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The CCL6 Promotes Innate Immunity via Immune Activation and Recruitment

This information is current as Ana L. Coelho, Matthew A. Schaller, Claudia F. Benjamim, of September 28, 2021. Amos Z. Orlofsky, Cory M. Hogaboam and Steven L. Kunkel J Immunol 2007; 179:5474-5482; ; doi: 10.4049/jimmunol.179.8.5474

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References This article cites 53 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/179/8/5474.full#ref-list-1 http://www.jimmunol.org/

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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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Chemokine CCL6 Promotes Innate Immunity via Immune Cell Activation and Recruitment1

Ana L. Coelho,2* Matthew A. Schaller,* Claudia F. Benjamim,† Amos Z. Orlofsky,‡ Cory M. Hogaboam,* and Steven L. Kunkel*

Septic syndrome is a consequence of innate immune failure. Recent studies showed that the CC chemokine CCL6 enhanced antimicrobial immunity during experimental sepsis through an unknown mechanism. The present study demonstrates that trans- genic CCL6 expression abolishes mortality in a septic peritonitis model via the modulation of resident peritoneal cell activation and, more importantly, through the recruitment of IFN-producing NK cells and killer dendritic cells into the peritoneum. Thus, CCL6 attenuates the immune failure during sepsis, in part, through a protective type 1- mediated mechanism. The Journal of Immunology, 2007, 179: 5474–5482. Downloaded from evere sepsis, defined as sepsis associated with acute organ , homing and development of lymphoid dysfunction, may result from exuberant innate immune tissue, and influencing the overall type 1/type 2 balance of an S and procoagulant responses to an (1). Despite immune response (3–5). Clinical studies have identified elevated important advances in antibiotic development and the improved levels of associated with human sepsis and acute effectiveness of critical care units with advanced ventilator sup- injury (6–9). Because chemokines are essential to the innate im-

port, the mortality due to sepsis has not dramatically changed over mune response, they have been considered therapeutic targets dur- http://www.jimmunol.org/ the past three decades (2). The present limitations for clinical in- ing sepsis. tervention in sepsis reflect, in part, our limited understanding of the CCL6 is a CC chemokine initially isolated from mouse bone mechanisms involved in the regulation of the innate immune re- marrow (10). This chemokine is mostly a chemoat- sponse during sepsis. It is now appreciated that the tractant in vitro and in vivo (11, 12), but it can also attract B cells, during sepsis is precariously balanced between pro- and anti- CD4ϩ and (11–13). CCL6 is a murine inflammatory mediators. If the patient fails to mount an effective chemokine with several functional homologues in humans includ- innate response, the clinical outcome could be an overwhelming ing macrophage inflammatory -1␥, CC chemokine F-18, infection. Conversely, should the immune system be improperly hemofiltrate CC chemokine-1 and hemofiltrate CC-2. These che- regulated, the patient may develop a systemic inflammatory re- mokines have an unusual genomic structure, with a unique second by guest on September 28, 2021 sponse syndrome (SIRS),3 characterized by the high expression of exon and the ability to bind to and activate CC chemokine several proinflammatory . SIRS may be just as lethal as 1 (14–19). Unlike all other chemokines, CCL6 is IL-4 inducible an overwhelming infection, thus the immune system initiates a but not LPS inducible (13, 20). Several studies have shown that compensatory anti-inflammatory response (CARS), which can lead CCL6 is produced in large quantities during inflammatory and re- to a syndrome leaving the patient’s immune system in a state of modeling disorders, including those that involve alveolar remod- immune suppression. CARS may increase the mortality of pa- eling, dermal wounding, allergic bronchopulmonary aspergillosis, tients; this host response can lead to an immunocompromised pa- bleomycin-induced pulmonary fibrosis, experimental demyelinat- tient and increase the susceptibility to secondary infection. ing diseases, and acute and chronic peritonitis (11, 14, 16, 21–24). Chemokines play important roles in several activities such as Recently, it was shown that rat microglia can express CCL6, and /angiostasis, and activation, this study shows a possible role of this protein in cell-cell com- munication (22). CCL6 is also up-regulated in the peritoneal fluid

