Rapid assay development for low input targeted proteomics using a versatile linear ion trap.

Advances in proteomics and mass spectrometry enable the study of limited cell populations, where high-mass accuracy instruments are typically required. While triple quadrupoles offer fast and sensitive low-mass accuracy measurements, these instruments are effectively restricted to targeted proteomics. Linear ion traps (LITs) offer a versatile, cost-effective alternative capable of both targeted and global proteomics. Here, we describe a workflow using a new hybrid quadrupole-LIT instrument that rapidly develops targeted proteomics assays from global data-independent acquisition (DIA) measurements without needing high-mass accuracy. Using an automated software approach for scheduling parallel reaction monitoring assays (PRM), we show consistent quantification across three orders of magnitude in a matched-matrix background. We demonstrate measuring low-level proteins such as transcription factors and cytokines with quantitative linearity below two orders of magnitude in a 1 ng background proteome without requiring stable isotope-labeled standards. From a 1 ng sample, we found clear consistency between proteins in subsets of CD4+ and CD8+ T cells measured using high dimensional flow cytometry and LIT-based proteomics. Based on these results, we believe hybrid quadrupole-LIT instruments represent an economical solution to democratizing mass spectrometry in a wide variety of laboratory settings.

Butein derivatives prevent obesity and improve insulin resistance through the induction of energy expenditure in high-fat diet-fed obese mice.

Obesity is a global public health problem and is related with fatal diseases such as cancer and cardiovascular and metabolic diseases. Medical and lifestyle-related strategies to combat obesity have their limitations. White adipose tissue (WAT) browning is a promising strategy for increasing energy expenditure in individuals with obesity. Uncoupling protein 1 (UCP1) drives WAT browning. We previously screened natural products that enable induction of Ucp1 and demonstrated that these natural products induced WAT browning and increased energy expenditure in mice with diet-induced obesity. In this study, we aimed to extensively optimise the structure of compound 1, previously shown to promote WAT browning. Compound 3 s exhibited a significantly higher ability to induce Ucp1 in white and brown adipocytes than did compound 1. A daily injection of compound 3 s at 5 mg/kg prevented weight gain by 13.6 % in high-fat diet-fed mice without any toxicological observation. In addition, compound 3 s significantly improved glucose homeostasis, decreased serum triacylglycerol levels, and reduced total cholesterol and LDL cholesterol levels, without altering dietary intake or physical activity. Pharmaceutical properties such as solubility, lipophilicity, and membrane permeability as well as metabolic stability, half-life (T1/2), and blood exposure ratio of i.p to i.v were significantly improved in compound 3 s when compared with those in compound 1. Regarding the mode of action of WAT browning, the induction of Ucp1 and Prdm4 by compounds 1 and 3 s was dependent on Akt1 in mouse embryonic fibroblasts. Therefore, this study suggests the potential of compound 3 s as a therapeutic agent for individuals with obesity and related metabolic diseases, which acts through the induction of WAT browning as well as brown adipose tissue activation.

Spatial and functional targeting of intratumoral Tregs reverses CD8+ T cell exhaustion and promotes cancer immunotherapy.

Intratumoral Tregs are key mediators of cancer immunotherapy resistance, including anti-programmed cell death (ligand) 1 [anti-PD-(L)1] immune checkpoint blockade (ICB). The mechanisms driving Treg infiltration into the tumor microenvironment (TME) and the consequence on CD8+ T cell exhaustion remain elusive. Here, we report that heat shock protein gp96 (also known as GRP94) was indispensable for Treg tumor infiltration, primarily through the roles of gp96 in chaperoning integrins. Among various gp96-dependent integrins, we found that only LFA-1 (αL integrin), and not αV, CD103 (αE), or ß7 integrin, was required for Treg tumor homing. Loss of Treg infiltration into the TME by genetic deletion of gp96/LFA-1 potently induced rejection of tumors in multiple ICB-resistant murine cancer models in a CD8+ T cell-dependent manner, without loss of self-tolerance. Moreover, gp96 deletion impeded Treg activation primarily by suppressing IL-2/STAT5 signaling, which also contributed to tumor regression. By competing for intratumoral IL-2, Tregs prevented the activation of CD8+ tumor-infiltrating lymphocytes, drove thymocyte selection-associated high mobility group box protein (TOX) induction, and induced bona fide CD8+ T cell exhaustion. By contrast, Treg ablation led to striking CD8+ T cell activation without TOX induction, demonstrating clear uncoupling of the 2 processes. Our study reveals that the gp96/LFA-1 axis plays a fundamental role in Treg biology and suggests that Treg-specific gp96/LFA-1 targeting represents a valuable strategy for cancer immunotherapy without inflicting autoinflammatory conditions.

