Anti-TNF therapy is central for treating ulcerative colitis, yet ∼40% are primary non-responders. To find pre-treatment signatures distinguishing anti-TNF non-responders from responders, we did serum cytokine profiling and laser microdissection transcriptomics on paired uninflamed and inflamed colonic epithelium and lamina propria from patients matched at baseline but differing in endoscopic outcome. Although inflammation drove more epithelial changes, the signatures separating non-responders from responders were found in uninflamed lamina propria, with elevated cell-cycle genes and a co-expression network enriched for humoral immune genes consistently higher expressed in non-responders. Cellular deconvolution localized this network to IgA plasma cells, confirmed by tissue staining, and the association was validated by pseudobulk analysis of an independent single-cell atlas. Baseline epithelial signatures or serum cytokines did not differ significantly between groups. These findings point to TNF-independent humoral activity already present in uninflamed mucosa as a candidate contributor to non-response, with implications for prediction and alternative therapies.

Abstract In the non-small cell lung cancer (NSCLC) tumor microenvironment (TME), tumor cells mediate inhibitory signals to key immune cells, promoting an immunosuppressive environment that facilitates tumor immune evasion. CD8 + cytotoxic T lymphocytes are central mediators of antitumor immunity, and tumors can be classified by immunohistochemistry (IHC) as immune-hot or immune-cold based on the abundance of tumor-infiltrating lymphocytes (TILs). Although cancer immunotherapy can boost lymphocyte functions and improve progression-free survival in patients with advanced NSCLC, only a minority of patients experience a durable clinical benefit. Tumor-associated macrophages (TAMs) are key immunoregulatory cells in the NSCLC microenvironment and capable of generating potent immunosuppressive signals. However, their functional roles in immune-hot versus immune-cold tumors remain poorly understood. In this study, we compared the transcriptional programs of TAMs from NSCLC patients with immune-hot or immune-cold tumors. We classified 11 surgically resected NSCLC tumors as immune‑hot or immune‑cold based on quantitative immunohistochemistry of CD4⁺ and CD8⁺ tumor‑infiltrating lymphocytes. TAMs were isolated from tumor and adjacent healthy tissue by fluorescence-activated cell sorting (FACS), followed by bulk RNA sequencing and differential gene expression analysis. TAMs from immune‑cold tumors exhibited a striking upregulation of genes involved in immunoglobulin-mediated immune responses. Additionally, these TAMs demonstrated increased expression of genes involved in extracellular matrix organization, including matrix metalloproteinases and collagen‑associated genes, suggesting enhanced matrix remodeling activity. These findings highlight TAMs’ potential contribution to immunosuppression, stromal remodeling, and impaired lymphocyte infiltration. The TAM-mediated pathways identified here may represent actionable targets for future immunotherapeutic strategies aimed at reshaping the tumor microenvironment.

Methylation of DNA is an epigenetic reversible process that regulates gene expression. Aberrant promoter methylation is associated with chronic inflammation; however, the genome-wide DNA promoter methylation signature in periodontitis remains unexplored. The present study is an epigenome-wide association study (EWAS) aimed at investigating salivary DNA methylation patterns within gene promoter regions in individuals with Stage III/IV periodontitis compared to healthy controls. Cases (all available individuals with Stage III/IV and probing pocket depths (PPD) > 5 mm, n = 50, 48

Objectives Comparative longitudinal analyses of cellular composition and peripheral blood gene expression in Rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and healthy pregnancies. Methods In total, 335 whole blood samples from 84 RA, SLE and healthy controls before pregnancy, at each trimester, 6 weeks, 6 months and 12 months post partum were analysed. We combined bulk and single cell RNA analyses for cell-type estimation, validated by flow cytometry, before combining this in a cell-type adjusted analysis for an improved resolution of unrecognised gene expression changes associated with RA and SLE pregnancies. Results Patients were well regulated throughout pregnancy, and few had pregnancy complications. In SLE, the interferon signature was augmented during pregnancy, and the pregnancy signature was continued post partum. An altered cell type composition strongly influences the profile. In the pregnancy signature, transcripts involved in galactosylation potentially altering the effector functions of autoantibodies became more evident. Several genes in the adjusted RA signature are expressed in mucosal associated invariant T cells. Conclusion We found distinct RA, SLE and pregnancy signatures, and no expression patterns could be attributed to medication or disease activity. Our results support the need for close postpartum follow-up of patients with SLE. Gene expression patterns in RA were closer to healthy controls than to SLE, and primarily became evident after cell-type adjustment. Adjusting for cell abundance unravelled gene expression signatures less associated with variation in cell-composition and highlighted genes with expression profiles associated with changes in specialised cell populations.

