Inactive Phenotype Research Articles

Abstract 13045: Myofibroblasts Revert to an Inactive Phenotype During Regression of Heart Failure

Introduction: The therapy of left ventricular mechanical unloading, such as transcatheter aortic valve replacement(TAVR)and left ventricular assist devices(LVAD)has significantly improved prognosis for patients with heart failure. However, the detailed cellular and/or molecular mechanisms of recovery of cardiac function remain unknown. Objective: We aimed to build a cellular atlas of the heart that experienced stress and de-stress process, with a focus on myofibroblasts, specifically its cell fate of differentiation and function. Methods: We conducted single-cell and ATAC sequencing on non-myocardial cells from mouse heart samples of pressure overload-induced heart failure and recovery, using TAC (transverse aortic arch constriction)/deTAC (with suture for the constriction removed 2 weeks after TAC) model to identify the dynamic changes. Lineage tracing technique was also used to gain further insight into cell fate and reciprocal interactions. Macrophage subgroup was cleared using various approaches to investigate the cross-talk between myofibroblast and macrophage. Results: We generated a TAC/deTAC mouse model which can faithfully simulate a reversal process of functioning for the heart that underwent a pressure overload stress. Multi-omics sequencing reveals the genetic changes and transcriptional regulation of cell populations at different stages. Among the different cell types, significant changes in the gene expression pattern were observed in fibroblasts. Aided by myofibroblast lineage tracing, flow cytometry examinations suggested that remaining in a quiescent state rather than showing a decrease in its number appeared as the main fate of myofibroblasts. Surprisingly, however, simultaneous pressure unloading and macrophage clearance resulted in a significant reduction in the number of myofibroblasts. Conclusions: With a reliable TAC/deTAC mouse model, our multi-omics data revealed dynamic changes in cell populations of the heart that underwent pressure overload followed by unloading, our data demonstrated a quiescent state rather than a reduction in number was the major fate for the activated myofibroblasts for which macrophages were involved. These findings provide a theoretical basis for further treatment.

Lymphocyte-Specific Protein Tyrosine Kinase Contributes to Spontaneous Regression of Liver Fibrosis mayby Interacting with Suppressor of Cytokine Signaling 1.

Quiescent hepatic stellate cells (qHSCs), converted to myofibroblasts, produce fibrous scars, which is an essential event during liver fibrogenesis. Clinical and experimental fibrosis undergo remarkable regression when the underlying etiological agent is removed. Some myofibroblasts revert to an inactive phenotype (iHSCs) during the regression of fibrosis. However, the mechanisms underlying HSC activation and reversal remain unclear. The present study demonstrated that the expression of lymphocyte-specific protein tyrosine kinase (LCK) was increased in fibrotic livers but decreased after spontaneous recovery in vivo and in vitro, which was correlated with the expression of α-smooth muscle actin (α-SMA) and type I collagen (COL-1). Further investigation indicated that specific knockdown of LCK by a recombination adeno-associated virus 9 (rAAV9) in C57BL/6 mice ameliorated liver fibrosis. Co-incubation of TGF-β1-induced HSC-T6 cells with LCK-siRNA inhibited cell proliferation and activation. Overexpression of LCK inhibited activated HSCs going to inactivated phenotype. Interestingly, we found that LCK may interact with suppressor of cytokine signaling 1 (SOCS1) and may influence the expression of p-JAK1 and p-STAT1/3. These data suggest that LCK may play a regulatory role in liver fibrosis by inhibiting SOCS1, indicating that LCK is a potential therapeutic target for liver fibrosis treatment.

Hyaluronic Acid in Synovial Fluid Prevents Neutrophil Activation in Spondyloarthritis

Spondyloarthritis (SpA) patients suffer from joint inflammation resulting in tissue damage, characterized by the presence of numerous neutrophils in the synovium and synovial fluid (SF). As it is yet unclear to what extent neutrophils contribute to the pathogenesis of SpA, we set out to study SF neutrophils in more detail. We analyzed the functionality of SF neutrophils of 20 SpA patients and 7 disease controls, determining ROS production and degranulation in response to various stimuli. In addition, the effect of SF on neutrophil function was determined. Surprisingly, our data show that SF neutrophils in SpA patients have an inactive phenotype, despite the presence of many neutrophil-activating stimuli such as GM-CSF and TNF in SF. This was not due to exhaustion as SF neutrophils readily responded to stimulation. Therefore, this finding suggests that one or more inhibitors of neutrophil activation may be present in SF. Indeed, when blood neutrophils from healthy donors were activated in the presence of increasing concentrations of SF from SpA patients, degranulation and ROS production were dose-dependently inhibited. This effect was independent of diagnosis, gender, age, and medication in the patients from which the SF was isolated. Treatment of SF with the enzyme hyaluronidase strongly reduced the inhibitory effect of SF on neutrophil activation, indicating that hyaluronic acid that is present in SF may be an important factor in preventing SF neutrophil activation. This finding provides novel insights into the role of soluble factors in SF regulating neutrophil function and may lead to the development of novel therapeutics targeting neutrophil activation via hyaluronic acid or associated pathways.

