TACI deficiency leads to alternatively activatedmacrophage phenotype and susceptibility toLeishmania infectionWindy R. Allmana,1, Ranadhir Deyb,1, Lunhua Liua, Shafiuddin Siddiquia, Adam S. Colemana, Parna Bhattacharyab,Masahide Yanoa, Kadriye Uslua, Kazuyo Takedac, Hira L. Nakhasib, and Mustafa Akkoyunlua,2

aLaboratory of Bacterial Polysaccharides, Division of Bacterial Parasitic and Allergenic Products, US Food and Drug Administration, Silver Spring, MD 20993;bDivision of Emerging and Transfusion Transmitted Diseases, US Food and Drug Administration, Silver Spring, MD 20993; and cMicroscopy and Imaging CoreFacility, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, MD 20993

Edited by Raif S. Geha, Children’s Hospital Boston, Boston, MA, and accepted by the Editorial Board June 15, 2015 (received for review November 11, 2014)

The TNF family member, transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), is a key moleculefor plasma cell maintenance and is required in infections whereprotection depends on antibody response. Here, we report thatcompared with WT mouse, TACI KO Μϕs expressed lower levels ofToll-like receptors (TLRs), CD14, myeloid differentiation primary re-sponse protein 88, and adaptor protein Toll/IL-1 receptor domain-containing adapter-inducing IFN-β and responded poorly to TLRagonists. Analysis of Μϕ phenotype revealed that, in the absenceof TACI, Μϕs adapt the alternatively activated (M2) phenotype.Steady-state expression levels for M2 markers IL-4Rα, CD206,CCL22, IL-10, Arg1, IL1RN, and FIZZ1 were significantly higher inTACI KO Μϕ than in WT cells. Confirming their M2 phenotype,TACI-KO Mϕs were unable to control Leishmania major infectionin vitro, and intradermal inoculation of Leishmania resulted in amore severe manifestation of disease than in the resistant C57BL/6strain. Transfer of WT Μϕs to TACI KO mice was sufficient to signif-icantly reduce disease severity. TACI is likely to influence Mϕ phe-notype by mediating B cell-activating factor belonging to the TNFfamily (BAFF) and a proliferation inducing ligand (APRIL) signalsbecause both these ligands down-regulated M2 markers in WTbut not in TACI-deficient Μϕs. Moreover, treatment of Μϕs withBAFF or APRIL enhanced the clearance of Leishmania from cells onlywhen TACI is expressed. These findings may have implications forunderstanding the shortcomings of host response in newbornswhere TACI expression is reduced and in combined variable immu-nodeficiency patients where TACI signaling is ablated.

TACI | BAFF | APRIL | macrophage | Leishmania

Transmembrane activator and calcium-modulator and cyclo-philin ligand interactor (TACI) is a member of the TNF family

molecules (1). It is a receptor for B-cell activating factor (BAFF)and a proliferation inducing ligand (APRIL). Although BAFF andAPRIL share a second receptor, B-cell maturation antigen(BCMA), BAFF-R only binds to BAFF, and heparan sulfateproteoglycans only engage APRIL. TACI is primarily expressedon mature B cells and mediates signals for Ig isotype switch andsecretion (2). Studies in TACI KO mice (3), combined variableimmune deficient (CVID) patients with mutations in TACI genetnfrsf13b (4), and newborns who express severely reduced B-cellTACI (5) all point to its pivotal role in determining antibody (Ab)development against T cell-independent type 2 (TI-2) antigens.In contrast to earlier publications (3), more recent reportsshowed diminished sustainment of plasma cells in response toT cell-dependent (TD) antigens in TACI KO mice (6). In-terestingly, Tsuji et al. reported that despite impaired plasma cellsurvival and reduced Ab response to TD antigens, TACI KOmice manifest enhanced clearance of the enteric pathogen Cit-robacter rodentium, presumably due to generation of higheravidity of Abs in the absence of TACI (7). Whereas TACI is wellestablished as a B-cell receptor, its involvement in innate im-

mune response is less clear. One study reported diminishedB-cell responses to Toll-like receptor (TLR)7 and TLR9 agonistsin CVID patients with TACI mutations (4). A second possible linkbetween TACI and innate immune system was suggested by Heand colleagues, who have shown that the TLR adaptor moleculemyeloid differentiation primary response protein 88 (MyD88) isdownstream of TACI in B cells (8). Other members of the innateimmune system such as dendritic cells (DCs) and monocytes areknown to be the main sources of circulating BAFF and APRIL,but TACI expression is limited to intracellular compartments inthese cells (9, 10). Although BAFF and APRIL can induce in-flammatory cytokine secretion in human DCs and monocytes,the significance of their activity remains to be understood (9, 10).Here, we investigated the role of TACI in innate immune

response and showed that TACI deficient macrophages (Mϕs)respond poorly to TLR agonists, and this ablated response islikely due to reduced expression of TLRs, CD14, MyD88, andadaptor protein Toll/IL-1 receptor domain-containing adaptor-inducing IFN-β (TRIF) in TACI KO cells. Furthermore, TACIKO Mϕs manifested alternatively activated Μϕ (M2) phenotypecharacterized by elevated levels of molecules associated with M2phenotype and impaired resistance to in vitro Leishmania majorinfection. Moreover, intradermal inoculation with L. major resulted

Significance

Here, we described a novel role for transmembrane activatorand calcium-modulator and cyclophilin ligand interactor (TACI)in determining Mϕ phenotype, a molecule that is previouslyknown to be important in B-cell responses. We found thatTACI-deficient mouse Mϕs manifest an M2 phenotype. We alsoobserved that, in WT mouse Mϕs, TACI mediates B-cell acti-vating factor- and a proliferation inducing ligand-induced sig-nals that favor M1 polarization and Leishmania clearance. InTACI-deficient mice, M2-polarized status of Mϕs was responsiblefor the diminished Th1 response and increased susceptibilityto Leishmania infection. These findings have implications inexplaining the propensity of infants to develop asthma andweak responses to vaccines because infant Mϕs are likely to beM2-skewed due to severely reduced expression of TACI.

Author contributions: W.R.A., R.D., H.L.N., and M.A. designed research; W.R.A., R.D.,L.L., S.S., A.S.C., P.B., M.Y., K.U., K.T., and M.A. performed research; W.R.A., R.D., L.L., S.S.,A.S.C., P.B., M.Y., K.T., H.L.N., and M.A. analyzed data; and W.R.A., R.D., L.L., S.S., andM.A. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. R.S.G. is a guest editor invited by the EditorialBoard.1W.R.A. and R.D. contributed equally to this work.2To whom correspondence should be addressed. Email: mustafa.akkoyunlu@fda.hhs.gov.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1421580112/-/DCSupplemental.

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in a more severe manifestation of disease in TACI KO mousethan the Leishmania-resistant WT C57BL/6 strain, and adap-tive transfer of Μϕs from the WT mouse was sufficient toreduce the severity of Leishmania induced cutaneous disease inthe TACI KO mouse. Comparison of the response of WT andTACI-deficient Μϕs revealed that TACI mediates ligand induceddown-regulation of molecules associated with M2 Mϕ phenotypeand up-regulation of some of the markers representative ofclassically activated (M1) phenotype. Collectively, these findingsextend the role of TACI from its well-defined involvement inB-cell homeostasis to Mϕ phenotype determination and resistanceto intracellular pathogens.

