Skip to main content

ORIGINAL RESEARCH article

Front. Pharmacol.
Sec. Experimental Pharmacology and Drug Discovery
Volume 15 - 2024 | doi: 10.3389/fphar.2024.1465776

Attenuation of neutrophil adhesion and formation of neutrophil extracellular traps by pooled human immune globulins

Provisionally accepted
Vidhya R Rao Vidhya R Rao 1,2Sana Iqbal Sana Iqbal 1Bradford A Young Bradford A Young 3Christine Mun Christine Mun 4,5Sandeep Jain Sandeep Jain 4,5Simon Kaja Simon Kaja 1,2*
  • 1 Loyola University Chicago, Chicago, Illinois, United States
  • 2 Edward Hines, Jr. VA Hospital, United States Department of Veterans Affairs, Hines, Illinois, United States
  • 3 Independent researcher, Annapolis, United States
  • 4 University of Illinois Chicago, Chicago, Illinois, United States
  • 5 Selagine, Inc., Chicago, Illinois, United States

The final, formatted version of the article will be published soon.

    Introduction: This study investigated the efficacy of pooled human immune globulins (Flebogamma ® DIF) to combat the formation of neutrophil extracellular traps (NETs) and NETosis, along with neutrophil adhesion to corneal epithelial cells in response to dry eye disease relevant stimuli.Methods: Human neutrophils were isolated by bead-based immunomagnetic depletion of non-target cells from human whole blood. NETosis was induced using phorbol 12-myristate 13-acetate (PMA) or anti-citrullinated histone 4 R3 antibody (H4R3 ACPA). Extracellular DNA was used as a surrogate biomarker of NETosis, and it was quantified using a 96-well, plate reader-based fluorescent assay and by confocal microscopy in 8-well chambers using the DNA dye, SYTOX TM Green. Neutrophils were labeled with calcein-AM and adhesion to human corneal epithelial cells was measured. The efficacy of a dose-range of pooled human immune globulin (Flebogamma® DIF, 0.01% -5%) was tested in all assays.Results: Pooled human immune globulins (Flebogamma® DIF) dose-dependently inhibited both PMA and H4R3 ACPA induced NETosis, with concentrations ³ 2.5% fully preventing release of extracellular DNA over a 2 to 16 hour time period. Similarly, Flebogamma® 5% DIF prevented NETosis against PMA (20 nM) and a dose range (0.1 -10 µg/mL) of H4R3 ACPA. Both PMA and H4R3 ACPA increased adhesion of neutrophils to corneal epithelial cells by 20% and 5%, respectively. Flebogamma® DIF treatment resulted in a dose-dependent reduction of neutrophil adhesion, with Flebogamma® 5% DIF reducing adhesion to baseline levels.Discussion: These findings show the dose-dependent efficacy of pooled human immune globulins, specifically Flebogamma ® DIF against experimentally and pathologically induced NETosis and neutrophil adhesion to corneal epithelial cells, in vitro. The results from this study support the continued clinical development of Flebogamma ® 5% DIF as a novel and efficacious treatment for the signs and symptoms of dry eye disease.

    Keywords: Ocular surface disease, Dry eye disease, anti-citrullinated protein autoantibodies, Neutrophil extracellular trap, pooled human immune globulin

    Received: 16 Jul 2024; Accepted: 22 Nov 2024.

    Copyright: © 2024 Rao, Iqbal, Young, Mun, Jain and Kaja. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence: Simon Kaja, Loyola University Chicago, Chicago, 60660, Illinois, United States

    Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.