Murine visceral leishmaniasis: IgM and polyclonal B-cell activation lead to disease exacerbation

Abstract

In visceral leishmaniasis, the draining LN (DLN) is the initial site for colonization and establishment of infection after intradermal transmission by the sand fly vector; however, little is known about the developing immune response within this site. Using an intradermal infection model, which allows for parasite visceralization, we have examined the ongoing immune responses in the DLN of BALB/c mice infected with Leishmania infantum. Although not unexpected, at early times post-infection there is a marked B-cell expansion in the DLN, which persists throughout infection. However, the characteristics of this response were of interest; as early as day 7 post-infection, polyclonal antibodies (TNP, OVA, chromatin) were observed and the levels appeared comparable to the specific anti-leishmania response. Although B-cell-deficient JhD BALB/c mice are relatively resistant to infection, neither B-cell-derived IL-10 nor B-cell antigen presentation appear to be primarily responsible for the elevated parasitemia. However, passive transfer and reconstitution of JhD BALB/c with secretory immunoglobulins, (IgM or IgG; specific or non-specific immune complexes) results in increased susceptibility to L. infantum infection. Further, JhD BALB/c mice transgenetically reconstituted to secrete IgM demonstrated exacerbated disease in comparison to WT BALB/c mice as early as 2 days post-infection. Evidence suggests that complement activation (generation of C5a) and signaling via the C5a receptor (CD88) is related to the disease exacerbation caused by IgM rather than cytokine levels (IL-10 or IFN-gamma). Overall these studies indicate that polyclonal B-cell activation, which is known to be associated with human visceral leishmaniasis, is an early and intrinsic characteristic of disease and may represent a target for therapeutic intervention.

Figure 1. Histology of infected lymph nodes

Immunohistochemical analyses of sections from the DLN of uninfected mice and L. infantum-infected mice (days 3 and 6, and at 3 months post-infection). DLN were stained with (A) CD3 (red; T cells) and B220 (green; B cells), (B) peanut agglutinin (germinal centers), or (C) FDC-M1 (follicular dendritic cells). There is an early and persistent B-cell expansion and apparent loss of T cells in the DLN. Germinal centers (GC) and follicular dendritic cells (FDC) are evident at day 6 and persist, through the chronic stage of the disease (3 months). Data are representative of at least 3 experiments.

Figure 2. Lymph node responses to L. infantum infection

Lymph node T and B lymphocyte responses of BALB/c mice intradermally infected with L.infantum. (A) Cellular proliferation was evaluated using BrdU incorporation. BrdU was injected intraperitoneally 4 hours prior to obtaining cells at the indicated times for FACS analysis. Results are shown as the % of BrdU+ T cells (CD3+) and % of BrdU+ B cells (CD19+) cells over time. B) Increases in cell numbers present in the DLN (total lymph node cells, B cells (CD19+), T cells (CD3+)). Data show mean ± SEM (n= 3–4/time point) and are representative of at least 2 experiments.

Figure 3. Early serum antibody responses in BALB/c mice infected with L. infantum are polyclonal

Sera were collected from mice at days 7, and 14 post-infection and assayed for antibody levels (total Ig (IgG and IgM)) to leishmanial (SLA) and non-leishmanial antigens ((TNP)₁₅-BSA, ovalbumin, chromatin). Statistical significance is indicated and was determined using Student’s t-test in comparison to uninfected controls for each antigen. Data show mean ± SEM (n=5 per group) and are representative of 2 experiments.

Figure 4. B-cell-derived IL-10 is insufficient to restore susceptibility

A) FACS analyses of the DLN cells from WT BALB/c mice (CD3+ T cells or CD19+ B cells) at 5 days post-infection with L. infantum. B cells and T cells contribute to IL-10 production early in infection. B) Bone marrow chimeric J_HD mice were employed to evaluate the effect of B-cell-derived IL-10 on disease progression. Shown are the parasite burdens 1 month after infection with L. infantum (DLN, spleen, liver) of J_HD mice reconstituted with B cells not producing IL-10 (20% IL-10 deficient + 80% J_HD bone marrow cells) or with IL-10-producing B cells (20% WT + 80% J_HD bone marrow cells). WT BALB/c mice were employed as controls. Data show mean ± SE (n=5 mice per group). No statistical differences were found between various groups of mice (p>0.2). C) Parasite burdens (LN, spleen, liver) and immune responses (Table 4) of BALB/c wild-type, J_HD and mice transgenically altered to have functional B cells but no circulating antibody (mIgM/J_HD transgenic mice) were compared at one month post-infection. Data are representative of 3 experiments and show mean ± SE (n=3 mice per group). *p=0.01, Student’s t-test.

