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Promotion of a Functional B Cell Germinal Center Response after Leishmania Species Co-Infection Is Associated with Lesion Resolution

Open AccessPublished:March 19, 2012DOI:https://doi.org/10.1016/j.ajpath.2012.01.012
      Co-infection of C3HeB/FeJ (C3H) mice with both Leishmania major and Leishmania amazonensis leads to a healed footpad lesion, whereas co-infection of C57BL/6 (B6) mice leads to non-healing lesions. This inability to heal corresponds to a deficiency in B cell stimulation of the macrophage-mediated killing of L. amazonensis in vitro and a less robust antibody response. The mechanism that leads to healing of these lesions is not completely known, although our studies implicate the B cell response as having an important effector function in killing L. amazonensis. To understand more completely this disparate clinical outcome to the same infection, we analyzed the draining lymph node germinal center B cell response between co-infected C3H and B6 mice. There were more germinal center B cells, more antibody isotype-switched germinal center B cells, more memory B cells, and more antigen-specific antibody-producing cells in co-infected C3H mice compared to B6 mice as early as 2 weeks postinfection. Interleukin (IL)-21 production and IL-21 receptor expression in both mouse strains, however, were similar at 2 weeks, suggesting that the difference in the anti-Leishmania response in these mouse strains may be due to differences in T follicular cell commitment or intrinsic B cell differences. These data support the idea that functional B cells are important for healing L. amazonensis in this infectious disease model.
      Leishmaniasis is a vector-borne disease caused by an obligate intracellular protozoan parasite of the genus Leishmania. Both Leishmania (L.) major and L. amazonensis cause cutaneous leishmaniasis, characterized by focal to multi-focal cutaneous ulcerations, which can occur after infection of a sand fly bite. Infection of C3HeB/FeJ (C3H) mice with L. major resolves within 8 to 12 weeks and is dependent on development of a polarized CD4+T helper 1 (Th1) immune response, which is critical for activation of infected macrophages to kill internalized parasites.
      • Afonso L.C.
      • Scott P.
      Immune responses associated with susceptibility of C57BL/10 mice to Leishmania amazonensis.
      Infection of the same mouse strain with L. amazonensis leads to large, non-healing lesions, and the immune response is not polarized to either a Th1 or Th2 response.
      • Afonso L.C.
      • Scott P.
      Immune responses associated with susceptibility of C57BL/10 mice to Leishmania amazonensis.
      • Ramer A.E.
      • Vanloubbeeck Y.F.
      • Jones D.E.
      Antigen-responsive CD4+ T cells from C3H mice chronically infected with Leishmania amazonensis are impaired in the transition to an effector phenotype.
      Prior infection of C3H mice with L. major leads to protection against subsequent L. amazonensis infection.
      • Vanloubbeeck Y.
      • Jones D.E.
      Protection of C3HeB/FeJ mice against Leishmania amazonensis challenge after previous Leishmania major infection.
      • Veras P.
      • Brodskyn C.
      • Balestieri F.
      • Freitas L.
      • Ramos A.
      • Queiroz A.
      • Barral A.
      • Beverley S.
      • Barral-Netto M.
      A dhfr-ts- Leishmania major knockout mutant cross-protects against Leishmania amazonensis.
      Using an in vitro model of Leishmania infection developed in our laboratory, we identified that CD4+ T cells and CD19+ B cells from L. major-infected C3H mice were necessary to kill L. amazonensis within infected macrophages.
      • Mukbel R.
      • Petersen C.A.
      • Jones D.E.
      Soluble factors from Leishmania major-specific CD4(+)T cells and B cells limit L. amazonensis amastigote survival within infected macrophages.
      More recently it was described that co-infection with both L. major and L. amazonensis in the same footpad led to significantly higher lesion size and parasite load in co-infected C57BL/6 (B6) mice when compared to C3H mice.
      • Gibson-Corley K.N.
      • Boggiatto P.M.
      • Mukbel R.M.
      • Petersen C.A.
      • Jones D.E.
      A deficiency in the B cell response of C57BL/6 mice correlates with loss of macrophage-mediated killing of Leishmania amazonensis.
      Using an in vitro assay with cell depletion and reconstitution it was determined that B cells from L. major-infected B6 mice fail to activate infected macrophages to kill L. amazonensis.
      • Gibson-Corley K.N.
      • Boggiatto P.M.
      • Mukbel R.M.
      • Petersen C.A.
      • Jones D.E.
      A deficiency in the B cell response of C57BL/6 mice correlates with loss of macrophage-mediated killing of Leishmania amazonensis.
      It was also shown that co-infected B6 mice did not make significant levels of Leishmania-antigen specific antibodies as compared to co-infected C3H mice, suggesting an important primary defect in the B cell response in B6 mice.
      • Gibson-Corley K.N.
      • Boggiatto P.M.
      • Mukbel R.M.
      • Petersen C.A.
      • Jones D.E.
      A deficiency in the B cell response of C57BL/6 mice correlates with loss of macrophage-mediated killing of Leishmania amazonensis.
      In addition, although studies have demonstrated that B cells contribute to the immunopathology of a non-healing response toward Leishmania infection, our findings suggest that there can be an important role for B cells during characteristically effective T-cell-mediated immunity.
      To understand the different biological response to an identical infectious challenge, we now show differences in the B cell germinal center response between C3H and B6 mice co-infected with L. major and L. amazonensis. There were significant differences in C3H versus B6 B cell responses during Leishmania co-infection. Co-infected C3H mice had increased germinal center B cells that were isotype switched, increased memory B cells, and increased antigen-specific antibody-producing cells as early as 2 weeks postinfection as compared to B6 mice. We also demonstrated that IL-21 production, location of IL-21 producing cells in the lymph node, and expression of the IL-21 receptor are similar, indicating that IL-21 expression does not necessarily translate to effective germinal center formation. Our findings establish a fundamental difference in the immune response to Leishmania infection between these two mouse strains and provide additional evidence that the B cell response, in part, determines immune effectiveness toward L. amazonensis infection.

