Pd-1基因敲除小鼠构建及初步表型验证

目的:细胞程序性死亡蛋白(programmed death ligand-1,PD-1)是机体T细胞的免疫检查点,也是肿瘤治疗的重要靶点。采用CRISPR/Cas9技术,利用非同源重组修复引入突变的方式,使基因蛋白读码框移码造成PD-1功能缺失,建立Pd-1基因敲除小鼠模型,为深入探究Pd-1基因功能及作用机制提供基础。方法:针对Pd-1基因2-4号外显子设计并合成2对sgRNA片段,与编码Cas9片段共同体外转录,通过受精卵显微注射方法将两者mRNA混合注射到C57BL/6小鼠受精卵中,经PCR产物测序鉴定获得F0代小鼠,之后与野生型C57BL/6小鼠交配获得F1代杂合子小鼠,F1代小鼠自交即获得F2代纯合子小鼠品系(Pd-1^-/-)。刀豆蛋白(concanavalin A,ConA)刺激Pd-1^-/-小鼠后,通过实时荧光定量PCR和流式细胞技术在mRNA和蛋白水平上分别检测Pd-1^-/-小鼠中Pd-1基因在转录和翻译过程中的表达情况,并通过ELISA方法检测Pd-1^-/-小鼠血清中IL-6、IFN-γ、IL12/IL23及TNF-α等因子的表达水平,初步分析Pd-1通路在T细胞反应调控中的作用机制及对免疫刺激的响应情况。结果:PCR及测序结果表明在小鼠基因组中Pd-1基因2-4号外显子被成功敲除;Real-Time PCR实验和流式检测结果显示:与野生型小鼠相比,Pd-1^-/-小鼠脾、肠系膜淋巴结、胸腺和血液各组织中Pd-1表达水平均显著降低;双抗夹心ELISA测定结果显示:Pd-1敲除后经ConA刺激,血清中IL-6和IFN-γ表达上调。结论:成功构建Pd-1基因敲除小鼠模型。Pd-1缺失能够上调IL-6和IFN-γ对ConA刺激的响应,增加ConA引起的炎症反应,为Pd-1的体内基因功能研究提供了新的小鼠模型和研究思路。     

IL-21 accelerates xenogeneic graft-versus-host disease correlated with increased B-cell proliferation

Graft-versus-host disease (GVHD) is a prevalent and potential complication of hematopoietic stem cell transplantation. An animal model, xenogeneic GVHD (X-GVHD), that mimics accurately the clinical presentation of GVHD would provide a tool for investigating the mechanism involved in disease pathogenesis. Murine models indicated that inhibiting IL-21 signaling was a good therapy to reduce GVHD by impairing T cell functions. We sought to investigate the effect of exogenous human IL-21 on the process of X-GVHD. In this study, human IL-21 was expressed by hydrodynamic gene delivery in BALB/c-Rag2^-/- IL-2Rγc^-/- (BRG) immunodeficient mice which were intravenously transplanted human peripheral blood mononuclear cells (hPBMCs). We found that human IL-21 exacerbated X-GVHD and resulted in rapid fatality. As early as 6 days after hPBMCs transplanted to BRG mice, a marked expansion of human CD19⁺ B cells, but not T cells, was observed in spleen of IL-21-treated mice. Compared with control group, IL-21 induced robust immunoglobulin secretion, which was accompanied by increased accumulation of CD19⁺ CD38^high plasma cells in spleen. In addition, we demonstrated that B-cell depletion was able to ameliorate X-GVHD. These results are the first to find in vivo expansion and differentiation of human B cells in response to IL-21, and reveal a correlation between the expansion of B cells and the exacerbation of xenogeneic GVHD. Our findings show evidence of the involvement of B cells in X-GVHD and may have implications in the treatment of the disease     

