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Updated: May 3, 2026
Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages
Published on: April 20, 2018
Elevated A20 contributes to age-dependent macrophage dysfunction in the lungs
Cecilia A Hinojosa1, Ramya Akula Suresh Babu1, Md M Rahman2
1Center for Airway Inflammation, Department of Microbiology and Immunology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.
Abstract:
Advanced age is associated with chronic low-grade inflammation (i.e. inflamm-aging) and poor macrophage function that includes a weak pro-inflammatory cytokine response to bacteria and diminished phagocytosis (i.e. age-dependent macrophage dysfunction [ADMD]). One reason for this is that ADMD is associated with poor NFκB and MAPK activation following Toll-like receptor stimulation. Herein, we tested the hypothesis that inflamm-aging induces production of A20, a cytosolic and homeostatic suppressor of the NFκB and MAPK signaling cascades that deubiquitinates (i.e. inactivates) the common upstream signaling molecule TRAF6, and this is responsible for ADMD. Western blots and immunohistochemistry comparing tissues from young, mature, and aged C57BL/6 mice indicated that A20 was strongly elevated in the lungs of aged mice but not in other tissues. Elevated A20 was also detected in alveolar macrophages (AM) from aged mice. In contrast CYLD, a second deubiquitinase that also negatively regulates the NFκB pathway was decreased with aging. Following co-incubation of AM with the bacteria Streptococcus pneumoniae, TRAF6 polyubiquitination was diminished in AM isolated from aged versus young mice. A20 production was inducible in the J774A.1 macrophage cell line and C57BL/6AM by overnight incubation with TNFα but not IL-6. Retrovirus-induced expression of A20 in J774A.1 cells resulted in their diminished production of IL-6 following exposure to S. pneumoniae but had no effect on levels of phagocytosis. Overnight incubation of AM from young mice with TNFα also resulted in a dampened IL-6 response to S. pneumoniae. Finally, dietary supplementation of aged mice with anti-inflammatory n-3 polyunsaturated fatty acids in the form of fish oil lowered lung A20 levels and enhanced resistance, including a 100-fold reduction in bacterial titers in the lungs, to experimental challenge with S. pneumoniae. We conclude that elevated A20 due to TNFα partially explains the ADMD phenotype and that ADMD is potentially reversible.
Insights
Elevated A20 levels in aged mice impair macrophage function, contributing to inflamm-aging. Fish oil supplementation reversed this, enhancing bacterial resistance and suggesting age-dependent macrophage dysfunction is reversible.
Area of Science:
- Immunology
- Aging Research
- Molecular Biology
Background:
- Advanced age is linked to chronic inflammation (inflamm-aging) and impaired macrophage function, termed age-dependent macrophage dysfunction (ADMD).
- ADMD is characterized by reduced pro-inflammatory cytokine response and phagocytosis, partly due to impaired NFκB and MAPK signaling.
- This dysfunction is associated with poor activation of key signaling pathways following Toll-like receptor stimulation.
Purpose of the Study:
- To test the hypothesis that elevated A20 production during inflamm-aging causes ADMD.
- To investigate the role of A20, a suppressor of NFκB and MAPK signaling, in age-related macrophage dysfunction.
- To explore potential therapeutic interventions for ADMD.
Main Methods:
- Comparative analysis of A20 and CYLD expression in lung tissues and alveolar macrophages (AM) from young, mature, and aged C57BL/6 mice using Western blots and immunohistochemistry.
- Assessment of TRAF6 polyubiquitination in AM after co-incubation with Streptococcus pneumoniae.
- In vitro studies using J774A.1 macrophage cell line and primary AM to investigate A20 inducibility by TNFα and IL-6, and the effect of A20 overexpression on cytokine production and phagocytosis.
- Evaluation of the impact of dietary fish oil supplementation in aged mice on lung A20 levels and resistance to S. pneumoniae challenge.
Main Results:
- A20 levels were significantly elevated in the lungs and AM of aged mice, while CYLD levels decreased.
- AM from aged mice exhibited diminished TRAF6 polyubiquitination upon bacterial challenge.
- A20 production was inducible by TNFα in macrophages, and its overexpression led to reduced IL-6 production in response to S. pneumoniae.
- Fish oil supplementation in aged mice reduced lung A20 levels and significantly enhanced bacterial clearance, improving resistance to S. pneumoniae infection.
Conclusions:
- Elevated A20, partly induced by TNFα, contributes to age-dependent macrophage dysfunction (ADMD).
- The study identifies A20 as a key mediator in the impaired immune response associated with aging.
- ADMD is potentially reversible, as demonstrated by the therapeutic effect of fish oil supplementation.
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