Macrophages Demonstrate Guanylate-Binding Protein-Dependent and Bacterial Strain-Dependent Responses to Francisella tularensis

Francisella tularensis is a facultative intracellular bacterium and the etiological agent of tularemia, a zoonotic disease. Infection of monocytic cells by F. tularensis can be controlled after activation with IFN-γ; however, the molecular mechanisms whereby the control is executed are incompletely understood. Recently, a key role has been attributed to the Guanylate-binding proteins (GBPs), interferon-inducible proteins involved in the cell-specific immunity against various intracellular pathogens. Here, we assessed the responses of bone marrow-derived murine macrophages (BMDM) and GBP-deficient BMDM to F. tularensis strains of variable virulence; the highly virulent SCHU S4 strain, the human live vaccine strain (LVS), or the widely used surrogate for F. tularensis, the low virulent F. novicida. Each of the strains multiplied rapidly in BMDM, but after addition of IFN-γ, significant GBP-dependent control of infection was observed for the LVS and F. novicida strains, whereas there was no control of the SCHU S4 infection. However, no differences in GBP transcription or translation were observed in the infected cell cultures. During co-infection with F. novicida and SCHU S4, significant control of both strains was observed. Patterns of 18 cytokines were very distinct between infected cell cultures and high levels were observed for almost all cytokines in F. novicida-infected cultures and very low levels in SCHU S4-infected cultures, whereas levels in co-infected cultures for a majority of cytokines showed intermediate levels, or levels similar to those of F. novicida-infected cultures. We conclude that the control of BMDM infection with F. tularensis LVS or F. novicida is GBP-dependent, whereas SCHU S4 was only controlled during co-infection. Since expression of GBP was similar regardless of infecting agent, the findings imply that SCHU S4 has an inherent ability to evade the GBP-dependent anti-bacterial mechanisms.

GDF15 Supports the Inflammatory Response of PdL Fibroblasts Stimulated by P. gingivalis LPS and Concurrent Compression

Periodontitis is characterized by bacterially induced inflammatory destruction of periodontal tissue. This also affects fibroblasts of the human periodontal ligaments (HPdLF), which play a coordinating role in force-induced tissue and alveolar bone remodeling. Excessive inflammation in the oral tissues has been observed with simultaneous stimulation by pathogens and mechanical forces. Recently, elevated levels of growth differentiation factor 15 (GDF15), an immuno-modulatory member of the transforming growth factor (TGFB) superfamily, were detected under periodontitis-like conditions and in force-stressed PdL cells. In view of the pleiotropic effects of GDF15 in various tissues, this study aims to investigate the role of GDF15 in P. gingivalis-related inflammation of HPdLF and its effect on the excessive inflammatory response to concurrent compressive stress. To this end, the expression and secretion of cytokines (IL6, IL8, COX2/PGE2, TNFα) and the activation of THP1 monocytic cells were analyzed in GDF15 siRNA-treated HPdLF stimulated with P. gingivalis lipopolysaccharides alone and in combination with compressive force. GDF15 knockdown significantly reduced cytokine levels and THP1 activation in LPS-stimulated HPdLF, which was less pronounced with additional compressive stress. Overall, our data suggest a pro-inflammatory role for GDF15 in periodontal disease and demonstrate that GDF15 partially modulates the force-induced excessive inflammatory response of PdLF under these conditions.

Platelet Endothelial Cell Adhesion Molecule 1 (CD31) Is Essential for Clostridium perfringens Beta-Toxin Mediated Cytotoxicity in Human Endothelial and Monocytic Cells

Beta toxin (CPB) is a small hemolysin beta pore-forming toxin (β-PFT) produced by Clostridium perfringens type C. It plays a central role in the pathogenesis of necro-hemorrhagic enteritis in young animals and humans via targeting intestinal endothelial cells. We recently identified the membrane protein CD31 (PECAM-1) as the receptor for CPB on mouse endothelial cells. We now assess the role of CD31 in CPB cytotoxicity against human endothelial and monocytic cells using a CRISPR/Cas9 gene knockout and an antibody blocking approach. CD31 knockout human endothelial and monocytic cells were resistant to CPB and CPB oligomers only formed in CD31-expressing cells. CD31 knockout endothelial and monocytic cells could be selectively enriched out of a polyclonal cell population by exposing them to CPB. Moreover, antibody mediated blocking of the extracellular Ig6 domain of CD31 abolished CPB cytotoxicity and oligomer formation in endothelial and monocytic cells. In conclusion, this study confirms the role of CD31 as a receptor of CPB on human endothelial and monocytic cells. Specific interaction with the CD31 molecule can thus explain the cell type specificity of CPB observed in vitro and corresponds to in vivo observations in naturally diseased animals.