ResultsTACI-Deficient Cells Respond Poorly to TLR Agonists. Previous studiesreported a diminished B-cell response from CVID patientswith TACI mutations to TLR7 and TLR9 agonists (4). Todirectly assess the involvement of TACI in response to TLRagonists, we measured TNF-α and IL-6 levels in the culturesupernatants of peritoneal Mϕs (pMϕ) and bone marrow-derived DCs (BMDC) from TACI KO and WT mice after stim-ulation with the TLR agonists LPS, CpG, peptidoglycan, PolyI:C, lipoteichoic acid, and ssRNA. All TLR agonists inducedsignificantly lower levels of TNF-α and IL-6 from TACI KOpMϕs than the WT cells (SI Appendix, Fig. S1A). With theexception of ssRNA-induced TNF-α and Poly I:C-induced IL-6,which remained low in both strains, all TLR agonists inducedsignificantly lower levels of TNF-α and IL-6 in TACI KOBMDCs than WT cells (SI Appendix, Fig. S1B). Similar resultswere obtained in bone marrow-derived Mϕs (BMDMs) (SI Ap-pendix, Fig. S1C). Furthermore, the decrease in cytokine se-cretion was preceded by impaired mRNA expression in CpG-,LPS-, or Poly I:C-stimulated TACI KO pMϕs, with the exceptionof IL-6 in CpG-stimulated cells (SI Appendix, Fig. S1D).Induction of costimulatory molecule expression is a crucial

component of TLR agonist activity on innate immune system.We therefore measured the expression of CD40, CD80, CD86,and MHCII molecules in TLR agonist-stimulated pMϕs. As ex-pected, WT pMϕs responded robustly to TLR agonist stimulation(SI Appendix, Fig. S2 A and B). In contrast, TACI KO pMϕs onlyinduced the expression of MHCII when stimulated with CpG andinduced the expression of CD86 when stimulated with LPS andPoly I:C. More importantly, all three TLR agonists failed to up-regulate CD40 or CD80 expression in TACI KO cells. These re-sults suggested that suboptimal response of TACI KO pMϕs toTLR agonists was not restricted to cytokine secretion.

TLR Agonists Manifest Ablated Adjuvant Activity in TACI KO Mice.Next, we assessed whether the TACI KO mouse response toTLR agonists was diminished in vivo as well. For this purpose,we immunized TACI KO and WT mice with tetanus (TT) aloneor TT with CpG, and measured serum IgG and IgM Ab levels 0,15, and 30 d after immunization. In agreement with recent re-ports (6), we also found that for TACI KO, Ab responses to theTD antigen, TT was significantly lower in TT-immunized TACIKO mice than the WT mice on all days tested (SI Appendix, Fig.S3A). As expected, CpG was able to significantly increase IgGand IgM Ab responses to TT in WT mice. In contrast, TACI KOmouse IgG and IgM Ab levels remained the same with or withoutCpG. We also examined the ability of LPS to induce inflam-matory cytokines in TACI KO mice. After i.p. injection of 0.75 mgof LPS, significantly reduced levels of serum IL-6 and TNF-α weremeasured in TACI KO mice compared with the WT mice (SIAppendix, Fig. S3B). Taken together, the in vivo data were inagreement with the results from in vitro experiments and sug-gested that TLR activity was severely impaired in TACI KO mice.

TACI KO Mϕs Have Reduced Expression of TLRs, TLR AdaptorMolecules, and CD14. One possibility for the impaired TACI KOresponse to TLR agonists could be the lower expression of TLRs(11). To test this possibility, we compared the levels of TLRs onTACI KO and WT pMϕs and found that the expression levelsand the frequencies of TLR9 and TLR4 (SI Appendix, Fig. S4 Aand B) expressing cells were less in TACI KO pMϕs than in WTcells. We also determined CD14 expression on pMϕs becauseCD14 is known to augment responses to TLR agonists and itsexpression is up-regulated on TLR stimulation (12). We foundthat after stimulation with CpG, LPS, and Poly I:C, the per-centage of CD14+ pMϕs was significantly lower in TACI KOcells than in WT cells (SI Appendix, Fig. S4 C and D). Thus,lower expression of TLRs and CD14 is likely to be responsiblefor the impaired response of TACI KO pMϕs to TLR agonists.A crucial step in successful TLR signaling is the colocalization

of TLRs with the adaptor molecules MyD88 and/or TRIF (13). Toexamine the activation events after TLR agonist stimulation, weincubated pMϕs with CpG for 3 h and evaluated TLR9 andMyD88 expression by confocal microscopy. Consistent with ourflow cytometry results, unstimulated TACI KO pMϕs had sig-nificantly lower TLR9 expression than WT mice (SI Appendix,Fig. S5). Confocal microscopy also illustrated that TACI KO pMϕshad significantly reduced MyD88 expression. Consequently, CpGstimulation resulted in limited colocalization of TLR9 andMyD88 in TACI KO pMϕs compared with WT pMϕs. Similarobservations were made when pMϕs were analyzed for TLR4and MyD88 or TRIF expression patterns after LPS stimulation(SI Appendix, Fig. S6 A and B). Finally, TLR3 and TRIF meanfluorescence intensity values were significantly reduced in TACIKO pMϕs compared with WT cells, and their colocalization andtranslocation were incomplete on Poly I:C stimulation (SI Ap-pendix, Fig. S6C). Jointly, both flow cytometry analysis and themicroscopic evaluations suggest lower TLR and adaptor moleculeexpression as the underlying cause of impaired TACI KO-pMϕresponse to TLR agonists.

TACI KO Mϕs Have an M2 Phenotype. In Mϕs, TLR-induced in-flammatory cytokine production and costimulatory moleculeexpression is an important component of the classically activated(M1) phenotype (14). Conversely, alternatively activated pMϕsare restricted in their ability to secrete inflammatory cytokinesand increase the expression of costimulatory molecules (15, 16).To examine whether ablated TLR activity in TACI KO pMϕs isassociated with the M2-skewed phenotype, we determined theexpression of a panel of representative M2 markers in TACI KOand WT cells. We found that cell surface IL-4Rα and CD206were expressed at significantly higher levels on TACI KO cellscompared with WT pMϕs (Fig. 1A). TGF-β is produced by M2Mϕs and inhibits proinflammatory responses and microbicidalactivity (17). Stimulation of TACI KO pMϕs with LPS, CpG, andPoly I:C resulted with a significant increase in TGF-β levels (Fig.1B). Among the three TLR agonists, only LPS significantly in-duced TGF-β secretion by WT pMϕs. Additional M2 markerswere evaluated by quantitative RT-PCR (qPCR). Of the eightgenes encoding for M2 markers, CCL22, IL-10, Arginase 1(Arg1), IL-1 receptor antagonist (IL1RN), and found in in-flammatory zone protein (FIZZ1) mRNA expression was sig-nificantly higher in TACI KO pMϕs compared with WT cells,while chitinase 3-like 3 (or Ym1) levels were comparable be-tween the two strains (Fig. 1C). TACI KO BMDMs also had aphenotype that was consistent with an M2 phenotype, althoughthere were differences in the expression of some of the markerstested. Similar to pMϕs, TACI KO BMDMs had significantlyhigher TGF-β in CpG- and Poly I:C-stimulated TACI KO cellscompared with WT cells, but LPS stimulation induced higherTGF-β in WT BMDMs (SI Appendix, Fig. S7A). The expressionof IL-4Rα was higher in TACI KO BMDM cells than WT cells,

