Microenvironmental and cell intrinsic factors governing human cDC2 differentiation and monocyte reprogramming.

cDC2s occur abundantly in peripheral tissues and arise from circulating blood cDC2s. However, the factors governing cDC2 differentiation in tissues, especially under inflammatory conditions, remained poorly defined. We here found that psoriatic cDC2s express the efferocytosis receptor Axl and exhibit a bone morphogenetic protein (BMP) and p38MAPK signaling signature. BMP7, strongly expressed within the lesional psoriatic epidermis, cooperates with canonical TGF-ß1 signaling for inducing Axl+cDC2s from blood cDC2s in vitro. Moreover, downstream induced p38MAPK promotes Axl+cDC2s at the expense of Axl+CD207+ Langerhans cell differentiation from blood cDC2s. BMP7 supplementation allowed to model cDC2 generation and their further differentiation into LCs from CD34+ hematopoietic progenitor cells in defined serum-free medium. Additionally, p38MAPK promoted the generation of another cDC2 subset lacking Axl but expressing the non-classical NFkB transcription factor RelB in vitro. Such RelB+cDC2s occurred predominantly at dermal sites in the inflamed skin. Finally, we found that cDC2s can be induced to acquire high levels of the monocyte lineage identity factor kruppel-like-factor-4 (KLF4) along with monocyte-derived DC and macrophage phenotypic characteristics in vitro. In conclusion, inflammatory and psoriatic epidermal signals instruct blood cDC2s to acquire phenotypic characteristics of several tissue-resident cell subsets.

SLCO4A1 expression is associated with activated inflammatory pathways in high-grade serous ovarian cancer.

Patients with high-grade serous ovarian cancer (HGSOC) have a very poor overall survival. Current therapeutic approaches do not bring benefit to all patients. Although genetic alterations and molecular mechanisms are well characterized, the molecular pathological conditions are poorly investigated. Solute carrier organic anion transporter family member 4A1 (SLCO4A1) encodes OATP4A1, which is an uptake membrane transporter of metabolic products. Its expression may influence various signaling pathways associated with the molecular pathophysiological conditions of HGSOC and consequently tumor progression. RNA sequencing of 33 patient-derived HGSOC cell lines showed that SLCO4A1 expression was diverse by individual tumors, which was further confirmed by RT-qPCR, Western blotting and immunohistochemistry. Gene Set Enrichment Analysis revealed that higher SLCO4A1 level was associated with inflammation-associated pathways including NOD-like receptor, adipocytokine, TALL1, CD40, NF-κB, and TNF-receptor 2 signaling cascades, while low SLCO4A1 expression was associated with the mitochondrial electron transport chain pathway. The overall gene expression pattern in all cell lines was specific to each patient and remained largely unchanged during tumor progression. In addition, genes encoding ABCC3 along with SLCO4A1-antisense RNA 1, were associated with higher expression of the SLCO4A1, indicating their possible involvement in inflammation-associated pathways that are downstream to the prostaglandin E2/cAMP axis. Taken together, increased SLCO4A1/OATP4A1 expression is associated with the upregulation of specific inflammatory pathways, while the decreased level is associated with mitochondrial dysfunction. These molecular pathophysiological conditions are tumor specific and should be taken into consideration by the development of therapies against HGSOC.

Singularity and Commonality in Response to SARS-CoV-2 in Lung and Colon Cell Models.

