Alzheimer's disease-related presenilins are key to intestinal epithelial cell function and gut immune homoeostasis.

OBJECTIVE: Mutations in presenilin genes are the major cause of Alzheimer's disease. However, little is known about their expression and function in the gut. In this study, we identify the presenilins Psen1 and Psen2 as key molecules that maintain intestinal homoeostasis. DESIGN: Human inflammatory bowel disease (IBD) and control samples were analysed for Psen1 expression. Newly generated intestinal epithelium-specific Psen1-deficient, Psen2-deficient and inducible Psen1/Psen2 double-deficient mice were used to dissect the functional role of presenilins in intestinal homoeostasis. RESULTS: Psen1 expression was regulated in experimental gut inflammation and in patients with IBD. Induced deletion of Psen1 and Psen2 in mice caused rapid weight loss and spontaneous development of intestinal inflammation. Mice exhibited epithelial barrier disruption with bacterial translocation and deregulation of key pathways for nutrient uptake. Wasting disease was independent of gut inflammation and dysbiosis, as depletion of microbiota rescued Psen-deficient animals from spontaneous colitis development but not from weight loss. On a molecular level, intestinal epithelial cells lacking Psen showed impaired Notch signalling and dysregulated epithelial differentiation. CONCLUSION: Overall, our study provides evidence that Psen1 and Psen2 are important guardians of intestinal homoeostasis and future targets for barrier-promoting therapeutic strategies in IBD.

ATP citrate lyase (ACLY)-dependent immunometabolism in mucosal T cells drives experimental colitis in vivo.

OBJECTIVE: Mucosal T cells play a major role in inflammatory bowel disease (IBD). However, their immunometabolism during intestinal inflammation is poorly understood. Due to its impact on cellular metabolism and proinflammatory immune cell function, we here focus on the enzyme ATP citrate lyase (ACLY) in mucosal T cell immunometabolism and its relevance for IBD. DESIGN: ACLY expression and its immunometabolic impact on colitogenic T cell function were analysed in mucosal T cells from patients with IBD and in two experimental colitis models. RESULTS: ACLY was markedly expressed in colon tissue under steady-state conditions but was significantly downregulated in lamina propria mononuclear cells in experimental dextran sodium sulfate-induced colitis and in CD4+ and to a lesser extent in CD8+ T cells infiltrating the inflamed gut in patients with IBD. ACLY-deficient CD4+ T cells showed an impaired capacity to induce intestinal inflammation in a transfer colitis model as compared with wild-type T cells. Assessment of T cell immunometabolism revealed that ACLY deficiency dampened the production of IBD-relevant cytokines and impaired glycolytic ATP production but enriched metabolites involved in the biosynthesis of phospholipids and phosphatidylcholine. Interestingly, the short-chain fatty acid butyrate was identified as a potent suppressor of ACLY expression in T cells, while IL-36α and resolvin E1 induced ACLY levels. In a translational approach, in vivo administration of the butyrate prodrug tributyrin downregulated mucosal infiltration of ACLYhigh CD4+ T cells and ameliorated chronic colitis. CONCLUSION: ACLY controls mucosal T cell immunometabolism and experimental colitis. Therapeutic modulation of ACLY expression in T cells emerges as a novel strategy to promote the resolution of intestinal inflammation.

Functional and spatial proteomics profiling reveals intra- and intercellular signaling crosstalk in colorectal cancer.

Precision oncology approaches for patients with colorectal cancer (CRC) continue to lag behind other solid cancers. Functional precision oncology-a strategy that is based on perturbing primary tumor cells from cancer patients-could provide a road forward to personalize treatment. We extend this paradigm to measuring proteome activity landscapes by acquiring quantitative phosphoproteomic data from patient-derived organoids (PDOs). We show that kinase inhibitors induce inhibitor- and patient-specific off-target effects and pathway crosstalk. Reconstruction of the kinase networks revealed that the signaling rewiring is modestly affected by mutations. We show non-genetic heterogeneity of the PDOs and upregulation of stemness and differentiation genes by kinase inhibitors. Using imaging mass-cytometry-based profiling of the primary tumors, we characterize the tumor microenvironment (TME) and determine spatial heterocellular crosstalk and tumor-immune cell interactions. Collectively, we provide a framework for inferring tumor cell intrinsic signaling and external signaling from the TME to inform precision (immuno-) oncology in CRC.

