Dysbiosis exacerbates colitis by promoting ubiquitination and accumulation of the innate immune adaptor STING in myeloid cells.

Alterations in the cGAS-STING DNA-sensing pathway affect intestinal homeostasis. We sought to delineate the functional role of STING in intestinal inflammation. Increased STING expression was a feature of intestinal inflammation in mice with colitis and in humans afflicted with inflammatory bowel disease. Mice bearing an allele rendering STING constitutively active exhibited spontaneous colitis and dysbiosis, as well as progressive chronic intestinal inflammation and fibrosis. Bone marrow chimera experiments revealed STING accumulation in intestinal macrophages and monocytes as the initial driver of inflammation. Depletion of Gram-negative bacteria prevented STING accumulation in these cells and alleviated intestinal inflammation. STING accumulation occurred at the protein rather than transcript level, suggesting post-translational stabilization. We found that STING was ubiquitinated in myeloid cells, and this K63-linked ubiquitination could be elicited by bacterial products, including cyclic di-GMP. Our findings suggest a positive feedback loop wherein dysbiosis foments the accumulation of STING in intestinal myeloid cells, driving intestinal inflammation.

Development of mammary luminal progenitor cells is controlled by the transcription factor STAT5A.

Mammary alveologenesis is abrogated in the absence of the transcription factors STAT5A/5B, which mediate cytokine signaling. To reveal the underlying causes for this developmental block, we studied mammary stem and progenitor cells. While loss of STAT5A/5B did not affect the stem cell population and its ability to form mammary ducts, luminal progenitors were greatly reduced and unable to form alveoli during pregnancy. Temporally controlled expression of transgenic STAT5A in mammary epithelium lacking STAT5A/5B restored the luminal progenitor population and rescued alveologenesis in a reversible fashion in vivo. Thus, STAT5A is necessary and sufficient for the establishment of luminal progenitor cells.

Interleukin-2 inhibits FMS-like tyrosine kinase 3 receptor ligand (flt3L)-dependent development and function of conventional and plasmacytoid dendritic cells.

Steady-state development of plasmacytoid dendritic cells (pDCs) and conventional dendritic cells (cDCs) requires the ligand for FMS-like tyrosine kinase 3 receptor (flt3L), but little is known about how other cytokines may also control this process. In this study, we show that IL-2 inhibits the development of both pDCs and cDCs from bone marrow cells under flt3L stimulation, by acting on lineage(-) flt3(+) precursors. This inhibition of DC development by IL-2 requires IL-2Rα and IL2Rß. IL-2Rα is specifically expressed in one stage of the DC precursor: the monocyte and DC progenitors (MDPs). Furthermore, more MDPs are found in flt3L-stimulated bone marrow cultures when IL-2 is present, suggesting that IL-2 may be inhibiting DC development at the MDP stage. Consistent with our in vitro findings, we observe that nonobese diabetic (NOD) mice, which express less IL-2 compared with diabetes-resistant NOD.Idd3/5 mice, have more splenic pDCs. Additionally, DCs developed in vitro in the presence of flt3L and IL-2 display reduced ability to stimulate T-cell proliferation compared with DCs developed in the presence of flt3L alone. Although the addition of IL-2 does not increase the apoptosis of DCs during their development, DCs developed in the presence of IL-2 are more prone to apoptosis upon interaction with T cells. Together our data show that IL-2 can inhibit both the development and the function of DCs. This pathway may have implications for the loss of immune tolerance: Reduced IL-2 signaling may lead to increased DC number and T-cell stimulatory capacity.

Postprandial inhibition of gastric ghrelin secretion by long-chain fatty acid through GPR120 in isolated gastric ghrelin cells and mice.