*Department of Pathology, University of Michigan, Ann Arbor, Michigan 48109; of mice following cecal ligation and puncture (CLP) (18, 25, 26). †Department of Pharmacology, Universidade Federal do Rio de Janeiro, Rio de In an experimental model of sepsis, recombinant CCL6 protected Janeiro, Brazil; ‡Albert Einstein College of Medicine, Yeshiva University, Bronx, mice from CLP-induced lethality and this protection was associ- NY 10461 ated with increased expression of TNF-␣, IL-13, and CCL2 (18). Received for publication March 30, 2007. Accepted for publication August 7, 2007. Also, exogenous CCL6 enhanced bacterial clearance and the The costs of publication of this article were defrayed in part by the payment of page phagocytic capacity of peritoneal (18). charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. In the present study, we investigated the mechanism(s) through 1 This work was supported by the National Institutes of Health (P50 HL 056402, P01 which this chemokine protects mice from severe sepsis. To this HL 031963, and R01 HL031237). end, we used mice transgenically overexpressing CLL6 (CCL6 Tg) 2 Address correspondence and reprint requests to Dr. Ana L. Coelho, Department of in the lung. Our data demonstrate that CCL6 Tg mice are ex- Pathology, University of Michigan, 109 Zina Pitcher Place, Ann Arbor, Michigan tremely resistant to the lethality following CLP. These mice ex- 48109. E-mail address: [email protected] hibited significantly increased levels of CCL2/MCP-1 at 4 h, and 3 Abbreviations used in this paper: SIRS, systemic inflammatory response syndrome; ␥ CARS, compensatory anti-inflammatory response; CLP, cecal ligation and puncture; IFN- at 24 and 72 h, after CLP in the peritoneal lavage fluid. CCL6 Tg, transgenically overexpressing CCL6; IKDC, IFN-producing killer den- IFN-␥ was identified as critical cytokine for the survival of CCL6 dritic cell; WT, wild type; AST, aspartate transaminase; ALT, alanine transaminase; Tg mice, because anti-IFN-␥ Ab abolished the protective effect of PC, peritoneal cavity; DC, ; MHCII, MHC class II. CCL6. Increased numbers of IFN-producing killer dendritic cells Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 (IKDCs) and NK cells were observed in the peritoneal cavity of www.jimmunol.org The Journal of Immunology 5475

FIGURE 2. CCL6 Tg mice clear more effectively than WT mice. At 72 h after CLP surgery, WT and CCL6 Tg mice (n ϭ 5 per group) were killed and and peritoneal fluids were harvested. Ten microliters of EDTA-treated blood (A) and peritoneal lavage fluids (B) were serially diluted and plated on thymic-shared Ag blood agar plates. Bacteria levels were expressed as CFU per 10 ␮l. The graphs represent pooled data from one to four independent studies showing similar results. The horizontal bar .p Ͻ 0.001 compared with WT mice ,ء .indicates the mean for each group

