LPS-induced cytokine production in human monocytes and macrophages.

Lipopolysaccharide (LPS) from Gram-negative bacteria is one of the most potent innate immune-activating stimuli known. Here we review the current understanding of LPS effects on human monocyte and macrophage function. We provide an overview of LPS signal transduction with attention given to receptor cooperativity and species differences in LPS responses, as well as the role of tyrosine phosphorylation and lysine acetylation in signalling. We also review LPS-regulated transcription, with emphasis on chromatin remodeling and primary versus secondary transcriptional control mechanisms. Finally, we review the regulation and function of LPS-inducible cytokines produced by human monocytes and macrophages including TNFα, the IL-1 family, IL-6, IL-8, the IL-10 family, the IL-12 family, IL-15 and TGFß.

Transient receptor potential melastatin 2 is required for lipopolysaccharide-induced cytokine production in human monocytes.

Transient receptor potential melastatin 2 (TRPM2) is a Ca(2+)-permeable nonselective cation channel that is stimulated by oxidative stress and specifically activated by intracellular ADP-ribose. Because TRPM2 is highly expressed in immunocytes, a role of this channel in inflammation processes has been proposed. The aim of the current study was to determine the function of TRPM2 in LPS-induced cytokine production of human monocytes. Incubation of human primary monocytes with LPS resulted in an upregulation of TRPM2 mRNA, protein, and of ADP-ribose-induced membrane currents. By using short hairpin RNA to downregulate TRPM2 expression in THP-1 monocytes, we demonstrate that TRPM2 is required for the LPS-induced production of IL-6, IL-8, IL-10, and TNF-alpha. Application of LPS led to a time-dependent increase in intracellular Ca(2+) concentrations in THP-1 cells that was clearly reduced by downregulation of TRPM2. Omission of extracellular Ca(2+) strongly decreased TNF-alpha production in TRPM2-expressing cells. Thus, TRPM2-mediated Ca(2+) entry is a central mechanism for LPS-induced cytokine production in monocytic cells. The identification of TRPM2 as a major player in this LPS-dependent process makes it a promising tool in modulating monocyte functions.

CD14 Is Involved in the Interferon Response of Human Macrophages to Rubella Virus Infection.

Macrophages (MΦ) as specialized immune cells are involved in rubella virus (RuV) pathogenesis and enable the study of its interaction with the innate immune system. A similar replication kinetics of RuV in the two human MΦ types, the pro-inflammatory M1-like (or GM-MΦ) and anti-inflammatory M2-like (M-MΦ), was especially in M-MΦ accompanied by a reduction in the expression of the innate immune receptor CD14. Similar to RuV infection, exogenous interferon (IFN) ß induced a loss of glycolytic reserve in M-MΦ, but in contrast to RuV no noticeable influence on CD14 expression was detected. We next tested the contribution of CD14 to the generation of cytokines/chemokines during RuV infection of M-MΦ through the application of anti-CD14 blocking antibodies. Blockage of CD14 prior to RuV infection enhanced generation of virus progeny. In agreement with this observation, the expression of IFNs was significantly reduced in comparison to the isotype control. Additionally, the expression of TNF-α was slightly reduced, whereas the chemokine CXCL10 was not altered. In conclusion, the observed downmodulation of CD14 during RuV infection of M-MΦ appears to contribute to virus-host-adaptation through a reduction of the IFN response.

Aryl Hydrocarbon Receptor Activation by Benzo[a]pyrene Prevents Development of Septic Shock and Fatal Outcome in a Mouse Model of Systemic Salmonella enterica Infection.

This study focused on immunomodulatory effects of aryl hydrocarbon receptor (AhR) activation through benzo[a]pyrene (BaP) during systemic bacterial infection. Using a well-established mouse model of systemic Salmonella enterica (S.E.) infection, we studied the influence of BaP on the cellular and humoral immune response and the outcome of disease. BaP exposure significantly reduced mortality, which is mainly caused by septic shock. Surprisingly, the bacterial burden in BaP-exposed surviving mice was significantly higher compared to non-exposed mice. During the early phase of infection (days 1-3 post-infection (p.i.)), the transcription of proinflammatory factors (i.e., IL-12, IFN-γ, TNF-α, IL-1ß, IL-6, IL-18) was induced faster under BaP exposure. Moreover, BaP supported the activity of antigen-presenting cells (i.e., CD64 (FcγRI), MHC II, NO radicals, phagocytosis) at the site of infection. However, early in infection, the anti-inflammatory cytokines IL-10 and IL-22 were also locally and systemically upregulated in BaP-exposed S.E.-infected mice. BaP-exposure resulted in long-term persistence of salmonellae up to day 90 p.i., which was accompanied by significantly elevated S.E.-specific antibody responses (i.e., IgG1, IgG2c). In summary, these data suggest that BaP-induced AhR activation is capable of preventing a fatal outcome of systemic S.E. infection, but may result in long-term bacterial persistence, which, in turn, may support the development of chronic inflammation.

