Granulocyte Adhesion Research Articles

Predictive models for stage and risk classification in head and neck squamous cell carcinoma (HNSCC).

Machine learning techniques are increasingly used in the analysis of high throughput genome sequencing data to better understand the disease process and design of therapeutic modalities. In the current study, we have applied state of the art machine learning (ML) algorithms (Random Forest (RF), Support Vector Machine Radial Kernel (svmR), Adaptive Boost (AdaBoost), averaged Neural Network (avNNet), and Gradient Boosting Machine (GBM)) to stratify the HNSCC patients in early and late clinical stages (TNM) and to predict the risk using miRNAs expression profiles. A six miRNA signature was identified that can stratify patients in the early and late stages. The mean accuracy, sensitivity, specificity, and area under the curve (AUC) was found to be 0.84, 0.87, 0.78, and 0.82, respectively indicating the robust performance of the generated model. The prognostic signature of eight miRNAs was identified using LASSO (least absolute shrinkage and selection operator) penalized regression. These miRNAs were found to be significantly associated with overall survival of the patients. The pathway and functional enrichment analysis of the identified biomarkers revealed their involvement in important cancer pathways such as GP6 signalling, Wnt signalling, p53 signalling, granulocyte adhesion, and dipedesis. To the best of our knowledge, this is the first such study and we hope that these signature miRNAs will be useful for the risk stratification of patients and the design of therapeutic modalities.

Shuxuening injection facilitates neurofunctional recovery via down-regulation of G-CSF-mediated granulocyte adhesion and diapedesis pathway in a subacute stroke mouse model

Post-stroke neural damage is a serious health concern which does not yet have an effective treatment. We have shown previously that Shuxuening injection (SXNI), a Ginkgo biloba extract-based natural medicine, protects brain after an acute ischemic stroke, but its efficacy for post-stroke recovery is not known. This study was to investigate whether SXNI can improve the prognosis of stroke at a subacute phase. Mice with cerebral ischemia-reperfusion injury (CIRI) were established by middle cerebral artery occlusion (MCAO), and drugs or saline were injected by the tail vein every 12 h after reperfusion. The therapeutic effect of SXNI was evaluated by survival rate, modified neurologic severity scores (mNSS), open-field test, locomotive gait patterns, cerebral infarction volume, brain edema and histopathological changes. Subsequently, a combined method of RNA-seq and Ingenuity® Pathway Analysis (IPA) was performed to identify key targets and pathways of SXNI facilitating the prognosis of stroke in mouse brain. The results of the transcriptome analysis were verified by real time reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), western blot (WB) and immunohistochemistry (IHC). The experimental results showed that in the new subacute stroke model, SXNI markedly improves the survival rate, neurological and motor functions and histopathological changes, and significantly reduces cerebral infarction and edema volume. RNA-seq analysis of subacute stroke mice with or without SXNI (3 mL/kg) indicated 963 differentially expressed genes (DEGs) with a fold change ≥ 1.5 and a P-value ≤ 0.01. IPA analysis of DEGs showed that granulocyte adhesion and diapedesis ranked first in the pathway ranking, and the most critical gene regulated by SXNI was G-csf. Simultaneously, RT-PCR, ELISA, WB and IHC results demonstrated that SXNI not only obviously reduced the mRNA expression levels of key genes G-csf, Sele and Mac-1 in this pathway, but also significantly decreased the protein expression levels of G-CSF in serum and E-selectin and MAC-1 in brain tissues. In summary, our research suggested that SXNI can exert a remarkable neurofunctional therapeutic effect on stroke mice via down-regulating G-CSF to inhibit granulocyte adhesion and diapedesis. This study provides experimental evidence that SXNI may fulfill the need for stroke medicine targeting specifically at the recovery stage.

SUN-603 Electronic Cigarette Exposure Induces Pro-Inflammatory Changes in Adipose Tissue in Apolipoprotein E (APOE) Knockout Mice

Electronic nicotine delivery systems or electronic cigarettes (e-cigarettes) are becoming exceptionally popular in the world as an alternative to conventional nicotine cigarettes, both in smokers and people who have never smoked. Nicotine can induce lipolysis in adipose tissue, leading to increased serum free fatty acids (FFA). Increased levels of FFA are one of the key elements in inducing a pro-inflammatory response and lead to ectopic lipid accumulation, lipotoxicity, mitochondrial dysfunction and metabolic disease. Our laboratory has shown that chronic e-cigarette exposure induces cardiac dysfunction, atherosclerosis and hepatic steatosis in the ApoE knockout (KO) model associated with increased levels of serum FFA. In this study, we investigated the role of adipose tissue in the metabolic changes associated with e-cigarette exposure. ApoE KO mice were exposed to saline, e-cigarette without nicotine [e-cigarette (0%)] and e-cigarette with 2.4% nicotine [e-cigarette (2.4%)] aerosol for 12 weeks. Western blot analyses from adipose tissue showed that mice treated with e-cigarette (2.4%) had decrease levels of SIRT1 when compared to mice treated with saline or e-cigarette (0%). Transcriptomic analysis of the differentially expressed genes shows a differential transcriptional response to e-cigarette (2.4%) in adipose tissue in comparison with e-cigarette (0%) or saline. The RNA-seq examination using ingenuity pathway analysis (IPA) software, revealed dysregulation of fibrosis, agranulocyte and granulocyte adhesion and diapedesis pathways in e-cigarette (2.4%) -exposed adipose tissue. Overall, we found an inflammatory phenotype associated with decreased levels of SIRT1 in adipose tissue of mice treated with e-cigarette (2.4%). Understanding the consequences of e-cigarette use on metabolic disease is directly relevant to the development of policies related to e-cigarette use.

