Human small intestine contains 2 functionally distinct regulatory T-cell subsets.

BACKGROUND: Regulatory T (Treg) CD4 cells in mouse gut are mainly specific for intestinal antigens and play an important role in the suppression of immune responses against harmless dietary antigens and members of the microbiota. However, information about the phenotype and function of Treg cells in the human gut is limited. OBJECTIVE: We performed a detailed characterization of Foxp3+ CD4 Treg cells in human normal small intestine (SI) as well as from transplanted duodenum and celiac disease lesions. METHODS: Treg cells and conventional CD4 T cells derived from SI were subjected to extensive immunophenotyping and their suppressive activity and ability to produce cytokines assessed. RESULTS: SI Foxp3+ CD4 T cells were CD45RA-CD127-CTLA-4+ and suppressed proliferation of autologous T cells. Approximately 60% of Treg cells expressed the transcription factor Helios. When stimulated, Helios-negative Treg cells produced IL-17, IFN-γ, and IL-10, whereas Helios-positive Treg cells produced very low levels of these cytokines. By sampling mucosal tissue from transplanted human duodenum, we demonstrated that donor Helios-negative Treg cells persisted for at least 1 year after transplantation. In normal SI, Foxp3+ Treg cells constituted only 2% of all CD4 T cells, while in active celiac disease, both Helios-negative and Helios-positive subsets expanded 5- to 10-fold. CONCLUSION: The SI contains 2 subsets of Treg cells with different phenotypes and functional capacities. Both subsets are scarce in healthy gut but increase dramatically in active celiac disease.

RvD1n-3 DPA Downregulates the Transcription of Pro-Inflammatory Genes in Oral Epithelial Cells and Reverses Nuclear Translocation of Transcription Factor p65 after TNF-α Stimulation.

Specialized pro-resolving mediators (SPMs) are multifunctional lipid mediators that participate in the resolution of inflammation. We have recently described that oral epithelial cells (OECs) express receptors of the SPM resolvin RvD1n-3 DPA and that cultured OECs respond to RvD1n-3 DPA addition by intracellular calcium release, nuclear receptor translocation and transcription of genes coding for antimicrobial peptides. The aim of the present study was to assess the functional outcome of RvD1n-3 DPA-signaling in OECs under inflammatory conditions. To this end, we performed transcriptomic analyses of TNF-α-stimulated cells that were subsequently treated with RvD1n-3 DPA and found significant downregulation of pro-inflammatory nuclear factor kappa B (NF-κB) target genes. Further bioinformatics analyses showed that RvD1n-3 DPA inhibited the expression of several genes involved in the NF-κB activation pathway. Confocal microscopy revealed that addition of RvD1n-3 DPA to OECs reversed TNF-α-induced nuclear translocation of NF-κB p65. Co-treatment of the cells with the exportin 1 inhibitor leptomycin B indicated that RvD1n-3 DPA increases nuclear export of p65. Taken together, our observations suggest that SPMs also have the potential to be used as a therapeutic aid when inflammation is established.

Topoisomerase IV tracks behind the replication fork and the SeqA complex during DNA replication in Escherichia coli.

Topoisomerase IV (TopoIV) is a vital bacterial enzyme which disentangles newly replicated DNA and enables segregation of daughter chromosomes. In bacteria, DNA replication and segregation are concurrent processes. This means that TopoIV must continually remove inter-DNA linkages during replication. There exists a short time lag of about 10-20 min between replication and segregation in which the daughter chromosomes are intertwined. Exactly where TopoIV binds during the cell cycle has been the subject of much debate. We show here that TopoIV localizes to the origin proximal side of the fork trailing protein SeqA and follows the movement pattern of the replication machinery in the cell.

Early transcriptional changes after UVB treatment in atopic dermatitis include inverse regulation of IL-36γ and IL-37.

Phototherapy with narrow-band Ultraviolet B (nb-UVB) is a major therapeutic option in atopic dermatitis (AD), yet knowledge of the early molecular responses to this treatment is lacking. The objective of this study was to map the early transcriptional changes in AD skin in response to nb-UVB treatment. Adult patients (n = 16) with AD were included in the study and scored with validated scoring tools. AD skin was irradiated with local nb-UVB on day 0, 2 and 4. Skin biopsies were taken before and after treatment (day 0 and 7) and analysed for genome-wide modulation of transcription. When examining the early response after three local UVB treatments, gene expression analysis revealed 77 significantly modulated transcripts (30 down- and 47 upregulated). Among them were transcripts related to the inflammatory response, melanin synthesis, keratinization and epidermal structure. Interestingly, the pro-inflammatory cytokine IL-36γ was reduced after treatment, while the anti-inflammatory cytokine IL-37 increased after treatment with nb-UVB. There was also a modulation of several other mediators involved in inflammation, among them defensins and S100 proteins. This is the first study of early transcriptomic changes in AD skin in response to nb-UVB. We reveal robust modulation of a small group of inflammatory and anti-inflammatory targets, including the IL-1 family members IL36γ and IL-37, which is evident before any detectable changes in skin morphology or immune cell infiltrates. These findings provide important clues to the molecular mechanisms behind the treatment response and shed light on new potential treatment targets.