耐力训练对ApoE-/-致AS小鼠IL-18和IL-10表达的影响

目的：深入观察耐力训练对载脂蛋白E基因敲除（ApoE^-/-）致动脉粥样硬化（AS）小鼠白介素18（IL-18）和白介素10（IL-10）的影响,探讨运动防治AS的可能机制。方法：选取8周龄雄性ApoE^-/-小鼠20只：随机分为2组（n=10）：AS模型组（AC组）和运动干预组（AE组）,AE组进行跑台耐力训练;选取8周龄C57BL/6J雄性小鼠10只作为正常对照组（CC组）。实验持续12周,取主动脉制作冰冻切片,分别用于观察主动脉AS斑块和病理变化以及主动脉IL-18、IL-10蛋白表达;采用ELISA法检测血清IL-18、IL-10水平。结果：①12周高脂膳食致ApoE^-/-小鼠发生典型的AS病变,耐力训练使AS斑块面积显著减少（P<0.01）,病变程度显著减轻。②与CC组比较,AC组、AE组小鼠血清IL-18和IL-10水平均显著升高（P<0.01）,且AC组IL-18/IL-10比值显著升高（P<0.01）。AE组血清IL-18水平及IL-18/IL-10比值均显著低于AC组（P<0.01）。③与CC组比较,AC组、AE组小鼠主动脉IL-10和IL-18蛋白表达均显著升高（P<0.01）,AE组IL-10表达显著高于AC组（P<0.05）,IL-18表达显著低于AC组（P<0.05）。结论：耐力训练通过降低血液和主动脉IL-18及提高IL-10水平,增强了主动脉血管抗炎能力,从而发挥抗AS的作用。     

Dependence of IL-4, IL-13, and nematode-induced alterations in murine small intestinal smooth muscle contractility on Stat6 and enteric nerves

IL-4 and IL-13 promote gastrointestinal worm expulsion in part through effects on nonlymphoid cells, such as intestinal smooth muscle cells. The roles of Stat6 in IL-4-, IL-13-, and parasitic nematode-induced effects on small intestinal smooth muscle contractility were investigated in BALB/c wild-type and Stat6-deficient mice treated with a long-lasting formulation of recombinant mouse IL-4 (IL-4C) or IL-13 for 7 days. Separate groups of BALB/c mice were infected with Nippostrongylus brasiliensis or were drug-cured of an initial Heligmosomoides polygyrus infection and later reinfected. Infected mice were studied 9 and 12 days after inoculation, respectively. Segments of jejunum were suspended in an organ bath, and responses to nerve stimulation and to acetylcholine and substance P in the presence and absence of tetradotoxin, a neurotoxin, were determined. Both IL-4 and IL-13 increased smooth muscle responses to nerve stimulation in wild-type mice, but the effects were greater in IL-13-treated mice and were absent in IL-13-treated Stat6-deficient mice. Similarly, hypercontractile responses to nerve stimulation in H. polygyrus- and N. brasiliensis-infected mice were dependent in part on Stat6. IL-13, H. polygyrus, and N. brasiliensis, but not IL-4, also increased contractility to acetylcholine by mechanisms that involved Stat6 and enteric nerves. These studies demonstrate that both IL-4 and IL-13 promote intestinal smooth muscle contractility, but by different mechanisms. Differences in these effects correlate with differences in the relative importance of these cytokines in the expulsion of enteric nematode parasites.     

IL-6 modulates hepatocyte proliferation via induction of HGF/p21cip1: regulation by SOCS3

The precise role of IL-6 in liver regeneration and hepatocyte proliferation is controversial and the role of SOCS3 in liver regeneration remains unknown. Here we show that in vitro treatment with IL-6 inhibited primary mouse hepatocyte proliferation. IL-6 induced p21cip1 protein expression in primary mouse hepatocytes. Disruption of the p21cip1 gene abolished the inhibitory effect of IL-6 on cell proliferation. Co-culture with nonparenchymal liver cells diminished IL-6 inhibition of hepatocyte proliferation, which was likely due to IL-6 stimulation of nonparenchymal cells to produce HGF. Finally, IL-6 induced higher levels of p21cip1 protein expression and a slightly stronger inhibition of cell proliferation in SOCS3+/- mouse hepatocytes compared to wild-type hepatocytes, while liver regeneration was enhanced and prolonged in SOCS3+/- mice. Our findings suggest that IL-6 directly inhibits hepatocyte proliferation via a p21cip1-dependent mechanism and indirectly enhances hepatocyte proliferation via stimulating nonparenchymal cells to produce HGF. SOCS3 negatively regulates liver regeneration.     