Selection and Validation of siRNAs Preventing Uptake and Replication of SARS-CoV-2.

In 2019, the novel highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak rapidly led to a global pandemic with more than 346 million confirmed cases worldwide, resulting in 5.5 million associated deaths (January 2022). Entry of all SARS-CoV-2 variants is mediated by the cellular angisin-converting enzyme 2 (ACE2). The virus abundantly replicates in the epithelia of the upper respiratory tract. Beyond vaccines for immunization, there is an imminent need for novel treatment options in COVID-19 patients. So far, only a few drugs have found their way into the clinics, often with modest success. Specific gene silencing based on small interfering RNA (siRNA) has emerged as a promising strategy for therapeutic intervention, preventing/limiting SARS-CoV-2 entry into host cells or interfering with viral replication. Here, we pursued both strategies. We designed and screened nine siRNAs (siA1-9) targeting the viral entry receptor ACE2. SiA1, (siRNA against exon1 of ACE2 mRNA) was most efficient, with up to 90% knockdown of the ACE2 mRNA and protein for at least six days. In vitro, siA1 application was found to protect Vero E6 and Huh-7 cells from infection with SARS-CoV-2 with an up to ∼92% reduction of the viral burden indicating that the treatment targets both the endosomal and the viral entry at the cytoplasmic membrane. Since the RNA-encoded genome makes SARS-CoV-2 vulnerable to RNA interference (RNAi), we designed and analysed eight siRNAs (siV1-8) directly targeting the Orf1a/b region of the SARS-CoV-2 RNA genome, encoding for non-structural proteins (nsp). As a significant hallmark of this study, we identified siV1 (siRNA against leader protein of SARS-CoV-2), which targets the nsp1-encoding sequence (a.k.a. 'host shutoff factor') as particularly efficient. SiV1 inhibited SARS-CoV-2 replication in Vero E6 or Huh-7 cells by more than 99% or 97%, respectively. It neither led to toxic effects nor induced type I or III interferon production. Of note, sequence analyses revealed the target sequence of siV1 to be highly conserved in SARS-CoV-2 variants. Thus, our results identify the direct targeting of the viral RNA genome (ORF1a/b) by siRNAs as highly efficient and introduce siV1 as a particularly promising drug candidate for therapeutic intervention.

Indol-3-Carbinol and Quercetin Ameliorate Chronic DSS-Induced Colitis in C57BL/6 Mice by AhR-Mediated Anti-Inflammatory Mechanisms.

Inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis, are multifactorial inflammatory disorders of the gastrointestinal tract, characterised by abdominal cramps, bloody diarrhoea, and anaemia. Standard therapies, including corticosteroids or biologicals, often induce severe side effects, or patients may develop resistance to those therapies. Thus, new therapeutic options for IBD are urgently needed. This study investigates the therapeutic efficacy and safety of two plant-derived ligands of the aryl hydrocarbon receptor (AhR), quercetin (Q), and indol-3-carbinol (I3C), using a translationally relevant mouse model of IBD. Q and I3C are administered by gavage to C57BL/6 wild-type or C57BL/6 Ahr-/- mice suffering from chronic colitis, induced by dextran sulphate sodium (DSS). The course of the disease, intestinal histopathological changes, and in-situ immunological phenotype are scored over 25 days. Our results show that both Q and I3C improved significantly clinical symptoms in moderate DSS colitis, which coincides with a significantly reduced histopathological score. Even in severe DSS colitis I3C, neither Q nor the therapy control 6-thioguanine (6-TG) can prevent a fatal outcome. Moreover, treatment with Q or I3C restored in part DSS-induced loss of epithelial integrity by induction of tight-junction proteins and reduced significantly gut inflammation, as demonstrated by colonoscopy, as well as by immunohistochemistry revealing lower numbers of neutrophils and macrophages. Moreover, the number of Th17 cells is significantly reduced, while the number of Treg cells is significantly increased by treatment with Q or I3C, as well as 6-TG. Q- or I3C-induced amelioration of colitis is not observed in Ahr-/- mice suggesting the requirement of AhR ligation and signalling. Based on the results of this study, plant-derived non-toxic AhR agonists can be considered promising therapeutics in IBD therapy in humans. However, they may differ in terms of efficacy; therefore, it is indispensable to study the dose-response relationship of each individual AhR agonist also with regard to potential adverse effects, since they may also exert AhR-independent effects.

