Abstract
The immune mechanisms by which early host-mycobacterium interaction leads to the development of severe tuberculosis (TB) remain poorly characterized in humans. Here, we demonstrate that severe TB in juvenile rhesus monkeys down-regulated many genes in the blood but up-regulated selected genes constituting gene networks of Th17 and Th1 responses, T cell activation and migration, and inflammation and chemoattractants in the pulmonary and lymphoid compartments. Overexpression (450–2740-fold) of 13 genes encoding inflammatory cytokines and receptors (IL-22, CCL27, MIP-1α, IP-10, CCR4, CCR5, and CXCR3), immune dysfunctional receptors and ligands (PD1 and PDL2), and immune activation elements (IL-3, IFN-β, TIM1, and TLR2) was seen in tissues, with low antigen-specific cellular responses. Thus, severe TB in macaques features unbalanced up-regulation of immune-gene networks without proportional increases in antigen-specific cellular responses.
Mycobacterium tuberculosis–induced tuberculosis (TB) is one of the 3 major killers among infectious diseases. Primary or reactivated M. tuberculosis infection can progress to severe or fatal TB, which is often characterized by large cavitary lung lesions, miliary TB, or meningitis. Children <6 years old infected with M. tuberculosis are at increased risk of developing severe TB compared with adults, and virtually all disseminated TB cases occur in children <2 years old [1]; in fact, pediatric TB represents a major cause of childhood morbidity and mortality worldwide, affecting >800,000 children each year and comprising ~10% of all TB cases [1].
The immune mechanisms by which early mycobacterium-host interaction leads to severe TB in humans remain poorly characterized. Immune cells and their cytokines and receptors may serve as host factors and affect immune activation or inflammation and immunity during TB. Although Th1, Th17, and regulatory T cells may play a role in the immune regulation of TB in humans, some host factors may contribute to the development of severe TB. Given the possibility that multiple genes or networks may play a role in the development of susceptibility or immunity to TB, studies of such complex gene networks may shed light on the pathogenesis of TB in humans [2, 3].
Studies from us and others have demonstrated that juvenile rhesus macaques are susceptible to severe or fatal M. tuberculosis infection [4] and thus may serve as a model to study host factors facilitating severe TB in humans. We therefore took advantage of the large-scale real-time quantitative system that we recently developed [5] and measured the expression of 138 immune genes after M. tuberculosis infection in juvenile rhesus monkeys. We found that severe TB in macaques induced unbalanced up-regulation of selected gene networks, causing overexpression of genes encoding for inflammatory cytokines and chemokines without proportional increases in antigen-specific cellular responses.
Methods
Four juvenile rhesus monkeys (2 years of age) were infected with the H37Rv strain of M. tuberculosis by aerosol, as described elsewhere [6]; 4 healthy, uninfected rhesus monkeys (5–8 years old) served as controls. The inhaled doses for the individual monkeys ranged from 400 to 500 cfu of M. tuberculosis. Four to 5 weeks after M. tuberculosis infection, 1 monkey was dying from respiratory distress and the other 3 were moribund as a result of the M. tuberculosis infection. The monkeys were immediately euthanized, and organs were carefully removed for immunologic and pathologic evaluation. Necropsy showed that all of the monkeys developed severe TB, characterized by miliary lesions in lungs. The data at week 4 for 2 monkeys and at week 5 for the other 2 monkeys were combined and are arbitrarily listed as being from week 5 after M. tuberculosis infection.
Lymph nodes and spleen were carefully teased to generate single-cell suspensions. Pieces of lung tissues were minced in RPMI 1640 medium, as described elsewhere [6], to collect single-cell suspensions (mainly lymphocytes and tissue macrophages). The single-cell suspensions and blood were used to isolate lymphocytes by Ficoll/diatrizoate gradient centrifugation for measuring the expression of genes and the numbers of antigen-specific interferon (IFN)–γ–producing cells.
