Modulation of NLRP3 inflammasomes activation contributes to improved survival and function of mesenchymal stromal cell spheroids.

Mesenchymal stem cells (MSCs) are ubiquitous multipotent cells that exhibit significant therapeutic potentials in a variety of disorders. Nevertheless, their clinical efficacy is limited owing to poor survival, low rate of engraftment, and impaired potency upon transplantation. Spheroidal three-dimensional (3D) culture of MSCs (MSC3D) has been proven to better preserve their in vivo functional properties. However, the molecular mechanisms underlying the improvement in MSC function by spheroid formation are not clearly understood. NLRP3 inflammasomes, a key component of the innate immune system, have recently been shown to play a role in cell fate decision of MSCs. The present study examined the role of NLRP3 inflammasomes in the survival and potency of MSC spheroids. We found that MSC3D led to decreased activation of NLRP3 inflammasomes through alleviation of ER stress in an autophagy-dependent manner. Importantly, downregulation of NLRP3 inflammasomes signaling critically contributes to the enhanced survival rate in MSC3D through modulation of pyroptosis and apoptosis. The critical role of NLRP3 inflammasome suppression in the enhanced therapeutic efficacy of MSC spheroids was further confirmed in an in vivo mouse model of DSS-induced colitis. These findings suggest that 3D culture confers survival and functional advantages to MSCs by suppressing NLRP3 inflammasome activation.

Geranylgeranyl pyrophosphate amplifies Treg differentiation via increased IL-2 expression to ameliorate DSS-induced colitis.

Blocking the mevalonate pathway for cholesterol reduction by using statin may have adverse effects including statin-induced colitis. Moreover, one of the predisposing factors for colitis is an imbalanced CD4+ T cell, which can be observed on the complete deletion of HMG-CoA reductase (HMGCR), a target of statins. In this study, we inquired geranylgeranyl pyrophosphate (GGPP) is responsible for maintaining the T-cell homeostasis. Following dextran sulfate sodium (DSS)-induced colitis, simvastatin increased the severity of disease, while cotreatment with GGPP, but not with cholesterol, reversed the disease magnitude. GGPP ameliorated DSS-induced colitis by increasing Treg cells. GGPP amplified Treg differentiation through increased IL-2/STAT 5 signaling. GGPP prenylated Ras protein, a prerequisite for extracellular signal-regulated kinase (ERK) pathway activation, leading to increased IL-2 production. Higher simvastatin dose increased the severity of colitis. GGPP ameliorated simvastatin-increased colitis by increasing Treg cells. Treg cells, which have the capacity to suppress inflammatory T cells and were generated through IL-2/STAT5 signaling, increased IL-2 production through prenylation and activation of the Ras/ERK pathway.

AMPK promotes antitumor immunity by downregulating PD-1 in regulatory T cells via the HMGCR/p38 signaling pathway.

BACKGROUND: AMP-activated protein kinase (AMPK) is a metabolic sensor that maintains energy homeostasis. AMPK functions as a tumor suppressor in different cancers; however, its role in regulating antitumor immunity, particularly the function of regulatory T cells (Tregs), is poorly defined. METHODS: AMPKα1fl/flFoxp3YFP-Cre, Foxp3YFP-Cre, Rag1-/-, and C57BL/6 J mice were used for our research. Flow cytometry and cell sorting, western blotting, immuno-precipitation, immuno-fluorescence, glycolysis assay, and qRT-PCR were used to investigate the role of AMPK in suppressing programmed cell death 1 (PD-1) expression and for mechanistic investigation. RESULTS: The deletion of the AMPKα1 subunit in Tregs accelerates tumor growth by increasing the expression of PD-1. Metabolically, loss of AMPK in Tregs promotes glycolysis and the expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), a key enzyme of the mevalonate pathway. Mechanistically, AMPK activates the p38 mitogen-activated protein kinase (MAPK) that phosphorylates glycogen synthase kinase-3ß (GSK-3ß), inhibiting the expression of PD-1 in Tregs. CONCLUSION: Our study identified an AMPK regulatory mechanism of PD-1 expression via the HMGCR/p38 MAPK/GSK3ß signaling pathway. We propose that the AMPK activator can display synergic antitumor effect in murine tumor models, supporting their potential clinical use when combined with anti-PD-1 antibody, anti-CTLA-4 antibody, or a HMGCR inhibitor.

Reactive oxygen species-responsive dual-targeted nanosystem promoted immunogenic cell death against breast cancer.

