Systemic lupus erythematosus (SLE) is prototypical autoimmune disease driven by pathological T cell-B cell interactions1,2. Expansion of T follicular helper (TFH) and T peripheral helper (TPH) cells, two T cell populations that provide help to B cells, is a prominent feature of SLE3,4. Human TFH and TPH cells characteristically produce high levels of the B cell chemoattractant CXCL13 (refs. 5,6), yet regulation of T cell CXCL13 production and the relationship between CXCL13+ T cells and other T cell states remains unclear. Here, we identify an imbalance in CD4+ T cell phenotypes in patients with SLE, with expansion of PD-1+/ICOS+ CXCL13+ T cells and reduction of CD96hi IL-22+ T cells. Using CRISPR screens, we identify the aryl hydrocarbon receptor (AHR) as a potent negative regulator of CXCL13 production by human CD4+ T cells. Transcriptomic, epigenetic and functional studies demonstrate that AHR coordinates with AP-1 family member JUN to prevent CXCL13+ TPH/TFH cell differentiation and promote an IL-22+ phenotype. Type I interferon, a pathogenic driver of SLE7, opposes AHR and JUN to promote T cell production of CXCL13. These results place CXCL13+ TPH/TFH cells on a polarization axis opposite from T helper 22 (TH22) cells and reveal AHR, JUN and interferon as key regulators of these divergent T cell states.

Disruptions in the gut epithelial barrier can lead to the development of chronic indications such as inflammatory bowel disease (IBD). Historically, barrier function has been assessed in cancer cell lines, which do not contain all human intestinal cell types, leading to poor translatability. To bridge this gap, we adapted human primary gut organoids grown as monolayers to quantify transcription factor phosphorylation, gene expression, cytokine production, and barrier function. In this work we describe and characterize a novel 96-well human gut organoid-derived monolayer system that enables quantitative assessment of candidate therapeutics. Normal human intestine differentiation patterns and barrier function were characterized and confirmed to recapitulate key aspects of in vivo biology. Next, cellular response to TNF-α (a central driver of IBD) was determined using a diverse cadre of quantitative readouts. We showed that TNF-α pathway antagonists rescued damage caused by TNF-α in a dose-dependent manner, indicating that this system is suitable for quantitative assessment of barrier modulating factors. Taken together, we have established a robust primary cell-based 96-well system capable of interrogating questions around mucosal response. This system is well suited to provide pivotal functional data to support translational target and drug discovery efforts.