Further characterization of the phenotype of mCry1/mCry2-deficient mice

Mice lacking cryptochromes (mCry1^-/- mCry2^-/-) were kept in a 16h light, 8h dark, light-dark (16:8 LD) cycle and were given additional pulses of light of different brightness, starting 2h after dark onset and lasting for 1h. The suppression of wheel running during these light pulses (i.e., masking) was compared to that of wild types. No evidence of any decrement in the masking response to light was detected. As well as studying masking, minor bouts of activity occurring in the main light portion of a light-dark cycle were quantified. One possible explanation of such predark activity is that some damped endogenous process is spared in mCry1/mCry2 double-knockout mice. (Chronobiology International, 18(4), 613-625, 2001)     

Selective expansion and enhanced anti-tumor effect of antigen-specific CD4+ T cells by retrovirus-mediated IL-15 expression

Mounting evidence has demonstrated that CD4⁺ T cells play an important role in anti-tumor immune responses. Thus, adoptive transfer of these cells may have great potential for anti-cancer therapy. However, due to the difficulty to generate sufficient tumor-specific CD4⁺ T cells, the use of CD4⁺ T cells in tumor therapy is limited. It has been found that IL-15 transfection enhances the proliferation and anti-tumor activity of tumor-specific CD8⁺ T cells, but the effect of IL-15 transfection on CD4⁺ T cells remains unknown. Here, the effects of retrovirus-mediated IL-15 expression in Ova-specific CD4⁺ T cells from Do11.10 mice were evaluated and it was discovered that IL-15 transfected CD4⁺ T cells expressed both soluble and membrane-bound IL-15. Retrovirus-mediated IL-15 expression led to a selective expansion of antigen-specific CD4⁺ T cells by inhibiting their apoptosis. Invivo IL-15 transfected CD4⁺ T cells were more effective in suppressing tumor growth than control retroviral vector transfected ones. To ensure the safety of the method, the employment of thymidine kinase gene made it possible to eliminate these transgenic CD4⁺ T cells following ganciclovir treatment. Together, we show that IL-15 transfection induced a selective expansion of antigen-specific CD4⁺ T cells ex vivo and enhanced their tumor-suppression effects in vivo. This has an important significance for improving the efficacy of adoptive T cell therapy.     

IL-12 inhibits thymic involution by enhancing IL-7- and IL-2-induced thymocyte proliferation

IL-12 has been reported to affect thymic T cell selection, but the role of IL-12 in thymic involution has not been studied. We found that in vivo, IL-12b knockout (IL-12b(-/-)) mice exhibited accelerated thymic involution compared with wild-type (WT) B6 mice. This is characterized by an increase in thymocytes with the early development stage phenotype of CD25(-)CD44(+)CD4(-)CD8(-) in aged IL-12b(-/-) mice. Histologically, there were accelerated degeneration of thymic extracellular matrix and blood vessels, a significantly decreased thymic cortex/medulla ratio, and increased apoptotic cells in aged IL-12b(-/-) mice compared with WT mice. There was, however, no apparent defect in thymic structure and thymocyte development in young IL-12(-/-) mice. These results suggest the importance of IL-12 in maintaining thymic integrity and function during the aging process. Surprisingly, in WT B6 mice, there was no age-related decrease in the levels of IL-12 produced from thymic dendritic cells. Stimulation of thymocytes with IL-12 alone also did not enhance the thymocyte proliferative response in vitro. IL-12, however, provided a strong synergistic effect to augment the IL-7 or IL-2 induced thymocyte proliferative response, especially in aged WT and IL-12b(-/-) mice. Our data strongly support the role of IL-12 as an enhancement cytokine, which acts through its interactions with other cytokines to maintain thymic T cell function and development during aging.     