The development of an optimal treatment modality to improve the therapeutic outcome of breast cancer patients is still difficult. Poor antigen presentation to T cells is a major challenge in cancer immunotherapy. In this study, a synergistic immunotherapy strategy for breast cancer incorporating immune cell infiltration, immunogenic cell death (ICD), and dendritic cell (DC) maturation through a reactive oxygen species (ROS)-responsive dual-targeted smart nanosystem (anti-PD-L1-TKNP) for the simultaneous release of DOX, R848, and MIP-3α in the tumor microenvironment is reported. Following local injection, anti-PD-L1-DOX-R848-MIP-3α/thioketal nanoparticle (TKNP) converts tumor cells to a vaccine owing to the combinatorial effect of DOX-induced ICD, R848-mediated immunostimulatory properties, and MIP-3α-induced immune cell recruitment in the tumor microenvironment. Intratumoral injection of anti-PD-L1-DOX-R848-MIP-3α/TKNP caused significant regression of breast cancer. Mechanistic studies reveal that anti-PD-L1-DOX-R848-MIP-3α/TKNP specifically targets tumor tissue, resulting in maximum exposure of calreticulin (CRT) and HMGB1 in tumors, and significantly enhances intratumoral infiltration of CD4+ and CD8+ T cells in tumors. Therefore, a combined strategy using dual-targeted ROS-responsive TKNP highlights the significant application of nanoparticles in modulating the tumor microenvironment and could be a clinical treatment strategy for effective breast cancer management.

Methionine consumption by cancer cells drives a progressive upregulation of PD-1 expression in CD4 T cells.

Programmed cell death protein 1 (PD-1), expressed on tumor-infiltrating T cells, is a T cell exhaustion marker. The mechanisms underlying PD-1 upregulation in CD4 T cells remain unknown. Here we develop nutrient-deprived media and a conditional knockout female mouse model to study the mechanism underlying PD-1 upregulation. Reduced methionine increases PD-1 expression on CD4 T cells. The genetic ablation of SLC43A2 in cancer cells restores methionine metabolism in CD4 T cells, increasing the intracellular levels of S-adenosylmethionine and yielding H3K79me2. Reduced H3K79me2 due to methionine deprivation downregulates AMPK, upregulates PD-1 expression and impairs antitumor immunity in CD4 T cells. Methionine supplementation restores H3K79 methylation and AMPK expression, lowering PD-1 levels. AMPK-deficient CD4 T cells exhibit increased endoplasmic reticulum stress and Xbp1s transcript levels. Our results demonstrate that AMPK is a methionine-dependent regulator of the epigenetic control of PD-1 expression in CD4 T cells, a metabolic checkpoint for CD4 T cell exhaustion.

AMPK suppresses Th2 cell responses by repressing mTORC2.

Allergic inflammation is a T helper 2 (Th2) cell-driven pathophysiological phenomenon, but the mechanism by which the metabolic cascade affects Th2 cell differentiation remains unclear. In this study, we investigated the roles of AMP-activated protein kinase (AMPK) and intracellular energy sensors in Th2 cell differentiation and the pathogenesis of allergic inflammation. Accordingly, T-cell-specific AMPK or Sirtuin 1 (Sirt1)-knockout mice were subjected to allergic inflammation, and their Th2 cell responses were investigated. The results demonstrated that inducing allergic inflammation in AMPK- and Sirt1-knockout mice increased Th2 cell responses and exacerbated allergic phenotypes. Furthermore, treatment with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMPK, ameliorated allergic inflammation in mice. Mechanistically, our findings revealed that AMPK repressed mechanistic target of rapamycin complex 2 (mTORC2), which downregulated the expression of suppressor of cytokine signaling 5 (SOCS5) in CD4+ T cells. In addition, the loss of AMPK signaling reduced SOCS5 expression and increased interleukin-4-STAT6-GATA3 axis-mediated Th2 cell differentiation. Finally, the T-cell-specific deletion of Rictor, a member of mTORC2, in Sirt1T-KO mice led to the reversal of allergic exacerbation to the level in control mice. Overall, our findings suggest that AMPK in CD4+ T cells inhibits the differentiation of Th2 cells by repressing mTORC2 and thus serves as a potential target for Th2 cell-associated diseases.

T-cell engaging poly(lactic-co-glycolic acid) nanoparticles as a modular platform to induce a potent cytotoxic immunogenic response against PD-L1 overexpressing cancer.

