Stratifying esophago-gastric cancer treatment using a patient-derived organoid-based threshold.

BACKGROUND AND AIMS: This study sought to determine the value of patient-derived organoids (PDOs) from esophago-gastric adenocarcinoma (EGC) for response prediction to neoadjuvant chemotherapy (neoCTx). METHODS: Endoscopic biopsies of patients with locally advanced EGC (n = 120) were taken into culture and PDOs expanded. PDOs' response towards the single substances of the FLOT regimen and the combination treatment were correlated to patients' pathological response using tumor regression grading. A classifier based on FLOT response of PDOs was established in an exploratory cohort (n = 13) and subsequently confirmed in an independent validation cohort (n = 13). RESULTS: EGC PDOs reflected patients' diverse responses to single chemotherapeutics and the combination regimen FLOT. In the exploratory cohort, PDOs response to single 5-FU and FLOT combination treatment correlated with the patients' pathological response (5-FU: Kendall's τ = 0.411, P = 0.001; FLOT: Kendall's τ = 0.694, P = 2.541e-08). For FLOT testing, a high diagnostic precision in receiver operating characteristic (ROC) analysis was reached with an AUCROC of 0.994 (CI 0.980 to 1.000). The discriminative ability of PDO-based FLOT testing allowed the definition of a threshold, which classified in an independent validation cohort FLOT responders from non-responders with high sensitivity (90%), specificity (100%) and accuracy (92%). CONCLUSION: In vitro drug testing of EGC PDOs has a high predictive accuracy in classifying patients' histological response to neoadjuvant FLOT treatment. Taking into account the high rate of successful PDO expansion from biopsies, the definition of a threshold that allows treatment stratification paves the way for an interventional trial exploring PDO-guided treatment of EGC patients.

Diet prevents the expansion of segmented filamentous bacteria and ileo-colonic inflammation in a model of Crohn's disease.

BACKGROUND: Crohn's disease (CD) is associated with changes in the microbiota, and murine models of CD-like ileo-colonic inflammation depend on the presence of microbial triggers. Increased abundance of unknown Clostridiales and the microscopic detection of filamentous structures close to the epithelium of Tnf ΔARE mice, a mouse model of CD-like ileitis pointed towards segmented filamentous bacteria (SFB), a commensal mucosal adherent bacterium involved in ileal inflammation. RESULTS: We show that the abundance of SFB strongly correlates with the severity of CD-like ileal inflammation in two mouse models of ileal inflammation, including Tnf ΔARE and SAMP/Yit mice. SFB mono-colonization of germ-free Tnf ΔARE mice confirmed the causal link and resulted in severe ileo-colonic inflammation, characterized by elevated tissue levels of Tnf and Il-17A, neutrophil infiltration and loss of Paneth and goblet cell function. Co-colonization of SFB in human-microbiota associated Tnf ΔARE mice confirmed that SFB presence is indispensable for disease development. Screening of 468 ileal and colonic mucosal biopsies from adult and pediatric IBD patients, using previously published and newly designed human SFB-specific primer sets, showed no presence of SFB in human tissue samples, suggesting a species-specific functionality of the pathobiont. Simulating the human relevant therapeutic effect of exclusive enteral nutrition (EEN), EEN-like purified diet antagonized SFB colonization and prevented disease development in Tnf ΔARE mice, providing functional evidence for the protective mechanism of diet in modulating microbiota-dependent inflammation in IBD. CONCLUSIONS: We identified a novel pathogenic role of SFB in driving severe CD-like ileo-colonic inflammation characterized by loss of Paneth and goblet cell functions in Tnf ΔARE mice. A purified diet antagonized SFB colonization and prevented disease development in Tnf ΔARE mice in contrast to a fiber-containing chow diet, clearly demonstrating the important role of diet in modulating a novel IBD-relevant pathobiont and supporting a direct link between diet and microbial communities in mediating protective functions. Video Abstract.

TiO2 nanoparticles abrogate the protective effect of the Crohn's disease-associated variation within the PTPN22 gene locus.

