Dependency of B-1 Cells in the Maintenance of Splenic Interleukin-10 Producing Cells and Impairment of Macrophage Resistance in Visceral Leishmaniasis

Abstract

Visceral leishmaniasis is a neglected disease caused by Leishmania protozoa parasites transmitted by infected sand fly vectors. This disease represents the second in mortality among tropical infections and is associated to a profound immunosuppression state of the host. The hallmark of this infection-induced host immunodeviation is the characteristic high levels of the regulatory interleukin-10 (IL-10) cytokine. In the present study, we investigated the role of B-1 cells in the maintenance of splenic IL-10 levels that could interfere with resistance to parasite infection. Using an experimental murine infection model with Leishmania (L.) infantum chagasi we demonstrated an improved resistance of B-1 deficient BALB/XID mice to infection. BALB/XID mice developed a reduced splenomegaly with diminished splenic parasite burden and lower levels of IL-10 secretion of purified splenocytes at 30 days post-infection, as compared to BALB/c wild-type control mice. Interestingly, we found that resident peritoneal macrophages isolated from BALB/XID mice were more effective to control the parasite load in comparison to cells isolated from BALB/c wild-type mice. Our findings point to a role of B-1 cells in the host susceptibility to visceral leishmaniasis.

Keywords: B-1 cells; Leishmania (L.) infantum chagasi; host protective responses; visceral leishmaniasis.

FIGURE 1

B-1 cell-deficient BALB/XID mice show resistance to visceral leishmaniasis. BALB/c and BALB/XID mice were intravenously injected with 5 × 10⁷ amastigotes of Leishmania (L.) infantum chagasi, and 30 days after infection the clinical signs of disease were examined. (A) Spleen/body relative weights are attenuated in B-1 cell-deficient BALB/XID mice. The body weight was measured in grams and the spleen/body relative weights (grams of organ weight × 100/grams of body weight) were determined in BALB/c and BALB/XID mice. (B) Decreased parasite burden in the spleen of BALB/XID mice. The vertical axis in the histogram represents the average parasite load from spleen or (C) liver tissues in Leishman–Donovan units of Stauber (LDU = number of amastigotes/1000 cell nuclei × organ weight in mg) obtained at 30 days post-infection (DPI). Data are means ± SE and represent the results of two independent experiments performed with five to six mice per group. Differences between groups are significant ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

FIGURE 2

Isotype and IgG subclass profiles of serum antibodies to Leishmania (L.) infantum chagasi of B-1 cell-deficient mice. Sera from wild type BALB/c and B-1 cell-deficient BALB/XID mice infected with 5 × 10⁷ amastigote forms of Leishmania (L.) infantum chagasi were collected at 30 DPI and the absorbance values of (A) antibody Isotypes (IgM, IgA and IgG); and (B) IgG antibody subclasses (IgG1, IgG2a, IgG2b and IgG3) were determined by ELISA using Leishmania (L.) infantum chagasi promastigote lysates as parasite antigens. Bars show levels of immunoglobulin isotypes and IgG subclasses as the individual absorbancy values of 1/100 diluted sera. Data represent the individual results for each group of mice obtained from two independent experiments.

FIGURE 3

Splenocytes from B-1 cell-deficient BALB/XID mice produce low levels of IL-10. BALB/c and BALB/XID mice were intravenously infected with 5 × 10⁷ amastigotes of Leishmania (L.) infantum chagasi, and 30 days after infection splenocytes were isolated and assayed for cytokine production upon parasite antigen (Ag) stimulation. After 3 days of in vitro culture with freeze and thawed lysates of Leishmania (L.) infantum chagasi promastigotes, as described elsewhere (Popi et al., 2012), splenocyte supernatants were harvested for determination of (A) TNF-α, (B) IFN-γ, (C) IL-10, and (D) TGF-β by ELISA. The y-axis represents the levels of cytokines, detected by specific ELISA assays, expressed in ng/ml. Data are means ± SE and represent the results of two independent experiments performed with five to six mice per group. Asterisks represent statistical significance between groups ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

FIGURE 4

Leishmania (L.) infantum chagasi infection of tissue-resident macrophages obtained from BALB/c and BALB/XID mice. Intraperitoneal macrophage cells purified from BALB/c and BALB/XID mice were cultured at 1.0 × 10⁵ cells/well in 48-well plates and infected with 10⁶ Leishmania (L.) infantum chagasi promastigotes at a 10:1 ratio of parasites:host cells in DMEM containing 10% FBS at 37°C. The infected cells were washed 4 h to remove extracellular parasites and then maintained for 3 days at 37°C to determine the intracellular amastigote load (A). Following intracellular parasitism, infected macrophages were cultured in Schneider medium supplemented with 20% FBS at 26°C for an additional 3 days to estimate the L. chagasi load by counting the promastigotes forms derived from released parasites (B). Histograms represent the means ± SE of total number of Leishmania (L.) infantum chagasi forms of triplicate assays and are representative from two independent experiments. Differences between groups are significant ^∗p < 0.05.