The Journal of Immunology

Myeloid Cell TRAF3 Regulates Immune Responses and Inhibits Inflammation and Tumor Development in Mice

Almin I. Lalani,*,† Carissa R. Moore,* Chang Luo,* Benjamin Z. Kreider,* Yan Liu,* Herbert C. Morse, III,‡ and Ping Xie*,x

Myeloid cells, including granulocytes, monocytes, macrophages, and dendritic cells, are crucial players in innate immunity and inflammation. These cells constitutively or inducibly express a number of receptors of the TNFR and TLR families, whose signals are transduced by TNFR-associated factor (TRAF) molecules. In vitro studies showed that TRAF3 is required for TLR-induced type I IFN production, but the in vivo function of TRAF3 in myeloid cells remains unknown. In this article, we report the generation and characterization of myeloid cell–specific TRAF3-deficient (M-TRAF32/2) mice, which allowed us to gain insights into the in vivo functions of TRAF3 in myeloid cells. We found that TRAF3 ablation did not affect the maturation or homeostasis of myeloid cells in young adult mice, even though TRAF3-deficient macrophages and neutrophils exhibited constitutive NF-kB2 activation. However, in response to injections with LPS (a bacterial mimic) or polyinosinic-polycytidylic acid (a viral mimic), M-TRAF32/2 mice exhibited an altered profile of production. M-TRAF32/2 mice immunized with T cell–independent and –dependent Ags displayed elevated T cell–independent IgG3 and T cell–dependent IgG2b responses. Interestingly, 15- to 22-mo-old M-TRAF32/2 mice spon- taneously developed chronic inflammation or tumors, often affecting multiple organs. Taken together, our findings indicate that TRAF3 expressed in myeloid cells regulates immune responses in myeloid cells and acts to inhibit inflammation and tumor development in mice. The Journal of Immunology, 2015, 194: 334–348.

