Molecular Analysis Of Leukocyte Activation By Chemoattractants

National Institute Of Allergy And Infectious Diseases

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Abstract

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 mechanisms mediated by the presence or absence of specific 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 FY25, we reported that chemokines kill bacteria by binding to membrane anionic phospholipids. Classically, chemokines coordinate leukocyte trafficking; however, many chemokines also have direct antibacterial activity in vitro. The bacterial killing mechanism of chemokines and the biochemical properties that define which members of the chemokine superfamily are antimicrobial had remained poorly understood before this paper. We reported that the antimicrobial activity of chemokines is defined by their ability to bind phosphatidylglycerol and cardiolipin, two anionic phospholipids commonly found in the bacterial plasma membrane. In eukaryotic cells cardiolipin is found only in mitochondrial membranes. We showed in this paper that only chemokines able to bind these two phospholipids kill bacteria and that they exert rapid bacteriostatic and bactericidal effects with a higher potency than the classic antimicrobial peptide β-defensin 3. Both biochemical and genetic interference with the chemokine-cardiolipin interaction impaired microbial growth arrest, bacterial killing, and membrane disruption by chemokines. Moreover, unlike conventional antibiotics, Escherichia coli failed to develop resistance when placed under increasing antimicrobial chemokine pressure in vitro, unlike for conventional antibiotics. Thus, we have identified cardiolipin and phosphatidylglycerol as binding partners for chemokines responsible for chemokine antimicrobial action in vitro. This work extended our discovery reported in a previous annual report that specific anionic phospholipids serve as a 3rd class of chemokine binding site (together with GPCRs and GAGs), and that chemokine binding to phosphatidylserine may be a find me signal for apoptotic cell clearance by macrophages. The work provides a potential platform for addressing the global problem of antimicrobial resistance using chemokine-derived antibiotics. 2.) In FY25, we posted a preprint in MedRxiv reporting the results of a Phase 1 trial in WHIM patients of plerixafor treatment given by continuous infusion using an insulin pump. WHIM syndrome is a primary immunodeficiency disorder caused by heterozygous gain-of-function mutations in chemokine receptor CXCR4. Patients typically have panleukopenia, including a type of neutropenia caused by neutrophil retention in bone marrow, presumably from exaggeration of CXCR4's normal function there. Consistent with this, the CXCR4 antagonist plerixafor mobilizes neutrophils and other leukocytes to the blood and represents potential targeted mechanism-based treatment. In this regard, in FY24 we had reported an investigator-initiated, single-center, quadruple-masked phase III crossover trial of plerixafor vs G-CSF, in which we compared the total infection severity score (TISS) as the primary endpoint in an intent-to-treat manner in 19 patients with WHIM who each received 12 months treatment with plerixafor and 12 months treatment with granulocyte CSF (G-CSF, the standard of care for severe congenital neutropenia). Plerixafor was nonsuperior to G-CSF for TISS (P = 0.54). In exploratory endpoints, plerixafor was noninferior to G-CSF for maintaining neutrophil counts of more than 500 cells/μL (P = 0.023) and was superior to G-CSF for maintaining lymphocyte counts above 1,000 cells/μL (P < 0.0001). Complete regression of a subset of large wart areas occurred only while on plerixafor (in 5 of 7 patients with major wart burdens at baseline). Transient rash occurred on plerixafor, and bone pain was more common on G-CSF. There were no significant differences in drug preference or quality of life or the incidence of drug failure or serious adverse events. Plerixafor was not superior to G-CSF in patients with WHIM for TISS, the primary endpoint. Together with wart regression and hematologic improvement, the infection severity results supported continued study of plerixafor as a potential treatment for WHIM syndrome. TRIAL REGISTRATION Clinicaltrials.gov NCT02231879. Plerixafor's short half-life requires twice daily injection. To develop a simpler, cheaper and potentially more effective method of drug delivery, in FY25 we reported a Phase 1 study of WHIM patients given plerixafor 0.02-0.08 mg/kg/d by continuous subcutaneous infusion using an OmniPod insulin pump, and assessed compliance as well as effects on blood leukocyte counts, infections, chronic skin conditions and adverse events. Six patients were treated for a total of 6.3 patient-years; one patient dropped out early for personal reasons. The drug infusion rate was adjusted to achieve a normal absolute lymphocyte count and an absolute neutrophil count >500 cells/μl in all patients. An average of 2.1 infections/patient-year occurred (range 0-4). Treatment of two infections involved brief hospitalization. On plerixafor, partial wart regression occurred in 3 of 4 patients, a single molluscum contagiosum infection regressed and a chronic post-Mohs surgical wound epithelialized. There were 3 serious adverse events, but none was attributable to the treatment. All patients preferred pump administration over syringe injection. Thus, in WHIM patients a continuous infusion pump may be a convenient, safe and potentially cost-effective means of delivering plerixafor chronically to correct panleukopenia and to improve chronic skin conditions. Clinicaltrials.gov NCT00967785. 3.) In FY25, we also reported a series of 3 collaborative papers with our NIH dermatology consultants defining the skin phenotypes in patients with WHIM syndrome. 4.) In FY25, we also contributed to work by our longtime collaborator Dr. Balabanian in France showing that CXCR4 signaling determines the fate of hematopoietic multipotent progenitors by stimulating mTOR activity and mitochondrial metabolism.