PI3Kγ Knockout Mice Research Articles

Genetic ablation of the isoform γ of PI3K decreases antidepressant efficacy of ketamine in male mice

About one-third of major depressive disorder (MDD) patients demonstrate unresponsiveness to classic antidepressants, and even the clinical efficacy of fast-acting drugs such as ketamine varies significantly among patients with treatment-resistant depression. Nevertheless, the lack of suitable animal models that mimic a possible ketamine-resistant phenotype challenges the understanding of resistance to drug treatment. In this study, we showed that PI3Kγ knock-out (KO) mice do not respond to classical doses of ketamine and classical antidepressants. PI3Kγ KO mice were unresponsive to both the rapid and sustained antidepressant-like effects of a single dose of ketamine in the forced swimming test. Additionally, they were unresponsive to the antidepressant-like effects induced by the tricyclic antidepressant imipramine and the selective serotonin reuptake inhibitor fluoxetine. However, acute pharmacological inhibition of PI3Kγ did not block the antidepressant-like effect of ketamine, showing that a chronic deficiency of the PI3Kγ-mediated pathway is necessary for the effects of classic doses of ketamine and antidepressants. Therefore, we propose that PI3Kγ participates in the antidepressant activity and is likely implicated in the neurobiology and phenotype observed in patients with MDD who demonstrate treatment resistance.

Abstract 18479: Lack of Catalytic and Non-Catalytic Function of PI3 Kinase Gamma in Mice Increases Hypoxia-Induced Pulmonary Hypertension

Rationale: Pulmonary hypertension (PH) is characterised by vasoconstriction and remodelling of pulmonary vessels, leading to elevated pulmonary arterial pressure and right ventricular (RV) hypertrophy. PI 3-kinase γ (PI3Kγ) is expressed in leukocytes, cardiomyocytes, and endothelial cells (EC) which are all involved in the pathogenesis of PH. The catalytic function of PI3Kγ is involved in leukocyte recruitment and EC proliferation which are important for vascular remodelling and maladaptive cardiac hypertrophy whereas its non-catalytic function affects cardiac contractility via cAMP signaling. Furthermore, inactivation of PI3Kγ impairs nitric oxide production by eNOS, which may lead to increased vascular resistance. Therefore, we investigated the role of both catalytic and non-catalytic functions of PI3Kγ in the pathogenesis of PH. Methods: PI3Kγ knockout mice (PI3Kγ -/- ), as well as mice with a catalytically inactive form of PI3Kγ (PI3Kγ KD/KD ) were exposed to hypoxia-induced PH (21 days at 10% O 2 hypoxia (HOX)). Systolic right ventricular pressure (RVSP) was measured using a Millar pressure catheter inserted via the jugular vein. RV hypertrophy was determined by Fulton's index. Results: PI3Kγ -/- mice showed significantly increased RVSP after hypoxia compared to WT controls (HOX WT 34.16±3.21 mmHg vs HOX PI3Kγ -/- 37.04±2.31 mmHg; p=0.0049). Already under normoxia (NOX), an increased RVSP was measured in PI3Kγ -/- mice (NOX WT 26.13±1.2mmHg vs. NOX PI3Kγ -/- 28.07±0.88 mmHg, p=0.0157). A significant increase in RVSP was also detected in PI3Kγ KD/KD (HOX WT 34.67±2.02 mmHg vs. HOX PI3Kγ KD/KD 37.95±1.39 mmHg; p=0.0228). Here, a significant increase in RV hypertrophy was evident for PI3Kγ KD/KD mice (HOX WT 0.38±0.06 vs. HOX PI3Kγ KD/KD 0.46±0.09; p=0.0037) which was not detected in PI3Kγ -/- compared to WT controls after hypoxia. Conclusion: The results show that both catalytic and non-catalytic inactivation of PI3Kγ in vivo do not counteract the pathogenesis of PH, but conversely enhance it. In this context, reduced phosphorylation of eNOS may play a crucial role, leading to increased vasoconstriction, whereas increased cAMP levels in PI3Kγ -/- may help the RV to adapt to the increased pressure. The exact mechanisms will be analysed in the future.

Hypoxia Sensing of β-Adrenergic Receptor Is Regulated by Endosomal PI3Kγ.