The High Mutational Sensitivity of ccdA Antitoxin Is Linked to Codon Optimality.

Deep mutational scanning studies suggest that synonymous mutations are typically silent and that most exposed, nonactive-site residues are tolerant to mutations. Here, we show that the ccdA antitoxin component of the Escherichia coli ccdAB toxin–antitoxin system is unusually sensitive to mutations when studied in the operonic context. A large fraction (∼80%) of single-codon mutations, including many synonymous mutations in the ccdA gene shows inactive phenotype, but they retain native-like binding affinity towards cognate toxin, CcdB. Therefore, the observed phenotypic effects are largely not due to alterations in protein structure/stability, consistent with a large region of CcdA being intrinsically disordered. E. coli codon preference and strength of ribosome-binding associated with translation of downstream ccdB gene are found to be major contributors of the observed ccdA mutant phenotypes. In select cases, proteomics studies reveal altered ratios of CcdA:CcdB protein levels in vivo, suggesting that the ccdA mutations likely alter relative translation efficiencies of the two genes in the operon. We extend these results by studying single-site synonymous mutations that lead to loss of function phenotypes in the relBE operon upon introduction of rarer codons. Thus, in their operonic context, genes are likely to be more sensitive to both synonymous and nonsynonymous point mutations than inferred previously.

Long-term in vivo two-photon imaging of the neuroinflammatory response to intracortical implants and micro-vessel disruptions in awake mice

Our understanding of biomaterials in the brain have been greatly enhanced by advancements in in vivo imaging technologies such as two-photon microscopy. However, when applied to chronic studies, two-photon microscopy enables high-resolution imaging only in superficial regions due to inflammatory responses introduced by the craniotomy and insertion of foreign biomaterials. Microprisms provide a unique vertical view from brain surface to ~1 mm deep or more (depending on the size of the microprisms) which may break through this limitation on imaging depth. Although microprism has been used in the field of neuroscience, the in vivo foreign body responses to the microprism implant have yet to be fully elucidated. This is of important concern in broader applications of this approach, especially for neuroinflammation-sensitive studies. In this work, we first assessed the activation of microglia/macrophages for 16 weeks after microprism implantation using two-photon microscopy in awake CX3CR1-GFP mice. The imaging window became clear from bleedings after ~2 weeks and the maximum imaging distance (in the horizontal direction) stabilized at around 500 μm after ~5 weeks. We also quantified the microglial morphology from week 3 to week 16 post-implantation. Compared to non-implant controls, microglia near the microprism showed higher cell density, smaller soma, and shorter and less branched processes in the early-chronic phase. After week 5, microglial morphology further than 100 μm from the microprism was generally similar to microglia in the control group. In addition, time-lapse imaging confirmed that microglial processes were surveying normally from week 3, even for microglia as close as 50 μm away. These morphological analyses and dynamic imaging results suggest that microglia around chronically implanted microprism eventually exhibit inactive phenotypes. Next, we examined microglial/macrophage responses following laser induced micro-vessel disruptions as an example application of microprism implantation for neuroinflammation related studies. Through the microprism, we captured microglial/macrophage polarization and migration, as well as blood flow changes after the insult for additional 16 weeks. To our surprise, microglia/macrophage aggregation around the insult site was sustained over the 16-week observation period. This work demonstrates the feasibility of using microprisms for long-term characterizations of inflammatory responses to other injuries including implantable devices at deeper depths than that achievable by conventional two-photon microscopy.

Impact of Staphylococcus aureus Small Colony Variants on Human Lung Epithelial Cells with Subsequent Influenza Virus Infection.

Human beings are exposed to microorganisms every day. Among those, diverse commensals and potential pathogens including Staphylococcus aureus (S. aureus) compose a significant part of the respiratory tract microbiota. Remarkably, bacterial colonization is supposed to affect the outcome of viral respiratory tract infections, including those caused by influenza viruses (IV). Since 30% of the world’s population is already colonized with S. aureus that can develop metabolically inactive dormant phenotypes and seasonal IV circulate every year, super-infections are likely to occur. Although IV and S. aureus super-infections are widely described in the literature, the interactions of these pathogens with each other and the host cell are only scarcely understood. Especially, the effect of quasi-dormant bacterial subpopulations on IV infections is barely investigated. In the present study, we aimed to investigate the impact of S. aureus small colony variants on the cell intrinsic immune response during a subsequent IV infection in vitro. In fact, we observed a significant impact on the regulation of pro-inflammatory factors, contributing to a synergistic effect on cell intrinsic innate immune response and induction of harmful cell death. Interestingly, the cytopathic effect, which was observed in presence of both pathogens, was not due to an increased pathogen load.