Abstract P2-06-01: Intrinsic transcriptomic differences of luminal progenitors and mature luminal cells of the normal breast

Abstract Epithelial cells in the normal breast are organized hierarchically into stem, luminal progenitor (LP) and mature luminal cells (LC) with distinct gene expression patterns. However, whether these gene expression differences are cell-intrinsic, driven through LP-LC cell interactions or due to stromal-luminal cell interactions in the breasts of healthy women is incompletely understood. Furthermore, prior studies on these cell populations did not take genetic ancestry into consideration despite widely reported genetic ancestry-dependent differences in the normal breast biology and breast cancer susceptibility/outcome. We isolated LP and LC breast epithelial cells from core breast biopsies of healthy women representing European, African American and Latino ancestry (five each). After a brief propagation (five passages) under epithelial reprogramming assay conditions, transcriptomes and DNA methylomes were examined using RNA- and Methylcapture-sequencing. Differences in gene expression (FDR<0.05, fold change >2) in LC cells compared to LP cells were observed, with 1581 and 609 genes up- and down-regulated, respectively. These data were enriched for signaling networks associated with cell cycle control of chromosomal replication, mitotic roles of polo-like kinase (down-regulated genes), granulocyte adhesion and diapedesis and LXR/RXR signaling (up-regulated genes). Despite marked gene expression differences between LC and LP cells, only 14 upregulated genes and four down-regulated genes showed DNA hypo- and hyper-methylation, respectively, in LC cells compared to LP cells, suggesting a minimal role for DNA methylation in differential gene expression between the two cell types. Furthermore, of the 2190 differentially expressed genes, 7% (158 genes) were regulated by estrogen, suggesting that differences in hormone responsiveness contribute to differential gene expression in LP and LC cells. To determine relationships between genes differentially methylated and expressed in LC and LP cells and breast cancer, we used publicly available databases, including TCGA. Interestingly, all genes hypermethylated and expressed at lower levels in LC cells were similarly hypermethylated and expressed at lower levels in breast cancers compared to normal breast, suggesting that these genes are minimally altered in breast cancer. Furthermore, in LC compared to LP cells, of thegenes hypomethylated and expressed at higher levels, only four genes (AIM1L, PRSS27, PTK6 and UNC13D) showed a similar pattern in breast tumors compared to normal breast. Of these, PTK6 is significantly overexpressed in luminal and HER2+ breast cancers compared to basal-like breast cancers, suggesting that PTK6+ cells are the preferred cell-of origin of luminal breast cancers. Taken together, this is the first study to demonstrate that transcriptomic data of normal breast cells from healthy women can be used to further define cell-of-origin of tumors and identify cancer-specific gene expression changes in breast cancer. Citation Format: Fang Fang, Poornima Bhat-Nakshatri, Manjushree Anjanappa, Dough Rusch, Aaron Buechlein, Dave Miller, Kenneth P Nephew, Harikrishna Nakshatri. Intrinsic transcriptomic differences of luminal progenitors and mature luminal cells of the normal breast [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-06-01.

OP11 Exposure to an inflammatory mix re-induces inflammation in organoids of ulcerative colitis patients, independent of the inflammatory state of the tissue of origin

Abstract Background Patient-derived intestinal organoids provide a powerful tool to unravel mechanisms underlying inflammatory bowel disease (IBD). Recently, we showed that organoids derived from inflamed regions in ulcerative colitis (UC) patients lose their inflammatory phenotype during ex vivo culture and were indistinguishable from organoids of non-inflamed regions in these patients.1 To study UC in an ex vivo model, we hypothesised that inflammation should be re-induced towards levels corresponding to the in vivo situation. In addition, we aimed to elucidate if organoids derived from inflamed regions are more sensitive towards inflammatory stimulation, compared with organoids from non-inflamed regions of UC patients and non-IBD controls. Methods Biopsies were obtained from 8 patients with active UC (endoscopic Mayo score of ≥2), both in inflamed and non-inflamed regions, and in 8 non-IBD controls. Crypts were isolated and cultured as organoids for at least four weeks. Organoids were subjected to a predefined inflammatory mix (MIX: 100 ng/ml TNF-α, 20 ng/ml IL-1β, 1 µg/ml Flagellin) or medium only (CTRL) for 24 h (Figure 1). RNA was extracted from organoids for RNA sequencing by Lexogen QuantSeq for Illumina. Differential gene expression and pathways were studied through DESeq2 and ingenuity pathway analysis (false discovery rate <0.05). Results Prior to inflammatory stimulation, principal component analysis (PCA) demonstrated separate clustering between organoids derived from non-IBD controls and UC patients. Exposure to the inflammatory mix induced transcriptional activation of inflammatory genes (CXCL1, DUOXA2, IL1β, IL8, IL23α, etc., all p < 0.001) and pathways in all conditions (Figure 2). However, organoids of non-IBD controls clustered separate from organoids of UC patients. Within organoids of UC patients (inflamed vs. non-inflamed origin), we observed no differentially expressed genes after inflammatory stimulation but organoids clustered per patient instead (Figure 3). Inflammatory markers in UC organoids reached transcriptional expression levels (CXCL1, CXCL2, IFNGR1, IL1β, DUOXA2, etc.) and activated pathways (antigen presentation, interferon signalling, granulocyte adhesion and diapedesis) similar to those observed in crypts derived from inflamed biopsies. Conclusion Inflammation can efficiently be (re-)induced in organoids from both UC and non-IBD origin. However, a different response was observed between organoids of non-IBD and UC origin. Of note, in UC organoids, the state of inflammation in the source tissue was irrelevant. In conclusion, we showed that it is essential to re-induce inflammation in patient-specific organoids, but there is no need to obtain biopsies from inflamed regions. Reference

