Immunoproteomics & macrophage mechanisms of human resistance to TST/IGRA conversion
CM. Anterasian1*, JD. Simmons1, DL. Swaney2, GJ. Peterson1, P. Benchek3, P. Van1, R. Gottardo1, CM. Stein3, H. Mayanja-Kizza4, J. Johnson5, J. Cox2, NJ. Krogan6, WH. Boom3, TR. Hawn1; 1University of Washington; 2University of California, Berkeley; 3Case Western Reserve University; 4Makerere University, Kampala, Uganda; 5Icahn School of Medicine, Mount Sinai;
6University of California, San Francisco
Background: Despite long-term and intense household exposure to Mycobacterium tuberculosis (Mtb), some individuals remain resistant to traditionally defined latent TB infection (LTBI) with persistently negative tuberculin skin tests and interferon-g release assays (“RSTR” phenotype). Global proteomics can be used to detect Mtb-induced changes in RSTR versus LTBI cellular biology, including pathways that are not transcriptionally regulated. We hypothesize that macrophage immunoproteomic responses to Mtb are genetically regulated and associated with Mtb clinical outcomes.
Methods: Primary human monocyte-derived macrophages (MDMs) from Ugandan individuals (RSTR n=20, LTBI n=19) were infected ex vivo with Mtb H37Rv. Protein abundance was measured at 24 hours by mass spectrometry. Differentially abundant proteins (DAPs) in RSTR versus LTBI macrophages were identified using a linear mixed effects model incorporating age, sex, and kinship. Ugandan cohort transcriptomic data from Mtb-infected monocytes and genomic data from Illumina MegaEx array were used to identify DAP polymorphisms associated with DAP mRNA expression and cytokine mRNA expression.
Results: We identified 46 DAPs that define the RSTR versus LTBI Mtb-induced proteome in MDMs (false-discovery rate (FDR) < 0.20). Enrichment analysis revealed vesicle-mediated transport (FDR <0.001) and leukocyte activation (FDR=0.009) as prominent DAP-mediated pathways. STRING analysis identified a central membrane-trafficking hub comprised of 3 RAB proteins, RAB14 (fold change(FC) -2.4, FDR <0.0001), RAB11B (FC +1.6, FDR < 0.0001), and RABGEF1 (FC +1.3, FDR = 0.04), the latter of which activates RAB5. We confirmed that RAB11B mRNA (FDR = 0.07; RSTR n=38, LTBI n= 40), but not RAB14 or RABGEF1 mRNA, was differentially expressed in RSTR versus LTBI Mtb-infected monocytes from the same cohort. Next, we assessed if genetic variation in these 3 RAB DAPs was associated with RAB mRNA expression and identified RABGEF1 polymorphism rs12536130 (p=0.03, RSTR n=38, LTBI n=42) as an expression quantitative trait locus (eQTL) associated with increased RABGEF1 expression concordant with proteomic data. rs12536130 was also associated with lower Mtb-induced IL1B (p=0.005) and IL6 (p=0.006) mRNA expression.
Conclusion: Using proteomic profiling of Mtb-infected RSTR and LTBI macrophages, we identified 46 DAPs, including 3 RAB GTPases, that are candidate genes regulating resistance to TST/IGRA conversion. In particular, RABGEF1 expression may be genetically regulated, resulting in increased RAB5 activation and decreased pro-inflammatory signaling in RSTR macrophages, which may differentially impact Mtb macrophage outcomes.