TIGIT can inhibit T cell activation via ligation-induced nanoclusters, independent of CD226 co-stimulation.

TIGIT is an inhibitory receptor expressed on lymphocytes and can inhibit T cells by preventing CD226 co-stimulation through interactions in cis or through competition of shared ligands. Whether TIGIT directly delivers cell-intrinsic inhibitory signals in T cells remains unclear. Here we show, by analysing lymphocytes from matched human tumour and peripheral blood samples, that TIGIT and CD226 co-expression is rare on tumour-infiltrating lymphocytes. Using super-resolution microscopy and other techniques, we demonstrate that ligation with CD155 causes TIGIT to reorganise into dense nanoclusters, which coalesce with T cell receptor (TCR)-rich clusters at immune synapses. Functionally, this reduces cytokine secretion in a manner dependent on TIGIT's intracellular ITT-like signalling motif. Thus, we provide evidence that TIGIT directly inhibits lymphocyte activation, acting independently of CD226, requiring intracellular signalling that is proximal to the TCR. Within the subset of tumours where TIGIT-expressing cells do not commonly co-express CD226, this will likely be the dominant mechanism of action.

Heterogeneity in extracellular vesicle secretion by single human macrophages revealed by super-resolution microscopy.

The diverse origins, nanometre-scale and invasive isolation procedures associated with extracellular vesicles (EVs) mean they are usually studied in bulk and disconnected from their parental cell. Here, we used super-resolution microscopy to directly compare EVs secreted by individual human monocyte-derived macrophages (MDMs). MDMs were differentiated to be M0-, M1- or M2-like, with all three secreting EVs at similar densities following activation. However, M0-like cells secreted larger EVs than M1- and M2-like macrophages. Proteomic analysis revealed variations in the contents of differently sized EVs as well as between EVs secreted by different MDM phenotypes. Super resolution microscopy of single-cell secretions identified that the class II MHC protein, HLA-DR, was expressed on ∼40% of EVs secreted from M1-like MDMs, which was double the frequency observed for M0-like and M2-like EVs. Strikingly, human macrophages, isolated from the resected lungs of cancer patients, secreted EVs that expressed HLA-DR at double the frequency and with greater intensity than M1-like EVs. Quantitative analysis of single-cell EV profiles from all four macrophage phenotypes revealed distinct secretion types, five of which were consistent across multiple sample cohorts. A sub-population of M1-like MDMs secreted EVs similar to lung macrophages, suggesting an expansion or recruitment of cells with a specific EV secretion profile within the lungs of cancer patients. Thus, quantitative analysis of EV heterogeneity can be used for single cell profiling and to reveal novel macrophage biology.

Internalization of the Membrane Attack Complex Triggers NLRP3 Inflammasome Activation and IL-1ß Secretion in Human Macrophages.

Interleukin 1ß (IL-1ß) plays a major role in inflammation and is secreted by immune cells, such as macrophages, upon recognition of danger signals. Its secretion is regulated by the inflammasome, the assembly of which results in caspase 1 activation leading to gasdermin D (GSDMD) pore formation and IL-1ß release. During inflammation, danger signals also activate the complement cascade, resulting in the formation of the membrane attack complex (MAC). Here, we report that stimulation of LPS-primed human macrophages with sub-lytic levels of MAC results in activation of the NOD-like receptor 3 (NLRP3) inflammasome and GSDMD-mediated IL-1ß release. The MAC is first internalized into endosomes and then colocalizes with inflammasome components; adapter protein apoptosis associated speck-like protein containing a CARD (ASC) and NLRP3. Pharmacological inhibitors established that MAC-triggered activation of the NLRP3 inflammasome was dependent on MAC endocytosis. Internalization of the MAC also caused dispersion of the trans-Golgi network. Thus, these data uncover a role for the MAC in activating the inflammasome and triggering IL-1ß release in human macrophages.

Identifying cell-enriched miRNAs in kidney injury and repair.