OBJECTIVE: Inflammatory bowel disease (IBD) is a multifactorial condition driven by genetic and environmental risk factors. A genetic variation in the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene has been associated with autoimmune disorders while protecting from the IBD subtype Crohn's disease. Mice expressing the murine orthologous PTPN22-R619W variant are protected from intestinal inflammation in the model of acute dextran sodium sulfate (DSS)-induced colitis. We previously identified food-grade titanium dioxide (TiO2, E171) as a neglected IBD risk factor. Here, we investigate the interplay of the PTPN22 variant and TiO2-mediated effects during IBD pathogenesis. DESIGN: Acute DSS colitis was induced in wild-type and PTPN22 variant mice (PTPN22-R619W) and animals were treated with TiO2 nanoparticles during colitis induction. Disease-triggering mechanisms were investigated using bulk and single-cell RNA sequencing. RESULTS: In mice, administration of TiO2 nanoparticles abrogated the protective effect of the variant, rendering PTPN22-R619W mice susceptible to DSS colitis. In early disease, cytotoxic CD8+ T-cells were found to be reduced in the lamina propria of PTPN22-R619W mice, an effect reversed by TiO2 administration. Normalisation of T-cell populations correlated with increased Ifng expression and, at a later stage of disease, the promoted prevalence of proinflammatory macrophages that triggered severe intestinal inflammation. CONCLUSION: Our findings indicate that the consumption of TiO2 nanoparticles might have adverse effects on the gastrointestinal health of individuals carrying the PTPN22 variant. This demonstrates that environmental factors interact with genetic risk variants and can reverse a protective mechanism into a disease-promoting effect.

Sortase A-Cleavable CD1d Identifies Sphingomyelins as Major Class of CD1d-Associated Lipids.

CD1d is an atypical MHC class I molecule which binds endogenous and exogenous lipids and can activate natural killer T (NKT) cells through the presentation of lipid antigens. CD1d surveys different cellular compartments including the secretory and the endolysosomal pathway and broadly binds lipids through its two hydrophobic pockets. Purification of the transmembrane protein CD1d for the analysis of bound lipids is technically challenging as the use of detergents releases CD1d-bound lipids. To address these challenges, we have developed a novel approach based on Sortase A-dependent enzymatic release of CD1d at the cell surface of live mammalian cells, which allows for single step release and affinity tagging of CD1d for shotgun lipidomics. Using this system, we demonstrate that CD1d carrying the Sortase A recognition motif shows unimpaired subcellular trafficking through the secretory and endolysosomal pathway and is able to load lipids in these compartments and present them to NKT cells. Comprehensive shotgun lipidomics demonstrated that the spectrum and abundance of CD1d-associated lipids is not representative of the total cellular lipidome but rather characterized by preferential binding to long chain sphingolipids and glycerophospholipids. As such, sphingomyelin species recently identified as critical negative regulators of NKT cell activation, represented the vast majority of endogenous CD1d-associated lipids. Moreover, we observed that inhibition of endolysosomal trafficking of CD1d surprisingly did not affect the spectrum of CD1d-bound lipids, suggesting that the majority of endogenous CD1d-associated lipids load onto CD1d in the secretory rather than the endolysosomal pathway. In conclusion, we present a novel system for the analysis of CD1d-bound lipids in mammalian cells and provide new insight into the spectrum of CD1d-associated lipids, with important functional implications for NKT cell activation.

Detecting drug resistance in pancreatic cancer organoids guides optimized chemotherapy treatment.

Drug combination therapies for cancer treatment show high efficacy but often induce severe side effects, resulting in dose or cycle number reduction. We investigated the impact of neoadjuvant chemotherapy (neoCTx) adaptions on treatment outcome in 59 patients with pancreatic ductal adenocarcinoma (PDAC). Resections with tumor-free margins were significantly more frequent when full-dose neoCTx was applied. We determined if patient-derived organoids (PDOs) can be used to personalize poly-chemotherapy regimens by pharmacotyping of treatment-naïve and post-neoCTx PDAC PDOs. Five out of ten CTx-naïve PDO lines exhibited a differential response to either the FOLFIRINOX or the Gem/Pac regimen. NeoCTx PDOs showed a poor response to the neoadjuvant regimen that had been administered to the respective patient in 30% of cases. No significant difference in PDO response was noted when comparing modified treatments in which the least effective single drug was removed from the complete regimen. Drug testing of CTx-naïve PDAC PDOs and neoCTx PDOs may be useful to guide neoadjuvant and adjuvant regimen selection, respectively. Personalizing poly-chemotherapy regimens by omitting substances with low efficacy could potentially result in less severe side effects, thereby increasing the fraction of patients receiving a full course of neoadjuvant treatment. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Microbiota-dependent activation of the myeloid calcineurin-NFAT pathway inhibits B7H3- and B7H4-dependent anti-tumor immunity in colorectal cancer.