Total RNA was isolated from peripheral blood mononuclear cells and lymphocytes from lungs, lymph nodes, and spleens by the TRIzol (Invitrogen) isolation method, as described elsewhere [5]. The large-scale real-time quantitation system was established and validated as described elsewhere [5]. Peripheral blood lymphocytes (PBLs) and lymphocytes from lungs and lymph nodes were assessed for their specific recognition of purified protein derivative (PPD) antigen and pools of overlapping early secretory antigenic target (ESAT) 6 or Ag85B peptides by enzyme-linked immunospot assay, as described elsewhere [5]. Student’s t test and nonparametric test were done as described elsewhere [5].
Results
To better understand the immunopathogenesis of severe M. tuberculosis infection seen in human adults and children, naive juvenile rhesus monkeys were infected with M. tuberculosis by aerosol and followed clinically for disease progression and immunologically for host responses. After M. tuberculosis infection, only 17 of 138 immune genes were up-regulated in blood lymphocytes from weeks 2–5 (table 1). In contrast, 29 selected immune genes (including IL-3, IL-12a, IL-16, IL-20, IL-22, IL-23a, IL-11R, MCP-1, MIP-1a, RANTES, CCR2, CCR3, CCR4, CCR5, CCR8, CCR9, CCR10, CCR11, CXCR4, GZMM, and TIM-1) were down-regulated during the entire 5-week experiment (P < .05) (tables 1 and 2). Importantly, the down-regulation of many immune genes was not attributed to lymphopenia, because the sizes of T cell populations slightly increased rather than decreased in the blood during the progression of M. tuberculosis infection (figure 1).
Table 2.
Expression levels of immune genes that were down-regulated in peripheral blood lymphocytes during progression of Mycobacterium tuberculosis infection.
| W2:W0 ratio | W3:W0 ratio | W5:W0 ratio | ||||
|---|---|---|---|---|---|---|
| Gene | Mean ± SD | P | Mean ± SD | P | Mean ± SD | P |
| IL-3 | 0.3 ± 0.2 | .001** | 0.2 ± 0.1 | <.001** | 0.2 ± 0.2 | <.001** |
| IL-12α | 0.5 ± 0.1 | .003** | 0.9 ± 0.2 | .196 | 0.5 ± 0.2 | .024* |
| IL-16 | 0.5 ± 0.4 | .044* | 0.5 ± 0.2 | .048* | 0.2 ± 0.1 | .011* |
| IL-20 | 0.4 ± 0.2 | .011* | 0.4 ± 0.1 | .007** | 0.3 ± 0.2 | .004** |
| IL-22 | 0.5 ± 0.2 | .005** | 0.5 ± 0.2 | .010* | 0.2 ± 0.1 | .001** |
| IL-23α | 0.3 ± 0.1 | <.001** | 0.5 ± 0.2 | .008** | 0.2 ± 0.1 | <.001** |
| IL-27 | 0.6 ± 0.2 | .001** | 0.7 ± 0.3 | .106 | 0.5 ± 0.3 | .006** |
| IFN-α | 0.7 ± 0.2 | .097 | 0.7 ± 0.2 | .071 | 0.3 ± 0.2 | .002** |
| IL-8Rα | 0.3 ± 0.2 | .030* | 0.6 ± 0.6 | .070 | 0.2 ± 0.1 | .026* |
| IL-11Rα | 0.4 ± 0.2 | .004** | 0.5 ± 0.1 | .011* | 0.3 ± 0.1 | .002** |
| IL-15Rα | 1.9 ± 1.0 | .236 | 4.3 ± 2.7 | .033* | 7.6 ± 8.6 | .041* |
| FRA | 0.4 ± 0.3 | .042* | 0.7 ± 0.5 | .108 | 0.2 ± 0.2 | .015* |
| MCP1 | 0.5 ± 0.5 | .029* | 0.6 ± 0.3 | .018* | 0.3 ± 0.2 | .001** |
