Molecular Analysis Of Leukocyte Activation By Chemoattractants

本项目的目的是确定血液白细胞迁移到炎症或感染的特定组织部位的分子机制和生物学背景。我们关注的是介导这一过程的化学引诱蛋白，并已经确定了部署在白细胞细胞表面的化学引诱受体大家族的成员。我们还发现了由病毒（包括疱疹病毒、痘病毒和艾滋病毒）制造的多种化学引诱剂和化学引诱剂受体模拟物的成员。我们将基因组学、分子生物学、细胞生物学和流行病学作为分析这些分子的主要方法。一个主要目标是确定个体化学引诱剂和化学引诱剂受体的特定疾病关联，以确定潜在的新治疗靶点。一个关键的策略是分析疾病模型中基因敲除小鼠的表型以及人类化学引诱系统基因突变患者的表型。

The aim of this project is to define the molecular mechanisms and biological contexts for blood leukocyte migration to specific tissue sites that are inflamed or infected. We have focused on chemoattractant proteins that mediate this process and have identified members of a large family of chemoattractant receptors that are deployed on the leukocyte cell surface. We have also identified members of a diverse group of chemoattractant and chemoattractant receptor mimics made by viruses, including herpesviruses, poxviruses and HIV. We use genomics, molecular biology, cell biology and epidemiology as the principle methods for analyzing these molecules. A major goal is to identify specific disease associations of individual chemoattractants and chemoattractant receptors, in order to identify potential new therapeutic targets. A key strategy is to analyze phenotypes of gene knockout mice in disease models as well as phenotypes in patients with mutations in human chemoattractant system genes. 1.) In FY19, we reported long-term data up to 5 years from a Phase 1 clinical trial of the safety of the CXCR4 antagonist plerixafor in G-CSF-intolerant patients with WHIM syndrome. The concept was to reduce CXCR4 signaling to normal, not zero, to minimize risk, a substantial concern given that Cxcr4-/- mice are non-viable. Two patients were being considered for transplant; one had advanced HPV+ HNSCC with extremely poor prognosis having failed radiation therapy. Instead, we treated all 3 patients in our still-open Phase 1 open-label study with low-dose plerixafor for 19-52 months, exceeding the longest time any patient outside of our cohort has ever received this drug by 51 months. We observed that myelofibrosis, panleukopenia, anemia and thrombocytopenia improved, wart burden and frequency of infection declined, HPV-associated HNSCC stabilized, and quality of life improved markedly. One patient had 17 different types of HPV isolated from his lower legs and a 6-year history of crippling chronic inflammatory lesions of the lower extremities related to recurrent cellulitis and folliculitis associated with Trichodysplasia spinulosum virus (TSV), which causes folliculitis in immunocompromised hosts. After several months of treatment, no HPVs or TSV could be detected and the lower leg lesions healed allowing the patient to return to normal ambulation and exercise tolerance. Adverse events were mainly infections attributable to the underlying immunodeficiency. Few infections occurred, and the patients all attested that the drug has changed their lives in tangible ways, particularly no longer having to contrive activities to avoid public exposure and the near occasions of infection. The patient with HNSCC improved to the point he became a surgical candidate for extensive reconstructive surgery needed to cure chronic osteomyelitis of the jaw that had complicated radiation necrosis from treatment for HNSCC. The operation required 9 hours and was complicated by flap failure and pneumonia from which he did not recover. We also reported separately the case of a WHIM patient who presented with monocular blindness due to infection with Toxoplasma gondii. 2.) In FY19, in a collaboration with Chris Bucks lab in the NCI, we reported results of a survey of viruses in our patients with WHIM syndrome. HPVs are divided into five genera designated Alpha, Beta, Gamma, Mu, and Nu. Prior to our study, 200 HPV types had been defined and classified into high and low risk types according to their ability to cause squamous cell carcinoma mostly in the anogenital tract, but also HNSCC. We used recently developed virion enrichment, rolling circle amplification and deep sequencing methods to identify circular DNA viruses present in skin swabs and/or wart biopsy samples from 48 patients with rare genetic immunodeficiencies, mainly WHIM syndrome and epidermodysplasia verruciformis (EV), as well as healthy individuals with or without warts or other types of skin lesions. We identified the complete genomes of 83 previously unknown HPV types and 35 incomplete genomes representing possible additional new types. HPV types in the Gamma genus were common in WHIM patients, whereas EV patients mainly shed Beta HPVs. Our results nearly double the number of known Gamma HPV types and suggest that WHIM patients are uniquely susceptible to Gamma HPV-associated skin warts. 3.) In FY19, we reported a new and safe method of universal allotransplantation in mice. The method uses an antibody drug conjugate (ADC) as a recipient conditioning agent, composed of the ribosome-targeted toxin saporin complexed to the cell-targeting agent anti-CD117 (c-kit) with the goal of selectively depleting hematopoietic stem cells without risk of genotoxicity. When mice are transiently immunosuppressed after ADC conditioning they engraft durably and at high levels when given bone marrow from a completely immunologically mismatched donor mouse. Establishment of hematopoietic chimerism in turn established a state of robust donor-specific immunologic tolerance as shown by durable acceptance of bone marrow donor type-mismatched but not third party mismatched skin allografts in the chimeric recipients. Importantly, we did not observe acute toxicity after administering the conditioning agent, nor did graft versus host disease occur. We have submitted a patent application to the US PTO disclosing this technology. 4.) In FY19, we reported results from a model of chronic Trypanosoma cruzi infection, the cause of Chagas disease. This infection causes life-threatening inflammatory diseases that develop over time in the heart, esophagus, and colon of some patients. C57BL/6 mice infected with the myotropic Colombiana strain of T. cruzi model many of the immunological and parasitological features of human infection but succumb to chronic paralyzing myositis and skeletal muscle vasculitis, not cardiomyopathy or gastrointestinal disease. We showed that T cell depletion in the chronic phase of this model increased tissue parasitism to acute-phase levels and induced neutrophilic skeletal muscle inflammation. Conversely, after daily treatment with the trypanocide benznidazole for 8weeks during the chronic phase, viable parasites were no longer detectable, myositis completely resolved, vasculitis was 80% reduced, fibrosis was reduced, and myofiber morphology normalized. After the drug was discontinued, parasitism rebounded, and immunopathology recurred. The parasite load was statistically strongly correlated with the severity of inflammation. Thus, both T cell immunity and trypanocidal pharmacotherapy suppress to very low levels, but do not cure, T. cruzi infection, which is necessary and possibly sufficient at very low levels to induce crippling chronic skeletal muscle myositis and vasculitis in the model.