Adaptor SKAP-55 Binds p21ras Activating Exchange Factor RasGRP1 and Negatively Regulates the p21ras-ERK Pathway in T-Cells

While the adaptor SKAP-55 mediates LFA-1 adhesion on T-cells, it is not known whether the adaptor regulates other aspects of signaling. SKAP-55 could potentially act as a node to coordinate the modulation of adhesion with downstream signaling. In this regard, the GTPase p21ras and the extracellular signal-regulated kinase (ERK) pathway play central roles in T-cell function. In this study, we report that SKAP-55 has opposing effects on adhesion and the activation of the p21ras -ERK pathway in T-cells. SKAP-55 deficient primary T-cells showed a defect in LFA-1 adhesion concurrent with the hyper-activation of the ERK pathway relative to wild-type cells. RNAi knock down (KD) of SKAP-55 in T-cell lines also showed an increase in p21ras activation, while over-expression of SKAP-55 inhibited activation of ERK and its transcriptional target ELK. Three observations implicated the p21ras activating exchange factor RasGRP1 in the process. Firstly, SKAP-55 bound to RasGRP1 via its C-terminus, while secondly, the loss of binding abrogated SKAP-55 inhibition of ERK and ELK activation. Thirdly, SKAP-55−/− primary T-cells showed an increased presence of RasGRP1 in the trans-Golgi network (TGN) following TCR activation, the site where p21ras becomes activated. Our findings indicate that SKAP-55 has a dual role in regulating p21ras-ERK pathway via RasGRP1, as a possible mechanism to restrict activation during T-cell adhesion

HPK1 Associates with SKAP-HOM to Negatively Regulate Rap1-Mediated B-Lymphocyte Adhesion

BACKGROUND: Hematopoietic progenitor kinase 1 (HPK1) is a Ste20-related serine/threonine kinase activated by a range of environmental stimuli including genotoxic stress, growth factors, inflammatory cytokines and antigen receptor triggering. Being inducibly recruited to membrane-proximal signalling scaffolds to regulate NFAT, AP-1 and NFkappaB-mediated gene transcription in T-cells, the function of HPK1 in B-cells to date remains rather ill-defined. METHODOLOGY/PRINCIPAL FINDINGS: By using two loss of function models, we show that HPK1 displays a novel function in regulating B-cell integrin activity. Wehi 231 lymphoma cells lacking HPK1 after shRNA mediated knockdown exhibit increased basic activation levels of Ras-related protein 1 (Rap1), accompanied by a severe lymphocyte function-associated antigen-1 (LFA-1) dependent homotypic aggregation and increased adhesion to intercellular adhesion molecule 1 (ICAM-1). The observed phenotype of enhanced integrin activity is caused downstream of Src, by a signalling module independent of PI3K and PLC, involving HPK1, SKAP55 homologue (SKAP-HOM) and Rap1-GTP-interacting adaptor molecule (RIAM). This alters actin dynamics and renders focal adhesion kinase (FAK) constitutively phosphorylated. Bone marrow and splenic B-cell development of HPK1(-/-) mice are largely unaffected, except age-related tendencies for increased splenic cellularity and BCR downregulation. In addition, naïve splenic knockout B-cells appear hyperresponsive to a range of stimuli applied ex vivo as recently demonstrated by others for T-cells. CONCLUSIONS/SIGNIFICANCE: We therefore conclude that HPK1 exhibits a dual function in B-cells by negatively regulating integrin activity and controlling cellular activation, which makes it an interesting candidate to study in pathological settings like autoimmunity and cancer

A 1-Year Prospective French Nationwide Study of Emergency Hospital Admissions in Children and Adults with Primary Immunodeficiency.

