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The Transcriptional Repressor BLIMP1 Curbs Host Defenses by Suppressing Expression of the CCL8

This information is current as Martina Severa, Sabina A. Islam, Stephen N. Waggoner, of October 1, 2021. Zhaozhao Jiang, Nancy D. Kim, Glennice Ryan, Evelyn Kurt-Jones, Israel Charo, Daniel R. Caffrey, Victor L. Boyartchuk, Andrew D. Luster and Katherine A. Fitzgerald J Immunol 2014; 192:2291-2304; Prepublished online 29 January 2014; doi: 10.4049/jimmunol.1301799 Downloaded from http://www.jimmunol.org/content/192/5/2291

Supplementary http://www.jimmunol.org/content/suppl/2014/01/28/jimmunol.130179 http://www.jimmunol.org/ Material 9.DCSupplemental References This article cites 78 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/192/5/2291.full#ref-list-1

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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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Transcriptional Repressor BLIMP1 Curbs Host Defenses by Suppressing Expression of the Chemokine CCL8

Martina Severa,*,† Sabina A. Islam,‡ Stephen N. Waggoner,x Zhaozhao Jiang,* Nancy D. Kim,‡ Glennice Ryan,* Evelyn Kurt-Jones,* Israel Charo,{ Daniel R. Caffrey,* Victor L. Boyartchuk,‖ Andrew D. Luster,‡ and Katherine A. Fitzgerald*,‖

The transcriptional repressor B –induced maturation 1 (BLIMP1) is a master regulator of B and T differentiation. To examine the role of BLIMP1 in innate immunity, we used a conditional knockout (CKO) of Blimp1 in myeloid cells and found that Blimp1 CKO mice were protected from lethal induced by Listeria monocytogenes. Transcriptome analysis of Blimp1 CKO identified the murine chemokine (C-C motif) 8, CCL8, as a direct target of Blimp1- mediated transcriptional repression in these cells. BLIMP1-deficient macrophages expressed elevated levels of Ccl8, and conse-

quently Blimp1 CKO mice had higher levels of circulating CCL8, resulting in increased in the peripheral , Downloaded from promoting a more aggressive antibacterial response. Mice lacking the Ccl8 were more susceptible to L. monocytogenes infection than were wild-type mice. Although CCL8 failed to recruit neutrophils directly, it was chemotactic for g/d T cells, and CCL8-responsive g/d T cells were enriched for IL-17F. Finally, CCL8-mediated enhanced clearance of L. monocytogenes was dependent on g/d T cells. Collectively, these data reveal an important role for BLIMP1 in modulating host defenses by suppressing expression of the chemokine CCL8. The Journal of Immunology, 2014, 192: 2291–2304. http://www.jimmunol.org/

lymphocyte–induced maturation protein 1 (BLIMP1) is differentiation, , and/or function of both CD4 and a transcriptional repressor critical for early embryonic CD8 T cells (9, 10). It also plays a central role in the differenti- B development in multiple species (1–3). BLIMP1 was first ation and function of (11) and sebocytes (12) and in identified as a factor that bound to the positive regulatory domains the tolerogenic function of DCs (13). The role of BLIMP1 in I and III of the IFN-b enhancer, where it was shown to compete macrophages and antimicrobial defenses, however, has not been with IFN regulatory factors to attenuate IFN-b gene transcription examined in detail. (4, 5). BLIMP1 is best studied, however, in the context of terminal Macrophages are one of the major mediators of inflammation and critical effector cells of innate immunity. They serve as host

differentiation of B to plasma cells (6–8) and in the by guest on October 1, 2021 cells for intracellular pathogens, including Listeria monocytogenes (14). L. monocytogenes is a Gram-positive facultative intracellular *Division of Infectious Diseases and Immunology, Department of Medicine, Univer- sity of Massachusetts Medical School, Worcester, MA 01605; †Dipartimento di bacterium, which causes disease in humans as a result of ingestion Malattie Infettive, Parassitarie ed Immuno-Mediata Malattie, Istituto Superiore di of contaminated foods. L. monocytogenes are generally ‡ Sanita`, 299-00161 Rome, Italy; Division of Rheumatology, and Immunol- limited; however, lethal infections can occur in immunocompro- ogy, Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02482; xDepartment of Pathology mised individuals, pregnant women, and neonates. Upon inges- and Program in Immunology and Virology, University of Massachusetts Medical tion, invade the intestinal epithelium, enter the draining School, Worcester, MA 01655; {Gladstone Institute of Cardiovascular Disease, Uni- versity of California, San Francisco, San Francisco CA 94158; and ‖Department of , and disseminate via the bloodstream to the liver and Cancer Research and Molecular Medicine, Centre of Molecular Inflammation Re- spleen (15). This rapid clearance of bacteria from the peripheral search, Norwegian University of Science and Technology, 7489 Trondheim, Norway blood is generally attributed to the resident liver macrophages— Received for publication July 11, 2013. Accepted for publication December 11, 2013. the Kupffer cells, which line the liver sinusoids—and to the This work was supported by the National Institutes of Health (Grants AI067497 to hepatocytes. Within the first 24 h, a coordinated interaction between K.A.F., R37AI040618 to A.D.L., and AI060991 to V.L.B.) and by Italy–USA Pro- neutrophils, g/d T cells, , and NK cells is instrumental in gram Grant 28C6 (to M.S.). preventing the bacterium from spreading and ensuring the survival The sequences presented in this article have been submitted to Omnibus (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE53145) under ac- of the host. L. monocytogenes is an excellent model system to cession number GSE53145. evaluate the role of innate immunity in antimicrobial defenses. Address correspondence and reprint requests to Dr. Katherine A. Fitzgerald, Univer- Several classes of germline-encoded pattern recognition receptors sity of Massachusetts Medical School, Division of Infectious Diseases and Immu- (PRRs) have been implicated in the recognition of L. mono- nology, LRB Room 308, Department of Medicine, 364 Plantation Street, Worcester, MA 01605. E-mail address: kate.fi[email protected] cytogenes (14, 16). TLR-2 plays a key role in bacterial recognition The online version of this article contains supplemental material. at the cell surface (17–19). Upon escape of intracellular bacteria into the cytosol, L. monocytogenes engages nucleotide-binding olig- Abbreviations used in this article: BLIMP1, B lymphocyte–induced maturation pro- tein 1; BMDM, –derived ; ChIP, chromatin immunoprecip- omerization domain (NOD) 2, members of the NACHT-, LRR- and itation; CKO, conditional knockout; GO, ; hpi, h postinfection; IRF, pyrin-domain–containing protein family, as well as the cytosolic IFN regulatory factor; KO, knockout; LLO, listeriolysin O; MOI, multiplicity of infection; NOD, nucleotide-binding oligomerization domain; PEC, peritoneal exu- DNA sensor AIM2 (absent in melanoma 2), all of which play date cell; PRR, pattern recognition ; qPCR, quantitative real-time RT-PCR; a role in the recognition of intracellular L. monocytogenes and RIP2, receptor-interacting - kinase 2; TSB, tryptic soy broth; unr, release of IL-1b through the activation of caspase-1 (20–23). In unrelated; WT, wild-type. addition, L. monocytogenes triggers type I IFN gene transcription via Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 STING (stimulator of IFN ) (24–28), TBK1 (TANK-binding www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301799 2292 BLIMP1-REGULATED CCL8 EXPRESSION IN HOST DEFENSE kinase 1), and IFN regulatory factor (IRF) 3 (29–31). Bacterial peritoneal cavities with 5 ml ice-cold PBS 3 d after i.p. injection with 3 ml DNA and the second messenger cyclic-di-AMP represent the L. thioglycollate (Remel, Lenexa, KS). Splenocytes were isolated by disrupt- monocytogenes products driving these responses (31–33). ing aseptically harvested spleens in RPMI 1640 10% FCS, using a 70-mm pore size mesh cell strainer (BD Biosciences) to obtain single-cell sus- In this study, we examined the role of BLIMP1 in regulating pensions. Whole-blood samples were obtained by cardiac puncture using the innate immune response using L. monocytogenes as a model 1-ml syringes containing EDTA and from tails in sterile 5-ml polypropylene system. We found that Blimp1 gene expression was rapidly in- tubes containing PBS with 10 mM EDTA. Lysis of RBCs was performed duced by this bacterium in macrophages as well as by a range of using RBC lysis buffer (Sigma-Aldrich, St. Louis, MO). Neutrophils were isolated from the peritoneal cavity (4 h after thioglycollate injection) or viral and bacterial pathogens. Because BLIMP1-deficient mice from peripheral blood by negative selection (Miltenyi Biotec). are embryonic lethal (6), we evaluated the role of BLIMP1 in in- nate immune defenses, using a myeloid-specific Blimp1 conditional Isolation and of bone marrow neutrophils and gd knockout (CKO) mouse. Blimp1 CKO mice were less susceptible T cells to L. monocytogenes infection than were littermate controls. We Freshly harvested bone marrow leukocytes from C57BL/6 mice were first focused this study on the role of BLIMP1 in macrophages. Blimp1 stained with PE-conjugated Ly6G Ab (BD Biosciences), and neutrophils deletion in macrophages had no effect on type I IFN gene tran- were then immunomagnetically isolated using EasySep PE selection kits (Stem Cell Technologies). Purity was assessed by flow cytometry scription but did affect transcription of several other genes, includ- (FACSCalibur). Pooled gd T cells were purified from splenocytes of age- ing the murine chemokine (C-C motif) ligand 8, CCL8, also called matched WT C57BL/6 and CCR82/2 mice. Splenocytes were labeled with MCP2. Chromatin immunoprecipitation (ChIP) assays demonstrated PE-conjugated Hamster Anti-Mouse gd TCR Ab (UC7-13D5; BD Bio- that Ccl8 was a direct BLIMP1 target gene. Increased levels of sciences) after preincubation with Anti-Mouse CD16/CD32 (BD Pharmingen), CCL8 led to higher numbers of circulating neutrophils in the and then magnetically purified with anti–PE-Ab (Miltenyi Biotec)–coated