      Materials and Methods

      Mice

      Female C57BL/6 (B6) and female C3HeB/FeJ (C3H) mice (6 to 8 weeks of age) were obtained either from Jackson Laboratories (Bar Harbor, Maine) or from an in-house breeding colony. Mice were maintained in a specific pathogen-free facility. Mice were infected with either 5 × 106 stationary-phase L. major, L. amazonensis or 2.5 × 106 L. major and 2.5 × 106 L. amazonensis promastigotes in 50 μL of PBS in the left hind footpad. All procedures involving animals were approved by the Institutional Animal Care and Use Committee at Iowa State University.

      Parasites and Antigens

      L. amazonensis (MHOM/BR/00/LTB0016) and L. major (MHOM/IL/80/Friedlin) promastigotes were grown in complete Grace's Insect medium (Atlanta Biologicals, Lawrenceville, GA) to stationary phase, harvested, washed in endotoxin-free PBS (Cellgro, Herdon, VA) and prepared to a concentration of 1 × 108 parasites/mL. Freeze-thawed Leishmania antigen was obtained from stationary phase promastigotes as previously described.
      • Jones D.E.
      • Ackermann M.R.
      • Wille U.
      • Hunter C.A.
      • Scott P.
      Early enhanced Th1 response after Leishmania amazonensis infection of C57BL/6 interleukin-10-deficient mice does not lead to resolution of infection.

      Lymph Node Cell Culture and Sorting

      Total lymph node (TLN) cells were obtained from the left popliteal lymph node draining the site of left footpad infection from C3H and B6 mice infected for 2 or 5 weeks with L. major, L. amazonensis, or co-infected with both species. Lymph nodes from each mouse were kept separate and harvested into 2 mL of complete tissue culture medium (RPMI 1640, 2 mmol/L l-glutamine, 100 U penicillin, 100 μg streptomycin/mL, 25 mmol/L HEPES, 0.05 μm 2-mercaptoethanol and 10% fetal bovine serum). A single cell suspension was created using a 2 mL tissue homogenizer. Cells were passed through a 40-μm nylon cell strainer (BD Falcon, Bedford, MA) and washed with 10 mL of complete tissue culture medium at 250 × g, 4°C for 10 minutes. After washing, the cells were resuspended in 0.5 mL complete tissue culture medium and counted.

      Flow Cytometry

      For analysis of surface molecule expression, 0.5 × 106 TLN cells were washed in 2 mL of fluorescence-activated cell sorting buffer (FACS) (0.1% sodium azide and 0.1% bovine serum albumin in phosphate buffer saline). Fcγ receptors were blocked with 10% purified rat anti-mouse CD16/CD32 antibody (BD Pharmingen, San Diego, CA) in 1 mg/mL rat IgG (Sigma, St. Louis, MO) for 20 minutes at 4°C to prevent nonspecific antibody binding. TLN cells were then incubated with appropriate primary antibody or isotype control for 30 minutes on ice. The antibodies used include phycoerythrin-labeled CD19(1D3), Cy5-labeled CD19 (1D3), biotin-labeled CD69, biotin-labeled IgM (b7-6), biotin-labeled IgD, (11–26) biotin-labeled major histocompatibility complex (MHC) class II (M5114 for B6 or I-Ak for C3H), fluorescein isothiocyanate-labeled CD86 (GL1), biotin-labeled CD40, fluorescein isothiocyanate-labeled peanut agglutinin (PNA), phycoerythrin-labeled CD23 (B2B4), Cy5-labeled B220 (6B2) phycoerythrin-labeled IL-21 receptor, and biotin-labeled CD95. CD69, CD95, IL-21 receptor and MHC class II (I-Ak) were purchased from eBiosciences (San Diego, CA), PE-CD19 was purchased from BD Pharmingen (San Diego, CA) and the remainder of antibodies were prepared as previously described.
      • Wolniak K.L.
      • Shinall S.M.
      • Waldschmidt T.J.
      The germinal center response.
      Following incubation, cells were washed twice in 2 mL of FACS buffer and incubated with appropriate secondary reagent, if necessary, for 30 minutes at 4°C. Secondary reagents included phycoerythrin-labeled streptavidin (as previously described
      • Wolniak K.L.
      • Shinall S.M.
      • Waldschmidt T.J.
      The germinal center response.
      ) and fluorescein isothiocyanate-labeled streptavidin (BD Pharmingen, San Diego, CA). After secondary incubation, cells were washed twice in 2 mL Fluorescence-activated cell sorting (FACS) buffer, fixed in 200 μL of 1% paraformaldehyde, and stored at 4°C in the dark until analysis. Then the analysis was performed on a BD FACScanto flow cytometer (Becton Dickinson, San Jose, CA) and data analysis was performed using FlowJo software V8.5.2 (Tree Star, Inc., Ashland, OR).