Selective BET-bromodomain inhibition by JQ1 suppresses dendritic cell maturation and antigen-specific T-cell responses.

Bromo- and extra-terminal domain (BET) inhibitors represent potential therapeutic approaches in solid and hematological malignancies that are currently analyzed in several clinical trials. Additionally, BET are involved in the epigenetic regulation of immune responses by macrophages and dendritic cells (DCs), that play a central role in the regulation of immune responses, indicating that cancer treatment with BET inhibitors can promote immunosuppressive effects. The aim of this study was to further characterize the effects of selective BET inhibition by JQ1 on DC maturation and DC-mediated antigen-specific T-cell responses. Selective BET inhibition by JQ1 impairs LPS-induced DC maturation and inhibits the migrational activity of DCs, while antigen uptake is not affected. JQ1-treated DCs show reduced ability to induce antigen-specific T-cell proliferation. Moreover, antigen-specific Tcells co-cultured with JQ1-treated DCs exhibit an inactive phenotype and reduced cytokine production. JQ1-treated mice show reduced immune responses in vivo to sublethal doses of LPS, characterized by a reduced white blood cell count, an immature phenotype of splenic DCs and Tcells and lower blood levels of IL-6. In our study, we demonstrate that selective BET inhibition by JQ1, a drug currently tested in clinical trials for malignant diseases, has profound effects on DC maturation and DC-mediated antigen-specific T-cell responses. These immunosuppressive effects can result in the induction of possible infectious side effects in cancer treatments. In addition, based on our results, these compounds should not be used in combinatorial regimes using immunotherapeutic approaches such as check point inhibitors, T-cell therapies, or vaccines.

Abstract 14: Computer-aided discovery of small-molecule inhibitors targeting neural transcription factor BRN2 in neuroendocrine prostate tumors

Abstract Therapies targeting tumor-fueling androgens have been mainstay treatments of advanced prostate cancer for almost 5 decades. While the androgen receptor (AR) inhibitor enzalutamide (ENZ) prolongs survival of castration-resistant prostate cancer patients (CRPC), ENZ-resistant (ENZR) tumors rapidly recur. Our laboratory has identified that ENZ resistance can occur both in the presence and absence of continued classical AR activity. Lack of AR activity is clinically relevant, as it has been estimated that up to 25% of men who die from advanced CRPC have non-AR driven disease and lethal AR inactive neuroendocrine and anaplastic phenotypes of CRPC emerge after hormone therapy. Recently our group identified the neural transcription factor BRN2 as a major clinically relevant driver of neuroendocrine prostate cancer (NEPC). Hence, using the integrated power of computational drug discovery platform and biologic testing, we identified first-in-field inhibitors for BRN2. First, a homology model of BRN2 protein was generated and used as an input to perform large-scale virtual screening and virtual hits were tested in a series of experiments. Cpd 18-14, a lead BRN2 inhibitor, exhibited profound inhibition of BRN2 in transcriptional assay and binding to the BRN2 in cellular and in vitro assays. Furthermore, Cpd18-14 reduced the expression of neuroendocrine genes SOX2 and NCAM1. Importantly, Cpd18-14 displayed antiproliferative activity specifically in patient-derived BRN2hi NCI-H660 and in house-developed 42DENZRcells while displaying no effect on BRN2low 16DCRPC and LNCaP cells. Further, the most stable binding orientation of Cpd18-14 was determined using molecular dynamics simulations and was used to carry out structure-based lead optimization. As a result, we developed several synthetic derivatives with significant improvement in antiproliferative activity and metabolic stability. Finally, Cpd18-94 reduced the growth of NCI-H660 and 42DENZR NEPC tumors significantly in xenograft models. We anticipate that the developed drug prototypes will lay a foundation for the development of small-molecule therapies capable of combating highly aggressive and lethal form of neuroendocrine tumors. Citation Format: Ravi Munuganti, Daksh Thaper, Adeleke Aguda, Soojin Kim, Olena Sivak, Amina Zoubeidi. Computer-aided discovery of small-molecule inhibitors targeting neural transcription factor BRN2 in neuroendocrine prostate tumors [abstract]. In: Proceedings of the AACR Special Conference on Advancing Precision Medicine Drug Development: Incorporation of Real-World Data and Other Novel Strategies; Jan 9-12, 2020; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_1):Abstract nr 14.

The mediating role of visceral adiposity in the relationship among schooling, physical inactivity, and unhealthy metabolic phenotype.

