Phagocytosis is a complex process that eliminates microbes and is performed by specialised cells such as macrophages. Toll-like receptor 4 (TLR4) is expressed on the surface of macrophages and recognizes Gram-negative bacteria. Moreover, TLR4 has been suggested to play a role in the phagocytosis of Gram-negative bacteria, but the mechanisms remain unclear. Here we have used primary human macrophages and engineered THP-1 monocytes to show that the TLR4 sorting adapter, TRAM, is instrumental for phagocytosis of Escherichia coli as well as Staphylococcus aureus. We find that TRAM forms a complex with Rab11 family interacting protein 2 (FIP2) that is recruited to the phagocytic cups of E. coli. This promotes activation of the actin-regulatory GTPases Rac1 and Cdc42. Our results show that FIP2 guided TRAM recruitment orchestrates actin remodelling and IRF3 activation, two events that are both required for phagocytosis of Gram-negative bacteria. The Gram-negative bacteria E. coli is the most common cause of severe human pathological conditions like sepsis. Sepsis is a clinical syndrome defined by pathological changes due to systemic inflammation, resulting in paralysis of adaptive T-cell immunity with IFN-β as a critical factor. TLR4 is a key sensing receptor of lipopolysaccharide on Gram-negative bacteria. Inflammatory signalling by TLR4 is initiated by the use of alternative pair of TIR-adapters, MAL-MyD88 or TRAM-TRIF. MAL-MyD88 signaling occurs mainly from the plasma membrane giving pro-inflammatory cytokines like TNF, while TRAM-TRIF signaling occurs from vacuoles like endosomes and phagosomes to give type I interferons like IFN-β. It has previously been shown that TLR4 can control phagocytosis and phagosomal maturation through MAL-MyD88 in mice, however, these data have been disputed and published before the role of TRAM was defined in the induction of IFN-β. A role for TRAM or TRIF in phagocytosis has not previously been reported. Here we describe a novel mechanism where TRAM and its binding partner Rab11-FIP2 control phagocytosis of E. coli and regulate IRF3 dependent production of IFN-β. The significance of these results is that we define Rab11-FIP2 as a potential target for modulation of TLR4-dependent signalling in different pathological states.

Corynebacterium diphtheriae is typically recognized as an extracellular pathogen. However, a number of studies revealed its ability to invade epithelial cells, indicating a more complex pathogen–host interaction. The molecular mechanisms controlling and facilitating internalization of C or. diphtheriae are poorly understood. In this study, we investigated the role of DIP0733 as virulence factor to elucidate how it contributes to the process of pathogen–host cell interaction. Based on in vitro experiments, it was suggested recently that the DIP0733 protein might be involved in adhesion, invasion of epithelial cells and induction of apoptosis. A corresponding Cor. diphtheriae mutant strain generated in this study was attenuated in its ability to colonize and kill the host in a Caenorhabditis elegans infection model system. Furthermore, the mutant showed an altered adhesion pattern and a drastically reduced ability to adhere and invade epithelial cells. Subsequent experiments showed an influence of DIP0733 on binding of Cor. diphtheriae to extracellular matrix proteins such as collagen and fibronectin. Furthermore, based on its fibrinogen-binding activity, DIP0733 may play a role in avoiding recognition of Cor. diphtheriae by the immune system. In summary, our findings support the idea that DIP0733 is a multi-functional virulence factor of Cor. diphtheriae.

Dual specificity phosphatases (DUSPs) have a well-known role as regulators of the immune response through the modulation of mitogen-activated protein kinases (MAPKs). Yet the precise interplay between the various members of the DUSP family with protein kinases is not well understood. Recent multi-omics studies characterizing the transcriptomes and proteomes of immune cells have provided snapshots of molecular mechanisms underlying innate immune response in unprecedented detail. In this study, we focus on deciphering the interplay between members of the DUSP family with protein kinases in immune cells using publicly available omics datasets. Our analysis resulted in the identification of potential DUSP-mediated hub proteins including MAPK7, MAPK8, AURKA, and IGF1R. Furthermore, we analyzed the association of DUSP expression with TLR4 signaling and identified VEGF, FGFR, and SCF-KIT pathway modules to be regulated by the activation of TLR4 signaling. Finally, we identified several important kinases including LRRK2, MAPK8, and cyclin-dependent kinases as potential DUSP-mediated hubs in TLR4 signaling. The findings from this study have the potential to aid in the understanding of DUSP signaling in the context of innate immunity. Further, this will promote the development of therapeutic modalities for disorders with aberrant DUSP signaling.

Innate immune signaling is essential to mount a fast and specific immune response to pathogens. Monocytes and macrophages are essential cells in the early response in their capacity as ubiquitous phagocytic cells. They phagocytose microorganisms or damaged cells and sense pathogen/damage-associated molecular patterns (PAMPs/DAMPs) through innate receptors such as Toll-like receptors (TLRs). We investigated a phenomenon where co-signaling from TLR2 and TLR8 in human primary monocytes provides a distinct immune activation profile compared to signaling from either TLR alone. We compare gene signatures induced by either stimulus alone or together and show that co-signaling results in downstream differences in regulation of signaling and gene transcription. We demonstrate that these differences result in altered cytokine profiles between single and multi-receptor signaling, and show how it can influence both T-cell and neutrophil responses. The end response is tailored to combat extracellular pathogens, possibly by modifying the regulation of IFNβ and IL12-family cytokines.

Dual specificity phosphatases (DUSPs) have a well-known role as regulators of the immune response through the modulation of mitogen activated protein kinases (MAPKs). Yet the precise interplay between the various members of the DUSP family with protein kinases is not well understood. Recent multi-omics studies characterizing the transcriptomes and proteomes of immune cells have provided snapshots of molecular mechanisms underlying innate immune response in unprecedented detail. In this study, we focused on deciphering the interplay between members of the DUSP family with protein kinases in immune cells using publicly available omics datasets. Our analysis resulted in the identification of potential DUSP-mediated hub proteins including MAPK7, MAPK8, AURKA, and IGF1R. Furthermore, we analyzed the association of DUSP expression with TLR4 signaling and identified VEGF, FGFR and SCF-KIT pathway modules to be regulated by the activation of TLR4 signaling. Finally, we identified several important kinases including LRRK2, MAPK8, and cyclin-dependent kinases as potential DUSP-mediated hubs in TLR4 signaling. The findings from this study has the potential to aid in the understanding of DUSP signaling in the context of innate immunity. Further, this will promote the development of therapeutic modalities for disorders with aberrant DUSP signaling.