umor necrosis factor receptor-associated factor 3 (TRAF3), system through interactions with additional adaptor , in- a member of the TRAF family of cytoplasmic adaptor cluding MyD88 and TRIF for TLR signaling, RIP2 for NLR T proteins,isusedinsignalingbyavarietyofimmune signaling, and MAVS for RLR signaling (3–5). The shared usage receptors, including the TNFR superfamily, TLRs, NOD-like of TRAF3 by such a variety of immune receptors is indicative of receptors (NLRs), and RIG-I–like receptors (RLRs) (1, 2). TRAF3 its broad functional roles in the immune system. binds directly to almost all members of the TNFR superfamily Mice made genetically deficient in TRAF3 (TRAF32/2)die that do not contain death domains, including CD40, BAFF-R, within 10 d of birth with severe progressive runting, illustrating TACI, BCMA, LT-bR, CD27, CD30, RANK, HVEM, EDAR, crucial developmental functions of TRAF3 (6). To circumvent XEDAR, 4-1BB (CD137), OX-40 (CD134), and GITR experimental limitations imposed by the early mortality of (TNFRSF18). TRAF3 is also indirectly recruited to the signaling TRAF32/2 mice and to explore the in vivo functions of TRAF3 in complexes of pattern recognition receptors of the innate immune various cell types of adult mice, we recently used a conditional targeting strategy to generate conditional TRAF3-deficient flox/flox *Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, (TRAF3 ) mice. This makes it possible to delete the Traf3 † NJ 08854; Graduate Program in Cellular and Molecular Pharmacology, Rutgers gene in specific cell types or tissues (7). Characterization of University, Piscataway, NJ 08854; ‡Laboratory of Immunogenetics, National Insti- tute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, conditional TRAF3-deficient mouse models revealed that TRAF3 x MD 20852; and Rutgers Cancer Institute of New Jersey, New Brunswick, NJ 08903 is critically involved in regulating multiple receptor signaling Received for publication June 17, 2014. Accepted for publication October 27, 2014. pathways in different immune cell types. We previously reported This work was supported by the Cancer Institute of New Jersey through National that specific ablation of TRAF3 in B lymphocytes results in Cancer Institute Grant P30CA072720 (to P.X.), a Rutgers Faculty Research Grant (to P.X.), the Arthur Herrmann Endowed Cancer Research Fund (to P.X.), and the Intra- marked peripheral B cell hyperplasia, due to remarkably pro- mural Research Program, National Institute of Allergy and Infectious Diseases, Na- longed survival of mature B cells independent of the B cell sur- tional Institutes of Health (to H.C.M.). vival factor BAFF, leading to the development of splenic marginal Address correspondence and reprint requests to Dr. Ping Xie, Department of Cell Biology and Neuroscience, Rutgers University, 604 Allison Road, Nelson Labs Room zone lymphomas (MZLs) or B1 lymphomas by 18 mo of age (7, B336, Piscataway, NJ 08854. E-mail address: [email protected] 8). These findings indicated that a major homeostatic function of The online version of this article contains supplemental material. TRAF3 in peripheral B cells is the promotion of spontaneous Abbreviations used in this article: BMDM, bone marrow–derived macrophage; CSR, apoptosis, a conclusion subsequently corroborated by Gardam and class switch recombination; DC, dendritic cell; DLBCL, diffuse large B cell lym- colleagues (9). In contrast, specific deletion of TRAF3 from the phoma; FL, follicular lymphoma; GC, germinal center; GI, gastrointestinal; IRF3, IFN regulatory factor 3; KLH, keyhole limpet hemocyanin; LMC, littermate control; T cell lineage leads to defective IgG1 responses to a T cell–de- LN, lymph node; LysM, lysozyme M; MCP, monocyte chemotactic ; MDSC, 2 2 pendent (TD) Ag and impaired T cell–mediated immunity to in- myeloid-derived suppressor cell; MLN, mesenteric LN; M-TRAF3 / , myeloid cell– specific TRAF3-deficient; MZL, splenic marginal zone lymphoma; NLR, NOD-like fection with Listeria monocytogenes because of compromised receptor; pDC, plasmacytoid DC; PEM, peritoneal exudate macrophage; polyI:C, TCR/CD28 signaling in both CD4 and CD8 T cells (10). In ad- polyinosinic-polycytidylic acid; RLR, RIG-I–like receptor; RT-qPCR, real-time quantitative PCR; SHM, somatic hypermutation; TD, T cell–dependent; TI, T cell– dition, recent evidence from other groups demonstrated that independent; TNP, trinitrophenyl; TRAF, TNFR-associated factor; Treg, regulatory TRAF3 regulates the effector function of regulatory T cells T cell. (Tregs) (11) and that TRAF3 is required for the development of Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 invariant NKT cells (12). Thus, TRAF3 plays distinct and pivotal www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401548 The Journal of Immunology 335 roles in regulating the development and function of different Southern Biotechnology Associates (Birmingham, AL). Neutrophil subsets of immune cells. purification kits were from Miltenyi Biotec (Auburn, CA). Tissue Myeloid cells, including granulocytes, monocytes, macro- culture supplements including stock solutions of sodium pyruvate, L-glutamine, nonessential amino acids, and HEPES (pH 7.55) were phages, and dendritic cells (DCs), are crucial determinants of innate from Invitrogen (Carlsbad, CA). DNA oligonucleotide primers were immunity and inflammation, and also play essential roles in Ag obtained from Integrated DNA Technologies (Coralville, IA). Alka- presentation, as well as the effector phase of adaptive immunity. line phosphatase substrates were purchased from Sigma-Aldrich These cells constitutively or inducibly express a number of (St. Louis, MO). receptors of the TNFR, TLR, NLR, and RLR families, whose Flow cytometry signals are regulated by TRAF3 (1, 2). Although in vitro evidence Single-cell suspensions were made from the spleen, bone marrow (BM), and indicates that TRAF3 is required for TLR-induced type I IFN peritoneal lavage. Immunofluorescence staining and FACS analyses were production (13, 14) and for CD40-induced IL-12 production in performed as previously described (7, 8). Erythrocytes from spleen were macrophages (15), the in vivo functions of TRAF3 in myeloid depleted with ACK lysis buffer. Cells (1 3 106) were blocked with rat cells remain unclear. In this study, we generated TRAF3flox/flox serum and FcR blocking Ab (2.4G2), and incubated with various Abs LysM+/Cre myeloid cell–specific TRAF3-deficient (M-TRAF32/2) conjugated to FITC, PE, PerCP, or Cy5 for multiple color fluorescence surface staining. Analyses of cell-surface markers included Abs to CD3, mice to evaluate the functions of TRAF3 in innate immunity and CD4, CD8, CD45R (B220), CD19, IgM, CD11b, Ly6C, Ly6G, CD115, F4/ inflammation mediated by myeloid cells. Cre expression driven by 80, Siglec-F, CD68, CD11c, CD317, NK1.1, CD49b, CD21, CD23, CD5, the lysozyme M (LysM) promoter mediates deletion of TRAF3 CD44, CD62L, CD69, CD80, and MHC class II. List-mode data were from neutrophils, eosinophils, basophils, monocytes, macro- acquired on a FACSCalibur (Becton Dickinson, Mountain View, CA) using Cell Quest software. The results were analyzed using FlowJo software phages, and monocyte-derived DCs, but not plasmacytoid DCs (Tree Star, San Carlos, CA). Forward scatter/side light scatter gating (pDCs) (16, 17). We report in this article that deletion of TRAF3 was used to identify live cells. in myeloid cells resulted in altered systemic responses to injec- tions with LPS (an agonist of TLR4) or polyinosinic-polycytidylic Culture of bone marrow–derived macrophages and peritoneal acid (polyI:C, an agonist of TLR3), as well as T cell–independent exudate macrophages 2/2 (TI) and TD Ags. Furthermore, we found that M-TRAF3 mice Bone marrow cells were harvested from 7- to 10-wk-old littermate control spontaneously experienced development of inflammation, infec- (LMC) or M-TRAF32/2 mice. Bone marrow–derived macrophages tion, and tumors between 15 and 22 mo of age. Taken together, (BMDMs) were prepared by culturing BM cells in complete RPMI 1640 our findings demonstrate obligatory and indispensable roles for medium (Invitrogen) supplemented with 20% conditioned medium from myeloid cell TRAF3 in inhibiting inflammation and tumor de- L929 cells overexpressing M-CSF for 7 d as described previously (18–20). For preparation of peritoneal exudate macrophages (PEMs), 7- to 10-wk- velopment. old LMC or M-TRAF32/2 mice were injected i.p. with 3 ml of 4% thi- oglycollate (Becton Dickinson), and cells were harvested by peritoneal lavage on day 4 postinjection as described previously (18). The cells were Materials and Methods washed, macrophages were allowed to adhere to the tissue culture plates 2/2 Generation of M-TRAF3 mice for 2 h, and nonadherent cells were removed. TRAF3flox/flox mice were generated as previously described (7). The TRAF3flox/flox line was backcrossed with C57BL/6J (B6) mice (Jackson Preparation of thioglycollate-elicited peritoneal neutrophils flox/flox Laboratory) for more than nine generations to generate TRAF3 mice Seven- to 10-wk-old LMC or M-TRAF32/2 mice were injected i.p. with on the B6 genetic background. These mice were subsequently bred with 3 ml of 4% thioglycollate (BD), and cells were harvested by peritoneal B6 mice transgenic for LysM-driven Cre expression (stock no. 4781; lavage at 18 h postinjection. Neutrophils were purified from peritoneal +/flox +/Cre Jackson Laboratory). TRAF3 LysM mice were backcrossed with cells using anti–Ly-6G-Biotin and anti-Biotin magnetic beads following flox/flox flox/flox +/Cre 2/2 TRAF3 B6 mice to generate TRAF3 LysM (M-TRAF3 ) the manufacturer’s protocol (Miltenyi). Purified neutrophils were mice. Mouse tails were screened by genomic PCR using primer sets (FC3 + resuspended in RPMI 1640 medium containing 5% FCS and aliquoted BT6) (7), (Lys-Com + Lys-WT), and (Lys-Com + Lys-Cre) (16) as de- for stimulation. scribed previously. Deletion of exons 1 and 2 of the TRAF3 gene in peritoneal macrophages was detected by genomic PCR using primers U7 TLR4 signaling and BT6 as previously described (7). All experimental mice for this study were subsequently produced by breeding of TRAF3flox/flox mice with Before stimulation with LPS, BMDMs or PEMs were cultured in 2.5% FCS TRAF3flox/floxLysM+/Cre mice, and TRAF3flox/flox littermates were used as medium for 2 h. BMDMs, PEMs, or neutrophils were stimulated with 100 controls for all experiments. All mice were kept in specific pathogen-free ng/ml LPS. Total protein lysates were prepared at different time points as conditions in the Animal Facility at Rutgers University and were used in previously described (21) for measurements of early signaling events by accordance with National Institutes of Health guidelines and under an immunoblot analysis. Total cellular RNA was extracted at different time animal protocol (Protocol #08-048) approved by the Animal Care and Use points for measurement of cytokine transcript levels by real-time quanti- Committee of Rutgers University. tative PCR (RT-qPCR) using TaqMan assay. Culture supernatants were collected at different time points for measurements of cytokine protein Abs and reagents levels by ELISA. Polyclonal rabbit Abs to TRAF1 (N19), TRAF3 (H122), TRAF6 (H274), TaqMan assays of Ifnb, Ifna4, Il12a, Il6, and Tnfa transcripts and RelB were from Santa Cruz Biotechnology (Santa Cruz, CA). Poly- clonal rabbit Ab to TRAF2 was from Medical and Biological Laboratories Total cellular RNA was extracted using TRIzol reagent (Invitrogen, (Nagoya, Japan). FITC-, PE-, or Cy5-labeled Abs against mouse CD3, CD4, Carlsbad, CA) according to the manufacturer’s protocol. cDNA was pre- CD8, CD45R (B220), CD19, IgM, CD11b, Ly6C, Ly6G, CD115, F4/80, pared from RNA using the High Capacity cDNA Reverse Transcription Kit Siglec-F, CD68, CD11c, NK1.1, CD49b, CD21, CD23, CD5, CD44, (Applied Biosystems, Foster City, CA). RT-qPCR was performed using CD62L, CD69, CD80, and MHC class II were purchased from eBioscience TaqMan Gene Assay (Applied Biosystems) as described previously (San Diego, CA). Anti-mouse IL-6, IL-12, and TNF-a ELISA Ab pairs (22). TaqMan primers and probes (FAM-labeled) specific for individual were also purchased from eBioscience. Anti-mouse IL-1b and IL-10 mouse were used in the PCR reaction to detect Ifnb, Ifna4, Il12a, ELISA Ab pairs were also purchased from R&D Systems (Minneapolis, Il6,orTnfa mRNA. Each reaction also included primers and a probe (VIC- MN). Polyclonal rabbit Abs against total or phosphorylated IFN regulatory labeled) specific for mouse Actb (b-actin) mRNA, which served as an factor 3 (IRF3), p38, ERK, JNK, IkBa, Akt, and NF-kB2 were from Cell endogenous control. Reactions were performed on a 7500 Fast Real-Time Signaling Technology (Beverly, MA). Anti-actin Ab was from Chemicon PCR System (Applied Biosystems). Relative mRNA expression levels of (Temecula, CA). HRP-labeled secondary Abs were from Jackson Immu- cytokines were analyzed using Sequencing Detection Software (Applied noresearch Laboratories (West Grove, PA). Alkaline phosphatase–conju- Biosystems) and the comparative cycle threshold (Ct; DDCt) method fol- gated polyclonal goat Abs specific for mouse Ig isotypes were from lowing the manufacturer’s procedures. 336 IN VIVO FUNCTIONS OF TRAF3 IN MYELOID CELLS