      Antigen-Specific Enzyme-Linked Immunosorbent Spot

      IgG1, IgG2a, and IgG2c enzyme-linked immunosorbent spot (ELISPOT) were performed on TLN cells. Immulon 2 plates (Fischer, Fair Lawn, NJ) were coated with 5 μg/mL of freeze-thawed Leishmania parasite antigen overnight at 4°C. After washing with PBS, commercially available biotinylated anti-IgG1, IgG2a, and IgG2c antibodies (Jackson ImmunoResearch West Grove, PA) were added at a 1:10,000 dilution in 5% fetal bovine serum overnight at 4°C. ELISPOT were developed using 2-amino-2-methyl-1-propanol (ICN Biomedicals Inc., Aurora, OH) and 5-bromo-4-chloro-3-indoly-phosphate (Fisher, Fair Lawn, NJ).
      For IL-21 ELISPOT, a similar procedure was used as previously described on TLN cells using the mouse IL-21 DuoSet kit, according to manufacturer's instructions (R&D Systems, Minneapolis, MN).

      Lymph Node Histopathology and Immunohistochemistry

      Popliteal lymph nodes from the left hind leg draining the site of infection were harvested and placed in cassettes in 10% neutral buffered formalin for histological and immunohistochemical analyses. Histological examination was performed on paraffin-embedded tissues cut at 5-μm thickness onto positively charged slides and stained with H&E. For immunohistochemistry, slides were de-paraffinized and blocked with 20% normal rat serum. The sections were then incubated with either an anti-mouse B220/CD45R antibody (BD Harlingen, San Diego, CA) at a concentration of 1:400, a biotin-labeled PNA (Vector Laboratories, Burlingame, CA) at a concentration of 1:500, or with IL-21 antibody (Millipore, Billerica, MA) at a concentration of 1:500 with 10% normal rat serum. The slides were rinsed with PBS and then incubated with biotin-labeled goat anti-rat IgG (KPL, Gaithersburg, MD) at a concentration of 1:300 in 10% normal donkey serum when appropriate. Slides were washed and incubated with peroxidase-conjugated streptavidin (BioGenex, San Ramon, CA) for 45 minutes. After 2 PBS washes, the color was developed with Nova Red (KPL, Gaithersburg, MD). The slides were then counterstained with Harris' hematoxylin, then dehydrated and mounted with coverslips. A semiquantitative scoring scale for PNA staining was used as defined by: 0, no PNA staining; 1, 1-2 PNA-positive germinal centers, 2, 3-4 PNA-positive germinal centers; 3, 5 or more PNA-positive germinal centers per lymph node section. All evaluations were made based on the average of one lymph node section from three animals and two separate experiments and assessed blindly by a board-certified veterinary pathologist.

      Statistics

      Statistical analysis was performed with Prism4 (GraphPad Software Inc., La Jolla, CA). Differences between groups were determined using two-way analysis of variance with Tukey post hoc test or a Mann-Whitney U-test when appropriate. P values <0.05 were considered statistically significant.

      Results

      Increased Germinal Center B Cells and Isotype Switched Germinal Center B Cells during Co-Infection of C3H Mice Compared to B6 Mice

