WBP1L is a broadly expressed transmembrane adaptor protein involved in regulating hematopoietic stem cell function and T cell development. It interacts with NEDD4-family E3 ubiquitin ligases and regulates important chemokine receptor CXCR4. Using tandem affinity purification coupled with mass spectrometry, we identified novel WBP1L interactions with the IFNγ receptor and the Cullin-RING ubiquitin ligases CRL1β-TrCP1/2. We found that WBP1L interaction with the IFNγ receptor serves to downregulate proximal IFNγ receptor signaling in female macrophages, while the interaction with CRL1β-TrCP1/2 ubiquitin ligases regulates WBP1L protein levels. Disrupting this interaction, as well as inhibiting proteasome activity or neddylation, increased WBP1L protein levels, demonstrating that CRL1β-TrCP1/2 ubiquitin ligases regulate WBP1L protein abundance. These data provide important insights into the mechanisms controlling WBP1L function.

• Klíčová slova
• Cullin-RING ubiquitin ligases, Interferon gamma receptor, Membrane adaptor proteins, NEDD4-Family ubiquitin ligases, WBP1L,
• MeSH
• adaptorové proteiny signální transdukční * metabolismus   MeSH
• HEK293 buňky MeSH
• hematopoéza * fyziologie   MeSH
• lidé MeSH
• makrofágy metabolismus   MeSH
• membránové proteiny * metabolismus   MeSH
• myši MeSH
• proteiny s repetitivními sekvencemi beta-transducinu * metabolismus   MeSH
• signální transdukce MeSH
• ubikvitinligasy * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční * MeSH
• membránové proteiny * MeSH
• proteiny s repetitivními sekvencemi beta-transducinu * MeSH
• ubikvitinligasy * MeSH

The knowledge about the contribution of the innate immune system to health and disease is expanding. However, to obtain reliable results, it is critical to select appropriate mouse models for in vivo studies. Data on genetic and phenotypic changes associated with different mouse strains can assist in this task. Such data can also facilitate our understanding of how specific polymorphisms and genetic alterations affect gene function, phenotypes, and disease outcomes. Extensive information is available on genetic changes in all major mouse strains. However, comparatively little is known about their impact on immune response and, in particular, on innate immunity. Here, we analyzed a mouse model of chronic multifocal osteomyelitis, an autoinflammatory disease driven exclusively by the innate immune system, which is caused by an inactivating mutation in the Pstpip2 gene. We investigated how the genetic background of BALB/c, C57BL/6J, and C57BL/6NCrl strains alters the molecular mechanisms controlling disease progression. While all mice developed the disease, symptoms were significantly milder in BALB/c and partially also in C57BL/6J when compared to C57BL/6NCrl. Disease severity correlated with the number of infiltrating neutrophils and monocytes and with the production of chemokines attracting these cells to the site of inflammation. It also correlated with increased expression of genes associated with autoinflammation, rheumatoid arthritis, neutrophil activation, and degranulation, resulting in altered neutrophil activation in vivo. Together, our data demonstrate striking effects of genetic background on multiple parameters of neutrophil function and activity influencing the onset and course of chronic multifocal osteomyelitis.

• Klíčová slova
• PSTPIP2, chronic multifocal osteomyelitis, genetic background, neutrophil,
• MeSH
• adaptorové proteiny signální transdukční genetika   MeSH
• aktivace neutrofilů genetika   MeSH
• cytoskeletální proteiny MeSH
• genetické pozadí * MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• neutrofily * imunologie   patologie   MeSH
• osteomyelitida * genetika   imunologie   patologie   MeSH
• přirozená imunita genetika   MeSH
• stupeň závažnosti nemoci MeSH
• zánět genetika   patologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• cytoskeletální proteiny MeSH
• Pstpip2 protein, mouse MeSH Prohlížeč

WW domain binding protein 1-like (WBP1L), also known as outcome predictor of acute leukemia 1 (OPAL1), is a transmembrane adaptor protein, expression of which was shown to correlate with ETV6-RUNX1 translocation and favorable prognosis in childhood leukemia. It has a broad expression pattern in hematopoietic and non-hematopoietic cells. Our previous work described WBP1L as a regulator of CXCR4 signaling and hematopoiesis. Here, we show that hematopoiesis in the mice with Wbp1l germline deletion is dysregulated, already at the level of hematopoietic stem cells and early progenitors. We further demonstrate that thymi of WBP1L-deficient mice are significantly enlarged and contain increased numbers of thymocytes of all subsets. This can potentially be explained by increased generation of multipotent progenitors 4 (MPP4) in the bone marrow, from which the thymus-seeding progenitors are derived. We also observed increases in multiple cell types in the blood. In addition, we show that WBP1L regulates hematopoietic stem cell functionality and leukocyte progenitor proliferation and gene expression during hematopoietic stem and progenitor cell transplantation, which contribute to more efficient engraftment of WBP1L-deficient cells. WBP1L thus emerges as a regulator of hematopoietic stem and progenitor cell function, which controls leukocyte numbers at the steady state and after bone marrow transplantation.