Impaired beta-adrenergic receptor (β1 and β2AR) function following hypoxia underlies ischemic heart failure/stroke. Activation of PI3Kγ (phosphoinositide 3-kinase γ) by beta-adrenergic receptor leads to feedback regulation of the receptor by hindering beta-adrenergic receptor dephosphorylation through inhibition of PP2A (protein phosphatase 2A). However, little is known about PI3Kγ feedback mechanism in regulating hypoxia-mediated β1 and β2AR dysfunction and cardiac remodeling. Human embryonic kidney 293 cells or mouse adult cardiomyocytes and C57BL/6 (WT) or PI3Kγ knockout (KO) mice were subjected to hypoxia. Cardiac plasma membranes and endosomes were isolated and evaluated for β1 and β2AR density and function, PI3Kγ activity and β1 and β2AR-associated PP2A activity. Metabolic labeling was performed to assess β1 and β2AR phosphorylation and epinephrine/norepinephrine levels measured post-hypoxia. Hypoxia increased β1 and β2AR phosphorylation, reduced cAMP, and led to endosomal accumulation of phosphorylated β2ARs in human embryonic kidney 293 cells and WT cardiomyocytes. Acute hypoxia in WT mice resulted in cardiac remodeling and loss of adenylyl cyclase activity associated with increased β1 and β2AR phosphorylation. This was agonist-independent as plasma and cardiac epinephrine and norepinephrine levels were unaltered. Unexpectedly, PI3Kγ activity was selectively increased in the endosomes of human embryonic kidney 293 cells and WT hearts post-hypoxia. Endosomal β1- and β2AR-associated PP2A activity was inhibited upon hypoxia in human embryonic kidney 293 cells and WT hearts showing regulation of beta-adrenergic receptors by PI3Kγ. This was accompanied with phosphorylation of endogenous inhibitor of protein phosphatase 2A whose phosphorylation by PI3Kγ inhibits PP2A. Increased β1 and β2AR-associated PP2A activity, decreased beta-adrenergic receptor phosphorylation, and normalized cardiac function was observed in PI3Kγ KO mice despite hypoxia. Compared to WT, PI3Kγ KO mice had preserved cardiac response to challenge with β1AR-selective agonist dobutamine post-hypoxia. Agonist-independent activation of PI3Kγ underlies hypoxia sensing as its ablation leads to reduction in β1- and β2AR phosphorylation and amelioration of cardiac dysfunction.

Inactivation of catalytic and non-catalytic PI3 kinase gamma functions enhances hypoxia-induced pulmonary hypertension in mice

Abstract Rationale Pulmonary hypertension (PH) is a pulmonary vascular disease characterised by chronically elevated pulmonary arterial mean pressure, increased pulmonary vascular resistance and right ventricular (RV) dysfunction and hypertrophy. The pathogenesis includes vasoconstriction of pulmonary vessels and pulmonary vascular remodelling. Phosphatidylinositol 3'-kinase γ (PI3Kγ) is highly expressed in leukocytes, endothelial cells (EC) and cardiomyocytes, which are involved in the pathogenesis of PH. The catalytic function of PI3Kγ has been shown to be associated with numerous processes that are potentially important for both vascular remodelling and maladaptive cardiac hypertrophy, including leukocyte recruitment and endothelial cell proliferation and survival. However, inhibition of non-catalytic PI3Kγ function results in reduced nitric oxide (NO) production by endothelial NO synthase (eNOS), which may lead to increased vascular resistance. Therefore, the aim of our study was to investigate the role of both catalytic and non-catalytic PI3Kγ functions in the pathogenesis of PH. Methods The importance of PI3Kγ in the pathogenesis of PH was analysed in vivo using the hypoxia-induced mouse model of PH (21 days at 10% O2 hypoxia (HOX)). PI3Kγ knockout mice (PI3Kγ−/−), as well as mice expressing a catalytically inactive form of PI3Kγ (PI3KγKD/KD) were investigated. To specifically inhibit the non-catalytic function of PI3Kγ, C57BL/6N mice were treated with a blocking peptide via intratracheal instillation and studied in the same model. Subsequently, systolic right ventricular pressure (RVSP) was measured using a Millar pressure catheter inserted via the jugular vein. In vitro, western blotting was used to investigate the phosphorylation of eNOS (Ser1177) in pulmonary ECs from wild type (WT), PI3Kγ−/− and PI3KγKD/KD mice. Results PI3Kγ−/− mice showed significantly increased RVSP after three weeks of hypoxia compared with WT controls (HOX WT 34.16±3.47mmHg vs. HOX PI3Kγ−/− 37.04±2.43mmHg; p=0.005; n≥7). A significant increase in RVSP was also detected in PI3KγKD/KD and blocking peptide-treated mice (HOX WT 34.67±2.02mmHg vs. HOX PI3KγKD/KD 37.95±1.39mmHg; p=0.023; n≥5 and HOX vehicle 34.19±2.74mmHg vs. HOX blocking peptide 37.61±2.46mmHg; p=0.011; n≥9). Heart rate, as well as systemic blood pressure, remained unchanged. Under normoxic conditions, no difference in RVSP between the groups could be measured. Interestingly, western blot detected reduced phosphorylation of eNOS after stimulation with fetal calf serum in PI3Kγ−/− ECs compared to WT and PI3KγKD/KD. Conclusion These results show that blunting of both catalytic and non-catalytic functions of PI3Kγ in vivo do not prevent the pathogenesis of PH, but conversely enhance hypoxia-induced elevation of RVSP. In this context, reduced phosphorylation of eNOS may play a crucial role, leading to increased vasoconstriction. Additional studies are required to evaluate the underlying mechanisms in detail. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): DFG Deutsche Forschungsgemeinschaft

Abstract P1067: IRS Mediated Recruitment Of PI3Kgamma By Insulin Inhibits Beta-adrenergic Receptor Function

