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.

Characterizing the Spectrum of Latent Mycobacterium tuberculosis in the Cynomolgus Macaque Model: Clinical, Immunologic, and Imaging Features of Evolution.

Mycobacterium tuberculosis infection outcomes have been described as active tuberculosis or latent infection but a spectrum of outcomes is now recognized. We used a nonhuman primate model, which recapitulates human infection, to characterize the clinical, microbiologic, and radiographic patterns associated with developing latent M. tuberculosis infection. Four patterns were identified. "Controllers" had normal erythrocyte sedimentation rate (ESR) without M. tuberculosis growth in bronchoalveolar lavage or gastric aspirate (BAL/GA). "Early subclinicals" showed transient ESR elevation and/or M. tuberculosis growth on BAL/GA for 60 days postinfection, "mid subclinicals" were positive for 90 days, and "late subclinicals" were positive intermittently, despite the absence of clinical disease. Variability was noted regarding granuloma formation, lung/lymph node metabolic activity, lung/lymph node bacterial burden, gross pathology, and extrapulmonary disease. Like human M. tuberculosis infection, this highlights the heterogeneity associated with the establishment of latent infection, underscoring the need to understand the clinical spectrum and risk factors associated with severe disease.

Characterization of natural killer cells in the blood and airways of cynomolgus macaques during Mycobacterium tuberculosis infection.

BACKGROUND: Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and kills more than 1.5 million people each year. METHODS: We examine the frequency and function of NK cells in the blood and airways over the course of Mtb infection in a TB macaque model and demonstrate differences in NK marker expression between the two compartments. Flow cytometry and intracellular cytokine staining were utilized to identify NK cell subsets (expressing NKG2A, CD56, or CD16) and function (IL-10, TNF, IL-2, IFN-g, IL-17, and CD107a). RESULTS: Blood and airway NK cell frequencies were similar during infection though there were differences in subset populations between blood and airway. Increased functional (cytokine/CD107a) parameters were observed in airway NK cells during the course of infection while none were seen in the blood. CONCLUSIONS: This study suggests that NK cells in the airway may play an important role in TB host response.

SIV Evolutionary Dynamics in Cynomolgus Macaques during SIV-Mycobacterium tuberculosis Co-Infection.

Co-infection with Mycobacterium tuberculosis (Mtb) and human immunodeficiency virus (HIV) is a worldwide public health concern, leading to worse clinical outcomes caused by both pathogens. We used a non-human primate model of simian immunodeficiency virus (SIV)-Mtb co-infection, in which latent Mtb infection was established prior to SIVmac251 infection. The evolutionary dynamics of SIV env was evaluated from samples in plasma, lymph nodes, and lungs (including granulomas) of SIV-Mtb co-infected and SIV only control animals. While the diversity of the challenge virus was low and overall viral diversity remained relatively low over 6-9 weeks, changes in viral diversity and divergence were observed, including evidence for tissue compartmentalization. Overall, viral diversity was highest in SIV-Mtb animals that did not develop clinical Mtb reactivation compared to animals with Mtb reactivation. Among lung granulomas, viral diversity was positively correlated with the frequency of CD4+ T cells and negatively correlated with the frequency of CD8+ T cells. SIV diversity was highest in the thoracic lymph nodes compared to other sites, suggesting that lymphatic drainage from the lungs in co-infected animals provides an advantageous environment for SIV replication. This is the first assessment of SIV diversity across tissue compartments during SIV-Mtb co-infection after established Mtb latency.

SIV and Mycobacterium tuberculosis synergy within the granuloma accelerates the reactivation pattern of latent tuberculosis.

Human immunodeficiency virus infection is the most common risk factor for severe forms of tuberculosis (TB), regardless of CD4 T cell count. Using a well-characterized cynomolgus macaque model of human TB, we compared radiographic, immunologic and microbiologic characteristics of early (subclinical) reactivation of latent M. tuberculosis (Mtb) infection among animals subsequently infected with simian immunodeficiency virus (SIV) or who underwent anti-CD4 depletion by a depletion antibody. CD4 depleted animals had significantly fewer CD4 T cells within granulomas compared to Mtb/SIV co-infected and Mtb-only control animals. After 2 months of treatment, subclinical reactivation occurred at similar rates among CD4 depleted (5 of 7 animals) and SIV infected animals (4 of 8 animals). However, SIV-induced reactivation was associated with more dissemination of lung granulomas that were permissive to Mtb growth resulting in greater bacterial burden within granulomas compared to CD4 depleted reactivators. Granulomas from Mtb/SIV animals displayed a more robust T cell activation profile (IFN-α, IFN-γ, TNF, IL-17, IL-2, IL-10, IL-4 and granzyme B) compared to CD4 depleted animals and controls though these effectors did not protect against reactivation or dissemination, but instead may be related to increased viral and/or Mtb antigens. SIV replication within the granuloma was associated with reactivation, greater overall Mtb growth and reduced Mtb killing resulting in greater overall Mtb burden. These data support that SIV disrupts protective immune responses against latent Mtb infection beyond the loss of CD4 T cells, and that synergy between SIV and Mtb occurs within granulomas.

