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 associations of loss of function mutations in the corresponding human genes in human disease cohorts. In FY16 we reported discoveries in the following areas: 1. Role in host defense against C. albicans and Ps. aeruginosa infection by the CXC chemokine receptor Cxcr1; 2. The primary immunodeficiency disease WHIM syndrome, 3. T. cruzi pathogenesis, and 4. mechanisms of allograft tolerance. 1.) In FY17, we filed a patent application on a novel method of universal allotransplantation in mice. This builds on earlier work of the lab showing that pre-treatment of allogeneic bone marrow recipients with the macrophage depleting agent clodronate durably enhances hematopoietic chimerism and promotes donor-specific skin allograft tolerance. Donor specific allograft tolerance is a holy grail in transplantation that, if achieved, could massively expand the number of potential organ donors and reduce the toxicity from immunosuppression associated with the procedure. It's known that establishing hematopoietic chimerism with the donor can facilitate donor-specific organ allograft tolerance. It's also thought that engraftment of stem cells requires vacant bone marrow niches. We therefore depleted bone marrow macrophages with clodronate in the recipient hoping to enhance donor engraftment and chimerism and hence skin allograft tolerance. We observed durable enhancement of chimerism in blood, spleen and bone marrow out to over 200 days, the latest time point tested. Although this work provided proof of principle for the general approach, it was associated with substantial toxicity and therefore could not be considered for clinical translation. In new work we collaborated with the Rossi lab from Children's Hospital/Harvard to demonstrate that an hematopoietic stem cell-directed immunotoxin binding to c-kit was safe, non-toxic and highly effective at promoting hematopoietic chimerism after bone marrow allotransplantation in mice across a major MHC-mismatch, and that this established a state of donor specific tolerance of skin allografts across the same mismatch. The work provides proof of principle in mice for a method of universal transplantation, and justifies further experimentation to optimize the protocol and to broaden it for application in disease models and other types of organ allografts. 2.) In FY17, we reported biochemical and structural characterization of the cytokine-like protein CYTL1. This protein was predicted by molecular modeling to fold like a chemokine, most closely to CCL2, and has been reported to act as an agonist at the CCL2 receptor CCR2. In the present study, we provide a new method for production of large amounts of recombinant CYTL1 as well as circular dichroism data consistent with chemokine-like structure. We also demonstrate that the protein is able to induce calcium flux responses in a chondrocyte cell line. The results are a prelude to definitive 3 dimensional structural determination of the protein. 3.) In FY17, we reported that the mouse cytomegalovirus (MCMV)-encoded chemokine MCK2 is able to bind to glycosaminoglycans in mouse cell lines and primary mouse salivary gland cells. MCK-2 has high homology to chemokines and appears to be important in MCMV virulence; however, its mechanism of action has remained elusive. The significance of this study is the demonstration that MCK-2 behaves like host chemokines as a GAG binding protein, and that this property governs the ability of the protein to oligomerize, which is an important structural determinant of chemokine action in vivo.
Showing the most recent 10 out of 72 publications