Individual plumage and integument scoring of laying hens on commercial farms: correlation with severe feather pecking and prognosis by visual scoring on flock level.

Various plumage and integument scoring methods are commonly used to deduce the occurrence of severe feather pecking and cannibalism in laying hens. The aim of our study was to provide evidence of correlations between the occurrence of severe feather pecking and our individual plumage scoring system used under practical conditions on commercial farms with non-beak-trimmed and beak-trimmed layers (study I). In second step, we aimed to verify whether the results of the elaborate individual scoring may be predicted with a visual scoring method based on the total body scores of groups of birds (study II). For study I we observed the pecking behavior and performed an individual plumage scoring at the beginning, in the middle, and at the end of a laying period on 8 commercial farms. For study II we performed both an individual and a visual plumage scoring on 49 flocks on 45 farms at the beginning of the laying period and on 43 flocks on 41 farms at the end of the laying period. Spearman's Rho revealed a correlation of the mean feather pecking rate with the total plumage score, the neck-back plumage score, and the total cannibalism score in all observation periods. A high feather pecking rate was correlated with severe plumage damage and the frequent occurrence of skin injuries. We conclude that both the total plumage score and the neck-back plumage score constitute a reliable indicator of the occurrence of severe feather pecking in the flocks assessed in this study. The results of study II suggest that the percental assessment of plumage damage on flock level in 3 categories ("visual score") leads to a good prognosis of the actual, individually assessed plumage score. Therefore, the application (and documentation) of the visual score on a regular basis can provide a good evaluation of the development of the plumage condition of the flock. The visual score presented in this study is suggested as a suitable instrument for self-evaluation programs on farms.

Comprehensive characterization of the prostate tumor microenvironment identifies CXCR4/CXCL12 crosstalk as a novel antiangiogenic therapeutic target in prostate cancer.

BACKGROUND: Crosstalk between neoplastic and stromal cells fosters prostate cancer (PCa) progression and dissemination. Insight in cell-to-cell communication networks provides new therapeutic avenues to mold processes that contribute to PCa tumor microenvironment (TME) alterations. Here we performed a detailed characterization of PCa tumor endothelial cells (TEC) to delineate intercellular crosstalk between TEC and the PCa TME. METHODS: TEC isolated from 67 fresh radical prostatectomy (RP) specimens underwent multi-omic ex vivo characterization as well as orthogonal validation of both TEC functions and key markers by immunohistochemistry (IHC) and immunofluorescence (IF). To identify cell-cell interaction targets in TEC, we performed single-cell RNA sequencing (scRNA-seq) in four PCa patients who underwent a RP to catalogue cellular TME composition. Targets were cross-validated using IHC, publicly available datasets, cell culture expriments as well as a PCa xenograft mouse model. RESULTS: Compared to adjacent normal endothelial cells (NEC) bulk RNA-seq analysis revealed upregulation of genes associated with tumor vasculature, collagen modification and extracellular matrix remodeling in TEC. PTGIR, PLAC9, CXCL12 and VDR were identified as TEC markers and confirmed by IF and IHC in an independent patient cohort. By scRNA-seq we identified 27 cell (sub)types, including endothelial cells (EC) with arterial, venous and immature signatures, as well as angiogenic tip EC. A focused molecular analysis revealed that arterial TEC displayed highest CXCL12 mRNA expression levels when compared to all other TME cell (sub)populations and showed a negative prognostic role. Receptor-ligand interaction analysis predicted interactions between arterial TEC derived CXCL12 and its cognate receptor CXCR4 on angiogenic tip EC. CXCL12 was in vitro and in vivo validated as actionable TEC target by highlighting the vessel number- and density- reducing activity of the CXCR4-inhibitor AMD3100 in murine PCa as well as by inhibition of TEC proliferation and migration in vitro. CONCLUSIONS: Overall, our comprehensive analysis identified novel PCa TEC targets and highlights CXCR4/CXCL12 interaction as a potential novel target to interfere with tumor angiogenesis in PCa.