• Klíčová slova
• T cell development, WBP1L, hematopoiesis, hematopoietic stem and progenitor cell transplantation, hematopoietic stem cells, transmembrane adaptor protein,
• MeSH
• hematopoetické kmenové buňky * fyziologie   MeSH
• hematopoéza * MeSH
• membránové proteiny * genetika   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• počet leukocytů MeSH
• regulace genové exprese MeSH
• thymocyty fyziologie   MeSH
• thymus * cytologie   fyziologie   MeSH
• transplantace hematopoetických kmenových buněk MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• membránové proteiny * MeSH
• Wbp1l protein, mouse MeSH Prohlížeč

Styrene-maleic acid (SMA) and similar amphiphilic copolymers are known to cut biological membranes into lipid nanoparticles/nanodiscs containing membrane proteins apparently in their relatively native membrane lipid environment. Our previous work demonstrated that membrane raft microdomains resist such disintegration by SMA. The use of SMA in studying membrane proteins is limited by its heterogeneity and the inability to prepare defined derivatives. In the present paper, we demonstrate that some amphiphilic peptides structurally mimicking SMA also similarly disintegrate cell membranes. In contrast to the previously used copolymers, the simple peptides are structurally homogeneous. We found that their membrane-disintegrating activity increases with their length (reaching optimum at 24 amino acids) and requires a basic primary structure, that is, (XXD)n, where X represents a hydrophobic amino acid (optimally phenylalanine), D aspartic acid, and n is the number of repeats of these triplets. These peptides may provide opportunities for various well-defined potentially useful modifications in the study of membrane protein biochemistry. Our present results confirm a specific character of membrane raft microdomains.

• Klíčová slova
• leukocyte, lipid raft, lymphocyte, membrane, membrane proteins, peptides,
• MeSH
• buněčná membrána metabolismus   chemie   MeSH
• buněčné linie MeSH
• lidé MeSH
• maleáty chemie   MeSH
• membránové mikrodomény metabolismus   chemie   MeSH
• membránové proteiny * chemie   metabolismus   MeSH
• peptidy * chemie   MeSH
• polystyreny chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Neutrophilic inflammation is a hallmark of many monogenic autoinflammatory diseases; pathomechanisms that regulate extravasation of damaging immune cells into surrounding tissues are poorly understood. Here we identified three unrelated boys with perinatal-onset of neutrophilic cutaneous small vessel vasculitis and systemic inflammation. Two patients developed liver fibrosis in their first year of life. Next-generation sequencing identified two de novo truncating variants in the Src-family tyrosine kinase, LYN, p.Y508*, p.Q507* and a de novo missense variant, p.Y508F, that result in constitutive activation of Lyn kinase. Functional studies revealed increased expression of ICAM-1 on induced patient-derived endothelial cells (iECs) and of β2-integrins on patient neutrophils that increase neutrophil adhesion and vascular transendothelial migration (TEM). Treatment with TNF inhibition improved systemic inflammation; and liver fibrosis resolved on treatment with the Src kinase inhibitor dasatinib. Our findings reveal a critical role for Lyn kinase in modulating inflammatory signals, regulating microvascular permeability and neutrophil recruitment, and in promoting hepatic fibrosis.

• MeSH
• dasatinib MeSH
• endoteliální buňky * metabolismus   MeSH
• fosforylace MeSH
• lidé MeSH
• neutrofily metabolismus   MeSH
• skupina kinas odvozených od src-genu * genetika   metabolismus   MeSH
• vaskulitida * genetika   MeSH
• zánět metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• dasatinib MeSH
• lyn protein-tyrosine kinase MeSH Prohlížeč
• skupina kinas odvozených od src-genu * MeSH