The Impact of Rubella Virus Infection on a Secondary Inflammatory Response in Polarized Human Macrophages.

Macrophages (MΦ) are known to exhibit distinct responses to viral and bacterial infection, but how they react when exposed to the pathogens in succession is less well understood. Accordingly, we determined the effect of a rubella virus (RV)-induced infection followed by an LPS-induced challenge on cytokine production, signal transduction and metabolic pathways in human GM (M1-like)- and M (M2-like)-MΦ. We found that infection of both subsets with RV resulted in a low TNF-α and a high interferon (IFN, type I and type III) release whereby M-MΦ produced far more IFNs than GM-MΦ. Thus, TNF-α production in contrast to IFN production is not a dominant feature of RV infection in these cells. Upon addition of LPS to RV-infected MΦ compared to the addition of LPS to the uninfected cells the TNF-α response only slightly increased, whereas the IFN-response of both subtypes was greatly enhanced. The subset specific cytokine expression pattern remained unchanged under these assay conditions. The priming effect of RV was also observed when replacing RV by IFN-ß one putative priming stimulus induced by RV. Small amounts of IFN-ß were sufficient for phosphorylation of Stat1 and to induce IFN-production in response to LPS. Analysis of signal transduction pathways activated by successive exposure of MΦ to RV and LPS revealed an increased phosphorylation of NFκB (M-MΦ), but different to uninfected MΦ a reduced phosphorylation of ERK1/2 (both subtypes). Furthermore, metabolic pathways were affected; the LPS-induced increase in glycolysis was dampened in both subtypes after RV infection. In conclusion, we show that RV infection and exogenously added IFN-ß can prime MΦ to produce high amounts of IFNs in response to LPS and that changes in glycolysis and signal transduction are associated with the priming effect. These findings will help to understand to what extent MΦ defense to viral infection is modulated by a following exposure to a bacterial infection.

Comparison of Three CD3-Specific Separation Methods Leading to Labeled and Label-Free T Cells.

T cells are an essential part of the immune system. They determine the specificity of the immune response to foreign substances and, thus, help to protect the body from infections and cancer. Recently, T cells have gained much attention as promising tools in adoptive T cell transfer for cancer treatment. However, it is crucial not only for medical purposes but also for research to obtain T cells in large quantities, of high purity and functionality. To fulfill these criteria, efficient and robust isolation methods are needed. We used three different isolation methods to separate CD3-specific T cells from leukocyte concentrates (buffy coats) and Ficoll purified PBMCs. To catch the target cells, the Traceless Affinity Cell Selection (TACS®) method, based on immune affinity chromatography, uses CD-specific low affinity Fab-fragments; while the classical Magnetic Activated Cell Sorting (MACS®) method relies on magnetic beads coated with specific high affinity monoclonal antibodies. The REAlease® system also works with magnetic beads but, in contrast to MACS®, low-affinity antibody fragments are used. The target cells separated by TACS® and REAlease® are "label-free", while cells isolated by MACS® still carry the cell specific label. The time required to isolate T cells from buffy coat by TACS® and MACS® amounted to 90 min and 50 min, respectively, while it took 150 min to isolate T cells from PBMCs by TACS® and 110 min by REAlease®. All methods used are well suited to obtain T cells in large quantities of high viability (>92%) and purity (>98%). Only the median CD4:CD8 ratio of approximately 6.8 after REAlease® separation differed greatly from the physiological conditions. MACS® separation was found to induce proliferation and cytokine secretion. However, independent of the isolation methods used, stimulation of T cells by anti CD3/CD28 resulted in similar rates of proliferation and cytokine production, verifying the functional activity of the isolated cells.

The noncoding RNA LINC00152 conveys contradicting effects in different glioblastoma cells.