| MIP-1α | 0.4 ± 0.2 | .005** | 0.5 ± 0.2 | .011* | 0.2 ± 0.1 | .004** |
| RANTES | 0.4 ± 0.2 | .005** | 0.5 ± 0.2 | .011* | 0.2 ± 0.1 | .004** |
| CCR2 | 0.5 ± 0.3 | .032* | 0.5 ± 0.1 | .013* | 0.3 ± 0.1 | .005** |
| CCR3 | 0.5 ± 0.3 | .007** | 0.5 ± 0.0 | .008** | 0.2 ± 0.1 | .001** |
| CCR4 | 0.6 ± 0.3 | .039* | 0.6 ± 0.1 | .005** | 0.3 ± 0.2 | .004** |
| CCR5 | 0.6 ± 0.3 | .073 | 0.4 ± 0.2 | .005** | 0.2 ± 0.2 | .001** |
| CCR6 | 0.4 ± 0.2 | .048* | 0.5 ± 0.2 | .008** | 0.3 ± 0.2 | .005** |
| CCR8 | 0.4 ± 0.1 | .001** | 0.4 ± 0.1 | <.001** | 0.2 ± 0.1 | <.001** |
| CCR9 | 0.5 ± 0.3 | .010* | 0.4 ± 0.2 | .010* | 0.2 ± 0.1 | .003** |
| CCR10 | 0.3 ± 0.3 | .008** | 0.5 ± 0.3 | .014* | 0.2 ± 0.2 | .002** |
| CCR11 | 0.6 ± 0.2 | .017* | 0.7 ± 0.1 | .015* | 0.3 ± 0.1 | .001** |
| CXCR4 | 0.6 ± 0.4 | .029* | 0.6 ± 0.1 | .013* | 0.3 ± 0.1 | .003** |
| B7–2 | 1.0 ± 0.6 | .412 | 0.7 ± 0.3 | .028* | 0.4 ± 0.4 | .005** |
| CTSγ | 0.9 ± 0.4 | .070 | 0.8 ± 0.4 | .016* | 0.6 ± 0.5 | .005** |
| GZMM | 0.5 ± 0.2 | <.001** | 0.5 ± 0.1 | .001** | 0.3 ± 0.2 | <.001** |
| TIM-1 | 0.3 ± 0.1 | .006** | 0.4 ± 0.2 | .005** | 0.2 ± 0.2 | .002** |
NOTE. Shown are the means of expression ratios (fold changes) for down-regulated immune genes at weeks (W) 2, 3, and 5 after pulmonary M. tuberculosis infection relative to preinfection (W0) in blood lymphocytes. The data represent results for 4 rhesus monkeys at each time point. Expression levels for all 138 immune genes are shown in table 1.
P < .05 and
P < .01.
Despite the down-regulation of many genes in the blood, remarkable up-regulation of selected immune genes was seen in lymphocytes isolated from lungs at week 5, when the monkeys exhibited severe TB (figure 2 and table 3). Fifty-three immune genes were up-regulated 6–1197-fold in lymphocytes from the lungs of the M. tuberculosis–infected monkeys, and 5 other genes were up-regulated <5-fold but with differences that were still statistically significant (P < .05) (tables 3 and 4). Thirty-five of these 58 immune genes exhibited significant up-regulation, compared with expression levels in lymphocytes from the lungs of healthy, uninfected monkeys (tables 3 and 4). Interestingly, most of these up-regulated genes appeared to be linked to Th1 commitment (IFN-γ, TNF-α, IL-2Rα, IL-2Rβ, IL-2Rβ, IP-10, TIM-1, and TIM-3), Th17 commitment (IL-17R, IL-22, IL-22Rβ [IL-10Rβ], IL-6, and TGFβ), T cell activation and migration (IL-3, IFN-α, IFN-β, TNF-α, IL-2Rγ, CD28, CTLA-4, B7-2, PD1, FASL, TRL-2, CCL27, CXCL11, CXCL12, IP-10, MIP-1α, MMP9, MCP4, CCR2, CCR3, CCR4, CCR5, CCR7, CCR8, CCR9, CCR11, XCR1, and CXCR3), and inflammation and chemoattractants (IL-1α, IL-6, IL-22, and TNF-α and the genes for those chemokines and chemokine receptors listed above for T cell activation and migration).
