ABSTRACT
The weak immunogenicity of gliomas presents a barrier for effective immunotherapy. Na/H exchanger isoform 1 (NHE1) maintains alkaline intracellular pH (pHi) of glioma cells and acidic microenvironment. In addition, NHE1 is expressed in tumor-associated microglia and tumor-associated macrophages (TAMs) and involved in protumoral communications between glioma and TAMs. Therefore, we hypothesize that NHE1 plays a role in developing tumor resistance and immunosuppressive tumor microenvironment. In this study, we investigated the efficacy of pharmacological inhibition of NHE1 on combinatorial therapies. Here we show that temozolomide (TMZ) treatment stimulates NHE1 protein expression in two intracranial syngeneic mouse glioma models (SB28, GL26). Pharmacological inhibition of NHE1 potentiated the cytotoxic effects of TMZ, leading to reduced tumor growth and increased median survival of mice. Blockade of NHE1 stimulated proinflammatory activation of TAM and increased cytotoxic T cell infiltration into tumors. Combining TMZ, anti-PD-1 antibody treatment with NHE1 blockade significantly prolonged the median survival in the mouse glioma model. These results demonstrate that pharmacological inhibition of NHE1 protein presents a new strategy for potentiating TMZ-induced cytotoxicity and increasing tumor immunogenicity for immunotherapy to improve glioma therapy.
Subject(s)
Glioma/drug therapy , Glioma/metabolism , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/metabolism , Sodium-Hydrogen Exchanger 1/metabolism , Temozolomide/pharmacology , Animals , Antibodies/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Female , Immunotherapy/methods , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Microglia/drug effects , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Cytotoxic/metabolism , Tumor Microenvironment/drug effectsABSTRACT
Na+ /H+ exchanger (NHE1) activation is required for multiple microglial functions. We investigated effects of selective deletion of microglial Nhe1 in Cx3cr1-CreER ;Nhe1f/f mice on neuroinflammation and tissue repair after ischemic stroke. Infarct volume was similar in corn oil or tamoxifen (Tam)-treated mice at 48 hr and 14 days post-stroke. However, the Tam-treated mice showed significantly higher survival rate and faster neurological function recovery during day 1-14 post-stroke. Deletion of microglial Nhe1 prevented the elevation of CD11b+ /CD45low-med microglia in the ischemic hemisphere at day 3 post-stroke, but stimulated expression of Ym1, CD68, TGF-ß, IL-10, decreased expression of CD86 and IL-1ß, and reduced GFAP+ reactive astrocytes. Moreover, at day 14 post-stroke, enhanced white matter myelination was detected in the microglial Nhe1 deleted mice. In comparison, neuronal Nhe1-null mice (the CamKII-Cre+/- ;Nhe1f/f mice) showed a significant reduction in both acute and subacute infarct volume, along with increased survival rate and moderate neurological function recovery. However, these neuronal Nhe1-null mice did not exhibit reduced activation of CD11b+ /CD45low-med microglia or CD11b+ /CD45hi macrophages in the ischemic brains, and they exhibited no reductions in white matter lesions. Taken together, this study demonstrated that deletion of microglial and neuronal Nhe1 had differential effects on ischemic brain damage. Microglial NHE1 is involved in pro-inflammatory responses during post-stroke brain tissue repair. In contrast, neuronal NHE1 activation is directly associated with the acute ischemic neuronal injury but not inflammation. Our study reveals that NHE1 protein is a potential therapeutic target critical for differential regulation of ischemic neuronal injury, demyelination and tissue repair.