Small noncoding RNAs, miRNAs (miRNAs), are emerging as important modulators in the pathogenesis of kidney disease, with potential as biomarkers of kidney disease onset, progression, or therapeutic efficacy. Bulk tissue small RNA-sequencing (sRNA-Seq) and microarrays are widely used to identify dysregulated miRNA expression but are limited by the lack of precision regarding the cellular origin of the miRNA. In this study, we performed cell-specific sRNA-Seq on tubular cells, endothelial cells, PDGFR-ß+ cells, and macrophages isolated from injured and repairing kidneys in the murine reversible unilateral ureteric obstruction model. We devised an unbiased bioinformatics pipeline to define the miRNA enrichment within these cell populations, constructing a miRNA catalog of injury and repair. Our analysis revealed that a significant proportion of cell-specific miRNAs in healthy animals were no longer specific following injury. We then applied this knowledge of the relative cell specificity of miRNAs to deconvolute bulk miRNA expression profiles in the renal cortex in murine models and human kidney disease. Finally, we used our data-driven approach to rationally select macrophage-enriched miR-16-5p and miR-18a-5p and demonstrate that they are promising urinary biomarkers of acute kidney injury in renal transplant recipients.

Corrected Super-Resolution Microscopy Enables Nanoscale Imaging of Autofluorescent Lung Macrophages.

Observing the cell surface and underlying cytoskeleton at nanoscale resolution using super-resolution microscopy has enabled many insights into cell signaling and function. However, the nanoscale dynamics of tissue-specific immune cells have been relatively little studied. Tissue macrophages, for example, are highly autofluorescent, severely limiting the utility of light microscopy. Here, we report a correction technique to remove autofluorescent noise from stochastic optical reconstruction microscopy (STORM) data sets. Simulations and analysis of experimental data identified a moving median filter as an accurate and robust correction technique, which is widely applicable across challenging biological samples. Here, we used this method to visualize lung macrophages activated through Fc receptors by antibody-coated glass slides. Accurate, nanoscale quantification of macrophage morphology revealed that activation induced the formation of cellular protrusions tipped with MHC class I protein. These data are consistent with a role for lung macrophage protrusions in antigen presentation. Moreover, the tetraspanin protein CD81, known to mark extracellular vesicles, appeared in ring-shaped structures (mean diameter 93 ± 50 nm) at the surface of activated lung macrophages. Thus, a moving median filter correction technique allowed us to quantitatively analyze extracellular secretions and membrane structure in tissue-derived immune cells.

A missense mutation in the MLKL brace region promotes lethal neonatal inflammation and hematopoietic dysfunction.

MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, MlklD139V, that alters the two-helix 'brace' that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of MlklD139V homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO).

Phenotypic Analysis of Human Lymph Nodes in Subjects With New-Onset Type 1 Diabetes and Healthy Individuals by Flow Cytometry.

Background: Ultrasound guided sampling of human lymph node (LN) combined with advanced flow cytometry allows phenotypic analysis of multiple immune cell subsets. These may provide insights into immune processes and responses to immunotherapies not apparent from analysis of the blood. Methods: Ultrasound guided inguinal LN samples were obtained by both fine needle aspiration (FNA) and core needle biopsy in 10 adults within 8 weeks of diagnosis of type 1 diabetes (T1D) and 12 age-matched healthy controls at two study centers. Peripheral blood mononuclear cells (PBMC) were obtained on the same occasion. Samples were transported same day to the central laboratory and analyzed by multicolour flow cytometry. Results: LN sampling was well-tolerated and yielded sufficient cells for analysis in 95% of cases. We confirmed the segregation of CD69+ cells into LN and the predominance of CD8+ Temra cells in blood previously reported. In addition, we demonstrated clear enrichment of CD8+ naïve, FOXP3+ Treg, class-switched B cells, CD56bright NK cells and plasmacytoid dendritic cells (DC) in LNs as well as CD4+ T cells of the Th2 phenotype and those expressing Helios and Ki67. Conventional NK cells were virtually absent from LNs as were Th22 and Th1Th17 cells. Paired correlation analysis of blood and LN in the same individuals indicated that for many cell subsets, especially those associated with activation: such as CD25+ and proliferating (Ki67+) T cells, activated follicular helper T cells and class-switched B cells, levels in the LN compartment could not be predicted by analysis of blood. We also observed an increase in Th1-like Treg and less proliferating (Ki67+) CD4+ T cells in LN from T1D compared to control LNs, changes which were not reflected in the blood. Conclusions: LN sampling in humans is well-tolerated. We provide the first detailed "roadmap" comparing immune subsets in LN vs. blood emphasizing a role for differentiated effector T cells in the blood and T cell regulation, B cell activation and memory in the LN. For many subsets, frequencies in blood, did not correlate with LN, suggesting that LN sampling would be valuable for monitoring immuno-therapies where these subsets may be impacted.

