The haematopoietic GTPase RhoH modulates IL3 signalling through regulation of STAT activity and IL3 receptor expression

<p>Abstract</p> <p>Background</p> <p>RhoH is a constitutively active member of the family of Rho GTPases. Its expression is restricted to the haematopoietic lineage, where it serves as a positive regulator for T cell selection and mast cell function and as a negative regulator for growth-related functions in other lineages. Here, we examined the activation of signal transducer and activator of transcription (STAT) proteins in response to stimulation with interleukin 3 (IL3).</p> <p>Results</p> <p>Using the murine IL3-dependent cell line BaF3 we investigated the influence of RhoH protein expression levels on IL3-mediated cellular responses. RhoH overexpressing cells showed lower sensitivity to IL3 and decreased STAT5 activation. SiRNA-mediated repression of <it>RhoH </it>gene expression led to an increase in proliferation and STAT5 activity which correlated with an increased number of IL3 receptor α chain molecules, also known as CD123, expressed at the cell surface. Interestingly, these findings could be reproduced using human THP-1 cells as a model system for acute myeloid leukaemia, where low RhoH levels are known to be an unfavourable prognostic marker. Overexpression of RhoH on the other hand caused an induction of STAT1 activity and western blot analysis revealed that activated STAT1 is phosphorylated on Tyr701. STAT1 is known to induce apoptosis or cell cycle arrest and we detected an upregulation of cyclin-dependent kinase inhibitors (CDKI) <it>p21^Cip1</it>and <it>p27^Kip1</it>in RhoH overexpressing BaF3 cells.</p> <p>Conclusions</p> <p>We propose that RhoH functions as a negative regulator for IL3-induced signals through modulation of the JAK-STAT pathway. High levels of RhoH allow the IL3-dependent activation of STAT1 causing decreased proliferation through upregulation of <it>p21^Cip1</it>and <it>p27^Kip1</it>. Low RhoH levels on the other hand led to an upregulation of IL3-dependent cell growth, STAT5 activity and an increase of CD123 surface expression, linking RhoH to a CD123/STAT5 phenotype that has been described in AML patients.</p

Staphylococcus massiliensis isolated from human blood cultures, Germany, 2017-2020

Clinical and laboratory data on newly described staphylococcal species is rare, which hampers decision-making when such pathogens are detected in clinical specimens. Here, we describe Staphylococcus massiliensis detected in three patients at a university hospital in southwest Germany. We report the discrepancy of microbiological fndings between matrix-assisted laser desorption/ionization time-of-fight mass spectrometry, 16S-rRNA polymerase chain reaction, and whole-genome sequencing for all three isolates. Our fndings highlight the diagnostic pitfalls pertinent to novel and non-model organisms in daily microbiological practice, in whom the correct identifcation is dependent on database accuracy

Bone Morphogenetic Protein (BMP)-7 expression is decreased in human hypertensive nephrosclerosis

Background: Bone Morphogenetic Protein (BMP)-7 is protective in different animal models of acute and chronic kidney disease. Its role in human kidneys, and in particular hypertensive nephrosclerosis, has thus far not been described. Methods: BMP-7 mRNA was quantified using real-time PCR and localised by immunostaining in tissue samples from normal and nephrosclerotic human kidneys. The impact of angiotensin (AT)-II and the AT-II receptor antagonist telmisartan on BMP-7 mRNA levels and phosphorylated Smad 1/5/8 (pSmad 1/5/8) expression was quantified in proximal tubular cells (HK-2). Functional characteristics of BMP-7 were evaluated by testing its influence on TGF-b induced epithelial-to-mesenchymal transition (EMT), expression of TGF-b receptor type I (TGF-bRI) and phosphorylated Smad 2 (pSmad 2) as well as on TNF-a induced apoptosis of proximal tubular cells. Results: BMP-7 was predominantly found in the epithelia of the distal tubule and the collecting duct and was less abundant in proximal tubular cells. In sclerotic kidneys, BMP-7 was significantly decreased as demonstrated by real-time PCR and immunostaining. AT-II stimulation in HK-2 cells led to a significant decrease of BMP-7 and pSmad 1/5/8, which was partially ameliorated upon co-incubation with telmisartan. Only high concentrations of BMP-7 (100 ng/ml) were able to reverse TNF-a-induced apoptosis and TGF-b-induced EMT in human proximal tubule cells possibly due to a decreased expression of TGF-bRI. In addition, BMP-7 was able to reverse TGF-b-induced phosphorylation of Smad 2. Conclusions: The findings suggest a protective role for BMP-7 by counteracting the TGF-b and TNF-a-induced negative effects. The reduced expression of BMP-7 in patients with hypertensive nephrosclerosis may imply loss of protection and regenerative potential necessary to counter the disease

Pasteurella multocida Toxin-Stimulated Osteoclast Differentiation Is B Cell Dependent ▿