Ghrelin is a gastric peptide hormone that controls appetite and energy homeostasis. Plasma ghrelin levels rise before a meal and fall quickly thereafter. Elucidation of the regulation of ghrelin secretion has been hampered by the difficulty of directly interrogating ghrelin cells diffusely scattered within the complex gastric mucosa. Therefore, we generated transgenic mice with ghrelin cell expression of green fluorescent protein (GFP) to enable characterization of ghrelin secretion in a pure population of isolated gastric ghrelin-expressing GFP (Ghr-GFP) cells. Using quantitative RT-PCR and immunofluorescence staining, we detected a high level of expression of the long-chain fatty acid (LCFA) receptor GPR120, while the other LCFA receptor, GPR40, was undetectable. In short-term-cultured pure Ghr-GFP cells, the LCFAs docosadienoic acid, linolenic acid, and palmitoleic acid significantly suppressed ghrelin secretion. The physiological mechanism of LCFA inhibition on ghrelin secretion was studied in mice. Serum ghrelin levels were transiently suppressed after gastric gavage of LCFA-rich lipid in mice with pylorus ligation, indicating that the ghrelin cell may directly sense increased gastric LCFA derived from ingested intraluminal lipids. Meal-induced increase in gastric mucosal LCFA was assessed by measuring the transcripts of markers for tissue uptake of LCFA, lipoprotein lipase (LPL), fatty acid translocase (CD36), glycosylphosphatidylinositol-anchored HDL-binding protein 1, and nuclear fatty acid receptor peroxisome proliferator-activated receptor-γ. Quantitative RT-PCR studies indicate significantly increased mRNA levels of lipoprotein lipase, glycosylphosphatidylinositol-anchored HDL-binding protein 1, and peroxisome proliferator-activated receptor-γ in postprandial gastric mucosa. These results suggest that meal-related increases in gastric mucosal LCFA interact with GPR120 on ghrelin cells to inhibit ghrelin secretion.

The G-protein-coupled receptor GPR40 directly mediates long-chain fatty acid-induced secretion of cholecystokinin.

BACKGROUND & AIMS: Long-chain fatty acid receptors G-protein-coupled receptor 40 (GPR40) (FFAR1) and GPR120 have been implicated in the chemosensation of dietary fats. I cells in the intestine secrete cholecystokinin (CCK), a peptide hormone that stimulates digestion of fat and protein, but these cells are rare and hard to identify. We sought to determine whether dietary fat-induced secretion of CCK is directly mediated by GPR40 expressed on I cells. METHODS: We used fluorescence-activated cell sorting to isolate a pure population of I cells from duodenal mucosa in transgenic mice that expressed green fluorescent protein under the control of the CCK promoter (CCK-enhanced green fluorescent protein [eGFP] bacterial artificial chromosome mice). CCK-eGFP cells were evaluated for GPR40 expression by quantitative reverse transcription polymerase chain reaction and immunostaining. GPR40(-/-) mice were bred with CCK-eGFP mice to evaluate functional relevance of GPR40 on long-chain fatty acid-stimulated increases in [Ca(2+)]i and CCK secretion in isolated CCK-eGFP cells. Plasma levels of CCK after olive oil gavage were compared between GPR40(+/+) and GPR40(-/-) mice. RESULTS: Cells that expressed eGFP also expressed GPR40; expression of GPR40 was 100-fold greater than that of cells that did not express eGFP. In vitro, linoleic, oleic, and linolenic acids increased [Ca(2+)]i; linolenic acid increased CCK secretion by 53% in isolated GPR40(+/+) cells that expressed eGFP. In contrast, in GPR40(-/-) that expressed eGFP, [Ca(2+)]i response to linoleic acid was reduced by 50% and there was no significant CCK secretion in response to linolenic acid. In mice, olive oil gavage significantly increased plasma levels of CCK compared with pregavage levels: 5.7-fold in GPR40(+/+) mice and 3.1-fold in GPR40(-/-) mice. CONCLUSIONS: Long-chain fatty acid receptor GPR40 induces secretion of CCK by I cells in response to dietary fat.

