inoculation. Confirming the we also provide evidence suggesting that IL-12p70, production by pathogenicity and viability of labeled parasites in vivo, we found that in- radioresistant (RR) cells, such as LCs, contributes to the genera- fection with PKH-labeled parasites did in fact result in chronic infection as tion of protection against L. major infection. Thus, the role of documented by parasite recovery from the DLNs of infected animals. Three days following intradermal injection of PKH-labeled L. major, mice CCR2 in the elicitation of protective immunity is 2-fold; on one were euthanized, and FACS was used to determine the proportion of hand, CCR2 modulates the amount of IL-12 produced by DCs, CD11c+ cells carrying PKH-labeled L. major in the DLNs. Cell sorting and + + and on the other hand, CCR2 regulates the migrating number of subsequent culture of PKH CD11c cells under conditions favoring par- Downloaded from IL-12p70 producing LCs. asite growth gave rise to L. major, indicating that PKH labeling of L. major did not significantly affect viability and pathogenicity (data not shown). We inferred that LCs constituted a large proportion of the population of PKH+CD11c+-labeled cells considering that the majority of these cells also Materials and Methods + + a2/low Materials expressed CD11b and DEC205 and were CD8 (data not shown). Furthermore, in vitro, PKH+CD11c+ cells promoted Ag-specific pro- liferation of purified T cells derived from the L. major-infected mice.

RPMI 1640 medium, medium 199, antibiotics, FBS, HEPES, PBS, and http://www.jimmunol.org/ trypsin were obtained from Invitrogen (Carlsbad, CA). Leukotriene C 4 LC migration to lymph nodes and NF-kB in vivo inhibition (LTC4) was from Cayman Chemical (Ann Arbor, MI), and recombinant CCL19 (ED50, 3–15 ng/ml; endotoxin , 0.1 ng/mg) and CXCL1 , The protocols described by Sato et al. (21) were used to track the migration (ED50, 3–15 ng/ml; endotoxin 1.0 EU/mg cytokine) were from R&D of FITC+ LCs, from the ear skin to the DLNs in uninfected animals. In brief, Systems (Minneapolis, MN). All other chemicals used were from Sigma- freshly prepared 5% FITC in 1:1 acetone/dibutylphthalate was painted on Aldrich (St. Louis, MO), and Abs were from BD Biosciences (San both sides of the ears (10 ml each) and on the abdomen (400 ml) (to quantify Diego, CA), unless stated otherwise. Intracellular staining for the number of FITC-positive DLNs following Flt3 ligand [Flt3L] treat- (CD207) was conducted using the clone 929F3.01 from Dendritics ment). The DLNs were harvested 48 h later and analyzed by FACS or frozen (Lyon, France). in OCT compound (Sakura Finetek, Torrance, CA), and 5-mm cryostat sections were prepared and examined by fluorescence microscopy (BX60 Mice, CCL19 blockade, and bone marrow transplantation by guest on September 27, 2021 Olympus microscope; Olympus, Melville, NY). Animals were given a 0.1- The Institutional Animal Care and Use Committee approved all protocols. ml i.p. and 10-ml intradermal (ear) injection of the NF-kB inhibitors pyr- The genetic inactivation of CCR2 mice and genotyping has been described rolidine dithiocarbamate (PDTC; 6 mg/ml) and MG-132 (5 mM) 1 h before previously (23). Wild-type (WT) and gene knockout (KO) mice were on FITC painting or ear explant experiments. the C57BL/6J background (n . 10 generations). In some experiments, we used WT and CCR2 KO mice of C57BL/6 3 129 background, and we Cell migration from ear skin explants obtained similar results to those in the C57BL/6J. Mice were born and bred As previously described (19, 21), ears were dissected, rinsed in 70% under specific pathogen-free conditions at the University of Texas Health ethanol with vigorous shaking, and allowed to dry for 15 min. The ventral Science Center (San Antonio, TX). SCID mice were purchased from Ta- 2 2 and dorsal sheets of ear skin were separated with a pair of fine forceps. The conic Farms (Germantown, NY). C57BL/6J IL-12p35 / mice were pur- two leaflets were transferred dermal side down for 24 or 72 h in a 10-cm chased from The Jackson Laboratory (Bar Harbor, ME). petri dish containing culture media. The loosely adherent populations of CCL19 Ab neutralization was performed in WT C57BL/6J, as described cells that spontaneously emigrated out of the dermal layers were recovered previously (16). Briefly, 50 mg purified goat IgG or neutralizing polyclonal by incubating the dermal layers in PBS containing 2 mg/ml glucose for Abs against CCL19 (R&D Systems) was administered 1 d prior to L. major 20 min at 37˚C. The collected cells were pooled together, filtered through infection and weekly thereafter. Ear thickness was recorded on a weekly a 70-mm nylon cell strainer, washed with PBS, and then stained. In these basis, mice were sacrificed 4 wk postinfection, and parasite burdens were experiments, LCs were identified as I-Ab bright and DEC-205+. In selected determined in DLNs. experiments, different compounds were added to the culture media of the For bone marrow (BM) transplantation, recipient mice received 1000 ear explants. cGy (lethal dose) irradiation from a 137Cs source 6–8 h before the cell transfer. Donor mice were euthanized, and BM was harvested, as described Generation BM-derived DC and in vitro infections below. BM cell suspensions were depleted of RBCs using RBC lysis buffer, resuspended in PBS, and transferred into recipient mice (10 3 106 BM-derived DCs (BMDCs) were generated as described previously (25). in 200 ml PBS) via tail vein injection. Recipient mice were infected 8 wk Briefly, BM was collected from 6- to 8-wk-old mice and cultured in the after BM transfer. presence of: recombinant murine (rm)GM-CSF (50 ng/ml) and rmIL-4 (1 ng/ml). BMDC cultures have been shown to contain a large proportion Parasites, infection, and tracking of infected LCs of mature DCs (25). For infection studies, BMDCs were cultured with live L. major L. major clone VI (MHOM/IL/80/Friedlin) was a gift from Dr. D.L. Sacks parasites in a 1:10 ratio. (Laboratory of Parasitic Diseases, National Institutes of Health, Bethesda, In vitro Ag presentation activity MD). The virulence of the L. major strain was maintained by propagating it in mice (21). Protocols similar to those used to prepare soluble Leishmania The Ag-specific syngeneic proliferation assays adapted for this experiment donovani Ags were used to make soluble L. major Ag (21). The intradermal have been described previously (21). WT micewere immunized with keyhole route was used to infect both ears with 2 3 106 or 1 3 107 stationary-phase limpet hemocyanin (KLH) and sacrificed 10–15 d postimmunization, and the L. major promastigotes resuspended in 20 ml PBS (21). Higher doses of DLNs were collected as a source of responding cells. KLH-responding parasites were used with similar results in short-term experiments. A dial- T cells (isolated using murine T cells, enrichment columns [R&D Systems]) thickness gauge caliper (Fred V. Fowler, Newton, MA) was used to measure were cocultured (1:4 ratio) with KLH-pulsed (200 mg/ml) BMDCs from WT ear thicknesses at weekly intervals for 5 wk (21). The parasites were grown or Ccr22/2 mice. After 48 h, culture supernatants were harvested, and cy- in medium 199 containing 20% FBS and supplemented with hemin, ade- tokine levels were measured by ELISA. The Journal of Immunology 3