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but CD206 was not (SI Appendix, Fig. S7B). Among the genesassociated with M2 phenotype, ccl22 and il10 was higher in TACIKO BMDMs, but arg1, il1rn, and ym1 were comparable betweenthe two strains (SI Appendix, Fig. S7C). The gene for FIZZ1 wasnot detected in BMDM from either mouse strains. These dataconfirmed the default M2 phenotype in TACI KO Mϕs, al-though there were variations in the expression of M2 markersdepending on the source of Mϕs.Macrophages can be polarized into the M1 or M2 phenotype

depending on the stimuli present in the microenvironment (14).To assess the plasticity of TACI KO Mϕs, cells were incubatedwith LPS/IFN-γ for M1 differentiation or IL-4/IL-13 for M2 dif-ferentiation (18). Whereas, TACI KO Mϕs were more sensitive

to IL-4/IL-13 stimulation than the WT cells, M1 stimulus had astronger effect on WT cells than on TACI KO Mϕs. After LPSand IFN-γ stimulation, TACI KO cells expressed significantlylower levels of MMP9 mRNA than WT cells, and the increase inTACI KO culture supernatant IL-12 p40 and nitrite concentrationswere significantly less compared with those in WT cells (Fig. 1D).Conversely, IL-4 and IL-13 stimulation led to a significantlyhigher CCL22, Arg1, Ym1, and FIZZ1 mRNA expression inTACI KO cells than WT cells (Fig. 1E). Expression of IL-10 andIL1RN mRNA was not different between the two mouse strains.Taken together, these data show that, in the absence of TACI,pMϕs default to an M2 phenotype. TACI cells also have a pro-pensity to express higher M2 markers following stimulation withIL-4/IL-13, and they respond poorly to M1 polarization signals.

Leishmania major Infection in TACI KO Mice. Control of infection byL. major, an intracellular parasite, requires M1 polarization ofMϕs, which enhances intracellular killing of parasite and theappropriate induction of a Th1 immune response (19). In gen-eral, the Th1 or Th2 polarization of T cells is a reflection of theM1 and M2 polarity of Mϕs, respectively (14). C57BL/6 mice aregenetically resistant to L. major and spontaneously resolveinfection by mounting Th1 response (20). Because TACI KOMϕs have a default M2 phenotype, we hypothesized that theywould be less able to control L. major infection. After 6 h ofincubation, the number of parasites inside the cells and thepercentage of infected cells were similar in both the mousestrains (Fig. 2B). This observation indicated that that thephagocytosis of parasites by TACI KO Mϕs was not impaired.However, after 2 and 4 d of infection, there was significantly en-hanced parasite growth in TACI KOMϕs compared with WT cells(Fig. 2 A and B). Moreover, the number of infected Mϕs was sig-nificantly higher in TACI KO cells than in WT cells. Leishmaniainfection induces the M1 phenotype-associated inducible nitricoxide synthase (iNOS or NOS2) molecules, which mediate invitro (21) and in vivo (22) nitric oxide-mediated parasite killing.Analysis of culture supernatants on day 2 of infection revealedthat Leishmania-infected WT Mϕs secreted significantly ele-vated levels of nitrite, the stable end product of nitric oxide,whereas TACI KO nitrite levels did not change over time (Fig.2C). These data indicate that, consistent with an M2 phenotype,the ability of Mϕs to kill intracellular pathogens is impaired inTACI-deficient cells.Next, we investigated whether the in vivo M2 microenviron-

ment would render TACI KO animals susceptible to L. majorinfection. TACI KO and WT mice were infected by intradermal(i.d.) inoculation of L. major parasite in the ear, and ear parasiteburden was assessed over a 12-wk period. Disease severity wasdetermined by analysis of lesion size and histological evaluationof ear sections. Although most of the ear pathology resolved inboth the strains by week 10, compared with WT mice, destructivechanges in TACI KO ears were more severe throughout theinfection (Fig. 2D), concomitant with significantly larger lesionsize in TACI KO mice between weeks 4 and 8 after infection(Fig. 2E). In histopathological evaluation (5 wk), the differencein lesion thickness was evident between the two mouse strains(Fig. 2F). In higher magnifications, TACI KO samples revealedmore infected cells, the majority of them being infiltrated Mϕs.Ear sections stained for iNOS at the same time point showedlimited iNOS production in TACI KO mice (Fig. 2G). Con-versely, and consistent with an inflammatory response, WTsamples had widespread iNOS staining. Accompanying the se-verity of disease, at 5 wk after infection, the number of parasitespresent in the ear and in the draining lymph node (LN) wassignificantly higher in TACI KO mice compared with WT mice(Fig. 2H). Despite the healed cutaneous lesion, Leishmaniapersisted in the ear and LN of both strains of mice. However,WT mice had significantly fewer parasites present in the ear and

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Fig. 1. Expression of M1 and M2 markers in Mϕs. (A) Shown in histogram isthe expression of IL-4Rα and CD206 in purified pMϕs determined by flowcytometry. Mean percentages ± SD of CD11b+IL-4Rα+ and CD11b+CD206+cells from three experiments are plotted. (B) Concentration of TGF-β in thesupernatants of pMϕs after CpG, LPS, or Poly I:C stimulation for 24 h. ELISAresults shown are the mean ± SD of six determinations from three in-dependent experiments. (C) Ex vivo mRNA expression of M2 markers bypMϕs. Relative gene expression normalized to GAPDH ± SD was plotted.(D) pMϕs were treated with 50 ng/mL IFN-γ and 100 ng/mL LPS (M1 stimulus)or left in media for 24 h. Fold differences in MMP9 mRNA expression com-pared with media were determined by qPCR. Culture supernatant IL-12p40levels were measured in ELISA, and nitrite production was measured usingGriess reagent. (E) TACI KO and WT pMϕs were treated with 15 ng/mL IL-4and 15 ng/mL IL-13 (M2 stimulus) or left in media for 24 h. Fold differences ofCCL22, IL-10, Arg1, IL1RN, Ym1, and FIZZ1 mRNA expression compared withmedia control were determined by qPCR. Mean ± SD was obtained fromthree independent experiments each with at least three mice per group.*P <0.05, **P < 0.01, and ***P < 0.001 for statistical differences betweenWT andTACI KO cells.

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LN by 12 wk after infection, whereas TACI KO mice failed todecrease parasite burden over time.

Host Response to Leishmania Challenge in TACI KO Mice. To de-termine whether the pronounced pathology, increased parasiteload, and limited inflammatory responses were accompanied byM2-polarized ear Mϕs, we assessed M2 markers in Mϕs isolatedfrom the ear. To facilitate the isolation of parasite-infected Mϕs,we inoculated TACI KO and WT mice ears with a red fluo-rescent protein (RFP)-expressing L. major (RFP-Leishmania)parasite (23). Two weeks after infection, Leishmania-infected

dermal Mϕs (RFP+CD11b+CD11c−Ly6G−) were isolated fromear and analyzed for M2 markers (SI Appendix, Fig. S8A). Thefrequency of dermal Mϕs was comparable between the two strains(SI Appendix, Fig. S8B). However, TACI KO mice had signifi-cantly more RFP-Leishmania+ dermal Mϕs than the WT mice(Fig. 3A). Among the M2-marker genes, arg1, il1rn, and ym1 wereexpressed significantly more in TACI KO dermal Mϕs than WTcells (Fig. 3B). Ccl22 and il10 genes were not detected in dermalMϕ from either strain. Thus, TACI KO dermal Mϕs preservedtheir M2 phenotype during Leishmania infection.Because the type of T helper response dictates the host re-

sistance or susceptibility to Leishmania infection (20), we ana-lyzed Th1 and Th2 populations at 2, 5, and 12 wk after theparasite challenge in the skin and the draining LNs by quanti-fying IFN-γ and IL-4 positive CD4+ T cells, respectively. In theskin, WT mouse IFN-γ–producing CD4+ cells were elevated by

