Interaction of Antigen 1 and CD40 Ligand Leads to IL-12 Production and Resistance in CD40-Deficient Mice Infected with major This information is current as of September 30, 2021. Ifeoma Okwor, Ping Jia and Jude E. Uzonna J Immunol 2015; 195:3218-3226; Prepublished online 24 August 2015; doi: 10.4049/jimmunol.1500922 http://www.jimmunol.org/content/195/7/3218 Downloaded from

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Material 2.DCSupplemental http://www.jimmunol.org/ References This article cites 46 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/195/7/3218.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Interaction of Macrophage Antigen 1 and CD40 Ligand Leads to IL-12 Production and Resistance in CD40-Deficient Mice Infected with Leishmania major

Ifeoma Okwor,* Ping Jia,† and Jude E. Uzonna*,†

Although some studies indicate that the interaction of CD40 and CD40L is critical for IL-12 production and resistance to cutaneous , others suggest that this pathway may be dispensable. In this article, we compared the outcome of Leishmania major in both CD40- and CD40L-deficient mice after treatment with rIL-12. We show that although CD40 and CD40L knockout (KO) mice are highly susceptible to L. major, treatment with rIL-12 during the first 2 wk of infection causes resolution of cutaneous lesions and control of parasite replication. Interestingly, although treated CD40 KO mice remained healed, developed long-term immunity, and were resistant to secondary L. major challenge, treated CD40L KO reactivated their lesion after Downloaded from cessation of rIL-12 treatment. Disease reactivation in CD40L KO mice was associated with impaired IL-12 and IFN-g production and a concomitant increase in IL-4 production by cells from lymph nodes draining the infection site. We show that IL-12 production by dendritic cells and via CD40L–macrophage Ag 1 (Mac-1) interaction is responsible for the sustained resistance in CD40 KO mice after cessation of rIL-12 treatment. Blockade of CD40L–Mac-1 interaction with anti–Mac-1 mAb led to spontaneous disease reactivation in healed CD40 KO mice, which was associated with impaired IFN-g response and loss of infection-induced immunity after secondary L. major challenge. Collectively, our data reveal a novel role of CD40L–Mac-1 http://www.jimmunol.org/ interaction in IL-12 production, development, and maintenance of optimal Th1 immunity in mice infected with L. major. The Journal of Immunology, 2015, 195: 3218–3226.