Flt3L treatment Results In a set of experiments, 10 d prior to infection, WT and Ccr22/2 mice Impaired LC migration in CCR2 KO mice is associated with received a daily i.p. dose of 10 mg recombinant human Flt3L, as previously reduced CCL19 production described (26, 27), and followed during a period of 7 wk. Groups of un- 2/2 infected animals were used for corroboration of the Flt3L effect on DCs by We have shown that compared with WT mice, Ccr2 mice FACS and analysis of DC migration using ear explants and FITC painting. exhibit decreased numbers of LCs migrating out of ear skin ex- plants (21). This reduced migration is seen despite comparable 2 2 Administration of rmIL-12 or LPS and measurement of numbers of resident LCs in the epidermis of WT and Ccr2 / endogenous heterodimeric IL-12p70 production mice (21). A similar outcome has been described in mice lacking CCR7 or its ligand CCL19, suggesting that the CCR7–CCL19 axis Mice were injected i.p. with 0.5 mg rmIL-12 (ED50 , 0.1 ng/ml, endotoxin , 0.1 ng/mg; PeproTech, Rocky Hill, NJ) or 100 mg LPS at the time of is essential in coordinating LC migration (6, 16, 17, 30). Thus, we infection and repeated at 24 and 48 h postinfection. The IL-12 adminis- hypothesized that there could be a connection between signals tration was a modification from a protocol described by Heinzel et al. (28). generated by CCR2 and the activation of the CCR7–CCL19 axis The administration period of IL-12 was shortened from 7 to 2 d given that that occurs during DC maturation. To test this notion, we first we wanted to capture the early release of this cytokine. A total of 1 mg/ml g , m measured the levels of expression of CCR7 and CCL19 mRNA in LPS and 10 ng/ml IFN- (ED50 0.1 ng/ml, endotoxin 0.1 ng/ g; Pe- 2/2 proTech) were used to induce IL-12p70 production in DLNs using pro- 24- and 72-h ear skin explants from WT and Ccr2 mice. tocols described previously (24). At different time points (6, 12, 24, and Compared with the WT mice, ear skin explants derived from 48 h poststimulation with LPS plus IFN-g), cultured supernatants were Ccr22/2 mice significantly expressed lower levels of CCL19 but collected. ELISA was used to determine IL-12p70 levels in the super- not CCR7 or CCL2, a control chemokine (Fig. 1A). Furthermore, natants, as described previously (24). Unless indicated otherwise, back- Downloaded from ground values obtained in the absence of stimulation are subtracted from this reduction in mRNA translated into significantly lower CCL19 2/2 the data presented. concentrations in the ear skin explants of Ccr2 com- pared with WT mice (Fig. 1B). Because DCs are a major source of NF-kB translocation in ear explants CCL19 in the ear explants (16), it seemed plausible that Ccr2-null

2/2 DCs had a reduced ability to produce this chemokine. Ears from WT and Ccr2 mice were split and immediately frozen (0 h) 2/2 or left in culture for 2 h and then frozen. Subsequently, ears were cut into Next, we surmised that if LC migration in Ccr2 mice was http://www.jimmunol.org/ small pieces and homogenized, and the nuclear proteins were extracted defective, because of lower levels of CCL19 in the explants, then using the nuclear protein extraction from Panomics (Fremont, CA) reconstitution of CCL19 to normal levels should ameliorate the following the manufacturer’s protocols. NF-kB was quantified in the nu- LC migration defect. To determine whether this was indeed the clear extracts using a previously validated beads-based system from case, we added CCL19 into the culture medium of the ear skin Panomics. The coefficient of variation for the assay was under 5% based 2/2 on the company’s data. The amount of NF-kB minus background signal explants of WT and Ccr2 mice and analyzed LC migration. We was normalized based on 1) the levels of the housekeeping gene tran- found that the addition of CCL19, but not the unrelated chemokine scription factor IID and 2) by the total amount of nuclear extracts origi- CXCL1, into the culture medium significantly increased the nally recovered from the ear explants. number of LCs that migrated out from ear explants of WT mice 2/2