Postinfusion PD-1+ CD8+ CAR T cells identify patients responsive to CD19 CAR T-cell therapy in non-Hodgkin lymphoma.

ABSTRACT: Chimeric antigen receptor (CAR) T-cell therapy has revolutionized treatment for relapsed/refractory B-cell non-Hodgkin lymphoma (NHL). Robust biomarkers and a complete understanding of CAR T-cell function in the postinfusion phase remain limited. Here, we used a 37-color spectral flow cytometry panel to perform high dimensional single-cell analysis of postinfusion samples in 26 patients treated with CD28 costimulatory domain containing commercial CAR T cells for NHL and focused on computationally gated CD8+ CAR T cells. We found that the presence of postinfusion Programmed cell death protein 1 (PD-1)+ CD8+ CAR T cells at the day 14 time point highly correlated with the ability to achieve complete response (CR) by 6 months. Further analysis identified multiple subtypes of CD8+ PD-1+ CAR T cells, including PD-1+ T cell factor 1 (TCF1)+ stem-like CAR T cells and PD-1+ T-cell immunoglobulin and mucin-domain containing-3 (TIM3)+ effector-like CAR T cells that correlated with improved clinical outcomes such as response and progression-free survival. Additionally, we identified a subset of PD-1+ CD8+ CAR+ T cells with effector-like function that was increased in patients who achieved a CR and was associated with grade 3 or higher immune effector cell-associated neurotoxicity syndrome. Here, we identified robust biomarkers of response to CD28 CAR T cells and highlight the importance of PD-1 positivity in CD8+ CAR T cells after infusion in achieving CR.

mRNA vaccines against SARS-CoV-2 induce divergent antigen-specific T-cell responses in patients with lung cancer.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant is highly transmissible and evades pre-established immunity. Messenger RNA (mRNA) vaccination against ancestral strain spike protein can induce intact T-cell immunity against the Omicron variant, but efficacy of booster vaccination in patients with late-stage lung cancer on immune-modulating agents including anti-programmed cell death protein 1(PD-1)/programmed death-ligand 1 (PD-L1) has not yet been elucidated. METHODS: We assessed T-cell responses using a modified activation-induced marker assay, coupled with high-dimension flow cytometry analyses. Peripheral blood mononuclear cells (PBMCs) were stimulated with various viral peptides and antigen-specific T-cell responses were evaluated using flow cytometry. RESULTS: Booster vaccines induced CD8+ T-cell response against the ancestral SARS-CoV-2 strain and Omicron variant in both non-cancer subjects and patients with lung cancer, but only a marginal induction was detected for CD4+ T cells. Importantly, antigen-specific T cells from patients with lung cancer showed distinct subpopulation dynamics with varying degrees of differentiation compared with non-cancer subjects, with evidence of dysfunction. Notably, female-biased T-cell responses were observed. CONCLUSION: We conclude that patients with lung cancer on immunotherapy show a substantial qualitative deviation from non-cancer subjects in their T-cell response to mRNA vaccines, highlighting the need for heightened protective measures for patients with cancer to minimize the risk of breakthrough infection with the Omicron and other future variants.

Selective suppression of de novo SARS-CoV-2 vaccine antibody responses in patients with cancer on B cell-targeted therapy.

