Quantifying NF-κB Activation by Flow Cytometry of IκBα Degradation.

Nuclear factor-κB (NF-κB) is a crucial pro-inflammatory transcription factor whose activation is of immense interest to immunology research. Estimation of NF-κB activation through flow cytometry is not possible due to the unavailability of robust flow cytometry antibodies that can bind to its phosphorylated, active, nuclear form. In this protocol, we describe a flow cytometry assay that measures the activation of the pro-inflammatory transcription factor NF-κB in stimulated immune cells by quantifying the degradation of its upstream regulator IκBα. We demonstrate the utility of this protocol by assessment of intracellular IκBα in human primary regulatory T cells experiencing TNFR2 agonism, a process previously reported to activate NF-κB in these cells. We also show that this assay may be applied to study NF-κB activation in other cell types, such as human primary T cells and THP-1 cell-derived macrophages, when induced by their corresponding inflammatory cues. Thus, this robust and reproducible protocol will be of interest to a wide range of scientists who aim to measure NF-κB activity in medium-to-high-throughput assays. © 2024 Wiley Periodicals LLC. Basic Protocol: Quantifying inflammatory activation by flow cytometry of IκBα degradation Support Protocol 1: Isolating and expanding human regulatory T cells Support Protocol 2: Calculating IC50 from flow cytometry data using Excel.

A protein kinase C α and ß inhibitor blunts hyperphagia to halt renal function decline and reduces adiposity in a rat model of obesity-driven type 2 diabetes.

Type 2 diabetes (T2D) and its complications can have debilitating, sometimes fatal consequences for afflicted individuals. The disease can be difficult to control, and therapeutic strategies to prevent T2D-induced tissue and organ damage are needed. Here we describe the results of administering a potent and selective inhibitor of Protein Kinase C (PKC) family members PKCα and PKCß, Cmpd 1, in the ZSF1 obese rat model of hyperphagia-induced, obesity-driven T2D. Although our initial intent was to evaluate the effect of PKCα/ß inhibition on renal damage in this model setting, Cmpd 1 unexpectedly caused a marked reduction in the hyperphagic response of ZSF1 obese animals. This halted renal function decline but did so indirectly and indistinguishably from a pair feeding comparator group. However, above and beyond this food intake effect, Cmpd 1 lowered overall animal body weights, reduced liver vacuolation, and reduced inguinal adipose tissue (iWAT) mass, inflammation, and adipocyte size. Taken together, Cmpd 1 had strong effects on multiple disease parameters in this obesity-driven rodent model of T2D. Further evaluation for potential translation of PKCα/ß inhibition to T2D and obesity in humans is warranted.

IL-13 Controls IL-33 Activity through Modulation of ST2.

IL-33 is a multifunctional cytokine that mediates local inflammation upon tissue damage. IL-33 is known to act on multiple cell types including group 2 innate lymphoid cells (ILC2s), Th2 cells, and mast cells to drive production of Th2 cytokines including IL-5 and IL-13. IL-33 signaling activity through transmembrane ST2L can be inhibited by soluble ST2 (sST2), which acts as a decoy receptor. Previous findings suggested that modulation of IL-13 levels in mice lacking decoy IL-13Rα2, or mice lacking IL-13, impacted responsiveness to IL-33. In this study, we used Il13 -/- mice to investigate whether IL-13 regulates IL-33 activity by modulating the transmembrane and soluble forms of ST2. In Il13 -/- mice, the effects of IL-33 administration were exacerbated relative to wild type (WT). Il13 -/- mice administered IL-33 i.p. had heightened splenomegaly, more immune cells in the peritoneum including an expanded ST2L+ ILC2 population, increased eosinophilia in the spleen and peritoneum, and reduced sST2 in the circulation and peritoneum. In the spleen, lung, and liver of mice given IL-33, gene expression of both isoforms of ST2 was increased in Il13 -/- mice relative to WT. We confirmed fibroblasts to be an IL-13-responsive cell type that can regulate IL-33 activity through production of sST2. This study elucidates the important regulatory activity that IL-13 exerts on IL-33 through induction of IL-33 decoy receptor sST2 and through modulation of ST2L+ ILC2s.

Relaxin Family Member Insulin-Like Peptide 6 Ameliorates Cardiac Fibrosis and Prevents Cardiac Remodeling in Murine Heart Failure Models.