CD4+ T cells persist for years in the human small intestine and display a TH1 cytokine profile.

Studies in mice and humans have shown that CD8+ T cell immunosurveillance in non-lymphoid tissues is dominated by resident populations. Whether CD4+ T cells use the same strategies to survey peripheral tissues is less clear. Here, examining the turnover of CD4+ T cells in transplanted duodenum in humans, we demonstrate that the majority of CD4+ T cells were still donor-derived one year after transplantation. In contrast to memory CD4+ T cells in peripheral blood, intestinal CD4+ TRM cells expressed CD69 and CD161, but only a minor fraction expressed CD103. Functionally, intestinal CD4+ TRM cells were very potent cytokine producers; the vast majority being polyfunctional TH1 cells, whereas a minor fraction produced IL-17. Interestingly, a fraction of intestinal CD4+ T cells produced granzyme-B and perforin after activation. Together, we show that the intestinal CD4+ T-cell compartment is dominated by resident populations that survive for more than 1 year. This finding is of high relevance for the development of oral vaccines and therapies for diseases in the gut.

The Long-lived Protein Degradation Assay: an Efficient Method for Quantitative Determination of the Autophagic Flux of Endogenous Proteins in Adherent Cell Lines.

Autophagy is a key player in the maintenance of cellular homeostasis in eukaryotes, and numerous diseases, including cancer and neurodegenerative disorders, are associated with alterations in autophagy. The interest for studying autophagy has grown intensely in the last two decades, and so has the arsenal of methods utilised to study this highly dynamic and complex process. Changes in the expression and/or localisation of autophagy-related proteins are frequently assessed by Western blot and various microscopy techniques. Such analyses may be indicative of alterations in autophagy-related processes and informative about the specific marker being investigated. However, since these proteins are part of the autophagic machinery, and not autophagic cargo, they cannot be used to draw conclusions regarding autophagic cargo flux. Here, we provide a protocol to quantitatively assess bulk autophagic flux by employing the long-lived protein degradation assay. Our procedure, which traces the degradation of 14C valine-labelled proteins, is simple and quick, allows for processing of a relatively large number of samples in parallel, and can in principle be used with any adherent cell line. Most importantly, it enables quantitative measurements of endogenous cargo flux through the autophagic pathway. As such, it is one of the gold standards for studying autophagic activity.

Rab7b modulates autophagic flux by interacting with Atg4B.

Autophagy (macroautophagy) is a highly conserved eukaryotic degradation pathway in which cytosolic components and organelles are sequestered by specialized autophagic membranes and degraded through the lysosomal system. The autophagic pathway maintains basal cellular homeostasis and helps cells adapt during stress; thus, defects in autophagy can cause detrimental effects. It is therefore crucial that autophagy is properly regulated. In this study, we show that the cysteine protease Atg4B, a key enzyme in autophagy that cleaves LC3, is an interactor of the small GTPase Rab7b. Indeed, Atg4B interacts and co-localizes with Rab7b on vesicles. Depletion of Rab7b increases autophagic flux as indicated by the increased size of autophagic structures as well as the magnitude of macroautophagic sequestration and degradation. Importantly, we demonstrate that Rab7b regulates LC3 processing by modulating Atg4B activity. Taken together, our findings reveal Rab7b as a novel negative regulator of autophagy through its interaction with Atg4B.

Novel steps in the autophagic-lysosomal pathway.