The systemic nature of COVID-19 with multiple extrapulmonary manifestations of disease, largely due to the wide tissue expression of SARS-CoV-2 major entry factors, as well as the patient-specific features of COVID-19 pathobiology, determine important directions for basic and translational research. In the current study, we addressed the questions of singularities and commonalities in cellular responses to SARS-CoV-2 and related SARS-CoV on the basis of compendium-wide analysis of publicly available transcriptomic datasets as part of the herein implemented multi-modular UNCOVIDING approach. We focused on cellular models attributed to the epithelial cells of the respiratory system, the Calu-3 cell line, and epithelial cells of the gastrointestinal tract, the Caco-2 cell line, infected with either SARS-CoV-2 or SARS-CoV. Here, we report the outcome of a comparative analysis based on differentially expressed genes in terms of perturbations and diseases, Canonical pathways, and Upstream Regulators. We furthermore performed compendium-wide analysis across more than 19,000 mRNASeq datasets and dissected the condition-specific gene signatures. Information was gained with respect to common and unique cellular responses and molecular events. We identified that in cell lines of colon or lung origin, both viruses show similarities in cellular responses; by contrast, there are cell type-specific regulators that differed for Calu-3 and Caco-2 cells. Among the major findings is the impact of the interferon system for lung Calu-3 cells and novel links to the liver- and lipid-metabolism-associated responses for colon Caco-2 cells as part of the extrapulmonary pathomechanisms in the course of COVID-19. Among differently expressed genes, we specifically dissected the expression pattern of the APOBEC family members and propose APOBEC3G as a promising intrinsic antiviral factor of the host response to SARS-CoV-2. Overall, our study provides gene expression level evidence for the cellular responses attributed to pulmonary and gastrointestinal manifestations of COVID-19.

AID and APOBECs as Multifaceted Intrinsic Virus-Restricting Factors: Emerging Concepts in the Light of COVID-19.

The AID (activation-induced cytidine deaminase)/APOBEC (apolipoprotein B mRNA editing enzyme catalytic subunit) family with its multifaceted mode of action emerges as potent intrinsic host antiviral system that acts against a variety of DNA and RNA viruses including coronaviruses. All family members are cytosine-to-uracil deaminases that either have a profound role in driving a strong and specific humoral immune response (AID) or restricting the virus itself by a plethora of mechanisms (APOBECs). In this article, we highlight some of the key aspects apparently linking the AID/APOBECs and SARS-CoV-2. Among those is our discovery that APOBEC4 shows high expression in cell types and anatomical parts targeted by SARS-CoV-2. Additional focus is given by us to the lymphoid structures and AID as the master regulator of germinal center reactions, which result in antibody production by plasma and memory B cells. We propose the dissection of the AID/APOBECs gene signature towards decisive determinants of the patient-specific and/or the patient group-specific antiviral response. Finally, the patient-specific mapping of the AID/APOBEC polymorphisms should be considered in the light of COVID-19.

Induction of the sphingosine-1-phosphate signaling pathway by TGF-ß1 during Langerhans-type dendritic cell differentiation.

Gene profiling revealed that the S1P signaling pathway is induced by TGF-ß1 during LC commitment of monocytopoietic cells. Constitutive-active TGF-ß1-S1P signaling seems to elevate the activation threshold of LCs and thereby prevent inappropriate and overshooting immune responses to microbial and physicochemical environmental signals. In turn, signals that lead to LC migration may disrupt this pathway via inhibiting S1P bioavailability.

The Immune Phenotype of Isolated Lymphoid Structures in Non-Tumorous Colon Mucosa Encrypts the Information on Pathobiology of Metastatic Colorectal Cancer.

The gut-associated lymphoid tissue represents an integral part of the immune system. Among the powerful players of the mucosa-associated lymphoid tissue are isolated lymphoid structures (ILSs), which as information centers, drive the local (and systemic) adaptive immune responses. Germinal center reactions, taking place within ILSs, involve the coordinated action of various immune cell types with a central role given to B cells. In the current study, we aimed at dissecting the impact of ILSs within non-tumorous colon tissue (NT) on the pathobiology of colorectal cancer (CRC) with metastasis in the liver (CRCLM). In particular, we focused on the immune phenotypes of ILSs and ectopic lymphoid structures (ELSs), built up at matching primary and metastatic tumor sites. We implemented an integrative analysis strategy on the basis of tissue image cytometry and clonality assessment to explore the immune phenotype of ILS/ELS at three tissue entities: NT, CRC, and CRCLM (69 specimens in total). Applying a panel of lineage markers used for immunostaining, we characterized and compared the anatomical features, the cellular composition, the activation, and proliferation status of ILSs and ELSs, and assessed the clinical relevance of staining-derived data sets. Our major discovery was that ILS characteristics at the NT site predefine the immune phenotype of ELSs at CRC and CRCLM. Thereby, B-cell-enriched (CD20) and highly proliferative (Ki67) ILSs and ELSs were found to be associated with improved clinical outcome in terms of survival and enabled patient stratification into risk groups. Moreover, the data revealed a linkage between B-cell clonality at the NT site and the metastatic characteristics of the tumor in the distant liver tissue. Consolidation of immunostaining-based findings with the results of compendium-wide transcriptomic analysis furthermore proposed CD27 as a novel marker of T follicular helper cells within lymphoid structures. Overall, the study nominates the ILS immune phenotype as a novel prognostic marker for patients with metastatic CRC.