t p DCt5Ct of cytokine 2 Ct of b actin: cance was determined with the unpaired test for two-tailed data. The values ,0.05 were considered significant, and p values ,0.001 were considered highly significant. DDCt5DCt of each sample 2 DCt of calibrator sample: For each cytokine assay, the calibrator sample was the one with lowest Results detectable RNA level (highest Ct value). Duplicate PCRs were performed 2/2 for each biologic sample. Validation of M-TRAF3 mice Previous in vitro studies indicated that TRAF3 regulates type I Cytokine ELISA IFN and proinflammatory cytokine production in myeloid cells (13, Concentrations of IL-6, IL-12, and TNF-a in culture supernatants or mouse 14). However, the in vivo functions of TRAF3 in myeloid cells sera were determined by quantitative ELISA using cytokine-specific coating had not been determined. To address this issue, we generated Abs and biotinylated detection Abs (eBioscience) as previously described 2/2 flox/flox (10, 22). Mouse serum levels of IL-10 and IL-1b were analyzed by ELISA M-TRAF3 mice by crossing TRAF3 mice with mice using cytokine-specific coating Abs and biotinylated detection Abs (R&D) as that express Cre under the control of endogenous LysM regulatory previously described (10, 22). Levels of IFN-b in mouse sera were measured elements (LysM-Cre mice), which allows the specific deletion of using the VeriKine-HS Mouse IFN-b Serum ELISA Kit (PBL Assay Sci- floxed in myeloid cells (16). Progeny were born at expected ence, Piscataway, NJ) following the manufacturer’s protocol. Mendelian frequencies with no developmental abnormalities In vivo responses to challenges with LPS or polyI:C noted, and they matured and bred normally. 2 2 LysM-driven Cre expression has been shown to induce specific Age- and sex-matched 8- to 12-wk-old LMC and M-TRAF3 / mice were injected i.p. with LPS (E. coli 0127:B8; 300 mg LPS/20 g body weight; and highly efficient deletion of loxP-flanked gene segments in Sigma-Aldrich) or polyI:C (200 mg polyI:C/20 g body weight; Invivogen). mature neutrophils and macrophages with a higher deletion effi- Sera were collected at 2 h and 6 h postinjection, and cytokine levels in sera ciency in neutrophils (16, 17, 29). In contrast, LysM-Cre–mediated were analyzed by ELISA as described earlier. deletion does not occur in T cells, B cells, NK cells, and pDCs, Immunoblot analysis despite the presence of LysM activity in a small population of the hematopoietic stem cells (30). Consistent with previous studies, Total protein lysates were prepared as described previously (21). Proteins we verified highly efficient deletion of the Traf3 gene and a cor- were separated by SDS-PAGE and immunoblotted with Abs to phos- phorylatedortotalIRF3,p38,ERK,JNK,Akt,IkBa,NF-kB2, RelB, responding loss of TRAF3 protein (80–90% reduction) in perito- 2 2 TRAF3, and actin. Immunoblot analysis was performed using Abs as neal macrophages and BMDMs prepared from M-TRAF3 / previously described (8, 21). Images of immunoblots were acquired using mice as determined by genomic PCR and Western blot analysis a low-light imaging system (LAS-4000 mini; FUJIFILM Medical Systems (Fig. 1A and data not shown). No deletion of Traf3 was observed USA, Stamford, CT) (8, 21). in splenic T and B cells of M-TRAF32/2 mice (data not shown). Immunizations and Ig ELISAs Interestingly, we found that TRAF1 and TRAF2 protein levels Basal serum levels of various Ig isotypes in naive 8- to 12-wk-old LMC and were modestly increased in TRAF3-deficient macrophages M-TRAF32/2 mice were analyzed by ELISA as previously described (7). (Fig. 1A). In contrast, TRAF6 protein levels were unchanged. We For TI Ab responses, age- and sex-matched 8- to 12-wk-old LMC and previously had observed modestly increased TRAF1 and TRAF2 2/2 M-TRAF3 mice were immunized i.p. with 50 mg trinitrophenyl (TNP)- protein levels in TRAF32/2 B cells and T cells (7, 10). These Ficoll (Biosource Technologies, Vacaville, CA), and sera were collected on findings raise the possibility that increased TRAF1 and TRAF2 day 7 after immunization. For TD Ab responses, age- and sex-matched 8- to 12-wk-old LMC and M-TRAF32/2 mice were injected i.p. with 100 mg may partially compensate for the loss of TRAF3 in these cell TNP-keyhole limpet hemocyanin (KLH; Biosource Technologies) in an types. Together, these data validated TRAF3flox/floxLysM+/Cre mice equal volume of Imject Alum (Thermo Scientific, Rockford, IL), and sera as M-TRAF32/2 mice. were collected on day 7 after immunization. Serum TNP-specific Ig iso- types were measured by ELISA as previously described (7). Plates were 2/2 read on a Versamax plate reader (Molecular Devices, Sunnyvale, CA), and Young adult M-TRAF3 mice have normal populations of results were analyzed by using SoftMax Pro 4.0 software as described lymphoid and myeloid cells previously (7). We previously reported that TRAF3 regulates mature B cell ho- Mouse disease monitoring and histology meostasis in secondary lymphoid organs (7). To investigate whether TRAF3 is involved in the maturation and homeostasis of Mice were monitored daily for signs of illness including weight loss, labored breathing, hunched posture, and paralysis or spontaneous tumor formation myeloid cells, we first examined the size and cell numbers of 2/2 indicated by enlarged lymph nodes (LNs) or abdomen (8). Necropsy and lymphoid organs of 8- to 12-wk-old M-TRAF3 and TRAF3- H&E staining of formalin-fixed, paraffin-embedded tissues were per- sufficient LMC mice (TRAF3flox/flox, LMC). We found that formed to assess the presence of phenotypic abnormalities as described M-TRAF32/2 mice had normal-sized spleens, LNs, and thymi, previously (7, 8, 23–28). Bright-field micrographs of stained sections were taken using a microscope (Olympus BX-51; Olympus America, Center and also had normal numbers of cells in the BM and peritoneum Valley, PA). (Supplemental Table I and data not shown). We next used flow cytometry to characterize lymphocyte and myeloid cell pop- Mouse cytokine protein array analyses ulations in the BM, spleen, and peritoneum. Our data demon- 2 2 Sera were collected from 15- to 22-mo-old LMC and diseased M-TRAF3 / strated that M-TRAF32/2 and LMC mice had similar proportions mice. Serum levels of cytokines and were measured using and numbers of splenic follicular and marginal zone B cell subsets a Mouse Cytokine Array Panel A kit (R&D, Minneapolis, MN) following the manufacturer’s instructions. Images of the blots were acquired, and quantitative and CD4 and CD8 T cells. This was also true for comparisons of analyses of cytokine or spots were performed using a low-light granulocytes, monocytes, and macrophages in the BM, spleen, and imaging system (LAS-4000 mini; FUJIFILM Medical Systems USA) (8, 21). peritoneal lavage (Fig. 1B, 1C, and Supplemental Table I). In- 2/2 + + Statistics terestingly, TRAF3 and LMC CD11b F4/80 macrophages in the spleen and peritoneal cavity also expressed comparable levels Statistical analyses were performed using Prism software (GraphPad, La of MHC class II, CD80, and CD86, suggesting that macrophages Jolla, CA). Survival curves were generated using the Kaplan–Meier method, and were compared using a log- (Mantel–Cox) test to determine were not activated as a result of TRAF3 deficiency in mice of this whether differences are significant. For direct comparison of cytokine or Ig age (data not shown). In addition, after i.p. injection of thio- isotype levels between LMC and M-TRAF32/2 mice, statistical signifi- glycollate, the numbers of neutrophils (analyzed at 2, 4, 6, 12, or The Journal of Immunology 337 338 IN VIVO FUNCTIONS OF TRAF3 IN MYELOID CELLS