umerous studies have shown that CD40–CD40L inter- . Interestingly, some studies suggest that CD40–CD40L action is essential for induction of effective cell-mediated interaction is not required for protection against L. major (8, 9). In N immunity to . CD40 is constitutively expressed addition, blockade of CD40–CD40L interaction in patients with on basophils, dendritic cells (DCs), B cells, and epithelial cells but due to did not can be induced on macrophages, endothelial cells, smooth muscle affect IFN-g production by T cells or progression of cutaneous + cells, and fibroblasts. CD40L, in contrast, is inducible on CD4 and lesion. Thus, the role of CD40–CD40L interaction in leishmaniasis by guest on September 30, 2021 CD8+ T cells, B cells, epithelial cells, eosinophils, monocytes, remains controversial and needs to be further delineated. macrophages, NK cells, and mast cells (1). Previous reports show Macrophage Ag 1 (Mac-1) is a b2 integrin that is present on that CD40–CD40L interaction is critical for the production of monocytes, neutrophils, and macrophages (10), and it plays an IL-12 and induction of optimal Th1 response and immunity to essential role in immunity by influencing adhesion and migration cutaneous leishmaniasis (2). Treatment of the susceptible BALB/c of phagocytic cells (11). It is composed of two chains, CD18 and mice with CD40 agonistic Ab led to healing postinfection with CD11b, a type 1 transmembrane receptor composed of extracel- Leishmania major (3). In contrast, injection of anti-CD40L antago- lular, transmembrane, and cytoplasmic domains (12). The cyto- nists led to susceptibility in the resistant C57BL/6, which was plasmic domain of CD11b is believed to be important for re- associated with reduced IL-12 production (4). Furthermore, defi- cognition of -associated molecular patterns such as ciency of CD40L or CD40 in the usually resistant C57BL/6 leads Mycobacterium tuberculosis oligosaccharides and Leishmania to susceptibility to L. major (5, 6) and Leishmania amazonensis (7) lipophosphoglycan (13). In contrast, the I domain near the N- terminal region is responsible for binding C3bi, fibrinogen, and other bacterial Ag (14). The binding ability of the C and I domains *Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba could explain the wide range of ligands bound by Mac-1 (14, 15). R3E 0J9, Canada; and †Department of Immunology, University of Manitoba, Winni- peg, Manitoba R3E 0T5, Canada Recent reports show that, in the absence of CD40, Mac-1 can bind Received for publication April 20, 2015. Accepted for publication July 28, 2015. to CD40L and mediate the production of proinflammatory cyto- kine leading to inflammation (10). However, whether Mac-1 can This work was supported by the Canadian Institutes for Health Research (CIHR; to J.E.U.), Research Manitoba (to J.E.U.), a CIHR-Frederic Banting and Charles Best bind to CD40L and regulate the outcome of immunity to L. major Doctoral Award (to I.O.), and the CIHR-International Infectious Disease and Global has not yet been determined. Health Training Program (I.O.). In this article, we compared the immune response and outcome Address correspondence and reprint requests to Dr. Jude E. Uzonna, Department of of L. major infection in CD40 and CD40L knockout (KO) mice Immunology, College of Medicine, University of Manitoba, 750 McDermot Avenue, Winnipeg, MB R3E 0T5, Canada. E-mail address: [email protected] and investigated the role of Mac-1–CD40L interaction in protec- The online version of this article contains supplemental material. tive immune response to L. major. Our data reveal striking dif- Abbreviations used in this article: BMDC, bone marrow–derived DC; BMDM, bone ferences in disease progression and immune response in IL- marrow–derived macrophage; DC, ; DTH, delayed-type hypersensitiv- 12–treated CD40 and CD40L KO mice infected with L. major. ity; KO, knockout; Mac-1, macrophage Ag 1; SLA, soluble Leishmania Ag; WT, Whereas rIL-12–treated CD40 KO mice remained healed, devel- wild type. oped long-term immunity, and were resistant to secondary L. major Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 challenge, rIL-12–treated CD40L KO reactivated their lesion after www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500922 The Journal of Immunology 3219 cessation of treatment. We show that these differences in disease Cytokine ELISAs pathogenesis were related, in part, to alternative utilization of IL-12p40, IFN-g, and IL-4 concentrations in cell culture supernatant fluids CD40L–Mac-1 pathway for continuous and sustained IL-12 were measured by sandwich ELISA using Ab pairs from BD Pharmingen production in CD40 KO mice. Thus, our studies reveal a criti- (San Jose, CA) according to manufacturer’s suggested protocols. The cal but redundant role of Mac-1–CD40L interaction in IL-12 hybridoma clones for the Ab pairs are: IL-12p40, C15.6 and C17.8; IFN-g, production and maintenance of optimal immunity to L. major R4-6A2 and XMG1.2; and IL-4, 11B11 and BVD6-24G2. infection. Generation of bone marrow–derived DCs and bone marrow– derived macrophages Materials and Methods Bone marrow–derived DCs (BMDCs) and bone marrow–derived macro- Mice phages (BMDMs) were generated from naive WT, CD40, and CD40L KO mice as described previously (17). In brief, bone marrow cells were iso- Six- to eight-week-old female wild type (WT) C57BL/6 mice were purchased from Charles River Laboratories (St-Constante, QC, Canada). CD40- and lated from the femur and tibia of mice and differentiated into macrophages CD40L-deficient mice on C57BL/6 background were purchased from The using complete medium supplemented with 30% L929 cell culture su- Jackson Laboratory (Sacramento, CA). Mice were housed in specific pernatant. BMDCs were differentiated in petri dishes in the presence of pathogen-free units at the Central Animal Care Services, University of 20 ng/ml rGM-CSF (Peprotech, Rocky Hill, NJ). Immature DCs were Manitoba. All mouse experiments were approved by the University of harvested on day 7 and assessed for the expression of CD11c, CD40, CD80, CD86, and MHC class II by flow cytometry. Manitoba Animal Care Committee in accordance with the regulation of the Canadian Council on Animal Care. Purification of splenic CD90.2+, CD11b+ (macrophages), and CD11c+ (dendritic) cells