microbeads. Isolated gd T cells were rested in 0.5% FCS RPMI overnight Downloaded from peripheral blood, promoting a more rapid and robust antibacterial prior to assays. Neutrophils (1 3 105 per well) and gd T cells (2.5 3 104 response, resulting in a better clearance of the pathogen. Mobili- per well) in RPMI 1640 containing BSA were respectively placed on the zation of neutrophils was mediated by CCL8-induced recruitment top of a 3-mm and a 5-mm pore size 96-well ChemoTx chemotaxis ap- of IL-17F–producing g/d T cells. Mice lacking Ccl8 were also paratus (Neuroprobe) in chemotaxis assays. Neutrophils and gd Tcellswere incubated for 1 h and 2 h, respectively, at 37˚C. In the assays, all more sensitive to L. monocytogenes infection than were wild-type nonmigrated cells were manually removed from the upper side of the filter, (WT) mice. whereas the migrated cells remained adherent to the lower side of the filter. Our study identifies a new regulatory mechanism in macro- The filters were stained with Hema 3 (Fisher Scientific) and mounted on http://www.jimmunol.org/ phages mediated by BLIMP1 and CCL8 expression in early host glass slides, and cell counts were determined by manual counting using a defenses against intracellular bacteria. BLIMP1 likely acts as a light microscope. In the gd assays, cell counts were determined in the lower wells by manual counting using an inverted microscope. gatekeeper of initial inflammatory responses following infection were placed in the lower wells of the chemotaxis apparatus in triplicate in all to prevent detrimental effects associated with excess inflammation assays. The number of cells migrating at each concentration of chemokine at the level of target tissues. was normalized to the number of cells migrating in the presence of media alone (chemokinesis) to calculate the chemotactic index. Materials and Methods Flow cytometry analysis Reagents and bugs The differentiation state of BMDMs and peritoneal macrophages was con- by guest on October 1, 2021 L. monocytogenes (clinical isolate 10403s) and its derivative mutant lis- firmed by staining for F4/80 and CD11b (BD Pharmingen, San Jose, CA), teriolysin O (LLO), Staphylococcus aureus (ATCC 25923), and Esche- using mAbs as direct conjugates and their isotype controls. Splenocytes and richia coli (ATCC 27325 [W3110]) were used at multiplicity of infection peripheral blood cells were stained with the indicated combination of the (MOI) 5. Sendai (Cantrell strain) was from Charles River Laboratories following fluorochrome-conjugated mAbs: CD19-PerCP, B220-APC, (Boston, MA) and was used at 300 hemagglutinating units per milliliter. CD11b-PerCP, F4/80-PE, Ly6G-PE, Ly6C-FITC, CD11c-FITC, CD4-APC, 3 5 Recombinant IL-1b, murine CCL8, CCL2, and CXCL2 were from Pepro- and CD8-PB (BD Pharmingen). Viable cells (2 10 ) in the lymphocyte tech (Rocky Hill, NJ). IL-1b was used at 100 ng/ml. The IL-1R antagonist gate, as defined according to side and forward scatters, were analyzed. Flow Kineret (or anakinra; Amgen) was used at 150ml/ml, a dose that inhibits cytometry was performed using an LSR II instrument (BD Biosciences), 100% of the IL-1 activity when used at 100 ng/ml. The inhibitors SB203580 and the results were analyzed using FlowJo software (TreeStar). and MG132, both used at 40 mM, were from Calbiochem (San Diego, CA). ELISA Mice Serum samples derived from naive and L. monocytogenes–infected WT or Prdm1flox/flox mice were generously provided by K. Calame (Columbia Blimp1 CKO mice and supernatants derived from cultures of untreated or University, New York, NY) (3) and were crossed with LysMCre mice (The L. monocytogenes–treated BMDMs were assayed for CCL8 levels, using Jackson Laboratory, Bar Harbor, ME) to generate Prdm1flox/floxLysMCre2 a specific ELISA kit (ANTIGENIX AMERICA) according to the manu- and Prdm1flox/floxLysMCre+ mice. C57BL/6 mice and IL-1R2/2 were facturer‘s instructions. 2/2 2/2 purchased from The Jackson Laboratory. TLR2 and MyD88 mice In vivo infections were obtained from S. Akira (Osaka University, Osaka, Japan). IRF32/2 mice were from T. Taniguchi, (University of Tokyo, Tokyo, Japan). Pretitered tryptic soy broth (TSB)–glycerol stocks of L. monocytogenes Receptor-interacting serine-threonine kinase 2 (RIP2)2/2 mice were from strain 10403S were stored at 280˚C. Prior to infection, 1-ml bacterial Genentech (San Francisco, CA). Ccl8/Ccl12 and Ccl12 knockout (KO) aliquots were recovered for 1 h at 37˚C in 9 ml TSB (BD Biosciences), were obtained from I. Charo (Gladstone Institute of Cardiovascular Disease, washed, and resuspended to the desired CFU in PBS. Mice were injected University of California, San Francisco, CA). In all experiments, 7- to 8-wk- i.v. with 3 3 104 CFU L. monocytogenes (strain 10403S) in 0.3 ml PBS. At old animals were used. All mouse strains were bred and maintained under defined time points, infected animals were killed by CO2 asphyxiation. specific pathogen–free conditions in the animal facilities at the University of Livers and spleens of infected animals were aseptically harvested, weighed, Massachusetts Medical School. All experiments involving live animals were and homogenized in 0.02% Triton X-100. Prior to homogenization, samples carried out in accordance with the guidelines set forth by the University of of infected livers were taken for slide preparation and staining with H&E or Massachusetts Medical School, Department of Animal Medicine and the Gram dye. Aliquots of serial 5-fold dilutions in sterile water were plated in Institutional Animal Care and Use Committee. g/d T cell–deficient mice duplicate on TSB agar (BD Biosciences) plates containing 10 mg/ml were obtained from The Jackson Laboratory, and CCR82/2 mice were from streptomycin. After overnight incubation, the number of bacteria per gram S. Lira (Mount Sinai School of Medicine, New York, NY). of tissue was determined by counting colonies at the appropriate dilution. Cell isolation Microarray hybridization and analysis Bone marrow–derived macrophages (BMDMs) were generated as described RNA (5 mg) was isolated from WT and Blimp1 CKO BMDM untreated (34). Peritoneal macrophages were obtained from mice by washing their or infected for 2 h with L. monocytogenes (MOI = 5). cDNA and cRNA The Journal of Immunology 2293 labeling and hybridization were performed by the Genomic Core Facility Columbia University, New York, NY) (41). qPCR was performed on (University of Massachusetts Medical School, Worcester, MA), using kits immunoprecipitated and input fractions from the immunoprecipitation. and GeneChip Scanner 3000 by Affymetrix (Santa Clara, CA). The gene The following set of primers was used to amplify the different regions chip mouse genome 430 2.0 Array (Affymetrix) was used. CEL files were of the Ccl8 promoter, as indicated: normalized in Bioconductor (35), using the GCRMA method (36). Ana- lyses were restricted to samples for which $ 25% of the fluorescence units Unrelated (unr) for 59-CATGTGCAAGGTTCTGGCTA-39, were . log2100 and the interquartile range was $ 0.5 on the log base 2 unr rev 59-GGCCTAAGTCTGGTCCCTTT-39; scale. Differentially regulated genes were identified using a moderated t +141 for 59-ACGCTAGCCTTCACTCCAAA-39, test (37). False discovery rate–adjusted p values were calculated using the 9 9 method of Benjamini and Hochberg (38). Gene ontology (GO) enrichment +141 rev 5 -GTGCCTGCTAAACCATTTCC-3 ; analysis was performed using the GOstats package (39). The data dis- 2288 for 59-GGGACCAGACTTAGGCCTTT-39, cussed in this publication have been deposited in the National Center for 2288 rev 59-GAAGGGCAAGCATCTGAAAC-39; Biotechnology Information’s Gene Expression Omnibus and are accessible 285 for 59-CTGATCCAGTGGGACTGCTT9, through GEO Series accession number GSE53145 at http://www.ncbi.nlm. 285 rev 59-GAAGGCTAGCGTCCTCTCAA-39. nih.gov/geo/query/acc.cgi?acc=GSE53145. RNA isolation and quantitative real-time PCR Transwell chemotaxis and intracellular staining of g/d DNase I–treated total RNA from BMDMs and peritoneal exudate cells T cells (PECs) was extracted with the RNeasy kit (QIAGEN, Valencia, CA) according to the manufacturer’s instructions. cDNA was synthesized, g/d T cells were first isolated, as described above, from pooled lymph and quantitative real-time RT-PCR (qPCR) analysis was performed on a nodes and spleens of 10–20 WT C57BL/6 mice and rested overnight in DNA engine Opticon 2 cycler (MJ Research, Watertown, MA), using the 0.5% FCS RPMI 1640 supplemented with HEPES, L-glutamate, nones- 6 SuperScript III Two-Step qRT-PCR Kit with SYBR Green (Invitrogen, sential amino acids, and Na pyruvate. The next day, g/dT cells (5 3 10 Downloaded from Carlsbad, CA). The specificity of amplification was assessed for each per milliliter in 100 ml) were harvested and placed in the upper chamber of sample by melting curve analysis. Relative quantification was performed Transwells (5-mm pore size polycarbonate membrane, 12-mm diameter) using standard curve analysis. The quantification data are presented as (Costar, Fisher) and 600 ml chemokine or media was placed in the lower a ratio to the b-actin level. The SEs (95% confidence limits) were calcu- chamber. Migration was allowed to proceed for 2–3 h at 37˚C and was lated using the Student t test. All gene expression data are presented as monitored after 2 h with an inverted microscope and compared with media a ratio of gene copy number per 100 copies of b-actin 6 SD. The fol- controls to determine the completion of the assay. A fraction of input total lowing primer pairs have been used: spleen and lymph node g/d T cells were set aside and simultaneously in-