Glioblastoma multiforme (GBM) is an extremely aggressive brain tumor, characterized by its high genetic heterogeneity. In search of novel putative therapeutic RNA targets we investigated the role of the oncogenic long noncoding RNA LINC00152 (CYTOR, and STAiR18) in A172 glioblastoma cells. Here, we are the first to describe, that LINC00152 unexpectedly acts in a tumor suppressive manner in this cell line. SiRNA-based knockdown of LINC00152 enhanced malignant tumor behaviors including proliferation, cell cycle entry, migration, and invasion, contradicting previous studies using U87-MG and LN229 glioblastoma cells. Furthermore, LINC00152 knockdown had no influence on survival of A172 glioblastoma cells. In a genome wide transcription analysis of A172 and U87-MG glioblastoma cells, we identified 70 LINC00152 target genes involved in locomotion, cell migration, and motility in A172 cells, whereas in U87-MG cells only 40 target genes were detected. The LINC00152-regulated genes found in A172 differed from those identified in U87-MG glioblastoma cells, none of them being regulated in both cell lines. These findings underline the strong genetic heterogeneity of glioblastoma and point to a potential, yet unknown risk addressing LINC00152 lncRNA as a prospective therapeutic target in GBM.

Nicotinamide Inhibits Self-renewal and Induces Granulocyte Differentiation of Multipotent Progenitor Cells.

Nicotinamide (NAM) a form of vitamin B3, is an essential precursor of NAD. This dinucleotide (pyridine nucleotide) participates in the regulation of fundamental processes including transcription, cell cycle progression and DNA repair. Here we assessed the effect of NAM on myeloid differentiation of the IL-3 dependent, multipotent hematopoietic progenitor cell line FDCP-Mix. We found that NAM reduces the pSTAT5 signaling response, cell cycling and self-renewal potential. It initiates an atypical program of myeloid differentiation that results in the emergence of granulocytic cells in the absence of added myeloid differentiation factors. NAM did not affect the expression the of cell surface granulocyte marker GR1 but led to a strong downregulation of MHC-II molecules. Taken together our data show that NAM induces a differentiation program in hematopoietic progenitors prompting them to undergo differentiation along the granulocyte path without reaching the status of fully developed granulocytes. Graphical abstract.

3D Scaffold-Based Macrophage Fibroblast Coculture Model Reveals IL-10 Dependence of Wound Resolution Phase.

Persistent inflammation and impaired repair in dermal wound healing are frequently associated with cell-cell and cell-matrix miscommunication. A direct coculture model of primary human myofibroblasts (MyoFB) and M-CSF-differentiated macrophages (M-Mɸ) in fibrillar three-dimensional Collagen I (Coll I) matrices is developed to study intercellular interactions. The coculture experiments reveal the number of M-Mɸ regulated MyoFB dedifferentiation in a dose-dependent manner. The amount of MyoFB decreases in dependence of the number of cocultured M-Mɸ, even in the presence of MyoFB-inducing transforming growth factor ß1 (TGF-ß1 ). Gene expression analysis of matrix proteins (collagen I, collagen III, ED-A-fibronectin) confirms the results of an altered MyoFB phenotype. Additionally, M-Mɸ is shown to be the main source of secreted cytokine interleukin-10 (IL-10), which is suggested to affect MyoFB dedifferentiation. These findings indicate a paracrine impact of IL-10 secretion by M-Mɸ on the MyoFB differentiation status counteracting the TGF-ß1 -driven MyoFB activation. Hence, the in vitro coculture model simulates physiological situations during wound resolution and underlines the importance of paracrine IL-10 signals by M-Mɸ. In sum, the 3D Coll I-based matrices with a MyoFB-M-Mɸ coculture form a highly relevant biomimetic model of late stages of wound healing.

NAD metabolites interfere with proliferation and functional properties of THP-1 cells.