Parasites and infection Downloaded from + + + L. major parasites (MHOM/IL/80/Friedlin) were grown in Grace’s insect Splenic CD90.2 , CD11b , and CD11c cells were isolated by positive + + + medium (Life Technologies) supplemented with 20% heat-inactivated selection using StemCell CD90.2 , CD11b , and CD11c cells EasySep FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomy- isolation kits, respectively, according to the manufacturer’s suggested pro- + cin. For infection, 7-d stationary-phase promastigotes were washed three tocols. The purities of the different cell populations were CD90.2 (98%), + + times in PBS and counted. Mice were infected by injecting 106 parasites CD11b (94%), and CD11c (87–93%). suspended in 50 ml PBS into the right hind footpad. Lesion development Coculture of T cells with splenic DCs and progression were monitored weekly by measuring the diameter of the http://www.jimmunol.org/ infected and uninfected footpads with Vernier calipers. Splenic CD90.2+ T cells were activated in vitro by stimulating with soluble ε Generation of healed WT, CD40L, and CD40 KO mice anti-CD3 and anti-CD28 (2 mg/ml) mAbs overnight. The next morning, the cells were washed and then cocultured with splenic CD11c+ or CD11b+ cells To obtain healed WT, we monitored infected mice (as described earlier) at 100 T cells to 1 CD11c+ or CD11b+ cells (100:1) for 48 h. until their cutaneous lesions were completely resolved (.12 wk). To generate healed CD40 and CD40L KO mice, we infected CD40 and In vitro stimulation of BMDCs and BMDMs and splenic CD40L KO mice as described earlier and treated with rIL-12 (0.3 mg) CD11c+ and CD11b+ cells with soluble CD40L, anti–Mac-1 intralesionally three times per week for the first 2 wk of infection. This mAb, anti-CD40 mAb, and LPS treatment leads initially to complete resolution of lesions in both KO mice, which is sustained only in CD40 KO mice, whereas disease recrudescence BMDMs, BMDCs, and splenic CD11b+ and CD11c+ cells (2 3 106 cells/ by guest on September 30, 2021 occurs in CD40L KO mice from 7 wk postinfection (see Results). In some ml were cultured in 96-well flat-bottom tissue culture plates (100 ml/well; experiments, rIL-12 treatment was continued until week 7 or restarted at Falcon) in the presence or absence of anti–Mac-1 Ab (5 mg/ml). In some weeks 5–7 postinfection (after the first 0–2 wk treatment) in the CD40L experiments, some wells were stimulated with sCD40L (2 mg/ml), anti- KO mice. CD40 mAb (5 mg/ml), or LPS (1 mg/ml). After 48 h, the culture super- natant fluids were collected and assayed for IL-12 by ELISA. Secondary L. major challenge and delayed-type hypersensitivity response Isolation of mononuclear cells from the footpad Healed WT and CD40 KO mice were challenged with 5 million L. major in Healed WT and CD40 KO mice were injected with anti–Mac-1 mAb or the contralateral footpad (left foot). Challenged mice were assessed for control Ig (100 mg/mouse) and then challenged with 5 million L. major in delayed-type hypersensitivity (DTH) response at 72 h postchallenge by the contralateral footpad the next day. After 7 d, mice were sacrificed and measuring the thickness of the challenged footpads with Vernier calipers. mononuclear cells were isolated from the footpads as previously described Unchallenged footpads were used as controls. Challenged mice were (18). The cells were stained directly ex vivo with fluorochrome-conjugated sacrificed at 3 wk postchallenge, and parasite burden was determined by Abs against CD3, CD4, CD8, and CD11b, and the expression of these limiting dilution analysis as previously described (16). markers was assessed by flow cytometry. In vivo blockade of Mac-1 signaling Statistical analysis To determine whether Mac-1 signaling is important for the maintenance of Student t test was used to compare the mean and SEM between two immunity in healed WT or CD40 KO mice, we injected anti–Mac-1 (clone groups. In some experiments, nonparametric one-way ANOVAwas used to M1/70) Ab or control Ig (human IgG1 100 mg/mouse) i.p. at 8 wk post- compare mean and SD of more than two groups. Tukey’s test was used infection, and lesion recrudescence was monitored weekly. To investigate where there was a significant difference in ANOVA. Differences were the role of Mac-1 signaling in secondary (memory) anti-Leishmania im- considered significant when p # 0.05. munity, we injected healed (.12 postinfection) WT and CD40 KO mice with anti–Mac-1 mAb (100 mg/mouse) and then challenged them with L. major in the contralateral footpad the next day. Challenged mice were Results sacrificed after 3 wk to determine parasite burden and immune response. Treatment with rIL-12 leads to healing in CD40 KO mice, but not CD40L KO mice In vitro recall response and intracellular cytokine staining IL-12 produced by DCs is important for the development of naive At various times postinfection, infected mice were sacrificed and the + draining popliteal lymph nodes were harvested and made into single-cell CD4 T cells into Th1 cells that mediate and maintain immunity suspensions. Cells were washed, suspended at 4 million/ml in complete in cutaneous leishmaniasis (19–21). Although some studies show medium (DMEM supplemented with 10% heat-inactivated FBS, 2 mM that CD40–CD40L interaction is critical for IL-12 and the de- glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin), and plated velopment of protective anti-Leishmania immunity (7), others at 1 ml/well in 24-well tissue culture plat (Falcon, VWR Edmonton, AB, Canada). Cells were stimulated with soluble Leishmania Ag (SLA; show that CD40L-deficient mice are capable of producing IL-12 50 mg/ml) for 72 h, and the supernatant fluids were collected and stored at and mounting a protective Th1 response against L. major infection 220˚C until assayed for cytokines by ELISA. (8, 9). To fully determine the role of CD40 and CD40L in IL-12 3220 Mac-1 REGULATES IMMUNITY TO CUTANEOUS LEISHMANIASIS production, and thus resistance to experimental L. major infection, to infection as evidenced by their inability to control lesion we compared disease progression and immune response in CD40 development (Fig. 1A, 1B) and parasite replication (Fig. 1C). and CD40L KO infected with L. major. Consistent with previous Treatment of infected CD40 and CD40L KO mice with rIL-12 reports, both CD40 and CD40L KO mice were highly susceptible during the first 2 wk of infection led to healing (lesion resolu- Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 1. Treatment with rIL-12 leads to healing in CD40 KO but not CD40L KO mice infected with L. major. WT, CD40 KO, and CD40L KO mice were infected with 1 3 106 stationary-phase L. major promastigotes in the right hind footpad. The CD40 and CD40L KO mice were treated intralesionally with rIL-12 (0.3 mg/mouse) three times per week for the first 2 wk, and the development and progression of cutaneous lesions (A and B) were monitored weekly by measuring the infected feet with Vernier calipers. At the indicated times, infected mice were sacrificed and parasite burden in the infected footpads was determined by limiting dilution (C). The draining lymph node cells were stimulated in vitro with SLA (50 mg/ml) for 3 d, and the levels of IL- 12p40 (D), IFN-g (E), and IL-4 (F) were determined by ELISA. In some experiments, CD40L KO mice were treated with rIL-12 (0.3 mg/mouse) three times per week continuously for 7 wk or for the first 2 wk and then restarted at 5–7 wk, and lesion progression was monitored (G). Results are representative of three (A–F) or two (G) independent experiments with similar results (n = 4–5 mice/group per experiment). *p , 0.05, **p , 0.01, ***p , 0.001. ND, not detectable; ns, not significant. The Journal of Immunology 3221 tion and parasite control) in both CD40 and CD40L KO mice CD40L KO mice (which express intact CD40 molecules) led to (Fig. 1A, 1B). However, whereas treated CD40 KO mice remained IL-12 production (Fig. 2A, 2C, 2D, 2F). Interestingly, sCD40L resistant for .14 wk, treated CD40L KO mice reactivated disease stimulation also led to IL-12 production (Fig. 2B, 2E) in CD40 starting around week 5–7 postinfection (Fig. 1A, 1B). The im- KO cells, suggesting that sCD40L must be binding to another paired parasite control and lesion recrudescence in rIL-12–treated molecule that is distinct from CD40. The ability of sCD40L to CD40L KO mice corresponded with significantly (p , 0.01– induce IL-12 production in cells from CD40 KO mice was sig- 0.001) lower IL-12 (Fig. 1D) and IFN-g (Fig. 1E) and significantly nificantly (p , 0.05–0.001) blocked by the addition of anti– (p , 0.001) higher IL-4 (Fig. 1F) production by their draining Mac-1 blocking Ab (Fig. 2B, 2E). Similar results were also ob- lymph node cells compared with the CD40 KO or WT mice. In- tained with BMDMs and BMDCs (Supplemental Fig. 1). Inter- terestingly, although continuous treatment of infected CD40L KO estingly, anti–Mac-1 mAb has no effect on LPS-induced IL-12 mice with rIL-12 resulted in lesion control and prevented disease production by CD11c+ and CD11b+ cells from CD40 KO mice reactivation, restarting rIL-12 treatment at weeks 5–7 after 3–4 wk (Fig. 2B, 2E), suggesting that this effect is specific to sCD40L cessation failed to prevent disease reactivation (Fig. 1G). Because stimulation. Taken together, these findings suggest that, in the continuous IL-12 is required for maintenance of anti-Leishmania absence of CD40, Mac-1 can interact with CD40L leading to IL- immunity in healed mice (19–21), these observations suggest the 12 production, and this may account for the difference in the existence of an alternative pathway for IL-12 production in CD40 outcome of infection after rIL-12 treatment in the CD40 KO and KO mice that is nonfunctional or absent in CD40L KO mice. They CD40L KO mice infected with L. major. further suggest that in the infected CD40L KO mice, exogenous Anti–Mac-1 blocking Ab inhibits activated T cell–mediated IL-12 treatment is important for initiating a protective immune Downloaded from IL-12 production by DCs response but is unable to prevent disease progression once lesion has developed. The preceding results show that anti–Mac-1 blocking Ab inhibits sCD40L-induced IL-12 production in vitro. Although CD40L is CD40-independent IL-12p40 production by macrophages and mainly expressed by activated CD4+ T cells, other cell types in- DCs is dependent on the presence of Mac-1 cluding eosinophils, basophils, mast cells, DCs, macrophages, and