Antioxidant and anti-inflammatory effects in lipopolysaccharide-induced THP-1 cells of coumarins from the bark of Hesperethusa crenulata R.

All parts of Thanakha (Hesperethusa crenulata R.) have been used as traditional skin care herbal material in Myanmar. In this study, coumarins from H. crenulata R. bark were isolated through solvent extraction, systematic solvent fractionation, and repeated column chromatography. Spectroscopic analyses using ESI–MS, 1D NMR (1H and 13C), 2D NMR (gHSQC and gHMBC), specific rotation, circular dichroism, and IR spectrometry revealed three coumarins 2R-7-hydroxy-8-(2,3-dihydroxy-3-methylbutyl)-coumarin (compound 1), peucedanol (compound 2), and methylpeucedanol (compound 3), which were first isolated from Thanakha tree. Antioxidant capacities of three coumarins decreased as follows: compound 2 > compound 3 > compound 1. Treatments of lipopolysaccharide-induced THP-1 human monocytic cells with compounds 2 and 3 at 378.8 μM and 359.7 μM inhibited tumor necrosis factor-α production by approximately 32.7% and 13.3%, respectively, compared with the negative control. In summary, these results suggest that Thanakha bark extracts can be used as a potent antioxidant and anti-inflammatory source for cosmetic ingredients.

Polystyrene microplastic particles induce endothelial activation

Due to its increasing production, durability and multiple applications, plastic is a material we encounter every day. Small plastic particles from the μm to the mm range are classified as microplastics and produced for cosmetic and medical products, but are also a result of natural erosion and decomposition of macroplastics. Although being omnipresent in our environment and already detected in various organisms, less is known about the effects of microplastics on humans in general, or on vascular biology in particular. Here we investigated the effects of carboxylated polystyrene microplastic particles (PS, 1 μm) on murine endothelial and immune cells, which are both crucially involved in vascular inflammation, using in vitro and in vivo approaches. In vitro, PS induced adhesion molecule expression in endothelial cells with subsequent adhesion of leukocytes both under static and flow conditions. In monocytic cells, PS enhanced pro-inflammatory cytokine expression and release. Accordingly, administering mice with PS led to enhanced aortic expression of cytokines and adhesion molecules. Furthermore, we identified neutrophils as the PS-clearing blood leukocyte population. The findings from this study for the first time indicate polystyrene microplastic as a new environmental risk factor for endothelial inflammation.

HIVEP1 Is a Negative Regulator of NF-κB That Inhibits Systemic Inflammation in Sepsis

Our previous work identified human immunodeficiency virus type I enhancer binding protein 1 (HIVEP1) as a putative driver of LPS-induced NF-κB signaling in humans in vivo. While HIVEP1 is known to interact with NF-ĸB binding DNA motifs, its function in mammalian cells is unknown. We report increased HIVEP1 mRNA expression in monocytes from patients with sepsis and monocytes stimulated by Toll-like receptor agonists and bacteria. In complementary overexpression and gene deletion experiments HIVEP1 was shown to inhibit NF-ĸB activity and induction of NF-ĸB responsive genes. RNA sequencing demonstrated profound transcriptomic changes in HIVEP1 deficient monocytic cells and transcription factor binding site analysis showed enrichment for κB site regions. HIVEP1 bound to the promoter regions of NF-ĸB responsive genes. Inhibition of cytokine production by HIVEP1 was confirmed in LPS-stimulated murine Hivep1-/- macrophages and HIVEP1 knockdown zebrafish exposed to the common sepsis pathogen Streptococcus pneumoniae. These results identify HIVEP1 as a negative regulator of NF-κB in monocytes/macrophages that inhibits proinflammatory reactions in response to bacterial agonists in vitro and in vivo.

