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Ex vivo effect of JAK inhibition on JAK-STAT1 pathway hyperactivation in patients with dominant-negative STAT3 mutations

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Abstract

Purpose

STAT1 gain-of-function (GOF) and dominant-negative (DN) STAT3 syndromes share clinical manifestations including infectious and inflammatory manifestations. Targeted treatment with Janus-kinase (JAK) inhibitors shows promising results in treating STAT1 GOF-associated symptoms while management of DN STAT3 patients has been largely supportive. We here assessed the impact of ruxolitinib on the JAK-STAT1/3 pathway in DN STAT3 patients’ cells.

Methods

Using flow cytometry, immunoblot, qPCR, and ELISA techniques, we examined the levels of basal STAT1 and phosphorylated STAT1 (pSTAT1) of cells obtained from DN STAT3, STAT1 GOF patients, and healthy donors following stimulation with type I/II interferons (IFNs) or interleukin (IL)-6. We also describe the impact of ruxolitinib on cytokine-induced STAT1 signaling in these patients.

Results

DN STAT3 and STAT1 GOF resulted in a similar phenotype characterized by increased STAT1 and pSTAT1 levels in response to IFNα (CD3+ cells) and IFNγ (CD14+ monocytes). STAT1-downstream gene expression and C-X-C motif chemokine 10 secretion were higher in most DN STAT3 patients upon stimulation compared to healthy controls. Ex vivo treatment with the JAK1/2-inhibitor ruxolitinib reduced cytokine responsiveness and normalized STAT1 phosphorylation in DN STAT3 and STAT1 GOF patient’ cells. In addition, ex vivo treatment was effective in modulating STAT1 downstream signaling in DN STAT3 patients.

Conclusion

In the absence of effective targeted treatment options for AD-HIES at present, modulation of the JAK/STAT1 pathway with JAK inhibitors may be further explored particularly in those AD-HIES patients with autoimmune and/or autoinflammatory manifestations.

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Data availability

The raw datasets generated and analyzed for this study and supporting the conclusions will be made available by the corresponding author without undue reservation and on reasonable request to any qualified researcher.

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Acknowledgements

The authors would like to thank the patients and their families for participating in this study.

Funding

This work was supported by the Job Research Foundation (NY, United States); Consejería de Salud de la Junta de Andalucía (SA0051/2020 to O.N.); Agencia de Innovación y Desarrollo de Andalucía (PI-0184-2018 to P.O.), Instituto de Salud Carlos III, Madrid, Spain [Sara Borrell, CD20/00124 to P.B.L, Juan Rodés JR18/00042 to P.O, FIS PI19/01471].

Author information

Author notes

  1. Pilar Blanco Lobo and Paloma Guisado-Hernández contributed equally to this manuscript.

  2. Peter Olbrich and Olaf Neth contributed equally.

Authors and Affiliations

  1. Pediatric Infectious Diseases, Rheumatology and Immunology Unit, Hospital Universitario Virgen del Rocío, Institute of Biomedicine of Seville (IBIS)/Universidad de Sevilla/CSIC, Red de Investigación Traslacional en Infectología Pediátrica RITIP, Av Manuel Siurot s/n, 41013, Seville, Spain

    Pilar Blanco Lobo, Paloma Guisado-Hernández, Isabel Villaoslada, Beatriz de Felipe, Olaf Neth & Peter Olbrich

  2. Pediatric Infectious Diseases and Immunodeficiency Unit, Hospital Universitario y Politécnico La Fe, Valencia, Spain

    Carmen Carreras & Hector Rodriguez

  3. Pediatric Infectology and Immunodeficiencies Unit, IBIMA, Department of Pediatrics, Hospital Regional Universitario Málaga, Malaga, Spain

    Begoña Carazo-Gallego

  4. Pediatric Infectious and Tropical Diseases Department, Hospital Universitario La Paz, CIBERINFEC, Carlos III Health Institute, Madrid, Spain

    Ana Méndez-Echevarria

  5. Immunology Unit, University Hospital Virgen del Rocio, Seville, Spain

    José Manuel Lucena & Pilar Ortiz Aljaro

  6. Servicio de Citometría y Separación Celular, Instituto de Biomedicina de Sevilla - IBiS/HUVR/US/CSIC, Seville, Spain

    María José Castro

  7. Institute of Biomedicine of Seville (IBiS) (HUVR, CSIC, Universidad de Sevilla), Seville, Spain

    José Francisco Noguera-Uclés

  8. Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Joshua D. Milner

  9. Laboratory of Clinical Immunology and Microbiology, Immunopathogenesis Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Katelyn McCann, Alexandra F. Freeman & Steven M. Holland

  10. Department of Medicine, Division of Allergy/Immunology, Washington University in St Louis, St Louis, MO, USA

    Ofer Zimmerman

  11. Fungal Pathogenesis Section, LCIM, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Michail S. Lionakis

Authors
  1. Pilar Blanco Lobo
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  2. Paloma Guisado-Hernández
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Contributions

PO and ON contributed to the conception of the work. PBL, PGH, and IV performed all sample processing and experiments. BdF organized sample ship**. CC, HR, BCG, AME, JML, POA, PO, and ON contributed to the diagnostic and inclusion of DN STAT3 patients. MJC contributed as technician of the cytometry core of the Institute of Biomedicine of Seville. JFNU contributed to the western blot performance analysis. All authors (PBL, PGH, IV, BdF, CC, HR, BCG, AME, JML, POA, MJC, JFNU, JDM, KM, OZ, AF, MSL, SMH, ON, and PO) contributed to the analysis or interpretation of the data, manuscript revision, read, and approved the submitted version. PBL wrote the first draft. PO and ON edited the manuscript.

Corresponding author

Correspondence to Olaf Neth.

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The study was approved by the Ethics Committee of the Hospitales Universitarios Virgen Macarena and Virgen del Rocío (0243-N-19).

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All patients, family members, and healthy volunteers provided written and signed informed consent at each Spanish participating center (Seville, Malaga, Valencia, and Madrid). The authors affirm that human research participants or their legal guardians provided informed consent for participation and publication of their individual details.

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The authors declare no competing interests.

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Lobo, P.B., Guisado-Hernández, P., Villaoslada, I. et al. Ex vivo effect of JAK inhibition on JAK-STAT1 pathway hyperactivation in patients with dominant-negative STAT3 mutations. J Clin Immunol 42, 1193–1204 (2022). https://doi.org/10.1007/s10875-022-01273-x

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