Insulin (INS) treatment results in impaired response to β-agonist isoproterenol (ISO) associated with phosphorylated β2-adrenergic receptor (β2AR) and impaired cAMP response. Although INS does not mediate dissociation of G-protein Gβγ subunits, yet surprisingly recruits phosphoinositide 3-kinase γ (PI3Kγ) to the plasma membrane. Correspondingly, knock-down of PI3Kγ (PI3Kγ KD) in HEK 293 or ablation in primary adult cardiomyocytes and fibroblasts (isolated from PI3Kγ knockout mice) abrogated β2AR phosphorylation reflecting a key role for PI3Kγ in retaining β2AR phosphorylation. Also, adult cardiomyocytes isolated from C57Bl6 mice showed significant loss of in vitro ISO-stimulated cardiomyocyte contraction upon INS pre-treatment which was remarkably preserved in the PI3Kγ knockout cardiomyocytes despite INS. As PI3Kγ is traditionally recruited to the βAR complex by the Gβγ subunits, we hypothesized that INS-mediates Gβγ-independent recruitment of PI3Kγ to the βAR complex underlying its dysfunction. INS stimulation is known to recruit insulin receptor substrate (IRS 1 & 2) to the receptor complex, and therefore, we tested whether PI3Kγ interacts with IRS 1/2 leading to Gβγ-independent recruitment of PI3Kγ to the β2AR complex. Immunoprecipitation PI3Kγ following INS showed that PI3Kγ interacts with IRS2 recruiting PI3Kγ to the β2AR complex bypassing the traditional Gβγ-dependent pathway. Since PI3Kγ KD resulted in loss of INS-mediated β2AR phosphorylation, we tested whether PI3Kγ inhibits protein phosphatase 2A (PP2A) function impairing β2AR de-phosphorylation. INS treatment resulted in significant loss of β2AR-associated PP2A activity which was rescued in PI3Kγ KD cells showing that PI3Kγ impairs PP2A function. Furthermore, our studies show that PI3Kγ phosphorylates the endogenous inhibitor of PP2A, I2PP2A which then robustly binds and inhibits PP2A activity impairing β2AR dephosphorylation. Consistently, CRISPR ablation of I2PP2A relieves this inhibition on PP2A leading to increased PP2A activity. This reduces accumulation of phosphorylated βARs despite the presence of PI3Kγ showing an underappreciated regulation of PP2A by insulin through non-canonical recruitment of PI3Kγ that impairs β2AR function.

Mesenchymal stem cell transplantation worsens intestinal inflammation and microenvironment in PI3Kγ-knockout mice

Considering the possible interaction between mesenchymal stem cells (MSCs) and PI3Kγ-associated drugs, we evaluated the efficacy and action mechanism of MSCs in the treatment of colitis in PI3Kγ-/- mice. Trinitro-benzene-sulfonic acid enema was used to create a colitis model, and MSCs were transplanted through the caudal vein to treat colitis in wild-type and PI3Kγ-/- mice. We sequenced microbial 16S rRNA genes in the colonic mucosa of PI3Kγ-/- and wild-type mice and quantified colonic IgA, IL-2, IL-10, IL-17A, occludin, and serum IgA. MSC transplantation led to a more serious reduction in the weight of trinitro-benzene-sulfonic acid-administered PI3Kγ-/- mice than that in wild-type mice. The disease activity index, pathological scoring, number of taxa in the colon, Berger–Parker index, I-index, proportion of Proteobacteria, and IgA level in the blood were higher in PI3Kγ-/- mice than in wild-type mice after MSC transplantation. The occludin and IL-10 levels in the colon tissues decreased before and after MSC transplantation in PI3Kγ-/- mice, whereas they were increased in wild-type mice The IL-17 level decreased in both wild-type and PI3Kγ-/- mice, with knockout mice showing a greater decrease. Therefore, MSC transplantation in PI3Kγ-/- mice led to increased numbers of exogenous pathogenic microorganisms and enhanced colitis that was difficult to relieve.

Phosphoinositide 3 Kinase γ Plays a Critical Role in Acute Kidney Injury.

Inflammatory cells contribute to the pathogenesis of renal ischemia-reperfusion injury (IRI). However, the signaling mechanisms underlying the infiltration of inflammatory cells into the kidney are not well understood. In this study, we examined the effects of phosphoinositide 3 kinase γ (PI3Kγ) on inflammatory cells infiltration into the kidney in response to ischemia-reperfusion injury. Compared with wild-type mice, PI3Kγ knockout mice displayed less IRI in the kidney with fewer tubular apoptotic cell. Furthermore, PI3Kγ deficiency decreased the number of infiltrated neutrophils, macrophages, and T cells in the kidney, which was accompanied by a decrease in the expression of pro-inflammatory cytokines in the kidney. Moreover, wild-type mice treated with AS-605240, a selective PI3Kγ inhibitor, displayed less tubular damage, accumulated fewer inflammatory cells, and expressed less proinflammatory molecules in the kidney following IRI. These results demonstrate that PI3Kγ has a critical role in the pathogenesis of kidney damage in IRI, indicating that PI3Kγ inhibition may serve as a potential therapeutic strategy for the prevention of ischemia-reperfusion-induced kidney injury.