      We previously demonstrated that C3H mice co-infected with L. major and L. amazonensis heal their footpad lesions by 10 to 12 weeks postinfection.
      • Gibson-Corley K.N.
      • Boggiatto P.M.
      • Mukbel R.M.
      • Petersen C.A.
      • Jones D.E.
      A deficiency in the B cell response of C57BL/6 mice correlates with loss of macrophage-mediated killing of Leishmania amazonensis.
      Co-infected C57BL/6 (B6) mice, in comparison, have persistent non-healing lesions (Figure 1) and a significantly higher footpad parasite burden (data not shown).
      • Gibson-Corley K.N.
      • Boggiatto P.M.
      • Mukbel R.M.
      • Petersen C.A.
      • Jones D.E.
      A deficiency in the B cell response of C57BL/6 mice correlates with loss of macrophage-mediated killing of Leishmania amazonensis.
      Using an in vitro co-culture assay, we have shown that B cells harvested from L. major-infected B6 mice cannot promote killing of intracellular L. amazonensis in contrast to B cells from L. major-infected C3H mice.
      • Gibson-Corley K.N.
      • Boggiatto P.M.
      • Mukbel R.M.
      • Petersen C.A.
      • Jones D.E.
      A deficiency in the B cell response of C57BL/6 mice correlates with loss of macrophage-mediated killing of Leishmania amazonensis.
      Based on these previous findings, we hypothesized that B cells from co-infected B6 are phenotypically and functionally different from B cells from co-infected C3H mice. We assessed the number of germinal center B cells and isotype-switched germinal center B cells via flow cytometric analysis using draining TLN cells from mice infected for 2 and 5 weeks with L. amazonensis, L. major or both species of parasites.
      Figure thumbnail gr1
      Figure 1Simultaneous co-infection with both Leishmania major and L. amazonensis allows for lesion resolution in co-infection of C3HeB/FeJ (C3H), but not C57BL/6 (B6) mice at 12 weeks postinfection. Mice with a single infection were inoculated with L. amazonensis (La) or L. major (Lm) stationary phase promastigotes, whereas co-infected mice (Co) were inoculated with Lm and La in the left hind footpad. Mice were infected for 12 weeks with weekly monitoring of lesion size. The lesion size was determined by measuring the infected footpad and comparing that to the non-infected footpad. Data are represented as the mean ± SEM of three separate experiments. *P ≤ 0.05.
      On entering the germinal center, B cells typically display PNA lectin and up-regulate CD95 surface expression.
      • Rabinowitz J.L.
      • Tsiagbe V.K.
      • Nicknam M.H.
      • Thorbecke G.J.
      Germinal center cells are a major IL-5-responsive B cell population in peripheral lymph nodes engaged in the immune response.
      There were significantly more germinal center positive (B220+, PNA+) B cells in the draining lymph nodes of co-infected C3H mice as compared to co-infected B6 mice at both 2 and 5 weeks postinfection (Figure 2A). As expected, naïve mice of both strains had negligible numbers of germinal center B cells (Figure 2A).
      Figure thumbnail gr2
      Figure 2Increased number of total germinal center B cells and germinal center B cells undergoing isotype switching in co-infection of C3HeB/FeJ (C3H) mice. C3H and C57BL/6 (B6) mice were infected with Leishmania amazonensis (La), L. major (Lm), or co-infected (Co) with both species of parasites. Total draining lymph node cells were harvested at 2 and 5 weeks postinfection. Cells were analyzed via fluorescence-activated cell sorting (FACS) and first gated on the B220+ population and analyzed for binding to peanut agglutinin (PNA) (A), PNA binding (B), surface expression of major histocompatibility complex (MHC) class II (C), and surface expression of CD86, as indicated by mean fluorescence intensity (MFI) (D). Cell number was determined based on the percentage of cells within the gated population and the total number of lymph node cells recovered. Data are represented as the mean ± SEM of three separate experiments. *P ≤ 0.05.
      The germinal center functions as the primary location for isotype switching of activated B cells.
      • Cozine C.L.
      • Wolniak K.L.
      • Waldschmidt T.J.
      The primary germinal center response in mice.
      To assess the population of B cells that have undergone isotype switching, we assessed the B220+, PNA+ cell populations for expression of IgM via FACS analysis of cells from the draining lymph nodes of L. amazonensis, L. major, and co-infected C3H and B6 mice. We determined that co-infected C3H mice have more germinal center B cells that are IgM and therefore have undergone isotype switching at 2 and 5 weeks postinfection (Figure 2B).
      On activation, B cells will also up-regulate surface expression of MHC class II, CD80, CD86, and CD40.
      • Leifeld L.
      • Trautwein C.
      • Dumoulin F.L.
      • Manns M.P.
      • Sauerbruch T.
      • Spengler U.
      Enhanced expression of CD80 (B7-1), CD86 (B7-2), and CD40 and their ligands CD28 and CD154 in fulminant hepatic failure.
      To assess B cell activation status, flow cytometry was used to determine surface expression of these markers. Draining lymph node cells were first gated on the B220+ (also known as CD45R) population and were then analyzed for B cell activation makers. Infection led to measurable activation in all infected groups, as determined by increases in mean fluorescent intensity over naïve controls for MHC class II (Figure 2C), CD86 (Figure 2D), or CD40 (data not shown) at either 2 or 5 weeks. All groups, however, expressed similar fold increases in mean fluorescent intensity over naïve controls, suggesting no measurable difference in overall B cell activation status between co-infected B6 and C3H mice.
      To confirm our flow cytometric findings regarding differences in B cell germinal center formation, we performed immunohistochemistry using anti-B220 and biotin-labeled PNA on draining lymph nodes of co-infected mice 2 weeks postinfection. The pattern of immunoreactivity for B220 demonstrated that lymph nodes from co-infected C3H mice have active cortices with multiple, large follicles, and distinct germinal center formation, as compared to co-infected B6 mice, which had less distinct follicles and rare germinal centers (Figure 3A, top panels). PNA staining confirmed there were more germinal centers in co-infected C3H mice, whereas draining lymph nodes of co-infected B6 mice had germinal centers were fewer in number (Figure 3, A [bottom panels] and B). Together the data indicate that the germinal center B cell response at both 2 and 5 weeks postinfection in co-infected B6 mice was less robust. It is known that the germinal center is the site in which B cells become either memory B cells or antibody-secreting plasma cells.
      • Allen C.D.
      • Okada T.
      • Cyster J.G.
      Germinal-center organization and cellular dynamics.
      These findings suggest that C3H mice co-infected with L. major and L. amazonensis have more memory B cells and/or more antibody-secreting cells.
      Figure thumbnail gr3
      Figure 3Increased number of germinal centers in the draining lymph node following co-infection of co-infection of C3HeB/FeJ (C3H) mice with Leishmania major and L. amazonensis. A: Photomicrographs of lymph node sections labeled with anti-mouse B220 and biotin-peanut agglutinin (PNA) from C3H and C57BL/6 (B6) mice co-infected for 2 weeks. Bar = 200 μm. Designated areas magnified in the insets which highlight PNA immunoreactive cells. Scale bars: 40 μm. B: Histological germinal center scores for PNA immunoreactivity at 2 weeks. Score is based on the number of PNA+ germinal centers within a single draining lymph node. Data are representative of two separate experiments ± SEM. *P = 0.0087.