BACKGROUND: The insulin/insulin-like growth factor/relaxin family represents a group of structurally related but functionally diverse proteins. The family member relaxin-2 has been evaluated in clinical trials for its efficacy in the treatment of acute heart failure. In this study, we assessed the role of insulin-like peptide 6 (INSL6), another member of this protein family, in murine heart failure models using genetic loss-of-function and protein delivery methods. METHODS AND RESULTS: Insl6-deficient and wild-type (C57BL/6N) mice were administered angiotensin II or isoproterenol via continuous infusion with an osmotic pump or via intraperitoneal injection once a day, respectively, for 2 weeks. In both models, Insl6-knockout mice exhibited greater cardiac systolic dysfunction and left ventricular dilatation. Cardiac dysfunction in the Insl6-knockout mice was associated with more extensive cardiac fibrosis and greater expression of fibrosis-associated genes. The continuous infusion of chemically synthesized INSL6 significantly attenuated left ventricular systolic dysfunction and cardiac fibrosis induced by isoproterenol infusion. Gene expression profiling suggests liver X receptor/retinoid X receptor signaling is activated in the isoproterenol-challenged hearts treated with INSL6 protein. CONCLUSIONS: Endogenous Insl6 protein inhibits cardiac systolic dysfunction and cardiac fibrosis in angiotensin II- and isoproterenol-induced cardiac stress models. The administration of recombinant INSL6 protein could have utility for the treatment of heart failure and cardiac fibrosis.

Modulation of the IL-33/IL-13 Axis in Obesity by IL-13Rα2.

In obesity, IL-13 overcomes insulin resistance by promoting anti-inflammatory macrophage differentiation in adipose tissue. Endogenous IL-13 levels can be modulated by the IL-13 decoy receptor, IL-13Rα2, which inactivates and depletes the cytokine. In this study, we show that IL-13Rα2 is markedly elevated in adipose tissues of obese mice. Mice deficient in IL-13Rα2 had high expression of IL-13 response markers in adipose tissue, consistent with increased IL-13 activity at baseline. Moreover, exposure to the type 2 cytokine-inducing alarmin, IL-33, enhanced serum and tissue IL-13 concentrations and elevated tissue eosinophils, macrophages, and type 2 innate lymphoid cells. IL-33 also reduced body weight, fat mass, and fasting blood glucose levels. Strikingly, however, the IL-33-induced protection was greater in IL-13Rα2-deficient mice compared with wild-type littermates, and these changes were largely attenuated in mice lacking IL-13. Although IL-33 administration improved the metabolic profile in the context of a high fat diet, it also resulted in diarrhea and perianal irritation, which was enhanced in the IL-13Rα2-deficient mice. Weight loss in this group was associated with reduced food intake, which was likely related to the gastrointestinal effects. These findings outline both potentially advantageous and deleterious effects of a type 2-skewed immune response under conditions of metabolic stress, and identify IL-13Rα2 as a critical checkpoint in adipose tissues that limits the protective effects of the IL-33/IL-13 axis in obesity.

Genetic deficiency of Wnt5a diminishes disease severity in a murine model of rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a common autoimmune disease characterized by chronic inflammation of the joints, leading to bone erosion and joint dysfunction. Despite the recent successes of disease-modifying anti-rheumatic drugs (DMARDs), there is still clinical need for understanding the development and molecular etiology of RA. Wnts are developmental morphogens whose roles in adult pathology are poorly characterized. Wnt5a is a member of the non-canonical family of Wnts that modulates a wide range of cell processes, including differentiation, migration, and inflammation. Wnt5a has been implicated as a possible contributor to arthritis and it is upregulated in synovial fibroblasts from RA patients. METHODS: We investigated the role of endogenous Wnt5a in RA. Tamoxifen-inducible, Wnt5a knockout (Wnt5a cKO) mice and littermate controls were monitored for arthritis development and joint pathology using the K/BxN serum transfer-induced arthritis (STIA) model. To explore a role of Wnt5a in osteoclast fusion, bone marrow-derived monocytes (BMDMs) were differentiated in vitro. RESULTS: Wnt5a cKO mice were resistant to arthritis development compared to control littermates as assessed by ankle thickness and histologic measurements. Some parameters of inflammation were reduced in the Wnt5a cKO mice, including the extent of polymononuclear cell infiltration and extra-articular inflammation. Wnt5a cKO mice also exhibited less cartilage destruction and a reduction in osteoclast activity with concomitant reduction in tartrate-resistant acid phosphatase (TRAP), cathepsin K (CTSK), macrophage colony-stimulating factor (MCSF), matrix metalloproteinase (MMP)2 and MMP9 in the arthritic joints. Treatment of BMDMs with Wnt5a enhanced osteoclast fusion and increased the expression of dendrocyte-expressed seven transmembrane protein (DCSTAMP) and MMP9, that are necessary for osteoclast formation and activity. CONCLUSIONS: These data suggest that Wnt5a modulates the development of arthritis by promoting inflammation and osteoclast fusion, and provide the first mouse genetic evidence of a role for endogenous Wnt5a in autoimmune disease.