PI3Kγ Mediates Microglial Proliferation and Cell Viability via ROS.

(1) Background: Rapid microglial proliferation contributes to the complex responses of the innate immune system in the brain to various neuroinflammatory stimuli. Here, we investigated the regulatory function of phosphoinositide 3-kinase γ (PI3Kγ) and reactive oxygen species (ROS) for rapid proliferation of murine microglia induced by LPS and ATP. (2) Methods: PI3Kγ knockout mice (PI3Kγ KO), mice expressing catalytically inactive PI3Kγ (PI3Kγ KD) and wild-type mice were assessed for microglial proliferation using an in vivo wound healing assay. Additionally, primary microglia derived from newborn wild-type, PI3Kγ KO and PI3Kγ KD mice were used to analyze PI3Kγ effects on proliferation and cell viability, senescence and cellular and mitochondrial ROS production; the consequences of ROS production for proliferation and cell viability after LPS or ATP stimulation were studied using genetic and pharmacologic approaches. (3) Results: Mice with a loss of lipid kinase activity showed impaired proliferation of microglia. The prerequisite of induced microglial proliferation and cell viability appeared to be PI3Kγ-mediated induction of ROS production. (4) Conclusions: The lipid kinase activity of PI3Kγ plays a crucial role for microglial proliferation and cell viability after acute inflammatory activation.

Impact of ambient temperature on inflammation-induced encephalopathy in endotoxemic mice\u2014role of phosphoinositide 3-kinase gamma

BackgroundSepsis-associated encephalopathy (SAE) is an early and frequent event of infection-induced systemic inflammatory response syndrome. Phosphoinositide 3-kinase γ (PI3Kγ) is linked to neuroinflammation and inflammation-related microglial activity. In homeotherms, variations in ambient temperature (Ta) outside the thermoneutral zone lead to thermoregulatory responses, mainly driven by a gradually increasing sympathetic activity, and may affect disease severity. We hypothesized that thermoregulatory response to hypothermia (reduced Ta) aggravates SAE in PI3Kγ-dependent manner.MethodsExperiments were performed in wild-type, PI3Kγ knockout, and PI3Kγ kinase-dead mice, which were kept at neutral (30 ± 0.5 °C) or moderately lowered (26 ± 0.5 °C) Ta. Mice were exposed to lipopolysaccharide (LPS, 10 μg/g, from Escherichia coli serotype 055:B5, single intraperitoneal injection)—evoked systemic inflammatory response (SIR) and monitored 24 h for thermoregulatory response and blood–brain barrier integrity. Primary microglial cells and brain tissue derived from treated mice were analyzed for inflammatory responses and related cell functions. Comparisons between groups were made with one-way or two-way analysis of variance, as appropriate. Post hoc comparisons were made with the Holm–Sidak test or t tests with Bonferroni’s correction for adjustments of multiple comparisons. Data not following normal distribution was tested with Kruskal-Wallis test followed by Dunn’s multiple comparisons test.ResultsWe show that a moderate reduction of ambient temperature triggers enhanced hypothermia of mice undergoing LPS-induced systemic inflammation by aggravated SAE. PI3Kγ deficiency enhances blood–brain barrier injury and upregulation of matrix metalloproteinases (MMPs) as well as an impaired microglial phagocytic activity.ConclusionsThermoregulatory adaptation in response to ambient temperatures below the thermoneutral range exacerbates LPS-induced blood–brain barrier injury and neuroinflammation. PI3Kγ serves a protective role in suppressing release of MMPs, maintaining microglial motility and reinforcing phagocytosis leading to improved brain tissue integrity. Thus, preclinical research targeting severe brain inflammation responses is seriously biased when basic physiological prerequisites of mammal species such as preferred ambient temperature are ignored.

Abstract IA26: Reprogramming tumor-associated macrophages by targeting PI3K-gamma with IPI-549