Chronic inflammation represents a major threat to human health since long-term systemic inflammation is known to affect distinct tissues and organs. Recently, solid evidence demonstrated that chronic inflammation affects hematopoiesis; however, how chronic inflammation affects hematopoietic stem cells (HSCs) on the mechanistic level is poorly understood. Here, we employ a mouse model of chronic multifocal osteomyelitis (CMO) to assess the effects of a spontaneously developed inflammatory condition on HSCs. We demonstrate that hematopoietic and nonhematopoietic compartments in CMO BM contribute to HSC expansion and impair their function. Remarkably, our results suggest that the typical features of murine multifocal osteomyelitis and the HSC phenotype are mechanistically decoupled. We show that the CMO environment imprints a myeloid gene signature and imposes a pro-inflammatory profile on HSCs. We identify IL-6 and the Jak/Stat3 signaling pathway as critical mediators. However, while IL-6 and Stat3 blockage reduce HSC numbers in CMO mice, only inhibition of Stat3 activity significantly rescues their fitness. Our data emphasize the detrimental effects of chronic inflammation on stem cell function, opening new venues for treatment.

• Klíčová slova
• IL-6/Jak/Stat3, chronic inflammation, chronic multifocal osteomyelitis, hematopoietic stem cells, niche,
• MeSH
• hematopoetické kmenové buňky metabolismus   MeSH
• hematopoéza MeSH
• interleukin-6 * genetika   metabolismus   MeSH
• lidé MeSH
• myši MeSH
• osteomyelitida MeSH
• signální transdukce MeSH
• transkripční faktor STAT3 genetika   metabolismus   MeSH
• zánět * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• interleukin-6 * MeSH
• STAT3 protein, human MeSH Prohlížeč
• transkripční faktor STAT3 MeSH

INTRODUCTION: Autoinflammatory diseases are characterized by dysregulation of innate immune system leading to spontaneous sterile inflammation. One of the well-established animal models of this group of disorders is the mouse strain Pstpip2cmo . In this strain, the loss of adaptor protein PSTPIP2 leads to the autoinflammatory disease chronic multifocal osteomyelitis. It is manifested by sterile inflammation of the bones and surrounding soft tissues of the hind limbs and tail. The disease development is propelled by elevated production of IL-1β and reactive oxygen species by neutrophil granulocytes. However, the molecular mechanisms linking PSTPIP2 and these pathways have not been established. Candidate proteins potentially involved in these mechanisms include PSTPIP2 binding partners, PEST family phosphatases (PEST-PTPs) and phosphoinositide phosphatase SHIP1. METHODS: To address the role of these proteins in PSTPIP2-mediated control of inflammation, we have generated mouse strains in which PEST-PTP or SHIP1 binding sites in PSTPIP2 have been disrupted. In these mouse strains, we followed disease symptoms and various inflammation markers. RESULTS: Our data show that mutation of the PEST-PTP binding site causes symptomatic disease, whereas mice lacking the SHIP1 interaction site remain asymptomatic. Importantly, both binding partners of PSTPIP2 contribute equally to the control of IL-1β production, while PEST-PTPs have a dominant role in the regulation of reactive oxygen species. In addition, the interaction of PEST-PTPs with PSTPIP2 regulates the production of the chemokine CXCL2 by neutrophils. Its secretion likely creates a positive feedback loop that drives neutrophil recruitment to the affected tissues. CONCLUSIONS: We demonstrate that PSTPIP2-bound PEST-PTPs and SHIP1 together control the IL-1β pathway. In addition, PEST-PTPs have unique roles in the control of reactive oxygen species and chemokine production, which in the absence of PEST-PTP binding to PSTPIP2 shift the balance towards symptomatic disease.

• Klíčová slova
• PEST-family phosphatases, PSTPIP2, SHIP1, autoinflammation, chronic multifocal osteomyelitis, neutrophils,
• MeSH
• adaptorové proteiny signální transdukční * metabolismus   MeSH
• cytoskeletální proteiny * metabolismus   MeSH
• myši MeSH
• neutrofily * MeSH
• osteomyelitida MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• zánět MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční * MeSH
• cytoskeletální proteiny * MeSH
• Pstpip2 protein, mouse MeSH Prohlížeč
• reaktivní formy kyslíku MeSH

