PI3K p85 ß regulatory subunit deficiency does not affect NK cell differentiation and increases NKG2D-mediated activation.

Activation of NK cells depends on a balance between activating and inhibitory signals. Class Ia PI3K are heterodimeric proteins with a catalytic and a regulatory subunit and have a central role in cell signaling by associating with tyrosine kinase receptors to trigger signaling cascades. The regulatory p85 subunit participates in signaling through NKG2D, one of the main activating receptors on NK cells, via its interaction with the adaptor protein DAP10. Although the effects of inhibiting catalytic subunits or deleting the regulatory p85α subunit have been studied, little attention has focused on the role of the p85ß subunit in NK cells. Using p85ß knockout mice, we found that p85ß deficiency does not alter NK cell differentiation and maturation in spleen or bone marrow. NK cells from p85ß-/- mice nonetheless produced more IFN-γ and degranulated more effectively when stimulated with anti-NKG2D antibody. These cells also degranulated and killed NKG2D ligand-expressing target cells more efficiently. We show that p85ß deficiency impaired NKG2D internalization, which could contribute to the activated phenotype. Decreasing p85ß subunit protein levels might thus constitute a therapeutic target to promote NK cell activity toward NKG2D ligand-expressing cells.

Recent findings on the role of natural killer cells in the pathogenesis of systemic lupus erythematosus.

Systemic lupus erythematosus is a chronic, multifactorial autoimmune disease of complex etiology, characterized by loss of tolerance to nuclear autoantigens, expansion of autoreactive T and B cell clones, polyclonal B cell activation that gives rise to hypergammaglobulinemia, and increased autoantibody production, as well as immune complex deposition and multiorgan tissue inflammation. As disease progresses, immune cells, mainly T cells and macrophages, infiltrate affected organs and amplify the local inflammatory response. Natural killer cells are large, granular lymphocytes that are an important link between the innate and adaptive immune systems; variations in their activity correlate with several autoimmune diseases. To date, the literature has disregarded natural killer cells as relevant modulators in systemic lupus erythematosus pathogenesis, as these cells are few in number and show a dysfunctional phenotype in patients with active systemic lupus erythematosus. This review focuses on research that could help define the role of natural killer cells in systemic lupus erythematosus and their function in regulating this autoimmune disorder in nonlymphoid organs.

NKG2D ligand overexpression in lupus nephritis correlates with increased NK cell activity and differentiation in kidneys but not in the periphery.

NK cells are a major component of the immune system, and alterations in their activity are correlated with various autoimmune diseases. In the present work, we observed an increased expression of the NKG2D ligand MICA in SLE patients' kidneys but not healthy subjects. We also show glomerulus-specific expression of the NKG2D ligands Rae-1 and Mult-1 in various murine SLE models, which correlated with a higher number of glomerular-infiltrating NK cells. As the role of NK cells in the immunopathogenesis of SLE is poorly understood, we explored NK cell differentiation and activity in tissues and organs in SLE-prone murine models by use of diseased and prediseased MRL/MpJ and MRL/lpr mice. We report here that phenotypically iNK cells accumulate only in the spleen but not in BM or kidneys of diseased mice. Infiltrating NK cells in kidneys undergoing a lupus nephritic process showed a more mature, activated phenotype compared with kidney, as well as peripheral NK cells from prediseased mice, as determined by IFN-γ and STAT5 analysis. These findings and the presence of glomerulus-specific NKG2D ligands in lupus-prone mice identify a role for NK cells and NKG2D ligands in the lupus nephritic process, which could aid in understanding their role in human SLE.

PI3K p110δ is expressed by gp38(-)CD31(+) and gp38(+)CD31(+) spleen stromal cells and regulates their CCL19, CCL21, and LTßR mRNA levels.