The Influence of Exogenous ATP on Functional Responses of Murine Bone Marrow Granulocytes

Extracellular ATP is a potent inflammatory mediator and regulator of neutrophil activity in an inflammatory site. The role of ATP in the regulation of bone marrow granulocyte functions remains unexplored. The aim of this work was to study effects of ATP on the cytoplasmic Ca2+ concentration ([Ca2+]i), generation of reactive oxygen species (ROS) and adhesion of bone marrow granulocytes. Changes of [Ca2+]i were estimated with Fura-2 AM ratiometric probe. To reveal cells with calcium activity in a population we developed an automated procedure based on the calculation of skewness and kurtosis of the [Ca2+]i value distribution. ROS generation was estimated by luminol-dependent chemiluminescence. Cell adhesion was evaluated by a standard absorbance (492 nm) method. The population of bone marrow granulocytes contained cells that generated calcium spikes in response to 10 μM ATP (35.2 ± 4.0%). The automated scanning allowed us to identify four groups of cells: those responding to ATP (23.4 ± 4.9%) or to the addition of Hanks’ solution (16.9 ± 2.5%) or to both ATP and Hanks’ solution (6.7 ± 1.5%), and cells not responding to either stimulus (53.1 ± 8.2%). ATP increased the adhesion of granulocytes, while apyrase, which hydrolyzes ATP, did not significantly change the adhesion. ATP did not influence the respiratory burst initiated by the bacterial peptide N-formyl-MLF (fMLF). Apyrase significantly enhanced the response to fMLF, and cytochalasin D inhibited this effect. In summary, exogenous ATP enhances adhesion and generation of calcium spikes in the population of bone marrow granulocytes. Endogenous ATP likely reduces respiratory burst through a mechanism involving the cytoskeleton. The studied granulocyte population contains cells with different ATP sensitivity.

62 Peripheral transcriptome response to embryo mortality in Holstein cows

Exactly how failure through conceptus-endometrial signalling on Day 16 of pregnancy causes early embryo mortality (EM) is yet unknown. It was hypothesised that EM pregnancies are associated with an impaired endocrine action of interferon tau (IFNT) from the conceptus on the corpus luteum (CL) and peripheral blood mononuclear cells (PBMC), in addition to paracrine failure in preventing the upregulation of oestradiol-mediated luteolytic signals in the endometrium. Cows not exposed to semen served as controls on Day 16 of the oestrous cycle (EC; n=7). Conceptuses were flushed on Day 16 of pregnancy and were then sorted based on quality. Normal (N, n=9) conceptuses were elongated and translucent, whereas EM conceptuses (n=6) lacked extensive elongation and were pink, red, or opaque. The RNA extracted from conceptuses, endometrium, CL, and PBMC was submitted to RNA-Seq analysis. Trimmed and aligned sequences were analysed in R using the DESEqn 2 package. Data were submitted to ingenuity pathway analysis with fold change ≥1.5 and P ≤ 0.05. Previously, we reported that N were longer (P<0.006) than EM conceptuses. The IFNT mRNA transcript raw counts (from RNA-Seq) were greater (P<0.0001) in N than in EM conceptuses. The IFNT protein concentrations (enzyme-linked immunosorbent assay) in uterine flushings were greater (P<0.004) in N compared with EC and tended (P<0.07) to be greater in EM compared with EC cows. Similarly, interferon stimulated gene 15 (ISG15) mRNA raw counts were greater (P<0.003) in N and tended (P<0.09) to be greater in EM endometrium compared with EC endometrium. Ingenuity pathway analysis revealed that T helper (Th1) cell activation, a key canonical pro-inflammatory pathway, was upregulated in EM compared with N conceptuses. Among the 15 upregulated endometrial genes in EM compared with N, 9 were associated with oestradiol action. In the current study, 7/9 PBMC (ISG15, MX2, OAS1, OAS2, IFI44, IFI6, and DDX58) and 3/3 CL (ZBP1, OAS1, and MX1) genes (ISGs) were upregulated in N versus EC and belonged to the interferon signalling canonical pathway. Key upstream regulators were IFNAR2 and IRF7 for PBMC and type I IFNs and ISG15 for CL. There were no differences in EM compared with N genes in PBMC. Conversely, 10 genes in the CL were upregulated for EM compared with N and aligned to pro-inflammatory/calcium binding (i.e. CAPS2, S100A12) and inhibition of adenylate cyclase (HTR1B) responses. The primary canonical pathways were granulocyte adhesion and diapedesis, and hypercytokinemia. Moroever, key upstream regulators were MAPK and TNF. In conclusion, ISGs are upregulated peripherally in PBMC and CL in response to normal pregnancy. When conceptuses are compromised, pro-inflammatory and calcium-mediated responses that promote a localised “cytokine storm” immune response are upregulated and may be coupled with disruption of steroidogenesis in the CL, which is mediated by adenylate cyclase. This research was supported by USDA-Agriculture and Food Research Initiative (AFRI)-National Needs Graduate and Postgraduate Fellowship (NNF) 2016-38420-25289 and the W3112 USDA-AFRI Ruminant Reproduction Project.

