Poster Session 6, 4:15 PM - 5:00 PM: Room 165 [D2]

Investigating the Nitric Oxide Production of Alveolar Macrophages at the Levels of Gene Expression, Protein Production, and Reactive Nitrogen Intermediates

Presenter: Mai Ha Phan

Faculty Sponsor: Alissa Rothchild

School: UMass Amherst

Research Area: Biochemistry and Molecular Biology

ABSTRACT

Mycobacterium Tuberculosis (Mtb), the causative agent of Tuberculosis, is an intracellular pathogen that primarily infects the lungs and remains a leading cause of infectious disease mortality worldwide. Alveolar macrophages (AMs) are tissue-resident innate immune cells that reside within the lung’s alveoli and serve as the first line of defense against inhaled pathogens such as Mtb. Macrophages produce nitric oxide (NO) as a key antimicrobial effector against Mtb.

Our results demonstrate that AMs exhibit significantly reduced Nos2 gene expression and NO production compared to bone marrow-derived macrophages (BMDMs) following stimulation, resulting in lower levels of reactive nitrogen intermediates. This diminished NO response may contribute to the impaired ability of AMs to control Mtb infection within the lung environment. To comprehensively assess NO production, we quantified it at multiple regulatory levels: (1) Griess assays were used to measure nitrite accumulation in culture supernatants as a readout of extracellular reactive nitrogen intermediates; (2) RT-qPCR was performed to assess gene expression of Nos2, which encodes inducible nitric oxide synthase (iNOS), the enzyme required for NO production; and (3) flow cytometry was used to measure intracellular iNOS protein production. Understanding the regulatory mechanisms that limit NO production in AMs may inform strategies to enhance lung-specific antimicrobial immunity against Mtb.

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