Morphological profiling of human T and NK lymphocytes by high-content cell imaging.

The immunological synapse is a complex structure that decodes stimulatory signals into adapted lymphocyte responses. It is a unique window to monitor lymphocyte activity because of development of systematic quantitative approaches. Here we demonstrate the applicability of high-content imaging to human T and natural killer (NK) cells and develop a pipeline for unbiased analysis of high-definition morphological profiles. Our approach reveals how distinct facets of actin cytoskeleton remodeling shape immunological synapse architecture and affect lytic granule positioning. Morphological profiling of CD8+ T cells from immunodeficient individuals allows discrimination of the roles of the ARP2/3 subunit ARPC1B and the ARP2/3 activator Wiskott-Aldrich syndrome protein (WASP) in immunological synapse assembly. Single-cell analysis further identifies uncoupling of lytic granules and F-actin radial distribution in ARPC1B-deficient lymphocytes. Our study provides a foundation for development of morphological profiling as a scalable approach to monitor primary lymphocyte responsiveness and to identify complex aspects of lymphocyte micro-architecture.

FODMAP Fingerprinting of Bakery Products and Sourdoughs: Quantitative Assessment and Content Reduction through Fermentation.

Fermentable oligo-, di-, and monosaccharides and polyols (FODMAPs) are associated with digestive disorders and with diseases such as irritable bowel syndrome. In this study, we determined the FODMAP contents of bread, bakery products, and flour and assessed the effectiveness of sourdough fermentation for FODMAP reduction. The fermentation products were analyzed to determine the DP 2-7 and DP >7 fructooligosaccharide (FOS) content of rye and wheat sourdoughs. FOSs were reduced by Acetobacter cerevisiae, Acetobacter okinawensis, Fructilactobacillus sanfranciscensis, and Leuconostoc citreum to levels below those in rye (-81%; -97%) and wheat (-90%; -76%) flours. The fermentation temperature influenced the sourdough acetic acid to lactic acid ratios (4:1 at 4 °C; 1:1 at 10 °C). The rye sourdough contained high levels of beneficial arabinose (28.92 g/kg) and mannitol (20.82 g/kg). Our study contributes in-depth knowledge of low-temperature sourdough fermentation in terms of effective FODMAP reduction and concurrent production of desirable fermentation byproducts.

RhoG deficiency abrogates cytotoxicity of human lymphocytes and causes hemophagocytic lymphohistiocytosis.

Exocytosis of cytotoxic granules (CG) by lymphocytes is required for the elimination of infected and malignant cells. Impairments in this process underly a group of diseases with dramatic hyperferritinemic inflammation termed hemophagocytic lymphohistiocytosis (HLH). Although genetic and functional studies of HLH have identified proteins controlling distinct steps of CG exocytosis, the molecular mechanisms that spatiotemporally coordinate CG release remain partially elusive. We studied a patient exhibiting characteristic clinical features of HLH associated with markedly impaired cytotoxic T lymphocyte (CTL) and natural killer (NK) cell exocytosis functions, who beared biallelic deleterious mutations in the gene encoding the small GTPase RhoG. Experimental ablation of RHOG in a model cell line and primary CTLs from healthy individuals uncovered a hitherto unappreciated role of RhoG in retaining CGs in the vicinity of the plasma membrane (PM), a fundamental prerequisite for CG exocytotic release. We discovered that RhoG engages in a protein-protein interaction with Munc13-4, an exocytosis protein essential for CG fusion with the PM. We show that this interaction is critical for docking of Munc13-4+ CGs to the PM and subsequent membrane fusion and release of CG content. Thus, our study illuminates RhoG as a novel essential regulator of human lymphocyte cytotoxicity and provides the molecular pathomechanism behind the identified here and previously unreported genetically determined form of HLH.

Publisher Correction: Human DEF6 deficiency underlies an immunodeficiency syndrome with systemic autoimmunity and aberrant CTLA-4 homeostasis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Human DEF6 deficiency underlies an immunodeficiency syndrome with systemic autoimmunity and aberrant CTLA-4 homeostasis.