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Fig. 2. Macrophage infection with L. major. (A–C) BMDMs were infectedwith stationary phase cultures of L. major promastigotes. At 6 h, 2 d, and 4 dafter infection, slides were fixed and stained. (A) Diff-Quick stain of WT andTACI KO Mϕs 4 d after infection is shown. Red arrows indicate intracellularLeishmania amastigotes. (B) Kinetics of parasite load in BMDMs. Resultsexpressed either as the mean number of parasites/infected Mϕs or as per-centage of Mϕs that were infected by Leishmania. *P < 0.05 and ***P <0.001 for statistical differences between WT and TACI KO mice. (C) Nitriteproduction in Leishmenia-infected BMDMs was measured in culture super-natants using Griess reagent. *P < 0.05 for statistical differences betweenWT and TACI KO mice. #P < 0.05 for statistical differences between 6 h and2 d. (D–H) The ears of WT and TACI KO mice were infected with 1 × 104

metacyclic promastigotes of Leishmania by i.d. injection. (D) Representativeear images 5 wk after i.d. infection with Leishmania. (E) Kinetics of ear lesionsize over the course of a 12-wk Leishmania infection in TACI KO (n = 9) andWT mice (n = 9). (F) Representative H&E-stained sections of WT and TACI KOears 5 wk after i.d. infection with Leishmania. (Upper) Magnification, 1.25×.(Lower) Magnification, 100×. Red arrows indicate intracellular Leishmaniaamastigotes. (G) Representative immunohistochemical staining of ear sec-tions showing iNOS expression at 5 wk of Leishmania infection. Shown at10×magnification. (H) Parasite load in ear or draining LN of TACI KO andWTmice 5 wk after infection with Leishmania. Results expressed as the geo-metric mean number of parasites per organ. ††P < 0.01 for statistical dif-ferences between 5 and 12 wk after infection. *P < 0.05, **P < 0.01, and***P < 0.001 for statistical differences between the WT and TACI KO mice.

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Fig. 3. Analysis of in vivo host response following L. major infection in TACIKO mice. (A and B) TACI KO and WT mice were infected i.d. with 2 × 105

metacyclic promastigotes of RFP-Leishmania. Two weeks after infection withRFP-Leishmania, infected dermal Mϕs (RFP-Leishmania+CD11b+Ly6G−CD11b−)were sorted from ear. Experiment was repeated three times with pooleddigests from four to six ears per experiment. (A) Mean percentage ± SD andMFI ± SD of RFP-Leishmania+ dermal Mϕs determined by flow cytometry areplotted. (B) Quantification of genes associated with M2 phenotype in RFP-Leishmania–infected dermal Mϕs. Relative gene expression compared withGAPDH ± SD was determined by qPCR. Mean values from three experimentsare plotted. (C and D) The ears of WT and TACI KO mice were infected with1 × 104 metacyclic promastigotes of Leishmania by i.d. injection. (C) Repre-sentative intracellular cytokine staining plots indicating the frequency ofTh1 (IFN-γ) and Th2 (IL-4) CD4+ T cells 2, 5, and 12 wk after infection foundwithin the Leishmania infected ear lesion. Freshly isolated ear-derived skincells were directly stained for IFN-γ or IL-4. (D) Kinetic analysis of meanpercentages ± SD of CD4+IL-4+ and CD4+IFN-γ+ cells from two experimentsare plotted as chart with 10 mice per group. *P < 0.05, **P < 0.01, and ***P <0.001 for statistical differences between TACI KO and WT mice.

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2 wk after infection, and their frequency gradually decreased onweeks 5 and 12 (Fig. 3 C and D). This decrease coincided withthe progressive improvement in the skin lesion after week 5 (Fig.2E). The frequency of skin IFN-γ+ T cells in TACI KO mouseremained low throughout the span of the experimental periodand was significantly less than the WT cells at 2 and 5 wk afterinfection. At 12 wk, the difference between the two mousestrains disappeared, when WT mouse IFN-γ+ T cells numbersdecreased to TACI KO levels. The frequency of IL-4–producingT cells remained unchanged in WT mice throughout the exper-imental period (Fig. 3 C and D). Suggesting the absence of acompensatory Th2 response, ablated Th1 development in TACIKO mice was not accompanied by an increase in IL-4+ T cells.We also measured serum anti-Leishmania IgG1 and IgG2c Ablevels because these isotypes reflect the phenotype of T helperresponse during L. major infection (24). Indicating a likely conse-quence of impaired IFN-γ–producing T cells in TACI KOmice, theserum anti-Leishmania IgG1/IgG2c ratio was higher in TACI KOmice than the WT mice (SI Appendix, Fig. S9A).The number of T-regulatory (Treg) cells (Foxp3+IL-10+CD4+

cells) in infected lesion has been reported to contribute toLeishmania persistence (25). To assess whether the elevated par-asite burden in TACI KO mice is accompanied by a heightenedTreg response, we analyzed the frequency of Treg cells in the skinlesions and found that Treg numbers were comparable betweenthe two mouse strains and remained unchanged throughout theexperimental period (SI Appendix, Fig. S9B). In vitro stimulationof draining LN cells with Leishmania antigen confirmed the sig-nificantly lower number of IFN-γ–producing T cells in TACI KOmouse compared with WT mice (SI Appendix, Fig. S9C). How-ever, the frequency of IFN-γ–producing T-cell numbers man-ifested a fluctuating kinetics in TACI KO mice. First, there wasa significant decrease at 5 wk compared with week 2 numbers.Subsequently, IFN-γ+ T-cell numbers were significantly more at12 wk than at 2 wk. Interestingly, this increase at 12 wk coincidedwith the alleviation of skin lesions (Fig. 2E) and a decrease inparasite count in the skin of TACI KO mice (Fig. 2H). As hasbeen shown for the WT strain previously (20) and coincidingwith the increase in IFN-γ+ T-cell numbers, the high numbersof IL-4+ T-cell numbers significantly decreased by 5 wk. Thekinetics of TACI KO mouse IL-4–producing T cells overlappedwith those of WT mice (SI Appendix, Fig. S9C).To assess whether TACI deficiency also results in an intrinsic

T-cell defect that prevents the expansion of Th1 cells, we deter-mined the generation of Th1, Th2, and Treg cells from naïveT cells (CD4+CD44lowCD62Lhigh) stimulated under Th1,Th2, and Treg polarizing conditions. Analysis of the mRNAexpression of lineage-specific markers excluded the possibilityof a defective Th1 cell polarization as the underlying cause ofthe inadequate Th1 response in TACI KO because both strainshad equal propensity to polarize to Th1, Th2, and Treg cells (SIAppendix, Fig. S10 A–C).Neutrophils play a key role during the initial stages of Leish-

mania infection after the deposition of parasite in the skin (23).As such, changes in neutrophil function may influence the infec-tion severity. To assess whether TACI deficiency alters neutrophilfunction, we measured the ability of TACI KO neutrophils tophagocytize polystyrene beads or RFP-Leishmania and foundthat TACI KO and WT neutrophils had a comparable numberof engulfed polystyrene beads and RFP-Leishmania parasites(SI Appendix, Fig. S11 A and B).