cubated at 37˚C during the course of the assay. At the end of the assay, http://www.jimmunol.org/ b-Actin for 59-TTGAACATGGCATTGTTACCAA-39, total input g/d T cells and CCL8- or CCL2-responsive migrated g/d T cells b-Actin rev 59-TGGCATAGAGGTCTTTACGGA-39; in the Transwells were harvested for intracellular staining and activated with 50 ng/ml PMA (Sigma-Aldrich) and 500 ng/ml ionomycin (Sigma- BLIMP1 for 59-GAGGATCTGACCCGA ATCAA-39, Aldrich) for 2 h in the presence of brefeldin A (BD Biosciences). At the BLIMP1 rev 59-GTTGCTGTGAGGCAACTTCA-39; end of the activation, cells were fixed and permeabilized with Fix & Perm CCL8 for 59-TAAGGCTCCAGTCACCTGCT-39, Medium A & B (Invitrogen), and then stained with anti–IL-17A Alexa CCL8 rev 59-TTCCAGCTTTGGCTGTCTCT-39; Fluor 647 (TC11-18H10.1; BioLegend) and anti–IL-17F Alexa Fluor 488 (eBio18F10; eBiosciences). Cells were acquired on a Becton-Dickinson Atm for 59-CCAGGCTTGAAGGACTGAAG-39, FACSCalibur and analyzed with FlowJo 8.8 software. Atm rev 59-TCACCACAATGCCATCTTGT-39;