Fabry disease is a rare disorder caused by variations in the alpha-galactosidase gene. To a degree, Fabry disease is manageable via enzyme replacement therapy (ERT). By understanding the molecular basis of Fabry nephropathy (FN) and ERT's long-term impact, here we aimed to provide a framework for selection of potential disease biomarkers and drug targets. We obtained biopsies from eight control individuals and two independent FN cohorts comprising 16 individuals taken prior to and after up to ten years of ERT, and performed RNAseq analysis. Combining pathway-centered analyses with network-science allowed computation of transcriptional landscapes from four nephron compartments and their integration with existing proteome and drug-target interactome data. Comparing these transcriptional landscapes revealed high inter-cohort heterogeneity. Kidney compartment transcriptional landscapes comprehensively reflected differences in FN cohort characteristics. With exception of a few aspects, in particular arteries, early ERT in patients with classical Fabry could lastingly revert FN gene expression patterns to closely match that of control individuals. Pathways nonetheless consistently altered in both FN cohorts pre-ERT were mostly in glomeruli and arteries and related to the same biological themes. While keratinization-related processes in glomeruli were sensitive to ERT, a majority of alterations, such as transporter activity and responses to stimuli, remained dysregulated or reemerged despite ERT. Inferring an ERT-resistant genetic module of expressed genes identified 69 drugs for potential repurposing matching the proteins encoded by 12 genes. Thus, we identified and cross-validated ERT-resistant gene product modules that, when leveraged with external data, allowed estimating their suitability as biomarkers to potentially track disease course or treatment efficacy and potential targets for adjunct pharmaceutical treatment.

Background The epithelium in the colonic mucosa is implicated in the pathophysiology of various diseases, including inflammatory bowel diseases and colorectal cancer. Intestinal epithelial organoids from the colon (colonoids) can be used for disease modeling and personalized drug screening. Colonoids are usually cultured at 18-21% oxygen without accounting for the physiological hypoxia in the colonic epithelium (3% to <1% oxygen). We hypothesize that recapitulating the in vivo physiological oxygen environment (i.e., physioxia) will enhance the translational value of colonoids as pre-clinical models. Here we evaluate whether human colonoids can be established and cultured in physioxia and compare growth, differentiation, and immunological responses at 2% and 20% oxygen. Methods Growth from single cells to differentiated colonoids was monitored by brightfield images and evaluated with a linear mixed model. Cell composition was identified by immunofluorescence staining of cell markers and single-cell RNA-sequencing (scRNA-seq). Enrichment analysis was used to identify transcriptomic differences within cell populations. Pro-inflammatory stimuli induced chemokines and Neutrophil gelatinase-associated lipocalin (NGAL) release were analyzed by Multiplex profiling and ELISA. Direct response to a lower oxygen level was analyzed by enrichment analysis of bulk RNA sequencing data. Results Colonoids established in a 2% oxygen environment acquired a significantly larger cell mass compared to a 20% oxygen environment. No differences in expression of cell markers for cells with proliferation potential (KI67 positive), goblet cells (MUC2 positive), absorptive cells (MUC2 negative, CK20 positive) and enteroendocrine cells (CGA positive) were found between colonoids cultured in 2% and 20% oxygen. However, the scRNA-seq analysis identified differences in the transcriptome within stem-, progenitor- and differentiated cell clusters. Both colonoids grown at 2% and 20% oxygen secreted CXCL2, CXCL5, CXCL10, CXCL12, CX3CL1 and CCL25, and NGAL upon TNF + poly(I:C) treatment, but there appeared to be a tendency towards lower pro-inflammatory response in 2% oxygen. Reducing the oxygen environment from 20% to 2% in differentiated colonoids altered the expression of genes related to differentiation, metabolism, mucus lining, and immune networks. Conclusions Our results suggest that colonoids studies can and should be performed in physioxia when the resemblance to in vivo conditions is important.