ObjectivesTo investigate the immune cell profiles of patients with systemic lupus erythematosus (SLE), and to identify longitudinal changes in those profiles over time.MethodsWe employed mass cytometry with 3 different panels of 38–39 markers (an immunophenotyping panel, a T cell/monocyte panel, and a B cell panel) in cryopreserved peripheral blood mononuclear cells (PBMCs) from 9 patients with early SLE, 15 patients with established SLE, and 14 controls without autoimmune disease. We used machine learning–driven clustering, flow self‐organizing maps, and dimensional reduction with t‐distributed stochastic neighbor embedding to identify unique cell populations in early SLE and established SLE. We used mass cytometry data of PBMCs from 19 patients with early rheumatoid arthritis (RA) and 23 controls to compare levels of specific cell populations in early RA and SLE. For the 9 patients with early SLE, longitudinal mass cytometry analysis was applied to PBMCs at enrollment, 6 months after enrollment, and 1 year after enrollment. Serum samples were also assayed for 65 cytokines using Luminex multiplex assay, and associations between cell types and cytokines/chemokines were assessed.ResultsLevels of peripheral helper T cells, follicular helper T (Tfh) cells, and several Ki‐67+ proliferating subsets (ICOS+Ki‐67+ CD8 T cells, Ki‐67+ regulatory T cells, CD19intermediateKi‐67high plasmablasts, and PU.1highKi‐67high monocytes) were increased in patients with early SLE, with more prominent alterations than were seen in patients with early RA. Longitudinal mass cytometry and multiplex serum cytokine assays of samples from patients with early SLE revealed that levels of Tfh cells and CXCL10 had decreased 1 year after enrollment. Levels of CXCL13 were positively correlated with levels of several of the expanded cell populations in early SLE.ConclusionTwo major helper T cell subsets and unique Ki‐67+ proliferating immune cell subsets were expanded in patients in the early phase of SLE, and the immunologic features characteristic of early SLE evolved over time.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathologic T cell-B cell interactions and autoantibody production. Defining the T cell populations that drive B cell responses in SLE may enable design of therapies that specifically target pathologic cell subsets. Here, we evaluated the phenotypes of CD4+ T cells in the circulation of 52 SLE patients drawn from multiple cohorts and identified a highly expanded PD-1hiCXCR5-CD4+ T cell population. Cytometric, transcriptomic, and functional assays demonstrated that PD-1hiCXCR5-CD4+ T cells from SLE patients are T peripheral helper (Tph) cells, a CXCR5- T cell population that stimulates B cell responses via IL-21. The frequency of Tph cells, but not T follicular helper (Tfh) cells, correlated with both clinical disease activity and the frequency of CD11c+ B cells in SLE patients. PD-1hiCD4+ T cells were found within lupus nephritis kidneys and correlated with B cell numbers in the kidney. Both IL-21 neutralization and CRISPR-mediated deletion of MAF abrogated the ability of Tph cells to induce memory B cell differentiation into plasmablasts in vitro. These findings identify Tph cells as a highly expanded T cell population in SLE and suggest a key role for Tph cells in stimulating pathologic B cell responses.

The androgen receptor (AR) is a member of the nuclear hormone receptor super family of transcription factors. Androgens play an essential role in the development, growth, and maintenance of male sex organs, as well as the musculoskeletal and central nervous systems. Yet with advancing age, androgens can drive the onset of prostate cancer, the second leading cause of cancer death in males within the United States. Androgen deprivation therapy (ADT) by pharmacologic and/or surgical castration induces apoptosis of prostate cells and subsequent shrinkage of the prostate and prostate tumors. However, ADT is associated with significant musculoskeletal and behavioral adverse effects. The unique pharmacological activity of selective androgen receptor modulator (SARM) MK-4541 recently has been reported as an AR antagonist with 5α-reductase inhibitor function. The molecule inhibits proliferation and induces apoptosis in AR positive, androgen dependent prostate cancer cells. Importantly, MK-4541 inhibited androgen-dependent prostate growth in male rats yet maintained lean body mass and bone formation following ovariectomy in female rats. In the present study, we evaluated the effects of SARM MK-4541 in the androgen-dependent Dunning R3327-G prostate carcinoma xenograft mouse model as well as on skeletal muscle mass and function, and AR-regulated behavior in mice. MK-4541 significantly inhibited the growth of R3327-G prostate tumors, exhibited anti-androgen effects on the seminal vesicles, reduced plasma testosterone concentrations in intact males, and inhibited Ki67 expression. MK-4541 treated xenografts appeared similar to xenografts in castrated mice. Importantly, we demonstrate that MK-4541 exhibited anabolic activity in androgen deficient conditions, increasing lean body mass and muscle function in adult castrated mice. Moreover, MK-4541 treatment restored general activity levels in castrated mice. Thus, MK-4541 exhibits an optimum profile as an adjuvant therapy to ADT which may provide potent anti-androgenic activity at the prostate yet protective activity on skeletal muscle and behavior in patients.