DCs and macrophages are the major cell types that produce IL-12 NK cells are also known to express CD40L under certain con- http://www.jimmunol.org/ during Leishmania infection (2, 22). Previous reports show that ditions (24). Previous reports show that the production of IL-12 by Mac-1 binds to CD40L and this interaction mediates inflammation DCs during Leishmania infection is dependent on the interaction (10) and leukocyte recruitment to atherogenic sites (23). Because of CD40 molecules (on DCs) with CD40L expressed on activated we observed that early treatment with rIL-12 results in long-term CD4+ T cells (2, 25, 26). Therefore, we assessed whether anti– healing in CD40 KO mice, but not CD40L KO mice, we hy- Mac-1 blocking Ab could inhibit activated CD4+ T cell–induced pothesized that the sustained resistance observed in CD40 KO IL-12 production by DCs in vitro. As for sCD40L, activated CD4+ mice is due to IL-12 production via the interaction of Mac-1 with T cells induced robust IL-12p40 production by BMDCs from WT, CD40L. Therefore, we purified CD11b+ and CD11c+ cells from CD40, or CD40L KO mice in vitro (Fig. 3A–C). Interestingly, + WT, CD40, and CD40L KO mice and stimulated them with sol- although anti–Mac-1 mAb inhibited CD4 T cell–induced IL- by guest on September 30, 2021 uble CD40L. As expected, stimulation of cells from WT and 12p40 production by DCs from CD40 KO mice (Fig. 3B), it has

