TACI deficiency leads to alternatively activated phenotype and susceptibility to Leishmania infection

Windy 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 Core Facility, 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- mune response is less clear. One study reported diminished modulator and cyclophilin ligand interactor (TACI), is a key molecule B-cell responses to Toll-like receptor (TLR)7 and TLR9 agonists for plasma cell maintenance and is required in infections where in CVID patients with TACI mutations (4). A second possible link protection depends on antibody response. Here, we report that between TACI and innate immune system was suggested by He compared with WT mouse, TACI KO Μϕs expressed lower levels of and colleagues, who have shown that the TLR adaptor molecule Toll-like receptors (TLRs), CD14, myeloid differentiation primary re- myeloid differentiation primary response 88 (MyD88) is sponse protein 88, and adaptor protein Toll/IL-1 receptor domain- downstream of TACI in B cells (8). Other members of the innate containing adapter-inducing IFN-β and responded poorly to TLR immune system such as dendritic cells (DCs) and monocytes are Μϕ agonists. Analysis of phenotype revealed that, in the absence known to be the main sources of circulating BAFF and APRIL, Μϕ of TACI, s adapt the alternatively activated (M2) phenotype. but TACI expression is limited to intracellular compartments in α Steady-state expression levels for M2 markers IL-4R , CD206, these cells (9, 10). Although BAFF and APRIL can induce in- CCL22, IL-10, Arg1, IL1RN, and FIZZ1 were significantly higher in flammatory secretion in human DCs and monocytes, TACI KO Μϕ than in WT cells. Confirming their M2 phenotype, the significance of their activity remains to be understood (9, 10). TACI-KO Mϕs were unable to control Leishmania major infection Here, we investigated the role of TACI in innate immune in vitro, and intradermal inoculation of Leishmania resulted in a response and showed that TACI deficient (Mϕs) more severe manifestation of disease than in the resistant C57BL/6 strain. Transfer of WT Μϕs to TACI KO mice was sufficient to signif- respond poorly to TLR agonists, and this ablated response is icantly reduce disease severity. TACI is likely to influence Mϕ phe- likely due to reduced expression of TLRs, CD14, MyD88, and notype by mediating B cell-activating factor belonging to the TNF adaptor protein Toll/IL-1 receptor domain-containing adaptor- β family (BAFF) and a proliferation inducing ligand (APRIL) signals inducing IFN- (TRIF) in TACI KO cells. Furthermore, TACI ϕ Μϕ because both these ligands down-regulated M2 markers in WT KO M s manifested alternatively activated (M2) phenotype but not in TACI-deficient Μϕs. Moreover, treatment of Μϕs with characterized by elevated levels of molecules associated with M2 BAFF or APRIL enhanced the clearance of Leishmania from cells only phenotype and impaired resistance to in vitro Leishmania major when TACI is expressed. These findings may have implications for infection. Moreover, intradermal inoculation with L. major resulted understanding the shortcomings of host response in newborns where TACI expression is reduced and in combined variable immu- Significance nodeficiency patients where TACI signaling is ablated. Here, we described a novel role for transmembrane activator TACI | BAFF | APRIL | macrophage | Leishmania and calcium-modulator and cyclophilin ligand interactor (TACI) in determining Mϕ phenotype, a molecule that is previously ransmembrane activator and calcium-modulator and cyclo- known to be important in B-cell responses. We found that Tphilin ligand interactor (TACI) is a member of the TNF family TACI-deficient mouse Mϕs manifest an M2 phenotype. We also molecules (1). It is a receptor for B-cell activating factor (BAFF) observed that, in WT mouse Mϕs, TACI mediates B-cell acti- and a proliferation inducing ligand (APRIL). Although BAFF and vating factor- and a proliferation inducing ligand-induced sig- APRIL share a second receptor, B-cell maturation antigen nals that favor M1 polarization and Leishmania clearance. In (BCMA), BAFF-R only binds to BAFF, and heparan sulfate TACI-deficient mice, M2-polarized status of Mϕs was responsible proteoglycans only engage APRIL. TACI is primarily expressed for the diminished Th1 response and increased susceptibility on mature B cells and mediates signals for Ig isotype switch and to Leishmania infection. These findings have implications in secretion (2). Studies in TACI KO mice (3), combined variable explaining the propensity of infants to develop asthma and immune deficient (CVID) patients with mutations in TACI weak responses to vaccines because infant Mϕs are likely to be tnfrsf13b (4), and newborns who express severely reduced B-cell M2-skewed due to severely reduced expression of TACI. TACI (5) all point to its pivotal role in determining antibody (Ab) development against T cell-independent type 2 (TI-2) antigens. 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., In contrast to earlier publications (3), more recent reports 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., and showed diminished sustainment of plasma cells in response to M.A. wrote the paper. T cell-dependent (TD) antigens in TACI KO mice (6). In- The authors declare no conflict of interest. terestingly, Tsuji et al. reported that despite impaired plasma cell This article is a PNAS Direct Submission. R.S.G. is a guest editor invited by the Editorial survival and reduced Ab response to TD antigens, TACI KO Board. mice manifest enhanced clearance of the enteric pathogen Cit- 1W.R.A. and R.D. contributed equally to this work. robacter rodentium, presumably due to generation of higher 2To whom correspondence should be addressed. Email: [email protected]. avidity of Abs in the absence of TACI (7). Whereas TACI is well This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. established as a B-cell receptor, its involvement in innate im- 1073/pnas.1421580112/-/DCSupplemental.