Bispecific nanoparticles (NPs) are conjugated with two antibodies that enhance T cell cytotoxicity by sequentially targeting CD3 and tumor-specific proteins. This interaction redirects T cells to specific tumor antigens and activates them to lyse tumor cells by blocking two different signaling pathways simultaneously. This study developed NP-based bispecific T-cell engagers (nanoBiTEs), which are R848-loaded bispecific poly(lactic-co-glycolic acid) NPs decorated with anti-CD3 antibody targeting T cells and anti-PD-L1 antibody targeting PD-L1 ligands (bis-R848-PLGA-NPs). Bis-R848-PLGA-NPs enhance the immunogenic response in destroying cancer cells by restoring the T cell effector functions. These interactions allow T cells to come in close proximity to the tumor cells. Finally, the release of R848 from PLGA-NPs activates dendritic cells, enhancing T cell activation. In vitro results show maximum internalization of bis-R848-PLGA-NPs in SK-OV3 and B16F10 cell lines, attributed to high PD-L1 expression in both cells. Furthermore, bis-R848-PLGA-NPs-treated CD8+ T cells exhibit a significantly increased total amount of CD8+/CD25+, CD8+/CD69+, and cytokine expression that leads to the robust inhibition of PD-L1 expressed cancer cells. Additionally, tumor growth is significantly inhibited by bis-R848-PLGA-NPs in the B16F10 xenograft mouse model and significantly enhanced intratumoral infiltration of CD4+ and CD8+ T cells, as well as tumor-infiltrated cytokines.

Engineering of hybrid spheroids of mesenchymal stem cells and drug depots for immunomodulating effect in islet xenotransplantation.

Immunomodulation is an essential consideration for cell replacement procedures. Unfortunately, lifelong exposure to nonspecific systemic immunosuppression results in immunodeficiency and has toxic effects on nonimmune cells. Here, we engineered hybrid spheroids of mesenchymal stem cells (MSCs) with rapamycin-releasing poly(lactic-co-glycolic acid) microparticles (RAP-MPs) to prevent immune rejection of islet xenografts in diabetic C57BL/6 mice. Hybrid spheroids were rapidly formed by incubating cell-particle mixture in methylcellulose solution while maintaining high cell viability. RAP-MPs were uniformly distributed in hybrid spheroids and sustainably released RAP for ~3 weeks. Locoregional transplantation of hybrid spheroids containing low doses of RAP-MPs (200- to 4000-ng RAP per recipient) significantly prolonged islet survival times and promoted the generation of regional regulatory T cells. Enhanced programmed death-ligand 1 expression by MSCs was found to be responsible for the immunomodulatory performance of hybrid spheroids. Our results suggest that these hybrid spheroids offer a promising platform for the efficient use of MSCs in the transplantation field.

LKB1-PTEN axis controls Th1 and Th17 cell differentiation via regulating mTORC1.

Immuno-environmental change triggers CD4+ T cell differentiation. T cell specialization activates metabolic signal pathways to meet energy requirements. Defective T cell-intrinsic metabolism can aggravate immunopathology in chronic diseases. Liver kinase B1 (LKB1) deletion in T cell or Treg cell results in systemic inflammatory symptoms, indicating a crucial role of LKB1 in T cells. However, the mechanism underlying the development of inflammation is unclear. In our study, LKB1-deficient T cells were differentiated preferentially into Th1 and Th17 cells in the absence of inflammation. Mechanistically, LKB1 directly binds and phosphorylates phosphatase and tensin homolog (PTEN), an upstream regulator of mammalian target of rapamycin complex 1 (mTORC1), which is independent of AMP-activated protein kinase (AMPK). As a result, LKB1 deficiency was associated with increased mTORC1 activity and hypoxia-inducible factor (HIF)1α-mediated glycolysis. Inhibition of glycolysis or biallelic disruption of LKB1 and HIF1α abrogated this phenotype, suggesting Th1- and Th17-biased differentiation in LKB1-deficient T cells was mediated by glycolysis. Our study indicates that LKB1 controls mTORC1 signaling through PTEN activation, not AMPK, which controls effector T cell differentiation in a T cell-intrinsic manner. KEY MESSAGES: • LKB1 maintains T cell homeostasis in a cell intrinsic manner. • Glycolysis is involved in the LKB1-mediated T cell differentiation. • LKB1 phosphorylates PTEN, not AMPK, to regulate mTORC1.

Combination chemotherapeutic and immune-therapeutic anticancer approach via anti-PD-L1 antibody conjugated albumin nanoparticles.