18 h postinjection) and macrophages (analyzed at day 3 or day 4 injection (Fig. 3A). Similarly, serum levels of IL-12 were also post injection) recruited to the peritoneal cavity were also com- significantly increased in M-TRAF32/2 mice at 2 h after injection parable between M-TRAF32/2 and LMC mice (data not shown). with polyI:C (Fig. 3B). In contrast, serum levels of the anti- Collectively, our findings indicate that specific ablation of TRAF3 inflammatory cytokine IL-10 were markedly decreased in in myeloid cells did not affect maturation, homeostasis, or mi- M-TRAF32/2 micecomparedwithLMCmiceat2and6hafterLPS gration of macrophages and neutrophils and, as expected, had no injection (Fig. 3A). These results suggest that TRAF3 inactivation effects on T and B cells. in myeloid cells alters cytokine production profiles to favor proin- 2 2 flammatory responses after in vivo challenges with LPS or polyI:C. Altered cytokine production in TRAF3 / BMDMs and PEMs in response to LPS stimulation Proximal signaling events downstream of LPS/TLR4 It was shown previously that LPS-induced type I IFN production is engagement in TRAF3-deficient BMDMs and neutrophils abolished, but IL-12 production is enhanced, in BMDMs derived To understand how TRAF3 deficiency affects LPS-induced cyto- from chimeric mice reconstituted with TRAF32/2 fetal liver cells kine production in myeloid cells, we investigated proximal sig- (13, 14). We therefore analyzed LPS-induced cytokine production naling events after TLR4 engagement by LPS in TRAF3-deficient in BMDMs and PEMs derived from M-TRAF32/2 mice by RT- BMDMs and neutrophils. Purified thioglycollate-elicited neu- qPCR and ELISA. Consistent with published studies, we found that trophils and BMDMs were stimulated with LPS, and total cellular LPS-induced expression of Ifnb and Ifna4 was almost abolished in proteins were prepared from both cell types at different time points. BMDMs and PEMs derived from M-TRAF32/2 mice (Fig. 2A, Phosphorylation of proximal signaling components of TLR4 in- 2B). Interestingly, production of IL-12 was enhanced by TRAF3 cluding IRF3, p38, ERK, JNK, Akt, and IkBa were examined by deficiency at both the transcript and protein levels (Fig. 2). In ad- immunoblot analyses. We found that LPS-induced phosphoryla- dition, although transcription of Il6 was not changed (Fig. 2A, 2B), tion of IRF3, the key transcription factor driving type I IFN ex- secretion of IL-6 was significantly increased in TRAF32/2 BMDMs pression, was markedly reduced in TRAF32/2 BMDMs (Fig. 4A). and PEMs (Fig. 2C, 2D). In contrast, LPS-induced production of We did not detect any induction of IRF3 phosphorylation by LPS TNF-a in either cell population was not affected by TRAF3 dele- in LMC or TRAF32/2 neutrophils (data not shown). We found tion (Fig. 2). Taken together, our results showed that in response to that LPS-induced phosphorylation of p38, ERK, JNK, and Akt LPS stimulation, TRAF3-deficient macrophages exhibited alter- was normal in TRAF32/2 BMDMs and neutrophils (Fig. 4A, 4B). ations in expression of some cytokines, but not others. These studies Similarly, activation of the classical NF-kB1 pathway was not validated M-TRAF32/2 mice as a suitable model to explore the affected by TRAF3 deletion in either cell type as measured by in vivo functions of myeloid cell TRAF3. phosphorylation and degradation of IkBa after stimulation with 2 2 LPS (Fig. 4A, 4B). We previously found that TRAF3 deficiency Enhanced proinflammatory responses in M-TRAF3 / mice resulted in constitutive NF-kB2 activation in B cells, leading to challenged with LPS or polyI:C prolonged B cell survival (7, 8). We therefore compared NF-kB2 Myeloid cells provide the first line of defense against bacterial and processing in TRAF32/2 and LMC BMDMs and neutrophils. viral infections by producing type I IFN and proinflammatory Interestingly, in the absence of stimulation, TRAF3-deficient cells cytokines. In light of our in vitro evidence for TRAF3 regulation of exhibited constitutive processing of NF-kB2 from the inactive cytokine production in BMDMs and PEMs, we investigated the precursor p100 to the active p52, which was as robust as that 2/2 in vivo responses of M-TRAF3 mice inoculated with LPS or observed in LMC cells after stimulation with LPS (Fig. 4A, 4B). polyI:C, which mimic aspects of bacterial and viral infections, However, in contrast with TRAF3-deficient B cells, constitutive respectively. We measured serum levels of IFN-b, as well as NF-kB2 processing did not result in prolonged survival or de- proinflammatory and anti-inflammatory cytokines, at different creased apoptosis in TRAF32/2 BMDMs or neutrophils (data not times after injection. Our results demonstrated that in response to shown). Collectively, our findings indicate that TRAF3 deficiency LPS, serum levels of the proinflammatory cytokines TNF-a and leads to constitutive activation of NF-kB2 in both BMDMs and 2/2 IL-1b were comparable in M-TRAF3 and LMC mice neutrophils, and specifically impairs LPS-induced activation of (Fig. 3A). In contrast with the reduced in vitro inducibility of Ifnb IRF3 in BMDMs, thereby modulating their cytokine production. 2/2 observed in LPS-simulated BMDMs and PEMs of M-TRAF3 2/2 mice, serum levels of IFN-b were not decreased in M-TRAF32/2 Enhanced TI and TD IgG responses in M-TRAF3 mice mice inoculated with LPS or polyI:C (Fig. 3A). The normal In addition to serving as primary players in innate immunity and in vivo production of IFN-b may reflect the lack of TRAF3 de- inflammation, myeloid cells also play important roles in Ag pre- letion in pDCs, which are the most potent producers of type I IFN sentation to activate adaptive immunity. We therefore measured after TLR ligation. Interestingly, levels of the proinflammatory basal Ig isotype levels in sera from 8- to 12-wk-old M-TRAF32/2 cytokines IL-6 and IL-12 in sera of M-TRAF32/2 mice were ele- and LMC mice, and found that the levels of IgM, IgG1, IgG2b, vated at 2 h, and IL-12 levels were also increased at 6 h after LPS IgG3, IgA, and IgE were similar in both cohorts (Fig. 5A). After