Muscle injury-induced thymosin β4 acts as a chemoattractant for myoblasts

Thymosin β4 (Tβ4) is a major intracellular G-actin-sequestering peptide. There is increasing evidence to support important extracellular functions of Tβ4 related to angiogenesis, wound healing and cardiovascular regeneration. We investigated the expression of 'Tβ4' and 'thymosin β10', a closely related peptide, during skeletal muscle regeneration in mice and chemotactic responses of myoblasts to these peptides. The mRNA levels of 'Tβ4' and 'thymosin β10' were up-regulated in the early stage of regenerating muscle fibres and inflammatory haematopoietic cells in the injured skeletal muscles of mice. We found that both Tβ4 and its sulphoxized form significantly accelerated wound closure and increased the chemotaxis of C2C12 myoblastic cells. Furthermore, we showed that primary myoblasts and myocytes derived from muscle satellite cells of adult mice were chemoattracted to sulphoxized form of Tβ4. These data indicate that muscle injury enhances the local production of Tβ4, thereby promoting the migration of myoblasts to facilitate skeletal muscle regeneration.     

Prolonged absence of myostatin reduces sarcopenia

Sarcopenia is a progressive age-related loss of skeletal muscle mass and strength. Parabiotic experiments show that circulating factors positively influence the proliferation and regenerative capacity of satellite cells in aged mice. In addition, we believe that negative regulators of muscle mass also serve to balance the signals that influence satellite cell activation and regeneration capacity with ageing. Myostatin, a negative regulator of pre- and postnatal myogenesis, inhibits satellite cell activation and muscle regeneration postnatally. To investigate the role of myostatin during age-related sarcopenia, we examined muscle mass and regeneration in young and old myostatin-null mice. Young myostatin-null muscle fibers were characterized by massive hypertrophy and hyperplasia and an increase in type IIB fibers, resulting in a more glycolytic muscle. With ageing, wild-type muscle became increasingly oxidative and fiber atrophy was prominent. In contrast no fiber type switching was observed and atrophy was minimal in aged myostatin-null muscle. The effect of ageing on satellite cell numbers appeared minimal, however, satellite cell activation declined significantly in both wild-type and myostatin-null muscles. In young mice, lack of myostatin resulted in increased satellite cell number and activation compared to wild-type, suggesting a greater propensity to undergo myogenesis, a difference maintained in the aged mice. In addition, muscle regeneration of myostatin-null muscle following notexin injury was accelerated and fiber hypertrophy and type were recovered with regeneration, unlike in wild-type muscle. In conclusion, a lack of myostatin appears to reduce age-related sarcopenia and loss of muscle regenerative capacity.     

Connexin39 deficient mice display accelerated myogenesis and regeneration of skeletal muscle

During muscle development and regeneration of skeletal muscle in mice connexin43 (Cx43) and connexin39 (Cx39) are specifically expressed: Cx43 in satellite cells and myoblasts, whereas Cx39 is exclusively expressed in myogenin-positive cells. We generated Cx39 deficient mice by replacing the coding region of the Gjd4 gene by DNA coding for the enhanced green fluorescent protein eGFP. Adult Cx39 deficient mice exhibit no obvious phenotypic alterations of skeletal muscle compared to wild type mice in the resting state. However, myogenesis in Cx39 deficient embryos is accelerated as indicated by increased myogenin expression on ED13.5 and ED16.5 and increased expression of Cx43 in developing skeletal muscle. In addition, the regeneration process of skeletal muscle in Cx39 deficient mice is accelerated as shown by a 2 day earlier onset of MyoD and myogenin expression, relative to wild type littermates. Interestingly, Cx43 expression was also upregulated in Cx39 deficient mice during regeneration of skeletal muscle. We hypothesize that Cx43 may compensate for the loss of Cx39 during myogenesis and regeneration.     

Effects of IL-10 and age on IL-6, IL-1beta, and TNF-alpha responses in mouse skeletal and cardiac muscle to an acute inflammatory insult.