WT Mouse Mϕs Alleviate Leishmania Infection in TACI KO Mice. Toexamine whether the extent of TACI expression on Mϕs con-tributes to disease severity during Leishmania infection, isolatedCD45.1+ WT or TACI KO Mϕs were adoptively transferred toTACI KO mice 1 d after infection of the ear. Skin CD45.1+Mϕswere detected in the ear as early as 1 d after transfer (SI Appendix,

Fig. S12). Ear pathology and parasite burden was monitored overa 5-wk period. Disease severity was determined by lesion sizeand histological evaluation of ear sections. Gross pathologicalchanges were most severe in TACI KO mice receiving TACI KOMϕs followed by the PBS-injected TACI KO mice (Fig. 4A). Inhistopathological evaluation (5 wk), lesion thickness was mark-edly less in WT Mϕ transferred TACI KO mice compared withTACI-deficient Mϕ or PBS-injected TACI KO mice (Fig. 4B). Inhigher magnifications, the presence of a lower number ofinfected Mϕs was apparent in TACI KO mice injected with WT

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Fig. 4. Adoptive transfer of WT Mϕs to TACI KO mice protects against cu-taneous leishmaniasis. Adherent pMϕs (CD11b+MHCII+Ly6G−CD11c− cells)were isolated from naïve WT or TACI KO mice and i.v. transferred (3 × 106

cells per mouse) to TACI KO mice that were infected with 1 × 104 metacyclicpromastigotes of Leishmania by i.d. injection the previous day. Figure labelindicates the strain of host/strain of Mϕs adoptively transferred. (A) Repre-sentative ear images 5 wk after i.d. infection with Leishmania. (B) Repre-sentative H&E-stained sections of WT and TACI KO ears 5 wk after i.d.infection with Leishmania (Upper and Lower at 4× and 100× magnification,respectively). (Insets) Magnified views of representative vacuoles containingLeishmania amastigotes depicted as cartoons. (C) Kinetics of ear lesion sizeover the course of a 5-wk Leishmania infection. Mean lesion size ± SD wasobtained from two independent experiments each with five mice per group.Statistical comparison can be found in SI Appendix, Table S1. (D) Parasiteload in ear or draining LN of TACI KO and WT mice 5 wk after infection withLeishmania. Results expressed as the geometric mean number of parasitesper ear (n = 10 mice per group). **P < 0.01 and ***P < 0.001 indicate forstatistical differences between mouse groups. (E) Representative intra-cellular cytokine staining plots indicating the frequency of Th1 (IFN-γ) andTh2 (IL-4) CD4+ T cells at 5 wk after infection found within the ear Leish-mania lesion. Freshly isolated ear-derived skin cells were directly stained forIFN-γ or IL-4. (F) Mean frequency ± SD of CD4+IL-4+ and CD4+ IFN-γ+ cellsfrom two experiments are plotted as chart with 10 mice per group. *P < 0.05for statistical differences between TACI KO and WT mice.

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Mϕs, in contrast to the PBS or TACI KO-Mϕ transferred TACIKO mice. Transfer of WT Mϕs into TACI KO mice also led to asignificant decrease in the week 3 and 5 lesion size comparedwith PBS-injected TACI KO animals (Fig. 4C) (SI Appendix,Table S1). Conversely, TACI-deficient Mϕs enhanced the lesionsize in the recipient TACI KO mice. In addition, the number ofparasites harvested from the ears of WT-Mϕ injected TACI KOmice was significantly lower than the TACI KO mice that re-ceived TACI deficient Mϕs or PBS (Fig. 4D). To evaluatewhether TACI expression in Mϕs contributed to the activation ofTh1 cells during Leishmania infection, we analyzed skin Th1 andTh2 populations 5 wk after the parasite challenge. Ex vivoevaluation of CD4+ cells in the skin lesions indicated that thetransfer of WT Mϕs was sufficient to significantly increase theamount of IFN-γ–producing CD4+ cells compared with PBS-injected TACI KO mice (Fig. 4 E and F). However, injection ofWT Mϕs did not lead to a significant change in the frequency ofTh2 (CD4+IL-4+) or Treg (Foxp3+IL-10+) cells (Fig. 4 E and Fand SI Appendix, Fig. S13 A and B). These data strongly suggestthat adoptive transfer of WT Mϕs to TACI KO mice was suffi-cient to reduce the severity of Leishmania infection and thatTACI expression by Mϕs is involved in the induction of aTh1 response.

TACI Ligands Regulate Phenotypic Changes in WT Mouse Mϕs. BothBAFF and APRIL are members of the TNF family of ligands andact as key mediators of B-cell maturation and homeostasis (26).They share the receptors TACI and BCMA, whereas BAFF-Rbinds exclusively to BAFF. Since the discovery of this receptor-ligand system, the primary focus of research has been on theirfunction in regulating B-cell responses. Our data thus far havesuggested that this system is also important for the determinationof Mϕ phenotype and function. To elucidate the mechanism ofthe BAFF system involvement in influencing Mϕ phenotype, wefirst determined the expression of BAFF system molecules inMϕs. We found that resident pMϕs express high levels of surfaceBAFF and APRIL, but little or no surface expression of BAFF-R,TACI, or BCMA (SI Appendix, Fig. S14A). However, at themRNA level, all members of the BAFF and APRIL system wereexpressed (SI Appendix, Fig. S14B). Despite the absence of cellsurface TACI, we detected cytoplasmic TACI in WT pMϕs usingimmunofluorescence microscopy (SI Appendix, Fig. S14C).Because TACI-deficient Mϕs manifest the M2 phenotype, we

hypothesized that TACI may be mediating signals that regulatethe Mϕ phenotype. We first assessed the effect of BAFF andAPRIL on the expression of molecules associated with the M2phenotype. We used IFN-γ and LPS as a control stimulus thatdown-regulates M2 markers (18) and measured the expression ofmRNA for CCL22, IL-10, Arg1, FIZZ1, Ym1, and IL1RN inBAFF- and APRIL-stimulated WT Mϕs. As with IFN-γ andLPS, both BAFF and APRIL induced significant down-regula-tion of all six genes by WT Mϕs (Fig. 5A). In contrast, other thanArg1, the expression of M2 markers remained unchanged inBAFF- and APRIL-stimulated TACI KO Mϕs (SI Appendix, Fig.S15). Next, we sought to determine whether the down-regulationof M2 markers is accompanied by an increase in M1 markers inWT cells stimulated with BAFF and APRIL. APRIL inducedsignificantly increased IL-6 secretion and elevated expression ofMMP9 mRNA and CD80 in WT cells (SI Appendix, Fig. S16A–C). The levels ofmmp9 and CD80 did not change in TACI KOcells after APRIL stimulation. However, APRIL did induce IL-6from TACI KO cells, although the level of IL-6 secreted byTACI KO cells was significantly less than the IL-6 secreted byWT cells. Unlike APRIL, BAFF was not able to induce IL-6secretion from either cells and only induced the expression ofMMP9 mRNA in WT cells. The small amount of IL-6 secreted inAPRIL-induced TACI KO cells may be due to signals mediatedthrough the other APRIL receptors: BCMA or heparan sulfate

proteoglycans. Alternatively, a yet to be identified APRILreceptor may be responsible for IL-6 secretion. Interestingly,neither APRIL nor BAFF induced other M1 markers, such asIL-12 p40, nitrite, and TNF-α, from WT or TACI KO cells (SIAppendix, Fig. S16D). Underscoring the M2-skewed phenotypeof TACI KO cells, the increase in five of six M1 markers wassignificantly less in TACI KO cells than those in WT cells afterstimulation with IFN-γ and LPS (SI Appendix, Fig. S16D). Bi-ological activities of BAFF and APRIL could not be attributedto a possible LPS contamination because BAFF was unable toinduce IL-6, and both the cytokines could not induce TNF-α,IL-12, and nitrite from Mϕs. Also, BAFF and APRIL had nearlyundetectable levels of LPS as measured in the limulus amebocytelysate assay (SI Appendix, Fig. S16E). Moreover, polymixin B was

