Classical Activation of HMC3 Human Brain Microglia by CyanobacteriumBrasilonemasp LPS: Interleukin-6 and Interferon-γ Production

Abstract ID 87471 Poster Board 209 Introduction Cosmopolitan Gram-negative cyanobacteria may contaminate freshwater by releasing toxins, such as lipopolysaccharide (LPS), thus affecting human and animal health (1). Although classical and alternative activation of rat microglia in vitro by cyanobacterial LPS has been reported (2), the possible activation of human microglia remains undetermined. We hypothesized that cyanobacterium Brasilonema sp. LPS will activate human brain microglia in vitro and the concomitant release of cytokines, specifically interleukin-6 (IL-6) and interferon-γ (IFN-γ), in a time- and concentration-dependent manner. Methods The human microglial clone 3 (HMC3) cell line (ATCC, Manassas, VA) was used for the experiments. LPS from cyanobacterium Brasilonema strain HT-58-2, from the Hawaii Culture Collection, was isolated and purified as described (3). HMC3 cells were treated with either E. coli LPS (026:B6, Difco Lab, Detroit, MI), used as a positive control, or cyanobacterium Brasilonema sp. LPS in a concentration-dependent manner for 18 hours at 35.9°C. After treatment, HMC3 cell-free supernatants were assayed with IL-6 and IFN-γ ELISAs (RayBiotech, Norcross, GA). Confocal immunofluorescence imaging was used to: (A) confirm HMC3 cells were microglia, using a primary mouse anti-human TMEM119 antibody (BioLegend, San Diego, CA) and a secondary Alex Fluor 594 goat anti-mouse IgG antibody (BioLegend); (B) determine HMC3 production of IL-6, using a primary rat anti-human IL-6 antibody (BioLegend) and a secondary Alex Fluor 488 goat anti-rat IgG antibody (BioLegend); and, (C) determine HMC3 production of IFN-γ, using a primary mouse anti-human IFN-γ antibody (RayBiotech) and a secondary donkey anti-mouse IgG FITC conjugate antibody (RayBiotech). Immunofluorescence confocal photography was performed using the Nikon A1R confocal laser inverted microscope (Nikon, Melville, NY). Results HMC3 cells treated with either E. coli LPS or cyanobacterium Brasilonema sp. LPS demonstrated a dose-dependent and statistically significant release of IL-6 and IFN-γ in cell-free supernatants. Furthermore, immunofluorescence confocal microscopy studies confirmed the presence of IL-6 as well as IFN-γ in LPS-treated HMC3 microglia cells. Conclusion Our current observations provide experimental support for our working hypothesis that an 18-hour in vitro stimulation of HMC3 cells with either E. coli LPS or cyanobacterium Brasilonema sp. LPS will result in classical activation of human brain microglia and concomitant production of IL-6 and IFN-γ. To our knowledge, this is the first report of cyanobacterial LPS toxicity to human microglia. Current ongoing studies in our laboratory will determine whether additional cytokines and chemokines are produced by HMC3 microglia cells that are treated with E. coli LPS or cyanobacterium Brasilonema sp. LPS. References (1) CDC: https://www.cdc.gov/habs/materials/factsheet-cyanobacterial-habs.html; (2) Mayer et al. Toxicol. Sci. 121(1): 63-72, 2011. PMID: 21362633; (3) Rexania et al. J. Med. Biotechnol. 3(1), 3–9, 2011. PMID: 23407691 Acknowledgements Support by the Chicago College of Osteopathic Medicine (CCOM) and the College of Graduate Studies, Midwestern University, and the University of Hawaii at Manoa is gratefully acknowledged. B. Zeka, T. Serbest, A. Fowler & T. Jasionowski's were CCOM Summer Research Fellowship recipients.