Adoptive Transfer of Immunomodulatory M2 Macrophages Prevents Type 1 Diabetes in NOD Mice

Macrophages are multifunctional immune cells that may either drive or modulate disease pathogenesis depending on their activation phenotype. Autoimmune type 1 diabetes (T1D) is a chronic proinflammatory condition characterized by unresolved destruction of pancreatic islets. Adoptive cell transfer of macrophages with immunosuppressive properties represents a novel immunotherapy for treatment of such chronic autoimmune diseases. We used a panel of cytokines and other stimuli to discern the most effective regimen for in vitro induction of immunosuppressive macrophages (M2r) and determined interleukin (IL)-4/IL-10/transforming growth factor-β (TGF-β) to be optimal. M2r cells expressed programmed cell death 1 ligand-2, fragment crystallizable region γ receptor IIb, IL-10, and TGF-β, had a potent deactivating effect on proinflammatory lipopolysaccharide/interferon-γ–stimulated macrophages, and significantly suppressed T-cell proliferation. Clinical therapeutic efficacy was assessed after adoptive transfer in NOD T1D mice, and after a single transfer of M2r macrophages, >80% of treated NOD mice were protected against T1D for at least 3 months, even when transfer was conducted just prior to clinical onset. Fluorescent imaging analyses revealed that adoptively transferred M2r macrophages specifically homed to the inflamed pancreas, promoting β-cell survival. We suggest that M2r macrophage therapy represents a novel intervention that stops ongoing autoimmune T1D and may have relevance in a clinical setting.Macrophages have critical functions in both innate and adaptive immune responses. They are present in almost every tissue, recognize exogenous/endogenous danger signals through pattern-recognition receptors, produce various cytokines/chemokines that orchestrate immune responses at the site of inflammation, and function as professional antigen-presenting cells (APCs).Two macrophage activation states have been defined in rodents and humans: “classically activated” (M1) cells have proinflammatory effector functions, and “alternatively activated” (M2) cells have anti-inflammatory properties. The existence of these different activation states implies prominent roles in different phases of an immunological response, i.e., inflammation versus resolution and tissue remodeling. M1 cells are identified by high expression of the enzyme inducible nitric oxide (NO) synthase, a potent respiratory burst, and secretion of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-12. The activation is induced by two signals, one toll-like receptor agonist, such as lipopolysaccharide (LPS), and one cytokine receptor–mediated signal, e.g., interferon-γ (IFN-γ) (1,2).We now understand that there are subpopulations of M2 macrophages, different types of activation leading to different functional phenotypes. M2 cells are generally characterized by secretion of anti-inflammatory cytokines such as IL-10 and by low or no secretion of proinflammatory cytokines. IL-4 was initially identified as an inducer of M2 macrophages (3), and it was later discerned that IL-4, in combination with IL-13, enhanced induction of wound-healing macrophages (M2a) (4). Further investigations determined that stimulation with glucocorticoids (e.g., dexamethasone), IL-10, immune complexes in combination with toll-like receptor agonists, and transforming growth factor-β (TGF-β) could induce at least two further distinct M2 polarization states with immunoregulatory properties (M2b and M2c) (1,5). We have previously studied macrophage phenotypes from autoimmune-resistant and -susceptible rodent strains and have determined that there is a diversity of M1 activation phenotypes; autoimmune-susceptible strains have a common phenotype that contributes to prolongation of inflammation instead of its resolution (6). Similar genetically determined aberrant macrophage phenotypes have also been reported for NOD mice (7,8) and in human type 1 diabetes (T1D) (9,10). This indicates that there is a genetic predisposition for autoimmune susceptibility regarding macrophage phenotype and implies that individuals with autoimmune diseases may lack the ability to generate a cellular phenotype important in the resolution of inflammation.Evidence of “immune regulatory” macrophage activity derives from cancer models in which tumor-associated macrophages have been reported to both suppress tumor immunity and promote tumor survival (5). Several in vitro (11,12) and in vivo disease studies (13–16) have investigated the regulatory role of macrophages in suppressing inflammation, the latter including models of multiple sclerosis, kidney disease, and spinal cord injury. Although these findings clearly indicate the important role of macrophages in the resolution of inflammation, there is no definitive consensus about their mechanism of action or optimal protocols for their induction.T1D is an autoimmune disease in which insulin-producing β-cells in the pancreas are attacked by leukocytes (macrophages and cluster of differentiation [CD]4⁺ and CD8⁺ T cells) (17), which leads to a subsequent loss of glucose control and acute complications. The most widely used animal model for investigation of T1D is the NOD mouse (18) in which diabetes spontaneously develops between 12 and 30 weeks of age after initial leukocyte infiltration into the pancreas (insulitis) between 3 and 5 weeks of age. Macrophages have been demonstrated to have a prominent role in T1D pathogenesis (19). Early M1 macrophage infiltration induces β-cell death and activates cytotoxic CD8⁺ T cells (20,21). Although many studies have provided convincing evidence of the destructive role of M1 macrophages in T1D, it was recently reported that transgenic NOD mice that did not spontaneously develop T1D expressed M2 macrophage–associated genes in the pancreas (22), and that the 10–20% of NOD mice that do not develop T1D possess protective macrophages with a phagocytic/immunosuppressive phenotype (23).Previous studies have investigated the use of cell transfer as an immunomodulatory therapy in NOD mice, with most reports using highly suppressive regulatory T cells (Tregs) (24,25). One drawback of this approach is that Treg antigen specificity is important for their suppressive ability (26), inferring that many Treg specificities might be required for the many pancreatic β-cell antigens implicated in T1D development. Adoptive cell transfer of tolerogenic dendritic cells has also been used in young NOD mice (5–8 weeks of age) during the early period of insulitis (27). For an optimal effect in a clinical setting, it would be advantageous to use cell therapy at the clinical debut of disease.The role of M2 macrophages in T1D and adoptive transfer of regulatory M2 macrophages as a cell therapy in T1D have never been reported before. In this study, we elucidated the ability of IL-4/IL-10/TGF-β to induce an immunosuppressive M2 NOD macrophage phenotype (M2r). When M2r macrophages were adoptively transferred into late-stage prediabetic NOD mice, the onset of T1D was significantly reduced; advanced imaging analysis revealed protection of residual pancreatic islets concomitant with infiltration of the transferred M2r cells.     