Abstract The PI3 kinases (PI3K) belong to a family of signal-transducing enzymes that mediate key cellular functions in cancer and immunity. The PI3K-gamma (γ) isoform is highly expressed in myeloid cells/macrophages and plays an important role in M2 macrophage polarization (Kaneda, Nature 2016). PI3K-γ knockout mice also demonstrated the role for PI3K-γ in tumor growth and immune tolerance (Gunderson, Cancer Discovery 2015; Kaneda, 2016). IPI-549 is an oral, potent, and selective inhibitor of PI3K-γ that shows single-agent antitumor activity in multiple murine tumor models (Kaneda, 2016) and enhanced antitumor activity and improved survival when combined with immune-checkpoint blockade (ICB) (De Henau, Nature 2016). This antitumor activity is dependent on the presence of both immune-suppressive tumor-associated CD11b+ myeloid cells and CD8+ cytotoxic T cells. IPI-549 can reduce the suppression of T-cell activity by both murine and human myeloid-derived suppressor cells in vitro and in vivo. IPI 549 treatment of tumor-bearing mice leads to a shift in tumor-associated myeloid cells from an immunosuppressive M2 phenotype to a proinflammatory M1 phenotype and an increased frequency of circulating tumor-specific T cells and tumor-infiltrating CD8+ T cells. In addition, preclinical tumor model systems show that resistance to ICB is directly mediated by the suppressive activity of infiltrating myeloid cells (De Henau, Nature 2016). Treatment of ICB-resistant 4T1 and B16GMCSF tumor-bearing mice with IPI-549 and anti-PD1 for 14 days led to a significant antitumor activity and an increased CD8+/T-reg cell ratio, confirming that selective pharmacologic targeting PI3K-γ by IPI-549 restores sensitivity to ICB. We are conducting a Ph 1/1b study IPI-549-01 (ClinicalTrials.gov NCT02637531) evaluating the safety, tolerability, pharmacokinetics, pharmacodynamics, and immunomodulatory activity of IPI-549 to determine its recommended phase 2 dose and preliminary clinical activity, as both a monotherapy and in combination with nivolumab, in patients with advanced solid tumors. Citation Format: Jeffery L. Kutok. Reprogramming tumor-associated macrophages by targeting PI3K-gamma with IPI-549 [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA26.

Phosphoinositide 3-kinase γ deficiency attenuates kidney injury and fibrosis in angiotensin II-induced hypertension.

We have shown that the CXCL16/CXCR6 axis plays a critical role in recruiting inflammatory cells and bone marrow-derived fibroblasts into the kidney leading to renal injury and fibrosis. However, the underlying signaling mechanisms are not known. In the present study, we examined the role of phosphoinositide-3 kinase γ (PI3Kγ) signaling in the recruitment of inflammatory cells and bone marrow-derived fibroblasts into the kidney and development of renal injury and fibrosis in an experimental model of hypertension induced by angiotensin II. Blood pressure was comparable between wild-type (WT) and PI3Kγ knockout (KO) mice at baseline. Angiotensin II treatment led to an increase in blood pressure that was similar between WT and PI3Kγ KO mice. Compared with WT mice, PI3Kγ KO mice were protected from angiotensin II-induced renal dysfunction and injury and developed less proteinuria. PI3Kγ deficiency suppressed bone marrow-derived fibroblast accumulation and myofibroblast formation in the kidney and inhibited total collagen deposition and extracellular matrix protein production in the kidney in response to angiotensin II. PI3Kγ deficiency inhibited the infiltration of F4/80+ macrophages and CD3+ T cells into the kidney and reduced gene expression levels of pro-inflammatory cytokines in the kidney following angiotensin II treatment. Finally, inhibition of PI3Kγ suppressed CXCL16-induced monocyte migration in vitro. These results indicate that PI3Kγ mediates the influx of macrophages, T cells and bone marrow-derived fibroblasts into the kidney resulting in kidney injury and fibrosis.

Degeneration of the intestinal microbial community in PI3Kγ-knockout mice

Background and aimPI3Kγ is closely related to inflammation and cardiovascular diseases and thus, PI3Kγ inhibitors are candidate drugs for the treatment of these disorders. Considering the potential effect of the intestinal microbiome on inflammation and cardiovascular diseases, this study aimed to identify characteristics of the intestinal microbial community under PI3Kγ deficiency, to help reveal the potential influence of PI3Kγ inhibitors mediated by the microbial community. MethodsExon 2 of the PI3Kγ gene was knocked out in a Balb/c mouse by using single-guide RNAs. Homozygous PI3Kγ-knockout (PI3Kγ–/–) mice were obtained by embryo transfer and hybridization. PI3Kγ–/– and wild-type (WT) mice were raised in the same specific pathogen-free conditions until 8 weeks of age. Then, colonic tissues and feces from the middle segment of the colon were collected and analyzed by Illumina MiSeq sequencing. Differences in intestinal microbial community between the PI3Kγ–/– and WT mice were detected by bioinformatics analysis. ResultsThe richness and alpha diversity of the colonic microbial community were decreased in PI3Kγ–/– mice. The alpha diversity of the microbial community in feces did not differ between PI3Kγ–/– and WT mice. The beta diversity of the microbial community in feces of PI3Kγ–/– mice was obviously different from that in WT mice, whereas the within-group variation in Bray-Curtis distances of the mucosal microbial community was significantly decreased in PI3Kγ–/– mice. The topological structure of the species-related network of the colonic microbial community in PI3Kγ–/– mice was more polarized. Finally, we predicted that PI3Kγ deficiency might affect the synthesis of some antibiotics, bile acid, and thiamine through effects on the microbial community. ConclusionsPI3Kγ dysfunction led to degeneration of the intestinal microbial community and might alter the synthesis of some antibiotics, bile acids, and thiamine. The usage of PI3Kγ inhibitors for inflammation and cardiovascular diseases might lead to knock-on effect on our organism through intestinal microbiota.