Two common polymorphic variants of OATP4A1 as potential risk factors for colorectal cancer.

Genetic variations in the organic-anion-transporting polypeptide (OATP)-encoding solute carrier of organic anions (SLCO) genes can promote cancer development and progression. The overexpression of solute carrier organic anion transporter family member 4A1 (OATP4A1), a transporter for steroid hormones, prostaglandins, and bile acids, has been previously associated with tumor recurrence and progression in colorectal cancer (CRC). Therefore, the present study aimed to investigate the association between 2 frequent single nucleotide polymorphisms (SNPs) in SLCO4A1 (rs34419428, R70Q; rs1047099G, V78I) and CRC predisposition. Following restriction fragment length polymorphism-PCR analysis in 178 patients with CRC [Union for International Cancer Control (UICC) stage I/II] and 65 healthy controls, no significant difference was observed in allele frequency and the number of heterozygous/homozygous individuals between the groups. Notably, the R70Q minor allele was identified to be associated with the V78I minor allele in the genome. Comparing of the individual genotypes of CRC patients to clinical data, including sex, UICC-stage and relapse revealed no increased risk for CRC. In addition, the OATP4A1 immunoreactivity assay in paraffin-embedded CRC and adjacent non-tumorous mucosa sections, examined using quantitative microscopy image analysis, did not reveal any association with these polymorphisms. No significant differences were observed in the expression levels, localization, and sodium fluorescein transport capacity among the OATP4A1 variants, which was studied using functional assays in Sf9-insect and A431 tumor cells overexpressing the 2 single and a double mutant OATP4A1 SNP variants. These results suggested that the 2 most frequent polymorphisms located in the first intracellular loop of OATP4A1 do not associate with CRC predisposition and tumor recurrence. They are unlikely to affect the outcome of CRC in patients.

Metabolism of Estrogens: Turnover Differs Between Platinum-Sensitive and -Resistant High-Grade Serous Ovarian Cancer Cells.

High-grade serous ovarian cancer (HGSOC) is currently treated with cytoreductive surgery and platinum-based chemotherapy. The majority of patients show a primary response; however, many rapidly develop drug resistance. Antiestrogens have been studied as low toxic treatment options for HGSOC, with higher response rates in platinum-sensitive cases. Mechanisms for this difference in response remain unknown. Therefore, the present study investigated the impact of platinum resistance on steroid metabolism in six established HGSOC cell lines sensitive and resistant against carboplatin using a high-resolution mass spectrometry assay to simultaneously quantify the ten main steroids of the estrogenic metabolic pathway. An up to 60-fold higher formation of steroid hormones and their sulfated or glucuronidated metabolites was observed in carboplatin-sensitive cells, which was reversible by treatment with interleukin-6 (IL-6). Conversely, treatment of carboplatin-resistant cells expressing high levels of endogenous IL-6 with the monoclonal anti-IL-6R antibody tocilizumab changed their status to "platinum-sensitive", exhibiting a decreased IC50 value for carboplatin, decreased growth, and significantly higher estrogen metabolism. Analysis of these metabolic differences could help to detect platinum resistance in HGSOC patients earlier, thereby allowing more efficient interventions.

Interrelations of Sphingolipid and Lysophosphatidate Signaling with Immune System in Ovarian Cancer.