Autophagy is the process by which portions of cytoplasm are enclosed by membranous organelles, phagophores, which deliver the sequestered cytoplasm to degradative autophagic vacuoles. Genes and proteins involved in phagophore manufacture have been extensively studied, but little is known about how mature phagophores proceed through the subsequent steps of expansion, closure and fusion. Here we have addressed these issues by combining our unique autophagic cargo sequestration assay (using the cytosolic enzyme lactate dehydrogenase as a cargo marker) with quantitative measurements of the lipidation-dependent anchorage and turnover of the phagophore-associated protein LC3. In isolated rat hepatocytes, amino acid starved to induce maximal autophagic activity, the two unrelated reversible autophagy inhibitors 3-methyladenine (3MA) and thapsigargin (TG) both blocked cargo sequestration completely. However, whereas 3MA inhibited LC3 lipidation, TG did not, thus apparently acting at a post-lipidation step to prevent phagophore closure. Intriguingly, the resumption of cargo sequestration seen upon release from a reversible TG block was completely suppressed by 3MA, revealing that 3MA not only inhibits LC3 lipidation but also (like TG) blocks phagophore closure at a post-lipidation step. 3MA did not, however, prevent the resumption of lysosomal LC3 degradation, indicating that phagophores could fuse directly with degradative autophagic vacuoles without carrying cytosolic cargo. This fusion step was clearly blocked by TG. Furthermore, density gradient centrifugation revealed that a fraction of the LC3-marked phagophores retained by TG could be density-shifted by the acidotropic drug propylamine along with the lysosomal marker cathepsin B, suggesting physical association of some phagophores with lysosomes prior to cargo sequestration.

Autophagic bulk sequestration of cytosolic cargo is independent of LC3, but requires GABARAPs.

LC3, a mammalian homologue of yeast Atg8, is assumed to play an important part in bulk sequestration and degradation of cytoplasm (macroautophagy), and is widely used as an indicator of this process. To critically examine its role, we followed the autophagic flux of LC3 in rat hepatocytes during conditions of maximal macroautophagic activity (amino acid depletion), combined with analyses of macroautophagic cargo sequestration, measured as transfer of the cytosolic protein lactate dehydrogenase (LDH) to sedimentable organelles. To accurately determine LC3 turnover we developed a quantitative immunoblotting procedure that corrects for differential immunoreactivity of cytosolic and membrane-associated LC3 forms, and we included cycloheximide to block influx of newly synthesized LC3. As expected, LC3 was initially degraded by the autophagic-lysosomal pathway, but, surprisingly, autophagic LC3-flux ceased after ~2h. In contrast, macroautophagic cargo flux was well maintained, and density gradient analysis showed that sequestered LDH partly accumulated in LC3-free autophagic vacuoles. Hepatocytic macroautophagy could thus proceed independently of LC3. Silencing of either of the two mammalian Atg8 subfamilies in LNCaP prostate cancer cells exposed to macroautophagy-inducing conditions (starvation or the mTOR-inhibitor Torin1) confirmed that macroautophagic sequestration did not require the LC3 subfamily, but, intriguingly, we found the GABARAP subfamily to be essential.

Dynamic Escherichia coli SeqA complexes organize the newly replicated DNA at a considerable distance from the replisome.

The Escherichia coli SeqA protein binds to newly replicated, hemimethylated DNA behind replication forks and forms structures consisting of several hundred SeqA molecules bound to about 100 kb of DNA. It has been suggested that SeqA structures either direct the new sister DNA molecules away from each other or constitute a spacer that keeps the sisters together. We have developed an image analysis script that automatically measures the distance between neighboring foci in cells. Using this tool as well as direct stochastic optical reconstruction microscopy (dSTORM) we find that in cells with fluorescently tagged SeqA and replisome the sister SeqA structures were situated close together (less than about 30 nm apart) and relatively far from the replisome (on average 200-300 nm). The results support the idea that newly replicated sister molecules are kept together behind the fork and suggest the existence of a stretch of DNA between the replisome and SeqA which enjoys added stabilization. This could be important in facilitating DNA transactions such as recombination, mismatch repair and topoisomerase activity. In slowly growing cells without ongoing replication forks the SeqA protein was found to reside at the fully methylated origins prior to initiation of replication.

Macroautophagic cargo sequestration assays.

Macroautophagy, the process responsible for bulk sequestration and lysosomal degradation of cytoplasm, is often monitored by means of the autophagy-related marker protein LC3. This protein is linked to the phagophoric membrane by lipidation during the final steps of phagophore assembly, and it remains associated with autophagic organelles until it is degraded in the lysosomes. The transfer of LC3 from cytosol to membranes and organelles can be measured by immunoblotting or immunofluorescence microscopy, but these assays provide no information about functional macroautophagic activity, i.e., whether the phagophores are actually engaged in the sequestration of cytoplasmic cargo and enclosing this cargo into sealed autophagosomes. Moreover, accumulating evidence suggest that macroautophagy can proceed independently of LC3. There is therefore a need for alternative methods, preferably effective cargo sequestration assays, which can monitor actual macroautophagic activity. Here, we provide an overview of various approaches that have been used over the last four decades to measure macroautophagic sequestration activity in mammalian cells. Particular emphasis is given to the so-called "LDH sequestration assay", which measures the transfer of the autophagic cargo marker enzyme LDH (lactate dehydrogenase) from the cytosol to autophagic vacuoles. The LDH sequestration assay was originally developed to measure macroautophagic activity in primary rat hepatocytes. Subsequently, it has found use in several other cell types, and in this article we demonstrate a further validation and simplification of the method, and show that it is applicable to several cell lines that are commonly used to study autophagy.