Subject(s)
CX3C Chemokine Receptor 1/metabolism , Demyelinating Diseases/etiology , Infarction, Middle Cerebral Artery/complications , Microglia/metabolism , Recovery of Function/physiology , Sodium-Hydrogen Exchanger 1/metabolism , White Matter/pathology , Animals , Brain/diagnostic imaging , Brain/drug effects , CX3C Chemokine Receptor 1/genetics , Calcium-Binding Proteins/metabolism , Demyelinating Diseases/drug therapy , Disease Models, Animal , Female , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Infarction, Middle Cerebral Artery/diagnostic imaging , Infarction, Middle Cerebral Artery/drug therapy , Macrophages/metabolism , Macrophages/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microfilament Proteins/metabolism , Microglia/drug effects , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Recovery of Function/drug effects , Sodium-Hydrogen Exchanger 1/genetics , Somatosensory Disorders/etiology , Tamoxifen/pharmacology , White Matter/diagnostic imagingABSTRACT
The WNK-SPAK kinase signaling pathway controls renal NaCl reabsorption and systemic blood pressure by regulating ion transporters and channels. A WNK3-SPAK complex is highly expressed in brain, but its function in this organ remains unclear. Here, we investigated the role of this kinase complex in brain edema and white matter injury after ischemic stroke. Wild-type, WNK3 knockout, and SPAK heterozygous or knockout mice underwent transient middle cerebral artery occlusion. One cohort of mice underwent magnetic resonance imaging. Ex-vivo brains three days post-ischemia were imaged by slice-selective spin-echo diffusion tensor imaging magnetic resonance imaging, after which the same brain tissues were subjected to immunofluorescence staining. A second cohort of mice underwent neurological deficit analysis up to 14 days post-transient middle cerebral artery occlusion. Relative to wild-type mice, WNK3 knockout, SPAK heterozygous, and SPAK knockout mice each exhibited a >50% reduction in infarct size and associated cerebral edema, significantly less demyelination, and improved neurological outcomes. We conclude that WNK3-SPAK signaling regulates brain swelling, gray matter injury, and demyelination after ischemic stroke, and that WNK3-SPAK inhibition has therapeutic potential for treating malignant cerebral edema in the setting of middle cerebral artery stroke.
Subject(s)
Brain Edema/genetics , Brain/pathology , Gene Deletion , Infarction, Middle Cerebral Artery/genetics , Protein Serine-Threonine Kinases/genetics , Animals , Brain/metabolism , Brain Edema/etiology , Brain Edema/pathology , Female , Infarction, Middle Cerebral Artery/complications , Infarction, Middle Cerebral Artery/pathology , Male , Mice, Inbred C57BL , Mice, Knockout , Myelin Sheath/genetics , Myelin Sheath/metabolism , Myelin Sheath/pathology , White Matter/metabolism , White Matter/pathologyABSTRACT
Microglia play important roles in extracellular matrix remodeling, tumor invasion, angiogenesis, and suppression of adaptive immunity in glioma. Na(+)/H(+) exchanger isoform 1 (NHE1) regulates microglial activation and migration. However, little is known about the roles of NHE1 in intratumoral microglial activation and microglia-glioma interactions. Our study revealed up-regulation of NHE1 protein expression in both glioma cells and tumor-associated Iba1(+) microglia in glioma xenografts and glioblastoma multiforme microarrays. Moreover, we observed positive correlation of NHE1 expression with Iba1 intensity in microglia/macrophages. Glioma cells, via conditioned medium or non-contact glioma-microglia co-cultures, concurrently upregulated microglial expression of NHE1 protein and other microglial activation markers (iNOS, arginase-1, TGF-ß, IL-6, IL-10 and the matrix metalloproteinases MT1-MMP and MMP9). Interestingly, glioma-stimulated microglia reciprocally enhanced glioma proliferation and migration. Most importantly, inhibition of microglial NHE1 activity via small interfering RNA (siRNA) knockdown or the potent NHE1-specific inhibitor HOE642 significantly attenuated microglial activation and abolished microglia-stimulated glioma migration and proliferation. Taken together, our findings provide the first evidence that NHE1 function plays an important role in glioma-microglia interactions, enhancing glioma proliferation and invasion by stimulating microglial release of soluble factors. NHE1 upregulation is a novel marker of the glioma-associated microglial activation phenotype. Inhibition of NHE1 represents a novel glioma therapeutic strategy by targeting tumor-induced microglial activation.