Bacterial sensing by intestinal tumor cells contributes to tumor growth through cell-intrinsic activation of the calcineurin-NFAT axis, but the role of this pathway in other intestinal cells remains unclear. Here, we found that myeloid-specific deletion of calcineurin in mice activated protective CD8+ T cell responses and inhibited colorectal cancer (CRC) growth. Microbial sensing by myeloid cells promoted calcineurin- and NFAT-dependent interleukin 6 (IL-6) release, expression of the co-inhibitory molecules B7H3 and B7H4 by tumor cells, and inhibition of CD8+ T cell-dependent anti-tumor immunity. Accordingly, targeting members of this pathway activated protective CD8+ T cell responses and inhibited primary and metastatic CRC growth. B7H3 and B7H4 were expressed by the majority of human primary CRCs and metastases, which was associated with low numbers of tumor-infiltrating CD8+ T cells and poor survival. Therefore, a microbiota-, calcineurin-, and B7H3/B7H4-dependent pathway controls anti-tumor immunity, revealing additional targets for immune checkpoint inhibition in microsatellite-stable CRC.

[Chronic or severe enteropathy and immunodeficiency: be prepared for a rara avis]. / Chronische oder schwere Darmerkrankung und Immundefekt: Manchmal ist es doch ein Kolibri.

In the work-up of chronic enteropathies an underlying inborn error of immunity (IEI) should be considered in certain cases. IEI are rare, but approximately 10% of patients may present with symptoms of inflammatory bowel disease (IBD), which is a much more common entity. Patients with IEI associated IBD may show extraintestinal symptoms or signs, and are often refractory to conventional anti-inflammatory treatment. In case of early-onset bowel inflammation and other intestinal or extraintestinal manifestations, an IEI should be excluded. A small fraction of monogenic IEI can be amenable to targeted therapies, or even corrected by allogeneic stem cell transplantation. Therefore, early diagnosis is crucial. This paper shows examples of clinical - gastrointestinal as well as extraintestinal - signs and findings which require immunological and possibly genetic workup.

Detailed Transcriptional Landscape of Peripheral Blood Points to Increased Neutrophil Activation in Treatment-Naïve Inflammatory Bowel Disease.

BACKGROUND AND AIMS: Inflammatory bowel disease [IBD] is a chronic relapsing disorder of the gastrointestinal tract, which generally manifests as Crohn's disease [CD] or ulcerative colitis [UC]. These subtypes are heterogeneous in terms of disease location and histological features, while sharing common clinical presentation, genetic associations and, thus, common immune regulatory pathways. METHODS: Using miRNA and mRNA coupled transcriptome profiling and systems biology approaches, we report a comprehensive analysis of blood transcriptomes from treatment-naïve [n = 110] and treatment-exposed [n = 177] IBD patients as well as symptomatic [n = 65] and healthy controls [n = 95]. RESULTS: Broadly, the peripheral blood transcriptomes of CD and UC patients were similar. However, there was an extensive gene deregulation in the blood of IBD patients, while only a slight deregulation in symptomatic controls, when compared with healthy controls. The deregulated mRNAs and miRNAs are mainly involved in the innate immunity and are especially enriched in neutrophil activation-related pathways. Oxidative phosphorylation and neutrophil activation-related modules were found to be differentially co-expressed among treatment-naïve IBD as compared to healthy controls. In the deregulated neutrophil activation-related co-expression module, IL1B was identified as the central gene. Levels of co-expression among IL1B and chemosensing receptor [CXCR1/2 and FPR1/2] genes were reduced in the blood of IBD patients when compared with healthy controls. CONCLUSIONS: Immune dysregulation seen in peripheral blood transcriptomes of treatment-naïve IBD patients is mainly driven by neutrophil activation.