Table 3.
Expression levels of immune genes that were up-regulated in lymphocytes of the lungs, bronchial lymph nodes (LNs), and spleen during severe tuberculosis.
| Infected-to-uninfected ratio | ||||||
|---|---|---|---|---|---|---|
| Lungs | LNs | Spleen | ||||
| Gene | Mean ± SD | P | Mean ± SD | P | Mean ± SD | P |
| IL-1α | 57.3 ± 20.0 | .046* | 335.4 ± 19.8 | .045* | 22.5 ± 5.2 | .086 |
| IL-1β | 24.1 ± 18.9 | .090 | 8.1 ± 4.5 | .030* | 1.2 ± 3.0 | .431 |
| IL-3 | 371.5 ± 224.8 | .033* | 50.1 ± 69.8 | .011* | 999.8 ± 230.4 | .027* |
| IL-6 | 21.1 ± 18.5 | .030* | 6.4 ± 17.5 | .046* | 7.7 ± 22.2 | .099 |
| IL-10 | 7.2 ± 5.5 | .127 | 4.6 ± 1.3 | .019* | 6.4 ± 2.6 | .044* |
| IL-16 | 8.3 ± 4.7 | .117 | 3.1 ± 2.2 | .053 | 4.2 ± 3.0 | .050 |
| IL-20 | 5.2 ± 5.9 | .182 | 9.3 ± 48.9 | .227 | 1.5 ± 5.1 | .327 |
| IL-22 | 220.3 ± 196.4 | .039* | 57.3 ± 104.0 | .030* | 986.3 ± 181.3 | .024* |
| IFN-α | 48.9 ± 40.7 | .156 | 3.1 ± 5.0 | .029* | 137.2 ± 36.1 | .109 |
| IFN-β | 1197.3 ± 296.4 | .038* | 39.7 ± 111.5 | .002** | 2603.8 ± 478.2 | .047* |
| IFN-γ | 24.4 ± 13.0 | .055 | 8.7 ± 6.3 | .092 | 2.4 ± 5.6 | .155 |
| TNF-α | 8.2 ± 3.2 | .072 | 12.4 ± 11.2 | .133 | 40.2 ± 9.2 | .079 |
| IL-1R1 | 9.6 ± 5.1 | .098 | 2.5 ± 2.1 | .146 | 12.6 ± 6.9 | .068 |
| IL-2Rα | 9.7 ± 18.8 | .175 | 0.8 ± 0.5 | .247 | 7.9 ± 34.4 | .214 |
| IL-2Rβ | 12.9 ± 5.5 | .020* | 1.8 ± 6.7 | .284 | 10.3 ± 14.4 | .046* |
| IL-2Rγ | 24.4 ± 19.8 | .021* | 7.5 ± 28.2 | .027* | 14.6 ± 24.8 | .002** |
| IL-4R | 2.6 ± 1.0 | .035* | 0.7 ± 5.1 | .389 | 4.7 ± 3.5 | .047* |
| IL-6R | 2.5 ± 0.5 | .021* | 2.1 ± 0.6 | .144 | 0.6 ± 1.4 | .360 |
| IL-10Rβ | 14.4 ± 6.9 | .041* | 1.6 ± 5.7 | .324 | 8.2 ± 7.1 | .026* |
| IL-13Rα1 | 63.5 ± 32.4 | .040* | 3.3 ± 3.2 | .191 | 40.8 ± 29.4 | .100 |
| IL-15Rα | 3.7 ± 1.6 | .049* | 0.3 ± 3.3 | .302 | 0.9 ± 3.1 | .442 |
| IL-17R | 2.7 ± 0.4 | .006** | 0.2 ± 3.4 | .128 | 0.6 ± 1.9 | .368 |
| CCL27 | 57.6 ± 112.4 | .032* | 44.1 ± 50.2 | .025* | 1767.1 ± 132.0 | .030* |
| CCL28 | 4.0 ± 13.8 | .239 | 7.1 ± 3.9 | .089 | 51.7 ± 7.4 | .048* |
| CXCL11 | 15.5 ± 6.2 | .085 | 0.7 ± 1.0 | .168 | 0.5 ± 1.9 | .304 |