E4094–E4103 | PNAS | Published online July 13, 2015 www.pnas.org/cgi/doi/10.1073/pnas.1421580112 Downloaded by guest on September 25, 2021 in a more severe manifestation of disease in TACI KO mouse TACI KO Mϕs Have Reduced Expression of TLRs, TLR Adaptor PNAS PLUS than the Leishmania-resistant WT C57BL/6 strain, and adap- Molecules, and CD14. One possibility for the impaired TACI KO tive transfer of Μϕs from the WT mouse was sufficient to response to TLR agonists could be the lower expression of TLRs reduce the severity of Leishmania induced cutaneous disease in (11). To test this possibility, we compared the levels of TLRs on the TACI KO mouse. Comparison of the response of WT and TACI KO and WT pMϕs and found that the expression levels TACI-deficient Μϕs revealed that TACI mediates ligand induced and the frequencies of TLR9 and TLR4 (SI Appendix, Fig. S4 A ϕ down-regulation of molecules associated with M2 Mϕ phenotype and B) expressing cells were less in TACI KO pM s than in WT ϕ and up-regulation of some of the markers representative of cells. We also determined CD14 expression on pM s because classically activated (M1) phenotype. Collectively, these findings CD14 is known to augment responses to TLR agonists and its extend the role of TACI from its well-defined involvement in expression is up-regulated on TLR stimulation (12). We found ϕ that after stimulation with CpG, LPS, and Poly I:C, the per- B-cell homeostasis to M phenotype determination and resistance + ϕ to intracellular pathogens. centage of CD14 pM s was significantly lower in TACI KO cells than in WT cells (SI Appendix, Fig. S4 C and D). Thus, Results lower expression of TLRs and CD14 is likely to be responsible for the impaired response of TACI KO pMϕs to TLR agonists. TACI-Deficient Cells Respond Poorly to TLR Agonists. Previous studies A crucial step in successful TLR signaling is the colocalization reported a diminished B-cell response from CVID patients of TLRs with the adaptor molecules MyD88 and/or TRIF (13). To with TACI mutations to TLR7 and TLR9 agonists (4). To examine the activation events after TLR agonist stimulation, we directly assess the involvement of TACI in response to TLR incubated pMϕs with CpG for 3 h and evaluated TLR9 and agonists, we measured TNF-α and IL-6 levels in the culture ϕ ϕ MyD88 expression by confocal microscopy. Consistent with our supernatants of peritoneal M s(pM) and bone marrow- flow cytometry results, unstimulated TACI KO pMϕshadsig- derived DCs (BMDC) from TACI KO and WT mice after stim- nificantly lower TLR9 expression than WT mice (SI Appendix, ulation with the TLR agonists LPS, CpG, peptidoglycan, Poly Fig. S5). Confocal microscopy also illustrated that TACI KO pMϕs I:C, lipoteichoic acid, and ssRNA. All TLR agonists induced had significantly reduced MyD88 expression. Consequently, CpG α significantly lower levels of TNF- and IL-6 from TACI KO stimulation resulted in limited colocalization of TLR9 and ϕ pM s than the WT cells (SI Appendix,Fig.S1A). With the MyD88 in TACI KO pMϕs compared with WT pMϕs. Similar exception of ssRNA-induced TNF-α and Poly I:C-induced IL-6, observations were made when pMϕs were analyzed for TLR4 which remained low in both strains, all TLR agonists induced and MyD88 or TRIF expression patterns after LPS stimulation significantly lower levels of TNF-α andIL-6inTACIKO (SI Appendix, Fig. S6 A and B). Finally, TLR3 and TRIF mean BMDCs than WT cells (SI Appendix,Fig.S1B). Similar results fluorescence intensity values were significantly reduced in TACI were obtained in bone marrow-derived Mϕs(BMDMs)(SI Ap- KO pMϕs compared with WT cells, and their colocalization and pendix,Fig.S1C). Furthermore, the decrease in cytokine se- translocation were incomplete on Poly I:C stimulation (SI Ap- cretion was preceded by impaired mRNA expression in CpG-, pendix,Fig.S6C). Jointly, both flow cytometry analysis and the LPS-, or Poly I:C-stimulated TACI KO pMϕs, with the exception microscopic evaluations suggest lower TLR and adaptor molecule of IL-6 in CpG-stimulated cells (SI Appendix,Fig.S1D). expression as the underlying cause of impaired TACI KO-pMϕ Induction of costimulatory molecule expression is a crucial response to TLR agonists. component of TLR agonist activity on innate immune system. We therefore measured the expression of CD40, CD80, CD86, TACI KO Mϕs Have an M2 Phenotype. In Mϕs, TLR-induced in- and MHCII molecules in TLR agonist-stimulated pMϕs. As ex- flammatory cytokine production and costimulatory molecule pected, WT pMϕs responded robustly to TLR agonist stimulation expression is an important component of the classically activated ϕ (SI Appendix,Fig.S2A and B). In contrast, TACI KO pMϕs only (M1) phenotype (14). Conversely, alternatively activated pM s induced the expression of MHCII when stimulated with CpG and are restricted in their ability to secrete inflammatory and increase the expression of costimulatory molecules (15, 16). induced the expression of CD86 when stimulated with LPS and ϕ Poly I:C. More importantly, all three TLR agonists failed to up- To examine whether ablated TLR activity in TACI KO pM sis regulate CD40 or CD80 expression in TACI KO cells. These re- associated with the M2-skewed phenotype, we determined the expression of a panel of representative M2 markers in TACI KO sults suggested that suboptimal response of TACI KO pMϕsto and WT cells. We found that cell surface IL-4Rα and CD206 TLR agonists was not restricted to cytokine secretion. were expressed at significantly higher levels on TACI KO cells compared with WT pMϕs (Fig. 1A). TGF-β is produced by M2 TLR Agonists Manifest Ablated Adjuvant Activity in TACI KO Mice. ϕ Next, we assessed whether the TACI KO mouse response to M s and inhibits proinflammatory responses and microbicidal activity (17). Stimulation of TACI KO pMϕs with LPS, CpG, and TLR agonists was diminished in vivo as well. For this purpose, INFLAMMATION

Poly I:C resulted with a significant increase in TGF-β levels (Fig. IMMUNOLOGY AND we immunized TACI KO and WT mice with tetanus (TT) alone 1B). Among the three TLR agonists, only LPS significantly in- or TT with CpG, and measured serum IgG and IgM Ab levels 0, duced TGF-β secretion by WT pMϕs. Additional M2 markers 15, and 30 d after immunization. In agreement with recent re- were evaluated by quantitative RT-PCR (qPCR). Of the eight ports (6), we also found that for TACI KO, Ab responses to the encoding for M2 markers, CCL22, IL-10, Arginase 1 TD antigen, TT was significantly lower in TT-immunized TACI (Arg1), IL-1 receptor antagonist (IL1RN), and found in in- KO mice than the WT mice on all days tested (SI Appendix, Fig. flammatory zone protein (FIZZ1) mRNA expression was sig- S3A). As expected, CpG was able to significantly increase IgG nificantly higher in TACI KO pMϕs compared with WT cells, and IgM Ab responses to TT in WT mice. In contrast, TACI KO while chitinase 3-like 3 (or Ym1) levels were comparable be- mouse IgG and IgM Ab levels remained the same with or without tween the two strains (Fig. 1C). TACI KO BMDMs also had a CpG. We also examined the ability of LPS to induce inflam- phenotype that was consistent with an M2 phenotype, although matory cytokines in TACI KO mice. After i.p. injection of 0.75 mg there were differences in the expression of some of the markers of LPS, significantly reduced levels of serum IL-6 and TNF-α were tested. Similar to pMϕs, TACI KO BMDMs had significantly measured in TACI KO mice compared with the WT mice (SI higher TGF-β in CpG- and Poly I:C-stimulated TACI KO cells Appendix,Fig.S3B). Taken together, the in vivo data were in compared with WT cells, but LPS stimulation induced higher agreement with the results from in vitro experiments and sug- TGF-β in WT BMDMs (SI Appendix, Fig. S7A). The expression gested that TLR activity was severely impaired in TACI KO mice. of IL-4Rα was higher in TACI KO BMDM cells than WT cells,

Allman et al. PNAS | Published online July 13, 2015 | E4095 Downloaded by guest on September 25, 2021 to IL-4/IL-13 stimulation than the WT cells, M1 stimulus had a AB30 ** + 25 stronger effect on WT cells than on TACI KO Mϕs. After LPS α 20 Cell # 15 and IFN-γ stimulation, TACI KO cells expressed significantly 10 5

% IL4R- lower levels of MMP9 mRNA than WT cells, and the increase in IL4Rα 0 TACI KO WT * TACI KO culture supernatant IL-12 p40 and nitrite concentrations 50 ** 40 *** * were significantly less compared with those in WT cells (Fig. 1D). Cell # 30 20 Conversely, IL-4 and IL-13 stimulation led to a significantly 10 % CD206+ higher CCL22, Arg1, Ym1, and FIZZ1 mRNA expression in CD206 0 TACI KO WT TACI KO cells than WT cells (Fig. 1E). Expression of IL-10 and C CCL22 IL-10 Arg1 IL1RN mRNA was not different between the two mouse strains. on on ion 80 *** 30 *** *** 60 Takentogether,thesedatashowthat,intheabsenceofTACI, ess essi 60 essi 20 pr pr pr ϕ

x 40 x 40 x pM s default to an M2 phenotype. TACI cells also have a pro- E 10 AE 20 AE 20 pensity to express higher M2 markers following stimulation with RNA RN 0 RN 0 0 m m TACI KO WT m TACI KO WT TACI KO WT IL-4/IL-13, and they respond poorly to M1 polarization signals. FIZZ1 Ym1 IL1RN on on on *** 250 8 80 *** Leishmania major Infection in TACI KO Mice. Control of infection by essi essi essi 200 6 60 pr pr 150 pr x x x 4 40 L. major, an intracellular parasite, requires M1 polarization of 100 AE AE 50 2 20 Mϕs, which enhances intracellular killing of parasite and the RN RN 0 0 0 mRNA E m TACI KO WT m TACI KO WT TACI KO WT appropriate induction of a Th1 immune response (19). In gen-