ketamine HCl (Abbot Laboratories) and 150 ␮g xylazine (Lloyd Labora-

FIGURE 1. CCL6 Tg mice have high levels of CCL6 in the lung and tories). Under sterile conditions, a 1–2 cm incision was made on the lower Downloaded from peritoneal cavity and they were protected against CLP-induced mortality. left abdomen and the cecum was exposed. The distal portion of the cecum Lung (A) and peritoneal (B) samples from WT and CCL6 Tg mice (n ϭ 5 was partial ligated with 3.0 silk suture and punctured (9 punctures) with a 21-gauge needle. The cecum was returned to the peritoneal cavity and the per group) were collected at 72 h after sham and CLP surgery and CCL6 incision was closed with surgical staples. Sham-operated mice were sub- concentrations were determined by ELISA as described in Materials and jected to a similar laparotomy without ligation and puncture. Mice were Methods. C, Mice were subjected to CLP or sham operation and the sur- rehydrated with 1 ml saline s.c. and placed on a heating pad until they vival of these mice was followed until day 7. The results are expressed as recovered from the anesthetic. Mice in both surgery groups were treated percentage of survival per day and each group has 10 mice. The data are with an antibiotic preparation containing Imipenem conjugated with Cila- http://www.jimmunol.org/ p Ͻ 0.05 as compared with sham statin (Primaxin I.V., 10 mg/kg, i.p.; Merck) beginning at 6 h after CLP ,ء .representative of three experiments group. §, p Ͻ 0.05 as compared with WT-CLP group. surgery and readministered every 12 h thereafter until day 3 postsurgery. Antibiotic therapy increased mouse survival to the 60–70% range, and these mice were used in the following experiments. Additionally, survival CLP-CCL6 Tg mice, suggesting these cells were the source of the experiments were performed without antibiotics, to address the role of observed increases in IFN-␥. Thus, these data support the concept CCL6 in mouse survival without antimicrobial intervention. that CCL6 modulates endogenous macrophages activation possi- In vivo experimental protocol bly via CCL2 and, more importantly, facilitated the recruitment of effector immune cells with the enhanced ability to generate IFN-␥. IFN-␥ neutralization. Wild-type (WT) and CCL6 Tg mice were treated i.p. with either control rabbit IgG (500 ␮g/mouse) or rabbit anti-murine by guest on September 28, 2021 IFN-␥-specific IgG (500 ␮g/mouse) 1 h before CLP surgery. The control Materials and Methods IgG and anti-IFN-␥ were purified from antiserum using a Protein A column Mice (Pierce). Anti-IFN-␥ antiserum was raised by immunizing New Zealand white rabbits with murine IFN-␥ (R&D Systems). The polyclonal Abs were Specific-pathogen-free, female, C57BL/6 mice (6 to 8 wk of age) were tittered by direct ELISA. The Abs did not cross-react with a number of obtained from Taconic Company, Germantown, NY. other murine cytokines, including CXC and CC chemokines, as seen in the fact that IFN-␥ ELISA established with the Abs did not detect any murine Generation of CCL6 transgenic mice cytokine at a concentration as high as 100 ng/ml. Mouse survival was A murine CCL6 minigene was constructed by PCR using CCL6 cDNA monitored in CLP groups containing ten mice for a total of 7 days. All (13) and genomic (27) clones. The resulting minigene consisted of the mouse survival studies were conducted a maximum of three experiments. complete CCL6 open reading frame, with intron-3 of the CCL6 in its NK cells depletion. WT and CCL6 Tg mice were treated i.p. with either natural location, followed by nt 1459–1643 of the vector RJB10A (28), isotype control (200 ␮g/mouse) or hamster anti-murine NK 1.1 (200 ␮g/ containing the bovine growth hormone polyadenylation signal. The PCR mouse), provided by Dr. T. Moore (University of Michigan, Ann Arbor, product was inserted into a pCRII vector (Invitrogen Life Technologies) in MI), beginning at 24 h prior CLP and readministrated at 48 h after CLP which the polylinker orientation was reversed by BstXI cleavage and re- surgery. Mouse survival was monitored in CLP groups containing ten mice ligation, and the sequence of the resulting construct (pCR-CCL6) was con- for a total of 7 days. All mouse survival studies were conducted a maxi- firmed. A 2.3-kb segment of the Clara cell 10kd gene promoter was iso- mum of three experiments. lated as a HindIII fragment from pRtCC10–2300 (29) (a gift from Jeffrey Whitsett, Children’s Hospital Research Foundation, Cincinnati, OH) and Clinical chemistry Hind inserted in the III site of pCR-CCL6. Transient protein expression Serum was separated from whole blood and the aspartate transaminase from this construct was verified by the immunostaining of transfected (AST) and alanine transaminase (ALT) levels were measured by Clinical H441 lung adenocarcinoma cells. The construct was linearized with NsiI Pathology at the University of Michigan Medical School using standard- and microinjected into FVB oocytes with the assistance of the Transgenic ized techniques. and ES Cell Core Facility of the Albert Einstein College of Medicine. Six founders were backcrossed to C57BL/6 and the hemizygous progeny as- Peritoneal cells culture sessed for CCL6 expression by both Western blot of bronchoalveolar la- vage and Northern blot of lung RNA. One positive line was chosen for Peritoneal cells were harvested from sham and CLP mice at 72 h after further study. The mice exhibited no gross or histologic abnormality. All surgery and suspended in RPMI 1640 (BioWhittaker) containing 5% FCS, mice were housed five per filter-topped cage and had free access to food 2mML-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin. Cells and water during their use in an experiment. The University of Michigan were placed in plastic 24-wells plates (1 ϫ 106 cell/well) and incubated at

Committee on Use and Care of Animals approved all animal studies outline 37°C in 5% CO2. After 24 h, supernatants were removed, clarified by in this manuscript. centrifugation, and analyzed by ELISA for IFN-␥ production. CLP experimental model Determination of CFU Animals were subjected to sham or CLP surgery as previously described Peritoneal lavages fluid and EDTA-treated blood from 72 h post-CLP were (30). In brief, mice were anesthetized with an i.p. injection of 2.25 mg placed on ice and serially diluted in sterile saline. A 10-␮l aliquot of each 5476 ROLE OF CCL6 IN INNATE IMMUNITY

FIGURE 3. CCL6 Tg mice demonstrated less CLP-induced liver injury. At 4, 24, and 72 h after sham and CLP surgery, WT and CCL6 Tg mice (n ϭ 4 per group) were bled and serum was iso- lated. Levels of AST (A) and ALT (B) in the sera were measured. The data are representative of p Ͻ 0.05 as compared with ,ء .three experiments sham group.

dilution was spread on thymic-shared Ag agar plates (BD Diagnostic Sys- nant murine cytokines/chemokines (R&D System) were used to gener- tems) and incubated at 37°C overnight. Colonies were counted and ex- ate standard curves and concentrations were expressed as ng or pg/ml. pressed as CFU/10 ␮l. Groups contained four to six mice and the experi- Experimental groups consisted of triplicate samples in vitro from three ments were repeated on three different occasions. Results were similar for independent experiments. each experiment and subsequent pooled.

