Whole-blood flow-cytometric analysis of antigen-specific CD4 T-cell cytokine profiles distinguishes active tuberculosis from non-active states

T-cell based IFN-γ release assays do not permit distinction of active tuberculosis (TB) from successfully treated disease or latent M. tuberculosis infection. We postulated that IFN-γ and IL-2 cytokine profiles of antigen-specific T cells measured by flow-cytometry ex vivo might correlate with TB disease activity in vivo. Tuberculin (PPD), ESAT-6 and CFP-10 were used as stimuli to determine antigen-specific cytokine profiles in CD4 T cells from 24 patients with active TB and 28 patients with successfully treated TB using flow-cytometry. Moreover, 25 individuals with immunity consistent with latent M. tuberculosis infection and BCG-vaccination, respectively, were recruited. Although the frequency of cytokine secreting PPD reactive CD4 T cells was higher in patients with active TB compared to patients with treated TB (median 0.81% vs. 0.39% of CD4 T cells, p?=?0.02), the overlap in frequencies precluded distinction between the groups on an individual basis. When assessing cytokine profiles, PPD specific CD4 T cells secreting both IFN-γ and IL-2 predominated in treated TB, latent infection and BCG-vaccination, whilst in active TB the cytokine profile was shifted towards cells secreting IFN-γ only (p<0.0001). Cytokine profiles of ESAT-6 or CFP-10 reactive CD4 T cells did not differ between the groups. Receiver operator characteristics (ROC) analysis revealed that frequencies of PPD specific IFN-γ/IL-2 dual-positive T cells below 56% were an accurate marker for active TB (specificity 100%, sensitivity 70%) enabling effective discrimination from non-active states. In conclusion, a frequency lower than 56% IFN-γ/IL-2 dual positive PPD-specific circulating CD4 T-cells is strongly indicative of active TB.     

Suboptimal activation of antigen-specific CD4+ effector cells enables persistence of M. tuberculosis in vivo

Adaptive immunity to Mycobacterium tuberculosis controls progressive bacterial growth and disease but does not eradicate infection. Among CD4+ T cells in the lungs of M. tuberculosis-infected mice, we observed that few produced IFN-γ without ex vivo restimulation. Therefore, we hypothesized that one mechanism whereby M. tuberculosis avoids elimination is by limiting activation of CD4+ effector T cells at the site of infection in the lungs. To test this hypothesis, we adoptively transferred Th1-polarized CD4+ effector T cells specific for M. tuberculosis Ag85B peptide 25 (P25TCRTh1 cells), which trafficked to the lungs of infected mice and exhibited antigen-dependent IFN-γ production. During the early phase of infection, ～10% of P25TCRTh1 cells produced IFN-γ in vivo; this declined to <1% as infection progressed to chronic phase. Bacterial downregulation of fbpB (encoding Ag85B) contributed to the decrease in effector T cell activation in the lungs, as a strain of M. tuberculosis engineered to express fbpB in the chronic phase stimulated P25TCRTh1 effector cells at higher frequencies in vivo, and this resulted in CD4+ T cell-dependent reduction of lung bacterial burdens and prolonged survival of mice. Administration of synthetic peptide 25 alone also increased activation of endogenous antigen-specific effector cells and reduced the bacterial burden in the lungs without apparent host toxicity. These results indicate that CD4+ effector T cells are activated at suboptimal frequencies in tuberculosis, and that increasing effector T cell activation in the lungs by providing one or more epitope peptides may be a successful strategy for TB therapy.     

