NOX family NADPH oxidases: roles in innate immunity and inflammatory disease

该计划探讨了先天免疫，促炎和NOX家族NADPH氧化酶的信号功能。目前的研究重点是主要在上皮细胞中表达的非吞噬性NADPH氧化酶（NOX 1，NOX 4，NOX 5，DUOX 1，DUOX 2），以及造血细胞中基于NOX 2和NOX 5的氧化酶。由上皮酶产生的有意的活性氧（ROS）响应于细胞因子、生长因子、激素以及危险和病原体相关的分子模式（DAMP和PAMP）而传递氧化还原信号。NOX酶还参与细胞迁移、增殖、肿瘤侵袭和转移、细胞分化、衰老、凋亡和微生物杀伤。 2019年，我们在三个研究领域探索了几种NOX家族NADPH氧化酶组分的功能：1）与免疫缺陷相关的NOX组分的遗传变体的研究，2）与炎性肠病相关的NOX缺陷的研究，和3）使用具有突变型TP 53和KRAS的条件性表达和已建立肿瘤的小鼠研究肿瘤微环境中NOX 4和NOX 2的相互作用和巨噬细胞样细胞系。 在与其他LCIM研究人员（Amy Hsu和Steven Holland）的合作中，我们描述了两种与几名患者的联合免疫缺陷相关的新型单等位基因RAC 2突变，导致严重的T和B细胞淋巴细胞减少症，骨髓功能障碍和反复呼吸道感染。全外显子组测序确定了RAC 2 E62 K和N92 T突变在几个病人的中性粒细胞表现出功能缺陷，包括自发激活，过量的超氧化物生成，异常巨胞饮和受损的趋化性反应甲酰基肽增强的标志物。我们设计这些RAC 2突变蛋白在转染的CHO-K1和COS-7细胞系中表达，以研究导致患者异常骨髓和淋巴表型的细胞和分子机制途径。用NOX 2组分和任一RAC 2突变蛋白重建的转染模型在静息和刺激的细胞中都表现出旺盛的ROS产生。过表达的突变蛋白还表现出增强的RAC 2-PAK 1结合和增加的活化磷酸-AKT，这是占主导地位的活性RAC 2分子的特征性反应，其解释了在转染模型和患者中性粒细胞中观察到的增强的膜皱褶和巨胞饮体形成。表达Rac 2 E62 K的转基因小鼠表现出相同的骨髓和淋巴疾病表型，包括B和T细胞淋巴细胞减少症、过量中性粒细胞超氧化物生成以及细胞骨架和迁移缺陷。总之，这些发现提供了新的见解RAC 2的作用，中性粒细胞功能和淋巴细胞生成不同的更广泛表达的RAC 1 GT3同源物。 其他工作是探索与炎症性肠病相关的几种NOX亚型的缺陷。在转染细胞模型中，当与其他支持NOX 1的辅因子共表达时，几种NOX 1变体显示部分或完全丧失超氧化物生成（与D. Kastner's group，NHGRI）。氧化酶活性的缺陷与转染的结肠上皮细胞中NOX 1依赖性细胞迁移减少相关。因此，与炎症性肠病相关的较低的NOX 1氧化酶活性可能反映了受损的NOX 1依赖性上皮屏障功能和对微生物暴露的增强的炎症反应。我们还探讨了通过全外显子组测序检测的早发性炎症性肠病患者中NOX 5缺失的功能影响（与K. Sullivan和J Kelsen，CHOP）。我们表明该突变不仅会损害NOX 5超氧化物的产生，而且对野生型NOX 5活性具有显性负效应。 我们对NOX参与癌症进展的研究源于观察结果，这些观察结果表明，在几种携带TP 53“热点”突变的上皮肿瘤中，TGF-β和SMAD 3对NOX 4的诱导作用增强，TP 53“热点”突变支持肿瘤细胞迁移、侵袭和血管生成，这些过程似乎都是NOX 4依赖性的。我们目前正在探索NOX 4和NOX 2在细胞培养模型中的肿瘤微环境中的炎症信号传导中的作用，以及在由于TP 53和KRAS突变的条件性敲入而发展为胰腺导管腺癌（PDAC）的小鼠中的作用。细胞培养研究表明，突变型p53介导N 0X 4依赖性的趋化因子（CCL 2和CCL 5）分泌，其不仅刺激肿瘤细胞迁移，而且募集巨噬细胞。反过来，巨噬细胞通过分泌CCL 5和TGF-β来促进肿瘤细胞动员，这可以通过针对这些因子的中和抗体来抑制。这些观察结果揭示了具有治疗干预潜力的新型NOX相关炎症途径。 在相关的整体动物研究中，我们检测到突变型p53表达转化的胰腺导管上皮细胞以及包裹腺癌的成纤维细胞层中的高NOX 4表达。相反，在腺癌周围的浸润性炎性细胞内观察到最高的NOX 2表达。因此，在PDAC中过表达的两种氧化酶在这些肿瘤组织中的不同细胞群中被检测到，并且似乎起到不同的作用，可以抑制或支持转移性疾病进展。正在进行的工作将研究Nox 4或Nox 2基因缺陷是否影响小鼠的PDAC进展或总生存期，以确定这些氧化酶是否代表抑制胰腺转移性疾病进展的有价值的靶点。

This program explores innate immune, pro-inflammatory, and signaling functions of NOX family NADPH oxidases. The current research focuses on non-phagocytic NADPH oxidases (NOX1, NOX4, NOX5, DUOX1, DUOX2) expressed primarily in epithelial cells, as well as NOX2 and NOX5-based oxidases in hematopoietic cells. Deliberate reactive oxygen species (ROS) production by the epithelial enzymes relays redox signals in responses to cytokines, growth factors, hormones, and danger- and pathogen-associated molecular patterns (DAMPs and PAMPs). NOX enzymes also participate in cell migration, proliferation, tumor invasiveness and metastasis, cell differentiation, senescence, apoptosis, and microbial killing. In 2019, we explored functions of several NOX family NADPH oxidase components in three areas of investigation: 1) studies on genetic variants of NOX components linked to immunodeficiencies, 2) studies on NOX defects associated with inflammatory bowel disease, and 3) studies on the interplay of NOX4 and NOX2 in the tumor microenvironment using mice with conditional expression of mutant TP53 and KRAS and established tumor and macrophage-like cell lines. In collaboration with other LCIM investigators (Amy Hsu and Steven Holland), we characterized two novel mono-allelic RAC2 mutations associated with combined immune deficiency in several patients, resulting in severe T- and B-cell lymphopenia, myeloid dysfunction, and recurrent respiratory infections. Whole exome sequencing identified RAC2 E62K and N92T mutations in several patients whose neutrophils exhibited functional defects, including enhanced markers of spontaneous activation, excess superoxide generation, abnormal macropinocytosis and impaired chemotaxis in response to formyl peptide. We engineered these RAC2 mutant proteins for expression in transfected CHO-K1 and COS-7 cell lines to investigate the cellular