Mpa2l for 59-TGGAGCAGCTGCATTATGTC-39, Statistical analysis by guest on October 1, 2021 Mpa2l rev 59-GCATTCTGGGTTTGTCACCT-39; Statistical analysis was calculated using a two-tailed Student t test, if not Arhgap25 for 59-ATGTCTGGGCTAAGGTGGTG-39, otherwise stated. A p value # 0.05 was considered statistically significant. Arhgap25 rev 59-ACCCTCAAGGATCCGTCTTT-39; Arhgap26 for 59-AAGACCATCAGCCCGTACAC-39, Results Arhgap26 rev 59-CTGGCCTGTCTACTGCTTCC-39; Regulation of Blimp1 gene expression following Pldn for 59-GGACTTCCCTGTGGACGATA-39, L. monocytogenes infection Pldn rev 59-AGCTTGGCGTGATAGTGCTT-39; While studying immune response genes in murine BMDMs infected Rasgrp1 for 5 9-TGTCACAGCTCCATCTCCAG-39, with bacterial and viral pathogens, we found that expression of the Rasgrp1 rev 59-TTCACCTTCCCATCTTCCAG-39; Prdm1 gene (which encodes BLIMP1) was markedly increased Tm7sf4 for 59-CCAAGGAGTCGTCCATGATT-39, upon infection with a variety of pathogens, including L. mono- Tm7sf4 rev 59-GGCTGCTTTGATCGTTTCTC-39; cytogenes, E. coli, S. aureus, the paramyxovirus Sendai, and the Tmem87a for 59-AATGTGGTGCACACTCCAAA-39, Gram (2)–derived LPS. In all cases, the induction was strikingly Tmem87a rev 59-AGGGCGAATAATCACAGCAC-39. rapid and transient, with expression peaking 2 h postinfection (hpi) (Fig. 1A). We also extended this time course to later time Cell extracts points, which revealed a second wave of inducible BLIMP1 ex- BMDMs were infected with L. monocytogenes 10403s at MOI 5. After 1 h pression at the 24-h time points (Supplemental Fig. 1A). We focused of infection, cells were washed once with 10% FCS/DMEM and then on L. monocytogenes as a model pathogen to understand both the treated with 10 mg/ml gentamicin to kill extracellular bacteria. Bone marrow molecular basis for BLIMP1 induction and the role of BLIMP1 in cells were then left for another 2 h, and nuclear lysates were prepared as antimicrobial defenses. L. monocytogenes is readily phagocytosed previously described (34). by macrophages, and once inside a host vacuole, the essential vir- Western blot analysis ulence factor LLO, a pore-forming toxin encoded by the hemolysin Nuclear cell extracts were separated by 7.5% SDS-PAGE gel and blotted (hly) gene, enables the bacterium to permeabilize the vacuolar onto nitrocellulose membranes. Blots were incubated with mouse mAbs membrane and enter the cytosol (42). Several classes of PRRs against murine BLIMP1 (Novus Biological) or b-actin (Sigma-Aldrich), as can sense L. monocytogenes–derived products at different stages indicated, and detected using an ECL system (Amersham, Piscataway, NJ). of infection. These include TLR2 (17–19, 43), NOD2 (20), NLRP3 ChIP assay (21), AIM2 (22, 23), STING, well as an as yet unidentified DNA ChIP experiments were performed essentially as described (40). Briefly, ∼ 4 3 sensor that turns on IFN-b gene transcription (28, 31). To under- 107 BMDMs were used to perform immunoprecipitation with rabbit poly- stand how Blimp1 gene induction was regulated, we monitored clonal recognizing the C terminus of BLIMP1 (a kind gift of K.L. Calame, L. monocytogenes–induced Blimp1 transcript levels in macrophages 2294 BLIMP1-REGULATED CCL8 EXPRESSION IN HOST DEFENSE Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. Blimp1 is rapidly induced by L. monocytogenes via TLR2 and cytosolic sensors. (A) RNA was isolated from BMDMs left untreated (un) or infected with L. monocytogenes (LM), E. coli, S. aureus (MOI = 5), or Sendai virus (SV) (300 hemagglutinating units) or treated with LPS (100 ng/ml) for the indicated times, and Blimp1 gene expression was quantified by qPCR. Data were normalized with b-actin and are representative of three independent experiments. (B) BMDMs from C57BL/6, IRF32/2, RIP22/2, TLR22/2, and IL1R2/2 mice were stimulated with L. monocytogenes for 2 h. mRNA expression for Blimp1 was measured by qPCR. One of three different experiments conducted singularly is shown. (C) RNA was isolated from L. mono- cytogenes–infected or IL-1b–treated BMDMs from C57BL/6, TLR22/2, IL1R2/2, or MyD882/2 mice or from TLR22/2 preincubated with IL-1R an- tagonist (IL1RA, 150 mg/ml). Blimp1 gene expression was quantified by real-time RT-PCR. Results are mean 6 SD of triplicate values and are representative of at least four experiments. (D) Lysates were prepared from BMDMs treated for 3 h with L. monocytogenes (MOI = 5) either before or after pretreatment (30 min) with an inhibitor for p38 MAPK (SB203580, 40 mM) or an inhibitor for NF-kB (MG132, 40 mM). BLIMP1 and b-actin expression was detected by Western blotting using the respective specific Abs. (E) Blimp1 gene expression was evaluated by real-time RT-PCR in RNA derived from C57BL/6, TLR22/2, and MyD882/2 macrophages and treated 2 h with MOI = 5 of either the WT or the LLO mutant L. monocytogenes strains. derived from mice with targeted deletions in components of these Indeed, L. monocytogenes infection of BMDMs lacking MyD88, signaling pathways. These included macrophages deficient in a key adaptor downstream of both TLR2 and IL-1R intracellular TLR2; in RIP2, the downstream effector of NOD1/2 signaling; in cascades, fails to induce Blimp1 expression. Furthermore, inhibi- IL-1R chain 1, which fails to signal in response to IL-1b released tion of p38 MAPK and NF-kB signaling using the respective spe- upon the activation of caspase-1; and finally in IRF3, the key cific inhibitors SB203580 or MG132 prevented L. monocytogenes– transducer of the cytosolic DNA sensing pathway. Induction of induced expression of BLIMP1 protein in macrophages (Fig. 1D). Blimp1 gene transcription upon L. monocytogenes infection was These data suggest that cell surface TLR2 and cytosolic PRRs, induced normally in macrophages lacking RIP2 and IRF3 but was which regulate IL-1b production, cooperate in control of Blimp1 reduced in both TLR22/2 and IL1R2/2 macrophages (Fig. 1B). transcription. In support of this idea, an LLO-deficient strain of Moreover, this response was completely lost in TLR22/2 mac- L. monocytogenes, which is not capable of entering the cytosol of rophages pretreated with the IL-1R antagonist, showing that for the host cell and, as a consequence, is not able to trigger IL-1b or a full induction of Blimp1 transcription, both these signaling type I IFN production, was less potent than WT bacteria in inducing pathways have to be activated by L. monocytogenes (Fig. 1C). Blimp1 transcription. This response to LLO-deficient bacteria was The Journal of Immunology 2295 entirely dependent on TLR2 because macrophages lacking TLR2 monocytic cell line suggested that BLIMP1 was important in the failed to upregulate Blimp1 (Fig. 1E). differentiation of myeloid cells (45). Flow cytometry analysis revealed normal numbers of macrophages, neutrophils, and den- Generation of a myeloid-specific Blimp1 CKO mouse dritic cells in the spleen of Blimp1 CKO mice (Fig. 3A). Similar Direct targeted disruption of the gene encoding BLIMP1 (Prdm1) numbers of elicited PECs were also obtained from WT and Blimp1 is lethal for mouse embryos (6). Therefore, to define the role of CKO mice after thioglycollate injection, and expression of CD11b this protein in the host response to microbial challenge, we gen- and F4/80 on these cells was equivalent between WT and CKO erated a myeloid-specific Blimp1 CKO mouse via lysozyme M-driven mice (data not shown). We also examined the differentiation of flox/flox Cre-mediated recombination (44). Prdm1 M-lysozyme Cre- Blimp1 CKO macrophages ex vivo from bone marrow. BMDMs flox/flox negative (6) and the Prdm1 M-lysozyme Cre-positive mice from WT and Blimp1 CKOs expressed similar levels of CD11b (hereafter referred to as WT and Blimp1 CKO mice, respectively) and F4/80 (Fig. 3B) and induced equivalent levels of NO synthase were developmentally normal and fertile. Efficient deletion of the in response to L. monocytogenes or after treatment with LPS (Fig. floxed Prdm1 sequence in the Blimp1 CKO macrophages was 3C). Moreover, L. monocytogenes grew at a comparable rate in confirmed by PCR analysis of BMDM genomic DNA (Fig. 2A, WT and Blimp1 CKO BMDMs (Fig. 3D). Collectively, these data 2B). Both the mRNA and protein were undetectable in BMDMs demonstrate that BLIMP1 is not required for the development or from Blimp1 CKO mice either before or after infection with normal functions of macrophages. L. monocytogenes (Fig. 2C, 2D, respectively). Similar results wereobtainedwhenelicitedPECsfromWTandBlimp1 CKO mice Blimp1 deficiency in myeloid cells protects mice from were examined ex vivo for the mRNA expression of the Blimp1 L. monocytogenes infection gene (Fig. 2E). M-lysozyme is expressed in both macrophages and To examine the role of BLIMP1 in innate immunity, we performed Downloaded from granulocytes, so we measured BLIMP-1 in both of these cell types. in vivo infection experiments using L. monocytogenes. We first Although BLIMP-1 was expressed in macrophages and dendritic monitored bacterial numbers in the spleens and livers 48 hpi (3 3 cells, its expression was very low by comparison in neutrophils 104 CFU delivered i.v.). Both organs in the Blimp1 CKO mice had (Supplemental Fig. 1B). We did observe efficient deletion of the lower bacterial loads than did WT littermate controls (Fig. 4A). floxed Prdm1 DNA sequence by PCR in neutrophils isolated from Whereas the bacterial load in both organs was comparable be- the peritoneal cavity of Blimp1 CKO mice (Supplemental Fig. 1C). tween WT and Blimp1 CKO mice 20 hpi, it progressively in- http://www.jimmunol.org/ Although BLIMP-1 was poorly expressed in resting neutrophils, its creased in WT, but not in Blimp1 CKO, spleens and livers at the expression was, however, inducible upon L. monocytogenes infec- later time points examined (Supplemental Fig. 1E). Furthermore, tion (Supplemental Fig. 1D). Blimp1 CKO mice had a greater rate of survival and quickly re- Because BLIMP1 is a key factor controlling the differentiation of covered from a dose of L. monocytogenes delivered i.v. that was many different cell types, we examined immune cell development lethal to most control mice (Supplemental Fig. 1F). Importantly, in Blimp1 CKO mice. As expected, no significant differences in we found no differences in bacterial load in liver or spleen when the numbers of B cells or T cells were observed when BLIMP1 C57BL/6 WT mice and C57BL/6 mice expressing M-lysozyme was deleted only in myeloid cells (Fig. 3A). Previous studies using Cre only were examined (Supplemental Fig. 1G), suggesting that overexpression of a truncated version of BLIMP1 in a human the Cre recombinase had no adverse effect in this system. by guest on October 1, 2021