Over the past few years the NAD-related compounds nicotinamide (NAM), nicotinamide riboside (NR) and 1-methylnicotinamide (MNA) have been established as important molecules in signalling pathways that contribute to metabolic functions of many cells, including those of the immune system. Among immune cells, monocytes/macrophages, which are the major players of inflammatory processes, are especially susceptible to the anti-inflammatory action of NAM. Here we asked whether NAM and the two other compounds have the potential to regulate differentiation and LPS-induced biological answers of the monocytic cell line THP-1. We show that treatment of THP-1 cells with NAM, NR and MNA resulted in growth retardation accompanied by enrichment of cells in the G0/G1-phase independent of p21 and p53. NAM and NR caused an increase in intracellular NAD concentrations and SIRT1 and PARP1 mRNA expression was found to be enhanced. The compounds failed to up-regulate the expression of the cell surface differentiation markers CD38, CD11b and CD14. They modulated the reactive oxygen species production and primed the cells to respond less effectively to the LPS induced TNF-α production. Our data show that the NAD metabolites interfere with early events associated with differentiation of THP-1 cells along the monocytic path and that they affect LPS-induced biological responses of the cell line.

CD14 Counterregulates Lipopolysacharide-Induced Tumor Necrosis Factor-α Production in a Macrophage Subset.

In response to GM-CSF or M-CSF, macrophages (MΦ) can acquire pro- or anti-inflammatory properties, respectively. Given the importance of CD14 and Toll-like receptor (TLR) 4 in lipopolysaccharide (LPS)-induced signaling, we studied the effect of anti-CD14 antibody mediated CD14 blockade on LPS-induced cytokine production, signal transduction and on the expression levels of CD14 and TLR4 in GM-MΦ and M-MΦ. We found M-MΦ to express higher levels of both surface antigens and to produce more interferon (IFN)-ß and interleukin-10, but less tumor necrosis factor (TNF)-α than GM-MΦ. Blockage of CD14 at high LPS concentrations increased the production of proinflammatory cytokines and decreased that of IFN-ß in M-MΦ but not in GM-MΦ. We show that phosphorylation states of signaling molecules of the MyD88 (myeloid differentiation primary response 88), TRIF (TIR-domain-containing adapter-inducing IFN-ß) and MAPK (mitogen-activated protein kinase) pathways are not altered in any way that would account for the cytokine overshoot reaction. However, CD14 blockage in M-MΦ decreased TLR4 and CD14 expression levels, regardless of the presence of LPS, indicating that the loss of the surface molecules prevented LPS from initiating TRIF signaling. As TNF-α synthesis was even upregulated under these experimental conditions, we suggest that TRIF is normally involved in restricting LPS-induced TNF-α overproduction. Thus, surface CD14 plays a decisive role in the biological response by determining LPS-induced signaling.

The orthologue of the "acatalytic" mammalian ART4 in chicken is an arginine-specific mono-ADP-ribosyltransferase.

BACKGROUND: Human ART4, carrier of the GPI-(glycosyl-phosphatidylinositol) anchored Dombrock blood group antigens, is an apparently inactive member of the mammalian mono-ADP-ribosyltransferase (ART) family named after the enzymatic transfer of a single ADP-ribose moiety from NAD+ to arginine residues of extracellular target proteins. All known mammalian ART4 orthologues are predicted to lack ART activity because of one or more changes in essential active site residues that make up the R-S-EXE motif. So far, no other function has been detected. RESULTS: Here we report the identification and characterisation of ART4 in chicken, which to our knowledge is the first true non-mammalian orthologue of a mammalian ART family member. The chicken ART4 gene has the same physical structure as its mammalian counterparts (three coding exons separated by two introns in phase 0 and phase 1, respectively) and maps to a region of conserved linkage synteny on chromosome 1. Its mRNA encodes a 289 amino acid protein with predicted N-terminal signal peptide and C-terminal GPI-anchor sequences and 47% sequence identity to human ART4. However, in striking contrast to its mammalian orthologues, the chicken protein contains an intact R-S-EXE motif. Upon ectopic expression in C-33A cells, recombinant chicken ART4 localized at the cell surface as a GPI-anchored, highly glycosylated protein, which displayed arginine-specific ART activity (apparent Km of the recombinant protein for etheno-NAD+ 1.0 +/- 0.18 microM). CONCLUSION: The avian orthologue of the "acatalytic" mammalian ART4 is a mono-ADP-ribosyltransferase with enzymatic activity comparable to that of other, catalytically active and GPI-anchored members of the mammalian ART family.

Identification of two regulatory binding sites which confer myotube specific expression of the mono-ADP-ribosyltransferase ART1 gene.