TACI KO eral, the Th1 or Th2 polarization of T cells is a reflection of the D MMP9 40 TACI KO WT ** TACI KO ϕ 500 WT M) 30 M1 and M2 polarity of M s, respectively (14). C57BL/6 mice are μ WT ** 400 20 genetically resistant to L. major and spontaneously resolve 300 200 10

Nitrite ( infection by mounting Th1 response (20). Because TACI KO 100 ** 0 Mϕs have a default M2 phenotype, we hypothesized that they Fold induction 0 Media IFN-γ+LPS Media IFN-γ+ LPS would be less able to control L. major infection. After 6 h of E CCL22 IL-10 Arg1 25 10 100 incubation, the number of parasites inside the cells and the ** *** TACI KO 20 8 80 percentage of infected cells were similar in both the mouse 15 6 60 WT 10 4 40 strains (Fig. 2B). This observation indicated that that the 5 2 20 0 0 0 phagocytosis of parasites by TACI KO Mϕs was not impaired. Fold induction Media IL-4+IL-13 Fold induction Media IL-4+IL-13 Fold induction Media IL-4+IL-13 However, after 2 and 4 d of infection, there was significantly en- FIZZ1 Ym1 IL1RN hanced parasite growth in TACI KO Mϕs compared with WT cells 50 *** 500 *** 6 40 400 (Fig. 2 A and B). Moreover, the number of infected Mϕs was sig- 30 300 4 20 200 2 nificantly higher in TACI KO cells than in WT cells. Leishmania 10 100 0 0 0 infection induces the M1 phenotype-associated inducible nitric Fold induction Fold induction Media IL-4+IL-13 Fold induction Media IL-4+IL-13 Media IL-4+IL-13 oxide synthase (iNOS or NOS2) molecules, which mediate in Fig. 1. Expression of M1 and M2 markers in Mϕs. (A) Shown in histogram is vitro (21) and in vivo (22) nitric oxide-mediated parasite killing. the expression of IL-4Rα and CD206 in purified pMϕs determined by flow Analysis of culture supernatants on day 2 of infection revealed cytometry. Mean percentages ± SD of CD11b+IL-4Rα+ and CD11b+CD206+ that Leishmania-infected WT Mϕs secreted significantly ele- cells from three experiments are plotted. (B) Concentration of TGF-β in the vated levels of nitrite, the stable end product of nitric oxide, supernatants of pMϕs after CpG, LPS, or Poly I:C stimulation for 24 h. ELISA whereas TACI KO nitrite levels did not change over time (Fig. results shown are the mean ± SD of six determinations from three in- 2C). These data indicate that, consistent with an M2 phenotype, dependent experiments. (C) Ex vivo mRNA expression of M2 markers by the ability of Mϕs to kill intracellular pathogens is impaired in ϕ ± pM s. Relative gene expression normalized to GAPDH SD was plotted. TACI-deficient cells. (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- Next, we investigated whether the in vivo M2 microenviron- pared with media were determined by qPCR. Culture supernatant IL-12p40 ment would render TACI KO animals susceptible to L. major levels were measured in ELISA, and nitrite production was measured using infection. TACI KO and WT mice were infected by intradermal Griess reagent. (E) TACI KO and WT pMϕs were treated with 15 ng/mL IL-4 (i.d.) inoculation of L. major parasite in the ear, and ear parasite and 15 ng/mL IL-13 (M2 stimulus) or left in media for 24 h. Fold differences of burden was assessed over a 12-wk period. Disease severity was CCL22, IL-10, Arg1, IL1RN, Ym1, and FIZZ1 mRNA expression compared with determined by analysis of lesion size and histological evaluation media control were determined by qPCR. Mean ± SD was obtained from of ear sections. Although most of the ear pathology resolved in three independent experiments each with at least three mice per group.*P < both the strains by week 10, compared with WT mice, destructive < < 0.05, **P 0.01, and ***P 0.001 for statistical differences between WT and changes in TACI KO ears were more severe throughout the TACI KO cells. infection (Fig. 2D), concomitant with significantly larger lesion size in TACI KO mice between weeks 4 and 8 after infection but CD206 was not (SI Appendix, Fig. S7B). Among the genes (Fig. 2E). In histopathological evaluation (5 wk), the difference associated with M2 phenotype, ccl22 and il10 was higher in TACI in lesion thickness was evident between the two mouse strains KO BMDMs, but arg1, il1rn, and ym1 were comparable between (Fig. 2F). In higher magnifications, TACI KO samples revealed more infected cells, the majority of them being infiltrated Mϕs. the two strains (SI Appendix, Fig. S7C). The gene for FIZZ1 was Ear sections stained for iNOS at the same time point showed not detected in BMDM from either mouse strains. These data ϕ limited iNOS production in TACI KO mice (Fig. 2G). Con- confirmed the default M2 phenotype in TACI KO M s, al- versely, and consistent with an inflammatory response, WT though there were variations in the expression of M2 markers samples had widespread iNOS staining. Accompanying the se- ϕ depending on the source of M s. verity of disease, at 5 wk after infection, the number of parasites Macrophages can be polarized into the M1 or M2 phenotype present in the ear and in the draining lymph node (LN) was depending on the stimuli present in the microenvironment (14). significantly higher in TACI KO mice compared with WT mice To assess the plasticity of TACI KO Mϕs, cells were incubated (Fig. 2H). Despite the healed cutaneous lesion, Leishmania with LPS/IFN-γ for M1 differentiation or IL-4/IL-13 for M2 dif- persisted in the ear and LN of both strains of mice. However, ferentiation (18). Whereas, TACI KO Mϕsweremoresensitive WT mice had significantly fewer parasites present in the ear and

E4096 | www.pnas.org/cgi/doi/10.1073/pnas.1421580112 Allman et al. Downloaded by guest on September 25, 2021 ϕ + + − − PNAS PLUS TACI KO dermal M s(RFP CD11b CD11c Ly6G ) were isolated from A B 500 100 * * WT ear and analyzed for M2 markers (SI Appendix, Fig. S8A). The 400 *** 80 frequency of dermal Mϕs was comparable between the two strains 300 60 * WT (SI Appendix,Fig.S8B). However, TACI KO mice had signifi- 200 40 + ϕ 100 20 cantly more RFP-Leishmania dermal M s than the WT mice

0 % of Infected Cells 0 Parasite/ 100 cells Parasite/ 100 (Fig. 3A). Among the M2-marker genes, arg1, il1rn, and ym1 were 6 hr 2 day 4 day 6 hr 2 day 4 day ϕ * expressed significantly more in TACI KO dermal M s than WT # TACI KO C 3 cells (Fig. 3B). Ccl22 and il10 genes were not detected in dermal WT ϕ ϕ TACI KO 2 M from either strain. Thus, TACI KO dermal M s preserved 1 their M2 phenotype during Leishmania infection. 0 Because the type of T helper response dictates the host re- 6 hr 2 day sistance or susceptibility to Leishmania infection (20), we ana- D WT TACI KO E lyzed Th1 and Th2 populations at 2, 5, and 12 wk after the 3 TACI KO * ** WT parasite challenge in the skin and the draining LNs by quanti- 2 fying IFN-γ and IL-4 positive CD4+ T cells, respectively. In the 5 wk ** γ– + 1 skin, WT mouse IFN- producing CD4 cells were elevated by

Lesion size (mm) 0 24681012 Weeks ABArg1 IL1RN FGWT TACI KO WT TACI KO n o i 200 *** ion 200