Modulation of intracellular calcium homeostasis blocks autophagosome formation.

Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stress-the calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca (2+)-ATPase inhibitor thapsigargin (TG)-potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.

Autophagic activity measured in whole rat hepatocytes as the accumulation of a novel BHMT fragment (p10), generated in amphisomes by the asparaginyl proteinase, legumain.

To investigate the stepwise autophagic-lysosomal processing of hepatocellular proteins, the abundant cytosolic enzyme, betaine:homocysteine methyltransferase (BHMT) was used as a probe. Full-length (45 kDa) endogenous BHMT was found to be cleaved in an autophagy-dependent (3-methyladenine-sensitive) manner in isolated rat hepatocytes to generate a novel N-terminal 10-kDa fragment (p10) identified and characterized by mass spectrometry. The cleavage site was consistent with cleavage by the asparaginyl proteinase, legumain and indeed a specific inhibitor of this enzyme (AJN-230) was able to completely suppress p10 formation in intact cells, causing instead accumulation of a 42-kDa intermediate. To prevent further degradation of p10 or p42 by the cysteine proteinases present in autophagic vacuoles, the proteinase inhibitor leupeptin had to be present. Asparagine, an inhibitor of amphisome-lysosome fusion, did not detectably impede either p42 or p10 formation, indicating that BHMT processing primarily takes place in amphisomes rather than in lysosomes. Lactate dehydrogenase (LDH) was similarly degraded primarily in amphisomes by leupeptin-sensitive proteolysis, but some additional leupeptin-resistant LDH degradation in lysosomes was also indicated. The autophagic sequestration of BHMT appeared to be nonselective, as the accumulation of p10 (in the presence of leupeptin) or of its precursors (in the additional presence of AJN-230) proceeded at approximately the same rate as the model autophagic cargo, LDH. The complete lack of a cytosolic background makes p10 suitable for use in a "fragment assay" of autophagic activity in whole cells. Incubation of hepatocytes with ammonium chloride, which neutralizes amphisomes as well as lysosomes, caused rapid, irreversible inhibition of legumain activity and stopped all p10 formation. The availability of several methods for selective targeting of legumain in intact cells may facilitate functional studies of this enigmatic enzyme, and perhaps suggest novel ways to reduce its contribution to cancer cell metastasis or autoimmune disease.

Sequestration assays for mammalian autophagy.

Macroautophagic activity is most directly and precisely measured by a cargo sequestration assay. Long-lived, cytosolic proteins that are degraded exclusively by the autophagic-lysosomal pathway, such as lactate dehydrogenase (LDH) are suitable as endogenous sequestration probes. Autophagic sequestration is measured as transfer of the protein from the soluble (cytosolic) to the sedimentable (organelle-containing) cell fraction, using leupeptin or other proteinase inhibitors to block inactivation and degradation of the protein inside autophagic vacuoles. A convenient separation method is electrodisruption of the cells, followed by sedimentation of the organelle fraction through a Nycodenz density cushion. A promising variant of the cargo assay is to use a protein probe that is processed by the autophagic-lysosomal pathway so as to generate an intravacuolar fragment. Because there is no cytosolic background, subcellular fractionation is unnecessary, allowing the use of the autophagic fragment assay to measure autophagic activity in whole cells. In hepatocytes, a small fragment, p10(BHMT), made by autophagic processing of the enzyme betaine:homocysteine methyltransferase, thus accumulates in an autophagy-dependent manner in the presence of leupeptin. Autophagic sequestration can also be measured by using exogenous cargo probes, such as radiolabeled di- and trisaccharides, which can be loaded into the cytosol of hepatocytes by reversible electrodisruption or mechanical stress. Raffinose is the preferable probe for measurement of autophagic activity, whereas sucrose (which can be hydrolyzed in amphisomes and lysosomes by added endocytosed invertase) and lactose (which is hydrolyzed in lysosomes by the endogenous beta-galactosidase) are useful for dissection of the various steps in the autophagic-lysosomal pathway and for studying autophagic-endocytic interactions. Furthermore, the intralysosomal hydrolysis of autophagocytosed lactose can be measured in whole cells (as formation of the hydrolysis product, galactose), thus providing a background-free assay (autophagic lactolysis) of the overall autophagic-lysosomal pathway.