FIGURE 2. Anti–Mac-1 blocking Ab blocks sCD40L-induced IL-12 production by splenic APCs from CD40L KO mice in vitro. CD11b+ (A–C) and CD11c+ (D–F) cells were isolated from spleens of WT, CD40 KO, and CD40L KO mice and stimulated with or without sCD40L (2 mg/ml), anti-CD40 mAb (5 mg/ml), or LPS (1 mg/ml) in vitro in the presence or absence of anti–Mac-1 blocking Ab (5 mg/ml). After 48 h, the culture supernatant fluids were collected and the levels of IL-12p40 were determined by ELISA. Results are representative of three independent experiments with similar results. **p , 0.01, ***p , 0.001. ND, not detectable; ns, not significant. 3222 Mac-1 REGULATES IMMUNITY TO CUTANEOUS LEISHMANIASIS

ing the CD40L–Mac-1 redundant pathway of resistance in L. major–infected CD40 KO mice. Treatment with anti–Mac-1 blocking Ab leads to disease reactivation in healed CD40 KO mice The preceding observations show that IL-12 production in a Mac- 1–dependent manner is responsible for maintaining resistance in CD40 KO mice. We reasoned that if resistance in IL-12–treated CD40 KO mice is dependent on IL-12 production through Mac-1 pathway, blockade of Mac-1 in these mice should lead to impaired IL-12 production and disease reactivation. Therefore, we injected anti–Mac-1 blocking mAb or control Ig into healed CD40 KO and WT mice and monitored the treated mice over time for disease reactivation. As shown in Fig. 4A, CD40 KO treated with anti– Mac-1 mAb spontaneously reactivated their cutaneous lesion within 1 wk posttreatment. This was associated with significantly (p , 0.01) higher parasite burden compared with those that re- ceived control Ig (Fig. 4B). Disease reactivation in anti–Mac-1–

treated healed CD40 KO was also associated with significantly Downloaded from (p , 0.01 and p , 0.001) lower IL-12 (Fig. 4C) and IFN-g (Fig. 4D) production by cells from the draining lymph nodes compared with those that received control Ig. In contrast, cells from the draining lymph nodes of anti–Mac-1–treated CD40 KO mice produced significantly higher levels of IL-4 than those given

control Ig (Fig. 4E). In contrast, anti–Mac-1 mAb treatment did http://www.jimmunol.org/ not result in disease reactivation, increased parasite burden, or altered cytokine production in healed WT mice (Fig. 4). Collec- tively, these results support our conclusion that Mac-1–dependent IL-12 production is responsible for the maintenance of resistance in IL-12–treated CD40 KO mice. Treatment of anti–Mac-1 blocking Ab does not affect cell migration into the infection site