as well as those of Ccr2 mice (Fig. 1C). However, the mag- by guest on September 27, 2021 RNase protection assay nitude of the increase induced by CCL19 was comparatively RNase protection assay (RPA) was performed as described previously (23, higher in ear explants derived from Ccr22/2 mice (Fig. 1C). Next, 29). Total RNA was isolated from homogenized ear explants and used to we asked whether this increase in LC migration by CCL19 was perform the RPA using RiboQuant Multiprobe RNase Protection Assay Kit (BD Biosciences). The data are presented as the ratio of the densitometric dependent on LTC4. The rationale for this line of inquiry was signals of a housekeeping gene L32 to the mRNA for the gene of interest. provided by two studies that showed that LTC4 is required to in- duce toward CCL19 in LCs (16) and that CCR2 ac- CCL19 ELISA tivation induces the release of LTC4 in a model of airway hyperreactivity (31). We found that addition of LTC did not en- CCL19 protein levels from homogenized ear explants at 6, 12, 24, and 72 h 4 were analyzed using a custom ELISA. Protein tissue lysates were prepared, hance the migration of LC from ear skin explants derived from 2/2 as described previously (24). CCL19 capture and detection Ab, strepta- Ccr2 mice (Fig. 1C). Taken together, these results suggest that vidin-conjugated HRP, as well as rmCCL19 were purchased from R&D the LC migration defect seen in Ccr22/2 mice may be due to Systems and used at recommended dilutions for the ELISA. To calculate reduced local upregulation of CCL19. the CCL19 protein ratio, total protein concentrations were measured with Bradford reagent (Sigma-Aldrich), using the microplate assay following the manufacturer’s instructions. Possible role of CCL2, the principal ligand of CCR2, and the induction of NF-kB–dependent gene transactivation of DC Ag-specific cytokine production in infected mice migration Single-cell suspensions (5 3 106/ml) were cultured with or without 50 mg/ We then focused on determining the possible upstream and down- ml soluble L. major Ag in 24-well plates, as described previously (21). stream mechanistic pathways that could account for the defect in After 48 h of culture, supernatants were harvested, and ELISA was used to 2/2 determine the IL-4, IL-5, and IFN-g protein levels. The range of detection CCL19 production and the related LC migration defect in Ccr2 by ELISAs for IL-4 was 2.0–125 and 15.6–1000 pg/ml for IL-5 and IFN-g. mice. We envisioned a scenario in which CCL2 released locally acts on its receptor CCR2 to induce the translocation of the transcription Statistical analysis and data modeling factor NF-kB into the nucleus. This transcription factor promotes DC migration manifested by the release of CCL19 and LC migra- Data represent the mean 6 SD. Groups were analyzed with Stata (StataCorp, College Station, TX) or SPSS (Chicago, IL) statistical software. According tion from the epidermis toward the DLNs. The following con- to the number of groups and the distribution (normally distributed or not), verging lines of evidence supported this line of reasoning. nonpaired t test, one-way ANOVA, Kruskal-Wallis, Mann-Whitney, or First, we found that, similar to the findings in Ccr22/2 mice, Fisher’s exact test were performed. Statistical significance was accepted at genetic inactivation of the CCR2 ligand CCL2 was also associated p , 0.05. Pathways Analysis Software (Ingenuity Systems, Redwood City, CA) was used to visualize potential pathways suggested from our data with abnormal LC migration (Fig. 1D). As expected, decreased LC and emerging from published literature by looking at interactions among migration was accompanied by a reduction in CCL19 mRNA (data CCR2, CCL2, CCL19, NF-kB, and IL-12. not shown). Furthermore, providing a more direct link between the 4 CCR2 IN DC MIGRATION AND Th1 RESPONSE

AB 1.2 20 WT NS WT

Ccr2-/- 16 Ccr2-/-

ts uni g/mg) 0.8 µ 12

8 otein ( otein

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0.4 **

Total pr Total

A r A mRN

0 0 CCL19 CCR7 CCL2 6h 12h 24h 72h C D Genotype Treatment DEC-205/I-Ab Ccr2 CCL19 CXCL1 LTC Migration 4 ** 60000 +/+ --- Normalized ++ +/+ - - 40000

0 1234 Total number number Total Fold increase Downloaded from 20000

-/- - - - Normalized 0 -/- - - WT CCL2

-/- - + - +

-/- - - http://www.jimmunol.org/

0 1234 Fold increase E F G

500 * ** 1.50 **

4 units 1.25

(pg/ml) (pg/ml) 400

3 1.00

elative in LN in 300 cells

0.75 by guest on September 27, 2021 + 2

200 0.50 mRNA r mRNA

CCL19 levels 1 0.25 100 FITC % 0.00 - +-+ -+ 1000 500 250 125 62.5 MG-132 PDTC CCL19

CCL2 (pg/ml) FIGURE 1. CCL19 and LC migration in Ccr22/2 mice. A, RPA assay for CCL19, CCR7, and CCL2 mRNA expression at 24 h in ear explants. Data presented as the ratio between the levels of CCL19, CCR7, and CCL2 mRNA to L-32 from five independent experiments with ear explants from two to three mice per experiment. B, CCL19 protein levels in ear explants. Levels of CCL19 are expressed in micrograms per milligram total protein. Samples were composed of at least two WT or Ccr22/2 ears per time point. C, Effects of exogenous administration of CCL19 on LCs migration out from ear explants. Recombinant mouse CCL19, CXCL1, or LTC4 was added to the culture medium. After 72 h, cells recovered from the medium were stained for I- Ab and DEC-205. The data represent the mean of three independent experiments, each consisting of at least three WT orCcr22/2 mice per treatment. Migration is shown as normalized values for each group regarding their unstimulated counterpart (numbers of migrated DCs from representative ex- periments are as follows: WT 32600, WT plus CCL19 67100, Ccr22/2 12418, and Ccr22/2 plus CCL19 68513). Dotted lines represent fold increase or decrease over media alone in WT or Ccr2-null mice (N). D, Migration of LCs (DEC-205+/I-Ab+) out of ear explants from WT and Ccl22/2 mice. A representative experiment of the three performed is shown. Each independent experiment performed included ears from at least three WT or Ccl22/2 mice. E, Dose-response induction of CCL19 production by CCL2. WT splenocytes were treated with increasing concentrations of CCL2, and after 24 h, the levels of CCL19 were measured in culture supernatants. A representative experiment of the three performed is shown. F, WT mice were pretreated with NF-kB chemical antagonists as described in Materials and Methods. Subsequently, their ears were painted with FITC/acetone. Migration of LCs from the skin to DLNs was quantified by assessing the percentage of FITC+ cells in LN cell suspensions. The majority of these cells also expressed I-Ab. A representative experiment from four performed for MG-132 and five for PDTC is shown. Vehicle (2) or active antagonists (+). Each experiment included a minimum of two mice. G, Ear explants of WT mice were treated with or without the NF-kB antagonist MG-132, and mRNA levels for CCL19 using RPA were quantified. pp , 0.05; ppp , 0.01.

