Sterol Regulatory Element-Binding Protein 2 (SREBP2) Activation after Excess Triglyceride Storage Induces Chemerin in Hypertrophic Adipocytes

Chemerin is an adipokine whose systemic concentration and adipose tissue expression is increased in obesity. Chemerin is highly abundant in adipocytes, yet the molecular mechanisms mediating its further induction in obesity have not been clarified. Adipocyte hypertrophy contributes to dysregulated adipokine synthesis, and we hypothesized that excess loading with free fatty acids (FFA) stimulates chemerin synthesis. Chemerin was expressed in mature adipocytes, and differentiation of 3T3-L1 cells in the presence of FFA further increased its level. TNF and IL-6 were induced by FFA, but concentrations were too low to up-regulate chemerin. Sterol regulatory element-binding protein 2 (SREBP2) was activated in these cells, indicative for cholesterol shortage. Suppression of cholesterol synthesis by lovastatin led to activation of SREBP2 and increased chemerin, and supplementation with mevalonate reversed this effect. Knockdown of SREBP2 reduced basal and FFA-induced chemerin. EMSA confirmed binding of 3T3-L1 adipocyte nuclear proteins to a SREBP site in the chemerin promotor. SREBP2 was activated and chemerin was induced in adipose tissue of mice fed a high-fat diet, and higher systemic levels seem to be derived from adipocytes. Lipopolysaccharide-mediated elevation of chemerin was similarly effective as induction by FFA, indicating that both mechanisms are equally important. Chemokine-like receptor 1 was not altered by the incubations mentioned above, and higher expression in fat of mice fed a high-fat diet may reflect increased number of adipose tissue-resident macrophages in obesity. In conclusion, the current data show that adipocyte hypertrophy and chronic inflammation are equally important in inducing chemerin synthesis

Flow cytometric analyses of human immune cells invading the infected footpad.

<p>(A) Infected footpad (3×10⁶<i>L. major</i>) of a humanized mouse is shown. (B) The invasion of human B cells (CD19) and human T cells (CD3) to the site of infection were determined (n = 3). (C) T cells were further characterized for CD4⁺ T helper and CD8⁺ cytotoxic T cell subsets and (D) their naïve (CD27⁺CD45RA⁺) and memory (CD27⁺CD45RA⁻) phenotype. Significances between groups were analyzed using Student's t test (* = p<0.05; ** = p<0.01, n = 3). Error bars represent means ± SEM (standard error of the mean).</p

Human-derived immune cells harbour <i>Leishmania</i> parasites in humanized mice.

<p>42 days after high dose infection (3×10⁶<i>L. major</i>), liver spleen and the site of infection were analyzed for the host parasite interaction. Cryosections of liver (A), and spleen (B) biopsies were stained for human CD45 (green), and <i>Leishmania</i> antigen (L-Ag, red). <i>L. major</i>-infected humanized mice appear in orange (CD45⁺ and L-Ag⁺ colocalization; white arrows). (C) Cryosections of footpad samples (site of infection) were stained for human CD68 (red), and L. antigen (L-Ag, green). The insert C-I highlights infected human CD68⁺ macrophages. The insert C-II highlights infected CD68⁻ cells. The blue-fluorescent DAPI nucleic acid stain was added to all staining (blue; nuclei). Bars represent 50 µm.</p

Flow cytometric analyses of human immune cells in the spleen affected by <i>L. major</i> infection.

<p>(A) Analyses of naïve (CD27⁺CD45RA⁺) and memory (CD27⁺CD45RA⁻) CD8⁺ (top) and CD4⁺ T cells in the spleen with (3×10⁶<i>L. major</i>; n = 9) or without infection (n = 4). (B) CFSE-labelled spleen cells isolated from <i>L. major</i>-infected humanized mice (3×10⁶<i>L. major</i>; n = 3) and control humanized mice (HM; without in vivo <i>L. major</i> infection; n = 2) re-stimulated with <i>Leishmania</i> major antigen (SLA), PMA or ConA for 72 hours. Cell samples were then analyzed for CD4⁺ T cell (light grey), CD8⁺ T cells (grey) and B cells proliferation (dark grey). Percentage of human T cells of three individual humanized mice samples were determined in C. (D) Human cytokine release from spleen cells (isolated from <i>L. major</i>-infected (3×10⁶) humanized mice; n = 3) restimulated with soluble <i>L. major</i> antigen (SLA), PMA or without restimulation after 72 hours. pb = peripheral blood. Significances between groups were analyzed in one-way (C) or two-way-Anova (A) (A* = p<0,05; A** = p<0,01; A**** = p<0,0001). Additionally significances between groups are marked with * (p<0,05), ** (p<0,01), and *** (p<0,001) analyzed with Tukey's Multiple Comparison Test (1-way-Anova) and Bonferroni posttest (2-way-Anova).</p

Humanized mice infected with <i>Leishmania major</i> showed dose dependent footpad swelling, weight loss, survival, and parasite infestation.