To explore the mediating role of visceral adiposity, as assessed by the visceral adiposity index (VAI), in the relationship among schooling, physical inactivity, and unhealthy metabolic phenotype (UMP). A cross-sectional population-based study was conducted with 854 adult individuals of both sexes from a Brazilian city. Data were collected through interviews, anthropometric evaluation, and clinical and laboratorial tests. We used multivariate path analysis, which simultaneously analyzes multiple relationships between variables. The analyses were adjusted by sex and age and stratified according to nutritional status. A positive direct effect of visceral adiposity on the UMP was observed for normal weight, standardized estimate (SE) = 0.632; confidence interval (CI 95%) = 0.547, 0.707) and overweight individuals (SE = 0.732; CI 95% = 0.647, 0.808), and negative direct effect of schooling on physical inactivity (normal weight: SE = -0.408; CI 95% = -0.550, -0.265) and overweight (SE = -0.304; CI 95% = -0.479, -0.129). Among normal-weight individuals, there was a positive direct effect (SE = 0.193; CI 95% = 0.059, 0.328) of physical inactivity on VAI. In relation to indirect effects, there was a mediating role of visceral adiposity in the association of schooling level and physical inactivity with the UMP only among normal-weight individuals. Visceral adiposity has a direct effect on the UMP regardless of nutritional status, and there is a mediating effect of VAI on the relationship among schooling, physical inactivity, and UMP in normal-weight individuals.

Abstract P3-08-20: The normal breast Active transcriptome associated with future breast cancer risk is driven by a dysregulated adipocyte microenvironment

Abstract Background: Previous studies of breast samples from cosmetic surgeries, benign biopsies associated with abnormal mammograms or cancer-adjacent tissue have identified at least two different transcriptional phenotypes of “normal” human breast tissue (Troester, 2009; Haakensen, 2011), including an “Active” phenotype linked to increased risk of later breast cancer mortality (Roman-Perez, 2012; Troester, 2016). This study compares breast histology and transcriptional phenotypes from healthy parous women with no prior history of breast disease who donated breast core biopsies for research and supplied reproductive histories enabling breast cancer risk calculation. Since the Active transcriptome phenotype was recently associated with increased mammary adipocyte content, we focused on the possibility that adipocyte activation contributes to the Active transcriptome and drives breast cancer risk. Methods: RNA from paraffin-embedded tissue sections sufficient for RNAseq analysis (~100ng) was extracted from 151/200 core biopsies donated to the Komen Tissue Bank by healthy, parous white women (age range: 27-66, median = 45) with no history of breast cancer. Questionnaire data enabled breast cancer risk (Gail) score calculation; and digitized H&E images were used for histologic analyses. A previously validated classifying signature was used in unsupervised hierarchical clustering to identify samples with Active (78/151) vs. Inactive (73/151) transcriptome phenotypes for comparison with donor risk factors, breast tissue composition, and expression of candidate genes and gene signatures. Results: Mean (+/-SD) BMI and Gail score values were 29.60 (+/-7.92) and 1.27 (+/- 1.34), respectively; BMI scores were not significantly different by phenotype, but Gail scores were significantly higher for donors with an Active phenotype (1.46 vs. 1.18; p=0.007, Wilcoxon rank-sum). Active normal breast tissue samples possessed significantly more (%) adipocyte nuclei (p=3.9e-11) and greater adipocyte size (p<0.01), with fewer (%) stromal (p=4.3e-7) and (%) epithelial (p=1.2e-10) nuclei. Differentially expressed genes between the Active and Inactive phenotypes were significantly enriched for gene sets (GSEA) involved in fat differentiation and metabolism (14% at FDR q≤10%). Signature scores for cAMP-dependent lipolysis (known to drive breast cancer progression), as well as white adipose tissue “browning,” were significantly higher (Wilcoxon p<0.01) for the Active samples, as were specific genes reflecting adipocyte activation (leptin, adiponectin), remodeling (CAV1, BNIP3), adipokine growth factors (IGF-1, FGF2), and pro-inflammatory fat signaling (IKBKG, CCL13). Conclusion: In this cohort of predominantly non-obese women without breast disease, ~50% express an Active transcriptome phenotype previously linked to later-life breast cancer mortality. While >80% of this donor cohort would not qualify for breast cancer chemoprevention, those with Active transcriptomes had significantly higher Gail scores supporting their increased future risk for breast cancer development. The Active breast transcriptome is strongly associated with increased adipocyte content, size, and overexpression of signatures and genes (including those previously linked to breast cancer progression) indicating a differentially activated adipocyte population. This dysregulated mammary adipocyte microenvironment not only appears to underlie the Active transcriptome phenotype but also precedes and potentially predicts the future histologic development of breast cancer. Citation Format: Christopher C Benz, Taekyu Kang, Christina Yau, Chris Wong, Yulia Newton, Charlie Vaske, Stephen C Benz, Gregor Krings, Roman Camarda, Jill E Henry, Josh Stuart, Mark Powell. The normal breast Active transcriptome associated with future breast cancer risk is driven by a dysregulated adipocyte microenvironment [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 P3-08-20.