The sphingolipid and lysophosphatidate regulatory networks impact diverse mechanisms attributed to cancer cells and the tumor immune microenvironment. Deciphering the complexity demands implementation of a holistic approach combined with higher-resolution techniques. We implemented a multi-modular integrative approach consolidating the latest accomplishments in gene expression profiling, prognostic/predictive modeling, next generation digital pathology, and systems biology for epithelial ovarian cancer. We assessed patient-specific transcriptional profiles using the sphingolipid/lysophosphatidate/immune-associated signature. This revealed novel sphingolipid/lysophosphatidate-immune gene-gene associations and distinguished tumor subtypes with immune high/low context. These were characterized by robust differences in sphingolipid-/lysophosphatidate-related checkpoints and the drug response. The analysis also nominates novel survival models for stratification of patients with CD68, LPAR3, SMPD1, PPAP2B, and SMPD2 emerging as the most prognostically important genes. Alignment of proprietary data with curated transcriptomic data from public databases across a variety of malignancies (over 600 categories; over 21,000 arrays) showed specificity for ovarian carcinoma. Our systems approach identified novel sphingolipid-lysophosphatidate-immune checkpoints and networks underlying tumor immune heterogeneity and disease outcomes. This holds great promise for delivering novel stratifying and targeting strategies.

Clinical Significance of Organic Anion Transporting Polypeptide Gene Expression in High-Grade Serous Ovarian Cancer.

High-grade serous ovarian cancer (HGSOC) is considered the most deadly and frequently occurring type of ovarian cancer and is associated with various molecular compositions and growth patterns. Evaluating the mRNA expression pattern of the organic anion transporters (OATPs) encoded by SLCO genes may allow for improved stratification of HGSOC patients for targeted invention. The expression of SLCO mRNA and genes coding for putative functionally related ABC-efflux pumps, enzymes, pregnane-X-receptor, ESR1 and ESR2 (coding for estrogen receptors ERα and ERß) and HER-2 were assessed using RT-qPCR. The expression levels were assessed in a cohort of 135 HGSOC patients to elucidate the independent impact of the expression pattern on the overall survival (OS). For identification of putative regulatory networks, Graphical Gaussian Models were constructed from the expression data with a tuning parameter K varying between meaningful borders (Pils et al., 2012; Auer et al., 2015, 2017; Kurman and Shih Ie, 2016; Karam et al., 2017; Labidi-Galy et al., 2017; Salomon-Perzynski et al., 2017; Sukhbaatar et al., 2017). The final value used (K = 4) was determined by maximizing the proportion of explained variation of the corresponding LASSO Cox regression model for OS. The following two networks of directly correlated genes were identified: (i) SLCO2B1 with ABCC3 implicated in estrogen homeostasis; and (ii) two ABC-efflux pumps in the immune regulation (ABCB2/ABCB3) with ABCC3 and HER-2. Combining LASSO Cox regression and univariate Cox regression analyses, SLCO5A1 coding for OATP5A1, an estrogen metabolite transporter located in the cytoplasm and plasma membranes of ovarian cancer cells, was identified as significant and independent prognostic factor for OS (HR = 0.68, CI 0.49-0.93; p = 0.031). Furthermore, results indicated the benefits of patients with high expression by adding 5.1% to the 12.8% of the proportion of explained variation (PEV) for clinicopathological parameters known for prognostic significance (FIGO stage, age and residual tumor after debulking). Additionally, overlap with previously described signatures that indicated a more favorable prognosis for ovarian cancer patients was shown for SLCO5A1, the network ABCB2/ABCB3/ABCC4/HER2 as well as ESR1. Furthermore, expression of SLCO2A1 and PGDH, which are important for PGE2 degradation, was associated with the non-miliary peritoneal tumor spreading. In conclusion, the present findings suggested that SLCOs and the related molecules identified as potential biomarkers in HGSOC may be useful for the development of novel therapeutic strategies.

Resveratrol Inhibits Key Steps of Steroid Metabolism in a Human Estrogen-Receptor Positive Breast Cancer Model: Impact on Cellular Proliferation.