Objective: Diabetes is a heterogeneous disease and a precise diagnosis of diabetes subgroups is necessary to initiate proper early treatment and clinical management of the disease. Circulating small RNAs (sRNAs) are potentially diagnostic biomarkers in diseases, including diabetes. Here we aimed to examine whether profiles of circulating sRNAs differed between patients with autoimmune and non-autoimmune diabetes and non-diabetic controls. Design: This cross-sectional case-control study included participants from the third survey of the HUNT study. Methods: We performed sRNA sequencing in serum from adult-onset type 1 diabetes (n = 51), type 2 diabetes (n = 50) and latent autoimmune diabetes in adult (LADA, n = 51), as well as non-diabetic HUNT3 participants as control group (n = 51). Differential expression analysis of the sRNAs was performed in R using limma-voom. Results: We identified differences in sRNA expression between autoimmune (type 1 diabetes and LADA) and non-autoimmune diabetes (type 2 diabetes) and between patients with diabetes and non-diabetic controls. Focusing on miRNA, we identified 10 differentially expressed mature miRNAs and 30 differentially expressed miRNA variants (isomiRs). We also identified significant changes within other sRNA classes, including a pronounced downregulation of a tRNA fragment in patients with diabetes compared to non-diabetic controls. We created cross-validated sRNA signatures based on the significant sRNAs that distinguished patients with diabetes from non-diabetic controls, and autoimmune from non-autoimmune diabetes, with high specificity and sensitivity. sRNA profiles did not distinguish between type 1 diabetes and LADA. Conclusions: Circulating sRNAs are differentially expressed between patients with diabetes and non-diabetic controls and between autoimmune and non-autoimmune diabetes.

Clear cell renal cell carcinoma (ccRCC) is the most common renal cancer. Identification of ccRCC likely to progress, despite an apparent low risk at the time of surgery, represents a key clinical issue. From a cohort of adult ccRCC patients (n = 443), we selected low-risk tumors progressing within a 5-years average follow-up (progressors: P, n = 8) and non-progressing (NP) tumors (n = 16). Transcriptome sequencing, miRNA sequencing and proteomics were performed on tissues obtained at surgery. We identified 151 proteins, 1167 mRNAs and 63 miRNAs differentially expressed in P compared to NP low-risk tumors. Pathway analysis demonstrated overrepresentation of proteins related to “LXR/RXR and FXR/RXR Activation”, “Acute Phase Response Signaling” in NP compared to P samples. Integrating mRNA, miRNA and proteomic data, we developed a 10-component classifier including two proteins, three genes and five miRNAs, effectively differentiating P and NP ccRCC and capturing underlying biological differences, potentially useful to identify “low-risk” patients requiring closer surveillance and treatment adjustments. Key results were validated by immunohistochemistry, qPCR and data from publicly available databases. Our work suggests that LXR, FXR and macrophage activation pathways could be critically involved in the inhibition of the progression of low-risk ccRCC. Furthermore, a 10-component classifier could support an early identification of apparently low-risk ccRCC patients.

SCOPE:The aim of this study is to explore the molecular mechanisms underlying the effect of replacing dietary saturated fat (SFA) with polyunsaturated fat (PUFA) on cardiovascular disease (CVD) risk using a whole transcriptome approach.METHODS AND RESULTS:Healthy subjects with moderate hypercholesterolemia (n = 115) are randomly assigned to a control diet (C-diet) group or an experimental diet (Ex-diet) group receiving comparable food items with different fatty acid composition for 8 weeks. RNA isolated from peripheral blood mononuclear cells (PBMCs) at baseline and after 8 weeks of intervention is analyzed by microarray technology (n = 95). By use of a linear regression model (n = 92), 14 gene transcripts are differentially altered in the Ex-diet group compared to the C-diet group. These include transcripts related to vascular smooth muscle cell proliferation, low-density lipoprotein receptor folding, and regulation of blood pressure. Furthermore, pathways mainly related to immune response and inflammation, signal transduction, development, and cytoskeleton remodeling, gene expression and protein function, are differentially enriched between the groups.CONCLUSION:Replacing dietary SFA with PUFA for 8 weeks modulates PBMC gene expression and pathways related to CVD risk in healthy subjects with moderate hypercholesterolemia.