      Increased Memory B Cells during Leishmania Co-Infection in C3H Mice Compared to B6 Mice

      We wanted to determine whether the difference in the number of germinal center B cells would lead to a downstream alteration in the memory B cell population between these two mouse strains after co-infection. Cells from the draining lymph nodes were analyzed via flow cytometry with anti-CD19 to identify B cells, anti-IgM, and anti-CD23. Anti-CD19, instead of anti-B220, was used to differentiate B cells from plasmacytoid dendritic cells, which also express B220. Nonswitched, mature B cells within the lymph node have been previously shown to express surface IgM and CD23, whereas memory B cells are CD19+, IgM, and CD23.
      • Wolniak K.L.
      • Shinall S.M.
      • Waldschmidt T.J.
      The germinal center response.
      At both 2 and 5 weeks after co-infection, we found that C3H mice had increased numbers of memory B cells as compared to B6 mice (Figure 4). These memory B cells include both germinal center memory B cells and postgerminal center switched memory B cells. The relatively low numbers of memory B cells and isotype switched effector B cells in co-infected B6 mice is consistent with poor germinal center formation in these mice.
      Figure thumbnail gr4
      Figure 4Increased number of memory B cells (B220+, IgM, and CD23) in the draining lymph node of co-infected C3HeB/FeJ (C3H) mice. C3H and C57BL/6 (B6) mice were infected with Leishmania amazonensis (La), L. major (Lm), or co-infected (Co) with both species of parasites. Total draining lymph node cells were harvested at 2 and 5 weeks postinfection. Via flow cytometry, surface marker expression was determined based on a CD19+ population. Surface expression of both IgM and CD23 was determined based on the percentage of cells within the gated population as compared to the total lymph node cells recovered. Data are representative of two separate experiments ± SEM. *P ≤ 0.05.

      Increased Antigen-Specific Antibody Production in Co-Infected C3H Mice Compared to Co-Infected B6 Mice

      To determine whether the observed differences in germinal center formation and B cell effector phenotype lead to differences in B cell antibody production, we analyzed the number of antigen-specific antibody-producing B cells during co-infection of C3H and B6 mice. ELISPOT analysis was performed on draining TLN cells for antigen-specific antibody production of IgG2a (C3H), IgG2c (B6), and IgG1 from draining lymph nodes of co-infected C3H and B6 mice. B6 mice express the Igh1-b allele, which encodes for and leads to IgG2c antibody isotype production, whereas C3H mice carry the Igh1-a allele, and therefore produce IgG2a.
      • Martin R.M.
      • Brady J.L.
      • Lew A.M.
      The need for IgG2c specific antiserum when isotyping antibodies from C57BL/6 and NOD mice.
      Co-infected C3H mice produce significantly more L. major-specific IgG2a than B6 mice produce L. major-specific IgG2c at 2 weeks postinfection (Figure 5A). No significant differences were noted in the production of antigen-specific IgG1 (Figure 5B). These results indicate a differential production of antigen-specific antibodies when C3H and B6 mice are compared at the 2-week time point. Not statistically significant, however, at 5 weeks postinfection there is a similar trend in which smaller numbers of antigen-specific antibodies in B6 mice are compared to C3H mice.
      Figure thumbnail gr5
      Figure 5Increased number of antigen-specific IgG2a-producing cells following co-infection in C3HeB/FeJ (C3H) mice. C3H and C57BL/6 (B6) mice were infected with Leishmania amazonensis (La), L. major (Lm), or co-infected (Co) with both species of parasites. Total draining lymph node cells were harvested at 2 and 5 weeks postinfection (please note scales). Number of IgG2a (C3H) and IgG2c (B6)-producing cells (A), and IgG1-producing cells (B), as determined by enzyme-linked immunosorbent spot (ELISPOT) analysis of total draining lymph node cells stimulated with freeze-thawed Leishmania promastigote antigen, as previously indicated. Data are represented as the mean ± SEM of three separate experiments. *P ≤ 0.05.