Plasma and urinary heme oxygenase-1 in AKI

AKI induces upregulation of heme oxygenase 1 (HO-1), which exerts cytoprotective effects and modulates the renal response to injury, suggesting that a biomarker of intrarenal HO-1 activity may be useful. Because HO-1 largely localizes to the endoplasmic reticulum and has no known secretory pathway, it is unclear whether plasma or urinary levels of HO-1 reflect intrarenal HO-1 expression. We measured plasma and urinary levels of HO-1 by ELISA during the induction and/or maintenance phases of four mouse models of AKI: ischemia/reperfusion, glycerol-induced rhabdomyolysis, cisplatin nephrotoxicity, and bilateral ureteral obstruction. In addition, we measured levels of HO-1 mRNA and protein in the renal cortex. Each AKI model increased renal HO-1 gene expression, which corresponded with release of HO-1 into plasma and urine by 4 hours. Over time, the magnitudes of plasma and urinary HO-1 paralleled renal cortical gene expression. AKI and the associated uremia did not seem to affect extrarenal HO-1 gene activity assessed in the liver, lung, and spleen. In iron-challenged, cultured proximal tubule cells, we observed a positive correlation between HO-1 mRNA level and HO-1 release. In humans, 10 patients with AKI demonstrated markedly higher levels of plasma and urine HO-1 levels than 10 critically ill patients without AKI or 20 patients with CKD or ESRD. In summary, these data suggest that plasma and urinary HO-1 levels may serve as biomarkers of AKI and intrarenal HO-1 gene activity.     

INZ-701 Prevents Ectopic Tissue Calcification and Restores Bone Architecture and Growth in ENPP1-Deficient Mice

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is the major enzyme that cleaves extracellular adenosine triphosphate (ATP) to generate pyrophosphate (PPi), an inorganic metabolite with potent anticalcification activity. Loss-of-function mutations cause hypopyrophosphatemia and lead to a state of ENPP1 deficiency, which has an acute infantile phase known as generalized arterial calcification of infancy (GACI) and a pediatric to adult phase known as autosomal-recessive hypophosphatemic rickets type 2 (ARHR2). ENPP1 deficiency manifests as ectopic calcification of multiple tissues, neointimal proliferation, premature mortality, impaired growth, and bone deformities. INZ-701, a human ENPP1-Fc protein, is in clinical development as an enzyme replacement therapy for the treatment of ENPP1 deficiency. The pharmacokinetic and pharmacodynamic profile and therapeutic effect of INZ-701 were investigated in Enpp1^asj/asj mice, a murine model of ENPP1 deficiency. Enpp1^asj/asj mice have undetectable plasma PPi, lower plasma phosphate, and higher FGF23 levels compared with wild-type (WT) mice. Enpp1^asj/asj mice on the acceleration diet, containing high phosphate and low magnesium, quickly develop clinical signs, including dehydration, rough hair coat, pinned ears, stiffed legs, and hunched back. Enpp1^asj/asj mice treated with vehicle had aforementioned clinical signs plus severe ectopic calcification in multiple tissues and bone defects, characteristics of the clinical phenotype observed in GACI and ARHR2 patients. Our results showed a durable PPi response for more than 3 days after a single dose of INZ-701. Treatment of ENPP1-deficient mice every other day with INZ-701 for 8 weeks restored circulating levels of PPi, prevented pathological calcification in all the tested organs, restored growth parameters, corrected bone defects, improved clinical signs, and decreased mortality in Enpp1^asj/asj mice, demonstrating the potential of INZ-701 to treat ENPP1 deficiency. © 2021 American Society for Bone and Mineral Research (ASBMR).     

