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.

Nuclear RNA-binding protein p54(nrb) and its murine homolog NonO are known to be involved in a variety of nuclear processes including transcription and RNA processing. Melanoma inhibitory activity (MIA) has been shown to play an essential role in the progression of malignant melanoma and to influence melanoma-associated molecules and pathways in the early tumor formation steps. Interestingly, recent studies suggest that MIA is a regulator of p54(nrb). Here, we show that p54(nrb) is strongly expressed and localized in the nucleus of both melanoma cell lines and melanoma tissue samples compared with normal human melanocytes or normal skin, respectively. Furthermore, all tested melanoma cell lines revealed strong p54(nrb) promoter activity. Treatment with MIA-specific small interfering RNAs showed an influence of MIA on p54(nrb) expression on both messenger RNA (mRNA) and protein level. Knockdown of p54(nrb) protein in melanoma cell lines led to reduced proliferation rates and to a strong decrease in their migratory potential. In addition, attachment to laminin and poly-l-lysine was significantly increased. We could identify Connexin-43 (Cx-43) as a downstream target molecule of p54(nrb) as knockdown of p54(nrb) resulted in enhanced Cx-43 mRNA and protein levels. As a confirmation of these findings, melanoma cell lines showed very low Cx-43 expression levels compared with melanocytes. Our results demonstrate that p54(nrb) is highly expressed in malignant melanoma and, as a MIA target molecule, it seems to be involved in the development and progression of malignant melanoma.

Leishmania pathogenesis is primarily studied using the disease-inducing promastigote stage of Leishmania major. Despite many efforts, all attempts so far have failed to culture the disease-relevant multiplying amastigote stage of L. major. Here, we established a stably growing axenic L. major amastigote culture system that was characterized genetically, morphologically, and by stage-specific DsRed protein expression. We found parasite stage-specific disease development in resistant C57BL/6 mice. Human neutrophils, as first host cells for promastigotes, do not take up amastigotes. In human macrophages, we observed an amastigote-specific complement receptor 3-mediated, endocytotic entry mechanism, whereas promastigotes are taken up by complement receptor 1-mediated phagocytosis. Promastigote infection of macrophages induced the inflammatory mediators TNF, CCL3, and CCL4, whereas amastigote infection was silent and resulted in significantly increased parasite numbers: from 7.1 ± 1.4 (after 3 h) to 20.1 ± 7.9 parasites/cell (after 96 h). Our study identifies Leishmania stage-specific disease development, host cell preference, entry mechanism, and immune evasion. Since the amastigote stage is the disease-propagating form found in the infected mammalian host, the newly developed L. major axenic cultures will serve as an important tool in better understanding the amastigote-driven immune response in leishmaniasis.

Myeloid-derived suppressor cells (MDSCsIn conclusion, we demonstrate that (i) genetic background defines MDSC differentiation; and (ii) Leishmania major parasites are able to modulate the suppressive effect of MDSCs in a strain-dependent manner.Keywords: Arginase-1 · Bone marrow · Inducible nitric oxide synthase · Leishmania major · Myeloid-derived suppressor cellsAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionMyeloid cells positive for the integrin α M chain CD11b are known to be important for host defense against a wide range Correspondence: Prof. Uwe Ritter e-mail: Uwe.Ritter@klinik.uni-regensburg.de of pathogens [1,2]. Paradoxically, CD11b + myeloid cells are also able to suppress ongoing antitumor immune responses [3]. These myeloid-derived suppressor cells (MDSCs) represent a heterogeneous cell population that can be further divided by the expression of lymphocyte antigen 6, locus C (Ly6C) and G (Ly6G Eur. J. Immunol. 2014. 44: 3295-3306 (ii) monocytic suppressor cells (Mo-MDSCs, CD11b + /Ly6C high / Ly6G − ) [3].In this context, it needs to be mentioned that other myeloid cells such as DCs, macrophages, and neutrophils can also express CD11b, Ly6C, and Ly6G depending on their stage of maturation [4]. Moreover, it has been described that blood vessel precursors also share markers used for MDSC identification and depletion [5]. Thus, a depletion of MDSCs with Gr-1 or Ly6C antibodies might be critical because it can also affect other subsets such as blood vessel progenitors and APCs [4,6]. As a result, the observed tumor regression might be the consequence of a reduced vasculogenesis rather than a pronounced T-cell response that is initiated in the absence of MDSCs [7,8].Due to the fact that different mouse strains were used to characterize the impact of MDSCs in cancer or infectious diseases, we addressed the question of whether MDSC differentiation and functionality is comparable between C57BL/6 and BALB/c mice. Those mouse strains were selected because they are known to develop different T-cell responses in experimental leishmaniasis [9]. Healing and elimination of parasites correlates with the ability to develop a T-cell response, which is characterized by an early IFN-γ production by Th1 cells and the expression of iNOS by activated macrophages [10]. Susceptibility of BALB/c mice is associated with diminished IFN-γ, but increased IL-4 production [10]. However, both genotypes show a massive infiltration of myeloid cells in the dermal compartment after infection with L. major [11]. Particularly, neutrophils and macrophages are detectable within hours at the site of infection and dominate the cellular infiltrate [12,13]. These phagocytes are not only involved in the elimination of parasites but are also pivotal for early replication of the obligatory intracellular parasite.Unfortunately, inflammatory macrophages and neutrophils share surface markers such as CD11b, Ly6G, and Ly6C that also define MDSCs [4]. In addition, macroph...

cBacterial infection with group B Streptococcus (GBS) represents a prominent threat to neonates and fetuses in the Western world, causing severe organ damage and even death. To improve current therapeutic strategies and to investigate new approaches, an appropriate in vivo model to study the immune response of a human immune system is needed. Therefore, we introduced humanized mice as a new model for GBS-induced sepsis. Humanized mice feature deficiencies similar to those found in neonates, such as lower immunoglobulin levels and myeloid cell dysfunction. Due to the husbandry in specific-pathogen-free (SPF) facilities, the human immune cells in these mice also exhibit a naive phenotype which mimics the conditions in fetuses/ neonates. Following infection, cytokine release and leukocyte trafficking from the bone marrow to the lymphoid organ (spleen) and into the peritoneum (site of infection) as well as bacterial spreading and clearance were traceable in the humanized mice. Furthermore, we investigated the effects of betamethasone and indomethacin treatment using this novel sepsis model. Although both drugs are commonly used in perinatal care, little is known about their effects on the neonatal immune system. Treatment of infected humanized mice not only induced the reduction of human leukocytes in the spleen but also increased the bacterial load in all analyzed organs, including the brain, which did not show infiltration of live GBS in untreated controls. These studies demonstrate the utility of the humanized mice as a new model to study an immature human immune response during bacterial infection and allow the investigation of side effects induced by various treatments.