Figure 4. B-cell-derived IL-10 is insufficient to restore susceptibility

A) FACS analyses of the DLN cells from WT BALB/c mice (CD3+ T cells or CD19+ B cells) at 5 days post-infection with L. infantum. B cells and T cells contribute to IL-10 production early in infection. B) Bone marrow chimeric J_HD mice were employed to evaluate the effect of B-cell-derived IL-10 on disease progression. Shown are the parasite burdens 1 month after infection with L. infantum (DLN, spleen, liver) of J_HD mice reconstituted with B cells not producing IL-10 (20% IL-10 deficient + 80% J_HD bone marrow cells) or with IL-10-producing B cells (20% WT + 80% J_HD bone marrow cells). WT BALB/c mice were employed as controls. Data show mean ± SE (n=5 mice per group). No statistical differences were found between various groups of mice (p>0.2). C) Parasite burdens (LN, spleen, liver) and immune responses (Table 4) of BALB/c wild-type, J_HD and mice transgenically altered to have functional B cells but no circulating antibody (mIgM/J_HD transgenic mice) were compared at one month post-infection. Data are representative of 3 experiments and show mean ± SE (n=3 mice per group). *p=0.01, Student’s t-test.

Figure 5. Secretory IgM causes disease exacerbation

Parasite burdens in mice transgenically altered to produce membrane and secretory IgM ({m+s}IgM/J_HD mice) were compared to that of wild type mice by limiting dilution analysis at A) 2 days and B) 1 month after infection with Leishmania infantum. Parasite burdens are shown for the DLN, spleen and liver, tissue sites. Data are representative of at least 2 experiments and are similar to what was found at other times post-infection (days 6, 10, 14, and 2 months). (Minimum of n=3 mice per group). *0.04≥p≥0.02; **0.002≥p≥0.001; Student’s t-test. C) In response to infection, B-cell activation and IgM production in {m+s}IgM/J_HD mice is elevated in comparison to WT mice. Data show summary of FACS analyses examining IgM+ (intracellular and extra-cellular), CD69+, CD19+ cells at five days post-infection. Data are representative of 2 experiments and show mean + SE (n=3).

Figure 5. Secretory IgM causes disease exacerbation

Parasite burdens in mice transgenically altered to produce membrane and secretory IgM ({m+s}IgM/J_HD mice) were compared to that of wild type mice by limiting dilution analysis at A) 2 days and B) 1 month after infection with Leishmania infantum. Parasite burdens are shown for the DLN, spleen and liver, tissue sites. Data are representative of at least 2 experiments and are similar to what was found at other times post-infection (days 6, 10, 14, and 2 months). (Minimum of n=3 mice per group). *0.04≥p≥0.02; **0.002≥p≥0.001; Student’s t-test. C) In response to infection, B-cell activation and IgM production in {m+s}IgM/J_HD mice is elevated in comparison to WT mice. Data show summary of FACS analyses examining IgM+ (intracellular and extra-cellular), CD69+, CD19+ cells at five days post-infection. Data are representative of 2 experiments and show mean + SE (n=3).

Figure 6. Reconstitution of J_HD mice with IgG or IgM antibodies restores wild type susceptibility to infection

To assess the role of antibody or antigen-antibody complexes, isolated IgG or IgM from naïve or chronically L. infantum-infected WT or ({m+s}IgM/J_HD Tg mice was employed. Additionally, IgG was isolated from BALB/c mice immunized with OVA. Mice were given 600 µg of purified antibody intraperitoneally, one day pre-infection, and subsequently, every 3 days for IgM, and every 7 days for IgG. OVA antigen was injected together at the time of infection in mice receiving anti-OVA antibody to create nonspecific antigen-antibody complexes. Parasite burdens were determined 3 weeks post-infection. Data show mean ± SE (n=3–5 mice per group). *0.05≥p≥0.01, **p≤0.002 in comparison to J_HD mice; Student’s t-test.

Figure 7. C5aR Antagonist peptide treatment ameliorates disease in wild type BALB/c and {m+s}IgM/J_HD Tg mice

BALB/c mice were treated with a C5a receptor antagonist C5aRa (Ac-Phe-[Orn-Pro-dCha-Trp-Arg]) or control peptide (Ac-Phe-[Orn-Pro-dCha-Ala-D-Arg]) on days -1 and 0, pre-infection and days 2 and 4 post-infection intradermally with 5×10⁵ L. infantum promastigotes. Parasite burdens were evaluated at day 6 post-infection in the DLN and liver. Parasite numbers were too low in the spleen at this early time point for evaluation. P values are for mice treated with C5a receptor antagonist in comparison to control mice. Data are representative of three independent experiments and show mean ± SE (n=3-5). **p=0.005, +p=0.06; Student’s t-test.