Reveal the Neutrophil: Elucidating the Role of a Neutrophil-Specific JAK2-V617F Mutation

Introduction: Myeloproliferative neoplasms (MPN) are characterized by monoclonal proliferation of myeloid cells. The clinical presentation of classic BCR-ABL negative MPN includes blood count changes, splenomegaly, chronic inflammation and, prognostically relevant, thrombosis. Within polycythemia vera, essential thrombocythemia and primary myelofibrosis, the most frequent genetic alteration is the point mutation V617F (VF) in the Jak2 gene. Due to an activation of leucocyte β1 and β2 integrins, in particular on neutrophils, JAK2-VF is driving thrombus formation (Edelmann et al., JCI, 2018). Thus, understanding the role of neutrophils within JAK2-VF-positive MPN is of special interest. We developed a murine JAK2-VF Catchup model of MPN that is characterized by the neutrophil-specific expression of JAK2-VF in combination with the fluorescent protein tdTomato allowing in-vivo two-photon microscopy. Neutrophil-specificity was achieved through a knock-in allele of Cre recombinase and tdTomato replacing exon 1 of Ly6G (Hasenberg et al., Nat Methods, 2015). Methods: The murine JAK2-VF Catchup model was developed by crossing JAK2+/loxP-VF-loxP mice and Ly6G+/Cre-tdTomato CatchupIVM-red mice. Experiments were performed in 12 weeks old JAK2+/loxP-VF-loxP Ly6G+/Cre-tdTomato CatchupIVM-red (JAK2+/VF Catchup) mice (n=5) and JAK2+/+ Ly6G+/Cre-tdTomato CatchupIVM-red (JAK2+/+ Catchup) mice (n=5) as wildtype (WT) controls. Whole blood cell counts and spleen weight were determined. Bone marrow (BM) cells were harvested from hind limbs followed by isolation of granulocytes using negative selection. In order to determine hematopoietic stem and progenitor cells (HSPCs), BM and spleen cell suspensions were analyzed by flow cytometry (FC). Using ICAM-1, VCAM-1, E-selectin and P-selectin, each with and without Fc-tag, adhesion properties of the isolated granulocytes were examined by either FC or microplate reader analysis. The expression of β1 integrin was further analyzed by FC. Results and Discussion: JAK2+/VF Catchup mice showed a slight but significant increase in platelet counts [1544±58.02 Gpt/l; WT: 1190±90.50 Gpt/l; p=0.011], with the mean value still remaining within the normal range. Hematocrit, red blood cell, white blood cell and neutrophil count as well as spleen weight were also within the normal range showing no differences from their WT controls. Interestingly, neutrophil and platelet counts of JAK2+/VF Catchup mice significantly correlated with one another [p=0.0251; R2=0.8529]. MPN-typical changes in number and composition of c-Kit+ HSPCs in either BM or spleen could not be found in JAK2+/VF Catchup mice. Directly compared to reliably MPN-developing Vav-1-Cre JAK2+/VF mice, JAK2+/VF Catchup mice showed significantly less megakaryocyte-erythroid progenitor (MEP), Lin- Sca1+ c-Kit+ (LSK) and multipotent progenitor cells (MPP) in the BM. In spleen of JAK2+/VF Catchup mice, the fractions of Lin- c-Kit+ (LK) and granulocyte-macrophage progenitor cells (GMP) were significantly lower compared to Vav-1-Cre JAK2+/VF mice. Thus, the neutrophil-specific JAK2-VF mutation does not lead to apparent numerical or compositional changes of HSPCs. These results show that a neutrophil-specific JAK2-VF mutation is not sufficient to induce a characteristic phenotype of JAK2-VF-positive MPN. The expression of β1 integrin on granulocytes was significantly increased in JAK2+/VF Catchup mice [fold change vs. control; 1.126±0.041; WT: 1.0±0.011; p=0.0194]. Unexpectedly, despite increased β1 integrin expression, the binding of granulocytes from JAK2+/VF Catchup mice to either ICAM-1, VCAM-1, E-selectin or P-selectin corresponded to that of their WT controls. Static adhesion and soluble ligand binding assays showed similar results. Since adhesion of granulocytes appeared unchanged in-vitro, we hypothesize that expression of JAK2-VF in neutrophils is not sufficient to overactivate integrins but secondary extrinsic signals (e.g. cytokines) may play a dominant role. Further experimentation is under way to elucidate this molecular mechanism. Conclusions: In order to dissect the properties of JAK2-VF-positive neutrophils, the JAK2-VF Catchup model represents a promising murine model of MPN. Herewith, the initiation of thrombosis and the development of other granulocyte-driven disease manifestations of JAK2-VF induced MPN can be thoroughly examined. Disclosures No relevant conflicts of interest to declare.

Significantly Upregulated Thrombo-Inflammatory Genes Are Normoregulated or Significantly Downregulated during Treatment with Interferon-Alpha2 in Patients with Philadelphia-Negative Chronic Myeloproliferative Neoplasms