Helminth species dependent effects on Th1 and Th17 cytokines in active tuberculosis patients and healthy community controls

Despite that the impact of different helminth species is not well explored, the current dogma states that helminths affect the Th1/Th2 balance which in turn affects the risk of tuberculosis (TB) reactivation and severity of disease. We investigated the influence of helminth species on cytokine profiles including IL-17A in TB patients and healthy community controls (CCs). In total, 104 newly diagnosed pulmonary TB patients and 70 HIV negative and QuantiFERON negative CCs in Gondar, Ethiopia were included following helminth screening by stool microscopy. Plasma samples and ex vivo stimulation of peripheral blood mononuclear cells (PBMCs) with purified protein derivative (PPD) and Staphylococcus enterotoxin B (SEB) was used to determine cytokine profiles by cytometric bead array. In CCs, Ascaris lumbricoides or Schistosoma mansoni infections were associated with an impaired Th1-type response (IFN-gamma, IL-6 and TNF-alpha) in PBMCs mainly with SEB stimulations, whereas in TB patients only hookworm infection showed a similar pattern. Among CCs, the IL-17A response in PBMCs stimulated with SEB was higher only for S. mansoni, whereas in TB patients, the elevated systemic IL-17A plasma level was significantly suppressed in hookworm infected TB patients compared to patients without helminth coinfection. Following treatment of TB and helminth infection there was a general decrease in ex vivio IL-10 and TNF-alpha production in unstimulated, PPD or SEB stimulated PBMCs that was the most pronounced and significant in TB patients infected with S. mansoni, whereas the follow-up levels of IFN-gamma and IL-17A was significantly increased only in TB patients without helminth coinfection from PBMCs stimulated mainly with SEB. In summary, in addition to confirming helminth specific effects on the Th1/Th2 response before and after TB treatment, our novel finding is that IL-17A was impaired in helminth infected TB patients especially for hookworm, indicating a helminth species-specific immunoregulatory effect on IL-17A which needs to be further investigated.     

Cellular Immune Responses to Cell Wall Peptidoglycan Associated Protein Antigens in Tuberculosis Patients and Healthy Subjects

We have isolated cell wall peptidoglycan associated proteins (CW-Pr) of Mycobacterium tuberculosis H₃₇Ra by chemical treatment with trifluoromethanesulfonic acid:anisole (2:1), which further resolved into 71, 60 and 45 kDa proteins on SDS-PAGE. A study was carried out to investigate the immunoreactivity of these proteins with blood samples from 4 categories, including 15 tuberculous patients (TB), 5 tuberculous patients on ATT (TBT), 10 PPD non-reactive healthy controls (HPPD^?) and 11 PPD reactive healthy controls (HPPD⁺). Comparing the proliferative responses to cell wall protein antigens, it was observed that the 71 kDa protein gave maximum stimulation with PBMCs from the TB and HPPD⁺ groups. The adherent PBMCs from the TB group also demonstrated enhanced phagocytosis, particularly in the presence of 71 and 45 kDa proteins, and the phagocytic index was significantly higher (P < 0.05) than the TBT group. However, PBMCs from of the groups recognized the 60 kDa cell wall antigen. Our results suggest that the 71 kDa protein from the cell wall of M. tuberculosis is highly immunogenic.     

Differential expression of granulysin and perforin by NK cells in cancer patients and correlation of impaired granulysin expression with progression of cancer

Granulysin has been identified as an effector molecule co-localized with perforin in the cytotoxic granules of cytotoxic T lymphocytes and natural killer (NK) cells, and has been reported to kill intracellular pathogens in infected cells in the presence of perforin and to induce a cytotoxic effect against tumor cells. The aim of the present study was to elucidate whether intracellular expression of granulysin and perforin by NK cells might be associated with progression of cancer. Flow cytometric analysis demonstrated high levels of perforin and granulysin expression by CD3(-) CD16(+) cells in healthy controls. In contrast, cancer patients exhibited significantly decreased levels of granulysin expression ( P<0.005), despite having equally high levels of perforin expression in comparison with healthy controls. The tumor-free patients expressed granulysin at levels similar to healthy controls, while the progressive tumor-bearing patients expressed remarkably lower levels of granulysin compared to healthy controls ( P<0.0001). Similarly, patients with an advanced performance status had significantly fewer granulysin-positive NK cells than healthy controls. Meanwhile, a considerable number of the tumor-bearing patients showed a decrease in the number of circulating NK cells, and a correlation between impaired granulysin expression and reduced circulating NK cells was observed. These findings suggest that the tumor-bearing patients with impaired granulysin expression were in an immunosuppressive state. In conclusion, impaired expression of granulysin by NK cells correlates with progression of cancer, and determination of granulysin expression might prove informative for assessing the immunological condition of cancer patients.     