FIGURE 2. Generation of prdm1flox/flox M-lysozime Cre+ mice. (A) Schematic representation of the locus targeted by M-lysozyme Cre within the Prdm1 gene, with indicated primer sites used for screening the de- letion (1–3). (B) Genomic DNA was isolated from BMDMs from C57BL/6, Prdm1flox/floxCre2 (WT), or Prdm1flox/floxCre+ (CKO) mice. PCR analysis for the Cre-targeted region on the Prdm1 gene was performed using a mix of primer 1, 2, and 3 on genomic DNA. Amplicon sizes are shown. (C–E) Lysozyme M-driven Cre-mediated deletion of Blimp1 was confirmed in untreated or L. monocytogenes–treated BMDMs from WT or Blimp1 CKO mice at the mRNA level by qPCR (C), at the protein level by Western blotting (D), or at the steady-state level in PECs from WT or Blimp1 CKO mice by qPCR (E). The p values were calculated by the two-tailed Student t test. *p , 0.001. 2296 BLIMP1-REGULATED CCL8 EXPRESSION IN HOST DEFENSE

FIGURE 3. Blimp1 deficiency does not influence normal macrophage function. (A) Flow cytometric anal- ysis of splenocytes in Blimp1 CKO mice. Single-cell suspensions were prepared from the spleens of 8-wk- old mice and stained as indicated. Viable cells (2 3 105) in the lym- phocyte gate, as defined according to side and forward scatters, were ana- lyzed. The numbers represent the percentage of cells contained in each gated region. (B) FACS analysis of CD11b and F4/80 expression in WT

or Blimp1 CKO BMDMs. A repre- Downloaded from sentative experiment is shown. The analysis was repeated four additional times. (C) Inducible NO synthase expression was evaluated by real- time RT-PCR in WT or Blimp1 CKO BMDMs treated with L. monocyto- genes (MOI = 5) or LPS (100 ng/ml) http://www.jimmunol.org/ for 4 h. (D) L. monocytogenes in- tracellular growth in WT or Blimp1 CKO BMDMs 1, 2, and 8 hpi. The average 6 SD of CFU counts obtained from BMDMs derived from six mice of each genotype is reported in the graph. BMDM lysate dilutions were plated overnight in duplicate on TSB agar plates containing streptomycin by guest on October 1, 2021 before counting. Reported values rep- resent means of CFU counts obtained in each condition 6 SD.

Histological analysis of liver sections revealed microabscesses supports this idea, because released from neutrophils are containing neutrophils and macrophages by 48 hpi and a further thought to control this process. increase in the number of these lesions by 72 hpi in WT animals (Fig. 4B [H&E staining], 4C), in line with bacterial counting The chemokine CCL8 is a target of BLIMP1-mediated described in Supplemental Fig. 1E. In contrast, very few micro- transcriptional repression abscesses were present in the Blimp1 CKO livers. The lesions in To elucidate the molecular mechanisms responsible for the height- the Blimp1 CKO mice, although present at higher numbers 20 hpi, ened clearance of L. monocytogenes in Blimp1 CKO mice, we did not increase in number during the course of the infection (Fig. focused on macrophages and compared the gene expression profile 4B [H&E staining], 4C). Notably, the lesions in Blimp1 CKO of WT and Blimp1-deficient macrophages before and after infec- livers were morphologically distinct from those found in the WT tion with L. monocytogenes. Supplemental Fig. 2A and Supple- mice. Neutrophils were present at what was presumably the site of mental Table I show genes with significantly different expression infection, surrounded by a zone rich in the ghostlike remnants of levels (p # 0.05) between WT and Blimp1 CKO BMDMs for either dead hepatocytes, characteristic of coagulative necrosis, a patho- untreated or infected cells, as evaluated by microarray analysis. The logical finding that results from a highly robust neutrophil response. set of genes was significantly enriched in a number of GO terms Gram staining of liver sections revealed large numbers of bacteria in (Supplemental Table II) related to macrophage activation and the microabscesses of WT livers but uncovered no bacteria in the . Comparison of transcriptomes of untreated WT Blimp1 CKO lesions (Fig. 4B, right panels [Gram staining]). These with untreated CKO macrophages identified a small number of observations suggested that there was a more robust and rapid genes that had significantly different expression levels. Impor- clearance of bacteria from the livers of Blimp1 CKO mice. The tantly, these observations revealed that deletion of Blimp1 had presence of hepatocytes in a state of coagulative necrosis also surprisingly little effect on global gene expression in macrophages The Journal of Immunology 2297 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021 FIGURE 4. Myeloid-specific Blimp1 CKO mice are protected from L. monocytogenes infection. (A)WTorBlimp1 CKO mice were infected with 3 3 104 CFU of L. monocytogenes i.v., and bacterial loads were quantified in spleens and livers 48 hpi. Results from three different experiments are expressed as mean 6 SEM. *p , 0.001 (two-tailed Student t test). (B) H&E staining (original magnification 310 and 340) and Gram staining (original magnification 3100) of WT or CKO livers 20, 48, and 72 hpi by L. monocytogenes. The experiment shown is representative of three experiments conducted separately. (C) Numbers of microabscesses per 20 fields of three different experiments were quantified and expressed as mean 6 SEM. *p , 0.05 (two-tailed Student t test). but rather identified a specific and restricted subset of genes that observed when an unr sequence at position 2618 of the Ccl8 were differentially expressed in macrophages in the absence of promoter was examined (Fig. 5D, upper and lower panels). These this factor. One gene in particular, Ccl8, encoding the chemokine data indicate that BLIMP1 directly binds to the Ccl8 promoter belonging to the C-C family, was expressed ∼ 17 times higher in in vivo. Blimp1-deficient than in WT macrophages, as measured by qPCR (Fig. 5A). The expression profile of seven additional genes was Critical role of CCL8 in antibacterial resistance to also confirmed by qPCR (Supplemental Fig. 2B). Ccl8 transcrip- L. monocytogenes tion levels were also significantly elevated in macrophages iso- Neutrophils play a key role in the early control of L. mono- lated from the peritoneum (PECs) of Blimp1 CKO mice in the cytogenes growth, appearing in the liver within the first 24–36 hpi absence of infection, compared with the levels found in WT mice (15). First generated in the bone marrow, neutrophils are elicited (Fig. 5B). Similarly, CCL8 protein levels were higher in the cul- into the peripheral blood and attracted to the liver, where they ture medium of untreated BMDMs and uninfected sera from contribute to antibacterial resistance by killing bacteria, lysing Blimp1 CKO mice, compared with WT littermate controls (Fig. 5C). infected host cells, stimulating antimicrobial activity of infected Sequence analysis of a genomic region spanning 1000 bp upstream macrophages, and inducing hepatocyte apoptosis (47). We spec- and 500 bp downstream of the Ccl8 transcription start site using ulated that the elevated expression of CCL8 in Blimp1 CKO mice the software MatInspector (http://www.genomatix.de/matinspector. could encourage egress of neutrophils from the bone marrow and html) (46) identified three BLIMP1 consensus binding sites, at promote their infiltration into the livers of L. monocytogenes– positions 2288, 285, and +141 (Fig. 5D, upper panel). ChIP anal- infected mice. Consistent with this hypothesis, both the bone ysis using an Ab to BLIMP1 (41) in macrophages derived from marrow and peripheral blood of uninfected Blimp1 CKO mice C57BL/6 mice revealed weak binding of BLIMP1 to both the 285 showed a higher percentage of CD11b+/Ly6G+ neutrophils than and +141 sites and a very strong binding to the 2288 site (Fig. 5D, did WT mice, whereas the percentage of CD11bint/Ly6Chi in- lower panel). Similar results were obtained when ChIP was per- flammatory monocytes remained constant in both compartments formed on infected BMDMs (data not shown). No binding was (Fig. 6A, 6B). Accordingly, in infected spleens of Blimp1 CKO 2298 BLIMP1-REGULATED CCL8 EXPRESSION IN HOST DEFENSE Downloaded from http://www.jimmunol.org/