The role of resveratrol (RES) in preventing breast cancer is controversial, as low concentrations may stimulate the proliferation of estrogen-receptor alpha positive (ERα+) breast cancer cells. As metabolism is the key factor in altering cellular estrogens, thereby influencing breast tumor growth, we investigated the effects of RES on the formation of estrogen metabolites, namely 4-androstene-3,17-dione (AD), dehydroepiandrosterone (DHEA), dehydroepiandrosterone-3-O-sulfate (DHEA-S), estrone (E1), estrone-3-sulfate (E1-S), 17ß-estradiol (E2), 17ß-estradiol-3-O-(ß-D-glucuronide) (E2-G), 17ß-estradiol-3-O-sulfate (E2-S), 16α-hydroxy-17ß-estradiol (estriol, E3), and testosterone (T) in ERα- MDA-MB-231 and ERα+ MCF-7 cells. Incubation of both of the cell lines with the hormone precursors DHEA and E1 revealed that sulfation and glucuronidation were preferred metabolic pathways for DHEA, E1 and E2 in MCF-7 cells, compared with in MDA-MB-231 cells, as the Vmax values were significantly higher (DHEA-S: 2873.0 ± 327.4 fmol/106 cells/h, E1-S: 30.4 ± 2.5 fmol/106 cells/h, E2-S: 24.7 ± 4.9 fmol/106 cells/h, E2-G: 7.29 ± 1.36 fmol/106 cells/h). RES therefore significantly inhibited DHEA-S, E1-S, E2-S and E2-G formation in MCF-7, but not in MDA-MB-231 cells (Kis: E2-S, 0.73 ± 0.07 µM < E1-S, 0.94 ± 0.03 µM < E2-G, 7.92 ± 0.24 µM < DHEA-S, 13.2 ± 0.2 µM). Suppression of these metabolites subsequently revealed twofold higher levels of active E2, concomitant with an almost twofold increase in MCF-7 cell proliferation, which was the most pronounced upon the addition of 5 µM RES. As the content of RES in food is relatively low, an increased risk of breast cancer progression in women is likely to only be observed following the continuous consumption of high-dose RES supplements. Further long-term human studies simultaneously monitoring free estrogens and their conjugates are therefore highly warranted to evaluate the efficacy and safety of RES supplementation, particularly in patients diagnosed with ERα+ breast cancer.

Reduced Levels of the Synaptic Functional Regulator FMRP in Dentate Gyrus of the Aging Sprague-Dawley Rat.

Fragile X mental retardation protein (FMRP) encoded by Fragile X mental retardation 1 (FMR1) gene is a RNA-binding regulator of mRNA translation, transport and stability with multiple targets responsible for proper synaptic function. Epigenetic silencing of FMR1 gene expression leads to the development of Fragile X syndrome (FXS) that is characterized by intellectual disability and other behavioral problems including autism. In the rat FXS model, the lack of FMRP caused a deficit in hippocampal-dependent memory. However, the hippocampal changes of FMRP in aging rats are not fully elucidated. The current study addresses the changes in FMRP levels in dentate gyrus (DG) from young (17 weeks) and aging (22 months) Sprague - Dawley rats. The aging animal group showed significant decline in spatial reference memory. Protein samples from five rats per each group were analyzed by quantitative proteomic analysis resulting in 153 significantly changed proteins. FMRP showed significant reduction in aging animals which was confirmed by immunoblotting and immunofluorescence microscopy. Furthermore, bioinformatic analysis of the differential protein dataset revealed several functionally related protein groups with individual interactions with FMRP. These include high representation of the RNA translation and processing machinery connected to FMRP and other RNA-binding regulators including CAPRIN1, the members of Pumilio (PUM) and CUG-BP, Elav-like (CELF) family, and YTH N(6)-methyladenosine RNA-binding proteins (YTHDF). The results of the current study point to the important role of FMRP and regulation of RNA processing in the rat DG and memory decline during the aging process.

Fasting metabolism modulates the interleukin-12/interleukin-10 cytokine axis.