| CXCL12 | 20.4 ± 162.5 | .035* | 10.3 ± 12.3 | .209 | 31.1 ± 152.0 | .042* |
| ETA-1 | 22.8 ± 92.4 | .044* | 1.6 ± 3.2 | .306 | 22.4 ± 42.9 | .160 |
| GCP2 | 6.9 ± 27.8 | .192 | 12.0 ± 7.2 | .083 | 182.0 ± 21.9 | .071 |
| IP-10 | 58.3 ± 69.0 | .041* | 46.1 ± 36.1 | .002** | 471.5 ± 45.3 | .010* |
| LARC | 6.4 ± 1.5 | .084 | UD | … | UD | … |
| MCP4 | 173.4 ± 50.8 | .027* | 3.4 ± 5.7 | .068 | 5.2 ± 19.7 | .145 |
| MIP-1α | 770.9 ± 414.4 | .039* | 520.2 ± 164.0 | .003** | 441.3 ± 246.6 | .018* |
| TECK | 5.2 ± 30.0 | .216 | 17.3 ± 10.9 | .077 | 175.7 ± 19.6 | .047* |
| CCR2 | 19.9 ± 12.7 | .160 | 28.8 ± 38.4 | .198 | 22.0 ± 5.5 | .073 |
| CCR3 | 42.9 ± 40.2 | .165 | 6.3 ± 7.7 | .035* | 129.9 ± 23.9 | .072 |
| CCR4 | 790.8 ± 512.4 | .036* | 502.8 ± 454.7 | .037* | 691.3 ± 359.5 | .008** |
| CCR5 | 124.2 ± 268.6 | .035* | 341.0 ± 222.2 | .012* | 2743.0 ± 411.8 | .039* |
| CCR7 | 16.4 ± 14.0 | .127 | 1.6 ± 4.3 | .305 | 3.1 ± 8.0 | .136 |
| CCR8 | 6.2 ± 2.3 | .081 | 1.5 ± 1.3 | .313 | 10.4 ± 2.0 | .035* |
| CCR9 | 170.4 ± 59.5 | .027* | 32.2 ± 26.2 | .009** | 374.0 ± 50.8 | .049* |
| CCR11 | 5.6 ± 2.4 | .109 | 0.4 ± 1.1 | .048 | 7.8 ± 1.1 | .029* |
| CXCR2 | 0.8 ± 0.6 | .335 | 11.0 ± 8.8 | .230 | 1.0 ± 0.6 | .500 |
| CXCR3 | 128.0 ± 81.2 | .049 | 17.4 ± 31.8 | .003** | 1039.2 ± 89.5 | .048* |
| XCR1 | 9.9 ± 9.9 | .168 | 0.6 ± 2.5 | .266 | 46.9 ± 8.9 | .105 |
| TGFβ1 | 3.8 ± 0.8 | .018* | 0.3 ± 4.7 | .287 | 0.5 ± 3.4 | .337 |
| VEGF | 18.1 ± 17.1 | .047* | 2.3 ± 7.2 | .163 | 213.9 ± 36.5 | .074 |
| PU.1 | 36.4 ± 19.1 | .046* | 3.6 ± 2.4 | .101 | 19.9 ± 20.2 | .062 |
| B7–2 | 34.3 ± 17.4 | .029* | 1.4 ± 2.6 | .298 | 23.6 ± 12.7 | .111 |
| CD28 | 688.2 ± 477.7 | .037* | 66.0 ± 250.9 | .026* | 79.5 ± 345.6 | .019* |
| CTLA4 | 14.0 ± 15.4 | .125 | 2.8 ± 4.3 | .220 | 27.4 ± 10.3 | .028* |
| ICOSL | 3.2 ± 2.5 | .114 | 18.3 ± 17.0 | .111 | 7.3 ± 11.2 | .095 |
| CTSG | 60.6 ± 27.8 | .032* | 5.3 ± 12.3 | .108 | 76.3 ± 31.4 | .087 |
| GZMM | 9.1 ± 7.9 | .140 | 1.6 ± 3.5 | .234 | 36.4 ± 6.4 | .025* |
| MMP-9 | 15.8 ± 8.0 | .087 | 2.4 ± 0.7 | .074 | 159.2 ± 22.4 | .053 |
| UPB43 | 25.9 ± 23.5 | .031* | 30.1 ± 29.0 | .016* | 31.5 ± 31.2 | .014* |