FIGURE 5. The Ccl8 gene is a direct target of BLIMP1-mediated transcriptional silencing. (A) Ccl8 expression was examined by qPCR in RNA isolated from untreated BMDMs from WT or Blimp1 CKO mice or those treated for 2 h with L. monocytogenes. Data were normalized with b-actin and expressed as means 6 SD. Data shown are representative of three independent experiments. *p = 0.001 (two-tailed Student t test). (B) Gene expression of Ccl8 was evaluated also in PECs derived from WT or Blimp1 CKO mice in the absence of infection by real-time RT-PCR. *p = 0.001 (two-tailed Student t test). (C) by guest on October 1, 2021 The CCL8 protein level was analyzed by ELISA in both culture media and sera derived from uninfected and L. monocytogenes–treated WT or Blimp1 CKO mice. *p , 0.05 (two-tailed Student t test). (D) Upper panel, Schematic representation and relative position of conserved BLIMP1 binding sites on the murine Ccl8 promoter and of an unr sequence. Lower panel, ChIP analysis of BMDMs using an Ab to BLIMP1 or preimmune serum is shown. Analysis of enrichment of selected sequences was performed by qPCR. The results are expressed as fold enrichment of signal in anti-BLIMP1 immunoprecipitates over that of preimmune serum. The graph shows one representative of two similar experiments. TSS, Transcription starting site. mice, a marked and statistically significant increase was observed in Blimp1 CKO mice: zones of hepatocytes in a state of coagu- in the percentage of neutrophils following L. monocytogenes in- lative necrosis surrounded infiltrating neutrophils (Fig. 6f, upper fection, compared with that observed in WT organs, whereas no panels). We also administered CCL2 (also called MCP1) i.v., a differences were observed for monocytes or macrophages (Fig. chemokine known to selectively induce migration of monocytes 6C). (47–49), and monitored the percentage in the blood of both CD11b+/ To test whether CCL8 would be responsible for this increased Ly6G+ neutrophils and CD11bint/Ly6Chi monocytes (Supplemental mobilization of neutrophils in Blimp1 CKO mice, we injected C57/ Fig. 2C). As expected, we found a significant increase in the Bl6 mice i.v. with two increasing doses of recombinant CCL8 for numbers, but no effect on the neutrophil population. 2dconsecutivelyandmonitoredCD11b+/Ly6G+ neutrophil To further define the role of CCL8 in antimicrobial resistance, numbers in the peripheral blood. The number of CD11b+/Ly6G+ we infected mice lacking Ccl8 with L. monocytogenes. Ccl8 single cells in mock-treated mice was very low, but this percentage KOs are not available; however, mice lacking Ccl8 and Ccl12 significantly increased in a dose-dependent manner with the ad- (also called MCP5) or mice lacking Ccl12 alone were studied ministration of CCL8 (Fig. 6D). Both CCL8- and mock-treated (48). Consistent with a role for CCL8 in the clearance of bacteria mice were then infected with L. monocytogenes, and bacterial during infection, the bacterial load was much higher in mice loads in the spleen and liver were examined 48 hpi. The bacterial lacking Ccl8/Ccl12 than in Ccl12 single KOs or C57BL/6 WT burden in both organs analyzed was much lower in the CCL8- mice (Fig. 7A). CCL8 was recently found to be highly expressed treated than in the mock-treated mice, and the numbers of bacteria in the , where it acts via the CCR8. CCL8 decreased proportionally with the increase of the chemokine dose responsiveness defined a population of highly differentiated, CCR8- (Fig. 6E). Histological analysis of liver sections by Gram staining expressing inflammatory Th2 cells enriched for IL-5 (50, 51). The confirmed these findings, revealing fewer bacteria-laden micro- ability of CCL8 to signal via CCR8 rather than CCR2 distinguishes abscesses in CCL8-treated mice than mock-treated animals (Fig. CCL8 from all other MCP chemokines. In an effort to understand 6F, lower panels). Most interestingly, lesions present in the livers the role of CCL8 in innate immune responses to L. monocytogenes, of CCL8-injected mice looked very similar to those we observed we first examined whether CCL8 was chemotactic for neutrophils The Journal of Immunology 2299 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 6. Role of CCL8 in antibacterial resistance and host defense against L. monocytogenes.(A and B) Bone marrow and peripheral blood were harvested from naive WT or Blimp1 CKO mice. Cells were stained for CD11b, Ly6G, or Ly6C, and their expression was evaluated in a large gate drawn in the live granulocyte/lymphocyte/monocyte population. Cytometric analysis of CD11b/Ly6G neutrophils (A) and CD11bint/Ly6Chi monocytes (B) from bone marrow (upper panels) or peripheral blood cells (lower panels) is shown. A representative experiment of three conducted separately is depicted. (C) CD11bint/Ly6Chi monocytes, CD11b+F480+ macrophages, and CD11b+Ly6G+ neutrophils derived from three WT or three CKO infected spleens were analyzed by FACS 20, 48, and 72 hpi. Mean percentages are shown. Error bars represent SEM. *p , 0.001 (two-tailed Student t test). (D) Percentages of CD11b/Ly6G-positive cells evaluated by FACS analysis in peripheral blood from WT mice mock treated or injected i.v. with 400 or 800 ng of recombinant CCL8 for 2 d consecutively are shown. Each bar represents an average of three to four mice from two independent experiments. Error bars represent SEM. *p = 0.012, **p , 0.001, as compared with mock controls (two-tailed Student t test). (E) Bacterial burdens from spleens and livers were calculated in mice mock treated or injected with 400 or 800 ng of recombinant CCL8 and then infected with L. monocytogenes i.v. (n = 3 mice per genotype). *p , 0.05 versus mock (two-tailed Student t test). (F) Representative H&E staining (original magnification 340) and Gram staining (original magnification 3100) of livers derived from mock-treated or CCL8-injected mice subsequently infected with L. monocytogenes i.v. Mice were sacrificed 48 hpi. 2300 BLIMP1-REGULATED CCL8 EXPRESSION IN HOST DEFENSE by monitoring the migration of neutrophils in response to this Genome-wide transcriptional profiling of WT and Blimp1-deficient chemokine using an in vitro chemotaxis assay. Neutrophils were macrophages identified the gene encoding the chemokine CCL8 as purified from bone marrow and tested for their ability to migrate differentially modulated in the presence or absence of BLIMP1. in response to CCL8 (Fig. 7B, 7C). We found that in contrast to Even in the absence of infection, Ccl8 was expressed at much MIP2 (also called CXCL2, an agonist of CXCR2 expressed on higher levels in Blimp1 CKO macrophages both at the transcrip- neutrophils), CCL8 was not chemotactic for neutrophils (52) (Fig. tional and at the protein levels. ChIP analysis indicated that 7B). Neutrophils can also be mobilized to the site of infection through BLIMP1 occupied the Ccl8 promoter in WT cells. Basal expres- IL-17–mediated events that stimulate granulopoiesis-inducing sion of BLIMP1 in unstimulated macrophages could bind ISRE and granulocyte-attracting chemokines (53). The major sites on target promoters and compete with ISGF3 or IRF tran- IL-17 producers at the early stage of Listeria infection in the liver scription factors for these sites, resulting in basal repression of these are g/d T cells (54), which also express CCR8 on their surface (55). genes. BLIMP1 binding sites present within the CCL8 promoter We therefore next investigated whether g/d T cells were the target bound by IRF factors, such as IRF1 or IRF7, would induce its of CCL8 action and found that CCL8 was chemotactic for these transcription. Because IRF1 and IRF7 are themselves regulated via cells (Fig. 7D, 7E). The migration of g/d T cells to CCL8 was ISGF3 binding sites, their