A crucial role of cell metabolism in immune cell differentiation and function has been recently established. Growing evidence indicates that metabolic processes impact both, innate and adaptive immunity. Since a down-stream integrator of metabolic alterations, mammalian target of rapamycin (mTOR), is responsible for controlling the balance between pro-inflammatory interleukin (IL)-12 and anti-inflammatory IL-10, we investigated the effect of upstream interference using metabolic modulators on the production of pro- and anti-inflammatory cytokines. Cytokine release and protein expression in human and murine myeloid cells was assessed after toll-like receptor (TLR)-activation and glucose-deprivation or co-treatment with 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) activators. Additionally, the impact of metabolic interference was analysed in an in-vivo mouse model. Glucose-deprivation by 2-deoxy-D-glucose (2-DG) increased the production of IL-12p40 and IL-23p19 in monocytes, but dose-dependently inhibited the release of anti-inflammatory IL-10. Similar effects have been observed using pharmacological AMPK activation. Consistently, an inhibition of the tuberous sclerosis complex-mTOR pathway was observed. In line with our in vitro observations, glycolysis inhibition with 2-DG showed significantly reduced bacterial burden in a Th2-prone Listeria monocytogenes mouse infection model. In conclusion, we showed that fasting metabolism modulates the IL-12/IL-10 cytokine balance, establishing novel targets for metabolism-based immune-modulation.

Activation of the ileal neuroendocrine tumor cell line P-STS by acetylcholine is amplified by histamine: role of H3R and H4R.

Neuroendocrine tumors may present with pseudoallergic reactions like diarrhea and idiopathic anaphylaxis. Here we present the P-STS human ileal neuroendocrine cell line as a model cell line for these tumors. Neuroendocrine markers and changes in cytoplasmic calcium concentration ([Ca2+]i) in response to several possible activators of 5-hydroxytryptamine (5-HT) release were analyzed. P-STS cells still expressed chromogranin A and synaptophysin after 2 years of culture. Tryptophan hydroxylase 1 mRNA and a low amount of 5-HT were also detected. Acetylcholine (ACh) caused a rise in [Ca2+]i. Somatostatin inhibited, whereas histamine (HA) but not the HA receptor ligand betahistine enhanced activation by ACh. The [Ca2+]i response to ACh/HA was inhibited by the HA receptor H3 (H3R) agonist methimepip and by the antidepressant imipramine. Further [Ca2+]i response studies indicated the presence of H4Rs and of a functional calcium sensing receptor. High or low affinity IgE receptor protein or mRNA were not detected. Taken together, neuroendocrine markers and response to intestinal neurotransmitters approve the P-STS cell line as a valuable model for enterochromaffin cells. Enhancement of their ACh-induced pro-secretory response by HA, with a role for H3R and H4R, suggests an amplifying role of neuroendocrine cells in allergen-induced diarrhea or anaphylaxis.

Sphingosine 1-phosphate signaling in bone remodeling: multifaceted roles and therapeutic potential.

INTRODUCTION: Sphingolipids belong to a complex class of lipid molecules that are crucially involved in the regulation of important biological processes including proliferation, migration and apoptosis. Given the significant progress made in understanding the sphingolipid pathobiology of several diseases, sphingolipid-related checkpoints emerge as attractive targets. Recent data indicate the multifaceted contribution of the sphingolipid machinery to osteoclast - osteoblast crosstalk, representing one of the pivotal interactions underlying bone homeostasis. Imbalances in the interplay of osteoblasts and osteoclasts might lead to bone-related diseases such as osteoporosis, rheumatoid arthritis, and bone metastases. Areas covered: We summarize and analyze the progress made in bone research in the context of the current knowledge of sphingolipid-related mechanisms regulating bone remodeling. Particular emphasis was given to bioactive sphingosine 1-phosphate (S1P) and S1P receptors (S1PRs). Moreover, the mechanisms of how dysregulations of this machinery cause bone diseases, are covered. Expert opinion: In the context of bone diseases, pharmacological interference with sphingolipid machinery may lead to novel directions in therapeutic strategies. Implementation of knowledge derived from in vivo animal models and in vitro studies using pharmacological agents to manipulate the S1P/S1PRs axes suggests S1PR2 and S1PR3 as potential drug targets, particularly in conjunction with technology for local drug delivery.

ICAM-1 Binding Rhinoviruses Enter HeLa Cells via Multiple Pathways and Travel to Distinct Intracellular Compartments for Uncoating.