As an integrin, Mac-1 plays an important role in adhesion and by guest on September 30, 2021 FIGURE 3. Anti–Mac-1 blocking Ab inhibits activated T cell–induced migration of cells into infection sites (11). Therefore, we wished IL-12 production by DCs from CD40 KO mice in vitro. BMDCs were generated from WT (A), CD40 KO (B), and CD40L KO (C) mice and to determine whether disease reactivation in healed CD40 KO cocultured with anti-CD3/anti-CD28 (1 mg/ml) activated CD4+ T cells mice after anti–Mac-1 Ab treatment was due to alteration in cell isolated from spleens of WT mice in the presence or absence of sCD40L composition and/or migration into the infection site in healed (2 mg/ml). In some experiments, BMDCs from WT mice were cocultured CD40 KO mice. Healed WT and CD40 KO mice were injected with activated CD4+ T cells isolated from spleens of WT, CD40 KO, or with Mac-1 blocking Ab, challenged with L. major after 24 h, and CD40L KO mice (D). After 48 h, the culture supernatant fluids were sacrificed 6 d later to assess cellular composition in the footpads. collected and the level of IL-12p40 was determined by ELISA. Results are There was no significant difference in the percentages of CD11b+ A C D representative of three ( – ) and two ( ) independent experiments with (Fig. 5A, 5B) and CD3+CD4+ and CD3+CD8+ T cells (Fig. 5C–E) , , similar results. **p 0.01, ***p 0.001. ns, not significant. in the footpads of both healed CD40 KO and WT mice treated with anti–Mac-1 or control Ig. Taken together, these results sug- no effect on IL-12p40 production by DCs from WT and CD40L gest that disease reactivation in healed CD40 KO after blockade KO (Fig. 3A, 3C), suggesting that binding of CD40L on T cells to of Mac-1 is not related to alteration in cell composition and/or Mac-1 on DCs is responsible for IL-12p40 production in CD40 migration into the infection sites. KO cells. To further confirm that the interaction of T cell–ex- pressed CD40L with Mac-1 on DCs leads to IL-12p40 production, Blockade of Mac-1 signaling leads to loss of established we cocultured DCs from WT mice with activated T cells from infection-induced immunity in CD40 KO mice WT, CD40 KO, and CD40L KO mice and assessed IL-12p40 Next, we investigated the impact of Mac-1 blockade on secondary production by ELISA. Unlike activated T cells from WT and immunity to L. major. Healed (.14 wk postinfection) CD40 KO CD40 KO mice that induced robust IL-12p40 production, CD40L and WT mice were treated with anti–Mac-1 blocking Ab 24 h KO T cells were unable to induce IL-12p40 production in DCs before challenge with virulent L. major. There was no significant in vitro (Fig. 3D), akin to anti–Mac-1 blockade. Collectively, these difference in DTH response and rapid control of parasite in healed results suggest that CD40L–Mac-1–induced IL-12 production is WT mice treated with either anti–Mac-1 mAb or control Ig a redundant pathway that is operational only in the absence of (Fig. 6A). In contrast, although healed CD40 KO mice treated functional CD40 molecule. Thus, in the presence of intact CD40 with control Ig displayed strong DTH response and rapid parasite signaling as in WT mice, CD40L–Mac-1 interaction is dispens- control akin to WT mice, CD40 KO mice treated with anti–Mac-1 able for IL-12p40 production by DCs. Interestingly, the expres- mAb blocking Ab had significantly (p , 0.001) lower DTH re- sion of CD40L on T cells from L. major–infected CD40 KO mice sponse (Fig. 6A), as well as significantly (p , 0.01) higher par- was upregulated after rIL-12 treatment (Supplemental Fig. 2). This asite burden (Fig. 6B). Further, the levels of IL-12 (Fig. 6C) and observation suggests that exogenous IL-12 is required for initiat- IFN-g (Fig. 6D) in the supernatant fluid of cells from CD40 KO The Journal of Immunology 3223 Downloaded from