Measurement of cytokines and chemokines by ELISA Flow cytometry analysis Downloaded from Concentrations of murine IFN-␥, IL-12 (p70), IL-10, IL-13, TNF-␣, Peritoneal cells were harvested from sham and CLP groups 72 h after CCL2, CCL3, CCL5, and CCL17 were measured in cell-free peritoneal lavage fluid and cell culture supernatants using a standardized sandwich surgery. RBC were lysed in lysis buffer and the remaining cells were ELISA previously described (31). In brief, 96-well microtiter plates washed in PBS and resuspended in PBS containing 1% BSA. Cells were (Nunc) were coated overnight at 4°C with mAb specific for the murine Fc blocked and stained with various Abs. For cells staining, we used cytokine and chemokine being measured (R&D System). Wells were Abs labeled with PE, FITC, PeCy5, or PeCy7 direct against the fol- washed with PBS plus 0.05% Tween 20 and nonspecific sites were lowing: CD11b, MHCII, CD19, IgM, CD3, CD4, CD8, NK 1.1 (all from blocked with 2% BSA in PBS for 90 min at 37°C. Plates were washed BD Pharmingen), CD49b (eBioscience), or F4/80 (abCam). For intra- http://www.jimmunol.org/ and samples were loaded and incubated at 37°C for 1 h. After washing, cellular staining, peritoneal cells were collected and placed in plastic ϫ 6 a secondary, biotinylated, cytokine/chemokine-specific polyclonal Ab 24-wells plates (2 10 cell/well) and incubated at 37°C in 5% CO2 (R&D Systems) was added for 30 min at 37°C. Plates were washed with protein transporter inhibitor Golgi Plug (BD Pharmingen) to pre- again and peroxidase-conjugated streptavidin (Bio-Rad) was added. vent the release of IFN-␥. After6hofincubation, cells were transferred Plates were washed and a chromogenic substrate (Bio-Rad) was added to 5 ml tubes and staining first for the surface markers described above and incubated at room temperature until fully developed. Reactions and then for IFN-␥ intracellular (BD Pharmingen). Cells were analyzed were stopped and read at 490 nm in an ELISA plate reader. Recombi- with a Beckman Coulter Cytomics FC500. by guest on September 28, 2021

FIGURE 4. Levels of chemokines and cytokines in peritoneal lavage fluids from sham and CLP operated mice. At 4 (A and D), 24 (B and E), and 72 h (C and F) post-CLP, WT and CCL6 Tg mice were sacrificed and 2 ml peritoneal lavages were performed. Concentrations of cytokines/chemokines in peritoneal fluids of WT mice (A, B, and C) and CCL6 Tg mice (D, E, and F) were measured using specific sandwich ELISAs. The data are representative of three separate experiments and -p Ͻ 0.05 as com ,ء .each group contained five mice pared with sham group. The Journal of Immunology 5477 Downloaded from

FIGURE 5. Ex vitro expression of IFN-␥ and IL-10 in cells isolated from the peritoneum of WT and CCL6 Tg mice. At 4 (A and D), 24 (B and E), and 72h(C and F) post-CLP, WT mice, and CCL6 Tg mice were sacrificed, peritoneal cell were isolated, placed in 24-wells plate (1 ϫ 106 cells/well), and ␥ incubated for 24 h at 37°C in 5% CO2. Concentrations of IL-10 (D, E, and F) and IFN- (A, B, and C) in cells culture supernatants were measured using p Ͻ 0.05 as compared with ,ء .specific sandwich ELISAs. The data are representative of three separate experiments and each group contained five mice sham-operated mice. §, p Ͻ 0.05 as compared with WT-CLP group. http://www.jimmunol.org/

