Immunoengineering

免疫工程

• 批准号: 10920194
• 负责人: Kaitlyn Sadtler
• 金额: $ 77.23万
• 依托单位: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10920194
• 关键词: 2019-nCoV; 3-Dimensional; Ablation; Adipose tissue; Adult; Antigen-Presenting Cells; Asthma; Autoimmunity; B-Lymphocytes; Biocompatible Materials; Biological Markers; Blood Vessels; Breast; CD19 gene; CD8-Positive T-Lymphocytes; CD8B1 gene; COVID-19; Categories; Cells; Cellular Infiltration; Cellularity; Collaborations; Color; Communication; Communities; Cross Presentation; Defect; Dendritic Cells; Development; Diabetic Foot Ulcer; Direct Lytic Factors; Extracellular Matrix; FOXP3 gene; Fatty acid glycerol esters; Fibrosis; Flow Cytometry; Foreign Bodies; Gene Expression Profile; Genes; Genetic Diseases; Greater sac of peritoneum; Hernia; Histologic; Human; Hypersensitivity; ITGAM gene; Image; Immune; Immune response; Immunity; Implant; In Vitro; Infiltration; Injections; Injury; Institutional Review Boards; Interleukin-10; Intramuscular; Journals; Light; Literature; Liver; Location; Macrophage; Maps; Mediator; Methanol; Methods; Microscopy; Modeling; Mus; Muscle; Muscle Fibers; National Institute of Biomedical Imaging and Bioengineering; Natural regeneration; Necrosis; Optics; Pathologic; Pattern; Physiologic Ossification; Plasma; Play; Polyethylenes; Population; Positioning Attribute; Prospective Studies; Publications; Regulatory T-Lymphocyte; Reporting; Research; Research Project Grants; Role; Sampling; Series; Severities; Skin injury; Specimen; Structure; Surface; T-Lymphocyte; Testing; Thick; Time; Tissues; Trauma; Trauma patient; Traumatic injury; United States National Institutes of Health; Up-Regulation; Visualization; Work; XCR1 gene; abdominal wall; adaptive immune response; biobank; bioscaffold; breast lumpectomy; calcification; eosinophil; follow-up; immune function; immunoengineering; immunoregulation; implantable device; implantation; injury recovery; interest; interstitial; intraperitoneal; machine learning method; mouse model; muscle regeneration; neurosurgery; neutrophil; novel; optical spectra; peripheral blood; post-trauma; preference; programs; quadriceps muscle; reconstruction; recruit; regenerative; repaired; response; response to injury; sample collection; scaffold; scavenger receptor; soft tissue; subcutaneous; tissue trauma; trend; volumetric muscle loss; wound

Finalizing SARS-CoV-2 Research We are finalizing the publication of our last works on COVID19 research which have all completed experimentation and are being prepared for submission to scientific journals. Please refer to prior report regarding COVID19 research projects. Identification of novel mediators of injury recovery and materials response Early in response to injury and biomaterial implantation, pro-fibrotic materials (polyethylene; PE) induced the recruitment of a strong neutrophil population in comparison to pro-regenerative materials (decellularized extracellular matrix; ECM) recruit a large proportion of eosinophils, commonly associated with allergy and asthma but previously described as important mediators of tissue development in the liver and muscle after cardiotoxin injection. When shifting focus to dendritic cells which have been largely ignored (with more emphasis on macrophages), we found that when an injury was treated with ECM biomaterials there was a preferential recruitment of CD103+XCR1+ type-1 conventional dendritic cells (cDC1s) that had atypical surface marker expression including an absence of CD8 and presence of CD301b, CD206, along with low levels of CD11b (Lokwani, Josyula, Ngo et al Accepted in Principle 2023). To determine if these trauma-associated dendritic cells (tDCs) were in fact cDC1s, we evaluated their presence in a Batf3-/- model (a common depletion model for cDC1s) and found ablation of these dendritic cells in the