The highly conserved Notch signaling pathway essentially participates in immunity through regulation of developmental processes and immune cell activity. In the adaptive immune system, the impact of the Notch cascade in T and B differentiation is well studied. In contrast, the function, and regulation of Notch signaling in the myeloid lineage during infection is poorly understood. Here we show that TLR signaling, triggered through LPS stimulation or in vitro infection with various Gram-negative and -positive bacteria, stimulates Notch receptor ligand Delta-like 1 (DLL1) expression and Notch signaling in human blood-derived monocytes. TLR activation induces DLL1 indirectly, through stimulated cytokine expression and autocrine cytokine receptor-mediated signal transducer and activator of transcription 3 (STAT3). Furthermore, we reveal a positive feedback loop between Notch signaling and Janus kinase (JAK)/STAT3 pathway during in vitro infection that involves Notch-boosted IL-6. Inhibition of Notch signaling by γ-secretase inhibitor DAPT impairs TLR4-stimulated accumulation of NF-κB subunits p65 in the nucleus and subsequently reduces LPS- and infection-mediated IL-6 production. The reduced IL-6 release correlates with a diminished STAT3 phosphorylation and reduced expression of STAT3-dependent target gene programmed death-ligand 1 (PD-L1). Corroborating recombinant soluble DLL1 and Notch activator oxaliplatin stimulate STAT3 phosphorylation and expression of immune-suppressive PD-L1. Therefore we propose a bidirectional interaction between Notch signaling and STAT3 that stabilizes activation of the transcription factor and supports STAT3-dependent remodeling of myeloid cells toward an immuno-suppressive phenotype. In summary, the study provides new insights into the complex network of Notch regulation in myeloid cells during in vitro infection. Moreover, it points to a participation of Notch in stabilizing TLR-mediated STAT3 activation and STAT3-mediated modulation of myeloid functional phenotype through induction of immune-suppressive PD-L1.

Pasteurella multocida toxin (PMT) is a protein toxin found in toxigenic strains of Pasteurella multocida. PMT is the causative agent for atrophic rhinitis in pigs, a disease characterized by loss of nasal turbinate bones due to an inhibition of osteoblast function and an increase in osteoclast activity and numbers. Apart from this, PMT acts as a strong mitogen, protects from apoptosis and has an impact on the differentiation and function of immune cells. Many signaling pathways have been elucidated, however, the effect of these signaling cascades as a means to subvert the host’s immune system are just beginning to unravel.

Antigen-presenting cells (APCs) regulate the balance of our immune response toward microbes. Whereas immunogenic APCs boost inflammation and activate lymphocytes, the highly plastic cells can switch into a tolerogenic/suppressive phenotype that dampens and resolves the response. Thereby the initially mediated inflammation seems to prime the switch of APCs while the strength of activation determines the grade of the suppressive phenotype. Recently, we showed that pathogen recognition receptor-mediated pro-inflammatory cytokines reprogram differentiating human blood monocytes in vitro toward an immunosuppressive phenotype through prolonged activation of signal transducer and activator of transcription (STAT) 3. The TLR7/8 ligand R848 (Resiquimod) triggers the high release of cytokines from GM-CSF/IL-4-treated monocytes. These cytokines subsequently upregulate T cell suppressive factors, such as programmed death-ligand 1 (PD-L1) and indolamin-2,3-dioxygenase (IDO) through cytokine receptor-mediated STAT3 activation. Here, we reveal an essential role for the microRNA (miR, miRNA) hsa-miR-99b/let-7e/miR-125a cluster in stabilizing the suppressive phenotype of R848-stimulated APCs on different levels. On the one hand, the miR cluster boosts R848-stimulated cytokine production through regulation of MAPkinase inhibitor Tribbles pseudokinase 2, thereby enhancing cytokine-stimulated activation of STAT3. One the other hand, the STAT3 inhibitor suppressor of cytokine signaling-1 is targeted by the miR cluster, stabilizing the STAT3-induced expression of immunosuppressive factors PD-L1 and IDO. Finally, hsa-miR-99b/let-7e/miR-125a cluster regulates generation of the suppressive tryptophan (Trp) metabolite kynurenine by targeting the tryptophanyl-tRNA synthetase WARS, the direct competitor of IDO in terms of availability of Trp. In summary, our results reveal the hsa-miR-99b/let-7e/miR-125a cluster as an important player in the concerted combination of mechanisms that stabilizes STAT3 activity and thus regulate R848-stimulated suppressive APCs.

Background: 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-negativePasteurella multocidaproduce a toxin (PMT) that constitutively activates the heterotrimeric G proteins Gα_q, Gα₁₃and Gα_iindependently of G protein-coupled receptors through deamidation. PMT is known to induce signalling events involved in cell proliferation, cell survival and cytoskeleton rearrangement.Results: 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.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.Conclusions: 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.

Background: 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). Results: 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 RhoH 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) p21 ^Cip1 and p27 ^Kip1 in RhoH overexpressing BaF3 cells. Conclusions: 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 p21 ^Cip1 and p27 ^Kip1 . 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.

Pasteurella multocida causes various diseases in a broad range of wild and domestic animals. Toxigenic strains of the serotypes A and D produce an AB protein toxin named Pasteurella multocida Toxin (PMT). PMT constitutively activates the heterotrimeric G protein subunits Gαq, Gα13 and Gαi through deamidation of a glutamine residue, which results in cytoskeletal rearrangements, increased proliferation and survival of the host cell. In human monocytes, PMT alters the lipopolysaccharide (LPS)-induced activation towards a phenotype that suppresses T cell activation. Here we describe that the toxin also modulates CD4-positive T helper (Th) cells directly. PMT amplifies the expansion of Th cells through enhanced cell cycle progression and suppression of apoptosis and manipulates the differentiation of Th subclasses through activation of Signal Transducers and Activators of Transcription (STAT) family members and induction of subtype-specific master transcription factors. A large population of toxin-treated T cells is double-positive for RORγt and Foxp3, the transcription factors expressed by Treg and Th17 cells, respectively. This suggests that these cells could have the potential to turn into Th17 cells or suppressive Treg cells. However, in terms of function the PMT-differentiated cells behave as inflammatory Th17 cells that produce IL-17 and trigger T cell proliferation.