Introduction: The Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are associated with a high risk of arterial and venous thrombosis, which are attributed to several mechanisms, including elevated blood cell counts per se, in vivo leukocyte and platelet activation with increased adhesion of granulocytes, monocytes and platelets to each other and to a dysfunctional endothelium. In recent years, evidence has accumulated that chronic inflammation is an important pathogenetic mechanism for MPN-disease development and disease progression, inducing increasing genomic instability in hematopoietic cells and thereby emergence of additional mutations of significance for myelofibrotic and leukemic transformation. Recent studies have shown several thrombo-inflammatory genes to be upregulated in patients with MPNs, likely contributing to the increased risk of thrombosis. Several studies have documented that long term treatment with interferon-alpha2 (IFN) is able to normalize elevated cell counts in concert with induction of a remarkable decrease in the JAK2V617F allele burden and accordingly impacting important thrombosis promoting factors in MPNs. Herein, using whole blood gene expression profiling we for the first time report that treatment with IFN is able to normoregulate or significantly downregulate upregulated thrombo-inflammatory genes in patients with MPNs. Methods: Eight patients with ET, 21 patients with PV, and 4 patients with PMF participated in the study. All patients received treatment with IFN, in the large majority in a dosage ranging from 45-90 ug x 1 sc/week. Gene expression microarray analysis of whole blood was performed before and after 3 months of treatment. Total RNA was purified from whole blood, amplified to biotin-labeled RNA, and hybridized to Affymetrix HG-U133 2.0 Plus chips. Results: We identified 6261, 10,008, and 2828 probe sets to be significantly differentially expressed in ET, PV, and PMF, respectively, in response to treatment with IFN (pvalue < 0.05). Six thrombo-inflammatory genes were investigated: F3, PADI4, SELP, SERPINE1, SLC2A1, and THBS1. In all patients groups, the 6 genes were significantly upregulated at baseline and either normoregulated or significantly downregulated during treatment with IFN (Figure 1). Discussion and Conclusions: Thrombosis contributes significantly to morbidity and mortality in MPNs. Despite treatment with conventional drugs (hydroxyurea, anagrelide) - the most used cytoreductive therapies worldwide - patients with MPNs are still suffering potentially life-threatening or life-invalidating thrombotic complications in the brain, heart, lungs and elsewhere. Therefore, there is an urgent need for studies that explore the pathogenetic mechanisms eliciting the thrombotic state and the impact of novel therapies, such as IFN, upon the thrombogenic factors which might be operative. Herein, we have for the first time shown that IFN significantly downregulates several thrombo-inflammatory genes, known to be the upregulated in patients with concurrent or previous thrombosis. Highly intriguing, we found that IFN significantly downregulated the PADI4 gene, which is required for neutrophil extracellular trap (NET) formation and thrombosis development. A most recent study has shown neutrophils from patients with MPNs to be associated with an increase in NET formation, which was blunted by ruxolitinib. This study also showed that JAK2V617F-driven MPN mouse models have a NET-rich, prothrombotic phenotype, highlighting NETosis to be yet another important thrombosis mechanism in MPNs. In conclusion, we have for the first time shown 3 months IFN-treatment to be associated with a significant downregulation of upregulated thrombo-inflammatory genes, including significant downregulation of the NETosis associated gene - PADI4. In the context of a significantly increased risk of thrombosis after the MPN-diagnosis with a particular increased risk at 3 months, our results of significant downregulation of these thrombo-inflammatory genes during IFN-therapy are of paramount importance and may signal an advantage of IFN over conventional cytoreductive therapies. Further studies are required to decipher the impact of IFN upon upregulated thrombo-inflammatory genes and if combination therapy with ruxolitinib may be even more efficacious. Figure 1 Disclosures Hasselbalch: Novartis: Research Funding; AOP Orphan Pharmaceuticals: Other: Data monitoring board. OffLabel Disclosure: Interferon-alpha for treatment of myeloproliferative neoplasms

Persistence of Inflammatory Phenotype in Residual Psoriatic Plaques in Patients on Effective Biologic Therapy

Many psoriasis patients treated with biologics do not achieve total skin clearance. These patients possess residual plaques despite ongoing biologic treatment. To elucidate mechanisms of plaque persistence despite overall good drug response, we studied 50 subjects: psoriasis patients with residual plaques treated with one of three different biologics, untreated patients, and healthy controls. Skin biopsies from all subjects were characterized using three methods: mRNA expression, histology, and FACS of hematopoietic skin cells. Although all three methods provided evidence of drug effect, gene expression analysis revealed the persistence of key psoriasis pathways in treated plaques, including granulocyte adhesion and diapedesis, T helper type17 activation pathway, and interferon signaling with no novel pathways emerging. Focal decreases in parakeratosis and keratinocyte proliferation and differential reduction in IL-17 producing CD103- T cells, but no change in CD103+ tissue-resident memory T cells were observed. Of note, antitumor necrosis factor increased the interferon signaling pathway already present. Interestingly mast cells were the dominant source of IL-22 in all psoriasis subjects. These data suggest that while subtle differences can be observed in drug-treated plaques, underlying biologic mechanisms are similar to those present in untreated psoriatic lesions.

682 Investigating the Pathogenesis of Ulcerative Colitis Through Meta-Analysis of Gene Expression Omnibus Data

INTRODUCTION: Ulcerative colitis (UC) is an inflammatory bowel disease that causes inflammation of the colon and rectum, alongside extra-colonic manifestations. The etiology is unclear but is thought to include such factors as genetic predisposition, immune regulation, microbiome, and environmental triggers. Though strides have been made in treatment, the failure of anti-IL13 drugs demonstrates our limited knowledge of UC pathology. Here we use meta-analysis to dissect UC pathogenesis. METHODS: We employed our STARGEO platform to search Gene Expression Omnibus and performed meta-analysis on 465 colon samples from active UC patients, using 326 healthy colon samples as a control. We then analyzed the signature in Ingenuity Pathway Analysis. RESULTS: Our analysis identified granulocyte adhesion and diapedesis, TREM1 signaling, and communication between innate and adaptive immune cells as top canonical pathways. TNF, IFNγ, and IL1 were top upstream regulators with predicted activation. We noted upregulation of the DUOX/DUOXA2 enzyme system, responsible for generating reactive oxygen species. We also found upregulation of calprotectin S100A8, which defines UC severity, and downregulation of the E3 ubiquitin ligase RNF5 responsible for controlling S100A8 expression. Impaired epithelial integrity was suggested by upregulation of matrix metalloproteinases MMP1 and MMP3 and down regulation of claudin 8. Furthermore, downregulation of aquaporin 8 and guanylin suggests loss of absorption. Upregulation of the amino acid transporter SLC6A14 and aldolase B may represent a compensation for loss of absorptive function. We also found metabolic dysfunction through downregulation of phosphoenolpyruvate carboxykinase 1, and UDP-glucuronosyltransferases UGT1A7/A5/A3. Lastly, we found downregulation of several miRNAs including mir-188, 192, 200a/b, 215, and 320. CONCLUSION: UC pathogenesis represents a multifactorial disease process that remains elusive. Here we suggest several mechanisms that may underpin UC development and susceptibility. In a recent study, inverse expression of RNF5 and S100A8 was found to coincide with UC severity and our results give credence to this correlation. Our results also demonstrate the parts that DUOX/DUOXA2 system and loss of epithelial and metabolic integrity have in UC. Lastly, miRNAs have an emerging role in UC and mimic therapy of the downregulated miRNAs we identified may prove efficacious. These results provide novel insights into UC pathophysiology and potential therapeutic avenues.