Epithelial X-Box Binding Protein 1 Coordinates Tumor Protein p53-Driven DNA Damage Responses and Suppression of Intestinal Carcinogenesis.

BACKGROUND & AIMS: Throughout life, the intestinal epithelium undergoes constant self-renewal from intestinal stem cells. Together with genotoxic stressors and failing DNA repair, this self-renewal causes susceptibility toward malignant transformation. X-box binding protein 1 (XBP1) is a stress sensor involved in the unfolded protein response (UPR). We hypothesized that XBP1 acts as a signaling hub to regulate epithelial DNA damage responses. METHODS: Data from The Cancer Genome Atlas were analyzed for association of XBP1 with colorectal cancer (CRC) survival and molecular interactions between XBP1 and p53 pathway activity. The role of XBP1 in orchestrating p53-driven DNA damage response was tested in vitro in mouse models of chronic intestinal epithelial cell (IEC) DNA damage (Xbp1/H2bfl/fl, Xbp1ΔIEC, H2bΔIEC, H2b/Xbp1ΔIEC) and via orthotopic tumor organoid transplantation. Transcriptome analysis of intestinal organoids was performed to identify molecular targets of Xbp1-mediated DNA damage response. RESULTS: In The Cancer Genome Atlas data set of CRC, low XBP1 expression was significantly associated with poor overall survival and reduced p53 pathway activity. In vivo, H2b/Xbp1ΔIEC mice developed spontaneous intestinal carcinomas. Orthotopic tumor organoid transplantation revealed a metastatic potential of H2b/Xbp1ΔIEC-derived tumors. RNA sequencing of intestinal organoids (H2b/Xbp1fl/fl, H2bΔIEC, H2b/Xbp1ΔIEC, and H2b/p53ΔIEC) identified a transcriptional program downstream of p53, in which XBP1 directs DNA-damage-inducible transcript 4-like (Ddit4l) expression. DDIT4L inhibits mechanistic target of rapamycin-mediated phosphorylation of 4E-binding protein 1. Pharmacologic mechanistic target of rapamycin inhibition suppressed epithelial hyperproliferation via 4E-binding protein 1. CONCLUSIONS: Our data suggest a crucial role for XBP1 in coordinating epithelial DNA damage responses and stem cell function via a p53-DDIT4L-dependent feedback mechanism.

Feather Pecking in Non-Beak-Trimmed and Beak-Trimmed Laying Hens on Commercial Farms with Aviaries.

Severe feather pecking (SFP) is a major animal welfare problem in layers. It results in pain and injuries in the affected animal. It was the aim of this study to gain insight into the actual pecking behavior of laying hens kept on commercial farms with flock sizes common in practice. We observed aggressive pecking and SFP in non-beak-trimmed and beak-trimmed flocks of laying hens and investigated possible influencing factors. The study took place on eight conventional farms in Germany with aviaries, including three farms with a free range and a winter garden, one with a free range and one with a winter garden. Pecking behavior was observed during three observational periods (OPs): OP 1, at the peak of the laying period between the 28th and 33rd week of life; OP 2, in the middle of the laying period between the 42nd and 48th week of life; and OP 3, at the end of the laying period between the 63rd and 68th week of life in one laying period. Videos were analyzed using behavior sampling and continuous recording. We found that SFP occurred in all flocks, but the pecking rate differed significantly between the flocks. SFP correlated positively with the number of hens per square meter of usable area, with statistical significance in the litter area (r = 0.564; p = 0.045). The multivariate analysis revealed that access to a winter garden or free range significantly reduced the SFP rate on perches (p = 0.001). The stocking density (number of birds per usable square meter) had a significant influence on the SPF rate in the nest-box area (p = 0.001). The hybrid line had a significant effect on the SFP rate on perches and in the nest-box area (p = 0.001 each). Lohmann Brown hens in mixed flocks had a higher SFP rate (significant in OP 2) than those in homogeneous flocks, indicating that mixed flocks may be a risk factor for SFP. Lohmann Brown hens pecked significantly less than Dekalb White hens in the litter area (p = 0.010) and in the nest-box area (p = 0.025) and less than Lohmann Selected Leghorn hens in the litter area (p = 0.010). Lohmann Brown and Lohmann Selected Leghorn hens showed increasing SFP rates during the laying period. All hybrid lines had significantly higher SFP rates in the litter area, followed by the nest-box area and perches. These findings emphasize the importance of providing enough litter, litter areas and environmental enrichment. We found a significant positive correlation between aggressive pecking and SFP-in OP 1: rho (Spearman) = 0.580, p < 0.001; OP 2: rho = 0.486, p = 0.002; and OP 3: rho = 0.482, p = 0.002 (n = 39) -indicating that SFP may lead to a higher stress level in the flock. Beak trimming reduced pecking rates but did not entirely prevent SFP. Instead of subjecting chicks to this potentially painful procedure, reasons for SFP should be addressed. In conclusion, our data suggest a positive influence of a lower stocking density and the provision of a winter garden or free range for additional space. The hybrid line had a significant influence on the feather-pecking rate on perches and the nest-box area. Aggressive pecking and severe feather pecking correlated positively. We assume that vigorous and painful AP were an additional stress factor, especially in non-beak-trimmed flocks, leading to more SFP in due course. Beak trimming had a reducing effect on SFP. However, our results showed that non-beak-trimmed flocks could be kept without major outbreaks of SFP.