** s ** s e ess r r 150 150 p xp x 5 wk 5 wk E 100 100 AE A N 50 N 50 R R 200 μm 200 μm m m 0 0 TACI KO WT TACI KO WT

†† H 6 6 TACI KO 10 TACI KO 10 60 Ym1 8 FIZZ1

** n 5 *** 5 * ** o WT on i 10 WT 10 i

*** *** s 4 †† 4 †† 6 10 10 40 3 3 10 xpress 10 xpres 4 E 2 2 E 10 10 1 A 20 1 NA 10 10 2 R RN 0 0 m 10 m

Parasite number/ Ear 10 5 wk 12 wk Parasite number/ LN 5 wk 12 wk 0 0 TACI KO WT TACI KO WT Fig. 2. Macrophage infection with L. major.(A–C) BMDMs were infected with stationary phase cultures of L. major promastigotes. At 6 h, 2 d, and 4 d CDTACI KO WT 20 after infection, slides were fixed and stained. (A) Diff-Quick stain of WT and TACI KO 15 WT TACI KO Mϕs 4 d after infection is shown. Red arrows indicate intracellular 2 wk 2.35 2.14 Leishmania amastigotes. (B) Kinetics of parasite load in BMDMs. Results 10 expressed either as the mean number of parasites/infected Mϕs or as per- ϕ < < 5.98 14.0 centage of M s that were infected by Leishmania.*P 0.05 and ***P 5 0.001 for statistical differences between WT and TACI KO mice. (C) Nitrite 0

production in Leishmenia-infected BMDMs was measured in culture super- Lesion in IL-4+ CD4+ % 1.24 1.18 2 wk 5 wk 12 wk natants using Griess reagent. *P < 0.05 for statistical differences between 5 wk # Post Infection WT and TACI KO mice. P < 0.05 for statistical differences between 6 h and 20 TACI KO 2d.(D–H) The ears of WT and TACI KO mice were infected with 1 × 104 5.30 9.79 * WT metacyclic promastigotes of Leishmania by i.d. injection. (D) Representative 15 * ear images 5 wk after i.d. infection with Leishmania.(E) Kinetics of ear lesion 10 size over the course of a 12-wk Leishmania infection in TACI KO (n = 9) and 2.79 3.14 = 12 wk WT mice (n 9). (F) Representative H&E-stained sections of WT and TACI KO 5 ears 5 wk after i.d. infection with Leishmania.(Upper) Magnification, 1.25×. 6.11 5.41 (Lower) Magnification, 100×. Red arrows indicate intracellular Leishmania 4 0 amastigotes. (G) Representative immunohistochemical staining of ear sec- IL- 2 wk 5 wk 12 wk IFN-γ Post Infection tions showing iNOS expression at 5 wk of Leishmania infection. Shown at

10× magnification. (H) Parasite load in ear or draining LN of TACI KO and WT INFLAMMATION

Fig. 3. Analysis of in vivo host response following L. major infection in TACI IMMUNOLOGY AND mice 5 wk after infection with Leishmania. Results expressed as the geo- KO mice. (A and B) TACI KO and WT mice were infected i.d. with 2 × 105 †† < metric mean number of parasites per organ. P 0.01 for statistical dif- metacyclic promastigotes of RFP-Leishmania. Two weeks after infection with < < ferences between 5 and 12 wk after infection. *P 0.05, **P 0.01, and RFP-Leishmania, infected dermal Mϕs(RFP-Leishmania+CD11b+Ly6G−CD11b−) < ***P 0.001 for statistical differences between the WT and TACI KO mice. were sorted from ear. Experiment was repeated three times with pooled digests from four to six ears per experiment. (A) Mean percentage ± SD and MFI ± SD of RFP-Leishmania+ dermal Mϕs determined by flow cytometry are LN by 12 wk after infection, whereas TACI KO mice failed to plotted. (B) Quantification of genes associated with M2 phenotype in RFP- decrease parasite burden over time. Leishmania–infected dermal Mϕs. Relative gene expression compared with GAPDH ± SD was determined by qPCR. Mean values from three experiments Host Response to Leishmania Challenge in TACI KO Mice. To de- are plotted. (C and D) The ears of WT and TACI KO mice were infected with termine whether the pronounced pathology, increased parasite 1 × 104 metacyclic promastigotes of Leishmania by i.d. injection. (C) Repre- load, and limited inflammatory responses were accompanied by sentative intracellular cytokine staining plots indicating the frequency of γ + M2-polarized ear Mϕs, we assessed M2 markers in Mϕs isolated Th1 (IFN- ) and Th2 (IL-4) CD4 T cells 2, 5, and 12 wk after infection found ϕ within the Leishmania infected ear lesion. Freshly isolated ear-derived skin from the ear. To facilitate the isolation of parasite-infected M s, cells were directly stained for IFN-γ or IL-4. (D) Kinetic analysis of mean we inoculated TACI KO and WT mice ears with a red fluo- percentages ± SD of CD4+IL-4+ and CD4+IFN-γ+ cells from two experiments rescent protein (RFP)-expressing L. major (RFP-Leishmania) are plotted as chart with 10 mice per group. *P < 0.05, **P < 0.01, and ***P < parasite (23). Two weeks after infection, Leishmania-infected 0.001 for statistical differences between TACI KO and WT mice.