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Fig. 5. The effect of BAFF and APRIL stimulation of Mϕs on M2 polarizationand parasite growth. (A) pMϕs from WT mouse were stimulated with BAFF,APRIL, IFN-γ, and LPS or left in media for 24 h. Fold differences of CCL22,IL-10, Arg1, FIZZ1, Ym1, and IL1RN mRNA expression compared with mediacontrol were determined by qPCR. Mean ± SD from six to nine samples fromthree independent experiments are shown. **P < 0.01 and ***P < 0.001 forstatistical differences between media and stimulated cells. (B) qPCR analysisof M2 gene expression in BAFF- or APRIL-stimulated Mϕs that had beentransfected with TACI siRNA or control siRNA. Relative gene expressionnormalized to GAPDH ± SD was plotted. Mean expression ± SD from fiveexperiments is shown. *P < 0.05, **P < 0.01, and ***P < 0.001 for statisticaldifferences between Control siRNA and TACI siRNA-treated cells. (C) pMϕsfrom WT or TACI KO mice were infected with stationary phase cultures ofL. major promastigotes. After 6 h, extracellular Leishmania were washedfrom cells, and BAFF or APRIL was added to infected pMϕs at various con-centrations. Mean number of parasites per infected pMϕs and the per-centage of pMϕs that were infected by Leishmania were determined 48 hlater. **P < 0.01 and ***P < 0.001 for statistical differences between mediaand cytokine-treated cells. Red asterisk indicates significant changes whenBAFF was added, whereas the blue asterisk indicates statistically significantchanges when APRIL was added to the cells.

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not able to inhibit APRIL-induced IL-6 secretion (SI Appendix,Fig. S16F), whereas boiling did (SI Appendix, Fig. S16G).The ligand dependence of TACI-mediated changes in the Mϕ

phenotype was also evaluated by silencing the TACI mRNA inWT Mϕs with siRNA. Transfection of WT cells with siRNA tar-geting the TACI gene resulted in a significant reduction in TACImRNA (SI Appendix, Fig. S17A) and protein (SI Appendix, Fig.S17B). Silencing of TACI mRNA did not affect the expression ofBAFF or APRIL in Mϕs (SI Appendix, Fig. S17C). Also, ablationof TACI expression did not automatically lead to a decrease inthe expression levels of M2 genes arg1, il1rn, ym1, fizz1, and ccl22(SI Appendix, Fig. S18). However, the ability of BAFF andAPRIL to down-regulate M2 genes was affected in siRNA-transfected Mϕs. The reduction in M2 genes after BAFF treat-ment was significantly less for all five M2 genes in TACI ablatedcells compared with cells transfected with control siRNA (Fig.5B). Suppression of TACI expression also led to a significantdecrease in the down-regulation of Arg1, IL1RN, FIZZ1, andCCL22 mRNA after APRIL stimulation. Reduction in ym1expression was also affected after APRIL stimulation of siRNA-transfected cells, but this decrease did not reach statisticalsignificance. Collectively, siRNA experiments indicate that TACIdeficiency does not spontaneously result in higher expressionof M2 markers in Mϕs. Instead, both the TACI KO andthe siRNA inhibition experiments support a role for TACIin ligand-mediated modulation of genes associated with Mϕsphenotype.Next, we assessed the biological significance of BAFF- and

APRIL-induced changes in Mϕ phenotype by monitoring Leish-mania survival in ligand-stimulated TACI KO and WT cells.Parasite count 6 h after the incubation of Mϕs with L. majorpromastigotes confirmed that cells from both the mouse strainscontained comparable numbers of parasites before stimulationwith BAFF and APRIL (SI Appendix, Fig. S19A). Stimulation ofLeishmania-infected Mϕs with BAFF or APRIL resulted in aconcentration-dependent decrease in the percentage of infectedcells and parasites per cell in WT Mϕs (Fig. 5C and SI Appendix,Fig. S19B). Supporting a role for TACI in ligand-mediatedparasite killing, parasite burden in TACI-deficient Mϕs was notaffected by APRIL or BAFF stimulation. Collectively, BAFFand APRIL stimulation experiments provide a mechanistic insightinto the Mϕ phenotypic changes and leishmaniacidal activitymediated by TACI.

ERK1/2-MAPK Pathway Is Involved in TACI-Mediated Signaling in Mϕs.Last, we investigated whether ligand stimulation is associatedwith a TACI-mediated signal transduction in Mϕs. We focusedon the extracellular signal-regulated kinases (ERK1/2)-mitogenactivated protein kinases (MAPK) pathway because this pathwayis shown to be involved in Mϕs polarization (27–29), and TACI isknown to mediate ERK1/2-MAPK signaling in B cells (30, 31).APRIL stimulation of WT pMϕs led to a significant increase inERK1/2 phosphorylation by 3 h, which peaked at 24 h (Fig. 6 Aand B). As it has been observed in gene expression analysis andLeishmania killing experiments, TACI-deficient pMϕs were notresponsive to APRIL-induced signaling. To determine whetherERK1/2-MAPK signaling is involved in APRIL-induced IL-6 se-cretion and CD80 expression, we stimulated pMϕs in the presenceof U0126, a selective inhibitor of MEK1/2, which is a signalingmolecule just upstream of ERK1/2. Treatment of pMϕs withU0126 abolished both constitutive and APRIL-induced phos-phorylation of ERK1/2 (Fig. 6C). Consequently, U0126 treat-ment blocked CD80 expression (Fig. 6D) and significantly reducedAPRIL-induced IL-6 secretion (Fig. 6E). Thus, signaling dataand biological activity experiments indicate a functional TACImolecule in Mϕs that is able to signal despite its intracellularsequestration.

DiscussionTACI is known to be crucial in the development and mainte-nance of plasma cells specific for TI-2 antigens by providing asecond signal besides the B-cell receptor engagement (3, 6, 31,32). Here, we identified a novel and fundamental role for TACIin influencing the Mϕ phenotype. First, Mϕs manifest pheno-typic and functional characteristics of the M2 phenotype in theabsence of TACI. Second, TACI deficiency renders otherwiseresistant C57BL/6 mice susceptible to Leishmania infection, andadoptive transfer of WT Mϕs into TACI KO mice is sufficient toincrease its resistance to Leishmania infection. Third, highlightingthe role of TACI in influencing pMϕ polarization, only TACI-expressing WT Mϕs respond to BAFF and APRIL stimulationby decreasing M2 markers and controlling in vitro Leishmaniainfection.Impaired response to TLR7 and TLR9 agonists on B cells

from CVID patients with tnfrsf13b mutations has been previouslyreported (4). Here, we extended these observations by investi-gating the in vitro and in vivo activities of a panel of TLR ago-nists in TACI KO cells, and we conclusively established thatTACI-deficient Mϕs respond poorly to TLR agonists. We alsofound that the expression levels of TLRs, CD14, and the adaptormolecules MyD88 and TRIF are significantly lower in TACI KOMϕs than in WT cells. Reduced expression of the TLR receptorcomplex and the adaptor molecules is likely to cause Mϕ re-sponse ablation to TLR agonists because it has been documentedthat the expression levels of TLRs determine the magnitude ofresponse to TLR agonists (11). Our data complement the previousreports demonstrating that TACI interacts with both TLRs (4)

