Phosphofructokinase P fine-tunes T regulatory cell metabolism, function, and stability in systemic autoimmunity.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by defective regulatory T (Treg) cells. Here, we demonstrate that a T cell-specific deletion of calcium/calmodulin-dependent protein kinase 4 (CaMK4) improves disease in B6.lpr lupus-prone mice and expands Treg cells. Mechanistically, CaMK4 phosphorylates the glycolysis rate-limiting enzyme 6-phosphofructokinase, platelet type (PFKP) and promotes aerobic glycolysis, while its end product fructose-1,6-biphosphate suppresses oxidative metabolism. In Treg cells, a CRISPR-Cas9-enabled Pfkp deletion recapitulated the metabolism of Camk4-/- Treg cells and improved their function and stability in vitro and in vivo. In SLE CD4+ T cells, PFKP enzymatic activity correlated with SLE disease activity and pharmacologic inhibition of CaMK4-normalized PFKP activity, leading to enhanced Treg cell function. In conclusion, we provide molecular insights in the defective metabolism and function of Treg cells in SLE and identify PFKP as a target to fine-tune Treg cell metabolism and thereby restore their function.

Interleukin 23 is elevated in the serum of patients with SLE.

Aim: Interleukin-23 (IL-23) is a cytokine that promotes the differentiation of T cells into pro-inflammatory Th17. We have previously shown that IL-23 is upregulated in systemic lupus erythematosus (SLE) patients and lupus prone mice. As SLE is highly heterogeneous, we asked whether IL-23 production correlates with different manifestations of the disease.Methods: We recruited 56 subjects who fulfilled the ACR criteria for SLE. Interleukin-23 was measured in the serum by ELISA.Results: IL-23 levels were positively correlated with the overall SLE disease activity as measured with the SLEDAI. Moreover, IL-23 correlated with the skin, renal domains of SLEDAI and arthritis but not with cytopenias or serositis. IL-23 did also correlate with anti-dsDNA antibody positivity and inversely correlated with C3 levels. We found no relationship between patients' demographics, prior disease manifestations, medications, or autoantibody profile and IL-23 levels. No immunomodulatory medication seemed to be affecting IL-23 levels suggesting that current medications used in SLE are not as effective in shutting down the IL-23/IL-17 axis. Conclusions: IL-23 levels track SLE disease activity mostly in the renal, skin and musculoskeletal domains. Our data suggest that IL-23 inhibitors may be helpful in combination with current standard of care in alleviating arthritis, renal and cutaneous manifestations of the disease.

T cell Metabolism in Lupus.

Abnormal T cell responses are central to the development of autoimmunity and organ damage in systemic lupus erythematosus. Following stimulation, naïve T cells undergo rapid proliferation, differentiation and cytokine production. Since the initial report, approximately two decades ago, that engagement of CD28 enhances glycolysis but PD-1 and CTLA-4 decrease it, significant information has been generated which has linked metabolic reprogramming with the fate of differentiating T cell in health and autoimmunity. Herein we summarize how defects in mitochondrial dysfunction, oxidative stress, glycolysis, glutaminolysis and lipid metabolism contribute to pro-inflammatory T cell responses in systemic lupus erythematosus and discuss how metabolic defects can be exploited therapeutically.

Pyruvate kinase M2 is requisite for Th1 and Th17 differentiation.

Th1 and Th17 are important in the pathogenesis of autoimmune diseases and they depend on glycolysis as a source of energy. T cell antigen receptor signaling phosphorylates a serine/threonine kinase, calcium/calmodulin-dependent protein kinase IV (CaMK4), and promotes glycolysis. Based on these findings we hypothesized that CaMK4 promotes glycolysis. Camk4-deficient CD4+ T cells and cells treated with a CaMK4 inhibitor had less glycolysis compared with their counterparts. Pull-down of CaMK4 and mass spectrometry identified pyruvate kinase muscle isozyme (PKM), the final rate-limiting enzyme in glycolysis, as a binding partner. Coimmunoprecipitation and Western blotting showed that CaMK4 interacts directly with PKM2. Camk4-deficient CD4+ T cells displayed decreased pyruvate kinase activity. Silencing or pharmacological inhibition of PKM2 reduced glycolysis and in vitro differentiation to Th1 and Th17 cells, while PKM2 overexpression restored Th17 cell differentiation. Treatment with a PKM2 inhibitor ameliorated experimental autoimmune encephalomyelitis and CD4+ T cells treated with PKM2 inhibitor or Pkm2-shRNA caused limited disease activity in an adoptive cell transfer model of experimental autoimmune encephalomyelitis. Our data demonstrate that CaMK4 binds to PKM2 and promotes its activity, which is requisite for Th1 and Th17 differentiation in vitro and in vivo. PKM2 represents a therapeutic target for T cell-dependent autoimmune diseases.

Novel Treatments in Lupus.

PURPOSE OF REVIEW: The standard treatment options for systemic lupus erythematosus (SLE) are focused on non-specific immunosuppression. Over the past few years, scientific studies and ongoing clinical trials have shifted the paradigm with rapid advances in developing biologics and small molecules. A number of monoclonal antibodies and small molecule inhibitors have been developed to target specific pathways involved in SLE. Many of these novel therapeutic agents are already being tested in clinical trials and they may 1 day reshape the landscape of SLE treatment. Herein we review potential future therapeutic options for SLE.

ApoE Receptor 2 Mediation of Trophoblast Dysfunction and Pregnancy Complications Induced by Antiphospholipid Antibodies in Mice.

