Benjamin Nota

Hobit and Blimp1 instruct a universal transcriptional program of tissue residency in lymphocytes

Transcription factors define tissue T cells The immune system fights microbial invaders by maintaining multiple lines of defense. For instance, specialized memory T cells [resident memory T cells (Trms)] colonize portals of pathogen entry, such as the skin, lung, and gut, to quickly halt reinfections. Mackay et al. now report that in mice, Trms as well as other tissue-dwelling lymphocyte populations such as natural killer cells share a common transcriptional program driven by the related transcription factors Hobit and Blimp1. Tissue residency and retention of lymphocytes require expression of Hobit and Blimp1, which, among other functions, suppress genes that promote tissue exit. Science, this issue p. 459 Tissue-dwelling lymphocyte populations share a common transcriptional signature. Tissue-resident memory T (Trm) cells permanently localize to portals of pathogen entry, where they provide immediate protection against reinfection. To enforce tissue retention, Trm cells up-regulate CD69 and down-regulate molecules associated with tissue egress; however, a Trm-specific transcriptional regulator has not been identified. Here, we show that the transcription factor Hobit is specifically up-regulated in Trm cells and, together with related Blimp1, mediates the development of Trm cells in skin, gut, liver, and kidney in mice. The Hobit-Blimp1 transcriptional module is also required for other populations of tissue-resident lymphocytes, including natural killer T (NKT) cells and liver-resident NK cells, all of which share a common transcriptional program. Our results identify Hobit and Blimp1 as central regulators of this universal program that instructs tissue retention in diverse tissue-resident lymphocyte populations.

Programs for the persistence, vigilance and control of human CD8+ lung-resident memory T cells

Tissue-resident memory T cells (TRM cells) in the airways mediate protection against respiratory infection. We characterized TRM cells expressing integrin αE (CD103) that reside within the epithelial barrier of human lungs. These cells had specialized profiles of chemokine receptors and adhesion molecules, consistent with their unique localization. Lung TRM cells were poised for rapid responsiveness by constitutive expression of deployment-ready mRNA encoding effector molecules, but they also expressed many inhibitory regulators, suggestive of programmed restraint. A distinct set of transcription factors was active in CD103+ TRM cells, including Notch. Genetic and pharmacological experiments with mice revealed that Notch activity was required for the maintenance of CD103+ TRM cells. We have thus identified specialized programs underlying the residence, persistence, vigilance and tight control of human lung TRM cells.

Profiling of apoptosis genes allows for clinical stratification of primary nodal diffuse large B-cell lymphomas.

Intrinsic resistance of lymphoma cells to apoptosis is a probable mechanism causing chemotherapy resistance and eventual fatal outcome in patients with diffuse large B cell lymphomas (DLBCL). We investigated whether microarray expression profiling of apoptosis related genes predicts clinical outcome in 46 patients with primary nodal DLBCL. Unsupervised cluster analysis using genes involved in apoptosis (n = 246) resulted in three separate DLBCL groups partly overlapping with germinal centre B‐lymphocytes versus activated B‐cells like phenotype. One group with poor clinical outcome was characterised by high expression levels of pro‐and anti‐apoptotic genes involved in the intrinsic apoptosis pathway. A second group, also with poor clinical outcome, was characterised by high levels of apoptosis inducing cytotoxic effector genes, possibly reflecting a cellular cytotoxic immune response. The third group showing a favourable outcome was characterised by low expression levels of genes characteristic for both other groups. Our results suggest that chemotherapy refractory DLBCL are characterised either by an intense cellular cytotoxic immune response or by constitutive activation of the intrinsic mediated apoptosis pathway with concomitant downstream inhibition of this apoptosis pathway. Consequently, strategies neutralising the function of apoptosis‐inhibiting proteins might be effective as alternative treatment modality in part of chemotherapy refractory DLBCL.

GATA1-Deficient Dendritic Cells Display Impaired CCL21-Dependent Migration toward Lymph Nodes Due to Reduced Levels of Polysialic Acid

Dendritic cells (DCs) play a pivotal role in the regulation of the immune response. DC development and activation is finely orchestrated through transcriptional programs. GATA1 transcription factor is required for murine DC development, and data suggest that it might be involved in the fine-tuning of the life span and function of activated DCs. We generated DC-specific Gata1 knockout mice (Gata1-KODC), which presented a 20% reduction of splenic DCs, partially explained by enhanced apoptosis. RNA sequencing analysis revealed a number of deregulated genes involved in cell survival, migration, and function. DC migration toward peripheral lymph nodes was impaired in Gata1-KODC mice. Migration assays performed in vitro showed that this defect was selective for CCL21, but not CCL19. Interestingly, we show that Gata1-KODC DCs have reduced polysialic acid levels on their surface, which is a known determinant for the proper migration of DCs toward CCL21.