FIGURE 4. Blockade of Mac-1 leads to spontaneous disease reactivation and impaired Th1 response in healed CD40 KO mice. WT and CD40 KO mice were infected with 1 3 106 L. major in the right hind footpads and allowed to heal by treating CD40 KO mice with rIL-12 during the first 2 wk of infection. Twelve weeks after primary infection when lesion was almost resolved in treated CD40 KO mice, mice were treated i.p. with Mac-1 blocking Ab http://www.jimmunol.org/ (100 mg/mouse) twice a week for 2 wk. Lesion development (A) in the primary infection site was measured weekly with Vernier calipers. Five weeks after the onset of anti–Mac-1 treatment, mice were sacrificed and parasite burden was determined by limiting dilution assay (B). The draining lymph node cells were stimulated with SLA (50 mg/ml) for 72 h, and the levels of IL-12p40 (C), IFN-g (D), and IL-4 (E) in the cell culture supernatant fluids were measured by ELISA. Results are representative of three independent experiments with similar results (n = 3–4 mice/group per experiment). Arrow indicates the onset of anti–Mac-1 treatment. **p , 0.01, ***p , 0.001. ND, not detectable; ns, not significant. treated with anti–Mac-1 blocking Ab were significantly (p , CD40L KO mice. We showed that blockade of Mac-1 signaling 0.01) lower than those from the control Ig–treated group. Taken using blocking anti–Mac-1 mAb in healed CD40 KO led to im- by guest on September 30, 2021 together, these results indicate that Mac-1 plays an important role paired IL-12 production, disease reactivation, and impaired Th1 in secondary immune response to L. major. response, suggesting that signaling via Mac-1 is responsible for IL-12 production and sustained immunity in CD40 KO mice. In- Discussion deed, we showed that anti–Mac-1 mAb blocked sCD40L-induced There have been conflicting reports on the role of CD40–CD40L IL-12 production by macrophages and DCs from CD40 KO, sug- interactions in immunity to experimental cutaneous leishmaniasis. gesting that the interaction of Mac-1 with CD40 is responsible for Even though a study showed that CD40L KO mice heal their low- IL-12 production in CD40 KO mice. dose L. major infection (9), another study showed that these mice IL-12 is a critical cytokine for both the development of primary are highly susceptible to L. amazonensis infection (7). Therefore, immunity and the maintenance of effector Th1 cells that mediate the primary aim of this study was to more precisely determine the secondary resistance to L. major. Although it is widely believed role of CD40 and CD40L interactions in primary and secondary that CD40–CD40L interaction is the primary pathway responsible immunity to L. major. We reasoned that if the interaction between for IL-12 production for optimal anti-Leishmania immunity, we CD40 and CD40L is the sole mechanism responsible for IL-12 and others have identified other alternative pathways such as production in L. major–infected mice, then the outcome of in- TRANCE-RANK (27) and LIGHT-HVEM (28, 29) that contribute fection after rIL-12 treatment should be the same in infected to IL-12 production and development of immunity to L. major. CD40 and CD40L KO mice. Surprisingly, we found that there are This raises the question as to which of the different pathways is differences in disease outcome and immune response between the most critical during primary and secondary/memory phases of CD40 and CD40L KO mice infected with L. major after treatment immunity to L. major. We believe that although all these other with rIL-12. Whereas CD40 KO mice treated with rIL-12 during pathways may be important for optimal immunity to L. major, the the first 2 wk of infection completely heal their footpad lesion current observations support the idea that in the absence of CD40– and are resistant to secondary challenge, CD40L KO mice reac- CD40L interaction, Mac-1–CD40L interaction plays an important tivated disease a few weeks after cessation of rIL-12 treatment. role in this resistance. This is supported by the fact that neither This inability to control infection in CD40L KO mice was asso- blockade of TRANCE–RANK interaction nor inhibition of LIGHT ciated with impaired IL-12 and IFN-g production by cells from signaling leads to spontaneous disease reactivation or loss of im- the spleens and lymph nodes draining the primary infection sites. munity in healed CD40 KO mice (data not shown). In contrast, Because continuous IL-12 production is indispensable for the blockade of Mac-1 led to both spontaneous disease reactivation initiation and maintenance of optimal cell-mediated immunity to (akin to IL-12 KO mice) and loss of infection-induced immunity L. major, the sustained immunity in rIL-12–treated CD40 KO in CD40 KO that healed their primary L. major infection. mice suggests the existence of an alternative pathway for IL-12 Although our in vitro and in vivo studies strongly suggest that production in these mice, which is absent or nonfunctional in Mac-1 interaction with CD40L is responsible for sustained IL-12 3224 Mac-1 REGULATES IMMUNITY TO CUTANEOUS LEISHMANIASIS Downloaded from http://www.jimmunol.org/

FIGURE 5. Blockade of Mac-1 does not affect cell migration into infection site. Healed WT and CD4 KO mice were treated with anti–Mac-1 or control Ig Abs (100 mg/mouse) 24 h before being challenged with virulent L. major in the footpad. All mice received another injection of anti–Mac-1 or control Abs on day 4 postchallenge. At day 7 postchallenge, mice were sacrificed and the percentages of CD32CD11b+ cells (A and B)andCD4+ and CD8+ T cells (gated on CD3+ by guest on September 30, 2021 cells) (C–E) were analyzed by flow cytometry. Results are representative of two independent experiments with similar results (n = 3 mice/group per experiment). production in mice lacking functional CD40, it is plausible that which is a primary receptor for fibronectin, a key molecule that other as yet untested pathways may be involved. For example, it has plays an important role in regulating inflammatory cytokine pro- been shown that sCD40L can bind to another integrin, a5b1 (30), duction by some cells (31, 32). However, we believe that Mac-1–