We assessed vaccine-induced antibody responses to the SARS-CoV-2 ancestral virus and Omicron variant before and after booster immunization in 57 patients with B cell malignancies. Over one-third of vaccinated patients at the pre-booster time point were seronegative, and these patients were predominantly on active cancer therapies such as anti-CD20 monoclonal antibody. While booster immunization was able to induce detectable antibodies in a small fraction of seronegative patients, the overall booster benefit was disproportionately evident in patients already seropositive and not receiving active therapy. While ancestral virus- and Omicron variant-reactive antibody levels among individual patients were largely concordant, neutralizing antibodies against Omicron tended to be reduced. Interestingly, in all patients, including those unable to generate detectable antibodies against SARS-CoV-2 spike, we observed comparable levels of EBV- and influenza-reactive antibodies, demonstrating that B cell-targeting therapies primarily impair de novo but not preexisting antibody levels. These findings support rationale for vaccination before cancer treatment.

Selective targeting of GARP-LTGFß axis in the tumor microenvironment augments PD-1 blockade via enhancing CD8+ T cell antitumor immunity.

BACKGROUND: Immune checkpoint blockade (ICB) has revolutionized cancer immunotherapy. However, most patients with cancer fail to respond clinically. One potential reason is the accumulation of immunosuppressive transforming growth factor ß (TGFß) in the tumor microenvironment (TME). TGFß drives cancer immune evasion in part by inducing regulatory T cells (Tregs) and limiting CD8+ T cell function. Glycoprotein-A repetitions predominant (GARP) is a cell surface docking receptor for activating latent TGFß1, TGFß2 and TGFß3, with its expression restricted predominantly to effector Tregs, cancer cells, and platelets. METHODS: We investigated the role of GARP in human patients with cancer by analyzing existing large databases. In addition, we generated and humanized an anti-GARP monoclonal antibody and evaluated its antitumor efficacy and underlying mechanisms of action in murine models of cancer. RESULTS: We demonstrate that GARP overexpression in human cancers correlates with a tolerogenic TME and poor clinical response to ICB, suggesting GARP blockade may improve cancer immunotherapy. We report on a unique anti-human GARP antibody (named PIIO-1) that specifically binds the ligand-interacting domain of all latent TGFß isoforms. PIIO-1 lacks recognition of GARP-TGFß complex on platelets. Using human LRRC32 (encoding GARP) knock-in mice, we find that PIIO-1 does not cause thrombocytopenia; is preferentially distributed in the TME; and exhibits therapeutic efficacy against GARP+ and GARP- cancers, alone or in combination with anti-PD-1 antibody. Mechanistically, PIIO-1 treatment reduces canonical TGFß signaling in tumor-infiltrating immune cells, prevents T cell exhaustion, and enhances CD8+ T cell migration into the TME in a C-X-C motif chemokine receptor 3 (CXCR3)-dependent manner. CONCLUSION: GARP contributes to multiple aspects of immune resistance in cancer. Anti-human GARP antibody PIIO-1 is an efficacious and safe strategy to block GARP-mediated LTGFß activation, enhance CD8+ T cell trafficking and functionality in the tumor, and overcome primary resistance to anti-PD-1 ICB. PIIO-1 therefore warrants clinical development as a novel cancer immunotherapeutic.

Genetic association-based functional analysis detects HOGA1 as a potential gene involved in fat accumulation.

Although there are a number of discoveries from genome-wide association studies (GWAS) for obesity, it has not been successful in linking GWAS results to biology. We sought to discover causal genes for obesity by conducting functional studies on genes detected from genetic association analysis. Gene-based association analysis of 917 individual exome sequences showed that HOGA1 attains exome-wide significance (p-value < 2.7 × 10-6) for body mass index (BMI). The mRNA expression of HOGA1 is significantly increased in human adipose tissues from obese individuals in the Genotype-Tissue Expression (GTEx) dataset, which supports the genetic association of HOGA1 with BMI. Functional analyses employing cell- and animal model-based approaches were performed to gain insights into the functional relevance of Hoga1 in obesity. Adipogenesis was retarded when Hoga1 was knocked down by siRNA treatment in a mouse 3T3-L1 cell line and a similar inhibitory effect was confirmed in mice with down-regulated Hoga1. Hoga1 antisense oligonucleotide (ASO) treatment reduced body weight, blood lipid level, blood glucose, and adipocyte size in high-fat diet-induced mice. In addition, several lipogenic genes including Srebf1, Scd1, Lp1, and Acaca were down-regulated, while lipolytic genes Cpt1l, Ppara, and Ucp1 were up-regulated. Taken together, HOGA1 is a potential causal gene for obesity as it plays a role in excess body fat development.