      IL-21 Production, Location of Production, and IL-21 Receptor Expression Are Similar between C3H and B6 Mice Co-Infected for 2 Weeks

      After B6 mice were co-infected in the footpad with both L. major and L. amazonensis, we observed an overall decrease in the number of lymph node germinal centers, isotype switched germinal center B cells, memory B cells, and antigen-specific IgG2a/c antibody-producing cells than co-infected C3H mice. We sought to determine whether these differences were intrinsic to B cell function or if they were due to differential production of IL-21 from the draining lymph node of co-infected C3H versus B6 mice. IL-21 is primarily produced by T follicular helper (Tfh) cells within germinal centers, but they can also be produced by T helper 17 cells and other lineages of CD4+ T cells.
      • Yi J.S.
      • Cox M.A.
      • Zajac A.J.
      Interleukin-21: a multifunctional regulator of immunity to infection.
      IL-21 has been shown to function in B cell proliferation and production of plasma cells.
      • Haynes N.M.
      Follicular associated T cells and their B-cell helper qualities.
      The important role of IL-21 in B cell function has been demonstrated in B cells deficient in IL-21 receptor expression, which have an impaired ability to undergo isotype switching and maintain germinal center organization.
      • Eto D.
      • Lao C.
      • DiToro D.
      • Barnett B.
      • Escobar T.C.
      • Kageyama R.
      • Yusuf I.
      • Crotty S.
      IL-21 and IL-6 are critical for diferent aspects of B cell immunity and redundantly induce optimal follicular helper CD4 T cell (Tfh) differentiation.
      • Fazilleau N.
      • Mark L.
      • McHeyzer-Williams L.J.
      • McHeyzer-Williams M.G.
      Follicular helper T cells: lineage and location.
      We performed IL-21 ELISPOT on TLN cells from co-infected C3H versus B6 mice. There was a significant increase in the number of IL-21-producing cells after co-infection of either B6 or C3H mice as compared to L. amazonensis infection alone. There was no difference in the number of IL-21 producing cells at 2 weeks postinfection between C3H and B6 mice with or without antigen stimulation (Figure 6A [data not shown]). IL-21 immunohistochemistry was used to confirm these results, and again we did not observe any differences in the distribution of IL-21 immunoreactive cells in the draining lymph nodes of co-infected C3H versus B6 mice (Figure 6C). Therefore, a paucity of IL-21 producing cells is not responsible for the differences we have described in germinal center B cell responses after co-infection of B6 mice versus C3H mice. We then determined if the expression of B cell IL-21 receptor was different between co-infected C3H and B6 mouse strains at 2 weeks postinfection. Using flow cystometry, we gated on B220+ cells and determined that expression of the IL-21 receptor was not significantly different between co-infection or single parasite infection (Figure 6B).
      Figure thumbnail gr6
      Figure 6No difference in the number of IL-21 producing cells, IL-21 receptor expression, or site of IL-21 production in draining lymph node cells from co-infected C3HeB/FeJ (C3H) and C57BL/6 (B6) mice. A: Number of IL-21-producing cells was determined by enzyme-linked immunosorbent spot (ELISPOT) analysis of total draining lymph node cells stimulated with freeze-thawed Leishmania promastigote antigen. C3H and B6 mice were infected with Leishmania amazonensis (La), L. major (Lm), or co-infected (Co) with both species of parasites. Total draining lymph node cells were harvested at 2 weeks postinfection. Data are represented as the mean ± SEM of two (for B6) or three (for C3H) separate experiments. B: Cells were first gated on the B220+ population and surface expression of IL-21 receptor was measured as indicated by mean fluorescence intensity (MFI). The MFI for each marker presented as fold increase over naïve control. Total draining lymph node cells were harvested at 2 weeks postinfection. Data are represented as the mean ± SEM of two separate experiments. C: Photomicrographs of lymph node sections labeled with anti-mouse IL-21 from C3H and B6 mice co-infected for 2 weeks. Bar = 50 μm.

