Gene Regulatory Circuits in Innate and Adaptive Immune Cells.

Cell identity and function largely rely on the programming of transcriptomes during development and differentiation. Signature gene expression programs are orchestrated by regulatory circuits consisting of cis-acting promoters and enhancers, which respond to a plethora of cues via the action of transcription factors. In turn, transcription factors direct epigenetic modifications to revise chromatin landscapes, and drive contacts between distal promoter-enhancer combinations. In immune cells, regulatory circuits for effector genes are especially complex and flexible, utilizing distinct sets of transcription factors and enhancers, depending on the cues each cell type receives during an infection, after sensing cellular damage, or upon encountering a tumor. Here, we review major players in the coordination of gene regulatory programs within innate and adaptive immune cells, as well as integrative omics approaches that can be leveraged to decipher their underlying circuitry.

PERK is a critical metabolic hub for immunosuppressive function in macrophages.

Chronic inflammation triggers compensatory immunosuppression to stop inflammation and minimize tissue damage. Studies have demonstrated that endoplasmic reticulum (ER) stress augments the suppressive phenotypes of immune cells; however, the molecular mechanisms underpinning this process and how it links to the metabolic reprogramming of immunosuppressive macrophages remain elusive. In the present study, we report that the helper T cell 2 cytokine interleukin-4 and the tumor microenvironment increase the activity of a protein kinase RNA-like ER kinase (PERK)-signaling cascade in macrophages and promote immunosuppressive M2 activation and proliferation. Loss of PERK signaling impeded mitochondrial respiration and lipid oxidation critical for M2 macrophages. PERK activation mediated the upregulation of phosphoserine aminotransferase 1 (PSAT1) and serine biosynthesis via the downstream transcription factor ATF-4. Increased serine biosynthesis resulted in enhanced mitochondrial function and α-ketoglutarate production required for JMJD3-dependent epigenetic modification. Inhibition of PERK suppressed macrophage immunosuppressive activity and could enhance the efficacy of immune checkpoint programmed cell death protein 1 inhibition in melanoma. Our findings delineate a previously undescribed connection between PERK signaling and PSAT1-mediated serine metabolism critical for promoting immunosuppressive function in M2 macrophages.

PRMT5 Promotes T follicular helper Cell Differentiation and Germinal Center Responses during Influenza Virus Infection.

Protein arginine methyltransferases (PRMTs) modify diverse protein targets and regulate numerous cellular processes; yet, their contributions to individual effector T cell responses during infections are incompletely understood. In this study, we identify PRMT5 as a critical regulator of CD4+ T follicular helper cell (Tfh) responses during influenza virus infection in mice. Conditional PRMT5 deletion in murine T cells results in an almost complete ablation of both Tfh and T follicular regulatory populations and, consequently, reduced B cell activation and influenza-specific Ab production. Supporting a potential mechanism, we observe elevated surface expression of IL-2Rα on non-T regulatory effector PRMT5-deficient T cells. Notably, IL-2 signaling is known to negatively impact Tfh differentiation. Collectively, our findings identify PRMT5 as a prominent regulator of Tfh programming, with potential causal links to IL-2 signaling.

Micro but mighty-Micronutrients in the epigenetic regulation of adaptive immune responses.

Micronutrients are essential small molecules required by organisms in minute quantity for survival. For instance, vitamins and minerals, the two major categories of micronutrients, are central for biological processes such as metabolism, cell replication, differentiation, and immune response. Studies estimated that around two billion humans worldwide suffer from micronutrient deficiencies, also known as "hidden hunger," linked to weakened immune responses. While micronutrients affect the immune system at multiple levels, recent studies showed that micronutrients potentially impact the differentiation and function of immune cells as cofactors for epigenetic enzymes, including the 2-oxoglutarate-dependent dioxygenase (2OGDD) family involved in histone and DNA demethylation. Here, we will first provide an overview of the role of DNA methylation in T cells and B cells, followed by the micronutrients ascorbate (vitamin C) and iron, two critical cofactors for 2OGDD. We will discuss the emerging evidence of these micronutrients could regulate adaptive immune response by influencing epigenetic remodeling.