Androgen conspires with the CD8+ T cell exhaustion program and contributes to sex bias in cancer.

Sex bias exists in the development and progression of nonreproductive organ cancers, but the underlying mechanisms are enigmatic. Studies so far have focused largely on sexual dimorphisms in cancer biology and socioeconomic factors. Here, we establish a role for CD8+ T cell-dependent antitumor immunity in mediating sex differences in tumor aggressiveness, which is driven by the gonadal androgen but not sex chromosomes. A male bias exists in the frequency of intratumoral antigen-experienced Tcf7/TCF1+ progenitor exhausted CD8+ T cells that are devoid of effector activity as a consequence of intrinsic androgen receptor (AR) function. Mechanistically, we identify a novel sex-specific regulon in progenitor exhausted CD8+ T cells and a pertinent contribution from AR as a direct transcriptional transactivator of Tcf7/TCF1. The T cell-intrinsic function of AR in promoting CD8+ T cell exhaustion in vivo was established using multiple approaches including loss-of-function studies with CD8-specific Ar knockout mice. Moreover, ablation of the androgen-AR axis rewires the tumor microenvironment to favor effector T cell differentiation and potentiates the efficacy of anti-PD-1 immune checkpoint blockade. Collectively, our findings highlight androgen-mediated promotion of CD8+ T cell dysfunction in cancer and imply broader opportunities for therapeutic development from understanding sex disparities in health and disease.

Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity.

The leptin receptor (LepR) acts as a signaling nexus for the regulation of glucose uptake and obesity, among other metabolic responses. The functional role of LepR under leptin-deficient conditions remains unclear. This study reports that epiregulin (EREG) governed glucose uptake in vitro and in vivo in Lepob mice by activating LepR under leptin-deficient conditions. Single and long-term treatment with EREG effectively rescued glucose intolerance in comparative insulin and EREG tolerance tests in Lepob mice. The immunoprecipitation study revealed binding between EREG and LepR in adipose tissue of Lepob mice. EREG/LepR regulated glucose uptake without changes in obesity in Lepob mice via mechanisms, including ERK activation and translocation of GLUT4 to the cell surface. EREG-dependent glucose uptake was abolished in Leprdb mice which supports a key role of LepR in this process. In contrast, inhibition of the canonical epidermal growth factor receptor (EGFR) pathway implicated in other EREG responses, increased glucose uptake. Our data provide a basis for understanding glycemic responses of EREG that are dependent on LepR unlike functions mediated by EGFR, including leptin secretion, thermogenesis, pain, growth, and other responses. The computational analysis identified a conserved amino acid sequence, supporting an evolutionary role of EREG as an alternative LepR ligand.

Treatment with soluble CD24 attenuates COVID-19-associated systemic immunopathology.