Altered microbial community structure in PI3Kγ knockout mice with colitis impeding relief of inflammation: Establishment of new indices for intestinal microbial disorder

Lipopolysaccharide stimulates the intestinal microbiome to activate phosphoinositide 3 kinase (PI3K) signaling via several pathways; however, the direct effect that PI3K has on the intestinal bacterial community remains unclear. Herein, we investigate changes in the colonic microbiome of colitis PI3Kγ-knockout (PI3Kγ−/−) mice. Additionally, the effect of anal administration of colonic irrigation fluid from control mice to those with colitis was examined. Microbial 16S rRNA genes from the colonic mucosa of PI3Kγ−/− and WT mice were sequenced using Illumina MiSeq platform, and colonic IgA, IL-2, IL-10, and IL-17A production was quantified by western blot analysis. Myeloperoxidase (MPO) activity was detected by absorbance via colorimetric analysis. From the results, two new indices were derived by dividing the bacterial community into invading taxa, common taxa, and vanishing taxa. These indices were used to estimate the degree of microbiome disorder in chronic experimental colitis models. PI3Kγ−/− mice showed slower remission of inflammation as assessed by the disease activity index，pathological score, IL-2, IL-17, IL-10, IgA expression and MPO activity. The unique and common taxa of wild-type and PI3Kγ−/− mice increased as colitis symptoms regressed. Continuous loss of commensal bacteria happened with the continuous invasion of exogenous bacteria in the intestinal mucosa of PI3Kγ‐−/− mice after colitis begin to aggravate. However, transplantation of normal intestinal microbiota to PI3Kγ−/− mice promoted remission of inflammation; while the microbial dysbiosis observed during PI3Kγ dysfunction aggravated the intestinal microbiome disorder and impeded colitis recovery. Thus, the PI3Kγ signaling pathway may regulate microbial community composition in the colon.

Induction of Neuronal PI3Kγ Contributes to Endoplasmic Reticulum Stress and Long-Term Functional Impairment in a Murine Model of Traumatic Brain Injury.

Phosphoinositide 3-kinase γ (PI3Kγ) expressed in immune cells is linked to neuroinflammation in several neurological diseases. However, the expression and role of PI3Kγ in preclinical traumatic brain injury (TBI) have not been investigated. In WT mice, we found that TBI induced rapid and extensive expression of PI3Kγ in neurons within the perilesional cortex and the ipsilateral hippocampal subfields (CA1, CA3), which peaked between 1 and 3days and declined significantly 7days after TBI. Intriguingly, the induction of neuronal PI3Kγ in these subregions of the brain spatiotemporally coincided with both the TBI-induced activation of the neuronal ER stress pathway (p-eIF2α, ATF4, and CHOP) and neuronal cell death (marked by TUNEL-positive neurons) 3days after TBI. Further, we show that the absence of PI3Kγ in knockout mice profoundly reduced the TBI-induced activation of the ER stress pathway and neuronal cell death. White matter disruption is a better predictor of long-term clinical outcomes than focal lesion size. We show that PI3Kγ deficiency not only reduced brain tissue loss but also alleviated white matter injury (determined by axonal injury and demyelination) up to 28days after TBI. Importantly, PI3Kγ-knockout mice exhibited greater functional recovery including forepaw use, sensorimotor balance and coordination, and spatial learning and memory up to 28days after TBI. These results unveil a previously unappreciated role for neuronal PI3Kγ in the regulation of ER stress associated with neuronal cell death, white matter damage, and long-term functional impairment after TBI.

Phosphatidyl Inositol 3 Kinase-Gamma Balances Antiviral and Inflammatory Responses During Influenza A H1N1 Infection: From Murine Model to Genetic Association in Patients.

Influenza A virus (IAV) infection causes severe pulmonary disease characterized by intense leukocyte infiltration. Phosphoinositide-3 kinases (PI3Ks) are central signaling enzymes, involved in cell growth, survival, and migration. Class IB PI3K or phosphatidyl inositol 3 kinase-gamma (PI3Kγ), mainly expressed by leukocytes, is involved in cell migration during inflammation. Here, we investigated the contribution of PI3Kγ for the inflammatory and antiviral responses to IAV. PI3Kγ knockout (KO) mice were highly susceptible to lethality following infection with influenza A/WSN/33 H1N1. In the early time points of infection, infiltration of neutrophils was higher than WT mice whereas type-I and type-III IFN expression and p38 activation were reduced in PI3Kγ KO mice resulting in higher viral loads when compared with WT mice. Blockade of p38 in WT macrophages infected with IAV reduced levels of interferon-stimulated gene 15 protein to those induced in PI3Kγ KO macrophages, suggesting that p38 is downstream of antiviral responses mediated by PI3Kγ. PI3Kγ KO-derived fibroblasts or macrophages showed reduced type-I IFN transcription and altered pro-inflammatory cytokines suggesting a cell autonomous imbalance between inflammatory and antiviral responses. Seven days after IAV infection, there were reduced infiltration of natural killer cells and CD8+ T lymphocytes, increased concentration of inflammatory cytokines in bronchoalveolar fluid, reduced numbers of resolving macrophages, and IL-10 levels in PI3Kγ KO. This imbalanced environment in PI3Kγ KO-infected mice culminated in enhanced lung neutrophil infiltration, reactive oxygen species release, and lung damage that together with the increased viral loads, contributed to higher mortality in PI3Kγ KO mice compared with WT mice. In humans, we tested the genetic association of disease severity in influenza A/H1N1pdm09-infected patients with three potentially functional PIK3CG single-nucleotide polymorphisms (SNPs), rs1129293, rs17847825, and rs2230460. We observed that SNPs rs17847825 and rs2230460 (A and T alleles, respectively) were significantly associated with protection from severe disease using the recessive model in patients infected with influenza A(H1N1)pdm09. Altogether, our results suggest that PI3Kγ is crucial in balancing antiviral and inflammatory responses to IAV infection.