Flow Cytometric Detection of the Ki-67 Proliferation Index and the Bcl-2 Anti-Apoptotic Index in Myelodysplastic Syndromes and Acute Myeloid Leukemia, and Their Clinical Implications

Introduction: Standardization of the detection and quantification of leukocyte differentiation markers by the EuroFlow Consortium has led to a major step forward in the integration of flow cytometry in classification of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). To further advance the integration and objectification of flow cytometry for characterization of these malignancies, more dynamic parameters assessing cell behavioral characteristics could prove useful, such as proliferative and (anti-)apoptotic markers. Proliferation and (anti-)apoptosis are processes that are tightly related to the pathogenesis, progression and chemo-/immunotherapy response of cancers. As a result, proliferation and (anti-)apoptotic markers have proven their value as objective parameters in the field of histopathology for diagnostic and prognostic applications in solid tumors and lymphoma. Although use of proliferative and (anti-)apoptotic markers as objective parameters in the diagnostic process of MDS and AML was studied in the past decades, this did not result in the incorporation of these biomarkers in their clinical diagnosis. The recent developments in flow cytometric analyses now allow the quantification of proliferative and (anti-)apoptotic fractions at the level of individual maturing bone marrow cells. Therefore, we aim to determine the Ki-67 proliferation indices and Bcl-2 anti-apoptotic indices in maturing bone marrow cells in order to assess whether these parameters could have future clinical implications for the diagnostic work-up of MDS and AML. Methods: Fifty bone marrow aspirates from femoral heads of non-malignant cases, 20 aspirates of MDS patients and 20 aspirates of AML were included in this study. Ten-color flow cytometry in combination with a software-based maturation tool was used for analysis of the Ki-67 proliferative and Bcl-2 anti-apoptotic indices of blast cells and during the erythro-, myelo-, and monopoiesis. Results: Ki-67 proliferative indices of blast cells and immature erythroid, myeloid and monocytic cells were significantly lower in MDS patients compared to the non-malignant cases, while the Bcl-2 anti-apoptotic indices were significantly elevated in these cells. Furthermore, the Bcl-2 anti-apoptotic indices were also increased in mature erythroid, myeloid and monocytic cells of MDS patients. The decreased Ki-67 proliferative indices and increased Bcl-2 anti-apoptotic indices in blast cells and erythroid, myeloid and monocytic cells were even more prominently observed in AML patients. Conclusions: The lowered Ki-67 proliferative indices and elevated Bcl-2 anti-apoptotic indices in blast cells and immature progenitor cells led to a better understanding of the pathophysiology of MDS and AML, and explained the low chemotherapy response of these patients. Side-effects of such therapies can also be explained by the Ki-67 proliferation indices and Bcl-2 anti-apoptotic indices. Moreover, the increase of the Bcl-2 anti-apoptotic fraction is an important factor in the progression of MDS to AML. Future studies on the clinical applications of these parameters for MDS and AML are necessary and can include many applications, such as prediction of chemo-/immunotherapy response, diagnostic and prognostic applications. Disclosures Ramaekers: Nordic-MUbio: Current Employment.

Miconazole Suppresses 27-Hydroxycholesterol-induced Inflammation by Regulating Activation of Monocytic Cells to a Proinflammatory Phenotype

Miconazole is effective in treating inflammatory skin conditions and has well-established antifungal effects. To elucidate the underlying mechanisms mediating its additional beneficial effects, we assessed whether miconazole influences the inflammation induced by 27-hydroxycholesterol (27OHChol), an oxygenated cholesterol derivative with high proinflammatory activity, using THP-1 monocytic cells. Miconazole dose-dependently inhibited the expression of proinflammatory markers, including CCL2 and CCR5 ligands such as CCL3 and CCL4, and impaired the migration of monocytic cells and CCR5-positive T cells. In the presence of 27OHChol, miconazole decreased CD14 surface levels and considerably weakened the lipopolysaccharide response. Furthermore, miconazole blocked the release of soluble CD14 and impaired the transcription of the matrix metalloproteinase-9 gene and secretion of its active gene product. Additionally, it downregulated the expression of ORP3 and restored the endocytic function of THP-1 cells. Collectively, these findings indicate that miconazole regulates the 27OHChol-induced expression of proinflammatory molecules in monocytic cells, thereby suppressing inflammation in an oxysterol-rich milieu.