Functional capacity of Mycobacterium tuberculosis-specific T cell responses in humans is associated with mycobacterial load

High Ag load in chronic viral infections has been associated with impairment of Ag-specific T cell responses; however, the relationship between Ag load in chronic Mycobacterium tuberculosis infection and functional capacity of M. tuberculosis-specific T cells in humans is not clear. We compared M. tuberculosis-specific T cell-associated cytokine production and proliferative capacity in peripheral blood from adults with progressively higher mycobacterial loads-that is, persons with latent M. tuberculosis infection (LTBI), with smear-negative pulmonary tuberculosis (TB), and smear-positive TB. Patients with smear-positive TB had decreased polyfunctional IFN-γ(+)IL-2(+)TNF-α(+) and IL-2-producing specific CD4 T cells and increased TNF-α single-positive cells, when compared with smear-negative TB and LTBI. TB patients also had increased frequencies of M. tuberculosis-specific CD8 T cells, compared with LTBI. M. tuberculosis-specific CD4 and CD8 T cell proliferative capacity was profoundly impaired in individuals with smear-positive TB, and correlated positively with ex vivo IFN-γ(+)IL-2(+)TNF-α(+) CD4 T cells, and inversely with TNF-α single-positive CD4 T cells. During 6 mo of anti-TB treatment, specific IFN-γ(+)IL-2(+)TNF-α(+) CD4 and CD8 T cells increased, whereas TNF-α and IFN-γ single-positive T cells decreased. These results suggest progressive impairment of M. tuberculosis-specific T cell responses with increasing mycobacterial load and recovery of responses during therapy. Furthermore, these data provide a link between specific cytokine-producing subsets and functional capacity of M. tuberculosis-specific T cells, and between the presence of specific CD8 T cells ex vivo and active TB disease. These data have potentially significant applications for the diagnosis of TB and for the identification of T cell correlates of TB disease progression.     

Enhanced T cell responsiveness to Mycobacterium bovis BCG r32-kDa Ag correlates with successful anti-tuberculosis treatment in humans

Th1 and Th2 cytokines play key role in protection from and pathogenesis of mycobacterial infection and their dynamic changes may predict clinical outcome of the patient. Patients with tuberculosis (TB) have a poorer cellular immune response to recombinant 32-kDa antigen (Ag) of Mycobacterium bovis (r32-kDa M. bovis) than do healthy volunteers. The basis for this observation was studied by evaluating the Th1 (gamma interferon [IFN-γ]) produced in response to the r32-kDa Ag M. bovis by peripheral blood mononuclear cells (PBMC) from patients with pulmonary TB (n = 20), extra-pulmonary TB (n = 13) and from healthy volunteers (n = 9). Recombinant 32-kDa M. bovis stimulated PBMC from TB patients produced significantly lower levels of IFN-γ at 0 month, and increased at 2–4, and 6 months of treatment and were highly significant (p < 0.000) compared to the responses in controls. The ratios of IFN-γ to IL-10 were low in patients newly diagnosed and improved both during and after treatment. The present study concludes that the levels of in vitro response to M. bovis BCG r32-kDa Ag leading to the specific release of IFN-γ increased after anti-tuberculosis treatment and seems to reflect the clinical status of the patient, thus reiterating the utility of this antigen in T cell based assays as a surrogate marker of cell mediated responses.     