Anticancer regimens have been substantially enriched through monoclonal antibodies targeting immune checkpoints, programmed cell death-1/programmed cell death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte antigen-4. Inconsistent clinical efficacy after solo immunotherapy may be compensated by nanotechnology-driven combination therapy. We loaded human serum albumin (HSA) nanoparticles with paclitaxel (PTX) via nanoparticle albumin-bound technology and pooled them with anti-PD-L1 monoclonal antibody through a pH-sensitive linker for targeting and immune response activation. Our tests demonstrated satisfactory preparation of paclitaxel-loaded, PD-L1-targeted albumin nanoparticles (PD-L1/PTX@HSA). They had small particle size (~200 nm) and polydispersity index (~0.12) and successfully incorporated each constituent. Relative to normal physiological pH, the formulation exhibited higher drug-release profiles favoring cancer cell-targeted release at low pH. Modifying nanoparticles with programmed cell death-ligand 1 increased cancer cell internalization in vitro and tumor accumulation in vivo in comparison with non-PD-L1-modified nanoparticles. PD-L1/PTX@HSA constructed by nanoparticle albumin-bound technology displayed successful tumor inhibition efficacy both in vitro and in vivo. There was successful effector T-cell infiltration, immunosuppressive programmed cell death-ligand 1, and regulatory T-cell suppression because of cytotoxic T-lymphocyte antigen-4 synergy. Moreover, PD-L1/PTX@HSA had low organ toxicity. Hence, the anti-tumor immune responses of PD-L1/PTX@HSA combined with chemotherapy and cytotoxic T-lymphocyte antigen-4 is a potential anti-tumor strategy for improving quantitative and qualitative clinical efficacy.

Preparation and evaluation of dabrafenib-loaded, CD47-conjugated human serum albumin-based nanoconstructs for chemoimmunomodulation.

The transmembrane proteins, CD47 and signal-regulatory protein α are overexpressed in cancer cells and macrophages, respectively, and facilitate the escape of cancer cells from macrophage-mediated phagocytosis. The immunomodulatory and targeting properties of CD47, the chemotherapeutic effects of dabrafenib (D), and the anti-programmed death-1 antibodies (PD-1) pave the way for effective chemoimmunomodulation-mediated anticancer combination therapy. In this study, CD47-conjugated, D-loaded human serum albumin (HSA) nanosystems were fabricated by modified nanoparticle albumin-bound technology. Cis-aconityl-PEG-maleimide (CA), an acid-labile linker, was used to conjugate D@HSA and CD47; the resultant CD47-CA@D@HSA exhibited tumor-specificity through receptor targeting, as well as preferential cleavage and drug release in the acidic tumor microenvironment (pH 5) compared to normal physiological pH conditions (pH 6.5, 7.4). The successful preparation of nanosized (∼220 nm), narrowly dispersed (∼0.13) CD47-CA@D@HSA was proven by physicochemical characterization. In vitro and in vivo internalization, accumulation, cytotoxicity, and apoptosis were observed to be higher with CD47-conjugated nanoconstructs, than with free D or non-targeted nanoconstructs. CD47-CA@D@HSA was found to promote the infiltration of cytotoxic T cells and tumor-associated macrophages into tumors and improve in vivo tumor inhibition. Administration in combination with PD-1 further improved antitumor efficacy by promoting immune responses that blocked the immune checkpoint. No signs of toxicity were seen in mice treated with the nanoconstructs; the formulation was, therefore, thought to be biocompatible and as having potential for clinical use. The targeted chemoimmunomodulation achieved by this combination therapy was found to combat major immunosuppressive facets, making it a viable candidate for use in the treatment of cancer.

Globular Adiponectin Inhibits Breast Cancer Cell Growth through Modulation of Inflammasome Activation: Critical Role of Sestrin2 and AMPK Signaling.

Adiponectin, an adipokine predominantly derived from adipose tissue, exhibits potent antitumor properties in breast cancer cells. However, its mechanisms of action remain elusive. Inflammasomes-intracellular multimeric protein complexes-modulate cancer cell growth in a complicated manner, as well as playing a role in the innate immune system. Herein, we examined the potential role of inflammasomes in the antitumor activity of adiponectin and found that globular adiponectin (gAcrp) significantly suppressed inflammasomes activation in breast cancer cells both in vitro and in vivo conditions, as determined by decreased expression of inflammasomes components, including NOD-like receptor pyrin domain-containing protein 3 (NLRP3) and the apoptosis-associated speck-like protein containing a CARD (ASC), and inhibition of interleukin-1ß and caspase-1 activation. Treatment with pharmacological inhibitors of inflammasomes caused decrease in cell viability, apoptosis induction, and G0/G1 cell cycle arrest, suggesting that inflammasomes activation is implicated in the growth of breast cancer cells. In addition, treatment with gAcrp generated essentially similar results to those of inflammasomes inhibitors, further indicating that suppression of breast cancer cell growth by gAcrp is mediated via modulation of inflammasomes. Mechanistically, gAcrp suppressed inflammasomes activation through sestrin2 (SESN2) induction, liver kinase B1 (LKB-1)-dependent AMP-activated protein kinase (AMPK) phosphorylation, and alleviation of endoplasmic reticulum (ER) stress. Taken together, these results demonstrate that gAcrp inhibits growth of breast cancer cells by suppressing inflammasomes activation, at least in part, via SESN2 induction and AMPK activation-dependent mechanisms.