Metformin Inhibits the Production of Reactive Oxygen Species from NADH:Ubiquinone Oxidoreductase to Limit Induction of Interleukin-1ß (IL-1ß) and Boosts Interleukin-10 (IL-10) in Lipopolysaccharide (LPS)-activated Macrophages.

Metformin, a frontline treatment for type II diabetes mellitus, decreases production of the pro-form of the inflammatory cytokine IL-1ß in response to LPS in macrophages. We found that it specifically inhibited pro-IL-1ß production, having no effect on TNF-α. Furthermore, metformin boosted induction of the anti-inflammatory cytokine IL-10 in response to LPS. We ruled out a role for AMP-activated protein kinase (AMPK) in the effect of metformin because activation of AMPK with A769662 did not mimic metformin here. Furthermore, metformin was still inhibitory in AMKPα1- or AMPKß1-deficient cells. The activity of NADH:ubiquinone oxidoreductase (complex I) was inhibited by metformin. Another complex I inhibitor, rotenone, mimicked the effect of metformin on pro-IL-1ß and IL-10. LPS induced reactive oxygen species production, an effect inhibited by metformin or rotenone pretreatment. MitoQ, a mitochondrially targeted antioxidant, decreased LPS-induced IL-1ß without affecting TNF-α. These results, therefore, implicate complex I in LPS action in macrophages.

The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism.

Mixed lineage kinase domain-like (MLKL) is a component of the "necrosome," the multiprotein complex that triggers tumor necrosis factor (TNF)-induced cell death by necroptosis. To define the specific role and molecular mechanism of MLKL action, we generated MLKL-deficient mice and solved the crystal structure of MLKL. Although MLKL-deficient mice were viable and displayed no hematopoietic anomalies or other obvious pathology, cells derived from these animals were resistant to TNF-induced necroptosis unless MLKL expression was restored. Structurally, MLKL comprises a four-helical bundle tethered to the pseudokinase domain, which contains an unusual pseudoactive site. Although the pseudokinase domain binds ATP, it is catalytically inactive and its essential nonenzymatic role in necroptotic signaling is induced by receptor-interacting serine-threonine kinase 3 (RIPK3)-mediated phosphorylation. Structure-guided mutation of the MLKL pseudoactive site resulted in constitutive, RIPK3-independent necroptosis, demonstrating that modification of MLKL is essential for propagation of the necroptosis pathway downstream of RIPK3.

Distinct mechanisms for induction and tolerance regulate the immediate early genes encoding interleukin 1ß and tumor necrosis factor α.

Interleukin-1ß and Tumor Necrosis Factor α play related, but distinct, roles in immunity and disease. Our study revealed major mechanistic distinctions in the Toll-like receptor (TLR) signaling-dependent induction for the rapidly expressed genes (IL1B and TNF) coding for these two cytokines. Prior to induction, TNF exhibited pre-bound TATA Binding Protein (TBP) and paused RNA Polymerase II (Pol II), hallmarks of poised immediate-early (IE) genes. In contrast, unstimulated IL1B displayed very low levels of both TBP and paused Pol II, requiring the lineage-specific Spi-1/PU.1 (Spi1) transcription factor as an anchor for induction-dependent interaction with two TLR-activated transcription factors, C/EBPß and NF-κB. Activation and DNA binding of these two pre-expressed factors resulted in de novo recruitment of TBP and Pol II to IL1B in concert with a permissive state for elongation mediated by the recruitment of elongation factor P-TEFb. This Spi1-dependent mechanism for IL1B transcription, which is unique for a rapidly-induced/poised IE gene, was more dependent upon P-TEFb than was the case for the TNF gene. Furthermore, the dependence on phosphoinositide 3-kinase for P-TEFb recruitment to IL1B paralleled a greater sensitivity to the metabolic state of the cell and a lower sensitivity to the phenomenon of endotoxin tolerance than was evident for TNF. Such differences in induction mechanisms argue against the prevailing paradigm that all IE genes possess paused Pol II and may further delineate the specific roles played by each of these rapidly expressed immune modulators.

The emerging role of metabolic regulation in the functioning of Toll-like receptors and the NOD-like receptor Nlrp3.