Here we describe the development of a human lung 'small airway-on-a-chip' containing a differentiated, mucociliary bronchiolar epithelium and an underlying microvascular endothelium that experiences fluid flow, which allows for analysis of organ-level lung pathophysiology in vitro. Exposure of the epithelium to interleukin-13 (IL-13) reconstituted the goblet cell hyperplasia, cytokine hypersecretion and decreased ciliary function of asthmatics. Small airway chips lined with epithelial cells from individuals with chronic obstructive pulmonary disease recapitulated features of the disease such as selective cytokine hypersecretion, increased neutrophil recruitment and clinical exacerbation by exposure to viral and bacterial infections. With this robust in vitro method for modeling human lung inflammatory disorders, it is possible to detect synergistic effects of lung endothelium and epithelium on cytokine secretion, identify new biomarkers of disease exacerbation and measure responses to anti-inflammatory compounds that inhibit cytokine-induced recruitment of circulating neutrophils under flow.

Prostate cancer (PCa) initially responds to inhibition of androgen receptor (AR) signaling, but inevitably progresses to hormone ablation-resistant disease. Much effort is focused on optimizing this androgen deprivation strategy by improving hormone depletion and AR antagonism. However we found that bicalutamide, a clinically used antiandrogen, actually resembles a selective AR modulator (SARM), as it partially regulates 24% of endogenously 5 alpha-dihydrotestosterone (DHT)-responsive genes in AR(+) MDA-MB-453 breast cancer cells. These data suggested that passive blocking of all AR functions is not required for PCa therapy. Hence, we adopted an active strategy that calls for the development of novel SARMs, which induce a unique gene expression profile that is intolerable to PCa cells. Therefore, we screened 3000 SARMs for the ability to arrest the androgen-independent growth of AR(+) 22Rv1 and LNCaP PCa cells but not AR(-) PC3 or DU145 cells. We identified only one such compound; the 4-aza-steroid, MK-4541, a potent and selective SARM. MK-4541 induces caspase-3 activity and cell death in both androgen-independent, AR(+) PCa cell lines but spares AR(-) cells or AR(+) non-PCa cells. This activity correlates with its promoter context- and cell-type dependent transcriptional effects. In rats, MK-4541 inhibits the trophic effects of DHT on the prostate, but not the levator ani muscle, and triggers an anabolic response in the periosteal compartment of bone. Therefore, MK-4541 has the potential to effectively manage prostatic hypertrophic diseases owing to its antitumor SARM-like mechanism, while simultaneously maintaining the anabolic benefits of natural androgens. (C) 2014 Elsevier Ltd. All rights reserved.

Myostatin is a highly conserved member of the transforming growth factor-β ligand family known to regulate muscle growth via activation of activin receptors. A fusion protein consisting of the extracellular ligand-binding domain of activin type IIB receptor with the Fc portion of human immunoglobulin G (ActRIIB-Fc) was used to inhibit signaling through this pathway. Here, we study the effects of this fusion protein in adult, 18-month-old, and orchidectomized mice. Significant muscle growth and enhanced muscle function were observed in adult mice treated for 3 days with ActRIIB-Fc. The ActRIIB-Fc-treated mice had enhanced fast fatigable muscle function, with only minor enhancement of fatigue-resistant fiber function. The ActRIIB-Fc-treated 18-month-old mice and orchidectomized mice showed significantly improved muscle function. Treatment with ActRIIB-Fc also increased bone mineral density and serum levels of a marker of bone formation. These observations highlight the potential of targeting ActRIIB receptor to treat age-related and hypogonadism-associated musculoskeletal degeneration.

Estrogen Receptor α (ERα) and Estrogen Receptor β (ERβ) are steroid nuclear receptors that transduce estrogen signaling to control diverse physiological processes linked to reproduction, bone remodeling, behavior, immune response and endocrine-related diseases. In order to differentiate between ERα and ERβ mediated effects in vivo, ER subtype selective biomarkers are essential. We utilized ERα knockout (AERKO) and ERβ knockout (BERKO) mouse liver RNA and genome wide profiling to identify novel ERα selective serum biomarker candidates. Results from the gene array experiments were validated using real-time RT-PCR and subsequent ELISA's to demonstrate changes in serum proteins. Here we present data that Lipopolysacharide Binding Protein (LBP) is a novel liver-derived ERα selective biomarker that can be measured in serum.