CD4CD8 Double Positive T cell responses during Mycobacterium tuberculosis infection in cynomolgus macaques.

BACKGROUND: Tuberculosis (TB) kills millions of people every year. CD4 and CD8 T cells are critical in the immune response against TB. T cells expressing both CD4 and CD8 (CD4CD8 T cells) are functionally active and have not been examined in the context of TB. METHODS: We examine peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage cells (BAL) and lung granulomas from 28 cynomolgus macaques during Mycobacterium tuberculosis (Mtb) infection. RESULTS: CD4CD8 T cells increase in frequency during early Mtb infection in PBMC and BAL from pre-infection. Peripheral, airway, and lung granuloma CD4CD8 T cells have distinct patterns and greater cytokine production than CD4 or CD8 T cells. CONCLUSION: Our data suggest that CD4CD8 T cells transient the blood and airways early during infection to reach the granulomas where they are involved directly in the host response to Mtb.

Diversity of Human and Macaque Airway Immune Cells at Baseline and during Tuberculosis Infection.

Immune cells of the distal airways serve as "first responders" of host immunity to the airborne pathogen Mycobacterium tuberculosis (Mtb). Mtb infection of cynomolgus macaques recapitulates the range of human outcomes from clinically silent latent tuberculosis infection (LTBI) to active tuberculosis of various degrees of severity. To further advance the application of this model to human studies, we compared profiles of bronchoalveolar lavage (BAL) cells of humans and cynomolgus macaques before and after Mtb infection. A simple gating strategy effectively defined BAL T-cell and phagocyte populations in both species. BAL from Mtb-naive humans and macaques showed similar differential cell counts. BAL T cells of macaques were composed of fewer CD4+cells but more CD8+ and CD4+CD8+ double-positive cells than were BAL T cells of humans. The most common mononuclear phagocyte population in BAL of both species displayed coexpression of HLA-DR, CD206, CD11b, and CD11c; however, multiple phagocyte subsets displaying only some of these markers were observed as well. Macaques with LTBI displayed a marked BAL lymphocytosis that was not observed in humans with LTBI. In macaques, the prevalence of specific mononuclear phagocyte subsets in baseline BAL correlated with ultimate outcomes of Mtb infection (i.e., LTBI versus active disease). Overall, these findings demonstrate the comparability of studies of pulmonary immunity to Mtb in humans and macaques. They also indicate a previously undescribed complexity of airway mononuclear phagocyte populations that suggests further lines of investigation relevant to understanding the mechanisms of both protection from and susceptibility to the development of active tuberculosis within the lung.

PET CT Identifies Reactivation Risk in Cynomolgus Macaques with Latent M. tuberculosis.

Mycobacterium tuberculosis infection presents across a spectrum in humans, from latent infection to active tuberculosis. Among those with latent tuberculosis, it is now recognized that there is also a spectrum of infection and this likely contributes to the variable risk of reactivation tuberculosis. Here, functional imaging with 18F-fluorodeoxygluose positron emission tomography and computed tomography (PET CT) of cynomolgus macaques with latent M. tuberculosis infection was used to characterize the features of reactivation after tumor necrosis factor (TNF) neutralization and determine which imaging characteristics before TNF neutralization distinguish reactivation risk. PET CT was performed on latently infected macaques (n = 26) before and during the course of TNF neutralization and a separate set of latently infected controls (n = 25). Reactivation occurred in 50% of the latently infected animals receiving TNF neutralizing antibody defined as development of at least one new granuloma in adjacent or distant locations including extrapulmonary sites. Increased lung inflammation measured by PET and the presence of extrapulmonary involvement before TNF neutralization predicted reactivation with 92% sensitivity and specificity. To define the biologic features associated with risk of reactivation, we used these PET CT parameters to identify latently infected animals at high risk for reactivation. High risk animals had higher cumulative lung bacterial burden and higher maximum lesional bacterial burdens, and more T cells producing IL-2, IL-10 and IL-17 in lung granulomas as compared to low risk macaques. In total, these data support that risk of reactivation is associated with lung inflammation and higher bacterial burden in macaques with latent Mtb infection.

Variability in tuberculosis granuloma T cell responses exists, but a balance of pro- and anti-inflammatory cytokines is associated with sterilization.

Lung granulomas are the pathologic hallmark of tuberculosis (TB). T cells are a major cellular component of TB lung granulomas and are known to play an important role in containment of Mycobacterium tuberculosis (Mtb) infection. We used cynomolgus macaques, a non-human primate model that recapitulates human TB with clinically active disease, latent infection or early infection, to understand functional characteristics and dynamics of T cells in individual granulomas. We sought to correlate T cell cytokine response and bacterial burden of each granuloma, as well as granuloma and systemic responses in individual animals. Our results support that each granuloma within an individual host is independent with respect to total cell numbers, proportion of T cells, pattern of cytokine response, and bacterial burden. The spectrum of these components overlaps greatly amongst animals with different clinical status, indicating that a diversity of granulomas exists within an individual host. On average only about 8% of T cells from granulomas respond with cytokine production after stimulation with Mtb specific antigens, and few "multi-functional" T cells were observed. However, granulomas were found to be "multi-functional" with respect to the combinations of functional T cells that were identified among lesions from individual animals. Although the responses generally overlapped, sterile granulomas had modestly higher frequencies of T cells making IL-17, TNF and any of T-1 (IFN-γ, IL-2, or TNF) and/or T-17 (IL-17) cytokines than non-sterile granulomas. An inverse correlation was observed between bacterial burden with TNF and T-1/T-17 responses in individual granulomas, and a combinatorial analysis of pair-wise cytokine responses indicated that granulomas with T cells producing both pro- and anti-inflammatory cytokines (e.g. IL-10 and IL-17) were associated with clearance of Mtb. Preliminary evaluation suggests that systemic responses in the blood do not accurately reflect local T cell responses within granulomas.