Hepatic ILC2 activity is regulated by liver inflammation-induced cytokines and effector CD4+ T cells

In immune-mediated hepatitis, type 2 innate lymphoid cells (ILC2) as well as effector CD4+ T cells have been shown to drive disease pathology. However, less is known about mechanisms involved in the regulation of ILC2 function during liver inflammation. We showed that in homeostasis, hepatic ILC2 constituted a very small population with a naive, inactive phenotype. During immune-mediated hepatitis, the cytokines IL-33 and IFNγ were expressed in liver tissue. IL-33 induced strong activation and expression of type 2 cytokines as well as IL-6 by hepatic ILC2 while IFNγ suppressed cytokine production. Interestingly, this inhibitory effect was overcome by IL-33. The phenotype of activated hepatic ILC2 were stable since they did not show functional plasticity in response to liver inflammation-induced cytokines. Moreover, hepatic ILC2 induced a Th2 phenotype in activated CD4+ T cells, which increased ILC2-derived cytokine expression via IL-2. In contrast, Th1 cells inhibited survival of ILC2 by production of IFNγ. Thus, hepatic ILC2 function is regulated by IL-33, IL-2, and IFNγ. While IL-33 and IL-2 support hepatic ILC2 activation, their inflammatory activity in immune-mediated hepatitis might be limited by infiltrating IFNγ-expressing Th1 cells.

AN INNOVATIVE PLATFORM BASED ON WEARABLE SENSOR TO QUANTIFY FRAILTY PHENOTYPES

This study evaluated an innovative wearable sensor based platform (instrumented trail-making task, iTMT) to quickly quantify frailty phenotypes, without the need of walking test. 61 older adults (age=72.8 ± 9.9years, BMI=27.4±4.9kg/m2) were recruited and assessed by Fried Frailty Criteria to determine frailty phenotypes. All subjects participated the iTMT test by standing in front of a computer, wearing a wearable sensor on the front lower shin. The sensor tracked the subject’s ankle movement and projected it on a computer-screen as a moving cursor at real-time. The subject rotated the ankle joint to navigate the cursor to reach 5 indexed circles (including numbers 1-to-3 and letters A&B placed in random orders) in the alternative order of numbers and letters. The iTMT required coordination of brain and body movement, testing subject’s cognitive-motor function. The sensor quantified ankle-rotation biomechanics during the test. All subjects completed the iTMT with average time less than 3 minutes. The ankle-rotation velocity generated during the test distinguished between the presence and absence of Slowness phenotype (Cohen’s effect size d=1.40, p<0.001). The decline of ankle-rotation velocity determined the presence of Exhaustion phenotype (d=0.98, p=0.003). The ankle-rotation power generated during the test determined the presence of Weakness phenotype (d=1.38, p<0.001). The ankle-velocity variability determined the presence of Inactivity phenotype (d=0.90, p<0.001). This study demonstrated the feasibility and validity of the iTMT to quantify frailty phenotypes. This new platform is time-efficient and doesn’t require walking test. It’s more practical for routine assessment in small and busy clinics among patients with mobility limitation.

Abstract 3748: Implantable tumor attracting niche models to study disseminated tumor cell biology

Abstract Metastasis is the leading cause of death in cancer but remains the most poorly understood aspect of tumor biology. This can be attributed to the lack of relevant experimental models that can recapitulate the complex and lengthy progression of metastatic relapse. Mouse models have been widely used to study metastasis, however they are limited for prolonged investigation of the post-dissemination phase of cancer cell biology due to the rapid onset of actively growing primary and secondary tumors. This limitation has left a critical gap in understanding the long-term bi-directional crosstalk between DTCs and their local microenvironment due to the relatively short experimental timeframe. Here we introduce an implantable tissue engineered metastasis model that can overcome the fundamental restrictions of existing mouse models and substantiate the role of the tumor microenvironment in the reactivation of dormant DTCs with molecular and cellular detail. Subcutaneous implantation of porous collagen-coated acrylamide scaffolds, fabricated as previously described (1-3), modulate the foreign body response to induce the formation of a robust vascularized tissue. During tissue formation and local inflammation, the scaffold generates a pre-metastatic niche, characterized by the recruitment of circulating tumor cells and given time, the formation of overt metastases. However, mice became moribund before meaningful investigation of dormant tumor cell biology could occur due to increasing tumor burden. We addressed this limitation via intact transplantation of the DTC microenvironment to tumor-free mice. Scaffolds have been maintained in secondary mice for up to 10 weeks. Compared to transplanted DTC-bearing lung tissue, scaffolds maintained an inactive phenotype for longer periods. At the end of this period, transplanted scaffolds captured three distinct DTC phenotypes: dormant, early colonization, and overt metastasis. Multiplex immunohistostaining was used to dissect the local microenvironment during the transition from single DTCs to overt metastasis. The local vasculature and Ly6G+ cells were observed to have differential roles during metastatic development. These experiments have been demonstrated in humanized NSG mouse models capable of generating scaffold niches containing human stromal, tumor (PC-3), and immune cells, as well as a spontaneous tumor model (MMTV-PyMT) with an intact immune system. Our results demonstrate the potential of a tissue engineering approach to generate pre-metastatic niches and investigate the transient activity of the DTC niche during tumor cell awakening and subsequent metastasis. We envision that implantable microenvironments will be an enabling tool to study DTC biology and aid in the development of anti-metastatic therapies.