      Discussion

      The immune factors required to heal cutaneous leishmaniasis caused by L. amazonensis are unknown. In this article, we demonstrated that co-infection with both L. major and L. amazonensis led to a healing phenotype in C3H mice, whereas B6 mice develop non-healing, persistent lesions (Figure 1).
      • Gibson-Corley K.N.
      • Boggiatto P.M.
      • Mukbel R.M.
      • Petersen C.A.
      • Jones D.E.
      A deficiency in the B cell response of C57BL/6 mice correlates with loss of macrophage-mediated killing of Leishmania amazonensis.
      Using an in vitro model that mimics co-infection, we have previously demonstrated that B cells from infected B6 mice do not function as effectively as B cells from C3H mice to kill intracellular L. amazonensis.
      • Gibson-Corley K.N.
      • Boggiatto P.M.
      • Mukbel R.M.
      • Petersen C.A.
      • Jones D.E.
      A deficiency in the B cell response of C57BL/6 mice correlates with loss of macrophage-mediated killing of Leishmania amazonensis.
      Here, for the first time we describe a significant difference in the germinal center B cell response between healing C3H and non-healing B6 mice co-infected with L. major and L. amazonensis, as early as 2 weeks postinfection. Germinal centers, formed within secondary lymphoid organs, are the site for early B cell expansion.
      • Allen C.D.
      • Okada T.
      • Cyster J.G.
      Germinal-center organization and cellular dynamics.
      After proliferation, signal-dependent isotype switching occurs within the germinal center changing the B cell receptor surface expression from IgD or IgM to IgG, IgA, or IgE.
      • Allen C.D.
      • Okada T.
      • Cyster J.G.
      Germinal-center organization and cellular dynamics.
      At 2 and 5 weeks following infection there were more germinal center B cells and more isotype switched germinal center B cells in healing co-infected C3H mice compared to B6 mice (Figure 2, A and B). We also observed increased germinal centers within the draining lymph nodes of co-infected C3H mice compared to co-infected B6 mice at 2 weeks postinfection (Figure 2, A and B). B cell memory is also formed in germinal centers,
      • Kalia V.
      • Sarkar S.
      • Gourley T.S.
      • Rouse B.T.
      • Ahmed R.
      Differentiation of memory B and T cells.
      and it was not surprising that there are more memory B cells at both 2 and 5 weeks post co-infection in C3H mice compared to B6 mice (Figure 3).
      Little has been documented regarding the germinal center response during Leishmania infection. Histological studies in mice have reported enlarged or hyperplastic germinal centers with apoptotic cells up to 40 days postinfection with L. amazonensis.
      • Abreu-Silva A.L.
      • Calabrese K.S.
      • Cupolilo S.M.
      • Cardoso F.O.
      • Souza C.S.
      • Goncalves da Costa S.C.
      Histopathological studies of visceralized Leishmania (Leishmania) amazonensis in mice experimentally infected.
      More recently, using L. major infection of BALB/c mice, it was determined that Tfh cells in germinal centers produce cytokines that influence the affinity and isotype of the antibody response.
      • Reinhardt R.L.
      • Liang H.E.
      • Locksley R.M.
      Cytokine-secreting follicular T cells shape the antibody repertoire.
      Our data indicate that there are significantly more antigen-specific IgG2a-producing cells in the draining lymph node of co-infected C3H mice as compared to antigen-specific IgG2c-producing cells from co-infected B6 mice at 2 weeks (Figure 5). These antibody isotypes are important, as they are the predominant antibodies produced during a polarized Th1 immune response.
      • Miles S.A.
      • Conrad S.M.
      • Alves R.G.
      • Jeronimo S.M.
      • Mosser D.M.
      A role for IgG immune complexes during infection with the intracellular pathogen Leishmania.
      The presence of robust germinal center formation in C3H mice versus B6 mice after co-infection and the downstream effector function of B cells, as measured by isotype switching, memory cell formation, and production of antigen-specific antibodies all suggest a robust B cell response is part of productive immunity and healing of co-infection with L. major and L. amazonensis.
      The role of B cells during infection with Leishmania is controversial. Some reports describe a negative effect of antibody production during Leishmania infection. When IgG-negative BALB/c mice were infected with L. major, there were smaller lesions with lower parasite loads compared to infected mice with IgG.
      • Miles S.A.
      • Conrad S.M.
      • Alves R.G.
      • Jeronimo S.M.
      • Mosser D.M.
      A role for IgG immune complexes during infection with the intracellular pathogen Leishmania.
      A more recent study showed L. amazonensis-infected mice, which lacked functional B cells, and therefore antibodies had a delayed onset of disease and developed small lesions.
      • Wanasen N.
      • Xin L.
      • Soong L.
      Pathogenic role of B cells and antibodies in murine Leishmania amazonensis infection.
      It has also been shown that there were limited infections with both L. amazonensis and L. pifanoi in the absence of circulating antibodies, and infection of Fc gamma receptor (Fc γR) knockout mice or Fc γRIII knockouts resulted in similarly limited lesions.