Deconvoluting tumor-infiltrating immune cells from RNA-seq data using quanTIseq.

Tumor-infiltrating immune cells comprise various cells of the innate and the adaptive immune system, which influence tumor growth and response to immunotherapy by exerting anti- and protumorigenic functions. Therefore, the quantification of tumor immune infiltrates is of paramount importance for cancer immunology and immunotherapy. We recently developed quanTIseq, a computational pipeline for the quantification of immune-cell fractions from bulk RNA sequencing (RNA-seq) data from blood or tumor samples. In this chapter, we show the capabilities of quanTIseq by analyzing two publicly available data sets. In the first example, we demonstrate how quanTIseq can be used to quantify circulating immune cells from preprocessed RNA-seq data and how to validate the results using matched flow cytometry data. In the second example, we analyze raw RNA-seq data from bulk tumor samples of melanoma patients collected before and on-treatment with kinase inhibitors to show how quanTIseq can be used to reveal the immunological effects of targeted and conventional drugs.

Correction to: Molecular and pharmacological modulators of the tumor immune contexture revealed by deconvolution of RNA-seq data.

It was highlighted that the original article [1] contained a typesetting mistake in the name of Noel Filipe da Cunha Carvalho de Miranda. This was incorrectly captured as Noel Filipe da Cunha Carvahlo de Miranda. It was also highlighted that in Fig. 3C the left panels Y-axis were cropped and in Fig. 5C, CD8 bar was cropped. This Correction article shows the correct Figs. 3 and 5. The original article has been updated.

Molecular and pharmacological modulators of the tumor immune contexture revealed by deconvolution of RNA-seq data.

We introduce quanTIseq, a method to quantify the fractions of ten immune cell types from bulk RNA-sequencing data. quanTIseq was extensively validated in blood and tumor samples using simulated, flow cytometry, and immunohistochemistry data.quanTIseq analysis of 8000 tumor samples revealed that cytotoxic T cell infiltration is more strongly associated with the activation of the CXCR3/CXCL9 axis than with mutational load and that deconvolution-based cell scores have prognostic value in several solid cancers. Finally, we used quanTIseq to show how kinase inhibitors modulate the immune contexture and to reveal immune-cell types that underlie differential patients' responses to checkpoint blockers.Availability: quanTIseq is available at http://icbi.at/quantiseq .

Modeling therapy resistance via the EGFR signaling pathway.

Mutations in KRAS are often associated with resistance to EGFR-targeting antibody therapy. Using comprehensive systems analyses, GNB5 has been identified as a potential target to overcome therapy resistance targeting the EGFR signaling pathways, whereby the AKT signaling pathway (PI3K) rather than the ERK signaling pathway (RAS) might be dominantly affected. Personalized mathematical modeling and simulations of this signaling pathway/network and respective perturbations are of great utility to customize therapy for patients.