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. 2022 Jul 13;11:e77457. doi: 10.7554/eLife.77457

Figure 3. Hypoxia modulates tissue-resident alveolar macrophage (TR-AM) cytokine production and metabolic response to lipopolysaccharide (LPS).

TR-AMs were incubated overnight (16 hr) under 21 or 1.5% O2, then stimulated with 20 ng/ml LPS for 6 hr while maintaining pretreatment conditions. For IL-1β measurements, 5 mM ATP was added to TR-AMs for 30 min following 6 hr LPS treatment to activate caspase 1, ensuring IL-1β release. (A) We measured cytokine (TNFα, IL-6, KC, CCL2, and IL-1β) levels in media using ELISA. Data represent at least three independent experiments; n = 3 per group. Significance was determined by unpaired, two-tailed t-test. (B) qPCR was used to measure mRNA expression (Tnfa, Il6, Kc, Ccl2, and Il1b). Gene expression was normalized to corresponding gene ct values in 21% group and represented as fold change using the ∆∆ct method. Data represent at least three independent experiments; n = 3 per group. Significance was determined by unpaired, two-tailed t-test. (C) Western blot analysis of whole-cell extracts at 6 and 24 hr post LPS treatment. (D) Extracellular acidification rate (ECAR) was measured in following acute LPS injection (final concentration: 20 ng/ml) in TR-AMs conditioned in 1.5% O2. (E) Capillary electrophoresis-mass spectrometry (CE-MS) metabolite heatmap for glycolytic intermediates. All error bars denote mean ± SD. *p<0.05.

Figure 3—source data 1. Changes in lipopolysaccharide (LPS)-induced expression of proIL-1β protein in tissue-resident alveolar macrophages (TR-AMs) under normoxia and hypoxia.
Uncropped Western blot images of proIL-1β protein in TR-AMs treated with LPS for 6 or 24 hr under normoxia or hypoxia.

Figure 3.

Figure 3—figure supplement 1. Hypoxia alters cytokine production in bone marrow-derived macrophages (BMDMs).

Figure 3—figure supplement 1.

BMDMs were incubated overnight (16 hr) under normoxia or 1.5% O2, then stimulated with lipopolysaccharide (LPS) (20 ng/ml) for 6 hr while maintaining pretreatment conditions. For IL-1β, 5 mM ATP was added to BMDMs for 30 min following 6 hr of LPS treatment to activate caspase 1, ensuring IL-1β release. (A) Sandwich ELISA was used to measure secreted cytokine (TNFα, IL-6, KC, CCL2, and IL-1β). Data represent at least three independent experiments; n = 3 per group. Significance was determined by unpaired, two-tailed t-test. All error bars denote mean ± SD. *p<0.05.
Figure 3—figure supplement 2. Lipopolysaccharide (LPS) induces an immediate increase in glycolysis in bone marrow-derived macrophages (BMDMs).

Figure 3—figure supplement 2.

Extracellular acidification rate (ECAR) was measured in normoxic BMDMs following acute LPS injection (final concentration: 20 ng/ml).