CEMIG: prediction of the cis-regulatory motif using the de Bruijn graph from ATAC-seq.

Sequence motif discovery algorithms enhance the identification of novel deoxyribonucleic acid sequences with pivotal biological significance, especially transcription factor (TF)-binding motifs. The advent of assay for transposase-accessible chromatin using sequencing (ATAC-seq) has broadened the toolkit for motif characterization. Nonetheless, prevailing computational approaches have focused on delineating TF-binding footprints, with motif discovery receiving less attention. Herein, we present Cis rEgulatory Motif Influence using de Bruijn Graph (CEMIG), an algorithm leveraging de Bruijn and Hamming distance graph paradigms to predict and map motif sites. Assessment on 129 ATAC-seq datasets from the Cistrome Data Browser demonstrates CEMIG's exceptional performance, surpassing three established methodologies on four evaluative metrics. CEMIG accurately identifies both cell-type-specific and common TF motifs within GM12878 and K562 cell lines, demonstrating its comparative genomic capabilities in the identification of evolutionary conservation and cell-type specificity. In-depth transcriptional and functional genomic studies have validated the functional relevance of CEMIG-identified motifs across various cell types. CEMIG is available at https://github.com/OSU-BMBL/CEMIG, developed in C++ to ensure cross-platform compatibility with Linux, macOS and Windows operating systems.

Supporting cognitive catch-up: The effects of cluster-randomized psychosocial stimulation interventions on preterm low birthweight children in rural China.

Improved survival of preterm low birthweight (LBW) infants due to advances in neonatal care has brought issues such as postnatal development trajectories to the foreground. This study pools evidence from three cluster-randomized experiments evaluating community-based psychosocial stimulation programs conducted from 2014 to 2017 that included 3571 rural Chinese children aged 6-24 months (51.1% male, 96.2% Han Chinese). The risk of severe cognitive delay was found to be 26.5 percentage points higher for preterm LBW children than for their peers at age 2.5, with a prevalence rate of 48.3%. Results show that psychosocial stimulation interventions can improve child cognitive development at scale, with beneficial impacts on child cognition disproportionately larger for preterm LBW children, helping them to catch up developmentally.

Anti-inflammatory effect of biologic therapy in patients with psoriatic disease: A prospective cohort FDG PET study.

AIM: The aim of the study was to evaluate the changes in central vascular inflammation measured by FDG PET and myocardial blood flow reserve (MFR) determined by 82Rb PET following therapy with biologic agents for 6 months in patients with psoriatic arthritis (PsA) and/or cutaneous psoriasis (PsO) (group 1) and compare with PsO subjects receiving non-biologic therapy (group 2) and controls (group 3). METHODS AND RESULTS: Target-to-background ratio (TBR) by FDG PET in the most diseased segment of the ascending aorta (TBRmax) was measured to assess vascular inflammation. 82Rb PET studies were used to assess changes in left ventricular MFR. A total of 34 participants were enrolled in the study (11 in group 1, 13 in group 2, and 10 controls). A significant drop in the thoracic aorta uptake was observed in the biologic-treated group (ΔTBRmax: - .46 ± .55) compared to the PsO group treated with non-biologic therapy (ΔTBRmax: .23 ± .67). Those showing response to biologic agents maintained MFR compared to who showed no response. CONCLUSION: In a cohort of psoriasis patients treated with biologics, FDG uptake in the thoracic aorta decreased over the study period. Patients who demonstrated a significant anti-inflammatory response on FDG PET imaging maintained their MFR compared to non-responders.

Anti-inflammatory effect of rosuvastatin in patients with HIV infection: An FDG-PET pilot study.