Transcription Profiling of Monocyte-Derived Macrophages Infected In Vitro With Two Strains of Streptococcus agalactiae Reveals Candidate Pathways Affecting Subclinical Mastitis in Cattle.

Macrophages are key cells of innate immune response and serve as the first line of defense against bacteria. Transcription profiling of bacteria-infected macrophages could provide important insights on the pathogenicity and host defense mechanisms during infection. We have examined transcription profiles of bovine monocyte-derived macrophages (bMDMs) isolated from the blood of 12 animals and infected in vitro with two strains of Streptococcus agalactiae. Illumina sequencing of RNA from 36 bMDMs cultures exposed in vitro to either one of two sequence types of S. agalactiae (ST103 or ST12) for 6 h and unchallenged controls was performed. Analyses of over 1,656 million high-quality paired-end sequence reads revealed 5,936 and 6,443 differentially expressed genes (p < 0.05) in bMDMs infected with ST103 and ST12, respectively, versus unchallenged controls. Moreover, 588 genes differentially expressed between bMDMs infected with ST103 versus ST12 were identified. Ingenuity pathway analysis of the differentially up-regulated genes in the bMDMs infected with ST103 revealed significant enrichment for granulocyte adhesion and diapedesis, while significant enrichment for the phagosome formation pathway was found among down-regulated genes. Moreover, Ingenuity pathway analysis of the differentially up-regulated genes in the bMDMs infected with ST12 showed significant enrichment for type 1/type 2 T helper cell activation, while the complement activation pathway was overrepresented in the down-regulated genes. Our study identified pathogen-induced regulation of key genes and pathways involved in the immune response of macrophages against infection but also likely involved in bacterial evasion of the host immune system. These results may contribute to better understanding of the mechanisms underlying subclinical infection such as bovine streptococcal mastitis.

Valuation of Activation Capabilities of SolidPhase Surfaces by the Rate of Adhesion of Blood Cells

When using the method of low-volume hemoperfusion (MOG) in the clinic, the therapeutic effect of which is to implement the mechanisms of hemomodulation and activation of the sorption procedure, it is important to take into account the activation properties of solid-phase granular drugs. Blood-contact interaction was carried out in bench conditions with the use of donated blood in rotary mode. Blood samples were taken before the experiment and after 5, 20, 40 and 60 minutes. Changes in blood cell and subcellular populations were evaluated using the Sysmex XT 1800i hematological analyzer (26 parameters), which made it possible to indirectly judge the activation of blood cells. 20 experiments were conducted. To analyze the activation functions of the hemocontact preparation SCT 6A HP, the speed-time adhesive profi le of blood cells on the sorbent was used. The maximum rate of adhesion was noted in the period of “5 min” from the beginning of contact. The rate of adhesion of granulocytes in all time intervals was signifi cantly higher than that of agranulocytes. The adhesion process can be an indicator of the activation of blood cells in contact with foreign surfaces, and serve as an evaluation criterion for the activation capabilities of these surfaces.

Elucidating the pathogenesis of esophageal adenocarcinoma through meta-analysis of public data.

71 Background: Esophageal adenocarcinoma (EAC) is the sixth most common cause of cancer-related deaths worldwide. It commonly arises in the setting of reflux disease and Barett’s esophagus. It remains incurable and holds a poor prognosis. Dissecting the genetic signature of EAC can pave new therapeutic avenues. Methods: We employed our Search Tag Analyze Resource (STARGEO) platform to conduct meta-analysis using the National Center for Biotechnology’s (NCBI) Gene Expression Omnibus (GEO). We tagged 151 tumor samples from EAC patients and 62 normal esophageal samples as a control. We then analyzed the signature in Ingenuity Pathway Analysis, restricting genes that showed statistical significance (p &lt; 0.05) and an absolute experimental log ratio greater than 0.15 between tumor and control. Results: Our analysis revealed granulocyte adhesion and diapedesis and FXR/RXR signaling as top canonical pathways. TGFB1, TNF, and beta-estradiol were top upstream regulators with predicted activation. TGFB1 and TNF expression have been correlated with poor prognosis in EAC. Also, beta-estradiol has tumorigenic activity in several cancers but has not been investigated in EAC. Among the top upregulated genes were oncogenic genes such as tetraspanin 8, the antiapoptotic factor OLFM4, and the protease cathepsin E (CTSE). SPINK1, a trypsin inhibitor with recently suggested role in cancer, and the choline transporter SLC44A4, a drug target for pancreatic cancer, were also upregulated. The most downregulated genes included alcohol dehydrogenase 7, associated with EAC in alcohol-drinkers, and the pro-apoptotic gene CRCT1. We also found downregulation of the serine peptidase inhibitor SPINK7. SPINK7 is involved in maintaining epithelial-barrier integrity and is implicated in eosinophilic esophagitis pathogenesis. Lastly, there was downregulation of the candidate tumor suppressor gene transglutaminase 3. Conclusions: Despite screening efforts, EAC incidence and mortality continues to increase as does the need for better treatment. This meta-analysis defines the significant gene expression changes within causal disease processes to provide markers for detection, prognostic insight, and therapeutic value for EAC.