Statistical analysis CFU cultured from plasma (Fig. 2A) and peritoneal wash samples All data are shown are means Ϯ SE and are representative of three separate (Fig. 2B) from CCL6 Tg mice were lower compared with WT experiments. The means between different treatments were compared by mice. These results were statistically significant for the PC samples ANOVA. When significance was detected, individual differences were an- ( p ϭ 0.05) but not for the blood samples despite a log difference alyzed using the Bonferroni’s t test for unpaired values. Statistical signif- in the means of these samples. icance was set at p Ͻ 0.05. Survival rates were expressed as percentages, 2 and a log rank test (␹ test) was used to detect differences in mouse CCL6 Tg mice have less liver injury induced by CLP survival. by guest on September 28, 2021 One of the major complications of sepsis is multiple organ failure, Results which often leads to death (1). To determine the ability of CCL6 Expression and biologic activity of CCL6 during the evolution to prevent CLP-induced liver injury, the levels of liver in of experimental sepsis the serum of sham- and CLP-operated mice were measured in WT and CCL6 Tg groups (Fig. 3). The AST (Fig. 3A) and ALT (Fig. CLP-induced sepsis exhibits many of the features of clinical sepsis 3B) levels of both groups were similar in both sham- and CLP- including SIRS and CARS. We previously demonstrated that ex- operated mice at 4 h after surgery. At 24 h post-CLP, the serum of ogenous CCL6 exerted a protective effect in CLP-induced sepsis, both CCL6 Tg and WT mice showed significantly higher levels of but the mechanism responsible for this protective effect was un- ALT and AST compared with sham-operated mice levels, but clear. In an attempt to understand the protective role of this che- CLP-CCL6 Tg mice exhibited lower AST and ALT levels (550 Ϯ mokine, CCL6 Tg and WT mice were subjected to experimental 70 and 110 Ϯ 10, respectively) than CLP-WT mice (780 Ϯ 50 and peritonitis. Fig. 1, A and B shows that the baseline and CLP-in- 200 Ϯ 32, respectively). At 72 h post-CLP, both AST and ALT duced CCL6 levels were significantly higher in the CCL6 Tg returned to baseline levels in CLP-CCL6 Tg mice, whereas these group compared with the WT group. At baseline these values were 7-fold higher in the lung (Fig. 1A) and 9-fold higher in the peri- toneal cavity (PC) (Fig. 1B). After CLP, these values were 6-fold higher in the lung (Fig. 1A) and 4-fold higher in the PC (Fig. 1B). 100 CCL6 Tg+control IgG To investigate the susceptibility of CCL6 Tg mice to CLP, WT, 75 and CCL6 Tg mice were submitted to sham and CLP without antibiotic treatment. Fig. 1C shows mouse survival following sham 50 and CLP. Ninety percent of the CCL6 Tg group was alive at day 25 * 7 after CLP, whereas only 20% of the WT group was alive at the CCL6 Tg+anti-IFN-γ same time. Together, these data suggest that increased levels of 0 0 1 2 3 4 5 6 7 CCL6 dramatically protected mice from severe sepsis. Days after CLP surgery CCL6 Tg mice had enhanced bacterial clearance and reduced FIGURE 6. IFN-␥ neutralization increase mortality associated with bacteremia after CLP CLP in CCL6 Tg mice. CCL6 Tg mice received either control IgG (500 ␮g/mouse) or anti-IFN-␥ (550 ␮g/mouse) 1 h before CLP surgery. The To determine whether the improved survival of CCL6 Tg mice survival of these mice was followed until day 7. The results are expressed was attributable to improved bacterial clearance, peritoneal, and as percentage of survival per day and each group has ten mice. The data are -p Ͻ 0.05 as compared with IgG ,ء .blood samples were collected to determine local and systemic bac- representative of three experiments terial loads, respectively (Fig. 2). At 72 h post-CLP, the bacteria CLP-operated mice. 5478 ROLE OF CCL6 IN INNATE IMMUNITY

FIGURE 7. Profile of peritoneal cell populations of WT and CCL6 Tg mice following sham and CLP sur- gery. Mice were killed 72 h after sham and CLP sur- gery and the cells were collected and processed for FACS analysis. The peritoneal cells were stained with Abs to specific cell markers following: macrophages, CD11bϩF4/80ϩMHCIIϩ (A); B1 lymphocytes, CD11bϩ IgMϩCD19ϩ (B); T lymphocytes, CD3ϩCD4ϩ (C) and CD3ϩCD8ϩ (D); NK cells, NK 1.1ϩCD49bϩ (E); and Downloaded from NKT cells, CD3ϩNK 1.1ϩ (F). The results are expressed as number of cells ϫ 106 per cavity and each group has three mice. The data are representative of three experi- p Ͻ 0.05 as compared with sham group. §, p Ͻ ,ء .ments 0.05 as compared with WT-CLP group. http://www.jimmunol.org/ by guest on September 28, 2021

levels remained elevated in CLP-WT mice. Thus, these data sug- 24 h and IFN-␥ and IL-10 levels were measured by ELISA. Con- gest that the CCL6 Tg mice recovered faster from the liver injury firming the trends showed in Fig. 4, cells isolated from the peri- caused by CLP. toneal cavity of CLP-WT mice generated higher levels of IFN-␥