Abstract 2448: Elucidating the metabolic plasticity of cancer by coupling gene regulation with metabolic pathways

Abstract Metabolic plasticity allows cancer cells to adjust their metabolic phenotypes to grow and metastasize in hostile environments. Both glycolysis and oxidative phosphorylation (OXPHOS) are adapted by cancer cells to meet their bioenergetic and biosynthetic requirements in a context-dependent manner. Despite the advance in studies focusing only on glycolysis or OXPHOS in cancer, it remains largely unknown how cancer cells orchestrate different metabolic phenotypes for better survival. To address this question, there is an urgent need to develop systemic approaches to quantitatively study the interplay between glycolysis and OXPHOS in cancer. Mathematical modeling approaches have been employed to elucidate cancer metabolic reprogramming. Constraint-based models including flux balance analysis based on conservation of mass have been the most widely used approaches to simulate cancer metabolism. In addition, modeling efforts have also been developed to identify anomalous gene activity involved in cancer metabolism. These computational studies offer a quantitative and dynamical perspective of cancer metabolism mostly focusing on either metabolic pathways or gene activities. However, the alteration of the metabolic activity is often coupled with the change in gene activity, and vice versa. Thus, to comprehensively characterize cancer metabolic reprogramming, a mathematical modeling framework integrating gene regulation with metabolic pathways is urgently needed. Here, we establish a theoretical framework to elucidate cancer metabolic plasticity through systems biology analysis of the coupling of gene regulation and metabolic pathways. Our modeling results demonstrate a direct association between the activities of AMPK and HIF-1, master regulators of OXPHOS and glycolysis respectively, with the activities of three metabolic pathways: glucose oxidation, glycolysis and fatty acid oxidation (FAO). Guided by the model, we develop metabolic pathway signatures to quantify the activities of glycolysis, FAO and the citric acid cycle of tumor samples by evaluating the expression levels of enzymes involved in corresponding processes. The association of AMPK/HIF-1 activity with metabolic pathway activity, predicted by the model and verified by analyzing the well-annotated metabolomic and transcriptomic data from a breast cancer patients’ cohort, is further validated by in vitro studies of aggressive triple negative breast cancer cell lines. We further investigate the existence of an aggressive hybrid metabolic phenotype that enables cancer cells metabolic plasticity for better survival and a metabolically inactive phenotype that may be employed by cancer cells under pressure. To the best of our knowledge, we are the first, or at least one of the first, to couple gene regulation with metabolic pathways to elucidate cancer metabolic plasticity. Citation Format: Dongya Jia, Mingyang Lu, Kwang Hwa Jung, Jun Hyoung Park, José N. Onuchic, Benny Abraham Kaipparettu, Herbert Levine. Elucidating the metabolic plasticity of cancer by coupling gene regulation with metabolic pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2448.

Liver-infiltrating CD11b\u2212CD27\u2212 NK subsets account for NK-cell dysfunction in patients with hepatocellular carcinoma and are associated with tumor progression