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Fig. 6. TACI-mediated ERK1/2 signaling in pMϕs. (A) pMϕs from TACI KO orWT mice were stimulated with APRIL, LPS, and IFN-γ for 0, 3, and 24 h. Totaland phosphorylated ERK1/2 molecules were detected by Western blotanalysis. One of three experiments with similar results is shown. (B) The ratioof the band density of phosphorylated ERK1/2 molecules to total ERK1/2 wascalculated for each condition. Mean fold induction in band density relativeto 0 h ± SD from three experiments is shown. **P < 0.01 for statistical dif-ferences between WT and TACI KO cells. ##P < 0.01 and ###P < 0.001 forstatistical differences between media and stimulated cells. (C–E) WT pMϕswere stimulated with APRIL in the presence or absence of U0126 inhibitor(20 nM) for 24 h. (C) Total and phosphorylated ERK1/2 molecules weredetected in Western blot analysis. One of two experiments with similar re-sults is shown. (D) CD80 expression was determined by flow cytometry. Arepresentative experiment out of three repeats with similar results is shown.The frequencies of CD80+ cells are shown in each histogram. Solid black lineindicates CD80 Ab staining, and filled histogram shows isotype controls Abstaining. (E) Culture supernatant IL-6 levels were measured by ELISA. Meanconcentrations ± SD from three experiments are shown. Data from threeexperiments ± SD were plotted. ***P < 0.001 for statistical differences be-tween U0126 treated and untreated cells. ###P < 0.001 for statistical differ-ences in expression between media and APRIL-stimulated cells.

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and MyD88 (8) and strengthen the burgeoning concept that TACIis needed for optimal TLR function.The differences between TACI KO and WT Mϕs are not

confined to their responses to TLRs. The profound reduction ofTLR agonist-induced inflammatory response can be interpretedas an attenuation of M1 phenotype (33, 34). Indeed, in additionto the dampened response to TLR agonists, significantly higherexpression of M2 phenotype-associated molecules is detected inTACI KO cells in comparison with WT cells. In vivo, the diversityand plasticity of Mϕs depend on the type of signals present intheir microenvironment (18). Using M1- and M2-polarizing sig-nals, we revealed that TACI KO Mϕ polarization to M1 pheno-type is limited, whereas its M2 polarization is enhanced. Thesedata collectively suggest that, on one hand, Mϕs adapt anM2 phenotype when TACI is absent, and on the other hand,M2-skewed TACI KO Mϕs have limited plasticity.The default M2 polarization of Mϕs in TACI KO mice suggests

a role for TACI in mediating signals that favor M1 polarization inMϕs. One possible mechanism by which TACI influences the Mϕphenotype might be through the interaction between TACI ex-pressed on B cells and the membrane exposed BAFF and APRILon Mϕs. This B-cell TACI-induced and membrane BAFF-andAPRIL-mediated “reverse signaling” in Mϕs was initially pro-posed based on assays using soluble TACI-fc molecules in whichmembrane BAFF- and APRIL-expressing cells were activated byTACI-fc (35–37). However, subsequent studies have shown thatthe Fc-receptor engagement by TACI-fc and BAFF-R-fc mole-cules, but not membrane BAFF or APRIL, is responsible for Mϕactivation (38). Additional evidence refuting the hypothesis ofreverse signaling induced by B-cell TACI molecules comes fromexperiments using B cell-deficient mice. Because B cells are theprimary source of membrane TACI, Mϕs from B-cell KO miceare expected to adopt the M2 phenotype because they would notbe exposed to reverse signaling. However, Andreu and colleaguesdemonstrated that B cell-deficient mouse Mϕs are polarized to-ward an M1 phenotype (39). Our experiments favor a direct sig-naling of Mϕ-TACI, induced by the soluble forms of ligands, bothBAFF and APRIL, as a mechanism responsible for the phenotypicchanges inflicted in Mϕs. First, we showed that, similar to the M1polarizing signal IFN-γ and LPS, BAFF and APRIL stimulation ofTACI-bearing WT Mϕs induces a profound reduction in theexpression of M2 markers, whereas TACI-deficient Mϕs are notresponsive. Second, by silencing of the TACI molecule in WTMϕs with siRNA, we reproduced the phenotypic changes ob-served in ligand-stimulated TACI KO cells. The siRNA experi-ments also explicate the ligand dependency of the phenotypicchanges observed in TACI KO cells, conceivably because WTMϕs do not spontaneously default to an M2 phenotype on TACIdown-regulation. Hence, constant in vivo encounters with BAFFand APRIL are likely to be responsible for the suppression ofM2 markers in WT mice, whereas TACI deficiency would pre-vent the BAFF- or APRIL-mediated suppressive signals, leadingto elevated M2 markers in TACI KO mice. Finally, a third pieceof evidence for the involvement of TACI in BAFF- and APRIL-mediated Mϕ activation emerges from the in vitro Leishmaniakilling experiments. These experiments revealed that both BAFFand APRIL are able to diminish Leishmania growth in WT Mϕs,as does the M1 polarizing signal IFN-γ and LPS. By contrast andunderscoring the role of TACI in mediating BAFF- and APRIL-induced signals, intracellular parasite load remains unchanged inBAFF- or APRIL-stimulated TACI KO Mϕs.The Mϕ polarization experiments suggested that BAFF and

APRIL activity is mediated through TACI despite its intracell-ular localization. Using human monocytes and DCs, Chang andcolleagues have shown that BAFF induces the secretion of pro-inflammatory cytokines through intracellular TACI (9, 10). Weprovided evidence that TACI-mediated events also involve signaltransduction as ERK1/2 is phosphorylated in APRIL-stimulated

pMϕs. The decision to focus on the ERK1/2-MAPK pathway wasbased on the fact that ERK1/2 phosphorylation has been shown tomediate M1 polarization of Mϕs (27–29), and we determined thatAPRIL induces the expression of M1 markers CD80, MMP9, andIL-6 (SI Appendix, Fig. S16 A–C). Supporting a role for the ERK1/2-MAPK pathway in transducing signals downstream of TACI,APRIL is able to induce ERK1/2 phosphorylation in WTMϕs butnot in TACI KO cells. Furthermore, the ERK1/2 inhibitor, U0126,significantly reduces IL-6 secretion and totally blocks the expres-sion of M1 markers CD80 and IL-6 in APRIL-stimulated WTMϕs. Interestingly, APRIL is able to induce IL-6 from TACI KOMϕs, although its levels remains significantly lower than the levelsof IL-6 secreted from WT cells. The absence of an abolished IL-6secretion, despite a total block in ERK1/2 phosphorylation, to-gether with the induction of IL-6 in TACI KO Mϕs, points to theinvolvement of an additional APRIL receptor in the secretion ofIL-6. Nevertheless, activation of ERK1/2-MAPK pathway is likelya result of either APRIL engaging intracellular TACI or APRILinducing the translocation of TACI on stimulation, as previouslysuggested by He and colleagues (8).Altogether, our ligand stimulation experiments indicate that