AIMS: This study aimed to evaluate markers of systemic as well as imaging markers of inflammation in the ascending aorta, bone marrow, and spleen measured by 18F-FDG PET/CT, in HIV+ patients at baseline and following therapy with rosuvastatin. METHODS AND RESULTS: Of the 35 HIV+ patients enrolled, 17 were randomized to treatment with 10 mg/day rosuvastatin and 18 to usual care for 6 months. An HIV- control cohort was selected for baseline comparison of serum inflammatory markers and monocyte markers of inflammation. 18F-FDG-PET/CT imaging of bone marrow, spleen, and thoracic aorta was performed in the HIV+ cohort at baseline and 6 months. While CD14++CD16- and CCR2 expressions were reduced, serum levels of IL-7, IL-8, and MCP-1 were elevated in the HIV+ population compared to the controls. There was a significant drop in FDG uptake in the bone marrow (TBRmax), spleen (SUVmax) and thoracic aortic (TBRmax) in the statin-treated group compared to the control group (bone marrow: - 10.3 ± 16.9% versus 5.0 ± 18.9%, p = .0262; spleen: - 9.8 ± 20.3% versus 11.3 ± 28.8%, p = .0497; thoracic aorta: - 19.1 ± 24.2% versus 4.3 ± 15.4%, p = .003). CONCLUSIONS: HIV+ patients had significantly markers of systemic inflammation including monocyte activation. Treatment with low-dose rosuvastatin in the HIV+ cohort significantly reduced bone marrow, spleen and thoracic aortic FDG uptake.

Circles of Life: linking metabolic and epigenetic cycles to immunity.

Metabolites are the essential substrates for epigenetic modification enzymes to write or erase the epigenetic blueprint in cells. Hence, the availability of nutrients and activity of metabolic pathways strongly influence the enzymatic function. Recent studies have shed light on the choreography between metabolome and epigenome in the control of immune cell differentiation and function, with a major focus on histone modifications. Yet, despite its importance in gene regulation, DNA methylation and its relationship with metabolism is relatively unclear. In this review, we will describe how the metabolic flux can influence epigenetic networks in innate and adaptive immune cells, with a focus on the DNA methylation cycle and the metabolites S-adenosylmethionine and α-ketoglutarate. Future directions will be discussed for this rapidly emerging field.

Generation of iPSC line NCHi015-A from a patient with truncus arteriosus carrying heterozygous variants in KMT2D and NOTCH1.

Truncus arteriosus (TA) is a congenital heart defect where one main blood vessel emerges from the heart, instead of individual aorta and pulmonary artreries. Peripheral mononuclear cells (PBMCs) of a male infant with TA were reporogrammed using Sendai virus. The resultant iPSC line (NCHi015-A) displayed normal colony formation, expressed pluripotency markers, and differentiated into cells from three germ layers. NCHi015-A was matched to the patient's genetic profile, had normal karyotype, retained genetic variants in KMT2D and NOTCH1, and tested negative for reprogramming transgene. This iPSC line can be used for studying congenital heart defects associated with genetic variants in KMT2D and NOTCH1.

3D-Bioprinted GelMA Scaffold with ASCs and HUVECs for Engineering Vascularized Adipose Tissue.

The purpose of tissue engineering is to reconstruct parts of injured tissues and to resolve the shortage of organ donations. However, the main concern is the limited size of engineered tissue due to insufficient oxygen and nutrition distribution in large three-dimensional (3D) tissue constructs. To provide better support for cells inside the scaffolds, the vascularization of blood vessels within the scaffold could be a solution. This study compared the effects of different culturing systems using human adipose tissue-derived stem/stromal cells (ASCs), human umbilical vein endothelial cells (HUVECs), and coculture of ASCs and HUVECs in 3D-bioprinted gelatin methacrylate (GelMA) hydrogel constructs. The in vitro results showed that the number of live cells was highest in the coculture of ASCs and HUVECs in the GelMA hydrogel after culturing for 21 days. Additionally, the tubular structure was the most abundant in the GelMA hydrogel, containing both ASCs and HUVECs. In the in vivo test, blood vessels were present in both the HUVECs and the coculture of ASCs and HUVECs hydrogels implanted in mice. However, the blood vessel density was the highest in the HUVEC and ASC coculture groups. These findings indicate that the 3D-bioprinted GelMA hydrogel coculture system could be a promising biomaterial for large tissue engineering applications.