| TIM1 | 98.5 ± 62.2 | .041* | 12.1 ± 37.3 | .044* | 864.0 ± 58.0 | .028* |
| TIM3 | 8.0 ± 20.1 | .020* | 7.2 ± 7.7 | .034* | 9.3 ± 7.9 | .075 |
| EMAP2 | 12.3 ± 13.1 | .171 | 0.5 ± 2.7 | .305* | 7.1 ± 7.5 | .110 |
| PD1 | 472.5 ± 298.4 | .035* | 40.1 ± 196.1 | .033* | 1119.3 ± 284.3 | .028* |
| FAS | 1.4 ± 0.3 | .160 | 0.5 ± 4.4 | .255 | 2.8 ± 0.3 | .045* |
| FASL | 409.9 ± 199.7 | .021* | 22.8 ± 142.3 | .044* | 185.4 ± 156.2 | .034* |
| PDL2 | 178.0 ± 101.7 | .036* | 0.3 ± 2.0 | .175 | 749.9 ± 111.6 | .193 |
| TLR2 | 173.3 ± 109.4 | .043* | 102.0 ± 30.9 | .014* | 457.9 ± 69.6 | .016* |
NOTE. Shown are the means of expression ratios (fold changes) for up-regulated immune genes in lymphocytes isolated at week 5 after infection relative to preinfection from the lungs, bronchial LNs, and spleens of Mycobacterium tuberculosis–infected rhesus monkeys (n = 4) vs. the corresponding gene in lymphocytes isolated from the same tissues of healthy, uninfected monkeys (n = 4). Expression levels for all 138 immune genes are shown in table 4.
P < .05 and
P < .01.
UD, undetectable.
The finding that many immune genes were down-regulated in the blood but remarkably up-regulated in lungs raised a question as to whether the pulmonary transcriptional responses were influenced by local or even remote lymphoid tissues. To address this question, we sought to examine changes in immune genes and cellular immune responses in bronchial lymph nodes and spleens. Up to 540-fold increases in the expression of 34 immune genes were demonstrated in lymphocytes from bronchial lymph nodes, and 24 of them were significantly up-regulated. Although the spleens from the monkeys displayed no or only a few microgranulomas (<2 mm), splenic lymphocytes appeared to express higher levels of selected immune genes than did those from M. tuberculosis–exposed lungs or bronchial lymph nodes. Forty-seven genes were up-regulated up to 2700-fold in splenic lymphocytes from infected monkeys compared with that in the normal controls, and 29 of them were significantly increased (tables 3 and 4). Of them, 12 genes were up-regulated >450-fold (table 3).