CCL2–CCR2 axis and CCL19, we found that the addition of CCL2 (1.47-fold increase from time 0 to 2 h; p= NS; n = 12 explants for to murine splenocytes induced CCL19 production (Fig. 1E), which each time point). Third, in vivo systemic treatment of mice with NF- was localized in DCs (data not shown). Second, 2 h after ear peeling kB inhibitors disrupted LC migration. We used two different com- there was a significant increase in nuclear NF-kB translocation in pounds routinely used for in vitro and in vivo studies (32–39). Both WTexplants (1.85-fold increase from time 0 to 2 h; p = 0.009; n =12 PDTC (32, 33) and MG-132 (40), administered to mice prior to explants for each time point). However, in Ccr22/2 explants, NF-kB epidermal FITC painting, inhibited LC migration from ear skin to translocation was blunted, failing to reach statistical significance DLNs (Fig. 1F). A similar effect was seen when inhibition of NF-kB The Journal of Immunology 5 was achieved by adding the inhibitors to cultured ear explants (data notion, we first asked whether CCR2-dependent signals influence not shown). the migration of LCs carrying L. major from the skin to the DLNs. Finally, we observed that chemical inhibitionof NF-kB resulted in To this end, we quantified the proportion of DCs carrying PKH- a reduction in CCL19 levels in the tissue (Fig. 1G) linking NF-kB labeled L. major from the epidermis into the DLNs of WT and and CCL19 and falling in line with our findings that LC migration in Ccr22/2 3 d after intradermal injection. We found that compared Ccr22/2 and Ccl22/2 mice is disrupted. We used two different with WT mice, Ccr22/2 DLNs contained a smaller proportion of inhibitors because in general chemical inhibitors are not 100% cells likely to constitute PKH-labeled LCs (Fig. 3A). specific. Therefore, PDTC and MG-142 could have other effects Next, we determined whether genetic inactivation of CCR2 af- beyond NF-kB inhibition that could also affect LC migration. fected the production of IL-12p70 by DCs. We first measured IL- In agreement with these data sets, it has been reported that the 12p70 production by DLN cells and splenocytes in vitro, because promoter region of the CCL19 gene has several NF-kB binding these lymphoid organs contain diverse populations of DCs, which sites (8, 41). have been shown to be an important source of this cytokine (42– 44). This observation was confirmed using intracellular staining Abnormal upregulation of costimulatory molecules in Ccr22/2 for IL-12, along with the surface staining for the DC marker DCs CD11c (data not shown). In this in vitro system, we found that LPS/IFN-g–induced production of IL-12p70 by splenocytes and The results presented so far suggest that CCL2-CCR2–mediated 2 2 lymph node (LN) cells from Ccr2 / mice was significantly lower induction of NF-kB gene transactivation could be required for than in WT mice (Fig. 3B,3C). Next, we sought to determine CCL19-driven LC migration from the skin to the DLNs. Next, we whether CCR2 also regulates IL-12p70 production and other DC Downloaded from tested the notion that absence of Ccr2 and the related impairment maturation processes triggered by exposure L. major. Indeed, us- of DC maturation could also be manifested by a failure to up- ing BMDCs as a model system, we found that L. major-induced regulate costimulatory molecules and MHC class II. To test this 2 2 upregulation of class II expression was reduced in Ccr2 / DCs notion, we studied DC differentiation and maturation occurring 2 2 compared with WT DCs (Fig. 3D). Likewise, Ccr2 / BMDCs in vitro from BM progenitors induced by GM-CSF and IL-4 (24, produced lower levels of IL-12p70 than WT DCs in response to 25). In this well-characterized in vitro model system, we found activation with live L. major (Fig. 3E). The reduction in IL-12p70 http://www.jimmunol.org/ that despite comparable numbers of CD11c+ DCs in cultures from 2/2 production seemed to be functionally relevant given that, com- WT and Ccr2 mice (Fig. 2A), the percentages of DCs ex- 2 2 pared with WT BMDCs, Ccr2 / BMDCs had a reduced capacity pressing costimulatory molecules and MHC class II were signif- to induce Ag-specific production of IFN-g by KLH-primed WT icantly lower in the KO mice (Fig. 2B–E). Overall, this difference T cells (Fig. 3F). Furthermore, there was a correlation between the was also evident when looking at the mean fluorescence intensity 2 2 defect in IL-12 production of DC Ccr2 / identified in vitro and values (mean fluorescence intensities in a representative experi- 2/2 in vivo. This was suggested by the observation that there was ment WT versus Ccr2 BMDCs included: CD80, 469 versus + b a reduced proportion of mature LCs producing IL-12 (CD205 I- 384; CD86, 264 versus 289; and I-A , 749 versus 443). This 2 2 Ab+IL-12+ cells) in the DLNs of L. major-infected Ccr2 / mice

finding is in line with the view that CCR2 participates in DC by guest on September 27, 2021 compared with infected WT mice (Fig. 3G). maturation in a broader context. We surmised that if the defective production of IL-12p70 in 2/2 2/2 Ccr2 DCs seen in vitro was an important determinant of sus- Susceptibility of Ccr2 mice to L. major infection is related ceptibility to L. major infection, then early exogenous adminis- to abnormal maturation of DC populations including LCs tration of this cytokine to Ccr22/2 mice, at the time of exposure to We postulated that genetic inactivation of CCR2 leads to suscep- L. major, would switch the susceptible phenotype to resistance. 2 2 tibility to L. major infection by disrupting LCs and other DC Supporting this posit, administration of rIL-12 to Ccr2 / mice maturation, such as migration and IL-12p70 production. To test this during the initial 3 d postinfection corrected their inability to