The mitochondrial stress-induced protein carboxyl-terminal alanine and threonine tailing (msiCAT-tailing) promotes glioblastoma tumorigenesis by modulating mitochondrial functions.

The rapid and sustained proliferation in cancer cells requires accelerated protein synthesis. Accelerated protein synthesis and disordered cell metabolism in cancer cells greatly increase the risk of translation errors. ribosome-associated quality control (RQC) is a recently discovered mechanism for resolving ribosome collisions caused by frequent translation stalls. The role of the RQC pathway in cancer initiation and progression remains controversial and confusing. In this study, we investigated the pathogenic role of mitochondrial stress-induced protein carboxyl-terminal terminal alanine and threonine tailing (msiCAT-tailing) in glioblastoma (GBM), which is a specific RQC response to translational arrest on the outer mitochondrial membrane. We found that msiCAT-tailed mitochondrial proteins frequently exist in glioblastoma stem cells (GSCs). Ectopically expressed msiCAT-tailed mitochondrial ATP synthase F1 subunit alpha (ATP5α) protein increases the mitochondrial membrane potential and blocks mitochondrial permeability transition pore (MPTP) formation/opening. These changes in mitochondrial properties confer resistance to staurosporine (STS)-induced apoptosis in GBM cells. Therefore, msiCAT-tailing can promote cell survival and migration, while genetic and pharmacological inhibition of msiCAT-tailing can prevent the overgrowth of GBM cells. Highlights: The RQC pathway is disturbed in glioblastoma (GBM) cellsmsiCAT-tailing on ATP5α elevates mitochondrial membrane potential and inhibits MPTP openingmsiCAT-tailing on ATP5α inhibits drug-induced apoptosis in GBM cellsInhibition of msiCAT-tailing impedes overall growth of GBM cells.

Inhibition of Wnt/ß-catenin signaling upregulates Nav 1.5 channels in Brugada syndrome iPSC-derived cardiomyocytes.

The voltage-gated Nav 1.5 channels mediate the fast Na+ current (INa ) in cardiomyocytes initiating action potentials and cardiac contraction. Downregulation of INa , as occurs in Brugada syndrome (BrS), causes ventricular arrhythmias. The present study investigated whether the Wnt/ß-catenin signaling regulates Nav 1.5 in human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). In healthy male and female iPSC-CMs, activation of Wnt/ß-catenin signaling by CHIR-99021 reduced (p < 0.01) both Nav 1.5 protein and SCN5A mRNA. In iPSC-CMs from a BrS patient, both Nav 1.5 protein and peak INa were reduced compared to those in healthy iPSC-CMs. Treatment of BrS iPSC-CMs with Wnt-C59, a small-molecule Wnt inhibitor, led to a 2.1-fold increase in Nav 1.5 protein (p = 0.0005) but surprisingly did not affect SCN5A mRNA (p = 0.146). Similarly, inhibition of Wnt signaling using shRNA-mediated ß-catenin knockdown in BrS iPSC-CMs led to a 4.0-fold increase in Nav 1.5, which was associated with a 4.9-fold increase in peak INa but only a 2.1-fold increase in SCN5A mRNA. The upregulation of Nav 1.5 by ß-catenin knockdown was verified in iPSC-CMs from a second BrS patient. This study demonstrated that Wnt/ß-catenin signaling inhibits Nav 1.5 expression in both male and female human iPSC-CMs, and inhibition of Wnt/ß-catenin signaling upregulates Nav 1.5 in BrS iPSC-CMs through both transcriptional and posttranscriptional mechanisms.

Fallopian tubal histogenesis of ovarian endometriosis-A study of folate receptor-alpha expression.