Gogadget: an R Package for Interpretation and Visualization of GO Enrichment Results

Gene expression profiling followed by gene ontology (GO) term enrichment analysis can generate long lists of significant GO terms. To interpret these results and get biological insight in the data, filtering and rearranging these long lists of GO terms might be desirable. The R package gogadget provides functions to modify GO analysis results, with a simple filter strategy. Furthermore, it groups redundant GO terms with hierarchical clustering and presents the results in a colorful heatmap. The filtered GO term enrichment results can also be exported by the package for subsequent analysis in Cytoscape Enrichment Map. The R package is freely available under the terms of the GNU GPLv3 at https://sourceforge.net/projects/gogadget/.

TLR4 and C5aR crosstalk in dendritic cells induces a core regulatory network of RSK2, PI3Kβ, SGK1, and FOXO transcription factors

Crosstalk between complement component 5a receptors (C5aRs) and TLRs in dendritic cells (DCs) occurs upon pathogen invasion; however, studies on C5aR and TLR crosstalk mainly focused on the modulating effect of C5a on TLR‐induced cytokine production. To elucidate the breadth of C5aR and TLR4 crosstalk, the effect of simultaneous treatment with C5a and LPS was investigated in human monocyte‐derived DCs (moDCs) 2 h after stimulation using whole transcriptome sequencing analysis. Although the effect of C5a on hallmark genes defining TLR4‐induced DC maturation was limited at this time point, RNA sequencing analysis revealed a great variety of novel C5a targets, of which many interfere with TLR4‐mediated immune activation. Analysis of functional relationships among these genes uncovered induction of a central immune regulatory network upon C5aR and TLR4 crosstalk, involving the transcription factors forkhead box (FOX)O1 and FOXO3 and the signaling molecules serum‐ and glucocorticoid‐inducible kinase (SGK1), ribosomal S6 kinase 2 (RSK2), and PI3Kβ. C5aR and TLR crosstalk, furthermore, yielded down‐regulation of mainly proinflammatory network branches, including IL‐12B, IL‐2Rα (IL‐2RA), and jagged 1 (JAG1) and cooperative induction of predominantly anti‐inflammatory network branches, including sphingosine kinase 1 (SPHK1), β2 adrenergic receptor (ADRB2), gastric inhibitory polypeptide receptor (GIPR), and four‐and‐a‐half Lin11, Isl‐1, and Mec‐3 domains protein 2 (FHL2). Together, these data point toward induction of generalized immune regulation of DC function. Motif enrichment analysis indicate a prominent role for basic leucine zipper (bZIP) and IFN regulatory factor 4 (IRF4) transcription factors upon C5aR and TLR4 crosstalk. Additionally, differences were observed in the modulating capacity of C5a on DCs in the absence or presence of a pathogen (TLR stimulus). Our findings shed new light on the depth and complexity of C5aR and TLR4 crosstalk and provide new foci of research for future studies.

Iron metabolism in critically ill patients developing anemia of inflammation : a case control study

BackgroundAnemia occurring as a result of inflammatory processes (anemia of inflammation, AI) has a high prevalence in critically ill patients. Knowledge on changes in iron metabolism during the course of AI is limited, hampering the development of strategies to counteract AI. This case control study aimed to investigate iron metabolism during the development of AI in critically ill patients.MethodsIron metabolism in 30 patients who developed AI during ICU stay was compared with 30 septic patients with a high Hb and 30 non-septic patients with a high Hb. Patients were matched on age and sex. Longitudinally collected plasma samples were analyzed for levels of parameters of iron metabolism. A linear mixed model was used to assess the predictive values of the parameters.ResultsIn patients with AI, levels of iron, transferrin and transferrin saturation showed an early decrease compared to controls with a high Hb, already prior to the development of anemia. Ferritin, hepcidin and IL-6 levels were increased in AI compared to controls. During AI development, erythroferrone decreased. Differences in iron metabolism between groups were not influenced by APACHE IV score.ConclusionsThe results show that in critically ill patients with AI, iron metabolism is already altered prior to the development of anemia. Levels of iron regulators in AI differ from septic controls with a high Hb, irrespective of disease severity. AI is characterized by high levels of hepcidin, ferritin and IL-6 and low levels of iron, transferrin and erythroferrone.

RNA sequencing of creatine transporter (SLC6A8) deficient fibroblasts reveals impairment of the extracellular matrix.