Macroscopic, histologic, and clinical assessment of acute graft-versus-host disease of the upper gastrointestinal tract within 6 weeks after allogeneic hematopoietic cell transplantation.

Acute graft-versus-host-disease (aGVHD) is the main cause of morbidity and nonrelapse mortality (NRM) following allogeneic hematopoietic cell transplantation (alloHCT). Nausea, vomiting, and anorexia after alloHCT can be early signs of aGVHD of the gastrointestinal tract (GIT) but may also reflect lasting mucosal damage or side effects of drugs. If upper GIT aGVHD is suspected, upper endoscopic evaluation and histological examination are crucial. Still, the interpretation of clinical symptoms, macroscopical alterations, and histological findings can be challenging. Therefore, we conducted a retrospective analysis on single-center data from 174 patients with suspected aGVHD of the upper GIT who underwent upper endoscopy within the first 6 weeks after alloHCT, to study the distribution of aGVHD-related histological findings in relation to clinical symptoms and macroscopic findings and to correlate the severity of changes with data on relapse and NRM. Our data suggest that biopsies of the duodenum reveal the severity of upper GIT aGVHD most accurately. While the histological grading correlated weakly with the severity of macroscopic changes, we found a tight correlation between histological and clinical grades of upper GIT aGVHD (p < 0.001). Although correlation of histological grading of upper GIT aGVHD with the risk for NRM missed statistical significance (HR 1.53, Lerner ≥1° versus <1º, p = 0.13), overall clinical aGVHD severity correlated with NRM (HR 4.3, IIIº-IVº versus 0-Iº, p < 0.01). In conclusion, biopsies from the duodenum are most sensitive in excluding aGVHD in patients with normal macroscopic findings at esophagogastroduodenoscopy. Clinical grading of aGVHD predicts NRM better than histological grading.

Deficiency in X-linked inhibitor of apoptosis protein promotes susceptibility to microbial triggers of intestinal inflammation.

Inflammatory bowel disease (IBD) is characterized by inappropriate immune responses to the microbiota in genetically susceptible hosts, but little is known about the pathways that link individual genetic alterations to microbiota-dependent inflammation. Here, we demonstrated that the loss of X-linked inhibitor of apoptosis protein (XIAP), a gene associated with Mendelian IBD, rendered Paneth cells sensitive to microbiota-, tumor necrosis factor (TNF)­, receptor-interacting protein kinase 1 (RIPK1)­, and RIPK3-dependent cell death. This was associated with deficiency in Paneth cell­derived antimicrobial peptides and alterations in the stratification and composition of the microbiota. Loss of XIAP was not sufficient to elicit intestinal inflammation but provided susceptibility to pathobionts able to promote granulomatous ileitis, which could be prevented by administration of a Paneth cell­derived antimicrobial peptide. These data reveal a pathway critical for host-microbial cross-talk, which is required for intestinal homeostasis and the prevention of inflammation and which is amenable to therapeutic targeting.

A global assessment of recent trends in gastrointestinal cancer and lifestyle-associated risk factors.