The extracellular calcium-sensing receptor is required for cholecystokinin secretion in response to L-phenylalanine in acutely isolated intestinal I cells.

The extracellular calcium-sensing receptor (CaSR) has recently been recognized as an L-amino acid sensor and has been implicated in mediating cholecystokinin (CCK) secretion in response to aromatic amino acids. We investigated whether direct detection of L-phenylalanine (L-Phe) by CaSR results in CCK secretion in the native I cell. Fluorescence-activated cell sorting of duodenal I cells from CCK-enhanced green fluorescent protein (eGFP) transgenic mice demonstrated CaSR gene expression. Immunostaining of fixed and fresh duodenal tissue sections confirmed CaSR protein expression. Intracellular calcium fluxes were CaSR dependent, stereoselective for L-Phe over D-Phe, and responsive to type II calcimimetic cinacalcet in CCK-eGFP cells. Additionally, CCK secretion by an isolated I cell population was increased by 30 and 62% in response to L-Phe in the presence of physiological (1.26 mM) and superphysiological (2.5 mM) extracellular calcium concentrations, respectively. While the deletion of CaSR from CCK-eGFP cells did not affect basal CCK secretion, the effect of L-Phe or cinacalcet on intracellular calcium flux was lost. In fact, both secretagogues, as well as superphysiological Ca(2+), evoked an unexpected 20-30% decrease in CCK secretion compared with basal secretion in CaSR(-/-) CCK-eGFP cells. CCK secretion in response to KCl or tryptone was unaffected by the absence of CaSR. The present data suggest that CaSR is required for hormone secretion in the specific response to L-Phe by the native I cell, and that a receptor-mediated mechanism may inhibit hormone secretion in the absence of a fully functional CaSR.

Novel Observations From Next-Generation RNA Sequencing of Highly Purified Human Adult and Fetal Islet Cell Subsets.

Understanding distinct gene expression patterns of normal adult and developing fetal human pancreatic α- and ß-cells is crucial for developing stem cell therapies, islet regeneration strategies, and therapies designed to increase ß-cell function in patients with diabetes (type 1 or 2). Toward that end, we have developed methods to highly purify α-, ß-, and δ-cells from human fetal and adult pancreata by intracellular staining for the cell-specific hormone content, sorting the subpopulations by flow cytometry, and, using next-generation RNA sequencing, we report the detailed transcriptomes of fetal and adult α- and ß-cells. We observed that human islet composition was not influenced by age, sex, or BMI, and transcripts for inflammatory gene products were noted in fetal ß-cells. In addition, within highly purified adult glucagon-expressing α-cells, we observed surprisingly high insulin mRNA expression, but not insulin protein expression. This transcriptome analysis from highly purified islet α- and ß-cell subsets from fetal and adult pancreata offers clear implications for strategies that seek to increase insulin expression in type 1 and type 2 diabetes.

Transcriptional analysis of intracytoplasmically stained, FACS-purified cells by high-throughput, quantitative nuclease protection.

Analyzing specialized cells in heterogeneous tissues is crucial for understanding organ function in health and disease. Thus far, however, there has been no convenient method for studying gene expression in cells purified by fluorescence-activated cell sorting (FACS) using intracellular markers. Here we show that the quantitative nuclease protection assay (qNPA) enables transcriptional analysis of intracytoplasmically stained cells sorted by FACS. Applying the method to mouse pancreatic islet-cell subsets, we detected both expected and unknown lineage-specific gene expression patterns. Some beta cells from pregnant animals were found to express Mafb, previously observed only in immature beta cells during embryonic development. The four 'housekeeping' genes tested were expressed in purified islet-cell subpopulations with a notable variability, dependent on both cell lineage and developmental stage. Application of qNPA to intracellularly stained, FACS-sorted cells should be broadly applicable to the analysis of gene expression in subpopulations of any heterogeneous tissue, including tumors.