Background: Ovary is a common organ site involved by endometriosis. We previously found that fallopian tube may contribute to the histogenesis of ovarian endometriosis. The finding was novel and requires further studies. We addressed this issue by examining a differentially expressed gene folate receptor alpha (FOLR1) and its protein (FRA) in this study. Results: A total of 144 tissue samples were studied. These included 32-paired tubal-endometrial-ovarian endometriosis samples (n = 96), 18 samples of ovarian endometriosis without corresponding fallopian tube or endometrium, and 30 ovarian tissue samples with ovarian surface epithelia but without endometriosis. Multiple comparisons among groups of ovarian endometriosis, normal fallopian tube and benign endometrium were performed. FOLR1 was highly expressed in the epithelia of fallopian tube and ovarian endometriosis, with paired endometrial samples showing a significantly lower level of expression. Similar differential studies for FRA protein were performed through Western blot and immunohistochemistry (IHC). The expression of folate receptor alpha at both mRNA and protein levels in the tissues (fallopian tube or ovarian endometriosis vs. the endometrium) were significantly different (p < 0.001). All ovarian surface mesothelial epithelia showed negative expression of FRA by IHC. Conclusion: The results further support that the fallopian tube may contribute to the development of ovarian endometriosis. Understanding the tubal contribution to ovarian endometriosis should ultimately contribute to ongoing investigative efforts aimed at identifying alternative ways to prevent and treat endometriosis. High level of FRA expression in the fallopian tube and endometriosis might be considered as potential tissue sites for targeted therapy.

Human theca arises from ovarian stroma and is comprised of three discrete subtypes.

Theca cells serve multiple essential functions during the growth and maturation of ovarian follicles, providing structural, metabolic, and steroidogenic support. While the function of theca during folliculogenesis is well established, their cellular origins and the differentiation hierarchy that generates distinct theca sub-types, remain unknown. Here, we performed single cell multi-omics analysis of primary cell populations purified from human antral stage follicles (1-3 mm) to define the differentiation trajectory of theca/stroma cells. We then corroborated the temporal emergence and growth kinetics of defined theca/stroma subpopulations using human ovarian tissue samples and xenografts of cryopreserved/thawed ovarian cortex, respectively. We identified three lineage specific derivatives termed structural, androgenic, and perifollicular theca cells, as well as their putative lineage-negative progenitor. These findings provide a framework for understanding the differentiation process that occurs in each primordial follicle and identifies specific cellular/molecular phenotypes that may be relevant to either diagnosis or treatment of ovarian pathologies.

Inhibition of Lck: evidence for a novel natural Src family kinase inhibitor.

Src family kinase (SFK) is a family of protein tyrosine kinases that play important roles in the development of various cancers. Here, we showed that a naturally occurring inhibitory factor of SFK can be extracted from the rat brain. This inhibitor strongly suppressed the activity of SFKs including Lck and Fyn. It did not inhibit other protein tyrosine kinases including Wee1 or serine/threonine kinases Mst2, Cdk5/p25, Cdk5/p35, and Cdk2/cyclin A. The inhibitor was not an ATPase, a phosphatase that dephosphorylates substrates of the SFK reaction, or a protease that degrades SFKs. Activity of mutant Lck with C-terminal tyrosine substituted with phenylalanine was also suppressed by the inhibitor to a similar extent of wild-type Lck, indicating that the inhibitor was not CSK. Gel filtration chromatography indicated that the molecular size of the prevalent form of this inhibitor was approximately 44 kDa.

Diffusion-weighted MR imaging of musculoskeletal tissues: incremental role over conventional MR imaging in bone, soft tissue, and nerve lesions.

Diffusion-weighted imaging is increasingly becoming popular in musculoskeletal radiology for its incremental role over conventional MR imaging in the diagnostic strategy and assessment of therapeutic response of bone and soft tissue lesions. This article discusses the technical considerations of diffusion-weighted imaging, how to optimize its performance, and outlines the role of this novel imaging in the identification and characterization of musculoskeletal lesions, such as bone and soft tissue tumors, musculoskeletal infections, arthritis, myopathy, and peripheral neuropathy. The readers can use the newly learned concepts from the presented material containing illustrated case examples to enhance their conventional musculoskeletal imaging and interventional practices and optimize patient management, their prognosis, and outcomes.

Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk.

Heart disease is the leading cause of morbidity and mortality worldwide. Due to their low cost, ease of handling, and abundance of transgenic strains, rodents have become essential models for cardiovascular research. However, spontaneous lethal cardiac arrhythmias that often cause mortality in heart disease patients are rare in rodent models of heart disease. This is primarily due to the species differences in cardiac electrical properties between human and rodents and poses a challenge to the study of cardiac arrhythmias using rodents. This protocol describes an approach to enable efficient transgene expression in mouse and rat ventricular myocardium using echocardiography-guided intramuscular injections of recombinant virus (adenovirus and adeno-associated virus). This work also outlines a method to enable reliable assessment of cardiac susceptibility to arrhythmias using isolated, Langendorff-perfused mouse and rat hearts with both adrenergic and programmed electrical stimulations. These techniques are critical for studying heart rhythm disorders associated with adverse cardiac remodeling after injuries, such as myocardial infarction.

Epigenetic remodeling by vitamin C potentiates plasma cell differentiation.

Ascorbate (vitamin C) is an essential micronutrient in humans. The severe chronic deficiency of ascorbate, termed scurvy, has long been associated with increased susceptibility to infections. How ascorbate affects the immune system at the cellular and molecular levels remained unclear. From a micronutrient analysis, we identified ascorbate as a potent enhancer for antibody response by facilitating the IL-21/STAT3-dependent plasma cell differentiation in mouse and human B cells. The effect of ascorbate is unique as other antioxidants failed to promote plasma cell differentiation. Ascorbate is especially critical during early B cell activation by poising the cells to plasma cell lineage without affecting the proximal IL-21/STAT3 signaling and the overall transcriptome. As a cofactor for epigenetic enzymes, ascorbate facilitates TET2/3-mediated DNA modification and demethylation of multiple elements at the Prdm1 locus. DNA demethylation augments STAT3 association at the Prdm1 promoter and a downstream enhancer, thus ensuring efficient gene expression and plasma cell differentiation. The results suggest that an adequate level of ascorbate is required for antibody response and highlight how micronutrients may regulate the activity of epigenetic enzymes to regulate gene expression. Our findings imply that epigenetic enzymes can function as sensors to gauge the availability of metabolites and influence cell fate decisions.

Tyk2/STAT3 signaling mediates beta-amyloid-induced neuronal cell death: implications in Alzheimer's disease.

One of the pathological hallmarks of Alzheimer's disease (AD) is deposition of extracellular amyloid-beta (Abeta) peptide, which is generated from the cleavage of amyloid precursor protein (APP). Accumulation of Abeta is thought to associate with the progressive neuronal death observed in AD. However, the precise signaling mechanisms underlying the action of Abeta in AD pathophysiology are not completely understood. Here, we report the involvement of the transcription factor signal transducer and activator of transcription 3 (STAT3) in mediating Abeta-induced neuronal death. We find that tyrosine phosphorylation of STAT3 is elevated in the cortex and hippocampus of APP/PS1 transgenic mice. Treatment of cultured rat neurons with Abeta or intrahippocampal injection of mice with Abeta both induces tyrosine phosphorylation of STAT3 in neurons. Importantly, reduction of either the expression or activation of STAT3 markedly attenuates Abeta-induced neuronal apoptosis, suggesting that STAT3 activation contributes to neuronal death after Abeta exposure. We further identify Tyk2 as the tyrosine kinase that acts upstream of STAT3, as Abeta-induced activation of STAT3 and caspase-3-dependent neuronal death can be inhibited in tyk2(-/-) neurons. Finally, increased tyrosine phosphorylation of STAT3 is also observed in postmortem brains of AD patients. Our observations collectively reveal a novel role of STAT3 in Abeta-induced neuronal death and suggest the potential involvement of Tyk2/STAT3 signaling in AD pathophysiology.