I29204

The NTM strains used in this study are listed in Table 1. All clinical samples are from CF patients while lettered samples are mixed morphology cultures from the same date and same patient. Bacteria were cultured at 37° C in 7H9 broth with OADC (Remel, R450605). For minimal media experiments, a modified 7H9 broth was used based on the methods described by Muñoz-Elías and McKinney [58 (link)], specifically, NTM were grown in 7H9 + 0.5% albumin, 0.085% NaCl, 0.05% Tween-80, and carbon substrate (10 mM short chain fatty acid) for the indicated number of days. For the pH adjusted minimal media, the media with all the above listed components was pH adjusted and then filter sterilized prior to SCFA addition and use. IA (Sigma-Aldrich, I29204) was resuspended in sterile ddH2O, pH adjusted (for the pH = 7 IA), filter sterilized, and added to the 7H9 media to the desired concentration. For pH-neutral IA experiments, IA received NaOH to bring the pH to a neutral range before being brought to the desired final volume and filter sterilized for addition to the 7H9 media. 4-Octyl itaconate (MedChem Express, HY-112675) was resuspended in DMSO and added to THP-1 cells at the desired concentrations. The short chain fatty acids (SCFA) acetate, propionate, and butyrate (Sigma-Aldrich, S2889, P5436, & 303410, respectively) were resuspended in sterile ddH20 and filter sterilized prior to use.

Intracellular itaconic acid levels were measured using high performance liquid chromatography. Pellets from 2 × 10⁷ macrophages were resuspended in PBS, pH 7.4 and lysed by three freeze-thaw cycles. Samples were bath-sonicated (5 min, room temperature) in two steps with an intermediate freeze step. After centrifugation (17,000 × g, 15 min, 4°C), debris-free lysates were transferred to new tubes. Proteins were then precipitated using 100 mM hydrochloric acid and centrifuged (17,000 × g, 15 min, 4°C). Remaining supernatants were injected into a column of 250 × 2 mm (Col Kromophase 100 C18 5.0 mm; Scharlau Chemie SA, Barcelona, Spain) and itaconate was quantified using UV detection at 210 mm. A standard curve (see Supplementary Figure 1A) was obtained using pure itaconic acid (0.50–500 μM) (I29204; Sigma-Aldrich). Results were normalized using the protein concentration of each sample.