Interferon gamma +874T/A polymorphism is associated with susceptibility to active pulmonary tuberculosis development in Tunisian patients

Interferon gamma (IFN-γ) is a key cytokine involved mainly in the defense against intracellular pathogens such as Mycobacterium tuberculosis. Given its key role in the control of tuberculosis (TB), in the present article we have investigated a possible association between IFN-γ gene single-nucleotide polymorphism linked to high and low producer phenotypes (IFN-γ [+874T(high)?→?A(low)]) (rs2430561) and risk development of active TB in Tunisian patients. Genomic DNA samples were obtained from 223 patients with active TB (168 pulmonary and 55 extrapulmonary cases) and 150 healthy blood donors. Genotypes were analyzed using polymerase chain reaction-restriction fragment length polymorphism method. The +874 AA genotype (low IFN-γ producer) was significantly associated with increased risk of developing of active pulmonary TB (odds ratio [OR]?=?2.18; 95% confidence intervals [CI], 1.33-3.57; P corrected for the number of genotypes [Pc]?=?0.003). By contrast, the AT genotype was found to be significantly associated with resistance to pulmonary TB (OR?=?0.46; 95% CI, 0.28-0.74; Pc?=?0.0018) and extrapulmonary TB development (OR?=?0.46; 95% CI, 0.23-0.91; Pc?=?0.045). Collectively, our data showed that the IFN-γ +874T/A polymorphism is a determinant in the resistance or susceptibility to the development of active TB in the studied population.     

Phenotypic and functional characterization of NK cells in human immune response against the dimorphic fungus Paracoccidioides brasiliensis

Besides their role in fighting viral infection and tumor resistance, recent studies have shown that NK cells also participate in the immune response against other infectious diseases. The aim of this study was to characterize the possible role of NK cells in the immune response against Paracoccidioides brasiliensis. Purified NK cells from paracoccidioidomycosis patients and healthy individuals were incubated with P. brasiliensis yeast cells or P. brasiliensis-infected monocytes, with or without the addition of recombinant IL-15. We found that NK cells from paracoccidioidomycosis patients exhibit a lower cytotoxic response compared with healthy individuals. NK cells are able directly to recognize and kill P. brasiliensis yeast cells, and this activity seems to be granule-dependent but perforin-independent, whereas the cytotoxicity against P. brasiliensis-infected monocytes is perforin-dependent. These results indicate that NK cells participate actively in the immune response against the P. brasiliensis infection either by directly destroying yeast cells or by recognizing and killing infected cells. Granulysin is the possible mediator of the cytotoxic effect, as the reduced cytotoxic activity against the yeast cells detected in patients with paracoccidioidomycosis is accompanied by a significantly lower frequency of CD56(+)granulysin(+) cells compared with that in healthy controls. Furthermore, we show that NK cells released granulysin in cultures after being stimulated by P. brasiliensis, and this molecule is able to kill the yeast cells in a dose-dependent manner. Another important finding is that stimulated NK cells are able to produce proinflammatory cytokines (IFN-γ and TNF-α) supporting their immunomodulatory role in the infection.     

Interleukin 24 as a novel potential cytokine immunotherapy for the treatment of Mycobacterium tuberculosis infection

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) remains a major global health problem. Interleukin 24 (IL-24) is a novel tumor suppressor and a unique member of the IL-10 family of cytokines. However, the in vivo immunological consequences of this cytokine's activity during Mtb infection are still unknown. We found that IL-24 concentration was significantly decreased in the sera of TB patients, and Mtb infection suppressed IL-24 expression of human peripheral blood mononuclear cells (PBMCs) in vitro. Furthermore, we used a mouse infection model utilizing the virulent Mtb H37Rv strain to demonstrate that the administration of exogenous IL-24 had a protective effect against the bacteria. We found that IL-24 could activate human CD8(+) T cells, driving CD8(+) T cells to produce interferon-γ (IFN-γ) and counteract TB. This activity was found to be dependent on early involvement of neutrophils in the mouse model. IL-24 strongly stimulated IFN-γ production mainly by signaling through the IL-24 receptors of human CD8(+) T cells. IL-12 secretion from neutrophils in response to IL-24 treatment might be a minor factor in activating human CD8(+) T cells to secrete IFN-γ. Suppression of IL-24 expression by Mtb infection might enhance susceptibility to infection and promote the development of chronic TB. This new information could potentially stimulate the development of a new cytokine-based immunotherapeutic approach using IL-24 to stimulate immunity against TB.     