While it has long been suspected that inflammation participates in the pathogenesis of metabolic disorders such as the insulin resistance that occurs in type 2 diabetes, recent work suggests that this is not the only important interaction between metabolism and inflammation. Inroads into the understanding of the relationship between metabolic pathways and inflammation are indicating that signaling by innate immune receptors such as TLR4 and Nlrp3 regulate metabolism. TLRs have been shown to promote glycolysis, whilst Nlrp3-mediated production of IL-1ß causes insulin resistance. A key role for the hypoxia-sensing transcription factor HIF1α in the functioning of macrophages activated by TLRs has also recently emerged. This review will assess recent evidence for these complex interactions and speculate on their importance for innate immunity and inflammation.

Activation of the NLRP3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced IL-1ß in type 2 diabetes.

Interleukin 1ß (IL-1ß) is an important inflammatory mediator of type 2 diabetes. Here we show that oligomers of islet amyloid polypeptide (IAPP), a protein that forms amyloid deposits in the pancreas during type 2 diabetes, triggered the NLRP3 inflammasome and generated mature IL-1ß. One therapy for type 2 diabetes, glyburide, suppressed IAPP-mediated IL-1ß production in vitro. Processing of IL-1ß initiated by IAPP first required priming, a process that involved glucose metabolism and was facilitated by minimally oxidized low-density lipoprotein. Finally, mice transgenic for human IAPP had more IL-1ß in pancreatic islets, which localized together with amyloid and macrophages. Our findings identify previously unknown mechanisms in the pathogenesis of type 2 diabetes and treatment of pathology caused by IAPP.

Suppressor of cytokine signaling 3 regulates CD8 T-cell proliferation by inhibition of interleukins 6 and 27.

Suppressor of cytokine signaling (SOCS) proteins regulate the intensity and duration of cytokine responses. SOCS3 is expressed in peripheral T cells, and recent reports have suggested that overexpression of SOCS3 modulates antigen- and/or costimulation-induced T-cell activation. To study the role of SOCS3 in the regulation of T-cell activation, we used a conditional gene-targeting strategy to generate mice that lack SOCS3 in T/natural killer T cells (Socs3(DeltaLck/DeltaLck) mice). SOCS3-deficient CD8 T cells showed greater proliferation than wild-type cells in response to T-cell receptor (TCR) ligation despite normal activation of signaling pathways downstream from TCR or CD28 receptors. Signaling in response to the gp130 cytokines interleukin (IL)-6 and IL-27 was prolonged in Socs3(DeltaLck/DeltaLck) T cells, and T cells from gp130(Y757F/Y757F) mice, in which the SOCS3-binding site on gp130 is ablated, showed a striking similarity to SOCS3-deficient CD8 T cells. Although the proliferative defect of Socs3(DeltaLck/DeltaLck) T cells was not rescued in the absence of IL-6, suppression of IL-27 signaling was found to substantially reduce anti-CD3-induced proliferation. We conclude that enhanced responses to TCR ligation by SOCS3-deficient CD8 T cells are not caused by aberrant TCR-signaling pathways but, rather, that increased IL-27 signaling drives unregulated proliferation in the absence of SOCS3.

SOCS2 can enhance interleukin-2 (IL-2) and IL-3 signaling by accelerating SOCS3 degradation.

Cytokine responses can be regulated by a family of proteins termed suppressors of cytokine signaling (SOCS) which can inhibit the JAK/STAT pathway in a classical negative-feedback manner. While the SOCS are thought to target signaling intermediates for degradation, relatively little is known about how their turnover is regulated. Unlike other SOCS family members, we find that SOCS2 can enhance interleukin-2 (IL-2)- and IL-3-induced STAT phosphorylation following and potentiate proliferation in response to cytokine stimulation. As a clear mechanism for these effects, we demonstrate that expression of SOCS2 results in marked proteasome-dependent reduction of SOCS3 and SOCS1 protein expression. Furthermore, we provide evidence that this degradation is dependent on the presence of an intact SOCS box and that the loss of SOCS3 is enhanced by coexpression of elongin B/C. This suggests that SOCS2 can bind to SOCS3 and elongin B/C to form an E3 ligase complex resulting in the degradation of SOCS3. Therefore, SOCS2 can enhance cytokine responses by accelerating proteasome-dependent turnover of SOCS3, suggesting a mechanism for the gigantism observed in SOCS2 transgenic mice.