Pasteurella multocida is a Gram-negative bacillus that infects a number of wild and domestic animals, causing respiratory diseases. Toxigenic Pasteurella multocida strains produce a protein toxin (PMT) that leads to atrophic rhinitis in swine due to enhanced osteoclastogenesis and the inhibition of osteoblast function. We show that PMT-induced osteoclastogenesis is promoted by an as-yet-uncharacterized B-cell population. The toxin, however, is not acting at the level of hematopoietic stem cells, since purified CD117+ cells from murine hematopoietic progenitor cells cultivated with PMT did not mature into osteoclasts. The early macrophages contained within this cell population (CD117+/CD11b+) did not further differentiate into osteoclasts but survived and were able to phagocytose. Within the CD117− population, however, we detected PMT-induced generation of a B220+/CD19+ and B220+/IgM+ B-cell population that was able to take up fluorescently labeled PMT. Using purified B-cell and macrophage populations, we show that these B cells are needed to efficiently generate osteoclasts from macrophages. Cells of the immune system are thought to affect osteoclast formation and function by secreting cytokines and growth factors. We show here that PMT-stimulated B cells produce elevated levels of the osteoclastogenic factors interleukin-1β (IL-1β), IL-6, tumor necrosis factor alpha, and receptor activator of nuclear factor receptor ligand (RANKL) compared to B cells generated through incubation with IL-7. These results suggest that the osteoclastic properties characteristic for PMT may result from a cross talk between bone cells and lymphoid cells and that B cells might be an important target of Pasteurella multocida

IL-1β As Mediator of Resolution That Reprograms Human Peripheral Monocytes toward a Suppressive Phenotype

During infection pathogen-associated molecular patterns activate immune cells to initiate a cascade of reactions leading to inflammation and the activation of the adaptive immune response culminating in the elimination of foreign pathogens. However, shortly after activation of the host defense machinery, a return to homeostasis is preferred to prevent inflammation-induced tissue damage. This switch from the initial immunogenic to the subsequent tolerogenic phase after clearance of the infection can be mediated through highly plastic peripheral monocytes. Our studies reveal that an early encounter with toll-like receptor 7/8-ligand R848 mediates a strong pro-inflammatory monocytic phenotype that primes its own reprogramming toward an immunosuppressive one. Previously, we showed that these R848-treated antigen-presenting cells (APCs) fail to activate allogeneic T cells and induce regulatory T cells (Tregs) through signal transducer and activator of transcription 3 (STAT3)-dependent PD-L1. Here, we further demonstrate that R848-treated APCs suppress CD3/CD28-mediated and dendritic cell-mediated T cell activation and that adenosine and indoleamine 2,3-dioxygenase/kynurenin pathways are involved in tolerance induction. Reprogramming of monocytes after R848 stimulation requires the pro-inflammatory cytokine IL-1β and a boosted IL-6 release. The subsequent autocrine prolonged activation of STAT3 induces direct upregulation of tolerogenic factors which finally downregulate proliferation of activated T cells and mediate Tregs. Thereby our study suggests that inflammatory cytokines, such as IL-1β and IL-6, should be considered as mediators of resolution of inflammation

Regulation of Toll-like receptor 4-mediated immune responses through <it>Pasteurella multocida</it> toxin-induced G protein signalling

<p>Abstract</p> <p>Background</p> <p>Lipopolysaccharide (LPS)-triggered Toll-like receptor (TLR) 4-signalling belongs to the key innate defence mechanisms upon infection with Gram-negative bacteria and triggers the subsequent activation of adaptive immunity. There is an active crosstalk between TLR4-mediated and other signalling cascades to secure an effective immune response, but also to prevent excessive inflammation. Many pathogens induce signalling cascades via secreted factors that interfere with TLR signalling to modify and presumably escape the host response. In this context heterotrimeric G proteins and their coupled receptors have been recognized as major cellular targets. Toxigenic strains of Gram-negative <it>Pasteurella multocida</it> produce a toxin (PMT) that constitutively activates the heterotrimeric G proteins Gα_q, Gα₁₃ and Gα_i independently of G protein-coupled receptors through deamidation. PMT is known to induce signalling events involved in cell proliferation, cell survival and cytoskeleton rearrangement.</p> <p>Results</p> <p>Here we show that the activation of heterotrimeric G proteins through PMT suppresses LPS-stimulated IL-12p40 production and eventually impairs the T cell-activating ability of LPS-treated monocytes. This inhibition of TLR4-induced IL-12p40 expression is mediated by Gα_i-triggered signalling as well as by Gβγ-dependent activation of PI3kinase and JNK.</p> <p>Taken together we propose the following model: LPS stimulates TLR4-mediated activation of the NFĸB-pathway and thereby the production of TNF-α, IL-6 and IL-12p40. PMT inhibits the production of IL-12p40 by Gα_i-mediated inhibition of adenylate cyclase and cAMP accumulation and by Gβγ-mediated activation of PI3kinase and JNK activation.</p> <p>Conclusions</p> <p>On the basis of the experiments with PMT this study gives an example of a pathogen-induced interaction between G protein-mediated and TLR4-triggered signalling and illustrates how a bacterial toxin is able to interfere with the host’s immune response.</p