Granulysin, a T cell product, kills bacteria by altering membrane permeability

Granulysin, a protein located in the acidic granules of human NK cells and cytotoxic T cells, has antimicrobial activity against a broad spectrum of microbial pathogens. A predicted model generated from the nuclear magnetic resonance structure of a related protein, NK lysin, suggested that granulysin contains a four alpha helical bundle motif, with the alpha helices enriched for positively charged amino acids, including arginine and lysine residues. Denaturation of the polypeptide reduced the alpha helical content from 49 to 18% resulted in complete inhibition of antimicrobial activity. Chemical modification of the arginine, but not the lysine, residues also blocked the antimicrobial activity and interfered with the ability of granulysin to adhere to Escherichia coli and Mycobacterium tuberculosis. Granulysin increased the permeability of bacterial membranes, as judged by its ability to allow access of cytosolic ss-galactosidase to its impermeant substrate. By electron microscopy, granulysin triggered fluid accumulation in the periplasm of M. tuberculosis, consistent with osmotic perturbation. These data suggest that the ability of granulysin to kill microbial pathogens is dependent on direct interaction with the microbial cell wall and/or membrane, leading to increased permeability and lysis.     

IFN-gamma-inducible protein 10 and pentraxin 3 plasma levels are tools for monitoring inflammation and disease activity in Mycobacterium tuberculosis infection

IFN-gamma-inducible protein 10 (IP-10/CXCL10) is a chemokine involved in delayed-type hypersensitivity and attraction of monocytes and activated T lymphocytes at inflammatory foci, whereas pentraxin 3 (PTX3) is part of the innate immune response. In the Republic of Guinea, 220 newly diagnosed, HIV-negative, pulmonary tuberculosis (TB) patients were studied together with 220 healthy household controls and 220 community controls. CXCL10 and PTX3 blood levels were assessed by ELISA at diagnosis, after 2 months and at the end of treatment. In untreated patients, both CXCL10 and PTX3 levels were higher (P < 0.0001) than in controls, although household controls had higher (P < 0.0001) CXCL10 and PTX3 levels than community controls, but lower (P < 0.0001) than those of patients. At the end of treatment, 186 cured patients showed reduction (P < 0.0001) in both CXCL10 and PTX3 levels. In 34 patients with treatment failure, both CXCL10 and PTX3 levels increased further. In five previously healthy households who developed TB during the follow-up and in two patients who relapsed after treatment, a remarkable increase in both CXCL10 and PTX3 plasma levels was observed. Active TB is associated with increased CXCL10 and PTX3 levels in the plasma. Although not specific for TB, measurement of these proteins may help the monitoring of disease activity and efficacy of therapy.     

Multiplex analysis of cytokines/chemokines as biomarkers that differentiate healthy contacts from tuberculosis patients in high endemic settings

Differentiation of latent tuberculosis infection (LTBI) from active disease is one of the crucial elements in the control of tuberculosis. Earlier in Indian population which is tuberculosis endemic, we identified that 10 Mycobacterium tuberculosis secreted protein fractions, induced IFN-γ response only in healthy contacts of TB patients (HCs) and not in tuberculosis patients (TB). These fractions were termed as “Contact Specific Fractions” (“CS” fractions) and found useful for differentiating HC from TB. Proteomic analysis revealed that “CS” fractions have 16 different proteins, of which three were novel T cell antigens. Using these “CS” fractions as stimulants, earlier IFN-γ, TNF-α and IL-4 cytokine responses were studied. In the present study, in order to identify the other useful cytokine biomarkers that were differentially expressed between HC and TB, Cytokine/chemokine response to “CS” fractions were analyzed using multiplex cytokine assay system. This preliminary investigation in our tuberculosis endemic population showed six cytokine (G-CSF, IL-6, IL-7, IL-8, IL-9, and PDGF) and one receptor antagonist (IL-1Ra) that were differentially expressed between HC and TB, for the first time. Especially IL-6 and PDGF were more promising biomarkers. IL-6 measurement identified seven as HC out of 10 HC analyzed. The measurement of PDGF identified eight as TB out of 10 TB tested. Studies are underway to further validate these biomarkers for the differentiation of LTBI from active tuberculosis.     