The injury-induced myokine insulin-like 6 is protective in experimental autoimmune myositis.

BACKGROUND: The idiopathic inflammatory myopathies represent a group of autoimmune diseases that are characterized by lymphocyte infiltration of muscle and muscle weakness. Insulin-like 6 (Insl6) is a poorly characterized member of the insulin-like/relaxin family of secreted proteins, whose expression is upregulated upon acute muscle injury. METHODS: In this study, we employed Insl6 gain or loss of function mice to investigate the role of Insl6 in a T cell-mediated model of experimental autoimmune myositis (EAM). EAM models in rodents have involved immunization with human myosin-binding protein C with complete Freund's adjuvant (CFA) emulsions and pertussis toxin. RESULTS: Insl6-deficiency in mice led to a worsened myositis phenotype including increased infiltration of CD4 and CD8 T cells and the elevated expression of inflammatory cytokines. Insl6-deficient mice show significant motor function impairment when tested with treadmill or Rotarod devices. Conversely, muscle-specific overexpression of Insl6 protected against the development of myositis as indicated by reduced lymphocyte infiltration in muscle, diminished inflammatory cytokine expression and improved motor function. The improvement in myositis by Insl6 could also be demonstrated by acute hydrodynamic delivery of a plasmid encoding murine Insl6. In cultured cells, Insl6 inhibits Jurkat cell proliferation and activation in response to phytohemagglutinin/phorbol 12-myristate 13-acetate stimulation. Insl6 transcript expression in muscle was reduced in a cohort of dermatomyositis and polymyositis patients. CONCLUSIONS: These data suggest that Insl6 may have utility for the treatment of myositis, a condition for which few treatment options exist.

Effects of adiponectin on calcium-handling proteins in heart failure with preserved ejection fraction.

BACKGROUND: Despite the increasing prevalence of heart failure with preserved ejection fraction (HFpEF) in humans, there remains no therapeutic options for HFpEF. Adiponectin, an adipocyte-derived cytokine, exerts cardioprotective actions, and its deficiency is implicated in the development of hypertension and HF with reduced ejection fraction. Similarly, adiponectin deficiency in HFpEF exacerbates left ventricular hypertrophy, diastolic dysfunction, and HF. However, the therapeutic effects of adiponectin in HFpEF remain unknown. We sought to test the hypothesis that chronic adiponectin overexpression protects against the progression of HF in a murine model of HFpEF. METHODS AND RESULTS: Adiponectin transgenic and wild-type mice underwent uninephrectomy, a continuous saline or d-aldosterone infusion and given 1.0% sodium chloride drinking water for 4 weeks. Aldosterone-infused wild-type mice developed HFpEF with hypertension, left ventricular hypertrophy, and diastolic dysfunction. Aldosterone infusion increased myocardial oxidative stress and decreased sarcoplasmic reticulum Ca(2+)-ATPase protein expression in HFpEF. Although total phospholamban protein expression was unchanged, there was a decreased expression of protein kinase A-dependent phospholamban phosphorylation at Ser16 and CaMKII (Ca(2+)/calmodulin-dependent protein kinase II)-dependent phospholamban phosphorylation at Thr17. Adiponectin overexpression in aldosterone-infused mice ameliorated left ventricular hypertrophy, diastolic dysfunction, lung congestion, and myocardial oxidative stress without affecting blood pressure and left ventricular EF. This improvement in diastolic dysfunction parameters in aldosterone-infused adiponectin transgenic mice was accompanied by the preserved protein expression of protein kinase A-dependent phosphorylation of phospholamban at Ser16. Adiponectin replacement prevented the progression of aldosterone-induced HFpEF, independent of blood pressure, by improving diastolic dysfunction and by modulating cardiac hypertrophy. CONCLUSIONS: These findings suggest that adiponectin may have therapeutic effects in patients with HFpEF.