BACKGROUND:Age-related sarcopenia is a disease state of loss of muscle mass and strength that affects physical function and mobility leading to falls, fractures, and disability. The need for therapies to treat age-related sarcopenia has attracted intensive preclinical research. To facilitate the discovery of these therapies, we have developed a non-invasive rat muscle functional assay system to efficiently measure muscle force and evaluate the efficacy of drug candidates.METHODS:The lower leg muscles of anesthetized rats are artificially stimulated with surface electrodes on the knee holders and the heel support, causing the lower leg muscles to push isometric pedals that are attached to force transducers. We developed a stimulation protocol to perform a fatigability test that reveals functional muscle parameters like maximal force, the rate of fatigue, fatigue-resistant force, as well as a fatigable muscle force index. The system is evaluated in a rat aging model and a rat glucocorticoid-induced muscle loss modelRESULTS:The aged rats were generally weaker than adult rats and showed a greater reduction in their fatigable force when compared to their fatigue-resistant force. Glucocorticoid treated rats mostly lost fatigable force and fatigued at a higher rate, indicating reduced force from glycolytic fibers with reduced energy reserves.CONCLUSIONS:The involuntary contraction assay is a reliable system to assess muscle function in rodents and can be applied in preclinical research, including age-related sarcopenia and other myopathy.

Effects of estradiol benzoate (EB) ER alpha-selective agonist propyl pyrazole triol (PPT) and ER beta-selective agonists diarylpropionitrile (DPN) and Compound 19 (C-19) on memory were investigated in OVX rats using object recognition (OR) and placement (OP) memory tasks Treatments were acute (behavior 4 h later) or sub chronic (daily injections for 2 days with behavior 48 h later) Objects were explored in sample trials (T1) and discrimination between sample (old) and new object/location in recognition trials (T2) was examined after 2-4 h inter-trial delays Subjects treated sub chronically with EB DPN and C-19 but not PPT discriminated between old and new objects and objects in old and new locations suggesting that at these doses and duration of treatments estrogenic interactions with ER beta contribute to enhancements in recognition memory Acute injections of DPN but not PPT immediately after T1 also enhanced discrimination for both tasks (C19 was not investigated) Effects of EB DPN and PPT on anxiety and locomotion measured on elevated plus maze and open field did not appear to account for the mnemonic enhancements Monoamines and metabolites were measured following DPN treatment in subjects that did not receive behavioral testing DPN was associated with alterations in monoamines in several brain areas indexed by the metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG) or the MHPG/norepinephrine (NE) ratio NE activity was increased by 60-130% in the prefrontal cortex (PFC) and ventral hippocampus and NE activity was decreased by 40-80% in the v diagonal bands and CA1 Levels of the dopamine (DA) metabolite homovanillic acid (HVA) Increased 100% in the PFC and decreased by 50% in the dentate gyrus following DPN treatment The metabolite of serotonin 5-hydroxyindole acetic acid (5-HIAA) was Increased in the PFC and CA3 by approximately 20% No monoaminergic changes were noted in striatum or medial septum Results suggest that ER beta mediates sub chronic and acute effects of estrogens on recognition memory and that memory enhancements by DPN may occur in part through alterations in monoaminergic containing systems primarily in PFC and hippocampus (C) 2010 Elsevier Inc All rights reserved