FIGURE 6. Blockade of Mac-1 abolishes in- fection-induced immunity and resistance to sec- ondary L. major challenge in healed CD40 KO mice. WT and CD40 KO mice were infected with 1 3 106 L. major and allowed to heal by treating CD40 KO mice with rIL-12. Healed mice and uninfected age-matched controls were injected with anti–Mac-1 mAb or control Ig (100 mg/mouse), challenged the next day in the contralateral feet with 5 3 106 L. major, and DTH response was measured after 72 h. (A) The mice further received weekly injections of anti– Mac-1 mAb or control Ig weekly and at 3 wk postchallenge were sacrificed to determine par- asite burden (B). At sacrifice, the draining lymph node cells were stimulated with SLA, and after 3 d, the levels of IL-12p40 (C) and IFN-g (D) in the cell culture supernatant fluids were de- termined by ELISA. Results presented are rep- resentative of two independent experiments with similar results (n = 3–4 mice/group per experi- ment). *p , 0.05, **p , 0.01. The Journal of Immunology 3225

CD40L interaction is responsible for the bulk of IL-12 produced the absence of CD40–CD40L interaction. Thus, during L. major in this system because addition of anti–Mac-1 mAb completely infection, CD40-CD40L signaling is the primary pathway for IL- abolished IL-12 secretion by DCs and macrophages from CD40 12 production. IL-12 production via the Mac-1–CD40L pathway KO mice after stimulation with sCD40L (Fig. 3). In line with this, becomes relevant only in the absence of CD40 molecules as is activated T cells from CD40L KO mice were unable to induce present in CD40 KO mice. Redundancy is an important and IL-12p40 production in DCs from WT mice in vitro. Interest- common occurrence in many biological systems because it pro- ingly, rIL-12 treatment dramatically upregulated the expression vides fail-safe mechanisms and backups that are necessary for of CD40L on CD3+ T cells from L. major–infected CD40 KO maintaining proper functioning of the . The redundancy in mice (Supplemental Fig. 2), suggesting that exogenous IL-12 may IL-12 production pathway may become particularly important in be required for initiating the CD40L–Mac-1 redundant pathway of individuals with genetic mutations in the CD40 gene. These resistance in L. major–infected CD40 KO mice. individuals have been shown to have recessive form of hyper- Our findings that Mac-1–CD40L interaction enhances IL-12p40 IgM syndrome similar to the disease seen in individuals with production is in disagreement with recently published data show- mutations in the CD40L gene (46). The CD40L–Mac-1 pathway ing that the uptake of Leishmania via Mac-1 leads to inhibition could be targeted in these individuals for therapeutic purposes. of IL-12 production in macrophages (33). This difference may In summary, our data show striking differences in the outcome be related, in part, to the experimental models and approach of disease and immune response in L. major–infected CD40 and between these studies. In this study, DCs and macrophages were CD40L KO mice after rIL-12 treatment. These differences were not infected with L. major in vitro. In addition, Ricardo-Carter related, in part, to alternative utilization of CD40L–Mac-1 path- et al. (33) merely assessed the effect of Mac-1– on LPS-induced way for continuous and sustained IL-12 production in infected Downloaded from IL-12 production in L. major–infected WT or Mac-1–deficient CD40 KO mice. Thus, our studies reveal a critical and redundant macrophages and did not assess the impact of Mac-1 deficiency on role of Mac-1 in IL-12 production and resistance to L. major T cell induction of IL-12 production in infected cells. The Mac- infection. 1–dependent inhibition of LPS-induced IL-12p40 production in infected macrophages was mediated by intracellular signals in- Disclosures volving the E26 transformation–specific pathway. It is plausible The authors have no financial conflicts of interest. http://www.jimmunol.org/ that Mac-1–CD40L interaction does not activate the E26 trans- formation–specific pathway, and thus would not inhibit IL-12 production. Although not assessed in this study, this proposal References is supported by the recent report that shows the interaction of 1. Croft, M., W. Duan, H. Choi, S. Y. Eun, S. Madireddi, and A. Mehta. 2012. TNF superfamily in inflammatory disease: translating basic insights. Trends Immunol. CD40L with all its receptors leads to activation of the MAPK 33: 144–152. pathway (34), which is a key intracellular signaling pathway that 2. Marovich, M. A., M. A. McDowell, E. K. Thomas, and T. B. Nutman. 2000. 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