Immune responses need to be controlled tightly to prevent autoimmune diseases, yet underlying molecular mechanisms remain partially understood. Here, we identify biallelic mutations in three patients from two unrelated families in differentially expressed in FDCP6 homolog (DEF6) as the molecular cause of an inborn error of immunity with systemic autoimmunity. Patient T cells exhibit impaired regulation of CTLA-4 surface trafficking associated with reduced functional CTLA-4 availability, which is replicated in DEF6-knockout Jurkat cells. Mechanistically, we identify the small GTPase RAB11 as an interactor of the guanine nucleotide exchange factor DEF6, and find disrupted binding of mutant DEF6 to RAB11 as well as reduced RAB11+CTLA-4+ vesicles in DEF6-mutated cells. One of the patients has been treated with CTLA-4-Ig and achieved sustained remission. Collectively, we uncover DEF6 as player in immune homeostasis ensuring availability of the checkpoint protein CTLA-4 at T-cell surface, identifying a potential target for autoimmune and/or cancer therapy.

CEBPE-Mutant Specific Granule Deficiency Correlates With Aberrant Granule Organization and Substantial Proteome Alterations in Neutrophils.

Specific granule deficiency (SGD) is a rare disorder characterized by abnormal neutrophils evidenced by reduced granules, absence of granule proteins, and atypical bilobed nuclei. Mutations in CCAAT/enhancer-binding protein-ε (CEBPE) are one molecular etiology of the disease. Although C/EBPε has been studied extensively, the impact of CEBPE mutations on neutrophil biology remains elusive. Here, we identified two SGD patients bearing a previously described heterozygous mutation (p.Val218Ala) in CEBPE. We took this rare opportunity to characterize SGD neutrophils in terms of granule distribution and protein content. Granules of patient neutrophils were clustered and polarized, suggesting that not only absence of specific granules but also defects affecting other granules contribute to the phenotype. Our analysis showed that remaining granules displayed mixed protein content and lacked several glycoepitopes. To further elucidate the impact of mutant CEBPE, we performed detailed proteomic analysis of SGD neutrophils. Beside an absence of several granule proteins in patient cells, we observed increased expression of members of the linker of nucleoskeleton and cytoskeleton complex (nesprin-2, vimentin, and lamin-B2), which control nuclear shape. This suggests that absence of these proteins in healthy individuals might be responsible for segmented shapes of neutrophilic nuclei. We further show that the heterozygous mutation p.Val218Ala in CEBPE causes SGD through prevention of nuclear localization of the protein product. In conclusion, we uncover that absence of nuclear C/EBPε impacts on spatiotemporal expression and subsequent distribution of several granule proteins and further on expression of proteins controlling nuclear shape.

Tristetraprolin binding site atlas in the macrophage transcriptome reveals a switch for inflammation resolution.

Precise regulation of mRNA decay is fundamental for robust yet not exaggerated inflammatory responses to pathogens. However, a global model integrating regulation and functional consequences of inflammation-associated mRNA decay remains to be established. Using time-resolved high-resolution RNA binding analysis of the mRNA-destabilizing protein tristetraprolin (TTP), an inflammation-limiting factor, we qualitatively and quantitatively characterize TTP binding positions in the transcriptome of immunostimulated macrophages. We identify pervasive destabilizing and non-destabilizing TTP binding, including a robust intronic binding, showing that TTP binding is not sufficient for mRNA destabilization. A low degree of flanking RNA structuredness distinguishes occupied from silent binding motifs. By functionally relating TTP binding sites to mRNA stability and levels, we identify a TTP-controlled switch for the transition from inflammatory into the resolution phase of the macrophage immune response. Mapping of binding positions of the mRNA-stabilizing protein HuR reveals little target and functional overlap with TTP, implying a limited co-regulation of inflammatory mRNA decay by these proteins. Our study establishes a functionally annotated and navigable transcriptome-wide atlas (http://ttp-atlas.univie.ac.at) of cis-acting elements controlling mRNA decay in inflammation.