BACKGROUND: Gastrointestinal (GI) cancers were responsible for 26.3% of cancer cases and 35.4% of deaths worldwide in 2018. This study aimed to analyze the global incidence, mortality, prevalence, and contributing risk factors of the 6 major GI cancer entities [esophageal cancer (EC), gastric cancer (GC), liver cancer (LC), pancreatic cancer (PC), colon cancer, and rectal cancer]. METHODS: Using the Global Cancer Observatory and the Global Health Observatory databases, we reviewed the current GI cancer incidence, prevalence, and mortality, analyzed the association of GI cancer prevalence with national human development indices (HDIs), identified the contributing risk factors, and estimated developing age- and sex-specific trends in incidence and mortality. RESULTS: In 2020, the trend in age-standardized rate of incidence of GI cancers closely mirrored that of mortality, with the highest rates of LC, EC, and GC in Asia and of colorectal cancer (CRC) and PC mainly in Europe. Incidence and mortality were positively, but the mortality-to-incidence ratio (MIR) was inversely correlated with the national HDI levels. High MIRs in developing countries likely reflected the lack of preventive strategies and effective treatments. GI cancer prevalence was highest in Europe and was also positively correlated with HDIs and lifestyle-associated risk factors, such as alcohol consumption, smoking, obesity, insufficient physical activity, and high blood cholesterol level, but negatively correlated with hypertension and diabetes. Incidences of EC were consistently and those of GC mostly decreasing, whereas incidences of CRC were increasing in most countries/regions, especially in the younger populations. Incidences of LC and PC were also increasing in all age-gender populations except for younger males. Mortalities were decreasing for EC, GC, and CRC in most countries/regions, and age-specific trends were observed in PC and LC with a decrease in the younger but an increase in the older population. CONCLUSIONS: On the global scale, higher GI cancer burden was accompanied, for the most part, by factors associated with the so-called Western lifestyle reflected by high and very high national HDI levels. In countries/regions with very high HDI levels, patients survived longer, and increasing GI cancer cases were observed with increasing national HDI levels. Optimizing GI cancer prevention and improving therapies, especially for patients with comorbid metabolic diseases, are thus urgently recommended.

The Colorectal Cancer Lipidome: Identification of a Robust Tumor-Specific Lipid Species Signature.

OBJECTIVE: Lipidomic changes were causally linked to metabolic diseases, but the scenario for colorectal cancer (CRC) is less clear. We investigated the CRC lipidome for putative tumor-specific alterations through analysis of 3 independent retrospective patient cohorts from 2 clinical centers, to derive a clinically useful signature. DESIGN: Quantitative comprehensive lipidomic analysis was performed using direct infusion electrospray ionization coupled with tandem mass spectrometry (ESI-MS/MS) and high-resolution mass spectrometry (HR-MS) on matched nondiseased mucosa and tumor tissue in a discovery cohort (n = 106). Results were validated in 2 independent cohorts (n = 28, and n = 20), associated with genomic and clinical data, and lipidomic data from a genetic mouse tumor model (Apc1638N). RESULTS: Significant differences were found between tumor and normal tissue for glycero-, glycerophospho-, and sphingolipids in the discovery cohort. Comparison to the validation collectives unveiled that glycerophospholipids showed high interpatient variation and were strongly affected by preanalytical conditions, whereas glycero- and sphingolipids appeared more robust. Signatures of sphingomyelin and triacylglycerol (TG) species significantly differentiated cancerous from nondiseased tissue in both validation studies. Moreover, lipogenic enzymes were significantly up-regulated in CRC, and FASN gene expression was prognostically detrimental. The TG profile was significantly associated with postoperative disease-free survival and lymphovascular invasion, and was essentially conserved in murine digestive cancer, but not associated with microsatellite status, KRAS or BRAF mutations, or T-cell infiltration. CONCLUSION: Analysis of the CRC lipidome revealed a robust TG-species signature with prognostic potential. A better understanding of the cancer-associated glycerolipid and sphingolipid metabolism may lead to novel therapeutic strategies.

Tumor-infiltrating plasmacytoid dendritic cells are associated with survival in human colon cancer.