b A CD11c BCCD40 I-A DCD80E CD86

49.0 79.6 *

32.4 * 48.6 * 21.0 * WT

42.6 50.3

6.5 11.3 8.8

-/- CCR2

FIGURE 2. Defective maturation of Ccr22/2 BMDCs. WT and Ccr22/2 BM cells were cultured in the presence of GM-CSF and IL-4 for a total of 7 d to generate BMDCs. Surface staining for the following DC maturation markers is shown. A, CD11c; B, CD40L; C, I-Ab; D, CD80; and E, CD86. A rep- resentative experiment with percentages of positive cells is shown. pp , 0.05, comparing the average of the percentages for individual markers. 6 CCR2 IN DC MIGRATION AND Th1 RESPONSE - Downloaded from http://www.jimmunol.org/ - by guest on September 27, 2021

FIGURE 3. IL-12 production, and response to L. major infection is defective in Ccr22/2 mice. A, Impaired migration of parasite-bearing LCs to the DLNs in Ccr22/2 mice. Absolute numbers of PKH+CD11c+ per LN of a representative experiment is shown. B and C, LPS/IFN-g–induced IL-12p70 production by LNs or splenocytes is decreased in CCR2-deficient mice. Results from one representative experiment out of two performed with five mice each are shown (mean 6 SD). D, Ccr22/2 BMDCs have diminished induction of class II molecules upon encounter of viable L. major promastigotes (1:5 ratio). E, Lower production of IL-12p70 by Ccr22/2 BMDCs in response to live L. major promastigotes challenges (1:10 ratio). Similar results were obtained in two independent ex- periments. F, KLH-pulsed BMDC T cells from Ccr22/2 mice have a reduced ability to induce the release of IFN-g protein compared with WT. Similar results were obtained in two independent experiments. G, Decreased production of IL-12p70 by Ccr22/2 by activated CD205 cells in the DLNs in 4-wk infected animals. In vitro stimulation of cells with known inductors of IL-12 restore the ability of these cells to produce this cytokine. pp , 0.05; ppp , 0.01. mount a protective response to L. major infection (Fig. 4A,4B). mice were equally susceptible to infection regardless of receiving Compared with untreated Ccr22/2 mice, rIL-12–treated Ccr22/2 either PBS or LPS (Fig. 4F). LPS administration was sufficient to mice had reduced ear swelling and reduced spleen parasite bur- induce systemic effects in WT mice, including DC mobilization dens at 6 wk postinfection (Fig. 4A,4B). Furthermore, Ag-specific from the marginal zone to T cell zone in the spleen (data not immune responses in IL-12–treated Ccr22/2 mice were charac- shown). This outcome was also in line with previous research terized by significantly higher Ag-specific IFN-g and lower IL-4 demonstrating the inability of LPS administration to change and IL-5 compared with untreated Ccr22/2 mice (Fig. 4C–E). a L. major-susceptible phenotype in mice in vivo (46). An important issue to address was the specificity of exogenous Potential role of RR/LC-derived IL-12 in the generation of 2/2 IL-12 administration for both the phenotype of Ccr2 mice and protective immunity against L. major infection the treatment itself. First, the observation that administration of rIL-12p70 did not have any measurable effect in WT mice (Fig. 4A, DCsubsetsvaryintheirabilitytoproduceIL-12p70,andconsequently, 4B) rules out a simple overall beneficial effect of IL-12p70 ad- they differ in their capability to polarize T cell responses toward Th1. ministration in resistant mice strains. Second, the effect of exoge- However, considering all the redundancy in the DC network, it is nous IL-12p70 administration in the phenotype of Ccr22/2 mice unclear whether the production of IL-12p70 by any DC subtype is was not a consequence of nonspecific inflammatory effects as indispensable for induction of protective immune responses. To infer 2/2 suggested by the lack of efficacy of treatment with LPS. In this that L. major susceptibility in Ccr2 mice is linked to a decreased experiment, mice received three doses of LPS or saline prior amount of IL-12 released by LCs, we first needed to demonstrate that to infection with L. major. Doses of LPS in this range are known to IL-12p70 production by LCs does play a nonredundant role in the promote DC migration and IL-12 production in vivo (45). Ccr22/2 generation of protective immune responses against L. major.Aiming The Journal of Immunology 7

*

FIGURE 4. Defective production of IL-12 by RR/LC may account for Ccr22/2 susceptibility to L. major infection. A and B, Administration of IL- 12 reverses the L. major-susceptible phenotype of Ccr22/2 mice. Exogenous administration of rmIL- 12inCCR2-deficientmicefor1dpriorand2dafter L. major infection corrected their susceptible phe- notype as measured by ear swelling and parasite * burden. Results are the mean 6 SD for five mice. A representative experiment of two independent ex- 2/2 periments is shown. Differences between Ccr2 ** - mice treated with or without IL-12: pp , 0.05; ppp , 0.01. C–E, Skewing from Th2 cytokine pro- * Downloaded from duction to a Th1 pattern in Ccr22/2 mice. Each symbol represents the Ag-specific cytokine levels produced from a single mouse. F,Treatmentwith LPSpriortoinfectionandevery24hfor2dpost- infection did not improve the L. major susceptibility 2/2 ** in Ccr2 mice. G, Lethal radiation of WT or IL- ** **

2 2 http://www.jimmunol.org/ 12p35 / miceandBMreconstitutionwithWTor ** IL-12p352/2 cells showed that IL-12 from the LC or RR compartment is necessary but not sufficient to control L. major infection. ppp , 0.01, com- paring WT + WT versus IL-12p352/2 +WTand WT + WT versus IL-12p352/2 + IL-12p352/2. Results are the mean 6 SD of four to five mice per group. H and I, T and B cell-deficient mice recipi- ents of Ccr22/2 (Scid + Ccr22/2)orWT(Scid + 2/2