Of the more than 150 human rhinovirus (RV) serotypes, 89 utilize intercellular adhesion molecule-1 (ICAM-1) for cell entry. These belong either to species A or B. We recently demonstrated that RV-B14 and RV-A89, despite binding this same receptor, are routed into distinct endosomal compartments for release of their RNA into the cytosol. To gain insight into the underlying mechanism we now comparatively investigate the port of entry, temperature-dependence of uncoating, and intracellular routing of RV-B3, RV-B14, RV-A16, and RV-A89 in HeLa cells. The effect of various drugs blocking distinct stages on the individual pathways was determined via comparing the number of infected cells in a TissueFaxs instrument. We found that RV-B14 and RV-A89 enter via clathrin-, dynamin-, and cholesterol-dependent pathways, as well as by macropinocytosis. Drugs interfering with actin function similarly blocked entry of all four viruses, indicating their dependence on a dynamic actin network. However, uniquely, RV-A89 was able to produce progeny when internalized at 20 °C followed by neutralizing the endosomal pH and further incubation at 37 °C. Blocking dynein-dependent endosomal transport prevented uncoating of RV-A16 and RV-A89, but not of RV-B3 and RV-B14, indicative for routing of RV-A16 and RV-A89 into the endocytic recycling compartment for uncoating. Our results call for caution when developing drugs aimed at targeting entry or intracellular trafficking of all rhinovirus serotypes.

Human skin dendritic cell fate is differentially regulated by the monocyte identity factor Kruppel-like factor 4 during steady state and inflammation.

BACKGROUND: Langerhans cell (LC) networks play key roles in immunity and tolerance at body surfaces. LCs are established prenatally and can be replenished from blood monocytes. Unlike skin-resident dermal DCs (dDCs)/interstitial-type DCs and inflammatory dendritic epidermal cells appearing in dermatitis/eczema lesions, LCs lack key monocyte-affiliated markers. Inversely, LCs express various epithelial genes critical for their long-term peripheral tissue residency. OBJECTIVE: Dendritic cells (DCs) are functionally involved in inflammatory diseases; however, the mechanisms remained poorly understood. METHODS: In vitro differentiation models of human DCs, gene profiling, gene transduction, and immunohistology were used to identify molecules involved in DC subset specification. RESULTS: Here we identified the monocyte/macrophage lineage identity transcription factor Kruppel-like factor 4 (KLF4) to be inhibited during LC differentiation from human blood monocytes. Conversely, KLF4 is maintained or induced during dermal DC and monocyte-derived dendritic cell/inflammatory dendritic epidermal cell differentiation. We showed that in monocytic cells KLF4 has to be repressed to allow their differentiation into LCs. Moreover, respective KLF4 levels in DC subsets positively correlate with proinflammatory characteristics. We identified epithelial Notch signaling to repress KLF4 in monocytes undergoing LC commitment. Loss of KLF4 in monocytes transcriptionally derepresses Runt-related transcription factor 3 in response to TGF-ß1, thereby allowing LC differentiation marked by a low cytokine expression profile. CONCLUSION: Monocyte differentiation into LCs depends on activation of Notch signaling and the concomitant loss of KLF4.

AID/APOBEC-network reconstruction identifies pathways associated with survival in ovarian cancer.

