Lactoferrin modulation of IL-12 and IL-10 response from activated murine leukocytes

Abstract

Lactoferrin possesses a wide range of immunomodulatory activities, including promotion of the delayed type hypersensitivity response (DTH) towards BCG (Bacillus Calmette Guerin) antigens. Addition of Lactoferrin as an adjuvant to the BCG vaccine was previously demonstrated to augment protection against subsequent mycobacterial challenge, with concomitant development of a strong T cell helper type 1 (TH1) immunity. Because generation of TH1 immunity is in large part dependent on the balance of monocytic pro- and anti-inflammatory cytokines, the effect of Lactoferrin on leukocytes was investigated. Lactoferrin enhanced proinflammatory responses in a dose-dependant manner from splenocyte and adherent (F4/80+) splenocyte populations, bone marrow derived monocytes (BMM), and J774A.1 cultured cells. In all scenarios tested, Lactoferrin induced a strong increase in the ratio of IL-12:IL-10 production from LPS stimulated cells. Examination of Lactoferrin effects on BCG infected J774A.1 cells and on BMM revealed similar immunomodulatory effects, with particularly strong increase in IL-12 production. Furthermore, immunization of mice with BCG admixed with Lactoferrin led to increased generation of CD4+ cells expressing IFN-gamma upon restimulation with BCG antigens. These results provide molecular evidence to support the role of Lactoferrin as an adjuvant candidate to augment development of DTH response to vaccine antigens.

Fig. 1

Examination of the effect of Lactoferrin on production of proinflammatory mediators from LPS stimulated splenocytes. Splenocytes were stimulated with LPS (400 ng/mL) with or without increasing concentrations of Lactoferrin (1, 10, 100 μg/mL). Supernatants were collected at 48 h and analyzed by ELISA for production of IL-12p40 and IL-10 (top) or TNF-α and IL-6 (bottom); average value with standard deviation shown. *P < 0.05

Fig. 2

Effects of Lactoferrin on cytokine production from macrophages stimulated with suboptimal concentration of LPS. J774A.1 murine macrophages were left untreated as media control (top), or stimulated with LPS at 1 ng/mL (bottom), with or without increasing concentrations of Lactoferrin (1, 10, 100 μg/mL). Supernatants were collected at 24 and 72 h and analyzed by ELISA for production of IL-12 (left), or IL-10 (right); average value with standard deviation shown. *P < 0.05, **P < 0.01

Fig. 3

Lactoferrin activity on inflammatory cytokine production from BCG stimulated macrophages. J774A.1 murine macrophages (top) or bone marrow derived macrophages (bottom) were infected with live BCG at MOI 10:1 with and without indicated concentrations of Lactoferrin. Supernatants collected were analyzed by ELISA for IL-12p40, and IL-10; average values with standard deviation shown. **P < 0.01, ***P < 0.001

Fig. 4

Immunization with BCG and Lactoferrin promotes increased IFN-γ production and high numbers of BCG antigen-specific CD4+ cells. Splenocytes from naïve mice, or from mice 8 weeks after immunization with BCG or BCG admixed with Lactoferrin, were stimulated in vitro with BCG antigens. IFN-γ production was measured by ELISA (top), expressed as average values with standard deviation for 6 mice (* P < 0.05). The same populations were restimulated in vitro with BCG antigens; flow cytometric analysis and intracellular staining (bottom) revealed increases in numbers of IFN-γ producing CD4+ cells in the BCG-Lactoferrin immunized population (right), compared to naïve (left) or BCG alone (middle) immunized groups. Representative flow data shown for splenocytes from four stimulated mice per group