Expression of PI3K Signaling Associated with T Cells in Psoriasis Is Inhibited by Seletalisib, a PI3Kδ Inhibitor, and Is Required for Functional Activity

The phosphoinositide 3-kinase (PI3K) pathway plays a key role in many cellular processes, including cell proliferation, survival, and protein synthesis, with the PI3K isoform, PI3Kδ, involved in normal T-cell development and function (Jarmin et al., 2008, Lucas et al., 2016, Vanhaesebroeck et al., 2012). Evidence to suggest that PI3K signaling might play a role in psoriasis comes from reports of increased expression of phosphorylated protein kinase B, mammalian target of rapamycin, and ribosomal protein S6 (pS6), which are downstream in the PI3K signaling pathway, in lesional psoriatic skin compared with nonlesional skin and healthy controls (Buerger et al., 2013, Madonna et al., 2012, Rosenberger et al., 2007). In addition, PI3Kδ knock-in mice, expressing a catalytically inactive form of PI3Kδ (p110δD910A/D910A), and PI3Kγ knockout mice are greatly protected from imiquimod-induced psoriasis-like skin inflammation compared with wild-type mice (Roller et al., 2012).

Noncanonical regulation of insulin-mediated ERK activation by phosphoinositide 3-kinase γ.

Classically Class IB phosphoinositide 3-kinase (PI3Kγ) plays a role in extracellular signal-regulated kinase (ERK) activation following G-protein coupled receptor (GPCR) activation. Knock-down of PI3Kγ unexpectedly resulted in loss of ERK activation to receptor tyrosine kinase agonists such as epidermal growth factor or insulin. Mouse embryonic fibroblasts (MEFs) or primary adult cardiac fibroblasts isolated from PI3Kγ knock-out mice (PI3KγKO) showed decreased insulin-stimulated ERK activation. However, expression of kinase-dead PI3Kγ resulted in rescue of insulin-stimulated ERK activation. Mechanistically, PI3Kγ sequesters protein phosphatase 2A (PP2A), disrupting ERK-PP2A interaction, as evidenced by increased ERK-PP2A interaction and associated PP2A activity in PI3KγKO MEFs, resulting in decreased ERK activation. Furthermore, β-blocker carvedilol-mediated β-arrestin-dependent ERK activation is significantly reduced in PI3KγKO MEF, suggesting accelerated dephosphorylation. Thus, instead of classically mediating the kinase arm, PI3Kγ inhibits PP2A by scaffolding and sequestering, playing a key parallel synergistic step in sustaining the function of ERK, a nodal enzyme in multiple cellular processes.

Abstract B032: The PI3K-γ inhibitor, IPI-549, increases antitumor immunity by targeting tumor-associated myeloid cells and remodeling the immune-suppressive tumor microenvironment