      • Buxbaum L.U.
      A detrimental role for IgG and Fc gammaR in Leishmania mexicana infection.
      • Kima P.E.
      • Constant S.L.
      • Hannum L.
      • Colmenares M.
      • Lee K.S.
      • Haberman A.M.
      • Shlomchik M.J.
      • McMahon-Pratt D.
      Internalization of Leishmania mexicana complex amastigotes via the Fc receptor is required to sustain infection in murine cutaneous leishmaniasis.
      In addition, L. mexicana infection of Fc γRIII−/− mice produced higher levels of IFN- γ.
      • Thomas B.N.
      • Buxbaum L.U.
      FcgammaRIII mediates immunoglobulin G-induced interleukin-10 and is required for chronic Leishmania mexicana lesions.
      In contrast, other studies have described a protective role for both B cells and antibodies during Leishmania infection. Scott et al
      • Scott P.
      • Natovitz P.
      • Sher A.
      B lymphocytes are required for the generation of T cells that mediate healing of cutaneous leishmaniasis.
      showed that eliminating B cells in neonatal mice using an anti-IgM antibody impaired the T cell-mediated immune response following L. major infection. Antibody production has also been shown to be a key factor for phagocytosis of L. major by dendritic cells and infected, B cell-deficient mice had larger lesions, higher parasite loads, lower IFN-γ production and a decreased T cell response.
      • Woelbing F.
      • Kostka S.L.
      • Moelle K.
      • Belkaid Y.
      • Sunderkoetter C.
      • Verbeek S.
      • Waisman A.
      • Nigg A.P.
      • Knop J.
      • Udey M.C.
      • von Stebut E.
      Uptake of Leishmania major by dendritic cells is mediated by Fc gamma receptors and facilitates acquisition of protective immunity.
      In our co-infection system, we have previously demonstrated an important role for B cells in vitro
      • Gibson-Corley K.N.
      • Boggiatto P.M.
      • Mukbel R.M.
      • Petersen C.A.
      • Jones D.E.
      A deficiency in the B cell response of C57BL/6 mice correlates with loss of macrophage-mediated killing of Leishmania amazonensis.
      and here we demonstrate that the in vivo B cell response is consistent with the hypothesis that these cells have an important role for cutaneous lesion resolution in C3H mice with L. amazonensis as part of a co-infection and that B cells from B6 mice do not play an effective role in co-infected animals.
      T cells have a critical role in germinal center formation and maintenance. Tfh cells are a T helper cell subset that is specialized in regulating the effector and memory responses of B cells.
      • Fazilleau N.
      • Mark L.
      • McHeyzer-Williams L.J.
      • McHeyzer-Williams M.G.
      Follicular helper T cells: lineage and location.
      Within the germinal center Tfh cells produce IL-21, which has been shown to promote B cell proliferation and production of plasma cells.
      • Yi J.S.
      • Cox M.A.
      • Zajac A.J.
      Interleukin-21: a multifunctional regulator of immunity to infection.
      Tfh cells not only produce IL-21, they can also produce interferon-γ, IL-4, or IL-17.
      • Fazilleau N.
      • Mark L.
      • McHeyzer-Williams L.J.
      • McHeyzer-Williams M.G.
      Follicular helper T cells: lineage and location.
      B cells express the IL-21 receptor and B cells deficient in this receptor have an impaired ability to undergo isotype switching and lose germinal center organization.
      • Fazilleau N.
      • Mark L.
      • McHeyzer-Williams L.J.
      • McHeyzer-Williams M.G.
      Follicular helper T cells: lineage and location.
      We hypothesized that differential IL-21 production could have been responsible for germinal center phenotypic differences between co-infected C3H and B6 mice. There was no difference, however, in the number of IL-21-producing cells at 2 weeks postinfection between these two mouse strains following antigenic stimulation (Figure 5A). IL-21 receptor expression was measured and again no differences in receptor expression were observed at 2 weeks postinfection in co-infected C3H versus B6 mice (Figure 5B). We also used immunohistochemistry for IL-21 and determined that IL-21 immunoreactive cells were present within the cortex, as well as within the subcapsular sinus and medullary sinuses (Figure 5C). These findings confirmed our ELISPOT data showing that there were no differences in IL-21 production in the two mouse strains. These results indicate that IL-21-producing cells may be necessary for robust germinal center formation and function, although IL-21 production per se is not sufficient to generate a germinal center response.
      Here we describe for the first time a difference in the draining lymph node germinal center B cell response between co-infected C3H and B6 mice. These findings are consistent with our in vitro data indicating that a productive B cell response is required for healing a co-infection with L. amazonensis and L. major in the mouse. Based on this, we expect that broadly effective anti-Leishmania vaccinations or treatments require not only a productive T cell response, but also a productive B cell response.

      Acknowledgments

      We thank the Histopathology Research Laboratory at the University of Iowa, as well as Lorraine Tygett and Deb Moore for their technical expertise and assistance.

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