Rac1抑制剂上调糖尿病小鼠肾小球nephrin的表达

目的：探讨Rac1抑制剂（NSC33766）对STZ诱导的糖尿病小鼠肾小球内nephrin水平的影响。方法：采用腹腔单剂注射链脲佐菌素（streptozotocin,STZ,150mg/kg）的方法建立小鼠糖尿病模型,检测NSC33766对正常和糖尿病小鼠尿白蛋白排泄率、血清肌酐水平的影响,采用PAS染色观察肾脏组织学的改变,透射电镜观察超微结构的改变;采用免疫组化观察NSC33766对肾组织内足细胞骨架蛋白nephrin表达的影响。结果：NSC33766降低糖尿病小鼠尿白蛋白排泄率;减少肾组织内系膜区细胞外基质的积聚和基底膜的厚度,改善肾小球滤过膜足突融合,上调nephrin表达,未发现NSC33766具有降低血糖的作用。结论：Rac1抑制剂NSC33766可能通过上调肾小球内nephrin水平,改善足细胞骨架的方式,对糖尿病肾脏损伤具有保护作用。     

Apocynin和DPI通过抑制NOD1信号通路在缺血性AKI中的保护作用

目的NOD样受体可促发炎症反应,夹竹桃麻素(apocynin)和二苯基碘鎓(DPI)均为氧化酶抑制剂。本研究观察在缺血性急性肾损伤中抑制氧化应激产生是否能通过NOD1信号通路减轻肾间质炎症反应与细胞凋亡。 方法将雄性Wistar大鼠随机分为4组：假手术(Sham)组、肾脏缺血再灌注(I/R)组、I/R ＋夹竹桃麻素(apocynin)组、I/R ＋二苯基碘鎓(DPI)组。通过Western印记法分别对肾组织核苷酸结合寡聚域样受体1(NOD1)、半胱天冬酶(caspase-1)及细胞核因子-κB(NF-κB)蛋白的表达进行检测;实时定量PCR法对NOD1mRNA的表达进行检测;HE染色法观察肾脏组织学改变;免疫组织化学法检测肾组织肿瘤坏死因子(TNF-ɑ)的表达;TUNEL法检测肾组织细胞凋亡。采用SPSS 22.0统计软包对实验数据进行统计学处理。 结果与Sham组比较,I/R组大鼠肾组织NOD1、caspase-1、NF-κB、TNF-ɑ蛋白表达增加(t＝16.81, t＝ 7.28, t＝ 11.08, t＝ 10.11;P<0.05);NOD1mRNA表达增加(t＝－7.93, P<0.05);HE染色表现为急性肾小管坏死,肾小管损伤评分明显增加(t＝－11.0, P<0.05);TUNEL染色显示缺血区凋亡细胞数目增加(t＝－18.38, P<0.05)。与I/R组比较,I/R＋ apocynin组的NOD1、caspase-1、NF-κB、TNF-ɑ蛋白表达减少(t＝－10.9, t＝－7.6, t＝－4.9, t＝－9.7;P<0.05);NOD1mRNA表达减少(t＝8.49, P<0.05);HE染色后者较前者急性肾小管坏死减轻,肾小管损伤评分减低(t＝－12, P<0.05);TUNEL染色显示缺血区凋亡细胞数目减少(t＝－11.3, P<0.05)。与I/R组比较,I/R＋DPI组的NOD1、caspase-1、NF-κB、TNF-ɑ蛋白表达减少(t＝－11.4, t＝－6.8, t＝－5.4, t＝－10.6, P<0.05);NOD1mRNA表达减少(t＝7.5, P<0.05);HE染色后者较前者急性肾小管坏死减轻,肾小管损伤评分减低(t＝－11, P<0.05);TUNEL染色显示缺血区凋亡细胞数目减少(t＝－10.8, P<0.05)。 结论抑制氧化应激可阻断NOD1样受体依赖的炎症途径与细胞凋亡,从而减轻肾缺血再灌注损伤。     