The role of p110δ PI3K in lymphoid cells has been studied extensively, showing its importance in immune cell differentiation, activation and development. Altered T cell localization in p110δ-deficient mouse spleen suggested a role for p110δ in non-hematopoietic stromal cells, which maintain hematopoietic cell segregation. We tested this hypothesis using p110δ(WT/WT) mouse bone marrow to reconstitute lethally irradiated p110δ(WT/WT) or p110δ(D910A/D910A) (which express catalytically inactive p110δ) recipients, and studied localization, number and percentage of hematopoietic cell subsets in spleen and lymph nodes, in homeostatic conditions and after antigen stimulation. These analyses showed diffuse T cell areas in p110δ(D910A/D910A) and in reconstituted p110δ(D910A/D910A) mice in homeostatic conditions. In these mice, spleen CD4(+) and CD8(+) T cell numbers did not increase in response to antigen, suggesting that a p110δ(D910A/D910A) stroma defect impedes correct T cell response. FACS analysis of spleen stromal cell populations showed a decrease in the percentage of gp38(-)CD31(+) cells in p110δ(D910A/D910A) mice. qRT-PCR studies detected p110δ mRNA expression in p110δ(WT/WT) spleen gp38(-)CD31(+) and gp38(+)CD31(+) subsets, which was reduced in p110δ(D910A/D910A) spleen. Lack of p110δ activity in these cell populations correlated with lower LTßR, CCL19 and CCL21 mRNA levels; these molecules participate in T cell localization to specific spleen areas. Our results could explain the lower T cell numbers and more diffuse T cell areas found in p110δ(D910A/D910A) mouse spleen, as well as the lower T cell expansion after antigen stimulation in p110δ(D910A/D910A) compared with p110δ(WT/WT) mice.

PI3K p110γ deletion attenuates murine atherosclerosis by reducing macrophage proliferation but not polarization or apoptosis in lesions.

Atherosclerosis is an inflammatory disease regulated by infiltrating monocytes and T cells, among other cell types. Macrophage recruitment to atherosclerotic lesions is controlled by monocyte infiltration into plaques. Once in the lesion, macrophage proliferation in situ, apoptosis, and differentiation to an inflammatory (M1) or anti-inflammatory phenotype (M2) are involved in progression to advanced atherosclerotic lesions. We studied the role of phosphoinositol-3-kinase (PI3K) p110γ in the regulation of in situ apoptosis, macrophage proliferation and polarization towards M1 or M2 phenotypes in atherosclerotic lesions. We analyzed atherosclerosis development in LDLR(-/-)p110γ(+/-) and LDLR(-/-)p110γ(-/-) mice, and performed expression and functional assays in tissues and primary cells from these and from p110γ(+/-) and p110γ(-/-) mice. Lack of p110γ in LDLR(-/-) mice reduces the atherosclerosis burden. Atherosclerotic lesions in fat-fed LDLR(-/-)p110γ(-/-) mice were smaller than in LDLR(-/-)p110γ(+/-) controls, which coincided with decreased macrophage proliferation in LDLR(-/-)p110γ(-/-) mouse lesions. This proliferation defect was also observed in p110γ(-/-) bone marrow-derived macrophages (BMM) stimulated with macrophage colony-stimulating factor (M-CSF), and was associated with higher intracellular cyclic adenosine monophosphate (cAMP) levels. In contrast, T cell proliferation was unaffected in LDLR(-/-)p110γ(-/-) mice. Moreover, p110γ deficiency did not affect macrophage polarization towards the M1 or M2 phenotypes or apoptosis in atherosclerotic plaques, or polarization in cultured BMM. Our results suggest that higher cAMP levels and the ensuing inhibition of macrophage proliferation contribute to atheroprotection in LDLR(-/-) mice lacking p110γ. Nonetheless, p110γ deletion does not appear to be involved in apoptosis, in macrophage polarization or in T cell proliferation.

Dimercaptosuccinic acid-coated magnetite nanoparticles for magnetically guided in vivo delivery of interferon gamma for cancer immunotherapy.

As radio- and chemotherapy-based cancer treatments affect both tumors and healthy tissue, cancer immunotherapy attempts to specifically enhance the natural immune response to tumor cells. In mouse models of cancer, we tested uniform dimercaptosuccinic acid (DMSA)-coated monodisperse magnetic nanoparticles as a delivery system for the anti-tumorigenic cytokine IFN-γ. IFN-γ-adsorbed DMSA-coated magnetic nanoparticles were targeted to the tumor site by application of an external magnetic field. We analyzed nanoparticle biodistribution before and after IFN-γ conjugation, as well as the efficiency of nanoparticle accumulation in tumors, IFN-γ release in the area of interest, and the effects of both on tumor development. At the tumor site, we observed a high degree of nanoparticle accumulation and of cytokine delivery, which led to increased T cell and macrophage infiltration and promoted an anti-angiogenic effect. The combined action led to a notable reduction in tumor size. Our findings indicate that IFN-γ-adsorbed DMSA-coated magnetite nanoparticles can be used as an efficient in vivo drug delivery system for tumor immunotherapy.