Ccr2 ) T cells exhibit similarly enhanced pro- by guest on September 27, 2021 tection against L. major infectioncomparedwith ** Scid mice that did not receive T cells (Scid). Results * are the mean 6 SD of three to four mice per group. * pp , 0.05; ppp , 0.01. **

to provide evidence for a role of LC-derived IL-12, we engineered IL-12p352/2 BM, and the fourth group comprised WT recipients of BM chimeric mice in which IL-12p70 production was deficient in WT BM. The final two groups served as controls because IL-12 either the BM-derived cell populations, the RR cell compartment, or production was blunted in both LCs and BMDCs (IL-12p352/2 in both compartments or was normal. →IL-12p352/2), or it was normal in both cell compartments The rationale for the use of BM chimeras mice was the knowl- (WT→WT). edge that a large proportion of LCs is derived from RR/LC pro- After allowing sufficient time for their reconstitution, chimeric genitors (47) and that therefore selective replacement of all BMDC mice were infected intradermally with L. major and followed over populations but not LCs could be accomplished in a lethally irra- 7 wk. Ear swelling and DLN parasite burdens were used as in- diated and syngeneically reconstituted host. Using WT and IL- dicators of protective immunity. Importantly, we found that the 12p352/2 mice as donor or recipients, the following four groups of proportion of LCs (CD11c+CD207+ or CD11c+DEC205+ cells) chimeric mice were created. The first group was lethally irradiated residing in the DLNs 7 wk postinfection was comparable across IL-12p352/2 recipients of WT mice BM (WT→IL-12p352/2). In all four groups of chimeras, indicating that genetic inactivation of these mice, LCs should be unable to produce IL-12p70; however, IL-12p352/2 was not influencing DC migration (data not shown). all other populations of BMDCs would release normal IL-12p70 As anticipated, the control groups WT→WTand IL-12p352/2→IL- levels. Group 2 consisted of lethally irradiated WT recipient mice 12p352/2 were completely resistant or susceptible to L. major in- of IL-12p352/2 BM (IL-12p352/2→WT). In these mice, normal fection, respectively (Fig. 4G, Table I). Interestingly, WT→IL- release of IL-12p70 is expected in LCs but disrupted in all BMDC 12p352/2 chimeras had significantly more ear swelling and higher populations. The third group consisted of IL-12p352/2recipients of DLN parasite burdens than WT→WT mice but less than IL-12p352/2 8 CCR2 IN DC MIGRATION AND Th1 RESPONSE

Table I. Sources of IL-12 production and susceptibility to L. major in mice receiving adoptive transplants

IL-12 from IL-12 from Groups Donor→Recipient RR/LCs BMDCs Log PB (LN) p , 0.05 AWT→WT Yes Yes 1.54 6 0.12 AvsC AvsD BWT→IL-12p35 No Yes 2.22 6 0.53 BvsC BvsD C IL-12p35→WT Yes No 3.40 6 0.81 NS D IL-12p35→IL-12p35 No No 4.64 6 0.63 NS PB, parasite burden.

→IL-12p352/2, suggesting that IL-12p70 derived from RR cells, such grating cells out of the ear explants (data not shown). However, as LCs, was necessary to elicit some degree of protective immunity this increase was not sufficient to change the susceptibly state of against L. major (Fig. 4G, Table I). Moreover, IL-12p352/2→WT and Ccr22/2 mice to L. major infection as PBS-treated and Flt3L- IL-12p352/2→IL-12p352/2 chimeras were equally susceptible to L. treated Ccr22/2 mice had a similar outcome following L. major major infection, suggesting that IL-12p70 produced by RR/LCs was infection (Table II, data not shown). insufficient for the elicitation of adequate immune responses against A possible interpretation of this finding is that although the numerical

L. major (Fig. 4G, Table I), and therefore, BM-derived populations DC/LC defect may be partially corrected by administering Flt3L to Downloaded from were an indispensable source of IL-12p70. Taken together, the results Ccr22/2 mice, increasing the availability of DCs seems ineffective from the BM chimeras suggest that IL-12p70 derived from RR cells, because their functional defects (abnormal IL-12p70 release and ex- namely LCs, contributes but is not sufficient for the elicitation of full pression of costimulatory molecules) remained unchanged. protection against intradermal L. major infection. On the basis of the results above, we surmised that if direct effects Discussion

