Effects of intranasal administration of a leptin-secreting Lactococcus lactis recombinant on food intake, body weight, and immune response of mice

Abstract

Leptin is an adipocyte-derived pleiotropic hormone that modulates a large number of physiological functions, including control of body weight and regulation of the immune system. In this work, we show that a recombinant strain of the food-grade lactic acid bacterium Lactococcus lactis (LL-lep) can produce and efficiently secrete human leptin. The secreted leptin is a fully biologically active hormone, as demonstrated by its capacity to stimulate a STAT3 reporter gene in HEK293 cells transfected with the Ob-Rb leptin receptor. The immunomodulatory activity of leptin-secreting L. lactis was evaluated in vivo by coexpression with the human papillomavirus type 16 E7 protein. In C57BL/6 mice immunized intranasally with a recombinant L. lactis strain coproducing leptin and E7 antigen, the adaptive immune response was significantly higher than in mice immunized with recombinant L. lactis producing only E7 antigen, demonstrating adjuvanticity of leptin. We then analyzed the effects of intranasally administered LL-lep in obese ob/ob mice. We observed that daily administration of LL-lep to these mice significantly reduced body weight gain and food intake. These results demonstrate that leptin can be produced and secreted in an active form by L. lactis and that leptin-producing L. lactis regulates in vivo antigen-specific immune responses, as well as body weight and food consumption.

FIG. 1.

Schematic representation of pSEC:lep vector and expression of leptin by L. lactis. (A) A 462-bp DNA fragment encoding mature human leptin was fused in frame with a DNA fragment containing the Usp45 signal peptide (SEC:Leptin), derived from the predominant L. lactis-secreted protein (67). In this plasmid, leptin expression is controlled by the nisin-inducible promoter (P_nisA) and harbors the Usp45 ribosome binding site and the rho-independent trpA transcription terminator (ter) (12) for clone stability. The pSEC:Lep carries the pC194 chloramphenicol resistance marker (cm) (34) (B) Strains LL and LL-lep were grown and induced with 10 ng/ml nisin for 1 h. After centrifugation, cell pellet and culture medium were treated as described in Materials and Methods. The antileptin antibody detected a protein in LL-lep culture supernatants with an apparent molecular mass identical to that of the commercial recombinant leptin. C, cell fraction; S, supernatant fraction. (C) Strains LL and LL-lep were grown as described in Materials and Methods. After centrifugation, leptin was immunoprecipitated from 1 ml of culture medium and immunodetected by Western blotting using antileptin antibody. (D) Strain LL or LL-lep culture supernatants were fractionated on 100-kDa centrifugal membranes as described previously (9). Leptin present in retentates (R) and eluates (E) was immunoprecipitated and detected by immunoblotting with antileptin antibody. Commercial leptin (Leptin) was used as a control in the assays.

FIG. 2.

Leptin expression by recombinant lactococci as a function of induction conditions. (A) Leptin quantification by ELISA of cell fractions or supernatant samples from LL-lep cultures induced (at an optical density at 600 nm of 0.6 U) with increasing concentrations of nisin (0 to 15 ng/ml). (B) Leptin quantification by ELISA of cell fractions or supernatant samples from LL-lep cultures induced (at an optical density at 600 nm of 0.6 U) with 10 ng/ml nisin at different times (0 to 24 h). The results of a representative experiment are shown.

FIG. 3.

In vitro biological activity of leptin produced by L. lactis. HEK293 cells were cotransfected with Ob-Rb receptor, STAT3-firefly luciferase reporter gene, and pcDNA3-Renilla luciferase. Twenty-four hours after transfection, cells were cultured for an additional 24 h in 0.5 ml of DMEM containing 1% serum and either 10 nM commercial leptin or 50 μl of L. lactis culture medium, from leptin-producing or -nonproducing strains. Results are expressed as ratios of activities for firefly luciferase over Renilla luciferase and represent the means of two independent experiments performed in triplicate.

FIG. 4.

Production levels of IFN-γ from spleen cells in mice immunized with live lactococci expressing human leptin and E7 antigen. Five C57BL/6 mice were intranasally immunized on days 0, 14, and 28 with strains LL, LL-E7, and LL-lep/E7. One week after the last immunization (day 35), splenocytes from immunized mice were pooled and stimulated in vitro with E7_30-67 peptide (MHC class II epitope) or E7_49-57 peptide (MHC class I epitope) for identification of IFN-γ-producing CD4⁺ and CD8⁺ T cells, respectively, by enzyme-linked immunospot assay. *, differences are statistically significant (P < 0.05).

FIG. 5.

Effects of daily intranasal administration of LL-Lep on food intake and body weight gain in ob/ob mice. ob/ob mice were inoculated daily with PBS or strain LL or LL-lep (1 × 10⁹ CFU/inoculum). Food intake and body weight gain were measured every day for 19 days. (A) The total amount of food ingested by the mice present in the same cage and receiving the same treatment was measured every day. The figure represents the evolution of cumulative food intake per mouse during the period of treatment. (B) Mean food intake per animal and per day. *, differences are statistically significant (P < 0.05); NS, difference is not significant. (C) Evolution of body weight gain during treatment. Differences are statistically significant (P < 0.05) between LL-lep- and LL-treated mice (*), between LL-lep- and PBS-treated mice (#), and between LL- and PBS-treated mice ($).