BACKGROUND: Mono-ADP-ribosyltransferase (ART) 1 belongs to a family of mammalian ectoenzymes that catalyze the transfer of ADP-ribose from NAD+ to a target protein. ART1 is predominantly expressed in skeletal and cardiac muscle. It ADP-ribosylates alpha7-integrin which together with beta1-integrin forms a dimer and binds to laminin, a protein of the extracellular matrix involved in cell adhesion. This posttranslational modification leads to an increased laminin binding affinity. RESULTS: Using C2C12 and C3H-10T 1/2 cells as models of myogenesis, we found that ART1 expression was restricted to myotube formation. We identified a fragment spanning the gene 1.3 kb upstream of the transcriptional start site as the functional promoter of the ART1 gene. This region contains an E box and an A/T-rich element, two conserved binding sites for transcription factors found in the promoters of most skeletal muscle specific genes. Mutating the DNA consensus sequence of either the E box or the A/T-rich element resulted in a nearly complete loss of ART1 promoter inducibility, indicating a cooperative role of the transcription factors binding to those sites. Gel mobility shift analyses carried out with nuclear extracts from C2C12 and C3H-10T 1/2 cells revealed binding of myogenin to the E box and MEF-2 to the A/T-rich element, the binding being restricted to C2C12 and C3H-10T 1/2 myotubes. CONCLUSION: Here we describe the molecular mechanism underlying the regulation of the ART1 gene expression in skeletal muscle cells. The differentiation-dependent upregulation of ART1 mRNA is induced by the binding of myogenin to an E box and of MEF-2 to an A/T-rich element in the proximal promoter region of the ART1 gene. Thus the transcriptional regulation involves molecular mechanisms similar to those used to activate muscle-specific genes.

Aryl hydrocarbon receptor activation by benzo(a)pyrene inhibits proliferation of myeloid precursor cells and alters the differentiation state as well as the functional phenotype of murine bone marrow-derived macrophages.

Intensive research during the past decade has highlighted the impact of the regulatory function of the aryl hydrocarbon receptor (AhR) in immunity. In this study, we focused on the influence of AhR activation on the differentiation of murine bone marrow-derived myeloid precursor cells into mature macrophages. Our results show that the activation of AhR by subtoxic doses of the AhR ligand benzo(a)pyrene (BaP) impaired the proliferation of bone marrow cells (BMCs) whereas the proportion of resulting adherent cells was not affected. Flow cytometric analysis revealed that the number of mature bone marrow-derived macrophages (BMMs) was significantly decreased by AhR activation. However, expression of the murine macrophage marker F4/80, the major histocompatibility complex class II (MHC-II) and the Fcγ receptor I (FcγRI/CD64) were upregulated on BaP-exposed BMMs in an AhR-dependent manner. Analysis of cytokine secretion after BMM activation with heat-killed (hk) salmonellae showed that BaP exposure resulted in suppressed secretion of interleukin (IL)-1ß, IL-6 and the chemokine CXC motif ligand 1 (CXCL1). In contrast, the release of tumor necrosis factor (TNF)-α and IL-10 was increased following BaP exposure. In addition, the production of antimicrobial nitric oxide (NO) was increased AhR-dependently. Bacterial stimulation of BaP exposed BMMs also induced the expression of MHC-II and CD64, while the expression of F4/80 was dramatically decreased. In summary, this study demonstrates for the first time that sustained exposure over 6 days of bone marrow-derived myeloid precursors to subtoxic doses of BaP critically interferes with differentiation and activation of BMMs. We could convincingly show that AhR-induced gene regulation is crucial for homeostasis of pro- and anti-inflammatory cytokines during macrophage activation.

Molecular mechanism of LPS-induced TNF-α biosynthesis in polarized human macrophages.

In response to environmental stimuli such as granulocyte-macrophage or macrophage colony stimulating factor (GM-CSF/M-CSF), macrophages (MΦ) can acquire distinct functional phenotypes that control inflammatory processes on the one hand and contribute to a broad spectrum of pathologies on the other. Potential intervention strategies will require an understanding of the signalling processes that are associated with macrophage polarization. In the present study, we show that M-MΦ produce more IFN-ß and IL-10 and a lot less TNF-α than do GM-MΦ in response to LPS. To define the molecular mechanisms that underlie the biosynthesis of TNF-α we carried out a detailed investigation of the LPS-induced activation of the canonical and non-canonical myeloid differentiation primary response 88 (MyD88)-dependent signal transduction pathways as well as the TIR-domain-containing adapter-inducing interferon-ß (TRIF)-dependent pathway. Our results show that all three pathways are activated in both cell types and that the activation is more pronounced in M-MΦ. While IL-10 was found to interfere with TNF-α production in M-MΦ, we exclude a decisive role for IFN-ß in this respect. Furthermore, we demonstrate that TNF-α mRNA is markedly destabilized in M-MΦ and that expression of the mRNA destabilizing protein tristetraprolin is greatly enhanced in these cells. Collectively, our study suggests that differential effects of LPS on TNF-α mRNA turnover and on signal transduction pathways influence the amount of TNF-α finally produced by GM-MΦ and M-MΦ.