FIGURE 1. Normal lymphocyte and myeloid cell populations in young adult M-TRAF32/2 mice. TRAF3flox/flox LMC and M-TRAF32/2 mice analyzed were 8–12 wk old. (A) Verification of TRAF3 deletion in BMDMs by Western blot analysis. Total cellular proteins were prepared from BMDMs derived from three pairs of LMC and M-TRAF32/2 mice. Proteins were immunoblotted for TRAF3, followed by TRAF2, TRAF1, TRAF6, and actin. (B) Representative FACS profiles of peritoneal lavage prepared from LMC and M-TRAF32/2 mice. Cells were stained with fluorochrome-conjugated Abs specific for CD11b, F4/80, and CD68, and then analyzed by a FACSCalibur. Peritoneal macrophages are identified as CD11b+F4/80+CD68+.(C) Rep- resentative FACS profiles of splenocytes prepared from LMC and M-TRAF32/2 mice. Cells were stained with fluorochrome-conjugated Abs specific for B220, CD3, CD11b, F4/80, CD11c, MHC class II, CD21, CD23, Ly6G, Siglec-F, and CD317 (PDCA-1), and then analyzed by a FACSCalibur. FACS profiles were scatter-gated on live cells. Gated cell populations include B cells, B220+CD32; T cells, CD3+B2202; red pulp macrophages, CD11b+F4/80+; conventional DCs, CD11c+MHC class II+; follicular B cells, B220+CD21IntCD23+; marginal zone (MZ) B cells, B220+CD21+CD23Int; neutrophils, CD11b+Ly6G+Siglec-F2; pDCs, B220+CD317 (PDCA-1)+. Similar results were observed in four additional experiments. The Journal of Immunology 339

FIGURE 2. Impaired type I IFN but enhanced IL-6 and IL-12 production in TRAF32/2 macrophages in response to LPS stimulation. (A and B) LPS-induced mRNA expression of cytokines in macrophages. BMDMs (A) or PEMs (B) derived from M-TRAF32/2 and LMC mice (7–10 wk old) were cultured in the absence or presence of 100 ng/ml LPS for 2 or 4 h. RNA was extracted, and real-time PCR was performed using TaqMan primers and probes specific for Ifnb, Ifna4, Il6, Il12a (p35), and Tnfa. Each reaction also included the probe (VIC-labeled) and primers for b- actin mRNA, which served as an endogenous control. Relative mRNA expression levels of each cytokine were analyzed using the Sequence Detection Software (Applied Biosystems) and the comparative Ct method. Graphs depict the results of three independent experi- ments with duplicate reactions in each experiment (mean 6 SD). (C and D) LPS-induced secretion of cytokines in macrophages. BMDMs (C) or PEMs (D) derived from M-TRAF32/2 and LMC mice (7–10 wk old) were cultured in the absence or presence of 100 ng/ml LPS for indicated time periods. The levels of IL- 6, IL-12, and TNF-a in culture supernatants were an- alyzed by ELISA. Graphs depict the results of three independent experiments with duplicate samples in each experiment (mean 6 SD). *p , 0.05 (t test, sig- nificantly different between LMC and M-TRAF32/2), **p , 0.01 (t test, very significantly different bet- ween LMC and M-TRAF32/2), ***p , 0.001 (t test, highly significantly different between LMC and M-TRAF32/2).

immunization with the TI Ag, TNP-Ficoll, M-TRAF32/2 mice Spontaneous inflammation, infection, and tumor development tended to have slightly greater levels of all TNP-specific isotypes in aging M-TRAF32/2 mice than LMC mice, although only IgG3 differences were significant In light of understandings that elevated proinflammatory cytokines (Fig. 5B). In contrast, after immunization with the TD Ag, TNP- contribute to inflammatory diseases, we hypothesized that KLH, only TNP-specific IgG2b levels were significantly increased M-TRAF32/2 mice should be predisposed to inflammation. We 2/2 in sera of M-TRAF3 as compared with LMC mice (Fig. 5C). thus monitored the health of aging M-TRAF32/2 and LMC mice. We Collectively, these results indicate that although baseline levels of first noticed that beginning around 10 mo of age, mortality of 2 2 serum Igs were similar in M-TRAF3 / and LMC mice, the M-TRAF32/2 mice was greatly accelerated over that of LMC (Fig. 6A). responses to TI and TD Ags were altered in M-TRAF32/2 mice. Histopathologic studies were performed on 22 M-TRAF32/2 mice

FIGURE 3. In vivo cytokine pro- duction in response to injection with LPS or polyI:C. Sex- and age- matched M-TRAF32/2 and LMC mice (8–12 wk old) were i.p. in- jected with (A) LPS (300 mg LPS/20 g body weight, n = 9 for each group) or (B) polyI:C (200 mg polyI:C/20 g body weight, n = 4 for each group), and sera were collected at 2 and 6 h postinjection. Serum levels of IFN-b, IL-6, IL-12, IL-10, IL-1b, and TNF-a were measured by ELISA. *p , 0.05 (t test, signifi- cantly different between LMC and M-TRAF32/2), **p , 0.01 (t test, very significantly different between LMC and M-TRAF32/2), ***p , 0.001 (t test, highly significantly differ- ent between LMC and M-TRAF32/2). 340 IN VIVO FUNCTIONS OF TRAF3 IN MYELOID CELLS

FIGURE 4. Altered signaling events in TRAF32/2 BMDMs and neutrophils in response to LPS stimulation. BMDMs (A) or thioglycollate-elicited peritoneal neutrophils (B) prepared from M-TRAF32/2 and LMC mice (8–12 wk old) were stimulated with 100 ng/ml LPS for indicated time periods. Total protein lysates were prepared, and signaling events of TLR4 were analyzed by immunoblot analysis. Proteins were immunoblotted for phosphorylated (P-) or total IRF-3, p38, ERK, JNK, Akt, IkBa, followed by NF- kB2 (p100 and p52), RelB, TRAF3, and actin. Results shown are representative of three independent experiments.