BACKGROUND: Plasmacytoid dendritic cells (pDCs) play a key role in the induction and maintenance of antitumor immunity. Conversely, they can act as tolerogenic DCs by inhibiting tumor-directed immune responses. Therefore, pDCs may profoundly influence tumor progression. To gain novel insights into the role of pDCs in colon cancer, we investigated the frequency and clinical relevance of pDCs in primary tumor tissues from patients with colon cancer with different clinicopathological characteristics. METHODS: Immunohistochemical stainings were performed to explore the frequency of tumor-infiltrating BDCA-2+ pDCs in patients with colon cancer. Statistical analyses were conducted to determine an association between the pDC density and clinicopathological characteristics of the patients. Furthermore, we used multiplex immunofluorescence stainings to evaluate the localization and phenotype of pDCs in stroma and tertiary lymphoid structures (TLS) of colon cancer tissues. RESULTS: An increased density of infiltrating pDCs was associated with lower Union for International Cancer Control (UICC) stages. Furthermore, a higher pDC frequency was significantly correlated with increased progression-free and overall survival of patients with colon cancer. Moreover, a lower number of coloncancer-infiltrating pDCs was significantly and independently linked to worse prognosis. In addition, we found that a proportion of pDCs shows a nuclear expression of the transcription factor interferon regulatory factor 7 (IRF7), which is characteristic for an activated phenotype. In various tumor stroma regions, IRF7+ pDCs were located in the neighborhood of granzyme B-expressing CD8+ T cells. Moreover, pDCs were identified as a novel component of the T cell zone of colon cancer-associated TLS, which are major regulators of adaptive antitumor immunity. A proportion of TLS-associated pDCs displayed a nuclear IRF7 expression and was preferentially located close to CD4+ T cells. CONCLUSIONS: These results indicate that higher densities of tumor-infiltrating pDCs are associated with prolonged survival of patients with colon cancer. Moreover, colon cancer-infiltrating pDCs may represent a novel prognostic factor. The colocalization of activated pDCs and T cells in tumor stroma and within TLS may contribute to the correlation between higher pDC densities and better prognosis. In addition, our findings may have implications for the design of novel immunotherapeutic strategies that are based on targeting colon cancer-infiltrating pDCs.

Gastric Carcinomas with Stromal B7-H3 Expression Have Lower Intratumoural CD8+ T Cell Density.

CD8+ T cells are the main effector cells of anti-cancer immune response that can be regulated by various costimulatory and coinhibitory molecules, including members of the B7 family. B7 homolog 3 (B7-H3) appears as a promising marker for immunotherapy; however, its significance in gastric cancer (GC) is unclear yet. We evaluated the spatial distribution of CD8+ T cells in relation to the expression of B7-H3 by double immunohistochemical staining. The level of B7-H3 intensity was scored manually (0-3) and dichotomized into B7-H3-low and B7-H3-high groups. The distribution and density of CD8+ T cells was analysed using whole slide digital imaging. B7-H3 was expressed mainly in the stromal compartment of GC (n = 73, 76% of all cases). Tumours with high expression of B7-H3 showed larger spatial differences of CD8+ T cells (86.4/mm2 in tumour centre vs. 414.9/mm2 in invasive front) when compared to B7-H3-low group (157.7/mm2 vs. 218.7/mm2, respectively) (p < 0.001). This study provides insight into the expression pattern of B7-H3 in GC of Western origin. In GCs with higher level of B7-H3 expression, CD8+ T cells were spatially suppressed in the tumour centre suggesting that B7-H3 might be involved in tumour escape mechanisms from the immune response.

SETDB1 is required for intestinal epithelial differentiation and the prevention of intestinal inflammation.

OBJECTIVE: The intestinal epithelium is a rapidly renewing tissue which plays central roles in nutrient uptake, barrier function and the prevention of intestinal inflammation. Control of epithelial differentiation is essential to these processes and is dependent on cell type-specific activity of transcription factors which bind to accessible chromatin. Here, we studied the role of SET Domain Bifurcated Histone Lysine Methyltransferase 1, also known as ESET (SETDB1), a histone H3K9 methyltransferase, in intestinal epithelial homeostasis and IBD. DESIGN: We investigated mice with constitutive and inducible intestinal epithelial deletion of Setdb1, studied the expression of SETDB1 in patients with IBD and mouse models of IBD, and investigated the abundance of SETDB1 variants in healthy individuals and patients with IBD. RESULTS: Deletion of intestinal epithelial Setdb1 in mice was associated with defects in intestinal epithelial differentiation, barrier disruption, inflammation and mortality. Mechanistic studies showed that loss of SETDB1 leads to de-silencing of endogenous retroviruses, DNA damage and intestinal epithelial cell death. Predicted loss-of-function variants in human SETDB1 were considerably less frequently observed than expected, consistent with a critical role of SETDB1 in human biology. While the vast majority of patients with IBD showed unimpaired mucosal SETDB1 expression, comparison of IBD and non-IBD exomes revealed over-representation of individual rare missense variants in SETDB1 in IBD, some of which are predicted to be associated with loss of function and may contribute to the pathogenesis of intestinal inflammation. CONCLUSION: SETDB1 plays an essential role in intestinal epithelial homeostasis. Future work is required to investigate whether rare variants in SETDB1 contribute to the pathogenesis of IBD.