Treatment of inflammatory bowel disease (IBD) is challenging, with a series of available drugs each helping only a fraction of patients. Patients may face time-consuming drug trials while the disease is active, thus there is an unmet need for biomarkers and assays to predict drug effect. It is well known that the intestinal epithelium is an important factor in disease pathogenesis, exhibiting physical, biochemical and immunologic driven barrier dysfunctions. One promising test system to study effects of existing or emerging IBD treatments targeting intestinal epithelial cells (IECs) is intestinal organoids (“mini-guts”). However, the fact that healthy intestinal epithelium is in a physiologically hypoxic state has largely been neglected, and studies with intestinal organoids are mainly performed at oxygen concentration of 20%. We hypothesized that lowering the incubator oxygen level from 20% to 2% would recapitulate better the in vivo physiological environment of colonic epithelial cells and enhance the translational value of intestinal organoids as a drug testing platform. In the present study we examine the effects of the key IBD cytokines and drug targets TNF/IL17 on human colonic organoids (colonoids) under atmospheric (20%) or reduced (2%) O2. We show that colonoids derived from both healthy controls and IBD-patients are viable and responsive to IBD-relevant cytokines at 2% oxygen. Because chemokine release is one of the important immunoregulatory traits of the epithelium that may be fine-tuned by IBD-drugs, we also examined chemokine expression and release at different oxygen concentrations. We show that chemokine responses to TNF/IL17 in organoids display similarities to inflamed epithelium in IBD-patients. However, inflammation-associated genes induced by TNF/IL17 were attenuated at low oxygen concentration. We detected substantial oxygen-dependent differences in gene expression in untreated as well as TNF/IL17 treated colonoids in all donors. Further, for some of the IBD-relevant cytokines differences between colonoids from healthy controls and IBD patients were more pronounced in 2% O2 than 20% O2. Our results strongly indicate that an oxygen concentration similar to the in vivo epithelial cell environment is of essence in experimental pharmacology.

During commercial saturation diving, divers live and work under hyperbaric and hyperoxic conditions. The myriads of bacteria that live in and on the human body must adjust to the resultant hyperbaric stress. In this study, we examined the shifts in bacterial content in the oral cavity of saturation divers, using a metagenomic approach to determine the diversity in the composition of bacterial phyla and genera in saliva from 23 male divers before, during, and immediately after 4 weeks of commercial heliox saturation diving to a working depth of circa 200 m. We found that the bacterial diversity fell during saturation, and there was a change in bacterial composition; with a decrease at the phylum level of obligate anaerobe Fusobacteria, and an increase of the relative abundance of Actinobacteria and Proteobacteria. At the genus level, Fusobacterium, Leptotrichia, Oribacterium, and Veillonella decreased, whereas Neisseria and Rothia increased. However, at the end of the decompression, both the diversity and composition of the microbiota returned to pre-dive values. The results indicate that the hyperoxic conditions during saturation may suppress the activity of anaerobes, leaving a niche for other bacteria to fill. The transient nature of the change could imply that hyperbaric heliox saturation has no lasting effect on the oral microbiota, but it is unknown whether or how a shift in oral bacterial diversity and abundance during saturation might impact the divers’ health or well-being.

Abstract Background It is unclear if smoking-related DNA methylation represents a causal pathway between smoking and risk of lung cancer. We sought to identify novel smoking-related DNA methylation sites in blood, with repeated measurements, and to appraise the putative role of DNA methylation in the pathway between smoking and lung cancer development. Methods We derived a nested case-control study from the Trøndelag Health Study (HUNT), including 140 incident patients who developed lung cancer during 2009–13 and 140 controls. We profiled 850 K DNA methylation sites (Illumina Infinium EPIC array) in DNA extracted from blood that was collected in HUNT2 (1995–97) and HUNT3 (2006–08) for the same individuals. Epigenome-wide association studies (EWAS) were performed for a detailed smoking phenotype and for lung cancer. Two-step Mendelian randomization (MR) analyses were performed to assess the potential causal effect of smoking on DNA methylation as well as of DNA methylation (13 sites as putative mediators) on risk of lung cancer. Results The EWAS for smoking in HUNT2 identified associations at 76 DNA methylation sites (P < 5 × 10–8), including 16 novel sites. Smoking was associated with DNA hypomethylation in a dose-response relationship among 83% of the 76 sites, which was confirmed by analyses using repeated measurements from blood that was collected at 11 years apart for the same individuals. Two-step MR analyses showed evidence for a causal effect of smoking on DNA methylation but no evidence for a causal link between DNA methylation and the risk of lung cancer. Conclusions DNA methylation modifications in blood did not seem to represent a causal pathway linking smoking and the lung cancer risk.