absence of BATF3, suggesting that they are cross-presentation capable cDC1-like cells. Pathologically, the Batf3-/- mice displayed increased intramuscular adipose and fibrosis along with stochastically positioned necrotic and calcified muscle fibers. This was mirrored with gene expression patterns where we saw increases in Col1a1 and Adipoq associated with fibrosis and white fat, and Acvr1, which is associated with a genetic disease in humans characterized by severe post-traumatic soft tissue ossification (FOP). Interestingly, we also saw increases in the expression of several genes associated with muscle regeneration including Gli1, suggesting a larger regulatory role of BATF3-dependent cells in post-trauma tissue development. As these cDC1s can communicate with CD8+ T cells, we investigated adaptive immune responses to trauma. Previous literature has associated a non-canonical CD8 regulatory cell in regulating autoimmunity, and thus our interest was piqued as these cells potentially playing an immunoregulatory role early in the response to wounding. We found a number of CD4+FoxP3+ regulatory T cells as well as CD4+ and CD8+ HELIOS+ induced Tregs (iTregs). We are continuing our exploration of these CD8 iTregs in trauma. We have multiple follow-up projects to the above referenced research that we will be continuing throughout FY24. Large volume imaging of biomaterial implantation in a mouse model In collaboration with AIM we developed a method to clear tissues and image biomaterial implants made of decellularized extracellular matrix with light sheet microscopy. Using a graded methanol series followed by DCM and DBE clearing, we generated thick (1cm), optically clear quadriceps muscles and peritoneal cavities to visualize the large volumetric structure around these biomaterial implants with autofluorescence alone. We could then reconstruct the tissue in 3D, and apply machine learning methods to categorize the tissue types based on the autofluorescence emission spectra. We were able to repeatably and accurately map muscle, ECM scaffold, interstitial space, and to a lesser extent blood vessels. The role of implant location in immune response to scaffolds Biomaterial scaffolds are used in a variety of tissue contexts. Decellularized extracellular matrix (ECM) scaffolds are used for abdominal wall repair during hernia reconstruction, dural repair after neurosurgery, breast filling after lumpectomy, diabetic foot ulcer treatment, skin injury reconstruction, and have been tested for the treatment of volumetric muscle loss (VML) and more significant tissue defects. When comparing intraperitoneal implant, versus subcutaneous implant, versus VML model, we found similarities and differences based on tissue location (DeStefano et al bioRxiv). Histologically, we say that there was a strong cellular infiltration into biomaterial implants in the subcutaneous space which was increased in the presence of an injury. With intraperitoneal implants, an increased cellularity was observed within the peritoneal cavity but infiltration in the material itself was decreased and limited to clusters around the periphery of the implant. In the VML we saw a strong preference for an M2-like macrophage polarization with enrichment of CD206 and CD301b expression on MHCII- macrophages. This supports prior work where the more M2-polarized macrophages were not antigen presenting (MHCII-) whereas those that expressed MHCII (suggesting communication with T cells) did not express as strong levels of these scavenger receptors. This peaked by 21 days post-injury with greater cell infiltration when compared to the IP and SQ models. In the IP model, there were two strong differences including a lower proportion of Ly6Chi macrophages which are associated with peripheral blood recruitment and correlates with previous findings of robust