transcription might also be held in check similar to that seen with CCL2, a known chemotactic factor for by BLIMP1. In the context of BLIMP1 CKO mice, therefore, these these cells (56). We next wanted to examine if the enhanced transcription factors are likely also expressed at elevated levels, clearance of bacteria in animals treated with exogenous CCL8 was which could lead to elevated CCL8 transcription, explaining this dependent on g/d T cells. In agreement with published studies, g/d high expression of CCL8 in uninfected BLIMP1 KO BMDMs. T cell–deficient mice had significantly higher bacterial loads in liver In an effort to link elevated CCL8 levels to the enhanced neu- andspleencomparedwiththeir WT counterparts following trophil response and heightened ability of Blimp1 CKO mice to Downloaded from L. monocytogenes infection (Fig. 7F). Although WT animals treated clear Listeria, we administered CCL8 in vivo to C57BL/6 mice with exogenous CCL8 cleared bacteria efficiently, bacterial loads in to mimic the elevated levels of the chemokine observed in the g/d T cell–deficient mice treated with CCL8 were unaltered. Blimp1 CKO mice. CCL8-treated C57BL/6 mice upon L. mono- Notably, the IL-17 cytokine family includes different members, cytogenes infection had significantly lower bacterial loads in the and three of them—IL-17A, IL-17F, and IL-17E (or IL-25)—have infected organs. The heightened clearance of this bacterium in re- been best characterized both in vivo and in vitro and have been sponse to CCL8 correlated with a higher percentage of neutrophils http://www.jimmunol.org/ shown to be proinflammatory in nature (57). In particular, whereas in the peripheral blood. Conversely, Ccl8 KO mice were consider- IL-17E induces Th2 cytokine production and , both ably more susceptible to L. monocytogenes infection, having higher IL-17A and IL-17F are involved in the recruitment of neutrophils. bacterial loads in both spleen and liver, than their WT counterparts. In models of mucoepithelial bacterial infections and autoimmu- On the basis of these observations, we hypothesized that under nity, IL-17A and IL-17F were both shown to play important roles normal circumstances, BLIMP1 acts to suppress the production in host defense but to have distinct activity in inflammatory pro- of CCL8 as a means to limit neutrophil responses and excess in- cesses and to be produced by different cell types (58). However, flammation, which can be deleterious to the host. We speculate that g/d T cells are able to produce both of these cytokines, amplifying the elevated expression of CCL8 in Blimp1 CKO mice could either by guest on October 1, 2021 Th17 responses (59). Hence, we evaluated in our model which directly or indirectly promote neutrophils to egress from the bone type of IL-17 was produced by CCL8-attracted g/d T cells. To this marrow and infiltrate the livers of L. monocytogenes–infected end, we first compared by intracellular staining IL-17A and IL- mice. Our study, however, demonstrates that neutrophils do not 17F expression in g/d T cells that migrate in response to CCL8 directly migrate in response to CCL8. Rather, we propose an in- after Transwell migration assays, compared with the total input direct mechanism involving CCL8 in neutrophil mobilization via population of g/d T cells isolated from WT spleen and lymph an IL-17–mediated feedback loop. The IL-17 cytokine family nodes (Fig. 8A, 8B). Our experimental setting shows that CCL8- comprises six isoforms, with IL-17A and its closest homolog IL- responding g/d T cells are the main producers of IL-17F, even if 17F best characterized in vivo and in vitro (57). Cellular sources of IL-17A was also expressed, in contrast to what was observed for IL-17 are mainly the innate immune cells a/b, g/d,andNKTcells g/d T cells responding to CCL2, which only weakly induced IL- (62), and the primary function of this cytokine is to curb infection 17F. We conclude from these studies, therefore, that the enhanced by bacterial and fungal pathogens (63, 64). Both IL-17A and IL-17F antibacterial effects associated with CCL8 are dependent on IL- play a particularly significant role in regulating neutrophil recruit- 17F2producing g/d T cells. ment and granulopoiesis (65–67) by inducing expression of factor- CSF and IL-6. In addition, the most strongly induced IL-17 target Discussion genes are neutrophil-recruiting chemokines, such as CXCL1, The role of BLIMP1 as a transcriptional repressor in embryonic CXCL5, and CXCL2 (68–70). development (1–3), as well as in the differentiation of B and g/d T cells are specialized innate T cells that signal through a T lymphocytes, is well established (9, 10, 60, 61). In this study we TCR formed by TCR-g and -d-chains (71, 72). These cells are have identified a novel role for BLIMP1 in the regulation of early rapidly activated, producing IFN-g, TNF-a, and IL-17 in several host defenses in macrophages. Using myeloid-specific Blimp1 mouse models of bacterial infection, including those of Myco- CKO mice, we found that mice lacking Blimp1 in myeloid cells bacterium bovis, Salmonella enterica, and L. monocytogenes (54, had greater survival than their WT counterparts following infec- 73, 74). It has been also recently shown that the negative regu- tion with L. monocytogenes. Analysis of the Blimp1 CKO–infec- lation of g/d T cell–mediated IL-17 production by type I IFN ted organs revealed few or no bacteria in the liver and spleen of induced by primary influenza virus infection is of key importance these animals compared with WT mice. The livers of CKO ani- for the increased susceptibility to secondary Streptococcus pneu- mals had lesions with considerable coagulative necrosis, a patho- moniae superinfection (75). Evidence confirms how the IL-17–g/d logical finding that results from a highly robust neutrophil response. T cell axis plays a pivotal role in resistance to bacterial infection. Both the bone marrow and the peripheral blood of uninfected Blimp1 In particular, for L. monocytogenes it was reported that IL-17 is CKO mice had a higher percentage of CD11b+/Ly6G+ neutrophils expressed in the liver of infected mice from an early stage of in- than did that of WT mice. fection and that the major IL-17–producing cells were g/d Tcells The Journal of Immunology 2301 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 7. g/d T cells are the cellular target of CCL8 chemotactic activity. (A) L. monocytogenes in vivo infection was performed in six different Ccl8/ 12 double KO or six Ccl12 single KO mice. Six sex- and age-matched C57BL/6 mice were used as controls. Infected spleens and livers were harvested 48 hpi, and bacterial loads were calculated. Data represent means 6 SD. *p = 0.001 (two-tailed Student t test). (B) Neutrophils purified from the bone marrow of WT mice were assayed for migration to mCCL8 and the positive control mCXCL2 in a Neuroprobe chemotaxis chamber. (C) Purity of neutrophils determined by Ly6G staining. (D) g/d T cells isolated from WT and Ccr8 2/2 mouse spleens were assayed for migration to mCCL8 and the positive control mCCL2 in a Neuroprobe chemotaxis chamber. (E) Purity of WT and Ccr82/2 g/d T cells determined by g/d TcR staining. Data in (B) through (E) are representative of three independent experiments. (F) L. monocytogenes in vivo infection was performed in g/d T cell KO or C57BL/6 WT mice that had received CCL8 i.v. Infected spleens and livers were harvested 48 hpi, and bacterial loads were calculated. Data are from two different experiments conducted separately. The p values were calculated by two-tailed Student t test. *p , 0.001 untreated versus CCL8-treated WT mice, **p , 0.05 untreated WT versus untreated gd KO mice. 2302 BLIMP1-REGULATED CCL8 EXPRESSION IN HOST DEFENSE