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) through direct lysis of infected lung epithelial cells, which releases damage-associated molecular patterns and induces a pro-inflammatory cytokine milieu causing systemic inflammation. Anti-viral and anti-inflammatory agents have shown limited therapeutic efficacy. Soluble CD24 (CD24Fc) blunts the broad inflammatory response induced by damage-associated molecular patterns via binding to extracellular high mobility group box 1 and heat shock proteins, as well as regulating the downstream Siglec10-Src homology 2 domain-containing phosphatase 1 pathway. A recent randomized phase III trial evaluating CD24Fc for patients with severe COVID-19 (SAC-COVID; NCT04317040) demonstrated encouraging clinical efficacy. METHODS: Using a systems analytical approach, we studied peripheral blood samples obtained from patients enrolled at a single institution in the SAC-COVID trial to discern the impact of CD24Fc treatment on immune homeostasis. We performed high dimensional spectral flow cytometry and measured the levels of a broad array of cytokines and chemokines to discern the impact of CD24Fc treatment on immune homeostasis in patients with COVID-19. RESULTS: Twenty-two patients were enrolled, and the clinical characteristics from the CD24Fc vs. placebo groups were matched. Using high-content spectral flow cytometry and network-level analysis, we found that patients with severe COVID-19 had systemic hyper-activation of multiple cellular compartments, including CD8+ T cells, CD4+ T cells, and CD56+ natural killer cells. Treatment with CD24Fc blunted this systemic inflammation, inducing a return to homeostasis in NK and T cells without compromising the anti-Spike protein antibody response. CD24Fc significantly attenuated the systemic cytokine response and diminished the cytokine coexpression and network connectivity linked with COVID-19 severity and pathogenesis. CONCLUSIONS: Our data demonstrate that CD24Fc rapidly down-modulates systemic inflammation and restores immune homeostasis in SARS-CoV-2-infected individuals, supporting further development of CD24Fc as a novel therapeutic against severe COVID-19.

Butein-Enriched Fractions of Butea monosperma (Lam.) Taub. Flower Decrease Weight Gains and Increase Energy Expenditure in High-Fat Diet-Induced Obese Mice.

Butea monosperma (Lam.) Taub. has been applied to treat inflammatory, metabolic, and infectious diseases. However, the antiobesity effects of B. monosperma (Lam.) Taub. flower (BMF) and the underlying mechanisms have not been determined. In this study, we analyzed the various extraction procedures, investigated the antiobesity effects, and identified the main chemical constituents of BMF. The BMF was subjected to acid hydrolysis in 5% H2SO4 in methanol at 50°C for 48 h and partitioned with ethyl acetate. The acid-hydrolyzed BMF ethyl acetate extracts (BMFE) strongly induced the expression of uncoupling protein 1 (Ucp1) and other thermogenic genes in C3H10T1/2 adipocytes. Daily oral administration of 70 mg/kg BMFE (BMFE70) to mice with diet-induced obesity resulted in less body weight gain, increased glucose tolerance, higher rectal temperature, and increased oxygen consumption. Qualitative and quantitative analyses along with treatments in Akt1 knockout mouse embryonic fibroblasts indicate that butein is a major active ingredient of BMFE, which stimulates Ucp1 gene expression. These data show the effects of butein-containing B. monosperma flower extract on thermogenesis and energy expenditure, further suggesting the potential role of BMFE as a functional ingredient in obesity and related metabolic diseases.

TREATMENT WITH SOLUBLE CD24 ATTENUATES COVID-19-ASSOCIATED SYSTEMIC IMMUNOPATHOLOGY.

BACKGROUND: SARS-CoV-2 causes COVID-19 through direct lysis of infected lung epithelial cells, which releases damage-associated molecular patterns (DAMPs) and induces a pro-inflammatory cytokine milieu causing systemic inflammation. Anti-viral and anti-inflammatory agents have shown limited therapeutic efficacy. Soluble CD24 (CD24Fc) is able to blunt the broad inflammatory response induced by DAMPs in multiple models. A recent randomized phase III trial evaluating the impact of CD24Fc in patients with severe COVID-19 demonstrated encouraging clinical efficacy. METHODS: We studied peripheral blood samples obtained from patients enrolled at a single institution in the SAC-COVID trial (NCT04317040) collected before and after treatment with CD24Fc or placebo. We performed high dimensional spectral flow cytometry analysis of peripheral blood mononuclear cells and measured the levels of a broad array of cytokines and chemokines. A systems analytical approach was used to discern the impact of CD24Fc treatment on immune homeostasis in patients with COVID-19. FINDINGS: Twenty-two patients were enrolled, and the clinical characteristics from the CD24Fc vs. placebo groups were matched. Using high-content spectral flow cytometry and network-level analysis, we found systemic hyper-activation of multiple cellular compartments in the placebo group, including CD8+ T cells, CD4+ T cells, and CD56+ NK cells. By contrast, CD24Fc-treated patients demonstrated blunted systemic inflammation, with a return to homeostasis in both NK and T cells within days without compromising the ability of patients to mount an effective anti-Spike protein antibody response. A single dose of CD24Fc significantly attenuated induction of the systemic cytokine response, including expression of IL-10 and IL-15, and diminished the coexpression and network connectivity among extensive circulating inflammatory cytokines, the parameters associated with COVID-19 disease severity. INTERPRETATION: Our data demonstrates that CD24Fc treatment rapidly down-modulates systemic inflammation and restores immune homeostasis in SARS-CoV-2-infected individuals, supporting further development of CD24Fc as a novel therapeutic against severe COVID-19. FUNDING: NIH.