tissue-resident macrophage population in the peritoneal cavity. These implants also recruited a very strong proportion of CD19+ B Cells which was in stark contrast to SQ and VML which did not recruit large numbers of B Cells. These B cells also expressed MHCII, suggesting that they could behave as antigen presenting cells. Continuing research on human immune response to traumatic injury We evaluated a panel of 59 biomarkers in the plasma of 1000 trauma patients alongside 50 healthy adults. Several interesting trends emerged, both confirming prior work and identifying new immune patterns of human traumatic injury. We found an upregulation of IL-10 which is known to increase in trauma patients, this had a correlation with trauma severity where those that ended up dying from their injuries were >70% positive for IL-10 whereas healthy controls had all but one sample return as undetected (2% positive). We are currently evaluating these markers in our mouse model of volumetric muscle loss to determine the potential correlates with human immunity and when during the course of injury recovery, we may see these similarities or differences occur. We hope for this work to be submitted for publication in FY24. We have established an MTA with the UMD Biorepository for a prospective study on discarded IRB-exempt human specimens. We have received our first sample and will continue to collect these at an estimated pace of 1 2 samples per month. We expect the first publication from this study to occur sometime in 2025 2026 pending sufficient sample collection

完成 SARS-CoV-2 研究 我们正在最后完成有关新冠病毒研究的最新著作的出版​​，这些著作已全部完成实验，并准备提交给科学期刊。请参阅之前有关 COVID19 研究项目的报告。 损伤恢复和材料反应的新型介质的鉴定 在对损伤和生物材料植入的早期反应中，与促再生材料（脱细胞细胞外基质；ECM）相比，促纤维化材料（聚乙烯；PE）诱导了强中性粒细胞群的募集，募集了大量嗜酸性粒细胞，通常与过敏和哮喘，但之前被描述为注射心脏毒素后肝脏和肌肉组织发育的重要介质。当将焦点转移到很大程度上被忽视的树突状细胞（更强调巨噬细胞）时，我们发现当用 ECM 生物材料治疗损伤时，会优先招募 CD103+XCR1+ 1 型传统树突状细胞 (cDC1s)，这些细胞具有非典型表面标志物表达，包括 CD8 缺失和 CD301b、CD206 存在，以及低水平的 CD11b（Lokwani、Josyula、Ngo 等）原则上于 2023 年接受）。为了确定这些创伤相关树突状细胞 (tDC) 是否实际上是 cDC1，我们评估了它们在 Batf3-/- 模型（cDC1 的常见耗竭模型）中的存在，并发现在没有 BATF3 的情况下这些树突状细胞被消融，这表明它们是具有交叉呈递能力的 CDC1 样细胞。病理学上，Batf3-/- 小鼠表现出肌内脂肪和纤维化增加，以及随机定位的坏死和钙化肌纤维。这与基因表达模式相一致，我们看到与纤维化和白色脂肪相关的 Col1a1 和 Adipoq 以及与人类以严重创伤后软组织骨化 (FOP) 为特征的遗传性疾病相关的 Acvr1 增加。有趣的是，我们还发现与肌肉再生相关的几个基因（包括 Gli1）的表达增加，表明 BATF3 依赖性细胞在创伤后组织发育中发挥更大的调节作用。由于这些 cDC1 可以与 CD8+ T 细胞通信，因此我们研究了对创伤的适应性免疫反应。先前的文献将非典型的 CD8 调节细胞与调节自身免疫联系起来，因此我们的兴趣被激起了，因为这些细胞可能在受伤反应的早期发挥免疫调节作用。我们发现了许多 CD4+FoxP3+ 调节性 T 细胞以及 CD4+ 和 CD8+ HELIOS+ 诱导的 Tregs (iTreg)。我们正在继续探索创伤中的这些 CD8 iTreg。我们有多个针对上述参考研究的后续项目，我们将在 2024 财年继续进行这些项目。 小鼠模型中生物材料植入的大体积成像 我们与 AIM 合作开发了一种方法，可通过光片显微镜对由脱细胞细胞外基质制成的生物材料植入物进行透明化和成像。使用分级甲醇系列，然后进行 DCM 和 DBE 透明化，我们生成了厚（1 厘米）、光学透明的股四头肌和腹腔，仅通过自发荧光即可可视化这些生物材料植入物周围的大体积结构。然后，我们可以以 3D 方式重建组织，并应用机器学习方法根据自发荧光发射光谱对组织类型进行分类。我们能够重复且准确地绘制肌肉、ECM 支架、间隙空间以及较小程度的血管图。 植入物位置在支架免疫反应中的作用 生物材料支架用于多种组织环境。脱细胞细胞外基质（ECM）支架用于疝气重建期间的腹壁修复、神经外科手术后的硬脑膜修复、肿瘤切除术后的乳房填充、糖尿病足溃疡治疗、皮肤损伤重建，并已被测试用于治疗体积肌肉损失（VML）以及更显着的组织缺陷。在比较腹膜内植入、皮下植入和 VML 模型时，我们发现了基于组织位置的相似点和差异（DeStefano 等人 bioRxiv）。从组织学上讲，我们说皮下空间的生物材料植入物中有强烈的细胞浸润，这种浸润在受伤时会增加。对于腹膜内植入物，观察到腹膜腔内细胞数量增加，但材料本身的渗透减少并仅限于植入物周边的簇。在 VML 中，我们看到对 M2 样巨噬细胞极化的强烈偏好，并且 MHCII 巨噬细胞上 CD206 和 CD301b 表达富集。这支持了之前的研究，其中更多 M2 极化的巨噬细胞不呈递抗原 (MHCII-)，而那些表达 MHCII（表明与 T 细胞通讯）的巨噬细胞不表达高水平的这些清道夫受体。与 IP 和 SQ 模型相比，这种情况在损伤后 21 天达到峰值，细胞浸润更大。在 IP 模型中，存在两个显着差异，包括 Ly6Chi 巨噬细胞比例较低，这与外周血募集相关，并与腹膜腔中强大的组织驻留巨噬细胞群的先前发现相关。这些植入物还招募了非常高比例的 CD19+ B 细胞，这与不招募大量 B 细胞的 SQ 和 VML 形成鲜明对比。这些 B 细胞还表达 MHCII，表明它们可以充当抗原呈递细胞。 人体对创伤的免疫反应的持续研究 我们评估了 1000 名创伤患者和 50 名健康成年人血浆中的 59 种生物标志物。出现了一些有趣的趋势，既证实了先前的工作，又确定了人类创伤性损伤的新免疫模式。我们发现 IL-10 的上调，已知在创伤患者中会增加，这与创伤严重程度相关，最终因受伤而死亡的患者的 IL-10 阳性率 >70%，而健康对照者只有一个样本以外的所有样本返回未检测到（2% 阳性）。我们目前正在小鼠体积肌肉损失模型中评估这些标记，以确定与人类免疫力的潜在相关性，并且在损伤恢复过程中，我们可能会看到这些相似或差异的发生。我们希望这项工作能够在 2024 财年提交出版。我们与 UMD 生物样本库建立了 MTA，对废弃的 IRB 豁免人体标本进行前瞻性研究。我们已收到第一个样本，并将继续以每月 1 2 个样本的估计速度收集这些样本。我们预计该研究的首次发表将于 2025 年 2026 年某个时候发表，等待足够的样本收集