Allman et al. PNAS | Published online July 13, 2015 | E4097 Downloaded by guest on September 25, 2021 2 wk after infection, and their frequency gradually decreased on Fig. S12). Ear pathology and parasite burden was monitored over weeks 5 and 12 (Fig. 3 C and D). This decrease coincided with a 5-wk period. Disease severity was determined by lesion size the progressive improvement in the skin lesion after week 5 (Fig. and histological evaluation of ear sections. Gross pathological γ+ 2E). The frequency of skin IFN- T cells in TACI KO mouse changes were most severe in TACI KO mice receiving TACI KO remained low throughout the span of the experimental period Mϕs followed by the PBS-injected TACI KO mice (Fig. 4A). In and was significantly less than the WT cells at 2 and 5 wk after histopathological evaluation (5 wk), lesion thickness was mark- infection. At 12 wk, the difference between the two mouse edly less in WT Mϕ transferred TACI KO mice compared with strains disappeared, when WT mouse IFN-γ+ T cells numbers ϕ B decreased to TACI KO levels. The frequency of IL-4–producing TACI-deficient M or PBS-injected TACI KO mice (Fig. 4 ). In T cells remained unchanged in WT mice throughout the exper- higher magnifications, the presence of a lower number of ϕ imental period (Fig. 3 C and D). Suggesting the absence of a infected M s was apparent in TACI KO mice injected with WT compensatory Th2 response, ablated Th1 development in TACI KO mice was not accompanied by an increase in IL-4+ T cells. We also measured serum anti-Leishmania IgG1 and IgG2c Ab levels because these isotypes reflect the phenotype of T helper A response during L. major infection (24). Indicating a likely conse- quence of impaired IFN-γ–producing T cells in TACI KO mice, the serum anti-Leishmania IgG1/IgG2c ratio was higher in TACI KO mice than the WT mice (SI Appendix,Fig.S9A). The number of T-regulatory (Treg) cells (Foxp3+IL-10+CD4+ B cells) in infected lesion has been reported to contribute to Leishmania persistence (25). To assess whether the elevated par- asite burden in TACI KO mice is accompanied by a heightened Treg response, we analyzed the frequency of Treg cells in the skin lesions and found that Treg numbers were comparable between the two mouse strains and remained unchanged throughout the experimental period (SI Appendix,Fig.S9B). In vitro stimulation of draining LN cells with Leishmania antigen confirmed the sig- nificantly lower number of IFN-γ–producing T cells in TACI KO CD mouse 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 was a significant decrease at 5 wk compared with week 2 numbers. Subsequently, IFN-γ+ T-cell numbers were significantly more at 12 wk than at 2 wk. Interestingly, this increase at 12 wk coincided E with the alleviation of skin lesions (Fig. 2E) and a decrease in parasite count in the skin of TACI KO mice (Fig. 2H). As has been shown for the WT strain previously (20) and coinciding with the increase in IFN-γ+ T-cell numbers, the high numbers of IL-4+ T-cell numbers significantly decreased by 5 wk. The kinetics of TACI KO mouse IL-4–producing T cells overlapped F with 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 naïve Tcells(CD4+CD44lowCD62Lhigh) stimulated under Th1, Th2, and Treg polarizing conditions. Analysis of the mRNA Fig. 4. Adoptive transfer of WT Mϕs to TACI KO mice protects against cu- expression of lineage-specific markers excluded the possibility taneous leishmaniasis. Adherent pMϕs (CD11b+MHCII+Ly6G−CD11c− cells) of a defective Th1 cell polarization as the underlying cause of were isolated from naïve WT or TACI KO mice and i.v. transferred (3 × 106 the inadequate Th1 response in TACI KO because both strains cells per mouse) to TACI KO mice that were infected with 1 × 104 metacyclic had equal propensity to polarize to Th1, Th2, and Treg cells (SI promastigotes of Leishmania by i.d. injection the previous day. Figure label Appendix,Fig.S10A–C). indicates the strain of host/strain of Mϕs adoptively transferred. (A) Repre- Neutrophils play a key role during the initial stages of Leish- sentative ear images 5 wk after i.d. infection with Leishmania.(B) Repre- mania infection after the deposition of parasite in the skin (23). 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, As such, changes in neutrophil function may influence the infec- respectively). (Insets) Magnified views of representative vacuoles containing tion severity. To assess whether TACI deficiency alters neutrophil Leishmania amastigotes depicted as cartoons. (C) Kinetics of ear lesion size function, we measured the ability of TACI KO neutrophils to over the course of a 5-wk Leishmania infection. Mean lesion size ± SD was phagocytize polystyrene beads or RFP-Leishmania and found obtained from two independent experiments each with five mice per group. that TACI KO and WT neutrophils had a comparable number Statistical comparison can be found in SI Appendix, Table S1.(D) Parasite of engulfed polystyrene beads and RFP-Leishmania parasites load in ear or draining LN of TACI KO and WT mice 5 wk after infection with (SI Appendix, Fig. S11 A and B). Leishmania. Results expressed as the geometric mean number of parasites per ear (n = 10 mice per group). **P < 0.01 and ***P < 0.001 indicate for WT Mouse Mϕs Alleviate Leishmania Infection in TACI KO Mice. To statistical differences between mouse groups. (E) Representative intra- ϕ cellular cytokine staining plots indicating the frequency of Th1 (IFN-γ)and examine whether the extent of TACI expression on M s con- Th2 (IL-4) CD4+ T cells at 5 wk after infection found within the ear Leish- tributes to disease severity during Leishmania infection, isolated + ϕ mania lesion. Freshly isolated ear-derived skin cells were directly stained for CD45.1 WT or TACI KO M s were adoptively transferred to IFN-γ or IL-4. (F) Mean frequency ± SD of CD4+IL-4+ and CD4+ IFN-γ+ cells TACI KO mice 1 d after infection of the ear. Skin CD45.1+ Mϕs from two experiments are plotted as chart with 10 mice per group. *P < 0.05 were detected in the ear as early as 1 d after transfer (SI Appendix, for statistical differences between TACI KO and WT mice.

E4098 | www.pnas.org/cgi/doi/10.1073/pnas.1421580112 Allman et al. Downloaded by guest on September 25, 2021 Mϕs, in contrast to the PBS or TACI KO-Mϕ transferred TACI A PNAS PLUS KO mice. Transfer of WT MϕsintoTACIKOmicealsoledtoa significant decrease in the week 3 and 5 lesion size compared with PBS-injected TACI KO animals (Fig. 4C)(SI Appendix, Table S1). Conversely, TACI-deficient Mϕs enhanced the lesion size in the recipient TACI KO mice. In addition, the number of parasites harvested from the ears of WT-Mϕ injected TACI KO mice was significantly lower than the TACI KO mice that re- ceived TACI deficient Mϕs or PBS (Fig. 4D). To evaluate whether TACI expression in Mϕs contributed to the activation of Th1 cells during Leishmania infection, we analyzed skin Th1 and Th2 populations 5 wk after the parasite challenge. Ex vivo evaluation of CD4+ cells in the skin lesions indicated that the B transfer of WT Mϕs was sufficient to significantly increase the amount of IFN-γ–producing CD4+ cells compared with PBS- injected TACI KO mice (Fig. 4 E and F). However, injection of WT Mϕs did not lead to a significant change in the frequency of Th2 (CD4+IL-4+)orTreg(Foxp3+IL-10+)cells(Fig.4E and F and SI Appendix,Fig.S13A and B). These data strongly suggest that adoptive transfer of WT Mϕs to TACI KO mice was suffi- cient to reduce the severity of Leishmania infection and that TACI expression by Mϕs is involved in the induction of a Th1 response.

TACI Ligands Regulate Phenotypic Changes in WT Mouse Mϕs. Both BAFF and APRIL are members of the TNF family of ligands and C act as key mediators of B-cell maturation and homeostasis (26). They share the receptors TACI and BCMA, whereas BAFF-R binds exclusively to BAFF. Since the discovery of this receptor- ligand system, the primary focus of research has been on their function in regulating B-cell responses. Our data thus far have suggested that this system is also important for the determination of Mϕ phenotype and function. To elucidate the mechanism of the BAFF system involvement in influencing Mϕ phenotype, we Fig. 5. The effect of BAFF and APRIL stimulation of Mϕs on M2 polarization first determined the expression of BAFF system molecules in and parasite growth. (A)pMϕs from WT mouse were stimulated with BAFF, Mϕs. We found that resident pMϕs express high levels of surface APRIL, IFN-γ, and LPS or left in media for 24 h. Fold differences of CCL22, BAFF and APRIL, but little or no surface expression of BAFF-R, IL-10, Arg1, FIZZ1, Ym1, and IL1RN mRNA expression compared with media TACI, or BCMA (SI Appendix,Fig.S14A). However, at the control were determined by qPCR. Mean ± SD from six to nine samples from < < mRNA level, all members of the BAFF and APRIL system were three independent experiments are shown. **P 0.01 and ***P 0.001 for statistical differences between media and stimulated cells. (B) qPCR analysis expressed (SI Appendix,Fig.S14B). Despite the absence of cell ϕ ϕ of M2 gene expression in BAFF- or APRIL-stimulated M s that had been surface TACI, we detected cytoplasmic TACI in WT pM susing transfected with TACI siRNA or control siRNA. Relative gene expression immunofluorescence microscopy (SI Appendix,Fig.S14C). normalized to GAPDH ± SD was plotted. Mean expression ± SD from five Because TACI-deficient Mϕs manifest the M2 phenotype, we experiments is shown. *P < 0.05, **P < 0.01, and ***P < 0.001 for statistical hypothesized that TACI may be mediating signals that regulate differences between Control siRNA and TACI siRNA-treated cells. (C)pMϕs the Mϕ phenotype. We first assessed the effect of BAFF and from WT or TACI KO mice were infected with stationary phase cultures of APRIL on the expression of molecules associated with the M2 L. major promastigotes. After 6 h, extracellular Leishmania were washed γ from cells, and BAFF or APRIL was added to infected pMϕs at various con- phenotype. We used IFN- and LPS as a control stimulus that ϕ down-regulates M2 markers (18) and measured the expression of centrations. Mean number of parasites per infected pM s and the per- centage of pMϕs that were infected by Leishmania were determined 48 h mRNA for CCL22, IL-10, Arg1, FIZZ1, Ym1, and IL1RN in < < ϕ γ later. **P 0.01 and ***P 0.001 for statistical differences between media BAFF- and APRIL-stimulated WT M s. As with IFN- and and cytokine-treated cells. Red asterisk indicates significant changes when LPS, both BAFF and APRIL induced significant down-regula- BAFF was added, whereas the blue asterisk indicates statistically significant INFLAMMATION

