CXCR4 Antagonism Corrects Peripheral Neutropenia and Mature Neutrophil Accumulation in Bone Marrow in a Pharmacological Mouse Model of CXCR2 Loss-of-Function
Authors: Nguyen Chi Huu , Johnson Robert , Zmajkovicova Katarina , Sekirnik Angelina , Taplin Sarah , Defontis Myriam , Bledsoe Jacob R. , Karlsson Lars , Taveras Arthur G. 
Published: December 2024

Introduction
The CXCR4 and CXCR2 chemokine receptor axes play pivotal but opposing roles in regulating neutrophil retention and release from the bone marrow (BM), respectively. Gain-of-function (GoF) variants in the gene encoding CXCR4 are associated with WHIM syndrome, an ultra-rare combined primary immunodeficiency disorder. Patients with WHIM syndrome frequently exhibit neutropenia, serious and/or frequent infections, accumulation of neutrophils in the BM, with distinctive morphology termed myelokathexis. Additional clinical manifestations in patients with WHIM syndrome may include warts, hypogammaglobulinemia and lymphocytopenia.1,2 Interestingly, patients with loss-of-function (LoF) variants in CXCR2 display phenotypic features similar to those observed in patients with WHIM syndrome, such as neutropenia, increased infection susceptibility, and myelokathexis.3,4 Mavorixafor, an orally bioavailable CXCR4 antagonist, has demonstrated clinically meaningful increases in absolute neutrophil and lymphocyte counts and concomitant reductions in infections in patients aged 12 years and older with WHIM syndrome, leading to its recent approval by the U.S. Food and Drug Administration.5 Whether CXCR4 antagonist therapy can similarly correct the common pathogenic phenotypes observed in patients with CXCR2 LoF variants as are seen in patients with WHIM syndrome has yet to be determined.
The current study aimed to develop a pharmacological mouse model of CXCR2 LoF, induced by a CXCR2 antagonist, in order to recapitulate the common phenotypes seen in patients diagnosed with WHIM syndrome, and those with CXCR2 deficiency, and to evaluate the ability of CXCR4 antagonism to correct the phenotypic defects in a CXCR2 LoF mouse model.
Methods
Female BALB/c mice (6 mice per group) were dosed with the CXCR2 antagonist navarixin (3 mg/kg) by oral gavage and subsequently the CXCR4 antagonist X4P-002 (10 mg/kg) or vehicle control daily for 7 days, 2 hours after the navarixin dose. Blood and BM were collected 4 hours after X4P-002 dose for enumeration and flow cytometry analysis. At day 7, blood and BM smears were prepared and stained with May-Grunwald Giemsa for morphological analysis.
Results
Mice with pharmacologically induced CXCR2 LoF recapitulated many of the phenotypic features observed in patients diagnosed with WHIM syndrome, and those with CXCR2 deficiency1-4, including peripheral blood neutropenia, accumulation of segmented neutrophils, and an increased myeloid/erythroid (M/E) ratio in BM. Furthermore, some neutrophils in BM and blood of CXCR2 LoF mice displayed a myelokathexis-like (MK) morphologic characteristic of that seen in WHIM syndrome, including nuclear hypersegmentation, an increased number of discernible nuclear lobes, connected by thinner strands of chromatin. Treatment with the CXCR4 antagonist X4P-002 resulted in the correction of these pathogenic features including normalization of absolute neutrophil count in peripheral blood, reversal of mature neutrophil accumulation in BM, and normalization of the M/E ratio in BM. Moreover, chronic dosing with the CXCR4 antagonist daily for 7 days appeared to reduce the frequency of MK neutrophils in BM as well as the number of mice with neutrophil myelokathexis in BM.
Conclusion
We have successfully developed a pharmacological mouse model of CXCR2 LoF, induced by a CXCR2 antagonist navarixin. Our CXCR2 LoF mice exhibit a phenotype similar to that observed in patients diagnosed with WHIM syndrome, including peripheral blood neutropenia, accumulation of segmented neutrophils, some displaying myelokathexis-like morphology, and an increased M/E ratio in BM, corrected by CXCR4 antagonist therapy. Our data suggest the potential for CXCR4 antagonist therapy to correct neutropenia and normalize clinical phenotypes consistent with WHIM syndrome in patients with CXCR2 LoF variants.
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