Sesamol Increases Ucp1 Expression in White Adipose Tissues and Stimulates Energy Expenditure in High-Fat Diet-Fed Obese Mice.

Sesamol found in sesame oil has been shown to ameliorate obesity by regulating lipid metabolism. However, its effects on energy expenditure and the underlying molecular mechanism have not been clearly elucidated. In this study, we show that sesamol increased the uncoupling protein 1 (Ucp1) expression in adipocytes. The administration of sesamol in high-fat diet (HFD)-fed mice prevented weight gain and improved metabolic derangements. The three-week sesamol treatment of HFD-fed mice, when the body weights were not different between the sesamol and control groups, increased energy expenditure, suggesting that an induced energy expenditure is a primary contributing factor for sesamol's anti-obese effects. Consistently, sesamol induced the expression of energy-dissipating thermogenic genes, including Ucp1, in white adipose tissues. The microarray analysis showed that sesamol dramatically increased the Nrf2 target genes such as Hmox1 and Atf3 in adipocytes. Moreover, 76% (60/79 genes) of the sesamol-induced genes were also regulated by tert-butylhydroquinone (tBHQ), a known Nrf2 activator. We further verified that sesamol directly activated the Nrf2-mediated transcription. In addition, the Hmox1 and Ucp1 induction by sesamol was compromised in Nrf2-deleted cells, indicating the necessity of Nrf2 in the sesamol-mediated Ucp1 induction. Together, these findings demonstrate the effects of sesamol in inducing Ucp1 and in increasing energy expenditure, further highlighting the use of the Nrf2 activation in stimulating thermogenic adipocytes and in increasing energy expenditure in obesity and its related metabolic diseases.

Amino acid-based compound activates atypical PKC and leptin receptor pathways to improve glycemia and anxiety like behavior in diabetic mice.

Differences in glucose uptake in peripheral and neural tissues account for the reduced efficacy of insulin in nervous tissues. Herein, we report the design of short peptides, referred as amino acid compounds (AAC) with and without a modified side chain moiety. At nanomolar concentrations, a candidate therapeutic molecule, AAC2, containing a 7-(diethylamino) coumarin-3-carboxamide side-chain improved glucose control in human peripheral adipocytes and the endothelial brain barrier cells by activation of insulin-insensitive glucose transporter 1 (GLUT1). AAC2 interacted specifically with the leptin receptor (LepR) and activated atypical protein kinase C zeta (PKCς) to increase glucose uptake. The effects induced by AAC2 were absent in leptin receptor-deficient predipocytes and in Leprdb mice. In contrast, AAC2 established glycemic control altering food intake in leptin-deficient Lepob mice. Therefore, AAC2 activated the LepR and acted in a cytokine-like manner distinct from leptin. In a monogenic Ins2Akita mouse model for the phenotypes associated with type 1 diabetes, AAC2 rescued systemic glucose uptake in these mice without an increase in insulin levels and adiposity, as seen in insulin-treated Ins2Akita mice. In contrast to insulin, AAC2 treatment increased brain mass and reduced anxiety-related behavior in Ins2Akita mice. Our data suggests that the unique mechanism of action for AAC2, activating LepR/PKCς/GLUT1 axis, offers an effective strategy to broaden glycemic control for the prevention of diabetic complications of the nervous system and, possibly, other insulin insensitive or resistant tissues.