tion of all six genes by WT Mϕs (Fig. 5A). In contrast, other than changes when APRIL was added to the cells. IMMUNOLOGY AND Arg1, the expression of M2 markers remained unchanged in BAFF- and APRIL-stimulated TACI KO Mϕs(SI Appendix,Fig. S15). Next, we sought to determine whether the down-regulation proteoglycans. Alternatively, a yet to be identified APRIL of M2 markers is accompanied by an increase in M1 markers in receptor may be responsible for IL-6 secretion. Interestingly, WT cells stimulated with BAFF and APRIL. APRIL induced neither APRIL nor BAFF induced other M1 markers, such as significantly increased IL-6 secretion and elevated expression of IL-12 p40, nitrite, and TNF-α, from WT or TACI KO cells (SI MMP9mRNAandCD80inWTcells(SI Appendix,Fig.S16 Appendix, Fig. S16D). Underscoring the M2-skewed phenotype A–C). The levels of mmp9 and CD80 did not change in TACI KO of TACI KO cells, the increase in five of six M1 markers was cells after APRIL stimulation. However, APRIL did induce IL-6 significantly less in TACI KO cells than those in WT cells after from TACI KO cells, although the level of IL-6 secreted by stimulation with IFN-γ and LPS (SI Appendix, Fig. S16D). Bi- TACI KO cells was significantly less than the IL-6 secreted by ological activities of BAFF and APRIL could not be attributed WT cells. Unlike APRIL, BAFF was not able to induce IL-6 to a possible LPS contamination because BAFF was unable to secretion from either cells and only induced the expression of induce IL-6, and both the cytokines could not induce TNF-α, MMP9 mRNA in WT cells. The small amount of IL-6 secreted in IL-12, and nitrite from Mϕs. Also, BAFF and APRIL had nearly APRIL-induced TACI KO cells may be due to signals mediated undetectable levels of LPS as measured in the limulus amebocyte through the other APRIL receptors: BCMA or heparan sulfate lysate assay (SI Appendix, Fig. S16E). Moreover, polymixin B was

Allman et al. PNAS | Published online July 13, 2015 | E4099 Downloaded by guest on September 25, 2021 not able to inhibit APRIL-induced IL-6 secretion (SI Appendix, AB 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 in WT Mϕs with siRNA. Transfection of WT cells with siRNA tar- geting the TACI gene resulted in a significant reduction in TACI mRNA (SI Appendix,Fig.S17A) and protein (SI Appendix, Fig. S17B). Silencing of TACI mRNA did not affect the expression of BAFF or APRIL in Mϕs(SI Appendix, Fig. S17C). Also, ablation C D of TACI expression did not automatically lead to a decrease in the expression levels of M2 genes arg1, il1rn, ym1, fizz1, and ccl22 (SI Appendix, Fig. S18). However, the ability of BAFF and APRIL to down-regulate M2 genes was affected in siRNA- E transfected Mϕs. The reduction in M2 genes after BAFF treat- ment was significantly less for all five M2 genes in TACI ablated cellscomparedwithcellstransfectedwithcontrolsiRNA(Fig. 5B). Suppression of TACI expression also led to a significant decrease in the down-regulation of Arg1, IL1RN, FIZZ1, and Fig. 6. TACI-mediated ERK1/2 signaling in pMϕs. (A)pMϕs from TACI KO or CCL22 mRNA after APRIL stimulation. Reduction in ym1 γ expression was also affected after APRIL stimulation of siRNA- WT mice were stimulated with APRIL, LPS, and IFN- for 0, 3, and 24 h. Total and phosphorylated ERK1/2 molecules were detected by Western blot transfected cells, but this decrease did not reach statistical analysis. One of three experiments with similar results is shown. (B) The ratio significance. Collectively, siRNA experiments indicate that TACI of the band density of phosphorylated ERK1/2 molecules to total ERK1/2 was deficiency does not spontaneously result in higher expression calculated for each condition. Mean fold induction in band density relative of M2 markers in Mϕs. Instead, both the TACI KO and to 0 h ± SD from three experiments is shown. **P < 0.01 for statistical dif- the siRNA inhibition experiments support a role for TACI ferences between WT and TACI KO cells. ##P < 0.01 and ###P < 0.001 for in ligand-mediated modulation of genes associated with Mϕs statistical differences between media and stimulated cells. (C–E)WTpMϕs phenotype. were stimulated with APRIL in the presence or absence of U0126 inhibitor (20 nM) for 24 h. (C) Total and phosphorylated ERK1/2 molecules were Next, we assessed the biological significance of BAFF- and ϕ detected in Western blot analysis. One of two experiments with similar re- APRIL-induced changes in M phenotype by monitoring Leish- sults is shown. (D) CD80 expression was determined by flow cytometry. A mania survival in ligand-stimulated TACI KO and WT cells. representative experiment out of three repeats with similar results is shown. Parasite count 6 h after the incubation of MϕswithL. major The frequencies of CD80+ cells are shown in each histogram. Solid black line promastigotes confirmed that cells from both the mouse strains indicates CD80 Ab staining, and filled histogram shows isotype controls Ab contained comparable numbers of parasites before stimulation staining. (E) Culture supernatant IL-6 levels were measured by ELISA. Mean with BAFF and APRIL (SI Appendix, Fig. S19A). Stimulation of concentrations ± SD from three experiments are shown. Data from three ± < Leishmania-infected Mϕs with BAFF or APRIL resulted in a experiments SD were plotted. ***P 0.001 for statistical differences be- tween U0126 treated and untreated cells. ###P < 0.001 for statistical differ- concentration-dependent decrease in the percentage of infected ences in expression between media and APRIL-stimulated cells. cells and parasites per cell in WT Mϕs (Fig. 5C and SI Appendix, Fig. S19B). Supporting a role for TACI in ligand-mediated parasite killing, parasite burden in TACI-deficient Mϕs was not Discussion affected by APRIL or BAFF stimulation. Collectively, BAFF TACI is known to be crucial in the development and mainte- and APRIL stimulation experiments provide a mechanistic insight nance of plasma cells specific for TI-2 antigens by providing a ϕ into the M phenotypic changes and leishmaniacidal activity second signal besides the B-cell receptor engagement (3, 6, 31, mediated by TACI. 32). Here, we identified a novel and fundamental role for TACI in influencing the Mϕ phenotype. First, Mϕs manifest pheno- ϕ ERK1/2-MAPK Pathway Is Involved in TACI-Mediated Signaling in M s. typic and functional characteristics of the M2 phenotype in the Last, we investigated whether ligand stimulation is associated absence of TACI. Second, TACI deficiency renders otherwise ϕ with a TACI-mediated signal transduction in M s. We focused resistant C57BL/6 mice susceptible to Leishmania infection, and on the extracellular signal-regulated kinases (ERK1/2)-mitogen adoptive transfer of WT Mϕs into TACI KO mice is sufficient to activated protein kinases (MAPK) pathway because this pathway increase its resistance to Leishmania infection. Third, highlighting ϕ – is shown to be involved in M s polarization (27 29), and TACI is the role of TACI in influencing pMϕ polarization, only TACI- known to mediate ERK1/2-MAPK signaling in B cells (30, 31). expressing WT Mϕs respond to BAFF and APRIL stimulation ϕ APRIL stimulation of WT pM s led to a significant increase in by decreasing M2 markers and controlling in vitro Leishmania ERK1/2 phosphorylation by 3 h, which peaked at 24 h (Fig. 6 A infection. and B). As it has been observed in gene expression analysis and Impaired response to TLR7 and TLR9 agonists on B cells Leishmania killing experiments, TACI-deficient pMϕs were not from CVID patients with tnfrsf13b mutations has been previously responsive to APRIL-induced signaling. To determine whether reported (4). Here, we extended these observations by investi- ERK1/2-MAPK signaling is involved in APRIL-induced IL-6 se- gating the in vitro and in vivo activities of a panel of TLR ago- cretion and CD80 expression, we stimulated pMϕs in the presence nists in TACI KO cells, and we conclusively established that of U0126, a selective inhibitor of MEK1/2, which is a signaling TACI-deficient Mϕs respond poorly to TLR agonists. We also molecule just upstream of ERK1/2. Treatment of pMϕs with found that the expression levels of TLRs, CD14, and the adaptor U0126 abolished both constitutive and APRIL-induced phos- molecules MyD88 and TRIF are significantly lower in TACI KO phorylation of ERK1/2 (Fig. 6C). Consequently, U0126 treat- Mϕs than in WT cells. Reduced expression of the TLR receptor ment blocked CD80 expression (Fig. 6D) and significantly reduced complex and the adaptor molecules is likely to cause Mϕ re- APRIL-induced IL-6 secretion (Fig. 6E). Thus, signaling data sponse ablation to TLR agonists because it has been documented and biological activity experiments indicate a functional TACI that the expression levels of TLRs determine the magnitude of molecule in Mϕs that is able to signal despite its intracellular response to TLR agonists (11). Our data complement the previous sequestration. reports demonstrating that TACI interacts with both TLRs (4)