We assessed the effects of subtype-selective ER agonists on monoamine levels in discrete regions of the female rat brain. Ovariectomized (ovx) rats were treated for 4 days with vehicle, 17β-estradiol (E; 0.05 mg/kg), an ERβ agonist (C19; 3 mg/kg) or an ERα agonist (PPT; 3 mg/kg) and samples from brain regions were assessed for monoamines and metabolites. We also assessed effects of ERβ modulation on baseline and fenfluramine-induced release of monoamines in hippocampus using microdialysis. In the first study, E and the ERα agonist increased norepinephrine in cortex and all three ER ligands increased it in the ventral hippocampus. Changes in levels of the noradrenergic metabolite, MHPG and the dopaminergic metabolite, DOPAC were noted in brain areas of ER ligand-treated animals. E also increased levels of 5HIAA in three brain areas. In the microdialysis study, there were no differences among groups in baseline levels of monoamines. However, E and the ERβ agonist increased levels of the dopaminergic metabolite, HVA following fenfluramine. In summary, activation of the two nuclear ERs with selective agonists affects monoamine and metabolite levels in discrete brain areas, a number of which are known to play key roles in cognitive and affective function.

Immunocytochemical studies have shown that nuclear and extranuclear estrogen receptors (ERs) are present in several extrahypothalamic brain regions. The goal of this study was to determine the subcellular location of functional ERs, particularly extranuclear ERs, by demonstrating (125)I-estradiol binding in the rat forebrain and medullary sections prepared for light and electron microscopic autoradiography. Some sections were immunocytochemically labeled with the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), prior to the autoradiographic procedure. By light microscopy, dense accumulations of silver grains denoting (125)I-estradiol binding were observed over cells in the ventromedial and arcuate hypothalamic nuclei, amygdala, and nucleus of the solitary tract. In sections labeled for TH, large accumulations of silver grains were admixed with TH-labeled processes in the medial nucleus of the amygdala and over TH-labeled perikarya in the medial and commissural nucleus of the solitary tract. Electron microscopic analyses were focused on the rostral ventrolateral medulla and the hippocampal CA1 region, two regions previously shown to have extranuclear ERs. In the rostral ventrolateral medulla, silver grains indicative of (125)I-estradiol binding were found within a few large terminals, affiliated with mitochondria. In the hippocampus, autoradiographic silver grains denoting (125)I-estradiol binding were associated with mitochondria in dendritic shafts or were near synaptic specializations on dendritic spines. These patterns of silver grain labeling were not seen in sections from rats that received (125)I-estradiol combined with cold estradiol. The association of (125)I-estradiol binding with pre- and postsynaptic profiles supports a functional role for nonnuclear ERs in brain.

Blood pressure in women increases after menopause, and sympathetic tone in female rats decreases with estrogen injections in the rostral ventrolateral medulla (RVLM) region that contains bulbospinal C1 adrenergic neurons and is involved in blood pressure control. We investigated the anatomical and physiological basis for estrogen effects in the RVLM. Neurons with α- or β-subtypes of estrogen receptor (ER) immunoreactivity (-ir) overlapped in distribution with tyrosine hydroxylase (TH)-containing C1 neurons. Immunoelectron microscopy revealed that ERα- and ERβ-ir had distinct cellular and subcellular distributions. ERα-ir was most commonly in TH-lacking profiles, many of which were axons and peptide-containing afferents that contacted TH-containing dendrites. ERα-ir was also in some TH-containing dendrites. ERβ-ir was most frequently in TH-containing somata and dendrites, particularly on endoplasmic reticula, mitochondria, and plasma membranes. In whole-cell patch clamp recordings from isolated bulbospinal RVLM neurons, 17β-estradiol dose-dependently reduced voltage-gated Ca++ currents, especially the long-lasting (L-type) component. This inhibition was reversed by washing or prevented by adding the non-subtype-selective ER antagonist ICI182780. An ERβ-selective agonist, but not an ERα-selective agonist, reproduced the Ca++ current inhibition. The data indicate that estrogens can modulate the function of RVLM C1 bulbospinal neurons either directly, through extranuclear ERβ, or indirectly through extranuclear ERα in selected afferents. Moreover, Ca++ current inhibition may underlie the decrease in sympathetic tone evoked by local 17β-estradiol application. These findings provide a structural and functional basis for the effects of estrogens on blood pressure control and suggest a mechanism for the modulation of cardiovascular function by estrogen in women.