PURPOSE: Patients with primary immunodeficiency (PID) are at risk of serious complications. However, data on the incidence and causes of emergency hospital admissions are scarce. The primary objective of the present study was to describe emergency hospital admissions among patients with PID, with a view to identifying "at-risk" patient profiles. METHODS: We performed a prospective observational 12-month multicenter study in France via the CEREDIH network of regional PID reference centers from November 2010 to October 2011. All patients with PIDs requiring emergency hospital admission were included. RESULTS: A total of 200 admissions concerned 137 patients (73 adults and 64 children, 53% of whom had antibody deficiencies). Thirty admissions were reported for 16 hematopoietic stem cell transplantation recipients. When considering the 170 admissions of non-transplant patients, 149 (85%) were related to acute infections (respiratory tract infections and gastrointestinal tract infections in 72 (36%) and 34 (17%) of cases, respectively). Seventy-seven percent of the admissions occurred during winter or spring (December to May). The in-hospital mortality rate was 8.8% (12 patients); death was related to a severe infection in 11 cases (8%) and Epstein-Barr virus-induced lymphoma in 1 case. Patients with a central venous catheter (n = 19, 13.9%) were significantly more hospitalized for an infection (94.7%) than for a non-infectious reason (5.3%) (p = 0.04). CONCLUSION: Our data showed that the annual incidence of emergency hospital admission among patients with PID is 3.4%. The leading cause of emergency hospital admission was an acute infection, and having a central venous catheter was associated with a significantly greater risk of admission for an infectious episode

Damage-induced reactive oxygen species enable zebrafish tail regeneration by repositioning of Hedgehog expressing cells.

Many aquatic vertebrates have a remarkable ability to regenerate limbs and tails after amputation. Previous studies indicate that reactive oxygen species (ROS) signalling initiates regeneration, but the mechanism by which this takes place is poorly understood. Developmental signalling pathways have been shown to have proregenerative roles in many systems. However, whether these are playing roles that are specific to regeneration, or are simply recapitulating their developmental functions is unclear. Here, we analyse zebrafish larval tail regeneration and find evidence that ROS released upon wounding cause repositioning of notochord cells to the damage site. These cells secrete Hedgehog ligands that are required for regeneration. Hedgehog signalling is not required for normal tail development suggesting that it has a regeneration-specific role. Our results provide a model for how ROS initiate tail regeneration, and indicate that developmental signalling pathways can play regenerative functions that are not directly related to their developmental roles

Potential immunosuppressive effects of Escherichia coli O157:H7 experimental infection on the bovine host

Background: Enterohaemorrhagic Escherichia coli (EHEC), like E. coli O157:H7 are frequently detected in bovine faecal samples at slaughter. Cattle do not show clinical symptoms upon infection, but for humans the consequences after consuming contaminated beef can be severe. The immune response against EHEC in cattle cannot always clear the infection as persistent colonization and shedding in infected animals over a period of months often occurs. In previous infection trials, we observed a primary immune response after infection which was unable to protect cattle from reinfection. These results may reflect a suppression of certain immune pathways, making cattle more prone to persistent colonization after re-infection. To test this, RNA-Seq was used for transcriptome analysis of recto-anal junction tissue and ileal Peyer's patches in nine Holstein-Friesian calves in response to a primary and secondary Escherichia coli O157: H7 infection with the Shiga toxin (Stx) negative NCTC12900 strain. Non-infected calves served as controls. Results: In tissue of the recto-anal junction, only 15 genes were found to be significantly affected by a first infection compared to 1159 genes in the ileal Peyer's patches. Whereas, re-infection significantly changed the expression of 10 and 17 genes in the recto-anal junction tissue and the Peyer's patches, respectively. A significant downregulation of 69 immunostimulatory genes and a significant upregulation of seven immune suppressing genes was observed. Conclusions: Although the recto-anal junction is a major site of colonization, this area does not seem to be modulated upon infection to the same extent as ileal Peyer's patches as the changes in gene expression were remarkably higher in the ileal Peyer's patches than in the recto-anal junction during a primary but not a secondary infection. We can conclude that the main effect on the transcriptome was immunosuppression by E. coli O157: H7 (Stx(-)) due to an upregulation of immune suppressive effects (7/12 genes) or a downregulation of immunostimulatory effects (69/94 genes) in the ileal Peyer's patches. These data might indicate that a primary infection promotes a re-infection with EHEC by suppressing the immune function