Non-small cell lung carcinoma (NSCLC) is one of the most commonly diagnosed cancers and a leading cause of cancer-related deaths. Immunotherapy with immune checkpoint inhibitors shows beneficial responses, but only in a proportion of patients. To improve immunotherapy in NSCLC, we need to map the immune checkpoints that contribute immunosuppression in NSCLC-associated immune cells and to identify novel pathways that regulate immunosuppression. Here, we investigated the gene expression profiles of intra-tumoral immune cells isolated from NSCLC patients and compared them to the expression profiles of their counterparts in adjacent healthy tissue. Transcriptome analysis was performed on macrophages, CD4+ and CD8+ T cells. The data was subjected to Gene Ontology (GO) term enrichment and weighted correlation network analysis in order to identify mediators of immunosuppression in the tumor microenvironment in NSCLC. Immune cells from NSCLC revealed a consistent differential expression of genes involved in interactions between myeloid cells and lymphocytes. We further identified several immunosuppressive molecules and pathways that may be activated in tumor-associated macrophages in NSCLC. Importantly, we report novel data on immune cell expression of the newly described CD200/CD200R1 pathway, and the leukocyte immunoglobulin-like receptors (LILRs), which may represent novel innate immune checkpoints, dampening the anti-tumor T cell immune response in NSCLC. Our study substantiates the importance of tumor-associated macrophages as a mediator of immunosuppression and a promising target for immunotherapy.

BACKGROUND:Microbiota is most likely essential in the pathogenesis of Crohn's disease (CD). Fecal diversion after ileocecal resection (ICR) protects against CD recurrence, whereas infusion of fecal content triggers inflammation. After ICR, the majority of patients experience endoscopic recurrence in the neoterminal ileum, and the ileal microbiome is of particular interest. We have assessed the mucosa-associated microbiome in the inflamed and noninflamed ileum in patients with CD.METHODS:Mucosa-associated microbiome was assessed by 16S rRNA sequencing of biopsies sampled 5 and 15 cm orally of the ileocecal valve or ileocolic anastomosis.RESULTS:Fifty-one CD patients and forty healthy controls (HCs) were included in the study. Twenty CD patients had terminal ileitis, with endoscopic inflammation at 5 cm, normal mucosa at 15 cm, and no history of upper CD involvement. Crohn's disease patients (n = 51) had lower alpha diversity and separated clearly from HC on beta diversity plots. Twenty-three bacterial taxa were differentially represented in CD patients vs HC; among these, Tyzzerella 4 was profoundly overrepresented in CD. The microbiome in the inflamed and proximal noninflamed ileal mucosa did not differ according to alpha diversity or beta diversity. Additionally, no bacterial taxa were differentially represented.CONCLUSIONS:The microbiome is similar in the inflamed and proximal noninflamed ileal mucosa within the same patients. Our results support the concept of CD-specific microbiota alterations and demonstrate that neither ileal sublocation nor endoscopic inflammation influence the mucosa-associated microbiome.