CD4 T cell depletion exacerbates acute Mycobacterium tuberculosis while reactivation of latent infection is dependent on severity of tissue depletion in cynomolgus macaques.

CD4 T cells are believed to be important in protection against Mycobacterium tuberculosis, but the relative contribution to control of initial or latent infection is not known. Antibody-mediated depletion of CD4 T cells in M. tuberculosis-infected cynomolgus macaques was used to study the role of CD4 T cells during acute and latent infection. Anti-CD4 antibody severely reduced levels of CD4 T cells in blood, airways, and lymph nodes. Increased pathology and bacterial burden were observed in CD4-depleted monkeys during the first 8 weeks of infection compared to controls. CD4-depleted monkeys had greater interferon (IFN)-γ expression and altered expression of CD8 T cell activation markers. During latent infection, CD4 depletion resulted in clinical reactivation in only three of six monkeys. Reactivation was associated with lower CD4 T cells in the hilar lymph nodes. During both acute and latent infection, CD4 depletion was associated with reduced percentages of CXCR3(+) expressing CD8 T cells, reported to be involved in T cell recruitment, regulatory function, and effector and memory T cell maturation. CXCR3(+) CD8 T cells from hilar lymph nodes had more mycobacteria-specific cytokine expression and greater coexpression of multiple cytokines compared to CXCR3(-) CD8 T cells. CD4 T cells are required for protection against acute infection but reactivation from latent infection is dependent on the severity of depletion in the draining lymph nodes. CD4 depletion influences CD8 T cell function. This study has important implications for human HIV-M. tuberculosis coinfection.

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.

Granule stores from cellubrevin/VAMP-3 null mouse platelets exhibit normal stimulus-induced release.

It is widely accepted that the platelet release reaction is mediated by heterotrimeric complexes of integral membrane proteins known as SNAREs (SNAP receptors). In an effort to define the precise molecular machinery required for platelet exocytosis, we have analyzed platelets from cellubrevin/VAMP-3 knockout mice. Cellubrevin/VAMP-3 has been proposed to be a critical v-SNARE for human platelet exocytosis; however, data reported here suggest that it is not required for platelet function. Upon stimulation with increasing concentrations of thrombin, collagen, or with thrombin for increasing time there were no differences in secretion of [3H]-5HT (dense core granules), platelet factor IV (alpha granules), or hexosaminidase (lysosomes) between null and wild-type platelets. There were no gross differences in bleeding times nor in agonist-induced aggregation measured in platelet-rich plasma or with washed platelets. Western blotting of wild-type, heterozygous, and null platelets confirmed the lack of cellubrevin/VAMP-3 in nulls and showed that most elements of the secretion machinery are expressed at similar levels. While the secretory machinery in mice was similar to humans, mice did express apparently higher levels of synaptobrevin/VAMP-2. These data show that the v-SNARE, cellubrevin/VAMP-3 is not a requirement for the platelet release reaction in mice.

SNAP-23 is a target for calpain cleavage in activated platelets.

The role of calpain in platelet function is generally associated with aggregation and clot retraction. In this report, data are presented to show that one component of the platelet secretory machinery, SNAP-23, is specifically cleaved by calpain in activated cells. Other proteins of the membrane fusion machinery, e.g. syntaxins 2 and 4 and alpha-SNAP, are not affected. In vitro studies, using permeabilized platelets, demonstrate that cleavage is time- and calcium-dependent. Analysis of SNAP-23 cleavage products suggests that the calpain cleavage site(s) is in the C-terminal third of the molecule potentially between the cysteine-rich acyl attachment sites and the C-terminal coiled-coil domain. The time course of cleavage is most consistent with late calpain-mediated events such as pp60(c-src) cleavage, but not early events such as protein-tyrosine phosphatase-1B activation. SNAP-23 cleavage is inhibited by calpeptin, calpastatin, calpain inhibitor IV, and E-64d, but not by caspase 3 inhibitor III or cathepsin inhibitor I. When tested for their effect on secretion, none of the calpain-specific inhibitors significantly affected release of soluble components from any of the three platelet granule storage pools. These results indicate that SNAP-23 cleavage occurs after granule release and therefore may play a role in affecting granule membrane exteriorization. This is consistent with the ultrastructural morphology of calpeptin-treated platelets after activation.