Time point differences in cytokine and chemokine levels in the peritoneal cavity of WT and CCL6 Tg mice: diminished early cytokine storm in CCL6 Tg group Previous studies have shown that the local and systemic levels of many cytokines and chemokines are markedly increased, follow- ing CLP surgery (32, 33). To establish the cytokine profile in CCL6 Tg mice, PC fluid from both sham and CLP groups was collected and analyzed in ELISA (Fig. 4). At 4 h after surgery, higher levels of IFN-␥ and IL-10 were detected in CLP-WT mice compared with CLP-CCL6 Tg mice (Fig. 4, A and D). At this same time, only CCL2 levels were significantly increased in the CLP- CCL6 Tg group when compared with WT mice (Fig. 4D). Levels of CCL3, CCL17, CCL2 and IL-10 were elevated in CLP-WT group when compared with CLP-CCL6 Tg group at 24 h post-CLP (Fig. 4, B and E). CCL2, CCL3, CCL17, and particularly, IL-10 levels were markedly higher in CLP-WT mice at 72 h after CLP (Fig. 4C). In contrast, at 72 h post-CLP, CLP-CCL6 Tg mice ex- hibited significantly higher levels of IFN-␥ (0.459 ng/ml vs 0.055 FIGURE 8. NK cell depletion had no significant effect on CCL6 Tg mice ng/ml) and IL-12 (0.225 ng/ml vs 0.054 ng/ml) compared with survival following CLP. WT and CCL6 Tg mice received either control IgG (200 ␮ ␮ CLP-WT group at this time after CLP (Fig. 4, C and F). g/mouse) or anti-NK 1.1 (200 g/mouse) 24 h before and 48 h after CLP sur- gery. At 72 h after CLP, cells from peritoneal cavity were collected and stained with Abs for the following specific cell markers: NK cells, NK 1.1ϩCD49bϩ (A) Cells from the peritoneal cavity of CCL6 Tg mice produce high ϩ ϩ ϩ levels of IFN-␥ and IKDCs, NK 1.1 CD49b MHCII (B). The survival of WT (C) and CCL6 Tg (D) mice was followed until day 6. The results are expressed as percentage of Peritoneal cells from WT and CCL6 Tg mice harvested at 4, 24, survival per day and each group has ten mice. The data are representative of three .p Ͻ 0.05 as compared with IgG-CLP-operated mice ,ء .and 72 h post-sham and post-CLP surgery were cultured in vitro for experiments The Journal of Immunology 5479

Peritoneal cell population in WT and CCL6 Tg mice at 72 h after CLP surgery To determine the type of cell contributing to increased IFN-␥ during sepsis, peritoneal cells were isolated 72 h postsurgery from WT and CCL6 Tg mice subjected to either sham or CLP surgery and analyzed by flow cytometer. Cells from the peritoneal cavity of sham and CLP mice were stained with Abs to specific mark- ers for macrophages (CD11bϩF4/80ϩMHCIIϩ), B1 lympho- FIGURE 9. IKDCs population is increased in CCL6 Tg mice after cytes (CD11bϩIgMϩCD19ϩ), T lymphocytes (CD3ϩCD4ϩ and CLP surgery. Mice were sacrified 72 h after sham or CLP surgery and ϩ ϩ ϩ ϩ CD3 CD8 ), NK cells (NK1.1 CD49b ), and NKT cells the peritoneal cells were collected and processed for FACS analysis. ϩ ϩ The peritoneal cells were stained for IKDCs specifics markers (CD3 NK1.1 ) (Fig. 7). There was no significant difference in NK1.1ϩCD49bϩMHCIIϩ. The results are expressed as number of cells ϫ the numbers of macrophages, B1 or T cells in WT and CCL6 Tg 106 per cavity and each group has three mice. The data are representative mice after CLP surgery (Fig. 7, A–D). In contrast, there was a p Ͻ 0.05 as compared with sham group. §, p Ͻ significant increase in the number of NK cells in CLP-CCL6 Tg ,ء .of three experiments 0.05 as compared with WT-CLP group. mice compared with CLP-WT mice (Fig. 7E). Additionally, the number of NKT cells was significantly lower in both sham- and CLP-operated CCL6 Tg mice compared with WT mice (Fig. 7F).

4 h after surgery compared with cells from CLP-CCL6 Tg mice Depletion of NK cells does not alter CCL6 Tg mice survival Downloaded from (Fig. 5A). However, at later time points IFN-␥ levels in cells from Activated NK cells are a major source of IFN-␥ during sepsis and CLP-WT mice decreased until 72 h when they were comparable to are therefore involved in the pathogenesis of sepsis (34, 35). To values expressed in cells from sham-WT mice. In contrast, IFN-␥ determine whether NK cells were involved in the immunomodu- expression in cells from CLP-CCL6 Tg mice increased with time latory role of CCL6 during severe sepsis, NK cells were depleted and was significantly higher than in all other experimental groups before CLP surgery. WT and CCL6 Tg mice received control IgG at 72 h (Fig. 5, A–C). In contrast, IL-10 expression showed a and anti-NK 1.1 Abs 24 h before and 48 h after CLP surgery. Fig. http://www.jimmunol.org/ reverse trend (Fig. 5, D–F): In cells from WT-CLP mice, IL-10 8A shows that the treatment with anti-NK1.1 before CLP com- significantly increased as a function of time between 4 and 72 h pletely depleted NK cells in both groups while 97% and 92% of while significantly decreasing in CLP-CCL6 Tg mice. These data IKDCs were depleted in WT and CCL6 Tg mice, respectively (Fig. suggested that IFN-␥ and IL-10 were expressed in a manner and at 8B). In WT mice (Fig. 8C), the NK cell depletion resulted in sig- levels conducive for clearance and survival from SIRS. nificant protection against CLP, while in CCL6 Tg mice (Fig. 8D) the NK depletion reduced survival, however these differences were Neutralization of IFN-␥ caused CLP-induced lethality in CCL6 not statistically significant. Tg mice by guest on September 28, 2021 In an attempt to determine whether the delayed increase in IFN-␥ IKDC are increased in CLP-CCL6 Tg mice levels in the peritoneal cavity of CCL6 Tg mice contributed to a NK cells and dendritic cells (DC) are central components of innate protective role of CCL6 against sepsis-induced mortality, CCL6 and adaptive immune responses. NK cells are believed to be the Tg mice were pretreated with control rabbit IgG or affinity-purified major producers of IFN-␥, which is secreted in response to IL-12 rabbit IgG specific for murine IFN-␥ 1 h before CLP surgery (Fig. (36). Upon stimulation, DCs are also able to produce cytokines, 6). When CCL6 Tg mice received anti-IFN-␥ before CLP, only such as IL-12 and TNF-␣. Recently, DCs have also been shown to 20% of these mice were alive at day 7 post-CLP compared with secrete IFN-␥ in response to IL-12 alone or in combination with mice pretreated with control IgG (90% of survival). These data IL-18 (36, 37). Recent studies have characterized a new subset of suggest a critical role for IFN-␥ in the protective effect observed in DC that share phenotypic and functional properties of both NK CCL6 Tg mice in our model of severe sepsis. cells and DCs. These cells express both NK cell (NK 1.1, CD49b)