BAFF and APRIL promote an M1 phenotype somewhat dif-ferently from the phenotype induced by the classical M1 stimu-lus, IFN-γ and LPS. A common feature between the classical M1signal IFN-γ and LPS and BAFF and APRIL is their suppressiveeffect on the expression of M2-markers. However, these twosignals differ in the induction of M1 markers. Unlike the classicalM1 signal, BAFF and APRIL induce only a limited number ofM1 markers. Nevertheless, elevated BAFF and APRIL levelsmay be contributing to the inflammatory responses by Mϕs (40).Supporting this hypothesis, Sutherland and colleagues found thatBAFF transgenic mice manifest exacerbated inflammatory diseaseand diminished allergic airway reaction (41). Likewise, APRILdeficiency results in enhanced Th2 response and increased lunginflammation in an asthma model (42). The fact that pediatricCVID patients with tnfrsf13b mutations are prone to asthmadevelopment may be another indication of a requirement forfunctional TACI to maintain the M1 phenotype (43).Macrophages are important in controlling intracellular in-

fections and they respond to microbial assault by expressing in-flammatory molecules (M1 markers). Conversely, M2 Mϕs areimpaired in killing of intracellular pathogens (18). In fact, path-ogens have evolved to induce the Mϕ M2 polarization to preventtheir elimination by the host (44). We were able to reiterate thefunctional properties of the M2 phenotype in Leishmania-infectedTACI KO Mϕs. Both TACI-deficient Mϕs and TACI KO miceare impaired in controlling Leishmania infection. Similar to find-ings from in vitro-infected Mϕs, immunohistochemical staining ofTACI KO mouse ear lesions shows limited NO activity, and Mϕsisolated from infected TACI KO ears express significantly moreArg1, IL1RN, and Ym1 mRNA than WT samples. Interestingly,unlike the susceptible BALB/c mouse (45), more severe mani-festation of infection is not accompanied by an increase in Th2response in TACI KO mice. Instead, TACI KO mice seem im-paired in mounting the protective Th1 response, as evidenced byreduced expansion of IFN-γ+CD4+ cells in the skin and drainingLNs throughout the course of the infection. This diminished Th1response is likely to be responsible for the persistence of parasitesin the skin and draining LNs until the 12th week of infection.Ablated Th1 response is not a result of an inherent defect in TACIKO CD4+ cells because TACI KO T cells respond to Th1 po-larizing stimuli as efficiently as WT T cells do. Although thepresence of M2 Mϕs may have led to the ablated Th1 response inTACI KO mice, a multitude of in vivo compensatory changesresulting in a response to TACI deficiency may have also con-tributed in preventing the expansion of Th1 cells. Our results pointto M2-skewed Mϕs responsible for the susceptibility of TACI KOmice to Leishmania, as the transfer of TACI+WTMϕs into TACI

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KO mice not only alleviates skin pathology but also results inreduced parasite burden in Leishmania-challenged TACI KOmice. Conversely, injection of TACI KO Mϕs exacerbates diseasein the recipient TACI KO mice. Thus, both phenotypes of Mϕsare sufficient to alter the infection severity in TACI KO mice.Other groups have shown that blocking M2 polarization affordsresistance in the susceptible BALB/c mouse (46, 47). To ourknowledge, our results provide the first direct evidence for a rolefor M2 Mϕs in the conversion of the resistant C57BL/6 strain intoa susceptible phenotype. The deficiency of IFN-γ or iNOS inC57BL/6 mouse results in a more severe manifestation of leish-maniasis, most probably because an elevated Th2 response is ac-companied by a total deficiency of Th1 response both in the IFN-γKOmice and the iNOS KOmice (48). The kinetics and severity ofLeishmania infection in TACI KO mice mimic those observed inTLR4- and TLR9-deficient C57BL/6 mice infected with Leish-mania, both exhibiting diminished dermal lesions at 8–10 wk afteran exacerbated manifestation of the disease at 4–6 wk afterchallenge (49, 50). The absence of a compensatory Th2 response,along with a low but detectable Th1 response, appears to be re-sponsible for the manifestation of a milder form of infection inTACI KO mouse compared with IFN-γ KO or iNOS KO mice.In conclusion, data presented in this study highlight an un-

recognized yet central function for TACI in modulating the Mϕphenotype. We propose that BAFF and APRIL control parasiticinfections by influencing the Mϕ phenotype during the earlystages of microbial assault as part of the innate immune re-sponse. The absence of a robust Th1 response in Leishmania-infected TACI KO mice is likely a result of the ablated M1 re-sponse. Of interest, a similar innate-like role for BAFF andAPRIL has been proposed in the rapid secretion of low affinityIgM and IgG3 Abs, where TACI engagement provides a secondsignal to B1 and Marginal Zone B cells specific for T cell-independent antigens, such as bacterial polysaccharides (26). Thediscovery of a role for TACI in influencing the Mϕ phenotypecan have implications for other conditions where TACI expres-sion or function is impaired. Two such instances are the newbornperiod (5) and CVID patients (4, 51). Characterization of theMϕ phenotype and function in these two populations will helpunveil whether their Mϕs also default to an M2 phenotype and, ifso, whether the phenotype of their Mϕ is responsible for con-ditions such as impaired responses to vaccines and increasedsusceptibility to pathogens.

Materials and MethodsEthics Statement. Mice were maintained and bred at the US Food and DrugAdministration (FDA) Animal Research Center. Experiments were carried outfollowing the Guide for the Care and Use of Laboratory Animals of theNational Institutes of Health (52). Experimental protocol (2002-31) was ap-proved by the Animal Care and Use Committee of the Center for BiologicsEvaluation and Research, US FDA.

Mice. Adult 6- to 8-wk-old C57BL/6 mice were purchased from The JacksonLaboratory. TACI KO mice on a C57BL/6 background were described pre-viously (3).

In Vivo L. major Infection. Experiments were carried out using different linesof L. major: L. major Friedlin strain FV1 (MHOM/IL/80/FN) and a stabletransfected line of L. major FV1 promastigotes expressing a red fluorescentprotein (RFP-Leishmania) (23). Parasites were grown at 26 °C in medium 199supplemented with 20% heat-inactivated FCS (Gemini Bio-Products). In-fective stage, metacyclic promastigotes of L. major were isolated from sta-tionary cultures (4–5 d old) by negative selection using peanut agglutinin(PNA; Vector Laboratories). For flow cytometric studies of dermal cells, micewere infected with the specified number of metacyclic promastigotes in theear dermis by i.d. injection in a volume of 10 μL. In parallel, control micereceived i.d. injection of DMEM in a volume of 10 μL.

Ear tissues were prepared as previously described (23). Briefly, the twosheets of infected ear dermis were separated and incubated in DMEMcontaining 0.2 mg/mL Liberase CI purified enzyme blend (Roche Diagnostics),and incubated for 1 h and 30 min at 37 °C. Digested tissue was cut into smallpieces and processed in a tissue homogenizer (Medimachine; Becton Dickenson).Tissue homogenates were washed with fresh DMEM containing 100 U/mLpenicillin and 100 μg/mL streptavidin and filtered through a 70-mm cellstrainer (Falcon Products). Cells were then labeled, and infected dermal Mϕs(RFP-Leishmania+CD11b+CD11c−Ly6G−) were isolated using a FACSARIA IIIcell sorter (BD Biosciences). Infected dermal Mϕs were processed for geneexpression analysis using qPCR as described in the previous section. In otherexperiments, cells were labeled to detect donor Mϕs (CD45.2+CD11b+MHCII+CD11c−Ly6G−CD45.1−) using a BD LSRFORTESSA (BD Biosciences).

Additional materials and methods are provided in the SI Appendix, SIMaterials and Methods.

ACKNOWLEDGMENTS. We thank Dr. Alain Debrabant (Center for BiologicsEvaluation and Research/US Food and Drug Administration) for providingL. major RFP parasites. This project is supported by intramural funds fromthe US Food and Drug Administration. W.R.A., A.S.C., K.U., and M.Y. were sup-ported by postdoctoral fellowships from the Oak Ridge Institute for Scienceand Education (Oak Ridge, TN).

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