FIGURE 8. mCCL8-responsive g/d T cells are enriched for IL-17F. (A and B) Intracytoplasmic staining was performed after Transwell migration assays to compare IL-17A and IL-17F expression by the total input population of g/d T cells isolated from WT spleen and lymph nodes and g/d T cells that migrate to mCCL8 and mCCL2. (A) Representative FACS plots of IL-17A and IL-17F expression by total input g/d T cells (left panel) and g/d T cells that migrate to mCCL8 (right panel) from three independent experiments are depicted. (B) Percentage of IL-17A+ and IL-17F+ cells among input spleen and lymph node g/d T cells, and g/d T cells that migrate to mCCL8 and mCCL2 expressed as mean 6 SEM. Data represent a total of three independent experiments for input and mCCL8-responsive g/d T cells, and two independent experiments for mCCL2-responsive g/d T cells. *p , 0.01 (two-tailed Student t test). Downloaded from expressing TCR Vg4orVg6 (54). Hamada et al. (54) also stated to lyse and release extracellular bacteria, this scenario may ne- that IL-17A–deficient mice fail to control bacterial growth in vivo. cessitate neutrophil cleanup. In the context of Blimp1 deletion, IL-17, therefore, is an effector molecule produced by TCR g/d neutrophil-mediated bacterial clearance, rather than inflammatory T cells, which is important in innate immunity against L. mono- monocyte-dependent mechanisms, appears to control enhanced cytogenes in the liver. Although murine CCL8 is a member of the bacterial clearance. http://www.jimmunol.org/ MCP family, until recently it has been poorly characterized. Our results describe a previously unknown mechanism involving Members of the C-C family of chemokines, such as CCL2 (also BLIMP1-dependent control of CCL8 in macrophages used by the called MCP1) and CCL7 (also called MCP3), are known to attract host to control L. monocytogenes infection. Our data demonstrate monocyte lineage cells bearing CCR2 on their surfaces (47, 48, that BLIMP1 normally acts as a gatekeeper to curb CCL8-induced 76). Luster and colleagues (51) identified CCR8 rather than CCR2 inflammation. Importantly, because BLIMP1 is also inducible in as the receptor for CCL8. Thus, in our study we finally demon- neutrophils and because LyzM Cre would also modulate BLIMP1 strated that the real target of CCL8 in the context of L. mono- levels in neutrophils, our data cannot exclude additional roles for cytogenes infection is g/d T cells, known to express CCR8 on their neutrophil-expressed BLIMP1 in this model. In the macrophage surface (55).g/d T cells are able to produce IL-17A and IL-17F, both scenario, however, it is not clear whether induction of Blimp1 by guest on October 1, 2021 involved in neutrophil recruitment, amplifying Th17 responses (59). transcription early during infection acts to minimize the release of Indeed, in this article we show that g/d T cells directly migrate in CCL8 to control indiscriminant cytolytic acivity of neutrophils response to CCL8 and through the release of IL-17, and in par- and minimize tissue damage or whether this is a result of the ticular IL-17F, likely encourage mobilization of neutrophils to the strategy of the pathogen to defeat host innate defenses. A common site of infection to promote a rapid innate immune response in feature of pathogens is their ability to evade or suppress innate situ. Even if the effects of removing g/d T cells or injecting CCL8 immunity, to avoid being eliminated and establish a successful are milder on CFU counts than those seen in both Ccl8/12 double infection. The ability of multiple pathogens to induce BLIMP1 KOs and Blimp1 CKOs, we believe that our findings together suggests that this may be a common strategy of immune evasion. support our model of CCL8-driven IL-17–g/d T cell–dependent Therapeutic manipulation of Blimp1 gene expression levels or resistance to bacterial infection. BLIMP1 protein function may therefore represent a novel means The importance of neutrophils in the early resistance of mice to to boost protective immunity in an early microbial infection. Fur- Listeria infection is still somewhat unclear. Early studies based thermore, dysregulation of BLIMP1 could exacerbate autoimmu- primarily on cell depletions with anti–Gr-1 (clone RB6-8C5), an nity by tipping the balance toward uncontrolled inflammation. Ab that binds to the cell surface markers Ly6C and Ly6G sug- In this regard, it is noteworthy that single-nucleotide polymor- gested that neutrophils played a critical role in reducing bacterial phisms in the Blimp1 gene have been linked to systemic lupus burden in the liver and spleen early during infection. Ly6C is erythematosus. Enhancing Blimp1 expression under these circum- expressed on neutrophils, monocytes, and subsets of CD8+ T cells, stances could therefore also represent a novel means of alleviating whereas Ly6G is expressed solely on neutrophils. Recent studies persistent inflammatory and autoimmune diseases. Our discovery using highly selective neutrophil depletion strategies indicated of the contribution of BLIMP1 to immune function reveals novel that neutrophils are important for control of L. monocytogenes in intervention points in the treatment of inflammatory diseases. the liver, but not the spleen, during infection (77). Additional studies, however, indicate that they are dispensable for control of Acknowledgments this bacterium and instead proposed that inflammatory monocytes We thank Kathryn Calame for providing Prdm1fl/fl mice and Anna Cerny (which were also depleted in the early anti-Gr1 studies) were for backcrossing these animals as well as for animal husbandry and essential for bacterial clearance (78). These disparate observations genotyping. may suggest that the role of neutrophils may depend on the dose of bacteria. At lower doses of bacteria, at which the bacteria are primarily intracellular, neutrophils may be less important, whereas Disclosures at higher doses of bacteria, which cause host cells heavily infected The authors have no financial conflicts of interest. The Journal of Immunology 2303

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