Interestingly, although patterns of up-regulated immune genes were different among lymphocyte populations from lungs, lymph nodes, and spleen, these 3 organs shared a set of 26 up-regulated immune genes (>6-fold or P < .05) (tables 3 and 4). Most of these up-regulated genes appeared to be linked to inflammation and/or chemotaxis (IL-1α, IL-6, IL-22, TNF-α, CCL2, CXCL12, IP-10, MIP-1α, CCR2, CCR3, CCR4, CCR5, CCR9, and CXCR3), Th1 and Th17 commitment (IL-22, TNF-α, IL-2Rγ, IP-10, TIM-1, and TIM-3), and immune activation (IL-3, IFN-α, IFN-β, TNF-α, IL-2Rγ, CD28, PD1, FASL, and TRL-2).
In contrast, mycobacterium-specific cellular responses were disproportional to the major up-regulation of immune genes. The numbers of PPD-specific IFN-γ–producing cells at 5 weeks after M. tuberculosis infection were actually low among PBLs and lymphocytes from lungs and local lymph nodes (figure 3). T cell responses to peptide pools of the early secreted protein ESAT6 and Ag85B were not detectable (data not shown).
Figure 3.
Immune responses of purified protein derivative (PPD)–specific interferon (IFN)–γ–producing cells during severe tuberculosis. A, PPD-specific IFN-γ–producing cells among peripheral blood lymphocytes (PBLs), determined by enzyme-linked immunospot assay. Shown are mean ± SE values after subtraction of the values for negative controls, which were exposed only to RPMI 1640 medium. B, Nos. of PPD-specific IFN-γ–producing cells among lymphocytes from lung tissues and bronchial lymph nodes (LNs) in rhesus monkeys with pulmonary M. tuberculosis infection (top) and in healthy, uninfected rhesus monkeys (bottom). ConA, concanavalin A.
Surprisingly, IL-22, CCL27, IP-1α, IP-10, CCR4, CCR5, CXCR3, PD1, PDL2, IL-3, IFN-β, TIM1, and TLR2 were mostly up-regulated, ranging from 450- to 2740-fold, in lymphocytes from lungs or lymph nodes and spleens (tables 3 and 4). Because the majority of cells used for gene analyses were T and B lymphocytes isolated by Ficoll centrifugation from lungs, lymph nodes, and spleen, the up-regulated genes might predominantly be expressed by T cells from the tissues [7]. Although the roles of all of these genes in TB have not been defined, these 13 genes could be grouped into 3 categories on the basis of the current knowledge of the individual molecules: (1) inflammatory cytokines and chemokines (IL-22, CCL27, MIP-1α, and IP-10) and chemokine receptors (CCR4, CCR5, and CXCR3); (2) immune dysfunctional receptors and ligands (PD1 and PDL2), as seen in HIV-1 infection [8]; and (3) immune activation elements (IL-3, IFN-β, TIM1, and TLR2).
Discussion
Severe TB in juvenile rhesus monkeys induced unbalanced up-regulation of gene networks between the blood and the lungs and between the blood and lymphoid tissues. The down-regulation of selected immune genes might result from the emigration of lymphocytes with migrating potential, because most down-regulated immune genes in the blood were indeed up-regulated in the lungs and lymphoid organs after M. tuberculosis infection. Whether this imbalance of immune gene networks between the blood and the lungs or the lymphoid compartments contributes to the insufficient development of antigen-specific cellular responses remains an open question.
M. tuberculosis–activated gene networks in tissues appear to be more inflammatory than bacille Calmette-Guérin (BCG)–induced gene networks in PBLs, as we recently reported ([5] and table 3), because BCG and M. tuberculosis are here considered different in terms of infectivity, transmission, tissue distribution, and disease. Tissue lymphocytes after M. tuberculosis aerosolization, but not PBLs after intravenous BCG vaccination, expressed remarkably high levels of transcripts that encode the inflammatory cytokines and receptors, such as IL-1α, IL-6, MCP4, MIP-1α, ETA-1, CXCL11, CXCL12, FASL, CCR2, CCR3, and CXCR3 ([5] and table 3). In addition, some genes encoding immune activating or proinflammatory cytokines and molecules (IL-3, IL-22, IFN-β, IFN-γ, IL-2Rβ, IL-2Rγ, IL-13Rα, CCL27, CCR4, CCR5, CCR9, TIM1, PD1, PDL2, and TLR2) were expressed at higher levels during severe TB than after BCG vaccination [5]. On the other hand, IL-8, IL-16, IL-18R, IL-20, TNF-R, MIP-1β, TECK, CCR1, CCR11, and ICOS were remarkably up-regulated (~248-fold) in BCG-elicited lymphocytes but were only slightly increased or not increased in lymphocytes derived from lungs or lymphoid tissues at 5 weeks after M. tuberculosis infection ([5] and table 3).