Transcriptome analysis unraveled potential mechanisms of resistance to Haemonchus contortus infection in Merino sheep populations bred for parasite resistance

Haemonchus contortus is one of the most pathogenic gastrointestinal nematodes in small ruminants. To understand molecular mechanisms underlying host resistance to this parasite, we used RNA-sequencing technology to compare the transcriptomic response of the abomasal tissue, the site of the host-parasite interaction, of Merino sheep bred to be either genetically resistant or susceptible to H. contortus infection. Two different selection flocks, the Haemonchus selection flock (HSF) and the Trichostrongylus selection flock (TSF), and each contains a resistant and susceptible line, were studied. The TSF flock was seemingly more responsive to both primary and repeated infections than HSF. A total of 127 and 726 genes displayed a significant difference in abundance between resistant and susceptible animals in response to a primary infection in HSF and TSF, respectively. Among them, 38 genes were significantly affected by infection in both flocks. Gene ontology (GO) enrichment of the differentially expressed genes identified in this study predicted the likely involvement of extracellular exosomes in the immune response to H. contortus infection. While the resistant lines in HSF and TSF relied on different mechanisms for the development of host resistance, adhesion and diapedesis of both agranulocytes and granulocytes, coagulation and complement cascades, and multiple pathways related to tissue repair likely played critical roles in the process. Our results offered a quantitative snapshot of changes in the host transcriptome induced by H. contortus infection and provided novel insights into molecular mechanisms of host resistance.

Cysteinyl leukotriene receptor 2 drives lung immunopathology through a platelet and high mobility box 1-dependent mechanism.

Cysteinyl leukotrienes (cysLTs) facilitate eosinophilic mucosal type 2 immunopathology, especially in aspirin-exacerbated respiratory disease (AERD), by incompletely understood mechanisms. We now demonstrate that platelets, activated through the type 2 cysLT receptor (CysLT2R), cause IL-33-dependent immunopathology through a rapidly inducible mechanism requiring the actions of high mobility box 1 (HMGB1) and the receptor for advanced glycation end products (RAGE). Leukotriene C4 (LTC4) induces surface HMGB1 expression by mouse platelets in a CysLT2R-dependent manner. Blockade of RAGE and neutralization of HMGB1 prevent LTC4-induced platelet activation. Challenges of AERD-like Ptges−/− mice with inhaled lysine aspirin (Lys-ASA) elicit LTC4 synthesis and cause rapid intrapulmonary recruitment of platelets with adherent granulocytes, along with platelet- and CysLT2R-mediated increases in lung IL-33, IL-5, IL-13, and bronchoalveolar lavage fluid HMGB1. The intrapulmonary administration of exogenous LTC4 mimics these effects. Platelet depletion, HMGB1 neutralization, and pharmacologic blockade of RAGE eliminate all manifestations of Lys-ASA challenges, including increase in IL-33, mast cell activation, and changes in airway resistance. Thus, CysLT2R signaling on platelets prominently utilizes RAGE/HMGB1 as a link to downstream type 2 respiratory immunopathology and IL-33-dependent mast cell activation typical of AERD. Antagonists of HMGB1 or RAGE may be useful to treat AERD and other disorders associated with type 2 immunopathology.

Whole Blood Gene Expression Reveals Specific Transcriptome Changes in Neonatal Encephalopathy

Background: Variable responses to hypothermic neuroprotection are related to the clinical heterogeneity of encephalopathic babies; hence better disease stratification may facilitate the development of individualized neuroprotective therapies. Objectives: We examined if whole blood gene expression analysis can identify specific transcriptome profiles in neonatal encephalopathy. Material and Methods: We performed next-generation sequencing on whole blood RNA from 12 babies with neonatal encephalopathy and 6 time-matched healthy term babies. Genes significantly differentially expressed between encephalopathic and control babies were identified. This set of genes was then compared to the host RNA response in septic neonates and subjected to pathway analysis. Results: We identified 950 statistically significant genes discriminating perfectly between healthy controls and neonatal encephalopathy. The major pathways in neonatal encephalopathy were axonal guidance signaling (p = 0.0009), granulocyte adhesion and diapedesis (p = 0.003), IL-12 signaling and production in macrophages (p = 0.003), and hypoxia-inducible factor 1α signaling (p = 0.004). There were only 137 genes in common between neonatal encephalopathy and bacterial sepsis sets. Conclusion: Babies with neonatal encephalopathy have striking differences in gene expression profiles compared with healthy control and septic babies. Gene expression profiles may be useful for disease stratification and for developing personalized neuroprotective therapies.