Exaggerated proinflammatory cytokine responses can be observed with aging, and reduced levels of the anti-inflammatory cytokine IL-10 may contribute to these responses. IL-10 can reduce IL-6, IL-1beta, and TNF-alpha expression in nonmuscle tissues; however, no studies have examined the combined effects of IL-10 and age on cytokine responses in skeletal and cardiac muscle. These experiments tested the hypothesis that the absence of IL-10, in vivo, is associated with greater IL-6, TNF-alpha, and IL-1beta responses to an inflammatory challenge in skeletal and cardiac muscle and that aging exaggerates these responses. We compared IL-6, IL-1beta, and TNF-alpha mRNA and protein levels in skeletal and cardiac muscle of young (4 mo) and mature (10-11 mo) wild-type (IL-10(+/+)) and IL-10 deficient (IL-10(-/-)) mice following LPS. Skeletal and cardiac IL-6 mRNA and protein were elevated by LPS for IL-10(+/+) and IL-10(-/-) mice with greater responses in the IL-10(-/-) mice (P < 0.01). In skeletal muscle these effects were greater in mature than young mice (P < 0.01). IL-1beta mRNA and protein responses to LPS were greater in cardiac muscle of young but not mature IL-10(-/-) mice compared with IL-10(+/+) (P < 0.01). However, IL-1beta responses were greater in mature than young mice, but only in IL-10(+/+) groups (P < 0.05). The absence of IL-10 was associated with higher TNF-alpha protein levels in cardiac muscle (P < 0.05). The results provide the first in vivo evidence that the absence of IL-10 is associated with a greater IL-6 response to LPS in skeletal and cardiac muscles, and in skeletal muscle aging further exaggerates these responses.     

The cell adhesion molecule M-cadherin is not essential for muscle development and regeneration

M-cadherin is a classical calcium-dependent cell adhesion molecule that is highly expressed in developing skeletal muscle, satellite cells, and cerebellum. Based on its expression pattern and observations in cell culture, it has been postulated that M-cadherin may be important for the fusion of myoblasts to form myotubes, the correct localization and function of satellite cells during muscle regeneration, and the specialized architecture of adhering junctions in granule cells of cerebellar glomeruli. In order to investigate the potential roles of M-cadherin in vivo, we generated a null mutation in mice. Mutant mice were viable and fertile and showed no gross developmental defects. In particular, the skeletal musculature appeared essentially normal. Moreover, muscle lesions induced by necrosis were efficiently repaired in mutant mice, suggesting that satellite cells are present, can be activated, and are able to form new myofibers. This was also confirmed by normal growth and fusion potential of mutant satellite cells cultured in vitro. In the cerebellum of M-cadherin-lacking mutants, typical contactus adherens junctions were present and similar in size and numbers to the equivalent junctions in wild-type animals. However, the adhesion plaques in the cerebellum of these mutants appeared to contain elevated levels of N-cadherin compared to wild-type animals. Taken together, these observations suggest that M-cadherin in the mouse serves no absolutely required function during muscle development and regeneration and is not essential for the formation of specialized cell contacts in the cerebellum. It seems that N-cadherin or other cadherins can largely compensate for the lack of M-cadherin.     

Functional importance of regional differences in localized gene expression of receptors for IL-13 in murine gut

IL-13 induces a STAT6-dependent hypercontractility of intestinal smooth muscle that is mediated by binding to the IL-13Ralpha1 component of the type 2 IL-4R that is linked to STAT6. IL-13 also binds to the IL-13Ralpha2 that is not linked to STAT6 and functions to limit the effects of IL-13 in vivo. In this study we assessed the contributions of regional and cellular differences in the distribution of the IL-13R components to the physiological regulation of smooth muscle function in wild-type mice and mice deficient in STAT6 or IL-13Ralpha2. The expression of IL-13 and IL-13Ralpha2 was higher in colon than in small intestine. Laser capture microdissection of specific cell types revealed that the expression of IL-13Ralpha2 was higher in the smooth muscle layer compared with levels in the epithelial cells of the mucosa. In contrast, there was a uniform distribution of IL-13alpha1 in smooth muscle, epithelia, and myenteric neurons. The significant hypercontractility of smooth muscle in mice deficient in IL-13Ralpha2, but not in STAT6, shows the physiological importance of IL-13 binding to IL-13Ralpha2. The pronounced differences in the expression of IL-13Ralpha2 suggest that the gut has developed sophisticated mechanisms for controlling the physiological and pathophysiological activities of IL-13.     