of 15–22 mo old. We found that 15 of the 22 (68.2%) mice displayed the mouse with hepatocellular adenoma, the liver was massively oc- inflammation, infection, or tumors involving multiple organs, features cupied by liver cancer cells in association with large cytoplasmic that were not observed in age-matched LMC mice (Fig. 6B, Table I). vacuoles (Supplemental Fig. 1). In mice with DLBCL, lymphoma Tumorsdevelopedin12mice,including 2 cases of histiocytic sarco- cells were responsible for marked expansion of the splenic white pulp mas (histiocytic neoplasms, tumors of a type of tissue-resident mac- and infiltration of the liver (Fig. 6C). In mice with inflammation, the rophages), 1 hepatocellular adenoma, and 9 B cell lymphomas majority of splenic lymphocytes were replaced with myeloid cells, (Table I). Lymphomas observed in M-TRAF32/2 mice were diffuse and granulocyte infiltration was observed in multiple organs, includ- large B cell lymphomas (DLBCLs) or follicular lymphomas (FLs). ing the liver, intestine, lung, and pancreas (Supplemental Fig. 2 and Six M-TRAF32/2 mice displayed inflammation involving multiple data not shown). In addition, glomerular damage was observed in the organs, including the liver, gastrointestinal (GI) tract, lung, kidney, kidneys of mice with inflammation (Supplemental Fig. 2). In mice pancreas, and heart (Table I). Three M-TRAF32/2 mice had infections with infection and inflammation, the splenic architecture was also in the intestine/colon, liver, and lung (Table I). Notably, seven disrupted by myeloid cells, and the liver contained clustered inflam- M-TRAF32/2 mice had internal hemorrhages and two had blistering matory cells, including histiocytes and necrotic areas (Fig. 6C). We (Table I). Interestingly, individual M-TRAF32/2 mice often had more observed two cases of bacterial abscesses (Fig. 6B), in which bacteria than one type of pathology. Examples include B cell lymphoma and lung were clearly identified in micrographs (Supplemental Fig. 3A, 3B). inflammation, pancreatitis and hepatocellular adenoma, and bacterial Interestingly, we also detected one case of infection by Entamoeba infection and hemorrhagic liver. Together, our findings indicate that muris, a strain of commensal protozoan parasite, in cecum (Sup- specific ablation of TRAF3 in myeloid cells contributed to spontaneous plemental Fig. 3C). Taken together, the splenic red pulp and white development of different types of tumors, inflammation, and infection. pulp were disrupted in mice with histiocytic sarcoma, DLBCL, inflam- mation, or infection, and other affected organs also exhibited infiltra- 2/2 Histopathologic features of M-TRAF3 mice with tion with tumor or myeloid cells. spontaneous inflammation, infection, or tumor development 2/2 Flow cytometry reveals drastically altered lymphocyte and Histologically, affected spleens of M-TRAF3 mice with 2/2 spontaneous inflammation, infection, or tumor development were myeloid cell populations in M-TRAF3 mice with most often characterized by a near-complete loss of normal ar- spontaneous inflammation, infection, or tumors chitecture (Fig. 6C). In mice with histiocytic sarcomas, tumor To detect potential alterations of lymphocyte and myeloid cell cells were identified as the major cell type in the spleen, and nodal populations underlying the earlier disease conditions, we per- areas of pure histiocytosis were also observed in the liver (Fig. 6C). In formed immunophenotypic analyses using flow cytometry. In The Journal of Immunology 341

FIGURE 5. Altered Ab responses in M-TRAF32/2 mice. LMC and M-TRAF32/2 mice analyzed were 8–12 wk old. (A) Basal serum titers of Ig isotypes. Sera from naive LMC and M-TRAF32/2 mice (n = 8 for each group) were tested for IgM, IgG1, IgG2b, IgG3, IgA, and IgE levels by ELISA. (B)TIAb responses. LMC and M-TRAF32/2 mice (n = 9 for each group) were immunized with the TI Ag TNP-Ficoll, and sera were collected on day 7 after immunization. Serum titers of anti-TNP IgM, IgG1, IgG2b, IgG3, IgA, and IgE were analyzed by ELISA. (C) TD Ab responses. LMC and M-TRAF32/2 mice (n = 12 for each group) were immunized with the TD Ag TNP-KLH in Alum. Sera were collected on day 7 after immunization. Serum titers of anti- TNP IgM, IgG1, IgG2b, IgG3, IgA, and IgE were measured by ELISA. Multiple serial dilutions of each serum sample were tested to ensure that the readout is within the linear range of the assay. *p , 0.05 (t test, significantly different from LMC). sharp contrast with the normal spleen size observed in young 8- to features of altered cell populations observed in spleens of different 12-wk-old M-TRAF32/2 mice or old LMC mice, diseased individual M-TRAF32/2 mice with spontaneous inflammation, M-TRAF32/2 mice had greatly enlarged spleens as evidenced infection, or tumor development include increased percentages of by significantly increased spleen weights (Fig. 7A). Similarly, CD11b+Gr-1+ myeloid cells and decreased percentages of CD3+ the splenic cell populations in M-TRAF32/2 mice with sponta- B2202 T lymphocytes (Fig. 7C). In mice with DLBCL, splenic B neous inflammation, infection, or tumor development were altered lymphoma cells were B220+CD212CD232 (Fig. 7D). These with increased populations of B2202CD32CD11b+/lowGr-1+/low results suggest that TRAF3 deficiency in myeloid cells promotes myeloid cells (Fig. 7B). Consistent with the histologic diagnoses, inflammation but also greatly compromises the ability of mice to the frequencies of normal B and T cells observed in LMC mice resist infections or control development of hematopoietic and were often diminished in diseased M-TRAF32/2 mice. In mice with solid tumors. histiocytic sarcomas, splenic lymphocytes were mainly replaced by 2 2 low low Aberrant production of cytokines and chemokines in diseased histiocytic tumor cells (B220 CD3 CD11b Gr-1 ;Fig.7B).In 2/2 mice with inflammation, most lymphocytes were replaced with M-TRAF3 mice a major population of Gr-1+CD11b+ myeloid cells in the spleen To gain deeper understandings of diseased M-TRAF32/2 mice, we (Fig. 7B). In mice with infections, splenic lymphocytes were also measured the serum levels of 40 cytokines and chemokines using replaced with CD11b+Gr-1+ myeloid cells or B2202CD32 a Mouse Cytokine Array assay kit. Our results demonstrated that CD11blowGr-1low histiocytes (Fig. 7B). In summary, common M-TRAF32/2 mice with spontaneous tumor development or 342 IN VIVO FUNCTIONS OF TRAF3 IN MYELOID CELLS

FIGURE 6. Gross and histopathologic features of M-TRAF32/2 mice with spontaneous tumor, inflammation, or infection. (A) Accelerated mortality of M-TRAF32/2 mice. Survival curves of LMC (CTL) and M-TRAF32/2 mice were generated using the Kaplan–Meier method. p , 0.001 as determined by the Mantel–Cox log-rank test. (B) Representative images of affected organs in diseased M-TRAF32/2 mice. (a) Massively enlarged mesenteric LN in a mouse with DLBCL (mouse ID: 237-2). (b) GI tract and mesenteric LN of a mouse with bacterial infection and inflammation (mouse ID: 228-5). Arrow indicates the large bacterial intestinal abscess. (c) Liver of a mouse with hepatocellular adenoma (mouse ID: 237-4). (C) Representative micrographs of the spleen and liver of diseased M-TRAF32/2 mice. Sections of the spleen and liver were stained with H&E, and representative micrographs of LMC and M-TRAF32/2 mice are shown for comparison. (a) Normal tissues from an LMC mouse. (b) Mouse (ID: 237-5) with extensive histiocytosis and increased erythroid activity in the spleen and areas of pure histiocytosis in the liver. (c) Mouse (ID: 233-5) with DLBCL showing marked enlargement of the splenic white pulp by tumor cells and large perivascular infiltrates with tumor cells in the liver. (d) Mouse (ID: 274-12) with infection showing inflammation and necrotic areas in the liver and marked red pulp hyperplasia of myeloid and erythroid elements in the spleen. inflammation/infection exhibited strikingly increased serum levels responses (33). Four chemokines, including CCL1, MCP-1 (CCL2), of a number of chemokines and cytokines, including CXCL-13, MCP-5 (CCL12), and IFN-g–induced protein 10 (CXCL10), all G-CSF, CCL1, IL-16, IL-17, IFN-g–induced protein 10, monocyte act as chemoattractants to recruit monocytes/macrophages, DCs, chemotactic protein (MCP)-1, MCP-5, CXCL9, tissue inhibitor of and lymphocytes to the sites of inflammation (34–40). Interest- metalloproteinases 1, and triggering receptor expressed on mye- ingly, triggering receptor expressed on myeloid cells 1, a trig- loid cells 1 (Fig. 8). Among these, G-CSF stimulates the bone gering receptor expressed on myeloid cells, plays a role in promoting marrow to produce granulocytes and release them into the blood- inflammatory responses mediated by monocytes/macrophages stream, and also promotes the survival, proliferation, differentiation, and neutrophils (41, 42). These cytokines and chemokines are and function of neutrophil precursors and mature neutrophils (31, also implicated in the pathogenesis of various inflammatory diseases 32). IL-17 is potent in inducing and mediating proinflammatory and tumor development (31–42), consistent with the phenotypes The Journal of Immunology 343