Th17-like transcriptional responses were apparent in the lungs and lymphoid organs during severe TB. Although genes associated with Th2 responses were not detectable, the expression of genes associated with Th17 responses was much higher than that of Th1-associated genes (low IFN-γ expression and no IL-2 expression). The gene encoding interleukin (IL)–22, a cytokine produced by Th17 cells, was up-regulated up to 1000-fold in the lungs, lymph nodes, and spleen. Consistently, IL-22Rβ (also known as IL-10Rβ) but not IL-10Rα (IL-22R is comprised of IL-22Rα and common IL-10Rβ [9]) was significantly up-regulated during M. tuberculosis but not BCG infection. The genes encoding IL-6 and transforming growth factor (TGF)–β cytokines for induction of Th17 cells were also up-regulated dramatically during severe TB ([5] and table 3). The role played by IL-22 in TB remains unknown, although IL-23, a cytokine that increases the numbers of Th17 cells, has been shown to enhance immunity to TB [10–12].
IL-22, IL-3, IFN-β, MIP-1α, CCL27, IP-10, CCR4, CCR5, CXCR3, TIM1, PDL, PDL2, and TLR2 were highly up-regulated, being increased from 450- to 2740-fold. IL-3, IFN-β, T cell immunoglobulin mucin (TIM) 1, and Toll-like receptor (TLR) 2 could serve as immune-activating molecules, despite the absence of definitive reports describing these molecules or their networks in TB. IL-22, macrophage inflammatory protein (MIP)–1α, CCL27, and IFN-γ–inducible protein (IP)–10 may represent the leading cytokines and chemokines in the inflammatory and chemoattractant networks that drive migration and accumulation of the immune cells expressing CCR4, CCR5, and CXCR3. Interestingly, MIP-1α but not MIP-1β were remarkably up-regulated during TB (table 3). We have previously reported that BCG induced expression of MIP-1β but not MIP-1α [5]. The absence of an increase in MIP-1β expression may favor M. tuberculosis infection because combined treatment with RANTES and MIP-1β but not with RANTES and MIP-1α can suppress intracellular growth of M. tuberculosis in vitro [13]. Thus, the overexpression of these 13 genes, together with the up-regulation of other genes, during M. tuberculosis infection might favor the development of inflammation and suppression of antigen-specific T cells. Elucidation of these genes and gene networks may help to identify biomarkers or regulatory pathways in the development of severe TB.
Supplementary Material
Progression of Mycobacterium tuberculosis infection induced subtle changes in the percentages (left) and absolute nos. (right) of T cell populations in the blood.
Development of severe tuberculosis (TB), characterized pathologically by miliary TB in both lungs, in juvenile rhesus monkeys infected with Mycobacterium tuberculosis by aerosol.
Acknowledgments
Financial support: National Institutes of Health (R01 grants HL64560 and RR13601 (both to Z.W.C.).
We thank the other members of the Chen Lab, for technical assistance.
Footnotes
Potential conflicts of interest: none reported.
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Associated Data
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Supplementary Materials
Progression of Mycobacterium tuberculosis infection induced subtle changes in the percentages (left) and absolute nos. (right) of T cell populations in the blood.
Development of severe tuberculosis (TB), characterized pathologically by miliary TB in both lungs, in juvenile rhesus monkeys infected with Mycobacterium tuberculosis by aerosol.