Single-Nucleus and In Situ RNA-Sequencing Reveal Cell Topographies in the Human Pancreas.

BACKGROUND & AIMS: Molecular evidence of cellular heterogeneity in the human exocrine pancreas has not been yet established because of the local concentration and cascade of hydrolytic enzymes that can rapidly degrade cells and RNA upon pancreatic resection. We sought to better understand the heterogeneity and cellular composition of the pancreas in neonates and adults in healthy and diseased conditions using single-cell sequencing approaches. METHODS: We innovated single-nucleus RNA-sequencing protocols and profiled more than 120,000 cells from pancreata of adult and neonatal human donors. We validated the single-nucleus findings using RNA fluorescence in situ hybridization, in situ sequencing, and computational approaches. RESULTS: We created the first comprehensive atlas of human pancreas cells including epithelial and nonepithelial constituents, and uncovered 3 distinct acinar cell types, with possible implications for homeostatic and inflammatory processes of the pancreas. The comparison with neonatal single-nucleus sequencing data showed a different cellular composition of the endocrine tissue, highlighting the tissue dynamics occurring during development. By applying spatial cartography, involving cell proximity mapping through in situ sequencing, we found evidence of specific cell type neighborhoods, dynamic topographies in the endocrine and exocrine pancreas, and principles of morphologic organization of the organ. Furthermore, similar analyses in chronic pancreatitis biopsy samples showed the presence of acinar-REG+ cells, a reciprocal association between macrophages and activated stellate cells, and a new potential role of tuft cells in this disease. CONCLUSIONS: Our human pancreas cell atlas can be interrogated to understand pancreatic cell biology and provides a crucial reference set for comparisons with diseased tissue samples to map the cellular foundations of pancreatic diseases.

Treatment with Allogenic Mesenchymal Stromal Cells in a Murine Model of Systemic Lupus Erythematosus.

OBJECTIVE: Pre-clinical and uncontrolled studies in patients with systemic lupus erythematosus (SLE) showed that mesenchymal stromal cells (MSCs) have a potential therapeutic role in refractory cases. The optimal therapeutic strategy in these patients remain to be elucidated. Our aim was to test the hypothesis that repeated administrations of 1×106/kg body weight of allogenic MSCs, that is a significantly lower dosage with respect to the fixed 1×106 MSC used in animal models, can be effective in improving the clinical course of a murine SLE model. METHODS: Bone marrow derived MSCs were obtained from 12-week-old C57BL/6J mice. Seventy-five 8 weeks old female NZ mice were randomly assigned to receive via caudal vein the following alternative treatments: 1) single infusion of 106 MSCs/kg body weight at 18 weeks of age (NZs18) or at at 22 weeks of age (NZs22); 2) multiple monthly infusions of 106 MSCs/kg body weight starting at 18 weeks of age (NZM18) or at 22 weeks of age (NZM22); 3) saline infusions (NZc) Fifteen 8 weeks old C57BL/6J mice (Envigo, Huntingdon, UK) were used as untreated controls (C). Weekly, body weight was recorded and twenty-four hour urines were collected by metabolic cages for each animal; proteinuria was detected by dipstick analysis. At sacrifice, peripheral blood samples were collected from mice and anti-dsDNA antibodies were detected by enzyme immunoassorbent assay (ELISA) method using commercial kits. At sacrifice, kidneys were analyzed for histopathology and immunohistochemical analysis for B220, CD4, MPO, CD4＋Foxp3, F40/80 infiltration was performed. RESULTS: Proteinuria occurrence was delayed NZS and NZM mice, no differences were observed in anti-dsDNA autoantibody titer among the groups at the different time-points; at 36 weeks, no significant differences were observed in term of nephritis scores. Inflammatory cells deposition (MPO and F4/80 positive cells) in NZM was significantly higher than in NZ and NZS. An overexpression of B lymphocytes (B220) was found in NZM while T regulatory cells (CD4＋ Foxp3＋ cells) were reduced in both NZS and NZM with respect to NZc. CONCLUSIONS: Overall, our study failed to show a positive effect of a treatment with murine MSCs in this model and, for some aspects, even deleterious results seem to be observed.