E4100 | www.pnas.org/cgi/doi/10.1073/pnas.1421580112 Allman et al. Downloaded by guest on September 25, 2021 and MyD88 (8) and strengthen the burgeoning concept that TACI pMϕs. The decision to focus on the ERK1/2-MAPK pathway was PNAS PLUS is needed for optimal TLR function. based on the fact that ERK1/2 phosphorylation has been shown to The differences between TACI KO and WT Mϕs are not mediate M1 polarization of Mϕs(27–29), and we determined that confined to their responses to TLRs. The profound reduction of APRIL induces the expression of M1 markers CD80, MMP9, and TLR agonist-induced inflammatory response can be interpreted IL-6 (SI Appendix,Fig.S16A–C). Supporting a role for the ERK1/ as an attenuation of M1 phenotype (33, 34). Indeed, in addition 2-MAPK pathway in transducing signals downstream of TACI, to the dampened response to TLR agonists, significantly higher APRIL is able to induce ERK1/2 phosphorylation in WT Mϕsbut expression of M2 phenotype-associated molecules is detected in not in TACI KO cells. Furthermore, the ERK1/2 inhibitor, U0126, TACI KO cells in comparison with WT cells. In vivo, the diversity significantly reduces IL-6 secretion and totally blocks the expres- and plasticity of Mϕs depend on the type of signals present in sion of M1 markers CD80 and IL-6 in APRIL-stimulated WT their microenvironment (18). Using M1- and M2-polarizing sig- Mϕs. Interestingly, APRIL is able to induce IL-6 from TACI KO nals, we revealed that TACI KO Mϕ polarization to M1 pheno- Mϕs, although its levels remains significantly lower than the levels type is limited, whereas its M2 polarization is enhanced. These of IL-6 secreted from WT cells. The absence of an abolished IL-6 data collectively suggest that, on one hand, Mϕs adapt an secretion, despite a total block in ERK1/2 phosphorylation, to- M2 phenotype when TACI is absent, and on the other hand, gether with the induction of IL-6 in TACI KO Mϕs, points to the M2-skewed TACI KO Mϕs have limited plasticity. involvement of an additional APRIL receptor in the secretion of The default M2 polarization of Mϕs in TACI KO mice suggests IL-6. Nevertheless, activation of ERK1/2-MAPK pathway is likely a role for TACI in mediating signals that favor M1 polarization in a result of either APRIL engaging intracellular TACI or APRIL Mϕs. One possible mechanism by which TACI influences the Mϕ inducing the translocation of TACI on stimulation, as previously phenotype might be through the interaction between TACI ex- suggested by He and colleagues (8). pressed on B cells and the membrane exposed BAFF and APRIL Altogether, our ligand stimulation experiments indicate that on Mϕs. This B-cell TACI-induced and membrane BAFF-and BAFF and APRIL promote an M1 phenotype somewhat dif- APRIL-mediated “reverse signaling” in Mϕs was initially pro- ferently from the phenotype induced by the classical M1 stimu- posed based on assays using soluble TACI-fc molecules in which lus, IFN-γ and LPS. A common feature between the classical M1 membrane BAFF- and APRIL-expressing cells were activated by signal IFN-γ and LPS and BAFF and APRIL is their suppressive TACI-fc (35–37). However, subsequent studies have shown that effect on the expression of M2-markers. However, these two the Fc-receptor engagement by TACI-fc and BAFF-R-fc mole- signals differ in the induction of M1 markers. Unlike the classical cules, but not membrane BAFF or APRIL, is responsible for Mϕ M1 signal, BAFF and APRIL induce only a limited number of activation (38). Additional evidence refuting the hypothesis of M1 markers. Nevertheless, elevated BAFF and APRIL levels reverse signaling induced by B-cell TACI molecules comes from may be contributing to the inflammatory responses by Mϕs (40). experiments using B cell-deficient mice. Because B cells are the Supporting this hypothesis, Sutherland and colleagues found that primary source of membrane TACI, Mϕs from B-cell KO mice BAFF transgenic mice manifest exacerbated inflammatory disease are expected to adopt the M2 phenotype because they would not and diminished allergic airway reaction (41). Likewise, APRIL be exposed to reverse signaling. However, Andreu and colleagues deficiency results in enhanced Th2 response and increased lung demonstrated that B cell-deficient mouse Mϕs are polarized to- inflammation in an asthma model (42). The fact that pediatric ward an M1 phenotype (39). Our experiments favor a direct sig- CVID patients with tnfrsf13b mutations are prone to asthma naling of Mϕ-TACI, induced by the soluble forms of ligands, both development may be another indication of a requirement for BAFF and APRIL, as a mechanism responsible for the phenotypic functional TACI to maintain the M1 phenotype (43). changes inflicted in Mϕs. First, we showed that, similar to the M1 Macrophages are important in controlling intracellular in- polarizing signal IFN-γ and LPS, BAFF and APRIL stimulation of fections and they respond to microbial assault by expressing in- TACI-bearing WT Mϕs induces a profound reduction in the flammatory molecules (M1 markers). Conversely, M2 Mϕs are expression of M2 markers, whereas TACI-deficient Mϕs are not impaired in killing of intracellular pathogens (18). In fact, path- responsive. Second, by silencing of the TACI molecule in WT ogens have evolved to induce the Mϕ M2 polarization to prevent Mϕs with siRNA, we reproduced the phenotypic changes ob- their elimination by the host (44). We were able to reiterate the served in ligand-stimulated TACI KO cells. The siRNA experi- functional properties of the M2 phenotype in Leishmania-infected ments also explicate the ligand dependency of the phenotypic TACI KO Mϕs. Both TACI-deficient Mϕs and TACI KO mice changes observed in TACI KO cells, conceivably because WT are impaired in controlling Leishmania infection. Similar to find- Mϕs do not spontaneously default to an M2 phenotype on TACI ings from in vitro-infected Mϕs, immunohistochemical staining of down-regulation. Hence, constant in vivo encounters with BAFF TACI KO mouse ear lesions shows limited NO activity, and Mϕs and APRIL are likely to be responsible for the suppression of isolated from infected TACI KO ears express significantly more M2 markers in WT mice, whereas TACI deficiency would pre- Arg1, IL1RN, and Ym1 mRNA than WT samples. Interestingly, INFLAMMATION