and molecular mechanistic pathways responsible for the patients' aberrant myeloid and lymphoid phenotypes. Transfected models reconstituted with NOX2 components and either RAC2 mutant protein demonstrated exuberant ROS generation in both resting and stimulated cells. The over-expressed mutant proteins also exhibited enhanced RAC2-PAK1 binding and increased activated phospho-AKT, responses characteristic of dominant active RAC2 molecules that account for the enhanced membrane ruffling and macropinosome formation observed in the transfected models and patients neutrophils. Transgenic mice expressing Rac2 E62K exhibited the same myeloid and lymphoid disease phenotypes, including B and T cell lymphopenia, excess neutrophil superoxide generation and cytoskeletal and migratory defects. Together, these findings provide novel insights on roles for RAC2 in neutrophil function and lymphopoiesis distinct from those of the more widely expressed RAC1 GTPase homologue. Other work is exploring defects in several NOX isoforms associated with inflammatory bowel disease. Several NOX1 variants show partial or complete loss of superoxide generation when co-expressed with the other NOX1-supportive cofactors in transfected cell models (collaboration with D. Kastner's group, NHGRI). The defects in oxidase activity were correlated with diminished NOX1-dependent cell migration in transfected colon epithelial cells. Thus, the lower NOX1 oxidase activities associated with inflammatory bowel disease may reflect compromised NOX1-dependent epithelial barrier functions and enhanced inflammatory responses to microbial exposure. We also explored functional effects of a deletion in NOX5 detected by whole exome sequencing in a patient with early onset inflammatory bowel disease (collaboration with K. Sullivan and J Kelsen, CHOP). We showed the mutation not only impairs NOX5 superoxide generation but has dominant-negative effects on wild type NOX5 activity. Our studies on NOX involvement in cancer progression originated from observations showing that NOX4 induction by TGF-beta and SMAD3 is enhanced in several epithelial tumors bearing TP53 'hot spot' mutations that support tumor cell migration, invasiveness, and angiogenesis, processes that all appear to be NOX4-dependent. We are currently exploring roles for NOX4 and NOX2 in inflammatory signaling within the tumor microenvironment in cell culture models and in mice that develop pancreatic ductal adenocarcinoma (PDAC) as a consequence of conditional knock-in of TP53 and KRAS mutations. The cell culture studies demonstrated that mutant p53 mediates NOX4-dependent secretion of chemokines (CCL2 and CCL5) that not only stimulate tumor cell migration, but also recruit macrophages. Macrophages, in turn, cross-talk by secreting CCL5 and TGF-beta to promote tumor cell mobilization, which can be inhibited by neutralizing antibodies against these factors. These observations have revealed novel NOX-related inflammatory pathways with potential for therapeutic intervention. In related whole animal studies, we detected high NOX4 expression in pancreatic ductal epithelial cells transformed by mutant p53 expression, as well as in the fibroblast layer encapsulating the adenocarcinoma. In contrast, highest NOX2 expression is observed within infiltrating inflammatory cells surrounding the adenocarcinoma. Thus, the two oxidases over-expressed in PDAC are detected in different cell populations in these tumor tissues and appear to serve distinct roles that can either suppress or support metastatic disease progression. Ongoing work will investigate whether Nox4 or Nox2 gene deficiencies affect PDAC progression or overall survival in mice to determine whether these oxidases represent worthwhile targets for suppressing pancreatic metastatic disease progression.