Divergent roles for adiponectin receptor 1 (AdipoR1) and AdipoR2 in mediating revascularization and metabolic dysfunction in vivo.

Adiponectin is a well described anti-inflammatory adipokine that is highly abundant in serum. Previous reports have found that adiponectin deficiency promotes cardiovascular and metabolic dysfunction in murine models, whereas its overexpression is protective. Two candidate adiponectin receptors, AdipoR1 and AdipoR2, are uncharacterized with regard to cardiovascular tissue homeostasis, and their in vivo metabolic functions remain controversial. Here we subjected AdipoR1- and AdipoR2-deficient mice to chronic hind limb ischemic surgery. Blood flow recovery in AdipoR1-deficient mice was similar to wild-type; however, revascularization in AdipoR2-deficient mice was severely attenuated. Treatment with adiponectin enhanced the recovery of wild-type mice but failed to rescue the impairment observed in AdipoR2-deficient mice. In view of this divergent receptor function in the hind limb ischemia model, AdipoR1- and AdipoR2-deficient mice were also evaluated in a model of diet-induced obesity. Strikingly, AdipoR1-deficient mice developed severe metabolic dysfunction compared with wild type, whereas AdipoR2-deficient mice were protected from diet-induced weight gain and metabolic perturbations. These data show that AdipoR2, but not AdipoR1, is functionally important in an in vivo model of ischemia-induced revascularization and that its expression is essential for the revascularization actions of adiponectin. These data also show that, in contrast to revascularization responses, AdipoR1, but not AdipoR2 deficiency, leads to diet-induced metabolic dysfunction, revealing that these receptors have highly divergent roles in vascular and metabolic homeostasis.

Cardiometabolic effects of adiponectin.

Over the past two decades, adiponectin has been studied in more than eleven thousand publications. A classical adipokine, adiponectin was among the first factors secreted from adipose tissue that were found to promote metabolic function. Circulating levels of adiponectin consistently decline with increasing body mass index. Clinical and basic science studies have identified adiponectin's cardiovascular-protective actions, providing a mechanistic link to the increased incidence of cardiovascular disease in obese individuals. While progress has been made in identifying receptors essential for the metabolic actions of adiponectin (AdipoR1 and AdipoR2), few studies have examined the receptor-mediated signaling pathways in cardiovascular tissues. T-cadherin, a GPI-anchored adiponectin-binding protein, was recently identified as critical for the cardiac-protective and revascularization actions of adiponectin. Adiponectin is abundantly present on the surfaces of vascular and muscle tissues through a direct interaction with T-cadherin. Consistent with this observation, adiponectin is absent from T-cadherin-deficient tissues. Since T-cadherin lacks an intracellular domain, additional studies would further our understanding of this signaling pathway. Here, we review the diverse cardiometabolic actions of adiponectin.

T-cadherin is essential for adiponectin-mediated revascularization.

Adipose tissue secretes protein factors that have systemic actions on cardiovascular tissues. Previous studies have shown that ablation of the adipocyte-secreted protein adiponectin leads to endothelial dysfunction, whereas its overexpression promotes wound healing. However, the receptor(s) mediating the protective effects of adiponectin on the vasculature is not known. Here we examined the role of membrane protein T-cadherin, which localizes adiponectin to the vascular endothelium, in the revascularization response to chronic ischemia. T-cadherin-deficient mice were analyzed in a model of hind limb ischemia where blood flow is surgically disrupted in one limb and recovery is monitored over 28 days by laser Doppler perfusion imaging. In this model, T-cadherin-deficient mice phenocopy adiponectin-deficient mice such that both strains display an impaired blood flow recovery compared with wild-type controls. Delivery of exogenous adiponectin rescued the impaired revascularization phenotype in adiponectin-deficient mice but not in T-cadherin-deficient mice. In cultured endothelial cells, T-cadherin deficiency by siRNA knockdown prevented the ability of adiponectin to promote cellular migration and proliferation. These data highlight a previously unrecognized role for T-cadherin in limb revascularization and show that it is essential for mediating the vascular actions of adiponectin.