Abstract The PI3 kinases (PI3K) belong to a family of signal-transducing enzymes that mediate key cellular functions in cancer and immunity. The PI3K-gamma (γ) isoform plays an important role in macrophage/myeloid cell function and migration, and a role for PI3K-γ in tumor growth and immune tolerance has been established in studies utilizing PI3K-γ knockout (KO) mice (Schmid et al., Cancer Cell, 2011; Gunderson et al., Cancer Discovery, 2015). We propose that pharmacological inhibition of PI3K-γ in myeloid cells can alter the tumor-immune microenvironment leading to enhanced antitumor T-cell responses. IPI-549 is an oral, potent, and selective inhibitor of PI3K-γ. Prior studies showed single agent antitumor activity in multiple murine tumor models, and enhanced antitumor activity and improved survival when combined with immune-checkpoint blockade. This antitumor activity is dependent on the presence of both immune-suppressive tumor-associated CD11b+ myeloid cells and CD8+ cytotoxic T cells. IPI-549 can reduce the T-cell-suppressive activity of both murine and human myeloid-derived suppressor cells in vitro (Kutok et al, 2015 CRI-CIMT-EATI-AACR Cancer Immunotherapy Meeting; De Henau et al, 2016 AACR Annual Meeting). We now show that IPI‑549 treatment of tumor‑bearing mice leads to a shift in tumor-associated myeloid cells from an immunosuppressive M2 phenotype to a proinflammatory M1 phenotype, characterized by reduced CD206 expression and enhanced expression of MHC class II and NOS2. Compared to vehicle-treated controls, short-term (9 days) treatment of CT26 tumor‑bearing animals with IPI‑549 revealed an increased frequency of circulating tumor-specific T cells, an increased percentage of tumor-infiltrating CD8+IFNγ+ T cells, and a reduced percentage of CD4+Foxp3+ regulatory T cells, leading to a trend towards increasing the CD8+/T-reg cell ratio. Treatment of 4T1 and B16GM tumor-bearing mice with IPI-549 for 14 days led to a significant increase in the CD8+/T-reg cell ratio. Together these data show that IPI-549 treatment leads to a proinflammatory tumor microenvironment. Importantly, gene and protein expression analysis of whole tumor tissue collected from IPI-549-treated mice revealed a cytotoxic T-cell signature characterized by increased production of proinflammatory cytokines, and enhanced expression costimulatory and coinhibitory genes relative to vehicle-treated animals. These findings indicate that IPI-549 increases antitumor immunity by remodeling the tumor-immune microenvironment via blockade of tumor-associated myeloid cells. In addition, the up-regulation of costimulatory and coinhibitory genes with IPI-549 treatment provides a mechanistic rationale for the observed combination activity with immune checkpoint inhibition. IPI-549 is currently in Phase I development, both as a single agent and in combination with an anti-PD-1 antibody, in solid tumors (ClinicalTrials.gov NCT02637531). Citation Format: Matthew Rausch, Jeremy Tchaicha, Thomas Tibbitts, Olivier De Henau, Sujata Sharma, Melissa Pink, Joseph Gladstone, Jennifer Proctor, Mark Douglas, Howard Stern, Taha Merghoub, Jedd Wolchok, Karen McGovern, Jeff Kutok, David Winkler. The PI3K-γ inhibitor, IPI-549, increases antitumor immunity by targeting tumor-associated myeloid cells and remodeling the immune-suppressive tumor microenvironment [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B032.

La inhibición de la PI3Kγ protege contra la miocardiopatía diabética en ratones

Introduction and objectivesCardiovascular diseases, including cardiomyopathy, are the major complications in diabetes. A deeper understanding of the molecular mechanisms leading to cardiomyopathy is critical for developing novel therapies. We proposed phosphoinositide3-kinase gamma (PI3Kγ) as a molecular target against diabetic cardiomyopathy, given the role of PI3Kγ in cardiac remodeling to pressure overload. Given the availability of a pharmacological inhibitor of this molecular target GE21, we tested the validity of our hypothesis by inducing diabetes in mice with genetic ablation of PI3Kγ or knock-in for a catalytically inactive PI3Kγ. MethodsMice were made diabetic by streptozotocin. Cardiac function was assessed by serial echocardiographic analyses, while fibrosis and inflammation were evaluated by histological analysis. ResultsDiabetes induced cardiac dysfunction in wild-type mice. Systolic dysfunction was completely prevented, and diastolic dysfunction was partially blocked, in both PI3Kγ knock-out and kinase-dead mice. Cardiac dysfunction was similarly rescued by administration of the PI3Kγ inhibitor GE21 in a dose-dependent manner. These actions of genetic and pharmacological PI3Kγ inhibition were associated with a decrease in inflammation and fibrosis in diabetic hearts. ConclusionsOur study demonstrates a fundamental role of PI3Kγ in diabetic cardiomyopathy in mice and the beneficial effect of pharmacological PI3Kγ inhibition, highlighting its potential as a promising strategy for clinical treatment of cardiac complications of diabetic patients.Full English text available from: www.revespcardiol.org/en

PI3Kγ Inhibition Protects Against Diabetic Cardiomyopathy in Mice

Introduction and objectivesCardiovascular diseases, including cardiomyopathy, are the major complications in diabetes. A deeper understanding of the molecular mechanisms leading to cardiomyopathy is critical for developing novel therapies. We proposed phosphoinositide3-kinase gamma (PI3Kγ) as a molecular target against diabetic cardiomyopathy, given the role of PI3Kγ in cardiac remodeling to pressure overload. Given the availability of a pharmacological inhibitor of this molecular target GE21, we tested the validity of our hypothesis by inducing diabetes in mice with genetic ablation of PI3Kγ or knock-in for a catalytically inactive PI3Kγ. MethodsMice were made diabetic by streptozotocin. Cardiac function was assessed by serial echocardiographic analyses, while fibrosis and inflammation were evaluated by histological analysis. ResultsDiabetes induced cardiac dysfunction in wild-type mice. Systolic dysfunction was completely prevented, and diastolic dysfunction was partially blocked, in both PI3Kγ knock-out and kinase-dead mice. Cardiac dysfunction was similarly rescued by administration of the PI3Kγ inhibitor GE21 in a dose-dependent manner. These actions of genetic and pharmacological PI3Kγ inhibition were associated with a decrease in inflammation and fibrosis in diabetic hearts. ConclusionsOur study demonstrates a fundamental role of PI3Kγ in diabetic cardiomyopathy in mice and the beneficial effect of pharmacological PI3Kγ inhibition, highlighting its potential as a promising strategy for clinical treatment of cardiac complications of diabetic patients.