OBJECTIVE: Pregnancies in women with the antiphospholipid syndrome (APS) are frequently complicated by fetal loss and intrauterine growth restriction (IUGR). How circulating antiphospholipid antibodies (aPL) cause pregnancy complications in APS is poorly understood. We sought to determine whether the low-density lipoprotein receptor family member apolipoprotein E receptor 2 (ApoER2) mediates trophoblast dysfunction and pregnancy complications induced by aPL. METHODS: Placental and trophoblast ApoER2 expression was evaluated by immunohistochemistry and immunoblotting. Normal human IgG and aPL were purified from healthy individuals and APS patients, respectively. The role of ApoER2 in aPL-induced changes in trophoblast proliferation and migration and in kinase activation was assessed using RNA interference in HTR-8/SVneo cells. The participation of ApoER2 in aPL-induced pregnancy loss and IUGR was evaluated in pregnant ApoER2(+/+) and ApoER2(-/-) mice injected with aPL or normal human IgG. RESULTS: We found that ApoER2 is abundant in human and mouse placental trophoblasts and in multiple trophoblast-derived cell lines, including HTR-8/SVneo cells. ApoER2 and its interaction with the cell surface protein ß2 -glycoprotein I were required for aPL-induced inhibition of cultured trophoblast proliferation and migration. In parallel, aPL antagonism of Akt kinase activation by epidermal growth factor in trophoblasts was mediated by ApoER2. Furthermore, in a murine passive-transfer model of pregnancy complications of APS, ApoER2(-/-) mice were protected from both aPL-induced fetal loss and aPL-induced IUGR. CONCLUSION: ApoER2 plays a major role in the attenuation of trophoblast function by aPL, and the receptor mediates aPL-induced pregnancy complications in vivo in mice. ApoER2-directed interventions can now potentially be developed to combat the pregnancy complications associated with APS.

Cholinergic receptors modulate immune complex-induced inflammation in vitro and in vivo.

Cholinergic neural output has been shown to modulate innate immune responses to infection, injury and ischemia through stimulation of α7 nicotinic acetylcholine receptors (α7nAChR) on mononuclear phagocytes. We tested the hypothesis that cholinergic neurotransmitters, similar to those released through activation of a neural reflex, regulate responses to products of the adaptive immune system, specifically immune complex (IC)-mediated activation of effector cells. In this study, we show that stimulation of α7nAChR on human polymorphonuclear neutrophils (PMNs) and blood mononuclear phagocytes in vitro attenuates C5aR- and FcγR-triggered generation of reactive oxygen species, expression of leukocyte markers involved in cell recruitment and adhesion, and release of TNF-α and other proinflammatory cytokines. We show that this pathway is operative in vivo. Ligation of cholinergic receptors blunts IC-triggered responses in the reverse peritoneal Arthus reaction in mice. The selective 7nAChR agonist GTS21 decreased PMN accumulation and release of cytokines and chemokines at sites of IC deposition. In addition, mice lacking α7nAChR had exaggerated responses to reverse peritoneal Arthus reaction characterized by increased infiltration of PMNs and elevated of levels of TNF-α and CXCL1 in peritoneal fluid compared with wild-type mice. Taken together, these findings suggest that cholinergic output has the potential to exert tonic inhibitory activity that dampens responses to ICs and C5a and thus may be a target to minimize tissue damage in autoimmune diseases.

Engraftment of peripheral blood mononuclear cells from systemic lupus erythematosus and antiphospholipid syndrome patient donors into BALB-RAG-2-/- IL-2Rγ-/- mice: a promising model for studying human disease.

OBJECTIVE: To construct a humanized mouse model of systemic lupus erythematosus (SLE) that resembles the human disease in order to define the pathophysiology and targets for treatments. METHODS: We infused peripheral blood mononuclear cells (PBMCs) from SLE patients into BALB- RAG-2-/- IL-2Rγ-/- double-knockout (DKO) mice, which lack T cells, B cells, and natural killer cells. PBMCs from 5 SLE patients and 4 normal donors were infused intravenously/intraperitoneally at a density of 3-5×10(6) cells per animal into nonirradiated 4-5-week-old mice. We evaluated the engraftment of human CD45+ cells and monitored the plasma levels of human IgG, anti-double-stranded DNA (anti-dsDNA) antibody, and anticardiolipin antibody (aCL), as well as proteinuria and kidney histology. RESULTS: There was 100% successful engraftment in 40 DKO mice infused with human PBMCs. In the PBMC fraction from SLE PBMC-infused DKO (SLE-DKO) mice and normal donor PBMC-infused DKO (ND-DKO) mice, an average of 41% and 53% human CD45+ cells, respectively, were observed at 4 weeks postengraftment, with 70-90% CD3+ cells. There were fewer CD3+CD4+ cells (mean±SEM 5.5±2.1%) and more CD3+CD8+ cells (79.4±3.6%) in the SLE-DKO mice as in the SLE patients from which the PBMCs were derived. CD19+ B cells and CD11c+ monocytic cells were found in the spleen, lung, liver, and bone marrow. There was no significant difference in plasma levels of human IgG and anti-dsDNA antibodies between SLE-DKO and ND-DKO mice. Levels of aCL were significantly higher in all SLE-DKO mice infused with PBMCs from an SLE patient who had high titers of aCL. SLE-DKO mice had proteinuria, human IgG deposits in the kidneys, and a shorter life span. In SLE-DKO mice engrafted with PBMCs from the aCL-positive patient, we found microthrombi and infiltration of CD3+, CD8+, and CD19+ cells in the glomeruli, recapitulating the human antiphospholipid syndrome in these mice. CONCLUSION: We established a novel humanized SLE-DKO mouse exhibiting many of the immunologic and clinical features of human SLE.