Benzo(a)pyrene attenuates the pattern-recognition-receptor induced proinflammatory phenotype of murine macrophages by inducing IL-10 expression in an aryl hydrocarbon receptor-dependent manner.

Polycyclic aromatic hydrocarbons such as benzo(a)pyrene (BaP) are environmental contaminants known to be immunosuppressive. Most effects of BaP towards immune cells are thought to be mediated through activation of the aryl hydrocarbon receptor (AhR). The AhR is a ligand-activated transcription factor, which plays a critical modulatory role in various cells during immune response. Macrophages are key players in innate immunity against intracellular bacteria and are discussed to be a target of AhR-mediated immune regulation. However, so far there is only incomplete knowledge about the effects of BaP on activated macrophages and whether these effects are AhR-dependent in each case. Using murine bone marrow-derived macrophages (BMMs) stimulated with heat-killed salmonellae as a source of different pathogen-associated molecular patterns (PAMPs) for stimulation of different pattern recognition receptors (PRRs) as an in-vitro model, we studied the immunomodulatory effects of low-dose BaP exposure. PRR-activated BMMs produced nitric oxide (NO) and a spectrum of proinflammatory cytokines, i.e. tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and IL-12 but also the anti-inflammatory cytokine IL-10. While BaP exposure suppressed the production of proinflammatory cytokines, the secretion of IL-10 was augmented. Moreover, BaP exposure increased the expression of major histocompatibility complex class II (MHC-II), CD14, Fcγ receptor I (FcγRI/CD64), or CD86, enhanced NO production and phagocytosis what may be beneficial for phagocytosis and killing of microbial pathogens. Of note, without PRR activation low-dose BaP exposure has little influence on the macrophage phenotype. BMMs from AhR-deficient (Ahr-/-) mice were widely refractory to BaP-induced modulation of cytokine production, surface marker expression, and functional properties in response to PAMPs stimulation, indicating that these effects are dependent on AhR. In summary, these data suggest that induction of AhR-mediated signalling pathways by BaP may attenuate the proinflammatory phenotype of PRR-activated BMMs, while activating IL-10-mediated anti-inflammatory properties but also enhancing uptake and killing of pathogens as well as antigen presentation. Together these features imply a favourable role of BaP exposure for macrophage functions in an ongoing immune response. However, the strong induction of IL-10 may lead to defective pathogen clearance and subsequently to chronic persistent infection. This concept suggests an inhibitory rather than a supporting influence of environmental BaP on immunity to infection or cancer and also emphasises the important regulatory role of AhR in immunity and inflammation.

A refined and translationally relevant model of chronic DSS colitis in BALB/c mice.

Inflammatory bowel diseases (IBD) are chronic relapsing disorders of the gastrointestinal tract. Several mouse models for IBD are available, but the acute dextran sulfate sodium (DSS)-induced colitis model is mostly used for preclinical studies. However, this model lacks chronicity and often leads to significant loss of mice. The aim of this study was to establish a refined and translationally relevant model of DSS chronic colitis in BALB/c mice. In the first part, we compared several standard therapeutic (ST) treatments for IBD in the acute DSS colitis model to identify the optimal treatment control for a DSS colitis model as compared to literature data. In the second part, we tested the two most effective ST treatments in a refined model of chronic DSS colitis. Cyclosporine A (CsA) and 6-thioguanine (6-TG) caused considerable reduction of clinical scores in acute DSS colitis. The clinical outcome was confirmed by the results for colon length and by histopathological evaluation. Moreover, CsA and 6-TG considerably reduced mRNA expression of several pro-inflammatory cytokines in spleen and colon. Both compounds also showed a substantial therapeutic effect in the refined model of chronic DSS colitis with regard to clinical scores and histopathology as well as the expression of inflammatory markers. The refined model of chronic DSS colitis reflects important features of IBD and is well suited to test potential IBD therapeutics.