Natural killer (NK) cells have a vital role in killing hepatocellular carcinoma (HCC) cells; however, the mechanism underlying tumor-infiltrating NK (TINK)-cell dysfunction remains poorly understood. Using flow cytometry staining, we precisely characterized the frequency, phenotype and function of NK subsets distinguished by CD27 and CD11b in 30 patients with HCC in comparison to 30 healthy controls. Interestingly, we found a substantial proportion of liver-infiltrating CD11b−CD27− (DN) NK subsets in tumor tissue from HCC patients. Remarkably, these relatively expanded DN NK subsets exhibited an inactive and immature phenotype. By detecting the expression of CD107a and interferon-gamma (IFN-γ) on NK subsets and NK cells, we demonstrated that DN NK subsets exhibited a poor cytotoxic capacity and deficient potential to produce IFN-γ in comparison to the other three subsets, which contributed to the dysfunction of TINK cells in HCC patients. In addition, we found that the presence of DN NK cells was closely associated with the clinical outcomes of HCC patients, as the frequency of DN NK cells among TINK cells was positively correlated with tumor stage and size. A large percentage of DN NK cells among TINK cells was an independent prognostic factor for lower survival in the 60-month follow-up period. In conclusion, a substantial proportion of CD11b−CD27−NK subsets among TINK cells accounts for NK-cell dysfunction in patients with HCC and is associated with tumor progression. Our study may provide a novel therapeutic target for the treatment of patients with HCC.

In utero induction of mixed chimerism results in deletion, receptor editing, and functional inactivation of antigen specific alloreactive B cells

Abstract Purpose B cell tolerance is critical for donor cell engraftment across immune barriers after in utero hematopoietic cell transplantation (IUHCT). Using our fetal transplant model we studied how B cells achieve tolerance to allogeneic MHC antigens. Methods 10×106 C57Bl/6-GFP (H2Kb) bone marrow (BM) cells were injected intravenously into gestational day 14 Balb/c (H2Kd) fetuses. Mice were sacrificed at 3 days (P3), 1 and 4 months of age and donor-specific B cell tolerance including deletion, receptor editing and functional inactivation were assessed. Age matched naïve C57Bl/6 and Balb/c mice served as controls. Results The % of alloantigen specific splenic B cells was significantly decreased in allogenic chimeras as compared to controls (p=0.016) consistent with clonal deletion. Alloreactive B cells that escape deletion can undergo receptor editing. There was a significant increase in the % of B cells undergoing editing (lambda light chain expression) at P3 in recipients of IUHCT compared to controls (10.8% vs. 8.4%; p=0.02). Alloreactive B cells can be functionally inactivated such that they no longer make antibodies or express IgM. In contrast to naïve Balb/c mice immunized with C57Bl/6 splenocytes, immunization of recipients of IUHCT failed to elicit an anti-donor antibody response (MFI fold change: IUHCT-0.89 naïve-2.77; p&amp;lt;0.05). Furthermore, IgM expression on alloantigen specific splenocytes in chimeric animals was lower than controls (MFI: chimeric-1206, control-1472 ; p=0.03). Conclusion B cell tolerance after IUHCT is achieved by partial deletion in alloreactive splenic B cells, receptor editing in early mature splenocytes, and the remainder of alloreactive B cells demonstrate a functionally inactive phenotype.

Hepatitis B Virus–Specific and Global T-Cell Dysfunction in Chronic Hepatitis B

T cells play a critical role in viral infection. We examined whether T-cell effector and regulatory responses can define clinical stages of chronic hepatitis B (CHB). We enrolled 200 adults with CHB who participated in the National Institutes of Health-supported Hepatitis B Research Network from 2011 through 2013 and 20 uninfected individuals (controls). Peripheral blood lymphocytes from these subjects were analyzed for T-cell responses (proliferation and production of interferon gamma and interleukin 10) to overlapping hepatitis B virus (HBV) peptides (preS, S, preC, core, and reverse transcriptase), influenza matrix peptides, and lipopolysaccharide. T-cell expression of regulatory markers FOXP3, programmed death-1, and cytotoxic T lymphocyte-associated antigen-4 was examined by flow cytometry. Immune measures were compared with clinical parameters, including physician-defined immune-active, immune-tolerant, or inactive CHB phenotypes, in a blinded fashion. Compared with controls, patients with CHB had weak T-cell proliferative, interferon gamma, and interleukin 10 responses to HBV, with increased frequency of circulating FOXP3(+)CD127(-) regulatory T cells and CD4(+) T-cell expression of programmed death-1 and cytotoxic T lymphocyte-associated antigen-4. T-cell measures did not clearly distinguish between clinical CHB phenotypes, although the HBV core-specific T-cell response was weaker in hepatitis B e antigen (HBeAg)(+) than HBeAg(-) patients (percent responders: 3% vs 23%; P = .00008). Although in vitro blockade of programmed death-1 or cytotoxic T lymphocyte-associated antigen-4 increased T-cell responses to HBV, the effect was weaker in HBeAg(+) than HBeAg(-) patients. Furthermore, T-cell responses to influenza and lipopolysaccharide were weaker in CHB patients than controls. HBV persists with virus-specific and global T-cell dysfunction mediated by multiple regulatory mechanisms, including circulating HBeAg, but without distinct T-cell-based immune signatures for clinical phenotypes. These findings suggest additional T-cell-independent or regulatory mechanisms of CHB pathogenesis that warrant further investigation.