Table I. Summary of histologic observations of M-TRAF32/2 mice at the age of 15–22 mo

Mice Examined (N =22) n Organs Involved Tumor Histiocytic sarcoma 2 Spleen, liver Hepatocellular adenoma 1 Liver B cell lymphoma (DLBCL or FL) 9 Spleen, cervical LNs, mesenteric LN, liver Inflammation 6 Liver, GI tract, lung, kidney, pancreas, heart Infection 3 GI tract, liver, lung Unknown Hemorrhage 7 Blister 2 Normal 7 Histologic diagnoses were made based on established criteria (23–28). Mice with DLBCL and FL were diagnosed histo- logically using criteria outlined in a consensus nomenclature of mouse lymphoid neoplasms (23). Malignancies of histiocytes (histiocytic sarcoma) were diagnosed histologically as described previously (24). Inflammation was diagnosed histologically using criteria of myeloid cell/lymphocyte infiltration in tissues as described previously (25). Cases diagnosed with inflammatory conditions exhibited expansions of immature myeloid cells, monocytes, and granulocytes in tissues outside the bone marrow and often in spleen or liver. Infections were diagnosed with evidence for bacterial or parasitic infection in different tissues, including visualization of bacteria and entamoeba as described previously (26–28). Individual mice could have more than one type of pathology. Examples include B cell lymphoma and lung inflammation, pancreatitis and hepatocellular adenoma, and bacterial infection and hemorrhagic liver. observed in our M-TRAF32/2 mice. Thus, aberrantly increased CD11b+Gr-1+ myeloid cells in aged M-TRAF32/2 mice. It serum levels of these cytokines and chemokines suggest that would thus be interesting to investigate whether and how TRAF3 dysregulation of myeloid cells actively contributes to the path- regulates the survival and/or proliferation of histiocytes and ogenesis of spontaneous inflammation, infection, and tumor de- CD11b+Gr-1+ myeloid cells. We speculate that TRAF3 deficiency 2 2 velopment observed in aging M-TRAF3 / mice. may gradually lead to prolonged survival or increased prolifera- tion of CD11b+Gr-1+ myeloid cells, causing chronic inflammation Discussion in M-TRAF32/2 mice. However, increasing evidence indicates In this study, we generated and characterized M-TRAF32/2 mice that both inflammation and tumors stimulate the expansion of to investigate the in vivo functions of TRAF3 in myeloid cells, CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) (45–48). central players in innate immunity and inflammation. Our results Therefore, it remains possible that the increased population of showed that 8- to 12-wk-old M-TRAF32/2 mice had normal CD11b+Gr-1+ cells observed in our study consists mostly of ex- lymphocyte and myeloid cell populations in various hematopoi- panded MDSCs as a consequence of spontaneous inflammation etic compartments, demonstrating that LysM-Cre–mediated and tumor development. TRAF3 ablation neither affects the development nor alters the The importance of TRAF3 in innate immunity is highlighted by homeostasis of myeloid cells in young adult mice. However, in the evidence that a variety of viral and bacterial proteins target response to challenge with LPS (a bacterial mimic) or polyI:C (a TRAF3 for inactivation. These include Lb(pro) of foot-and-mouth viral mimic), M-TRAF32/2 mice exhibited an altered profile of disease virus, X protein (HBx) of hepatitis B virus, UL36 of HSV 1 2 cytokine production. After immunization, M-TRAF32/2 mice (HSV-1), YopJ of the Gram bacterium Yersinia pestis, Tat protein displayed elevated TI IgG3 as well as TD IgG2b responses. In- of HIV-1, Gn protein of NY-1 hantavirus, and M protein of severe terestingly, 15- to 22-mo-old M-TRAF32/2 mice spontaneously acute respiratory syndrome coronavirus (1). All these pathogen developed chronic inflammation or tumors with some cases proteins target TRAF3, and thus inhibit IRF3 phosphorylation and showing two pathologic conditions affecting multiple organs. type I IFN production (1). Consistent with these findings, TRAF3 Together, our findings indicate that myeloid cell TRAF3 regulates mediates type I IFN production and viral resistance in BMDMs, immune responses and is required for inhibiting inflammation and DCs, and TLR2-reprogrammed macrophages (13, 14, 49–51). In tumor development in mice. this study, we verified previous observations that LPS-induced It has been shown that TRAF3 regulates the homeostasis of IRF3 phosphorylation and type I IFN production are impaired in 2 2 multiple cell types through different mechanisms. Specific deletion TRAF3 / BMDMs, and extended these findings to PEMs. Para- of TRAF3 from B cells leads to vastly prolonged survival of mature doxically, in vivo IFN-b production was not affected in young adult 2 2 B cells because of constitutive activation of the NIK–NF-kB2 M-TRAF3 / mice injected with LPS or polyI:C, which is likely due pathway (7, 9). Although ablation of TRAF3 from T cells does not to compensation by TRAF3-sufficient pDCs, the most potent producers affect the homeostasis of CD4 or CD8 T cells (9, 10), the fre- of type I IFN. Regardless of this, innate immunity is evidently altered quency and number of Tregs are increased in T-TRAF32/2 mice by TRAF3 deletion in myeloid cells, as demonstrated by the spon- 2 2 and Treg-specific TRAF32/2 mice (10, 11). TRAF3 promotes IL- taneous development of inflammation and infection in M-TRAF3 / 15–mediated survival and proliferation in invariant NKT cells mice .15 mo. Bacterial or entamoeba infections observed in aged 2 2 (12). TRAF3 also regulates the development of medullary thy- M-TRAF3 / mice are most likely caused by opportunistic strains of mic epithelial cells by affecting the LTbR–NF-kB2 and CD40– commensal microbiota (termed “pathobionts”) (52–54), which may NF-kB2 pathways (43). In osteoclast precursor cells, TRAF3 trigger TRAF3-dependent signaling pathways via TLRs or NLRs inhibits osteoclast formation by suppressing RANK–NF-kB2 in macrophages, neutrophils, and DCs. For example, lipopeptido- signaling (44). In this study, we also observed constitutive NF- phosphoglycan of entamoeba has been shown to induce signaling kB2 activation in TRAF3-deficient macrophages and neutrophils. through TLR2 and TLR4, whereas DNA of entamoeba triggers sig- Interestingly, although the homeostasis of macrophages and neu- naling via TLR9 in macrophages (55). Similarly, muramyl dipeptide trophils was normal in young adult M-TRAF32/2 mice, we and meso-diaminopimelic acid of commensal bacteria stimulate detected malignant histiocytosis and frequent expansion of signaling through NOD1 and NOD2, respectively (56, 57). Defective 344 IN VIVO FUNCTIONS OF TRAF3 IN MYELOID CELLS

