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FK506 impairs neutrophil migration that results in increased polymicrobial sepsis susceptibility

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Abstract

Objective

This study aimed to investigate the effects of FK506 on experimental sepsis immunopathology. It investigated the effect of FK506 on leukocyte recruitment to the site of infection, systemic cytokine production, and organ injury in mice with sepsis.

Methods

Using a murine cecal ligation and puncture (CLP) peritonitis model, the experiments were performed with wild-type (WT) mice and mice deficient in the gene Nfat1 (Nfat1−/−) in the C57BL/6 background. Animals were treated with 2.0 mg/kg of FK506, subcutaneously, 1 h before the sepsis model, twice a day (12 h/12 h). The number of bacteria colony forming units (CFU) was manually counted. The number of neutrophils in the lungs was estimated by the myeloperoxidase (MPO) assay. The expression of CXCR2 in neutrophils was determined using flow cytometry analysis. The expression of inflammatory cytokines in macrophage was determined using ELISA. The direct effect of FK506 on CXCR2 internalization was evaluated using HEK-293T cells after CXCL2 stimulation by the BRET method.

Results

FK506 treatment potentiated the failure of neutrophil migration into the peritoneal cavity, resulting in bacteremia and an exacerbated systemic inflammatory response, which led to higher organ damage and mortality rates. Failed neutrophil migration was associated with elevated CXCL2 chemokine plasma levels and lower expression of the CXCR2 receptor on circulating neutrophils compared with non-treated CLP-induced septic mice. FK506 did not directly affect CXCL2-induced CXCR2 internalization by transfected HEK-293 cells or mice neutrophils, despite increasing CXCL2 release by LPS-treated macrophages. Finally, the CLP-induced response of Nfat1−/− mice was similar to those observed in the Nfat1+/+ genotype, suggesting that the FK506 effect is not dependent on the NFAT1 pathway.

Conclusion

Our data indicate that the increased susceptibility to infection of FK506-treated mice is associated with failed neutrophil migration due to the reduced membrane availability of CXCR2 receptors in response to exacerbated levels of circulating CXCL2.

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Acknowledgements

This study was supported by the São Paulo Research Foundation (FAPESP) (grants: 13/08216-2) and the National Council of Technological and Scientific Development (CNPq–Brazil). VFB and LSG hold a PhD scholarship from FAPESP (grants: 17/18264-5 and 20/08109-5).

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Author notes

  1. Vanessa de Fátima Borges and Leticia Selinger Galant contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmacology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil

    Vanessa de Fátima Borges, Leticia Selinger Galant, Alexandre Kanashiro, Fernanda Vargas e Silva Castanheira, Filipe Camargo Rodrigues, Camila Meirelles de Souza Silva, Ayda Henriques Schneider, Guilherme Cesar Martelossi Cebinelli, Mikhael Haruo Fernandes de Lima, Thiago Mattar Cunha, José Carlos Farias Alves-Filho & Fernando de Queiroz Cunha

  2. Center for Research in Inflammatory Diseases, CRID, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil

    Vanessa de Fátima Borges, Leticia Selinger Galant, Alexandre Kanashiro, Fernanda Vargas e Silva Castanheira, Valter Vinícius Silva Monteiro, Filipe Camargo Rodrigues, Camila Meirelles de Souza Silva, Ayda Henriques Schneider, Guilherme Cesar Martelossi Cebinelli, Mikhael Haruo Fernandes de Lima, Thiago Mattar Cunha, José Carlos Farias Alves-Filho & Fernando de Queiroz Cunha

  3. Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil

    Valter Vinícius Silva Monteiro, Diego Ângelo Duarte & Claudio Miguel da Costa Neto

  4. National Cancer Institute (INCA), Rio de Janeiro, Brazil

    João Paulo de Biaso Viola

  5. Department of Biophysics and Pharmacology, Institute of Biosciences, University of São Paulo State (UNESP), Botucatu, São Paulo, Brazil

    André Sampaio Pupo

Authors
  1. Vanessa de Fátima Borges
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  2. Leticia Selinger Galant
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Contributions

VFB, LSG, AK, and FQC designed the study. VFB, LSG, AK, FVSC, VVSM, DAD, FCR, AHS, GCMC, CMSS, and MHFL performed the mouse experiments. VFB, LSG, AK, and FQC wrote the manuscript. VFB, LSG, AK, FVSC, VVSM, AHS, GCMC, CMSS, MHFL, JPBV, TMC, CMCN, JCFAF, ASP, and FQC reviewed the manuscript.

Corresponding author

Correspondence to Fernando de Queiroz Cunha.

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Borges, V., Galant, L.S., Kanashiro, A. et al. FK506 impairs neutrophil migration that results in increased polymicrobial sepsis susceptibility. Inflamm. Res. 72, 203–215 (2023). https://doi.org/10.1007/s00011-022-01669-w

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