Tissue-infiltrating plasma cells are an important source of carboxylesterase 2 contributing to the therapeutic efficacy of prodrugs.

Carboxylesterase 2 (CES-2) is instrumental for conversion of ester-containing prodrugs in cancer treatment. CES-2 expression was analyzed by immunohistochemistry in colorectal cancer (CRC) compared to colonic inflammation as well as in liver and peripheral blood. In CRC, tumor grades showed no correlation with levels of CES-2 expression, which was heterogeneous within these tumors. Cellular infiltrates in the immediate tumor vicinity expressed high levels of CES-2. Thus, tissue adjacent to the tumor was a substantial source of CES-2 with high expression in plasma cells. CES-2(high) plasma cells were abundantly found in the colon of patients with inflammatory bowel disease. CES-2 expression is strong in hepatocytes of normal livers, while CES-2 expression in peripheral blood mononuclear cells of healthy donors was overall low at protein and mRNA levels. In summary, the conversion of ester-containing prodrugs by CES-2 is mainly to occur in the periphery, during liver passage and in the colon after enterohepatic recirculation. We here demonstrated plasma cells as strong producers of CES-2. Further studies should elucidate the role of CES-2(+) plasma cells in intestinal inflammation and cancer.

A guide to histomorphological evaluation of intestinal inflammation in mouse models.

Histomorphology remains a powerful routine evaluating intestinal inflammation in animal models. Emphasizing the focus of a given animal study, histopathology can overstate differences between established models. We aimed to systematize histopathological evaluation of intestinal inflammation in mouse models facilitating inter-study comparisons. Samples of all parts of the intestinal tract from well-established mouse models of intestinal inflammation were evaluated from hematoxylin/eosin-stained sections and specific observations confirmed by subsequent immunohistochemistry. Three main categories sufficiently reflected the severity of histopathology independent of the localization and the overall extent of an inflammation: (i) quality and dimension of inflammatory cell infiltrates, (ii) epithelial changes and (iii) overall mucosal architecture. Scoring schemata were defined along specified criteria for each of the three categories. The direction of the initial hit proved crucial for the comparability of histological changes. Chemical noxes, infection with intestinal parasites or other models where the barrier was disturbed from outside, the luminal side, showed high levels of similarity and distinct differences to changes in the intestinal balance resulting from inside events like altered cytokine responses or disruption of the immune cell homeostasis. With a high degree of generalisation and maximum scores from 4-8 suitable scoring schemata accounted specific histopathological hallmarks. Truly integrating demands and experiences of gastroenterologists, mouse researchers, microbiologists and pathologists we provide an easy-to-use guideline evaluating histomorphology in mouse models of intestinal inflammation. Standard criteria and definitions facilitate classification and rating of new relevant models, allow comparison in animal studies and transfer of functional findings to comparable histopathologies in human disease.

Splenic proliferative lymphoid nodules distinct from germinal centers are sites of autoantigen stimulation in immune thrombocytopenia.

To understand more specific abnormalities of humoral autoimmunity, we studied 31 spleens from immune thrombocytopenia (ITP) patients and 36 control spleens. Detailed analysis identified at least 2 different splenic structures accommodating proliferating B cells, classic germinal centers (GCs), and proliferative lymphoid nodules (PLNs). PLNs were characterized by proliferating Ki67(+) B cells close to follicular dendritic cells (FDCs) and lacked polarization into dark and light zones. As opposed to cells in GCs, proliferating B cells in PLN lacked expression of Bcl6. In both PLNs and GCs of ITP spleens, the density of T cells was significantly reduced. Both T follicular helper cells (T(FH)) and regulatory T cells were reduced within PLNs of ITP spleens suggesting a defect of tolerance related to a loss of T-cell control. Within PLNs of ITP, but not controls, abundant platelet glycoprotein (GP) IIb/IIIa autoantigens was found in IgM containing immune complexes tightly bound to FDCs and closely approximated to proliferating B cells. GPIV was found less often, but not in the same PLNs as GPIIb/IIIa. Autoantigens were not found in the GCs of ITP or controls indicating that PLNs are the sites of autoantigen stimulation in ITP potentially related to a lack of control by T cells and/or the present autoantigen.