FIGURE 7. Abnormal lymphocyte and myeloid cell populations in the spleen of diseased M-TRAF32/2 mice. (A) Enlarged spleens of M-TRAF32/2 mice (age: 15–22 mo old). Graph depicts spleen weights of age-matched LMC and M-TRAF32/2 mice (n = 15 for each group of mice). (B) Representative FACS profiles of splenocytes of age-matched LMC and diseased M-TRAF32/2 mice. Splenocytes were stained with fluorochrome-conjugated B220, CD3, CD11b, Gr-1, CD21, and CD23 Abs, and then analyzed by a FACSCalibur. FACS profiles were scatter-gated on live cells. Gated cell populations include B cells, B220+CD32; T cells, CD3+B2202; myeloid cells, CD11b+Gr-1+; a distinct population of histiocytes (gated with a circle in the third and fifth panels): CD11blowGr-1low. M-TRAF32/2 mice shown include 237-2, DLBCL; 237-5, histiocytosis; 237-1, inflammation; 274-12, infection and inflammation. FACS profiles shown are representative of two cases of DLBCL, two cases of histiocytosis, two cases of inflammation, and two cases of infection, respectively. (C) Increased percentage of CD11b+Gr-1+ myeloid cells and decreased percentage of CD3+B2202 T cells in diseased M-TRAF32/2 mice. Graphs depict the percentages of CD11b+Gr-1+ myeloid cells or CD3+B2202 T cells of age-matched LMC and diseased M-TRAF32/2 mice (n = 8 for each group of mice) analyzed by FACS. The p values (t test) between LMC and M-TRAF32/2 mice are shown. (D) Representative FACS profiles of B220+ splenic B cells of M-TRAF32/2 mice with DLBCL. Follicular B cells are identified as B220+CD21IntCD23+, and marginal zone (MZ) B cells are identified as B220+CD21+CD23Int. The small population (6.91%) of B220+CD212CD232 observed in LMC are immature and activated B cells, and the major population (41.34% in mouse ID 237-5 and 68.48% in mouse ID 233-5) of B220+CD212CD232 observed in M-TRAF32/2 mice are predominantly DLBCL cells. type I IFN production in TRAF32/2 myeloid cells in response to TLR be required for the proper control of commensal microbiota-triggered or NLR signaling may occasionally allow colonization of commensal innate immune responses and inflammation in myeloid cells. bacteria or entamoeba after opportunistic penetration of protective Myeloid cells not only mediate innate immunity and inflam- mucosal and epithelial barriers in the gut. Thus, TRAF3 appears to mation, but also act as APCs in adaptive immunity. In this study, we The Journal of Immunology 345

FIGURE 8. Aberrant serum cytokine and chemokine levels of diseased M-TRAF32/2 mice. (A) Cytokine and chemokine protein array blots of age-matched LMC and diseased M-TRAF32/2 mice. Cytokines and chemokines in mouse sera were detected using the Mouse Cytokine Array Assay kit (R&D Systems) fol- lowing the manufacturer’s protocol. In each blot, combined sera of three mice (70 ml serum of each mouse) were used for the cytokine array assay. Sera of M-TRAF32/2 mice with tumor examined include 237- 5 (histiocytosis), 228-7 (DLBCL), and 274-11 (FL). Sera of M-TRAF32/2 mice with inflammation and in- fection examined include 228-5, 237-1, and 274-12. (B) Quantification of cytokine and chemokine levels of diseased M-TRAF32/2 mice measured by the cytokine protein array analyses. Each cytokine or chemokine spot on the blots in (A) was quantitated using a low- light imaging system, and the results are presented graphically. The amount of each cytokine or chemokine in diseased M-TRAF32/2 mice (tumor or inflamma- tion) was relative to the mean of the intensity of cor- responding LMC1 and LMC2 spots. Each cytokine or chemokine has duplicate detection spots. Graph depicts the fold change (M-TRAF32/2/LMC) of each cytokine or chemokine (mean). Bold numeric labels indicate the spots that are strikingly different between M-TRAF32/2 mice with tumor or inflammation/infection and LMC in (A) and the corresponding cytokine/chemokine quanti- tation data in (B).

found that a TI Ag stimulated increased IgG3 response, whereas tumor surveillance. In this regard, B cells are especially suscep- a TD Ag induced increased IgG2b response in M-TRAF32/2 mice, tible to genetic alterations due to the unique features of B cell which may result from increased levels of proinflammatory formation and development, which includes V(D)J recombination, cytokines in myeloid cells. It has been previously shown that IL- somatic hypermutation (SHM), and class switch recombination 12 enhances TI IgG3 responses (58). IL-6 selectively promotes (CSR) of Ig genes. All of these processes produce dsDNA breaks, IgG2b production in germinal center (GC) B cells, and TD IgG2b which increase the risk for genomic instability in B cells (65–67). In- responses are selectively impaired in IL-62/2 mice (59, 60). In deed, unlike MZLs and B1 lymphomas observed in B-TRAF32/2 addition, IL-6 induces the differentiation, expansion, and main- mice that do not involve SHM or CSR (8), the DLBCLs and FLs tenance of T follicular helper cells by inducing Bcl6 expression identified in M-TRAF32/2 mice originate from GC or post-GC (61–63). Decreased T follicular helper cell number has been B cells, which are undergoing or have gone through GC-related shown to correlate with decreased TD IgG2b response (64). events, including SHM and CSR. B cells that have acquired onco- Therefore, increased levels of IL-12 and IL-6 likely mediate in- genic alterations during GC passage may escape the compromised creased TI IgG3 and TD IgG2b responses, respectively. We pre- tumor surveillance and develop into malignant lymphomas in viously reported that specific deletion of TRAF3 in B cells leads M-TRAF32/2 mice. Alternatively, the chronic inflammatory envi- to hypergammaglobulinemia, increased IgM and IgG responses ronment of M-TRAF32/2 mice, a strong risk factor for cancer, may after immunization with a TI Ag, but normal IgG1 responses to induce mutations that facilitate malignant transformation of TRAF3- a TD Ag (7). In contrast, specific deletion of TRAF3 in T cells sufficient cells (e.g., hepatocytes and B cells), stimulate tumor causes defective IgG1 responses to a TD Ag because of impaired growth, and promote angiogenesis to accelerate tumor progression, CD4 Th cell activation (10). Interestingly, specific ablation of invasion, and metastasis (68–71). Consistent with this notion, we did TRAF3 in Tregs results in moderately enhanced IgG2b responses detect strikingly increased levels of 11 cytokines and chemokines in to a TI Ag and markedly increased IgG1 and IgG2b responses to M-TRAF32/2 mice with tumors. Furthermore, chronic inflammation a TD Ag, because of decreased induction of follicular regulatory may induce the generation and expansion of CD11b+Gr-1+ MDSCs, CD4 T cells after immunization (11). Therefore, TRAF3 defi- which, in turn, suppress the antitumor immune responses mounted ciency in different immune cell types modulates Ab responses in by NK cells and CD8 cytotoxic T cells (45–48). MDSCs can also distinct manners. recruit and activate Tregs to further inhibit antitumor responses One particularly interesting finding of our study is spontaneous (45–48). Taken together, the evidence presented in this article tumor development in older M-TRAF32/2 mice. Notably, ma- suggests that TRAF3 is a tumor suppressor gene not only in lignant transformation was not only detected in TRAF3-deficient B cells, but also in myeloid cells. histiocytes, a type of tissue-resident macrophages, but was also Considering that TRAF3 is used in signaling by many immune observed in other cell types that are TRAF3 sufficient, including receptors (1, 2), it will be especially interesting to further decode B cells and hepatocytes. In contrast, tumor development is limited the signaling pathways that lead to spontaneous inflammation and to TRAF3-deficient B cells but is not observed in other TRAF3- tumor development in M-TRAF32/2 mice. In the absence of in- sufficient cell types in B-TRAF32/2 mice (8). 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