of CCR2 on T cells could be ruled out, then at least two interrelated In this study, we show that CCR2-dependent signals triggered by http://www.jimmunol.org/ 2 2 APC factors might underlie the susceptibility in Ccr2 / mice to L. bindingofitsligandCCL2andsubsequentnucleartranslocationofNF- major infection. The first factor being a reduced amount of IL- kB are required for DC maturation and LC migration from the epi- 2 2 12p70 produced by Ccr2 / DC populations including LCs (Figs. dermis to the DLNs. Interestingly, CCR2-dependent signals con- 3A,3B,3E,3F,4G, Table I). The second, a reduced number of LCs tribute to the production of CCL19, upregulation of costimulatory in the DLN, would factor the production of lower amounts of IL- molecules, and the production of IL-12 (a proposed model is shown in 12p70 (Fig. 3C,3G) and reduced support adequate T cell activa- Fig. 5). In particular, the later process was found to be critical for the tion. To address these factors, the following sets of experiments generation of protective immune responses against L. major infection were conducted. in resistant mice. Taken together, these data extend our previous re- To rule out the possibility that intrinsic defects in T cells were ports that the susceptibility of Ccr22/2 mice to L. major infection by guest on September 27, 2021 2 2 responsible for abnormalities in Ccr2 / mice, we conducted could, in fact, be secondary to the inability of LCs to produce IL-12 adoptive T cell transfer studies from WT or Ccr2-null mice into and a failure to mount a protective Th1 response. SCID mice. As shown in Fig. 4H and 4I, T cells from WT or The role of the chemokine system in DC biology is classically seen 2 2 Ccr2 / mice were comparable in their ability to protect SCID as being critical for cell migration. However, increasing evidence mice against the infectious challenge. This finding is in line with suggests that in addition to their role in chemotaxis, chemokines and 2 2 our previous report that WT and Ccr2 / T cells are comparable their receptors also regulate DC maturation(50). For instance, human in their ability to polarize into Th1/Th2 cells (48). DCs treated with neutralizing Abs against the chemokine receptors Next, we attempted to gather evidence for a nonredundant role of CCR1 or CCR3 reduced their ability to activate T cells in vitro (51). 2 2 the reduced number of LCs arriving in the DLNs in Ccr2 / mice’s In mice, CCL19 was found to induce maturation of DCs, resulting in susceptibility to L. major infection. We successfully increased upregulation of costimulatory molecules, production of proinfla- 2 2 only the number of DCs migrating in Ccr2 / mice without mmatory cytokines, and enhancement of DCs’ ability to activate changing the functional maturation defect (e.g., ability to produce T cells (52). More recently, evidence consistent with our findings IL-12p70; data not shown) using 10-d treatment with Flt3L (Table suggests that CCR2-dependent signals are critical for DC maturation II). Administration of Ftl3L is a well characterized approach to (22). The authors showed that genetic inactivation of CCR2 in mu- expand the multiple subsets of DCs, including LCs and dermal rine DCs lead to reduced expression of costimulatory molecules DCs (49). Flt3L treatment partially increased the detection of upon activation with LPS, reduced allostimulatory capacity, and 2 2 FITC+ LCs in the DLNs of Ccr2 / mice (Table II) and LC mi- abnormal migration (22). Unfortunately, the mechanisms underlying these observations were not reported. In this paper, we extend those Table II. Increased numbers of DCs with Flt3L treatment does not observations by revealing the potential role of the CCL2, CCR2’s change susceptibility toward L. major in Ccr22/2 mice main ligand CCL2, and of NF-kB–mediated signals, as well as the critical role of CCR2 in regulation of IL-12 production by maturating Flt3L LN FITC+ (%) Log PB (Spleen) DCs. We also present data supporting the in vivo relevance of these observations in the context of a specific DC subset, namely LCs, and WT 2 19/19 (100) 0.0 6 0.0 WT + 15/19 (78) 0.0 6 0.0 test the relevance of our findings in an in vivo infectious model. Ccr22/2 2 6/22 (28) 0.9 6 1.07* A role for the CCL2–CCR2 axis in LC migration/maturation is 2 2 Ccr2 / + 11/25 (44) 3.7 6 0.6** also supported by three recent reports. First, human CCL2 trans- LN FITC+ cells indicate the number of slides positive for FITC cells over the total genic mice exhibit an acceleration of migration of LC from the number of analyzed slides in the DLNs. epidermis into the DLN, following sensitization with haptens, an pp = 0.054, difference between WT and Ccr22/2; ppp = 0.001, comparing WT and Ccr22/2 mice receiving Flt3L. Results are the mean 6 SD for five mice. effect that is accompanied with increased expression of class II PB, parasite burden. and costimulatory molecules (53). Second, CCL2 has been shown The Journal of Immunology 9

CCL2 NF-kB is linked to reduced production of CCL19, a critical chemo- kine for LC migration (16), and further highlight the critical role of the

ERK 1/2 CCL2–CCR2 axis in the induction of NF-kB. Moreover, the data CCR7 provide a mechanistic backdrop to the observation that chemical CCR2 antagonism of CCR2 in vivo reduces the magnitude of delayed-type hypersensitivity responses (61), an immunological process that is ERK highly dependent on DC migration from the skin to the DLNs (2, 60). CCL19 Thus, collectively our data suggest that CCL2-dependent signals IRF1 acting via CCR2 lead to NF-kB translocation to the nucleus and

Akt transactivation of the CCL19 gene (Fig. 5). This possibility is further IL12B supported by work documenting that both human and mouse CCL19 promoters have several functional NF-kB binding sites (8, 41). NF-kB (complex) Our data also indicate that many of the phenotypic consequences FIGURE 5. Potential molecular underpinnings of CCR2’s contribution of CCR2 inactivation may be due to deficits in NF-kB activation. to DC maturation: pathway analysis and visualization. A model was Indeed, in IL-12p70 production in DCs evidently occurs in re- generated using Ingenuity Pathways Analysis software from published literature and screening of direct (e.g., CCL2→CCR2) or indirect (e.g., sponse to signals that require NF-kB activation (7, 62, 63). CCL2→NF-kB) interactions emerging by the data presented in this paper, The observation that inactivation of CCR2 is associated with involving CCR2, CCL2, CCL19, NF-kB, and IL-12. Our model suggests abnormal production of IL-12p70 by DCs in vitro supports previous 2 2

/ Downloaded from that the interaction between CCR2 and its cognate ligand CCL2 induces reports documenting abnormal IL-12p70 production in Ccr2 the expression of upregulated concurrently as part of DC maturation, mice (64), as well as the fact that chemokines are increasingly such as CCR7, CCL19, and IL-12B (IL-12p40), possibly via induction of recognized as regulators of the production of this cytokine by DCs NF-kB transactivation. It also suggests other potential mediators are in- (65). By contrast, in this study, we show the relevance of reduced volved in the pathway. production of IL-12p70 in Ccr22/2 mice, in vivo, as highlighted by the demonstration that administration of IL-12p70 prior to