vent the BAFF- or APRIL-mediated suppressive signals, leading unlike the susceptible BALB/c mouse (45), more severe mani- IMMUNOLOGY AND to elevated M2 markers in TACI KO mice. Finally, a third piece festation of infection is not accompanied by an increase in Th2 of evidence for the involvement of TACI in BAFF- and APRIL- response in TACI KO mice. Instead, TACI KO mice seem im- mediated Mϕ activation emerges from the in vitro Leishmania paired in mounting the protective Th1 response, as evidenced by killing experiments. These experiments revealed that both BAFF reduced expansion of IFN-γ+CD4+ cells in the skin and draining and APRIL are able to diminish Leishmania growth in WT Mϕs, LNs throughout the course of the infection. This diminished Th1 as does the M1 polarizing signal IFN-γ and LPS. By contrast and response is likely to be responsible for the persistence of parasites underscoring the role of TACI in mediating BAFF- and APRIL- in the skin and draining LNs until the 12th week of infection. induced signals, intracellular parasite load remains unchanged in Ablated Th1 response is not a result of an inherent defect in TACI BAFF- or APRIL-stimulated TACI KO Mϕs. KO CD4+ cells because TACI KO T cells respond to Th1 po- The Mϕ polarization experiments suggested that BAFF and larizing stimuli as efficiently as WT T cells do. Although the APRIL activity is mediated through TACI despite its intracell- presence of M2 Mϕs may have led to the ablated Th1 response in ular localization. Using human monocytes and DCs, Chang and TACI KO mice, a multitude of in vivo compensatory changes colleagues have shown that BAFF induces the secretion of pro- resulting in a response to TACI deficiency may have also con- inflammatory cytokines through intracellular TACI (9, 10). We tributed in preventing the expansion of Th1 cells. Our results point provided evidence that TACI-mediated events also involve signal to M2-skewed Mϕs responsible for the susceptibility of TACI KO transduction as ERK1/2 is phosphorylated in APRIL-stimulated mice to Leishmania, as the transfer of TACI+ WT MϕsintoTACI

Allman et al. PNAS | Published online July 13, 2015 | E4101 Downloaded by guest on September 25, 2021 KO mice not only alleviates skin pathology but also results in Materials and Methods reduced parasite burden in Leishmania-challenged TACI KO Ethics Statement. Mice were maintained and bred at the US Food and Drug mice. Conversely, injection of TACI KO Mϕs exacerbates disease Administration (FDA) Animal Research Center. Experiments were carried out in the recipient TACI KO mice. Thus, both phenotypes of Mϕs following the Guide for the Care and Use of Laboratory Animals of the are sufficient to alter the infection severity in TACI KO mice. National Institutes of Health (52). Experimental protocol (2002-31) was ap- Other groups have shown that blocking M2 polarization affords proved by the Animal Care and Use Committee of the Center for Biologics resistance in the susceptible BALB/c mouse (46, 47). To our Evaluation and Research, US FDA. knowledge, our results provide the first direct evidence for a role for M2 Mϕs in the conversion of the resistant C57BL/6 strain into Mice. Adult 6- to 8-wk-old C57BL/6 mice were purchased from The Jackson a susceptible phenotype. The deficiency of IFN-γ or iNOS in Laboratory. TACI KO mice on a C57BL/6 background were described pre- C57BL/6 mouse results in a more severe manifestation of leish- viously (3). maniasis, most probably because an elevated Th2 response is ac- companied by a total deficiency of Th1 response both in the IFN-γ In Vivo L. major Infection. Experiments were carried out using different lines KO mice and the iNOS KO mice (48). The kinetics and severity of of L. major: L. major Friedlin strain FV1 (MHOM/IL/80/FN) and a stable Leishmania infection in TACI KO mice mimic those observed in transfected line of L. major FV1 promastigotes expressing a red fluorescent TLR4- and TLR9-deficient C57BL/6 mice infected with Leish- protein (RFP-Leishmania) (23). Parasites were grown at 26 °C in medium 199 mania, both exhibiting diminished dermal lesions at 8–10 wk after supplemented with 20% heat-inactivated FCS (Gemini Bio-Products). In- – fective stage, metacyclic promastigotes of L. major were isolated from sta- an exacerbated manifestation of the disease at 4 6wkafter – challenge (49, 50). The absence of a compensatory Th2 response, tionary cultures (4 5 d old) by negative selection using peanut agglutinin along with a low but detectable Th1 response, appears to be re- (PNA; Vector Laboratories). For flow cytometric studies of dermal cells, mice were infected with the specified number of metacyclic promastigotes in the sponsible for the manifestation of a milder form of infection in ear dermis by i.d. injection in a volume of 10 μL. In parallel, control mice TACI KO mouse compared with IFN-γ KO or iNOS KO mice. received i.d. injection of DMEM in a volume of 10 μL. In conclusion, data presented in this study highlight an un- Ear tissues were prepared as previously described (23). Briefly, the two recognized yet central function for TACI in modulating the Mϕ sheets of infected ear dermis were separated and incubated in DMEM phenotype. We propose that BAFF and APRIL control parasitic ϕ containing 0.2 mg/mL Liberase CI purified enzyme blend (Roche Diagnostics), infections by influencing the M phenotype during the early and incubated for 1 h and 30 min at 37 °C. Digested tissue was cut into small stages of microbial assault as part of the innate immune re- pieces and processed in a tissue homogenizer (Medimachine; Becton Dickenson). sponse. The absence of a robust Th1 response in Leishmania- Tissue homogenates were washed with fresh DMEM containing 100 U/mL infected TACI KO mice is likely a result of the ablated M1 re- penicillin and 100 μg/mL streptavidin and filtered through a 70-mm cell sponse. Of interest, a similar innate-like role for BAFF and strainer (Falcon Products). Cells were then labeled, and infected dermal Mϕs APRIL has been proposed in the rapid secretion of low affinity (RFP-Leishmania+CD11b+CD11c−Ly6G−) were isolated using a FACSARIA III IgM and IgG3 Abs, where TACI engagement provides a second cell sorter (BD Biosciences). Infected dermal Mϕs were processed for gene signal to B1 and Marginal Zone B cells specific for T cell- expression analysis using qPCR as described in the previous section. In other independent antigens, such as bacterial polysaccharides (26). The experiments, cells were labeled to detect donor Mϕs (CD45.2+CD11b+ discovery of a role for TACI in influencing the Mϕ phenotype MHCII+CD11c−Ly6G−CD45.1−) using a BD LSRFORTESSA (BD Biosciences). can have implications for other conditions where TACI expres- Additional materials and methods are provided in the SI Appendix, SI sion or function is impaired. Two such instances are the newborn Materials and Methods. period (5) and CVID patients (4, 51). Characterization of the Mϕ phenotype and function in these two populations will help ACKNOWLEDGMENTS. We thank Dr. Alain Debrabant (Center for Biologics unveil whether their Mϕs also default to an M2 phenotype and, if Evaluation and Research/US Food and Drug Administration) for providing ϕ L. major RFP parasites. This project is supported by intramural funds from so, whether the phenotype of their M is responsible for con- the US Food and Drug Administration. W.R.A., A.S.C., K.U., and M.Y. were sup- ditions such as impaired responses to vaccines and increased ported by postdoctoral fellowships from the Oak Ridge Institute for Science susceptibility to pathogens. and Education (Oak Ridge, TN).

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