Murine host response to Neisseria gonorrhoeae upper genital tract infection reveals a common transcriptional signature, plus distinct inflammatory responses that vary between reproductive cycle phases

BackgroundThe emergence of fully antimicrobial resistant Neisseria gonorrhoeae has led global public health agencies to identify a critical need for next generation anti-gonococcal pharmaceuticals. The development and success of these compounds will rely upon valid pre-clinical models of gonorrhoeae infection. We recently developed and reported the first model of upper genital tract gonococcal infection. During initial characterization, we observed significant reproductive cycle-based variation in infection outcome. When uterine infection occurred in the diestrus phase, there was significantly greater pathology than during estrus phase. The aim of this study was to evaluate transcriptional profiles of infected uterine tissue from mice in either estrus or diestrus phase in order to elucidate possible mechanisms for these differences.ResultsGenes and biological pathways with phase-independent induction during infection showed a chemokine dominant cytokine response to Neisseria gonorrhoeae. Despite general induction being phase-independent, this common anti-gonococcal response demonstrated greater induction during diestrus phase infection. Greater activity of granulocyte adhesion and diapedesis regulators during diestrus infection, particularly in chemokines and diapedesis regulators, was also shown. In addition to a greater induction of the common anti-gonococcal response, Gene Set Enrichment Analysis identified a diestrus-specific induction of type-1 interferon signaling pathways.ConclusionsThis transcriptional analysis of murine uterine gonococcal infection during distinct points in the natural reproductive cycle provided evidence for a common anti-gonococcal response characterized by significant induction of granulocyte chemokine expression and high proinflammatory mediators. The basic biology of this host response to N. gonorrhoeae in estrus and diestrus is similar at the pathway level but varies drastically in magnitude. Overlaying this, we observed type-1 interferon induction specifically in diestrus infection where greater pathology is observed. This supports recent work suggesting this pathway has a significant, possibly host-detrimental, function in gonococcal infection. Together these findings lay the groundwork for further examination of the role of interferons in gonococcal infection. Additionally, this work enables the implementation of the diestrus uterine infection model using the newly characterized host response as a marker of pathology and its prevention as a correlate of candidate vaccine efficacy and ability to protect against the devastating consequences of N. gonorrhoeae-associated sequelae.

Immune and biochemical responses in hemolymph and gills of the Patagonian freshwater mussel Diplodon chilensis, against two microbiological challenges: Saccharomyces cerevisiae and Escherichia coli.

Immune cell characterization, immunological response and the associated gill oxidative balance were studied in the Patagonian freshwater mussel, Diplodon chilensis, using two microbiological immunostimulant models: Saccharomyces cerevisiae and Escherichia coli. Mussels were collected out of the breeding season in Paimún Lake and acclimated in the laboratory. Two exposure experiments were performed during two consecutive weeks: (1) mussels challenged with 500 yeast cells mL-1; and (2) mussels challenged with 1000 bacteria cells mL-1. Microorganisms were added in the water every two days, alternating with 6000 lyophilized cells of the green algae Scenedesmus vacuolatus mL-1. A control group, fed with S. vacuolatus, was set for each treatment. Morphological cell characterization was carried out in adherent hemocytes of D. chilensis hemolymph under control conditions. The most important cell type observed were the hyalinocytes (representing ca. 98% of the circulating cells), agranular cells with non-central polymorphic nucleus surrounded by cytoplasm; granulocytes (cells with cytoplasmic granules and non-central rounded nucleus) represented ca. 2%. Another two cell types were occasionally detected, binucleated hyalinocytes and hemoblast-like cells but were not considered for the analyses. Both adherent hyalinocytes and granulocytes exhibit phagocytic activity towards Congo red stained yeast, which was two-fold higher in granulocytes than in hyalinocytes, regardless of the applied challenge. Total hemocyte counts were diminished in mussels challenged with S. cerevisiae or E. coli. Hydrolytic and defense cellular enzyme activities were analyzed only for hyalinocytes. Both, S. cerevisiae and E. coli increased acid phosphatase activity. E. coli challenge diminished hemocyte lysosomal membrane stability and increased humoral phenoloxidase activity, while S. cerevisiae challenge did not affect any of these variables. Mussels challenged with E. coli showed increased gill antioxidant response without oxidative damage, while those challenged with S. cerevisiae showed no change in these variables.

Transcriptomic signatures reveal immune dysregulation in human diabetic and idiopathic gastroparesis

BackgroundCellular changes described in human gastroparesis have revealed a role for immune dysregulation, however, a mechanistic understanding of human gastroparesis and the signaling pathways involved are still unclear.MethodsDiabetic gastroparetics, diabetic non-gastroparetic controls, idiopathic gastroparetics and non-diabetic non-gastroparetic controls underwent full-thickness gastric body biopsies. Deep RNA sequencing was performed and pathway analysis of differentially expressed transcripts was done using Ingenuity®. A subset of differentially expressed genes in diabetic gastroparesis was validated in a separate cohort using QT-PCR.Results111 genes were differentially expressed in diabetic gastroparesis and 181 in idiopathic gastroparesis with a log2fold difference of | ≥ 2| and false detection rate (FDR) < 5%. Top canonical pathways in diabetic gastroparesis included genes involved with macrophages, fibroblasts and endothelial cells in rheumatoid arthritis, osteoarthritis pathway and differential regulation of cytokine production in macrophages and T helper cells by IL-17A and IL-17F. Top canonical pathways in idiopathic gastroparesis included genes involved in granulocyte adhesion and diapedesis, agranulocyte adhesion and diapedesis, and role of macrophages, fibroblasts and endothelial cells in rheumatoid arthritis. Sixty-five differentially expressed genes (log2fold difference | ≥ 2|, FDR < 5%) were common in both diabetic and idiopathic gastroparesis with genes in the top 5 canonical pathways associated with immune signaling. 4/5 highly differentially expressed genes (SGK1, APOLD1, CXCR4, CXCL2, and FOS) in diabetic gastroparesis were validated in a separate cohort of patients using RT-PCR. Immune profile analysis revealed that genes associated with M1 (pro inflammatory) macrophages were enriched in tissues from idiopathic gastroparesis tissues compared to controls (p < 0.05).ConclusionsDiabetic and idiopathic gastroparesis have both unique and overlapping transcriptomic signatures. Innate immune signaling likely plays a central role in pathogenesis of human gastroparesis.