Metabolites enhance innate resistance to human Mycobacterium tuberculosis infection

To determine the mechanisms that mediate resistance to Mycobacterium tuberculosis (M. tuberculosis) infection in household contacts (HHCs) of patients with tuberculosis (TB), we followed 452 latent TB infection–negative (LTBI–) HHCs for 2 years. Those who remained LTBI– throughout the study were identified as nonconverters. At baseline, nonconverters had a higher percentage of CD14+ and CD3–CD56+CD27+CCR7+ memory-like natural killer (NK) cells. Using a whole-transcriptome and metabolomic approach, we identified deoxycorticosterone acetate as a metabolite with elevated concentrations in the plasma of nonconverters, and further studies showed that this metabolite enhanced glycolytic ATP flux in macrophages and restricted M. tuberculosis growth by enhancing antimicrobial peptide production through the expression of the surface receptor sialic acid binding Ig-like lectin–14. Another metabolite, 4-hydroxypyridine, from the plasma of nonconverters significantly enhanced the expansion of memory-like NK cells. Our findings demonstrate that increased levels of specific metabolites can regulate innate resistance against M. tuberculosis infection in HHCs of patients with TB who never develop LTBI or active TB.

MDMs (n = 6) were infected with H37Rv at an MOI of 2.5. After 2 h, the supernatant was 55 aspirated, and the MDMs were lysed. The supernatant was centrifuged to pellet the bacteria, and 56 the pellets were added to the cell lysates. The bacterial suspensions were ultrasonically dispersed, 57 serially diluted, and plated in triplicate on 7H10 agar. The number of resultant colonies was 58 counted after 3 weeks. The p values were derived using an unpaired two-tailed independent t test. 59 The mean values and SDs are shown for the number of CFUs per well. PBMCs from LTBI-healthy donors (n = 5) were cultured in the presence or absence of γ-Mtb.

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After 72  and 4-hydroxypyridine was quantified in the plasma sample of nonconverters (n = 10) and 106 converters (n = 10) at baseline and follow-up using LC-MS.  fluorescence intensity) of Siglec-14 was determined by flow cytometry. The mean ± SD is shown.

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The p values were determined by one-way ANOVA with Tukey's multiple comparisons test.

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Transcriptome and bioinformatics analysis. 285 Methods for data normalization and analysis are based on the use of "internal standards" that 286 characterize some aspects of the system's behavior, such as technical variability, as presented 287 elsewhere (1, 2). Created initially for the analysis of microarray data, these standards were slightly 288 modified to be applicable to the RNA-seq data analysis. Differential gene expression analysis 289 included the following steps. 1. Construction of the 'reference group' by identifying a group of 290 genes expressed above background with inherently low variability as determined by an F test. The

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'reference group' presents an internal standard of equal expression. As such, the 'reference group' 292 is used to assess the inherent variability resulting from technical factors alone (technological 293 variation). By creating an estimate of the technological variation, we were able to select a group algorithm were used to identify the most discriminating metabolites for group comparisons.

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Heatmaps were generated using metabolites that were significantly different across the groups 326 (p<0.05). Metabolite data analyses were performed using MetaboAnalyst.

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Metabolite concentration determination and cytotoxicity assay. 328 Each metabolite was tested for its cytotoxicity against macrophages using a 3-(4,5-  supernatant was centrifuged to pellet the bacteria, and the pellets were added to the cell lysates.

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The bacterial suspensions were ultrasonically dispersed, serially diluted, and plated in triplicate on 378 7H10 agar. The number of colonies was counted after 3 weeks.

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Apo-Direct TUNEL assay. 380 Apoptosis was confirmed by the determination of single DNA strand breaks using an Apo-Direct  Autophagy detection assay. 396 Autophagy was detected using an LC-3 antibody-based assay kit (Millipore's FlowCellect) 397 according to the manufacturer's instructions. In brief, MDMs were infected with Mtb H37Rv at 398 an MOI of 2.5. After 72 h, macrophages were harvested using PBS-EDTA and washed once in 1X 399 assay buffer (provided in the kit) followed by centrifugation at 300 g for 5 min. The pellet was 400 suspended in 1X autophagy reagent B and immediately centrifuged at 300 g for 5 min. The 401 supernatant was aspirated, and the pellet was again resuspended in 95 µl of 1X assay buffer + 5 µl 402 of 20X anti-LC-3/FITC antibody for 30 min at room temperature in the dark. The cells were 403 washed once with 1X assay buffer to remove unbound antibody followed by centrifugation at 300 404 g for 5 min and then resuspended in 1X assay buffer, and then the data were acquired by FACS.

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Mitochondrial stress test assay. 406 MDMs were freshly prepared from the PBMCs of LTBI-nonexposed healthy donors and plated 407 in XF96 plates. The mito-stress test (Agilent Technologies; Cat: 103015-100) was performed on a 408 Seahorse Xfe96 Analyzer (Agilent, Santa Clara, CA). Cells were cultured in XF RPMI medium,