Potential role of myeloid cell/eosinophil-derived IL-17 in LPS-induced endotoxin shock

IL-17RA is a shared receptor subunit for several cytokines of the IL-17 family, including IL-17A, IL-17C, IL-17E (also called IL-25) and IL-17F. It has been shown that mice deficient in IL-17RA are more susceptible to sepsis than wild-type mice, suggesting that IL-17RA is important for host defense against sepsis. However, it is unclear which ligands for IL-17RA, such as IL-17A, IL-17C, IL-17E/IL-25 and/or IL-17F, are involved in the pathogenesis of sepsis. Therefore, we examined IL-17A, IL-17E/IL-25 and IL-17F for possible involvement in LPS-induced endotoxin shock. IL-17A-deficient mice, but not IL-25- or IL-17F-deficient mice, were resistant to LPS-induced endotoxin shock, as compared with wild-type mice. Nevertheless, studies using IL-6-deficient, IL-21Rα-deficient and Rag-2-deficient mice, revealed that neither IL-6 and IL-21, both of which are important for Th17 cell differentiation, nor Th17 cells were essential for the development of LPS-induced endotoxin shock, suggesting that IL-17A-producing cells other than Th17 cells were important in the setting. In this connection, IL-17A was produced by macrophages, DCs and eosinophils after LPS injection. Taken together, these findings indicate that IL-17A, but not IL-17F or IL-25, is crucial for LPS-induced endotoxin shock. In addition, macrophages, DCs and eosinophils, but not Th17 cells or γδ T cells, may be sources of IL-17A during LPS-induced endotoxin shock.     

Necdin mediates skeletal muscle regeneration by promoting myoblast survival and differentiation

Regeneration of muscle fibers that are lost during pathological muscle degeneration or after injuries is sustained by the production of new myofibers. An important cell type involved in muscle regeneration is the satellite cell. Necdin is a protein expressed in satellite cell-derived myogenic precursors during perinatal growth. However, its function in myogenesis is not known. We compare transgenic mice that overexpress necdin in skeletal muscle with both wild-type and necdin null mice. After muscle injury the necdin null mice show a considerable defect in muscle healing, whereas mice that overexpress necdin show a substantial increase in myofiber regeneration. We also find that in muscle, necdin increases myogenin expression, accelerates differentiation, and counteracts myoblast apoptosis. Collectively, these data clarify the function and mechanism of necdin in skeletal muscle and show the importance of necdin in muscle regeneration.     

Oxidative modification of low-density lipoprotein and immune regulation of atherosclerosis

Oxidized low-density lipoprotein (oxLDL) is thought to promote atherosclerosis through complex inflammatory and immunologic mechanisms that lead to lipid dysregulation and foam cell formation. Recent findings suggested that oxLDL forms complexes with β₂-glycoprotein I (β₂GPI) and/or C-reactive protein (CRP) in the intima of atherosclerotic lesions. Autoantibodies against oxLDL/β₂GPI complexes occur in patients with systemic lupus erythematosus (SLE) and/or antiphospholipid syndrome (APS) and significantly correlate with arterial thrombosis. IgG autoantibodies having similar specificity emerged spontaneously in non-immunized NZW × BXSB F1 mice, an animal model of APS, and a monoclonal autoantibody (WB-CAL-1; IgG2a) against complexed β₂GPI (oxLDL/β₂GPI complexes) was derived from the same mice. WB-CAL-1 significantly increased the in vitro uptake of oxLDL/β₂GPI complexes by macrophages. This observation strongly suggests that such IgG autoantibodies are pro-atherogenic. In contrast, IgM anti-oxLDL natural antibodies found in the atherosclerosis-prone mice (ApoE^-/- and LDL-R^-/- mice) have been proposed to be anti-atherogenic (protective). The presence of IgG anti-oxLDL antibodies in humans has been documented in many publications but their exact clinical significance remains unclear. In this article, we review recent progress in our understanding of the mechanisms involved in oxidation of LDL, formation of oxLDL complexes, and antibody mediated-immune regulation of atherogenesis.