BACKGROUND: Building up of pathway-/disease-relevant signatures provides a persuasive tool for understanding the functional relevance of gene alterations and gene network associations in multifactorial human diseases. Ovarian cancer is a highly complex heterogeneous malignancy in respect of tumor anatomy, tumor microenvironment including pro-/antitumor immunity and inflammation; still, it is generally treated as single disease. Thus, further approaches to investigate novel aspects of ovarian cancer pathogenesis aiming to provide a personalized strategy to clinical decision making are of high priority. Herein we assessed the contribution of the AID/APOBEC family and their associated genes given the remarkable ability of AID and APOBECs to edit DNA/RNA, and as such, providing tools for genetic and epigenetic alterations potentially leading to reprogramming of tumor cells, stroma and immune cells. RESULTS: We structured the study by three consecutive analytical modules, which include the multigene-based expression profiling in a cohort of patients with primary serous ovarian cancer using a self-created AID/APOBEC-associated gene signature, building up of multivariable survival models with high predictive accuracy and nomination of top-ranked candidate/target genes according to their prognostic impact, and systems biology-based reconstruction of the AID/APOBEC-driven disease-relevant mechanisms using transcriptomics data from ovarian cancer samples. We demonstrated that inclusion of the AID/APOBEC signature-based variables significantly improves the clinicopathological variables-based survival prognostication allowing significant patient stratification. Furthermore, several of the profiling-derived variables such as ID3, PTPRC/CD45, AID, APOBEC3G, and ID2 exceed the prognostic impact of some clinicopathological variables. We next extended the signature-/modeling-based knowledge by extracting top genes co-regulated with target molecules in ovarian cancer tissues and dissected potential networks/pathways/regulators contributing to pathomechanisms. We thereby revealed that the AID/APOBEC-related network in ovarian cancer is particularly associated with remodeling/fibrotic pathways, altered immune response, and autoimmune disorders with inflammatory background. CONCLUSIONS: The herein study is, to our knowledge, the first one linking expression of entire AID/APOBECs and interacting genes with clinical outcome with respect to survival of cancer patients. Overall, data propose a novel AID/APOBEC-derived survival model for patient risk assessment and reconstitute mapping to molecular pathways. The established study algorithm can be applied further for any biologically relevant signature and any type of diseased tissue.

A detailed proteomic profiling of plasma membrane from zebrafish brain.

Zebrafish (Danio rerio) is a well-established model organism in developmental biology and disease modeling. In recent years, an increasing amount of studies used zebrafish to analyze the genetic changes underlying various neurological disorders. The brain plasma membrane proteome represents the major subsets of signaling proteins and promising drug targets, but is often understudied due to traditional experimental difficulties including problems with solubility, detergent removal, or low abundance. Here, we report a comprehensive dataset of the proteins identified in the enriched plasma membrane of the zebrafish brain by applying sequential trypsin/chymotrypsin digestion with multidimensional LC-MS/MS. A total number of 97 017 peptide groups corresponding to 9201 proteins were identified. These were annotated in various molecular functions or neurological disorders. The dataset of the current study provides a useful data source for further utilizing zebrafish in basic and clinical neuroscience.

Exploring the role of sphingolipid machinery during the epithelial to mesenchymal transition program using an integrative approach.

The epithelial to mesenchymal transition (EMT) program is activated in epithelial cancer cells and facilitates their ability to metastasize based on enhanced migratory, proliferative, anti-apoptotic, and pluripotent capacities. Given the fundamental impact of sphingolipid machinery to each individual process, the sphingolipid-related mechanisms might be considered among the most prominent drivers/players of EMT; yet, there is still limited knowledge. Given the complexity of the interconnected sphingolipid system, which includes distinct sphingolipid mediators, their synthesizing enzymes, receptors and transporters, we herein apply an integrative approach for assessment of the sphingolipid-associated mechanisms underlying EMT program. We created the sphingolipid-/EMT-relevant 41-gene/23-gene signatures which were applied to denote transcriptional events in a lung cancer cell-based EMT model. Based on defined 35-gene sphingolipid/EMT-attributed signature of regulated genes, we show close associations between EMT markers, genes comprising the sphingolipid network at multiple levels and encoding sphingosine 1-phosphate (S1P)-/ceramide-metabolizing enzymes, S1P and lysophosphatidic acid (LPA) receptors and S1P transporters, pluripotency genes and inflammation-related molecules, and demonstrate the underlying biological pathways and regulators. Mass spectrometry-based sphingolipid analysis revealed an EMT-attributed shift towards increased S1P and LPA accompanied by reduced ceramide levels. Notably, using transcriptomics data across various cell-based perturbations and neoplastic tissues (24193 arrays), we identified the sphingolipid/EMT signature primarily in lung adenocarcinoma tissues; besides, bladder, colorectal and prostate cancers were among the top-ranked. The findings also highlight novel regulatory associations between influenza virus and the sphingolipid/EMT-associated mechanisms. In sum, data propose the multidimensional contribution of sphingolipid machinery to pathological EMT and may yield new biomarkers and therapeutic targets.