FIGURE 10. IKDCs are producing IFN-␥ in CCL6 Tg mice after CLP surgery. Peritoneal cells were isolated from WT-CLP and CCL6 Tg-CLP groups at 72 h after surgery, stained and analyzed by flow cytometry. Peri- toneal cells were stained with Abs to specific cell markers for IKDCs, NK 1.1ϩCD49bϩMHCIIϩ (A), NK cells, NK1.1ϩCD49bϩ (B) and T lympho- cytes, CD3ϩCD8ϩ (C). All cells were also stained for IFN-␥ intracellular. The data are representative of two separate experiments and each group p Ͻ 0.05 as ,ء .contained five mice compared with total number of the cells type group. §, p Ͻ 0.05 as com- pared with WT-CLP group. 5480 ROLE OF CCL6 IN INNATE IMMUNITY and DC (CD11c, MHC class II (MHCII)) surface markers and duction concomitant with a decrease of IL-12 and TNF production produce significant amounts of IFN-␥ and IL-12 in response to (43). Several studies demonstrated that CCL2 is produced early in CpG (38). To determine whether IKDCs were present in the response to endotoxin challenge, returning to baselines levels after peritoneal cavity of WT and CCL6 Tg sham- and CLP-operated 48 h (18, 32, 42). In our model, CCL2 peaked at 4 h post-CLP in mice, cells from the peritoneal cavity of sham and CLP mice CCL6 Tg mice, which was coincident with low levels of IL-12 and were stained for NK1.1, CD49b, and MHCII markers and an- IFN-␥. However, at 24 and 72 h, the CCL6 Tg mice presented very alyzed by flow cytometry. The number of IKDCs in CCL6 Tg low levels of CCL2. Previous studies demonstrated that CCL6 mice was higher than in WT mice regardless of the surgical neutralization decreased CCL2 production in the lung (16, 20). intervention (sham and CLP) (Fig. 9). A 3-fold increase in the However, previous studies showed that the neutralization of CCL6 number of IKDCs was seen when CLP-CCL6 Tg mice were increased CCL2 levels in their model of acute peritonitis (23). In compared with CLP-WT mice. These data suggest that the pro- CLP-CCL6 Tg mice, CCL6 levels were elevated at 24 and 72 h tective role of CCL6 during severe sepsis may be related to the post-CLP and very low levels of CCL2 were found at these time effect of this chemokine on IKDC recruitment into the perito- points, suggesting that CCL6 may inhibit CCL2 production. Pre- neal cavity after CLP surgery. vious studies demonstrated that CCL2 increases the production of IL-10 in experimental models of sepsis (42, 43). IL-10 has been ␥ IKDCs are the main cell type producing IFN- in CCL6 Tg identified as a crucial modulator of the inflammatory response in mice sepsis and it is an important cytokine mediator of sepsis-induced To investigate whether the IKDCs were producing IFN-␥, perito- immunosupression (44). Prolonged immune suppression is char- neal cells from WT and CCL6 Tg mice were harvested 72 h after acterized by defects in Ag presentation, macrophage paralysis, T Downloaded from CLP, stained for NK 1.1, CD49b, and MHCII markers and intra- cell anergy, suppressed proliferation, and increased T cell cellular IFN-␥ and then analyzed by flow cytometer. Fig. 10A and apoptosis, which may be partially attributable to the shows that 63.3% of total IKDCs (NK1.1ϩCD49bϩMHCIIϩ) biological effects of IL-10. Indeed, in our model, the high and were positive for IFN-␥ in CCL6 Tg mice. To investigate whether sustained levels of CCL2 and IL-10 in CLP-WT mice correlated other cell types were producing IFN-␥, peritoneal cells were also with poor survival rates. Interestingly, we observed a gradual in- stained for NK1.1/CD49b (NK cells) and CD3/CD8 (T lympho- crease of IFN-␥ and IL-12 in CLP-CCL6 Tg mice with time post- http://www.jimmunol.org/ cytes) and IFN-␥. Only 1.6% of total NK cells and 1.8% of total CLP. IFN-␥ strongly stimulates /macrophages, increas- CD8ϩ T lymphocytes were positive for IFN-␥ in CCL6 Tg mice ing their microbicidal activity, Ag presentation function, and (Fig. 10, B and C). Other cell types such as macrophages, B1 cells, production of proinflammatory cytokines on contact with micro- and T lymphocytes CD4ϩ were negative for IFN-␥ in WT and bial stimuli. Genetics defects in the IFN-␥ receptor system have CCL6 Tg mice at 72 h after CLP (data not shown). These data been described in patients with vaccine-associated