732 Background: Historically, 30 % of patients with localized kidney cancer develop distant metastases during follow-up. There is an urgent need to improve the individual risk assessment for clear cell renal cell carcinoma (ccRCC) patients. We therefore aim to characterize the gene expression profile of low-risk patients both with and without progressive disease to define predictive outcome candidate markers. Methods: Formalin-fixed tissue blocks from ccRCC patients (n=24, eight progressors and 16 non-progressors) with a low Leibovich score were collected. Patients had a mean age of 65 years (5 females and 19 males). The non-progressors were matched 2:1 to the progressors for gender, age, pT tumor stage, size, Fuhrman grade, and eGFR. Total RNA was extracted(miRNeasy FFPE Kit, Qiagen) and sequenced (TruSeq RNA Access Library Kit, Illumina). RNA-seq results were analyzed by ingenuity pathway analysis, K Nearest Neighbors algorithm, and survival analysis. Results: 1167 differentially expressed genes (abs.FC≥2, p≤0.05) were detected. Progressors overexpressed genes related to cancer, B-cell infiltration and other immune-system related pathways. Principal component analyses and hierarchical clustering depicted a systematic transcriptomic difference between progressors and non-progressors. Combinations of up to 10 genes were evaluated as classifiers. The AGAP2-AS1 mRNA classified 23 out of 24 samples correctly, without the need for a larger gene panel. The trend of expression was confirmed with RT-PCR.The correlation between sample status as either progressor or non-progressor and AGAP2-AS1 level was R2 =0.69, p <0.01. Patients were split into groups based on AGAP2-AS1 expression (cut-off log2cpm>1), where higher expression correlated with shorter survival; Wilcoxon (p<0.0001),Log-rank test (p<0.0001), Hazard ratio; 9.24E-11. Immunohistochemistry of AGAP2, USP10 and KI-67 confirmed results from the mRNA level. Conclusions: RNA-seq results show a transcriptomic difference between low-risk ccRCC progressors and low-risk non-progressors. AGAP2-AS1 may serve as a potential classifier for the identification of low-risk progressors.

Proton pump inhibitor use is associated with an increased risk of gastric cancer, which may be mediated by hypergastrinemia. Spasmolytic polypeptide-expression metaplasia (SPEM) has been proposed as a precursor of gastric cancer. We have examined the effects of the gastrin receptor antagonist netazepide (NTZ) or vehicle on the gastric corpus mucosa of H+/K+ATPase beta subunit knockout (KO) and wild-type (WT) mice. The gastric corpus was evaluated by histopathology, immunohistochemistry (IHC), in situ hybridization (ISH) and whole-genome gene expression analysis, focusing on markers of SPEM and neuroendocrine (NE) cells. KO mice had pronounced hypertrophy, intra- and submucosal cysts and extensive expression of SPEM and NE cell markers in the gastric corpus, but not in the antrum. Numerous SPEM-related genes were upregulated in KO mice compared to WT mice. NTZ reduced hypertrophia, cysts, inflammation and NE hyperplasia. However, NTZ neither affected expression of SPEM markers nor of SPEM-related genes. In conclusion, NTZ prevented mucosal hypertrophy, cyst formation and NE cell hyperplasia but did not affect SPEM. The presence of SPEM seems unrelated to the changes caused by hypergastrinemia in this animal model.

The benefits of physical activity in cardiovascular diseases have long been appreciated. However, the molecular mechanisms that trigger and sustain the cardiac benefits of exercise are poorly understood, and it is anticipated that unveiling these mechanisms will identify novel therapeutic targets. In search of these mechanisms we took advantage of unbiased RNA-sequencing (RNA-seq) technology to discover cardiac gene targets whose expression is disrupted in heart failure (HF) and rescued by exercise in a rat model. Upon exhaustive validation in a separate rat cohort (qPCR) and human datasets, we shortlisted 16 targets for a cell-based screening, aiming to evaluate whether targeted disruption of these genes with silencing RNA would affect the abundance of a CVD biomarker (BNP, B-type natriuretic peptide) in human cardiomyocytes. Overall, these experiments showed that Proline Dehydrogenase (PRODH) expression is reduced in human failing hearts, rescued by exercise in a rat model of HF, and its targeted knockdown increases BNP expression in human cardiomyocytes. On the other hand, overexpression of PRODH increases the abundance of metabolism-related gene transcripts, and PRODH appears to be crucial to sustain normal mitochondrial function and maintenance of ATP levels in human cardiomyocytes in a hypoxic environment, as well as for redox homeostasis in both normoxic and hypoxic conditions. Altogether our findings show that PRODH is a novel molecular target of exercise in failing hearts and highlight its role in cardiomyocyte physiology, thereby proposing PRODH as a potential experimental target for gene therapy in HF.