Toll-like receptor (TLR) signaling relies on Toll/interleukin-1 receptor homology (TIR) domain-containing adaptor proteins that recruit downstream signaling molecules to generate tailored immune responses. In addition, the palmitoylated transmembrane adaptor protein family member Scimp acts as a non-TIR-containing adaptor protein in macrophages, scaffolding the Src family kinase Lyn to enable TLR phosphorylation and proinflammatory signaling responses. Here we report the existence of a smaller, naturally occurring translational variant of Scimp (Scimp TV1), which is generated through leaky scanning and translation at a downstream methionine. Scimp TV1 also scaffolds Lyn, but in contrast to full-length Scimp, it is basally rather than lipopolysaccharide (LPS)-inducibly phosphorylated. Macrophages from mice that selectively express Scimp TV1, but not full-length Scimp, have impaired sustained LPS-inducible cytokine responses. Furthermore, in granulocyte macrophage colony-stimulating factor-derived myeloid cells that express high levels of Scimp, selective overexpression of Scimp TV1 enhances CpG DNA-inducible cytokine production. Unlike full-length Scimp that localizes to the cell surface and filopodia, Scimp TV1 accumulates in intracellular compartments, particularly the Golgi. Moreover, this variant of Scimp is not inducibly phosphorylated in response to CpG DNA, suggesting that it may act via an indirect mechanism to enhance TLR9 responses. Our findings thus reveal the use of alternative translation start sites as a previously unrecognized mechanism for diversifying TLR responses in the innate immune system.

• Klíčová slova
• Adaptor protein, CpG DNA, Toll-like receptor, alternative translation start site, macrophage, protein translation,
• MeSH
• DNA metabolismus   MeSH
• makrofágy metabolismus   MeSH
• myši MeSH
• signální transdukce * MeSH
• skupina kinas odvozených od src-genu metabolismus   MeSH
• toll-like receptory * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• skupina kinas odvozených od src-genu MeSH
• toll-like receptory * MeSH

BACKGROUND: Inborn errors of immunity are genetic disorders characterized by various degrees of immune dysregulation that can manifest as immune deficiency, autoimmunity, or autoinflammation. The routine use of next-generation sequencing in the clinic has facilitated the identification of an ever-increasing number of inborn errors of immunity, revealing the roles of immunologically important genes in human pathologies. However, despite this progress, treatment is still extremely challenging. OBJECTIVE: We sought to report a new monogenic autoinflammatory disorder caused by a de novo activating mutation, p.Tyr515∗, in hematopoietic cell kinase (HCK). The disease is characterized by cutaneous vasculitis and chronic pulmonary inflammation that progresses to fibrosis. METHODS: Whole-exome sequencing, Sanger sequencing, mass spectrometry, and western blotting were performed to identify and characterize the pathogenic HCK mutation. Dysregulation of mutant HCK was confirmed ex vivo in primary cells and in vitro in transduced cell lines. RESULTS: Mutant HCK lacking the C-terminal inhibitory tyrosine Tyr522 exhibited increased kinase activity and enhanced myeloid cell priming, migration and effector functions, such as production of the inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α, and production of reactive oxygen species. These aberrant functions were reflected by inflammatory leukocyte infiltration of the lungs and skin. Moreover, an overview of the clinical course of the disease, including therapies, provides evidence for the therapeutic efficacy of the Janus kinase 1/2 inhibitor ruxolitinib in inflammatory lung disease. CONCLUSIONS: We propose HCK-driven pulmonary and cutaneous vasculitis as a novel autoinflammatory disorder of inborn errors of immunity.

• Klíčová slova
• SRC-family kinase, autoinflammation, cutaneous vasculitis, hematopoietic cell kinase, inborn error of immunity, inflammatory cytokines, pulmonary hemorrhage, reactive oxygen species, ruxolitinib,
• MeSH
• lidé MeSH
• plíce MeSH
• protoonkogenní proteiny c-hck genetika   metabolismus   MeSH
• protoonkogenní proteiny genetika   MeSH
• skupina kinas odvozených od src-genu * genetika   MeSH
• vaskulitida * genetika   patologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• HCK protein, human MeSH Prohlížeč
• protoonkogenní proteiny c-hck MeSH
• protoonkogenní proteiny MeSH
• skupina kinas odvozených od src-genu * MeSH

Cells communicate with their environment via surface receptors, but nanoscopic receptor organization with respect to complex cell surface morphology remains unclear. This is mainly due to a lack of accessible, robust and high-resolution methods. Here, we present an approach for mapping the topography of receptors at the cell surface with nanometer precision. The method involves coating glass coverslips with glycine, which preserves the fine membrane morphology while allowing immobilized cells to be positioned close to the optical surface. We developed an advanced and simplified algorithm for the analysis of single-molecule localization data acquired in a biplane detection scheme. These advancements enable direct and quantitative mapping of protein distribution on ruffled plasma membranes with near isotropic 3D nanometer resolution. As demonstrated successfully for CD4 and CD45 receptors, the described workflow is a straightforward quantitative technique to study molecules and their interactions at the complex surface nanomorphology of differentiated metazoan cells.

• MeSH
• buněčná membrána metabolismus   MeSH
• nanotechnologie * MeSH
• receptory buněčného povrchu * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• receptory buněčného povrchu * MeSH