bacterial infec- show that IKDCs are the key cells producing IFN-␥ in CCL6 Tg tions, demonstrating the importance of IFN-␥-mediated immunity mice at 72 h post-CLP surgery. in human host defense against intracellular pathogens (45, 46). In addition, the important role of IFN-␥ in the pathogenesis of LPS- Discussion induced shock was confirmed using mice deficient for the IFN-␥ by guest on September 28, 2021 Dysregulation of the immune system occurs during severe sepsis, receptor (47, 48). In this study, we show the crucial role of IFN-␥ leading to a rapid death due to the development of multiorgan in CCL6-Tg mice during severe sepsis. These mice expressed sig- failure and an increase in complications due to long-term immu- nificantly elevated levels of IFN-␥ at 72 h after CLP surgery and nosupression (39–41). CCL6, a CC chemokine, is present in a 90% survival was observed. This increase in IFN-␥ was important variety of inflammatory and remodeling disorders (11, 14, 16, 18, for the survival of these mice because, 80% of CCL6 Tg mice died 24). We previously showed that the immunoneutralization of following anti- IFN-␥ Ab treatment. In contrast, the neutralization CCL6 enhanced the sepsis-related mortality. In contrast, the ad- of IFN-␥ protected WT mice from sepsis-induced mortality sug- ministration of recombinant CCL6 to mice immediately after CLP gesting that the rapid increase of IFN-␥ levels observed at 4 h after surgery significantly improved the survival of these mice (18). In CLP can be deleterious and increase the mortality of these mice this study, we show that mice overexpressing CCL6 are protected (data not shown). from mortality following CLP. In our model of severe sepsis (with IFN-␥ is produced primarily by NK cells and a certain subpopu- no antibiotic treatment), these mice presented an impressive 90% lation of T lymphocytes (49). In this study, we demonstrated that survival rate vs a 20% survival in WT mice. The CCL6 Tg mice a NK cell population is increased in CLP-CCL6 Tg mice compared exhibited higher levels of CCL6, in the lung and PC, at 24 h post- with CLP-WT mice. To determine whether NK cells were playing CLP (data not shown) and the levels remained elevated even at a role in the protective role of CCL6 during severe sepsis, we 72 h after CLP as shown in Fig. 1. These data agree with other depleted NK cells in WT and CCL6 Tg mice. NK cells did not studies demonstrating that the production of CCL6 remains ele- appear to provide a protective role, because we observed only a vated several days after septic peritonitis (23, 24). In our model, slight decrease (not statistically significant) in the survival rate of this sustained increase of CCL6 levels seemed to be important in CCL6 Tg mice. However, the treatment with anti-NK1.1 Ab did regulating the deleterious effects induced by sepsis, because, in not completely deplete IKDCs population, suggesting that there is addition to improved survival, the CLP-CCL6 mice present an still a small population of IKDCs that may be producing IFN-␥. improved recovery from liver injury compared with CLP-WT This potentially could explain why there is not a significant dif- mice. This protective effect is likely caused by an increase in ference in the CLP-CCL6 Tg survival rate. In contrast, the treat- CCL6-mediated cytokine cascades, which provides an apparent ment with anti-NK Ab was protective for WT mice against sepsis- cytokine environment for both microorganism clearance and reg- induced mortality. Some studies demonstrated a detrimental role ulate the local inflammatory response. for NK cells in experimental models of sepsis (50, 51). Activated Previous studies demonstrated that CCL2 has a protective role NK cells are a main source of IFN-␥ during sepsis and therefore during septic peritonitis (32, 42). Mice treated with CCL2 were likely are involved in the pathogenesis of sepsis (52). Indeed, de- protected against a lethal dose of LPS or bacteria, and the mech- pletion of NK cells in septic mice offers protection against cyto- anism of protection seems to be by way of increased IL-10 pro- kine- and LPS-induced shock (34, 52). The Journal of Immunology 5481

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