Development of molecular and pharmacological switches for chimeric antigen receptor T cells.

The use of chimeric antigen receptor (CAR) T cell technology as a therapeutic strategy for the treatment blood-born human cancers has delivered outstanding clinical efficacy. However, this treatment modality can also be associated with serious adverse events in the form of cytokine release syndrome. While several avenues are being pursued to limit the off-target effects, it is critically important that any intervention strategy has minimal consequences on long term efficacy. A recent study published in Science Translational Medicine by Dr. Hudecek's group proved that dasatinib, a tyrosine kinase inhibitor, can serve as an on/off switch for CD19-CAR-T cells in preclinical models by limiting toxicities while maintaining therapeutic efficacy. In this editorial, we discuss the recent strategies for generating safer CAR-T cells, and also important questions surrounding the use of dasatinib for emergency intervention of CAR-T cell mediated cytokine release syndrome.

Thermogenic crosstalk occurs between adipocytes from different species.

Visceral obesity increases risks for all-cause mortality worldwide. A small population of thermogenic adipocytes expressing uncoupling protein-1 (Ucp1) regulates energy dissipation in white adipose tissue (WAT) depots. Thermogenic adipocytes subsets decrease obesity in mice, but their efficacy has not been tested in obese large animals. Here we enclosed murine subcutaneous adipocytes with and without engineered thermogenic response in biocompatible microcapsules and implanted them into the left and right side of the visceral falciform depot in six obese dogs. After 28 days of treatment, dogs have markedly reduced waist circumference, body weight, and fat mass. Ucp1 expression in canine WAT was increased at sites implanted with thermogenic vs. wild type murine adipocytes. This site-specific thermogenic remodeling of canine tissue by thermogenic murine adipocytes suggests evolutionary conserved paracrine regulation of energy dissipation across species. These findings have translational potential aimed to reduce deleterious WAT depots in humans and pets.

The Emerging Roles of Endoplasmic Reticulum Stress in Balancing Immunity and Tolerance in Health and Diseases: Mechanisms and Opportunities.

The endoplasmic reticulum (ER) is an organelle equipped with mechanisms for proper protein folding, trafficking, and degradation to maintain protein homeostasis in the secretory pathway. As a defense mechanism, perturbation of ER proteostasis by ER stress agents activates a cascade of signaling pathways from the ER to the nucleus known as unfolded protein response (UPR). The primary goal of UPR is to induce transcriptional and translational programs to restore ER homeostasis for cell survival. As such, defects in UPR signaling have been implicated as a key contributor to multiple diseases including metabolic diseases, degenerative diseases, inflammatory disorders, and cancer. Growing evidence support the critical role of ER stress in regulating the fate as well as the magnitude of the immune response. Moreover, the availability of multiple UPR pharmacological inhibitors raises the hope that targeting UPR can be a new strategy for immune modulation and immunotherapy of diseases. This paper reviews the principal mechanisms by which ER stress affects immune cell biology and function, with a focus of discussion on UPR-associated immunopathology and the development of potential ER stress-targeted therapeutics.

Mechanisms underlying UCP1 dependent and independent adipocyte thermogenesis.

The growing focus on brown adipocytes has spurred an interest in their potential benefits for metabolic diseases. Brown and beige (or brite) adipocytes express high levels of uncoupling protein 1 (Ucp1) to dissipate heat instead of generating ATP. Ucp1 induction by stimuli including cold, exercise, and diet increases nonshivering thermogenesis, leading to increased energy expenditure and prevention of obesity. Recently, studies in adipocytes have indicated the existence of functional Ucp1-independent thermogenic regulators. Furthermore, substrate cycling involving creatine metabolites, cold-induced N-acyl amino acids, and oxidized lipids in white adipocytes can increase energy expenditure in the absence of Ucp1. These studies emphasize the need for a better understanding of the mechanisms governing energy expenditure in adipocytes and their potential applications in the prevention of human obesity and metabolic diseases.