Characterization of the Protective Role of Regulatory T Cells in Experimental Periapical Lesion Development and Their Chemoattraction Manipulation as a Therapeutic Tool

IntroductionThe pathogenesis of periapical lesions is determined by the balance between host proinflammatory immune response and counteracting anti-inflammatory and reparative responses, which include regulatory T cells (Tregs) as potential immunoregulatory agents. In this study, we investigated (in a cause-and-effect manner) the involvement of CCL22-CCR4 axis in Treg migration to the periapical area and the role of Tregs in the determination of outcomes in periapical lesions. MethodsPeriapical lesions were induced in C57Bl/6 (wild-type) and CCR4KO mice (pulp exposure and bacterial inoculation) and treated with anti–glucocorticoid-induced TNF receptor family regulated gene to inhibit Treg function or alternatively with CCL22-releasing, polylactic-glycolic acid particles to induce site-specific migration of Tregs. After treatment, lesions were analyzed for Treg influx and phenotype, overall periapical bone loss, and inflammatory/immunologic and wound healing marker expression (analyzed by real-time polymerase chain reaction array). ResultsTreg inhibition by anti–glucocorticoid-induced TNF receptor family regulated gene or CCR4 depletion results in a significant increase in periapical lesion severity, associated with upregulation of proinflammatory, T-helper 1, T-helper 17, and tissue destruction markers in parallel with decreased Treg and healing marker expression. The local release of CCL22 in the root canal system resulted in the promotion of Treg migration in a CCR4-dependent manner, leading to the arrest of periapical lesion progression, associated with downregulation of proinflammatory, T-helper 1, T-helper 17, and tissue destruction markers in parallel with increased Treg and healing marker expression. ConclusionsBecause the natural and CCL22-induced Treg migration switches active lesion into inactivity phenotype, Treg chemoattractant may be a promising strategy for the clinical management of periapical lesions.

(In)activity-related neuroplasticity in brainstem control of sympathetic outflow: unraveling underlying molecular, cellular, and anatomical mechanisms.

More people die as a result of physical inactivity than any other preventable risk factor including smoking, high cholesterol, and obesity. Cardiovascular disease, the number one cause of death in the United States, tops the list of inactivity-related diseases. Nevertheless, the vast majority of Americans continue to make lifestyle choices that are creating a rapidly growing burden of epidemic size and impact on the United States healthcare system. It is imperative that we improve our understanding of the mechanisms by which physical inactivity increases the incidence of cardiovascular disease and how exercise can prevent or rescue the inactivity phenotype. The current review summarizes research on changes in the brain that contribute to inactivity-related cardiovascular disease. Specifically, we focus on changes in the rostral ventrolateral medulla (RVLM), a critical brain region for basal and reflex control of sympathetic activity. The RVLM is implicated in elevated sympathetic outflow associated with several cardiovascular diseases including hypertension and heart failure. We hypothesize that changes in the RVLM contribute to chronic cardiovascular disease related to physical inactivity. Data obtained from our translational rodent models of chronic, voluntary exercise and inactivity suggest that functional, anatomical, and molecular neuroplasticity enhances glutamatergic neurotransmission in the RVLM of sedentary animals. Collectively, the evidence presented here suggests that changes in the RVLM resulting from sedentary conditions are deleterious and contribute to cardiovascular diseases that have an increased prevalence in sedentary individuals. The mechanisms by which these changes occur over time and their impact are important areas for future study.

Reversion of hepatic stellate cell to a quiescent phenotype: From myth to reality?

Myofibroblasts produce the fibrous scar in hepatic fibrosis. In the carbon tetrachloride (CCl(4)) model of liver fibrosis, quiescent hepatic stellate cells (HSC) are activated to become myofibroblasts. When the underlying etiological agent is removed, clinical and experimental fibrosis undergoes a remarkable regression with complete disappearance of these myofibroblasts. Although some myofibroblasts apoptose, it is unknown whether other myofibroblasts may revert to an inactive phenotype during regression of fibrosis. We elucidated the fate of HSCs/myofibroblasts during recovery from CCl(4)and alcohol-induced liver fibrosis using Cre-LoxP-based genetic labeling of myofibroblasts. Here we demonstrate that half of the myofibroblasts escape apoptosis during regression of liver fibrosis, down-regulate fibrogenic genes, and acquire a phenotype similar to, but distinct from, quiescent HSCs in their ability to more rapidly reactivate into myofibroblasts in response to fibrogenic stimuli and strongly contribute to liver fibrosis. Inactivation of HSCs was associated with up-regulation of the anti-apoptotic genes Hspa1a/b, which participate in the survival of HSCs in culture and in vivo.