Mucosal and systemic antigen-specific antibody responses correlate with protection against active tuberculosis in nonhuman primates.

BACKGROUND: Increasing evidence supports that antibodies can protect against active tuberculosis (TB) but knowledge of potentially protective antigens, especially in the airways, is limited. The main objective of this study was to identify antigen-specific airway and systemic immunoglobulin isotype responses associated with the outcome of controlled latent Mycobacterium tuberculosis (Mtb) infection (LTBI) versus uncontrolled infection (TB) in nonhuman primates. METHODS: In a case-control design, using non-parametric group comparisons with false discovery rate adjustments, we assessed antibodies in 57 cynomolgus macaques which, following low-dose airway Mtb infection, developed either LTBI or TB. We investigated airway and systemic IgG, IgA, and IgM responses in paired bronchoalveolar lavage and plasma samples prior to, two-, and 5-6-months post Mtb infection using an antigen-unbiased approach with Mtb glycan and proteome-wide microarrays. FINDINGS: Macaques that developed LTBI (n = 36) had significantly increased airway and plasma IgA reactivities to specific arabinomannan (AM) motifs prior to Mtb infection compared to those that developed TB (n = 21; p < 0.01, q < 0.05). Furthermore, LTBI macaques had higher plasma IgG reactivity to protein MTB32A (Rv0125) early post Mtb infection (p < 0.05) and increasing airway IgG responses to some proteins over time. INTERPRETATION: Our results support a protective role of pre-existing mucosal (lung) and systemic IgA to specific Mtb glycan motifs, suggesting that prior exposure to nontuberculous mycobacteria could be protective against TB. They further suggest that IgG to Mtb proteins early post infection could provide an additional protective mechanism. These findings could inform TB vaccine development strategies. FUNDING: NIH/NIAID AI117927, AI146329, and AI127173 to JMA.

Alcohol-induced alterations in hepatic microtubule dynamics can be explained by impaired histone deacetylase 6 function.

UNLABELLED: We have been using polarized, hepatic WIF-B cells to examine ethanol-induced liver injury. These cells polarize in culture and maintain numerous liver-specific activities including the ability to metabolize alcohol. Previously, we found that microtubules were more highly acetylated and more stable in ethanol-treated WIF-B cells and that increased microtubule acetylation required ethanol metabolism and was likely mediated by acetaldehyde. This study was aimed at identifying the mechanism responsible for increased microtubule acetylation. We examined the expression of two known microtubule deacetylases, histone deacetylase 6 (HDAC6) and Sirtuin T2 (SirT2), in WIF-B cells. Immunoblotting, immunofluorescence microscopy, and assays using the SirT2 inhibitor nicotinamide revealed that WIF-B cells do not express SirT2. In contrast, HDAC6 was highly expressed in WIF-B cells. Addition of trichostatin A (TSA), an HDAC6 inhibitor, induced microtubule acetylation to the same extent as in ethanol-treated cells (approximately threefold). Although immunofluorescence labeling revealed that HDAC6 distribution did not change in ethanol-treated cells, immunoblotting showed HDAC6 protein levels slightly decreased. HDAC6 solubility was increased in nocodazole-treated cells, suggesting impaired microtubule binding. Direct microtubule binding assays confirmed this hypothesis. The decreased microtubule binding was partially prevented by 4-methyl pyrazole, indicating the effect was in part mediated by acetaldehyde. Interestingly, HDAC6 from ethanol-treated cells was able to bind and deacetylate exogenous tubulin to the same extent as control, suggesting that ethanol-induced tubulin modifications prevented HDAC6 binding to endogenous microtubules. CONCLUSION: We propose that lower HDAC6 levels combined with decreased microtubule binding lead to increased tubulin acetylation in ethanol-treated cells.

Microtubule acetylation and stability may explain alcohol-induced alterations in hepatic protein trafficking.

UNLABELLED: We have been using polarized hepatic WIF-B cells to examine ethanol-induced liver injury. Previously, we determined microtubules were more highly acetylated and more stable in ethanol-treated WIF-B cells. We proposed that the ethanol-induced alterations in microtubule dynamics may explain the ethanol-induced defects in membrane trafficking that have been previously documented. To test this, we compared the trafficking of selected proteins in control cells and cells treated with ethanol or with the histone deacetylase 6 inhibitor trichostatin A (TSA). We determined that exposure to 50 nM TSA for 30 minutes induced microtubule acetylation ( approximately 3-fold increase) and stability to the same extent as did ethanol. As shown previously in situ, the endocytic trafficking of the asialoglycoprotein receptor (ASGP-R) was impaired in ethanol-treated WIF-B cells. This impairment required ethanol metabolism and was likely mediated by acetaldehyde. TSA also impaired ASGP-R endocytic trafficking, but to a lesser extent. Similarly, both ethanol and TSA impaired transcytosis of the single-spanning apical resident aminopeptidase N (APN). For both ASGP-R and APN and for both treatments, the block in trafficking was internalization from the basolateral membrane. Interestingly, no changes in transcytosis of the glycophosphatidylinositol-anchored protein, 5'-nucleotidase, were observed, suggesting that increased microtubule acetylation and stability differentially regulate internalization. We further determined that albumin secretion was impaired in both ethanol-treated and TSA-treated cells, indicating that increased microtubule acetylation and stability also disrupted this transport step. CONCLUSION: These results indicate that altered microtubule dynamics explain in part alcohol-induced defects in membrane trafficking.