The Selective Serotonin Reuptake Inhibitor Fluoxetine Directly Inhibits Osteoblast Differentiation and Mineralization During Fracture Healing in Mice

Chronic use of selective serotonin reuptake inhibitors (SSRIs) for the treatment of depression has been linked to osteoporosis. In this study, we investigated the effect of chronic SSRI use on fracture healing in two murine models of bone regeneration. First, we performed a comprehensive analysis of endochondral bone healing in a femur fracture model. C57/BL6 mice treated with fluoxetine, the most commonly prescribed SSRI, developed a normal cartilaginous soft‐callus at 14 days after fracture and demonstrated a significantly smaller and biomechanically weaker bony hard‐callus at 28 days. In order to further dissect the mechanism that resulted in a smaller bony regenerate, we used an intramembranous model of bone healing and revealed that fluoxetine treatment resulted in a significantly smaller bony callus at 7 and 14 days postinjury. In order to test whether the smaller bony regenerate following fluoxetine treatment was caused by an inhibition of osteogenic differentiation and/or mineralization, we employed in vitro experiments, which established that fluoxetine treatment decreases osteogenic differentiation and mineralization and that this effect is serotonin‐independent. Finally, in a translational approach, we tested whether cessation of the medication would result in restoration of the regenerative potential. However, histologic and μCT analysis revealed non‐union formation in these animals with fibrous tissue interposition within the callus. In conclusion, fluoxetine exerts a direct, inhibitory effect on osteoblast differentiation and mineralization, shown in two disparate murine models of bone repair. Discontinuation of the drug did not result in restoration of the healing potential, but rather led to complete arrest of the repair process. Besides the well‐established effect of SSRIs on bone homeostasis, our study provides strong evidence that fluoxetine use negatively impacts fracture healing. © 2017 American Society for Bone and Mineral Research.     

Monoclonal Antibody Specific for TIRC7 Induces Donor-specific Anergy and Prevents Rejection of Cardiac Allografts in Mice

T cell immune response c-DNA (TIRC7) is up-regulated during the early stages of T-cell activation in response to alloantigens. In this study, we analyzed the effects of newly developed monoclonal antibodies (mAb) against TIRC7 in acute cardiac allograft rejection. Fully vascularized heterotopic allogeneic heart transplantation was performed in mice across a full-mismatch barrier (C57Bl/10 into CBA). Recipients received seven injections (day 0-7) of a novel anti-TIRC7 mAb or remained untreated. Graft survival, histology and ex vivo lymphocyte functions were tested. Targeting of TIRC7 with an anti-TIRC7 mAb diminishes lymphocyte infiltration into grafts resulting in delay of morphological graft damage and prolongation of allograft survival. The lymphocytes from anti-TIRC7 mAb-treated animals exhibit hypo-responsiveness without evidence of lymphocyte depletion against the donor allo-antigens. Proliferation and expression of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) were down-regulated while interleukin-4 (IL-4) and IL-10 expression were spared. Moreover, anti-TIRC7 mAb enhanced up-regulation of CTLA-4 expression but suppressed up-regulation of CD25 on stimulated lymphocytes in vitro and in vivo. Ligation of TIRC7 has important effects on the regulation of co-stimulatory signaling pathways associated with suppressing of T-cell activation. Targeting of TIRC7 may therefore provide a novel therapeutic approach for modulating T cell immune responses during organ transplantation.     

Role of the CDK Inhibitor p27 (Kip1) in Mammary Development and Carcinogenesis: Insights from Knockout Mice

The cyclin-dependent kinase inhibitor p27 (Kip1) is an important cell cycle regulatory gene in breast cancer, and decreased p27 expression is associated with poor prognosis. Some investigations of its role in mammary development have demonstrated reduced cyclin D1 expression and consequent lack of lobuloalveolar development, but others have found increased cyclin E-Cdk2 activity and increased proliferation balanced by increased apoptosis. It is unclear at present why these apparently divergent results have been obtained. Mice with reduced p27 gene dosage alone do not develop mammary carcinomas but do display substantially shorter tumor latency upon overexpression of erbB2, consistent with a role for p27 as a mammary tumor suppressor gene. In this review we summarize these and other data addressing the role of p27 in normal mammary epithelium and experimental models of mammary carcinogenesis.