CD40 ligand deficiency causes functional defects of peripheral neutrophils that are improved by exogenous IFN-? - 06/08/18

Doi : 10.1016/j.jaci.2018.02.026

Otavio Cabral-Marques, PhD ^a,^b,^{∗
These authors contributed equally to this work.}, Tabata Takahashi França, MSc ^a,^{∗
These authors contributed equally to this work.}, Ashraf Al-Sbiei, PhD ^c, Lena Friederike Schimke, MD ^a,^b, Taj Ali Khan, PhD ^a,^d, Claudia Feriotti, PhD ^a, Tania Alves da Costa, PhD ^a, Osvaldo Reis Junior, PhD ^e, Cristina Worm Weber, MD ^f, Janaíra Fernandes Ferreira, MD ^g, Fabiola Scancetti Tavares, MD ^h, Claudia Valente, MD ^h, Regina Sumiko Watanabe Di Gesu, MD ⁱ, Asif Iqbal, PhD ^j, Gabriela Riemekasten, MD ^b, Gustavo Pessini Amarante-Mendes, PhD ^k, José Alexandre Marzagão Barbuto, PhD ^a,^l, Beatriz Tavares Costa-Carvalho, MD, PhD ^m, Paulo Vitor Soeiro Pereira, PhD ^a,ⁿ, Maria J. Fernandez-Cabezudo, PhD ^o, Vera Lucia Garcia Calich, PhD ^a, Luigi D. Notarangelo, MD ^p, Troy R. Torgerson, MD, PhD ^q, Basel K. al-Ramadi, PhD ^c, Hans D. Ochs, MD ^q, Antonio Condino-Neto, MD, PhD ^a,^⁎
^a Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
^j Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, Brazil
^k Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
^l Cell and Molecular Therapy Center, NETCEM, University of São Paulo, São Paulo, Brazil
^b Department of Rheumatology, University Lübeck, Lübeck, Germany
^c Department of Medical Microbiology & Immunology, College of Medicine and Health Sciences, UAE University, Al Ain, United Arab Emirates
^o Department of Biochemistry, College of Medicine and Health Sciences, UAE University, Al Ain, United Arab Emirates
^d Department of Microbiology, Kohat University of Science and Technology, Kohat, Pakistan
^e Central Laboratory of High Performance Technologies (LaCTAD), State University of Campinas, Campinas, Brazil
^f Pediatric Allergy & Immunology Clinic, Caxias do Sul, Brazil
^g Albert Sabin Hospital, Fortaleza, Brazil
^h Pediatric Immunology Clinic, Unit of Pediatrics, Hospital de Base do Distrito Federal, Asa Sul, Brazil
ⁱ Division of Allergy and Immunology, Department of Pediatrics, Conceicão Children Hospital, Porto Alegre, Brazil
^m Division of Allergy-Immunology and Rheumatology, Department of Pediatrics, Federal University of São Paulo, São Paulo, Brazil
ⁿ Department of Pathology, Federal University of Maranhao, São Luis, Brazil
^p Laboratory of Clinical Immunology and Microbiology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md
^q Department of Pediatrics, University of Washington School of Medicine, and Seattle Children's Research Institute, Seattle, Wash

^∗Corresponding author: Antonio Condino-Neto, MD, PhD, Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, 1730 Lineu Prestes Ave, São Paulo 05508-000, Brazil.Department of ImmunologyInstitute of Biomedical SciencesUniversity of São Paulo1730 Lineu Prestes AveSão Paulo05508-000Brazil

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Abstract

Background

Patients with X-linked hyper-IgM syndrome caused by CD40 ligand (CD40L) deficiency often present with episodic, cyclic, or chronic neutropenia, suggesting abnormal neutrophil development in the absence of CD40L-CD40 interaction. However, even when not neutropenic and despite immunoglobulin replacement therapy, CD40L-deficient patients are susceptible to life-threatening infections caused by opportunistic pathogens, suggesting impaired phagocyte function and the need for novel therapeutic approaches.

Objectives

We sought to analyze whether peripheral neutrophils from CD40L-deficient patients display functional defects and to explore the in vitro effects of recombinant human IFN-γ (rhIFN-γ) on neutrophil function.

Methods

We investigated the microbicidal activity, respiratory burst, and transcriptome profile of neutrophils from CD40L-deficient patients. In addition, we evaluated whether the lack of CD40L in mice also affects neutrophil function.

Results

Neutrophils from CD40L-deficient patients exhibited defective respiratory burst and microbicidal activity, which were improved in vitro by rhIFN-γ but not soluble CD40L. Moreover, neutrophils from patients showed reduced CD16 protein expression and a dysregulated transcriptome suggestive of impaired differentiation. Similar to CD40L-deficient patients, CD40L knockout mice were found to have impaired neutrophil responses. In parallel, we demonstrated that soluble CD40L induces the promyelocytic cell line HL-60 to proliferate and mature by regulating the expression of genes of the same Gene Ontology categories (eg, cell differentiation) when compared with those dysregulated in peripheral blood neutrophils from CD40L-deficient patients.

Conclusion

Our data suggest a nonredundant role of CD40L-CD40 interaction in neutrophil development and function that could be improved in vitro by rhIFN-γ, indicating a potential novel therapeutic application for this cytokine.

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Key words : Neutrophils, CD40 ligand, cell development, IFN-γ

Abbreviations used : APC, CD40L, CD62L, CFU, CGD, DEG, DMSO, FITC, fMLP, G-CSF, GPI, MFI, NET, PE, PEC, PMA, Q-VD-OPh, rhIFN-γ, RNAseq, ROS, sCD40L, TLR

Plan

Methods

Subjects

Analysis of human neutrophil function

Neutrophil responses in CD40L-deficient mice

Statistical analysis

Results

ROS production by neutrophils from CD40L-deficient patients is reduced, with possible implications for their killing activity

rhIFN-γ improves defective respiratory burst and microbicidal activity of neutrophils from CD40L-deficient patients

Dysregulated transcriptome of neutrophils from CD40L-deficient patients is improved by in vitro treatment with rhIFN-γ

Neutrophils from CD40L-deficient patients exhibit an immature phenotype

Impaired neutrophil immune responses in CD40L^−/− mice resemble defects shown by neutrophils of CD40L-deficient patients

sCD40L accelerates differentiation of the human promyelocytic cell line HL-60

Discussion

Supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; grant 2012/50515-4 to O.C.-M. and grant 2012/51745-3 and 2016/22158-3 to A.C.-N.), the Jeffrey Modell Foundation (to A.C.-N.), and PENSI Institute and Ministério da Saúde do Brasil (PRONAS/PDC 2015 and 25000.077928/2015-06 to A.C.-N.). The animal studies were supported by a UAEU Program for Advanced Research grant (no. 31M193) from the Office of Research and Sponsored Projects, UAE University (to B.K.a.-R.).

Disclosure of potential conflict of interest: T. R. Torgerson has consultant arrangements with Shire Pharmaceuticals, CSL Behring, UCB Pharmaceuticals, and ADMA Biologics and has received grants from the National Institutes of Health. The rest of the authors declare that they have no relevant conflicts of interest.