<p>Humanized mice (A, C, E) and BALB/c (B, D, F) mice (at the age of 3 months) were infected with different numbers of <i>L. major</i> parasites and were weekly monitored for footpad swelling (A; n = 11 and B n = 9), weight changes (C; n = 11), survival (D; n = 15). The amount of <i>L. major</i> parasites was normalized to human β-actin in 3×10⁶<i>L. major</i> infected humanized mice (D; n = 7 in spleen, liver, lung and bone marrow; n = 6 in kidney; n = 4 in footpad; n = 3 in lymph node). Three 3×10⁶<i>L. major</i> infected BALB/c mice served as control and parasite load was normalized to mouse β-actin (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001741#pntd-0001741-g001" target="_blank">Figure 1F</a>). Error bars represent means ± SEM (standard error of the mean). Significances between groups were analyzed in one-way Anova (A** = p = 0,0013). Additionally significances between groups are marked with ** (p<0,01) analyzed with Tukey's Multiple Comparison Test. Survival curve was analyzed by Log-rank (Mantel-Cox) Test (* = p<0,05).</p

Leishmania major Infection in Humanized Mice Induces Systemic Infection and Provokes a Nonprotective Human Immune Response

Background Leishmania (L.) species are the causative agent of leishmaniasis. Due to the lack of efficient vaccine candidates, drug therapies are the only option to deal with cutaneous leishmaniasis. Unfortunately, chemotherapeutic interventions show high toxicity in addition to an increased risk of dissemination of drug-resistant parasites. An appropriate laboratory animal based model is still missing which allows testing of new drug strategies in the context of human immune cells in vivo. Methodology/Principal Findings Humanized mice were infected subcutaneously with stationary phase promastigote L. major into the footpad. The human immune response against the pathogen and the parasite host interactions were analyzed. In addition we proved the versatility of this new model to conduct drug research studies by the inclusion of orally given Miltefosine. We show that inflammatory human macrophages get infected with Leishmania parasites at the site of infection. Furthermore, a Leishmania-specific human-derived T cell response is initiated. However, the human immune system is not able to prevent systemic infection. Thus, we treated the mice with Miltefosine to reduce the parasitic load. Notably, this chemotherapy resulted in a reduction of the parasite load in distinct organs. Comparable to some Miltefosine treated patients, humanized mice developed severe side effects, which are not detectable in the classical murine model of experimental leishmaniasis. Conclusions/Significance This study describes for the first time L. major infection in humanized mice, characterizes the disease development, the induction of human adaptive and innate immune response including cytokine production and the efficiency of Miltefosine treatment in these animals. In summary, humanized mice might be beneficial for future preclinical chemotherapeutic studies in systemic (visceral) leishmaniasis allowing the investigation of human immune response, side effects of the drug due to cytokine production of activated humane immune cells and the efficiency of the treatment to eliminate also not replicating (“hiding”) parasites

data_sheet_1.pdf

<p>Resistant mouse strains mount a protective T cell-mediated immune response upon infection with Leishmania (L.) parasites. Healing correlates with a T helper (Th) cell-type 1 response characterized by a pronounced IFN-γ production, while susceptibility is associated with an IL-4-dependent Th2-type response. It has been shown that dermal dendritic cells are crucial for inducing protective Th1-mediated immunity. Additionally, there is growing evidence that C-type lectin receptor (CLR)-mediated signaling is involved in directing adaptive immunity against pathogens. However, little is known about the function of the CLR Dectin-1 in modulating Th1- or Th2-type immune responses by DC subsets in leishmaniasis. We characterized the expression of Dectin-1 on CD11c⁺ DCs in peripheral blood, at the site of infection, and skin-draining lymph nodes of L. major-infected C57BL/6 and BALB/c mice and in peripheral blood of patients suffering from cutaneous leishmaniasis (CL). Both mouse strains responded with an expansion of Dectin-1⁺ DCs within the analyzed tissues. In accordance with the experimental model, Dectin-1⁺ DCs expanded as well in the peripheral blood of CL patients. To study the role of Dectin-1⁺ DCs in adaptive immunity against L. major, we analyzed the T cell stimulating potential of bone marrow-derived dendritic cells (BMDCs) in the presence of the Dectin-1 agonist Curdlan. These experiments revealed that Curdlan induces the maturation of BMDCs and the expansion of Leishmania-specific CD4⁺ T cells. Based on these findings, we evaluated the impact of Curdlan/Dectin-1 interactions in experimental leishmaniasis and were able to demonstrate that the presence of Curdlan at the site of infection modulates the course of disease in BALB/c mice: wild-type BALB/c mice treated intradermally with Curdlan developed a protective immune response against L. major whereas Dectin-1^−/− BALB/c mice still developed the fatal course of disease after Curdlan treatment. Furthermore, the vaccination of BALB/c mice with a combination of soluble L. major antigens and Curdlan was able to provide a partial protection from severe leishmaniasis. These findings indicate that the ligation of Dectin-1 on DCs acts as an important checkpoint in adaptive immunity against L. major and should therefore be considered in future whole-organism vaccination strategies.</p