to have potent chemoattractant activity for murine LCs in vitro infection corrects the L. major-susceptible phenotype but does not http://www.jimmunol.org/ (54). And finally immature DCs respond to CCL2 (55). affect the response of WT mice. Another line of convergent evidence supporting a role for NF-kB The observation that treatment with LPS prior to infection, translocation by the CCR2 ligand CCL2 is highlighted in recent a stimulus known to promote DC maturation and mobilization (66), work from our laboratory (29). Using an EMSA-based approach, we was not able to induce resistance in Ccr22/2 mice suggests that in found CCL2-induced nuclear translocation of NF-kB in astrocytes. the absence of CCR2, DCs are incapable of overcoming using the The effect of CCL2 on NF-kB translocation was 1) time dependent, nonspecific stimuli. Thus, effective signals via CCR2 might be peaking at 120 min after the addition of CCL2; 2) dose dependent indispensable. Aside from CCL2, other CCR2 ligands could with maximal effects at 100 ng/ml for the dose-range tested; and 3) provide these signals as suggested by our observation that CCL2- dependent predominantly on the p65 component of NF-kB (29). We null mice are resistant to L. major infection (67) and that CCL2 by guest on September 27, 2021 also demonstrated that CCL2-induced NF-kB translocation was signals are not critical for IL-12p70 production in vivo (64). markedly diminished in the absence of CCR2. (29). Our experiments using lethal radiation and BM transplantation A logical extension of this work was to investigate the functional suggests that IL-12p70 released by a cell type with RR progenitors, implications of CCL2-CCR2 activation of NF-kB in DC biology. such as LCs contributes, but is not sufficient, to induce protective NF-kB is highly expressed by DCs, and it mediates key DC func- immunity against L. major. This finding allows us to infer that the tions (6). Indeed, in line with our findings, previous reports have susceptible phenotype in Ccr22/2 mice could be related, in part, to highlighted that NF-kB strongly enhances DC longevity (56); well- abnormalities in IL-12p70 production by LCs and other DC pop- known DC-activating mediators such CD40L promote maturation via ulations. Importantly, the contribution of CCR2 to the generation of sustained activation of NF-kB (57); and inhibition of NF-kB activation protective immune responses goes beyond its role in LC migration blocks maturation of DCs, in terms of upregulation of MHC and and involves the modulation of maturation processes. Indeed, costimulatory molecules (9, 58). solely increasing the numbers of DCs available in Ccr22/2 prior to The contribution of NF-kB to DC migration has been less studied L. major infection using Flt3L failed to change their susceptibility. ex vivo and in vivo. We were able to document blunting in Ccr22/2 Our work confirms and further extends the role of DCs (68), spe- skin explants of the rapid upregulation of NF-kB that occurred cifically LCs, in the generation of immune responses against L. following ear peeling in WT explants. Highlighting further the im- major, including the observation that LCs produce IL-12p70 when portance of this transcription factor, we found that administration of exposed to L. major parasites (43, 69) and that IL-12p70 released in the NF-kB chemical antagonists PDTC and MG-132 to mice sig- the early stages of infection is indispensable for the induction of nificantly reduced LC migration in the FITC-painted model. Cer- protective immune responses against L. major (70). It has also been tainly, a major concern with the use of the chemical antagonists is shown that production of IL-12p70 by mature DCs is required to the lack of specificity. However, our finding of reduced LC migra- generate protective immune responses against L. major, and work tion in mice receiving PDTC or MG-132 is further supported by by Wiethe et al. (71) showed that immature DCs induce Th2 po- reports indicating that the proteasome inhibitor-based (N-benzy- larization and are susceptibility to L. major infection as is likely to loxycarbonyl-Ile-Glu[O-tert-butyl]-Ala-leucinal [PSI]) or adeno- be the case in Ccr22/2 mice. viral- mediated antagonism of NF-kB blocks DC maturation events, Although the relevance of LCs for the generation of protective such as upregulation of costimulatory molecules and production of immune responses against L. major seems to be well established, cytokines (59). some reports challenge this notion (72). For instance, skin-derived Moreover,verysimilartowhatweshowinthecurrentreport,NF-kB DCs, including LCs and dermal DCs, were found to migrate blockade using decoy oligodeoxynucleotides in vivo was shown to poorly to LNs after L. major infection and have been shown to reduce LC migration and impair T cell responses (60). Nonetheless, play a minor role in early T cell activation (73). Moreover, resi- our findings extend these observations by revealing that blockade of dent DCs in LNs (rather than LCs) were responsible for the 10 CCR2 IN DC MIGRATION AND Th1 RESPONSE induction of protective immune responses (74). Of note, in these 19. Larsen, C. P., R. M. Steinman, M. Witmer-Pack, D. F. Hankins, P. J. Morris, and reports, the infectious challenge was administered via the s.c. J. M. Austyn. 1990. Migration and maturation of Langerhans cells in skin transplants and explants. J. Exp. Med. 172: 1483–1493. route. By contrast, our study used the intradermal route. Clearly, 20. Macatonia, S. E., S. C. Knight, A. J. Edwards, S. Griffiths, and P. Fryer. 1987. the use of the intradermal route more likely engaged LCs and led Localization of antigen on lymph node dendritic cells after exposure to the contact sensitizer fluorescein isothiocyanate: functional and morphological to differential immune responses (67, 75). Furthermore, the route studies. J. Exp. Med. 166: 1654–1667. of administration is critical given that LCs promote T cell re- 21. Sato, N., S. K. Ahuja, M. Quinones, V. Kostecki, R. L. Reddick, P. C. Melby, sponses to skin Ag, but only under defined conditions (76) and W. A. Kuziel, and S. S. Ahuja. 2000. CC (CCR)2 is required for Langerhans cell migration and localization of T helper cell type 1 (Th1)- also through our work indicating that the effects of the CCL2– inducing dendritic cells: absence of CCR2 shifts the Leishmania major-resistant CCR2 axis are modulated by the route of infectious challenge phenotype to a susceptible state dominated by Th2 cytokines, B cell outgrowth, (67). The doses of parasite used also can be a critical factor. The and sustained neutrophilic inflammation. J. Exp. Med. 192: 205–218. 22. Fiorina, P., M. Jurewicz, A. Vergani, A. Augello, J. Paez, V. Ricchiuti, V. Tchipachvili, doses of L. major used in this study were significantly higher than M. H. Sayegh, and R. Abdi. 2008. Phenotypic and functional differences between wild- expected during human infection and did not use a live vector type and CCR2–/– dendritic cells: implications for islet transplantation. 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