Loss of hepatic Mboat7 leads to liver fibrosis.

OBJECTIVE: The rs641738C>T variant located near the membrane-bound O-acyltransferase domain containing 7 (MBOAT7) locus is associated with fibrosis in liver diseases, including non-alcoholic fatty liver disease (NAFLD), alcohol-related liver disease, hepatitis B and C. We aim to understand the mechanism by which the rs641738C>T variant contributes to pathogenesis of NAFLD. DESIGN: Mice with hepatocyte-specific deletion of MBOAT7 (Mboat7Δhep) were generated and livers were characterised by histology, flow cytometry, qPCR, RNA sequencing and lipidomics. We analysed the association of rs641738C>T genotype with liver inflammation and fibrosis in 846 NAFLD patients and obtained genotype-specific liver lipidomes from 280 human biopsies. RESULTS: Allelic imbalance analysis of heterozygous human liver samples pointed to lower expression of the MBOAT7 transcript on the rs641738C>T haplotype. Mboat7Δhep mice showed spontaneous steatosis characterised by increased hepatic cholesterol ester content after 10 weeks. After 6 weeks on a high fat, methionine-low, choline-deficient diet, mice developed increased hepatic fibrosis as measured by picrosirius staining (p<0.05), hydroxyproline content (p<0.05) and transcriptomics, while the inflammatory cell populations and inflammatory mediators were minimally affected. In a human biopsied NAFLD cohort, MBOAT7 rs641738C>T was associated with fibrosis (p=0.004) independent of the presence of histological inflammation. Liver lipidomes of Mboat7Δhep mice and human rs641738TT carriers with fibrosis showed increased total lysophosphatidylinositol levels. The altered lysophosphatidylinositol and phosphatidylinositol subspecies in MBOAT7Δhep livers and human rs641738TT carriers were similar. CONCLUSION: Mboat7 deficiency in mice and human points to an inflammation-independent pathway of liver fibrosis that may be mediated by lipid signalling and a potentially targetable treatment option in NAFLD.

Shotgun lipidomics-based characterization of the landscape of lipid metabolism in colorectal cancer.

Solid tumors are characterized by global metabolic alterations which contribute to their growth and progression. Altered gene expression profiles and plasma lipid composition suggested a role for metabolic reprogramming in colorectal cancer (CRC) development. However, a conclusive picture of CRC-associated lipidome alterations in the tumor tissue has not emerged. Here, we determined molar abundances of 342 species from 20 lipid classes in matched biopsies of CRC and adjacent normal mucosa. We demonstrate that in contrast to previous reports, CRC shows a largely preserved lipidome composition that resembles that of normal colonic mucosa. Important exceptions include increased levels of lyso-phosphatidylinositols in CRC and reduced abundance of ether phospholipids in advanced stages of CRC. As such, our observations challenge the concept of widespread alterations in lipid metabolism in CRC and rather suggest changes in the cellular lipid profile that are limited to selected lipids involved in signaling and the scavenging of reactive oxygen species.

Sterile activation of invariant natural killer T cells by ER-stressed antigen-presenting cells.

Invariant NKT (iNKT) cells have the unique ability to shape immunity during antitumor immune responses and other forms of sterile and nonsterile inflammation. Recent studies have highlighted a variety of classes of endogenous and pathogen-derived lipid antigens that can trigger iNKT cell activation under sterile and nonsterile conditions. However, the context and mechanisms that drive the presentation of self-lipid antigens in sterile inflammation remain unclear. Here we report that endoplasmic reticulum (ER)-stressed myeloid cells, via signaling events modulated by the protein kinase RNA-like ER kinase (PERK) pathway, increase CD1d-mediated presentation of immunogenic endogenous lipid species, which results in enhanced iNKT cell activation both in vitro and in vivo. In addition, we demonstrate that actin cytoskeletal reorganization during ER stress results in an altered distribution of CD1d on the cell surface, which contributes to enhanced iNKT cell activation. These results define a previously unidentified mechanism that controls iNKT cell activation during sterile inflammation.