Creatine transporter (SLC6A8) deficiency is the most common cause of cerebral creatine syndromes, and is characterized by depletion of creatine in the brain. Manifestations of this X‐linked disorder include intellectual disability, speech/language impairment, behavior abnormalities, and seizures. At the moment, no effective treatment is available. In order to investigate the molecular pathophysiology of this disorder, we performed RNA sequencing on fibroblasts derived from patients. The transcriptomes of fibroblast cells from eight unrelated individuals with SLC6A8 deficiency and three wild‐type controls were sequenced. SLC6A8 mutations with different effects on the protein product resulted in different gene expression profiles. Differential gene expression analysis followed by gene ontology term enrichment analysis revealed that especially the expression of genes encoding components of the extracellular matrix and cytoskeleton are altered in SLC6A8 deficiency, such as collagens, keratins, integrins, and cadherins. This suggests an important novel role for creatine in the structural development and maintenance of cells. It is likely that the (extracellular) structure of brain cells is also impaired in SLC6A8‐deficient patients, and future studies are necessary to confirm this and to reveal the true functions of creatine in the brain.

Integrated proteomic analysis of tumor necrosis factor α and interleukin 1β-induced endothelial inflammation

The vascular endothelium provides a unique interaction plane for plasma proteins and leukocytes in inflammation. The pro-inflammatory cytokines Tumor Necrosis Factor α (TNFα) and interleukin 1β (IL-1β) have a profound effect on endothelial cells, which includes increased levels of adhesion molecules and a disrupted barrier function. To assess the endothelial response to these cytokines at the protein level, we evaluated changes in the whole proteome, cell surface proteome and phosphoproteome after 24 h of cytokine treatment. The effects of TNFα and IL-1β on endothelial cells were strikingly similar and included changes in proteins not previously associated with endothelial inflammation. Temporal profiling revealed time-dependent proteomic changes, including a limited number of early responsive proteins such as adhesion receptors ICAM1 and SELE. In addition, this approach uncovered a greater number of late responsive proteins, including proteins related to self-antigen peptide presentation, and a transient increase in ferritin. Peptide-based cell surface proteomics revealed extensive changes at the cell surface, which were in agreement with the whole proteome. In addition, site-specific changes within ITGA5 and ICAM1 were detected. Combined, our integrated proteomic data provide detailed information on endothelial inflammation, emphasize the role of the extracellular matrix therein, and include potential targets for therapeutic intervention. SIGNIFICANCE: Pro-inflammatory cytokines induce the expression of cell adhesion molecules in vascular endothelial cells. These molecules mediate the adhesion and migration of immune cells across the vessel wall, which is a key process to resolve infections in the underlying tissue. Dysregulation of endothelial inflammation can contribute to vascular diseases and the vascular endothelium is therefore an attractive target to control inflammation. Current strategies targeting endothelial adhesion molecules, including PECAM, CD99, ICAM1 and VCAM1 do not completely prevent transmigration. To identify additional therapeutic targets, we mapped the endothelial proteome after pro-inflammatory cytokine treatment. In addition to the whole proteome, we assessed the surface proteome to focus on cell adhesion molecules, and the phosphoproteome to uncover protein activation states. Here, we present an integrated overview of affected processes which further improves our understanding of endothelial inflammation and may eventually aid in therapeutic intervention of imbalanced inflammation.

Differences between Platelets Derived from Neonatal Cord Blood and Adult Peripheral Blood Assessed by Mass Spectrometry

It has been proposed that differences may exist between umbilical cord blood (CB) platelets and adult peripheral blood (APB) platelets, including altered protein levels of the main platelet integrins. We have now compared the protein expression profiles of CB and APB platelets employing a label-free comparative proteomics approach. Aggregation studies showed that CB platelets effectively aggregate in the presence of thromboxane A2 analogue, collagen, and peptide agonists of the proteinase-activated receptors 1 and 4. In agreement with previous studies, higher concentrations of the agonists were required to initiate aggregation in the CB platelets. Mass spectrometry analysis revealed no significant difference in the expression levels of critical platelet receptors like glycoprotein (GP)Ib, GPV, GPIX, and integrin αIIbβ3. This was confirmed using flow cytometry-based approaches. Gene ontology enrichment analysis revealed that elevated proteins in CB platelets were in particular enriched in proteins contributing to mitochondrial energy metabolism processes. The reduced proteins were enriched in proteins involved in, among others, platelet degranulation and activation. In conclusion, this study reveals that the CB and APB platelets are distinct. In particular, changes were observed for proteins that belong to metabolic and energy generation processes and not for the critical adhesive platelet integrins and glycoproteins.