The assessment of host and bacterial proteins in sputum from active pulmonary tuberculosis

Pulmonary tuberculosis (TB) is caused by Mycobacterium tuberculosis. The protein composition of sputum may reflect the immune status of the lung. This study aimed to evaluate the protein profiles in spontaneous sputum samples from patients with active pulmonary TB. Sputum samples were collected from patients with pulmonary TB and healthy controls. Western blotting was used to analyze the amount of interleukin 10 (IL-10), interferon-gamma (IFN-γ), IL-25, IL-17, perforin-1, urease, albumin, transferrin, lactoferrin, adenosine deaminase (also known as adenosine aminohydrolase, or ADA), ADA-2, granzyme B, granulysin, and caspase-1 in sputum. Results of detection of IL-10, IFN-γ, perforin-1, urease, ADA2, and caspase-1, showed relatively high specificity in distinguishing patients with TB from healthy controls, although sensitivities varied from 13.3% to 66.1%. By defining a positive result as the detection of any two proteins in sputum samples, combined use of transferrin and urease as markers increased sensitivity to 73.2% and specificity to 71.1%. Furthermore, we observed that the concentration of transferrin was proportional to the number of acid-fast bacilli detected in sputum specimens. Detection of sputum transferrin and urease was highly associated with pulmonary TB infection. In addition, a high concentration of transferrin detected in sputum might correlate with active TB infection. This data on sputum proteins in patients with TB may aid in the development of biomarkers to assess the severity of pulmonary TB.     

Regulation of Mycobacterium-specific mononuclear cell responses by 25-hydroxyvitamin D3

The active vitamin D metabolite, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), has been shown to be an important regulator of innate and adaptive immune function. In addition, synthesis of 1,25(OH)(2)D(3) from 25-hydroxyvitamin D(3) (25(OH)D(3)) by the enzyme 1α-hydroxylase in monocytes upon activation by TLR signaling has been found to regulate innate immune responses of monocytes in an intracrine fashion. In this study we wanted to determine what cells expressed 1α-hydroxylase in stimulated peripheral blood mononuclear cell (PBMC) cultures and if conversion of 25(OH)D(3) to 1,25(OH)(2)D(3) in PBMC cultures regulated antigen-specific immune responses. Initially, we found that stimulation of PBMCs from animals vaccinated with Mycobacterium bovis (M. bovis) BCG with purified protein derivative of M. bovis (M. bovis PPD) induced 1α-hydroxylase gene expression and that treatment with a physiological concentration of 25(OH)D(3) down-regulated IFN-γ and IL-17F gene expression. Next, we stimulated PBMCs from M. bovis BCG-vaccinated and non-vaccinated cattle with M. bovis PPD and sorted them by FACS according to surface markers for monocytes/macrophages (CD14), B cells (IgM), and T cells (CD3). Sorting the PBMCs revealed that 1α-hydroxylase expression was induced in the monocytes and B cells, but not in the T cells. Furthermore, treatment of stimulated PBMCs with 25(OH)D(3) down-regulated antigen-specific IFN-γ and IL-17F responses in the T cells, even though 1α-hydroxylase expression was not induced in the T cells. Based on evidence of no T cell 1α-hydroxylase we hypothesize that activated monocytes and B cells synthesize 1,25(OH)(2)D(3) and that 1,25(OH)(2)D(3) down-regulates antigen-specific expression of IFN-γ and IL-17F in T cells in a paracrine fashion.     

Granulysin-expressing CD4+ T cells as candidate immune marker for tuberculosis during childhood and adolescence

Background

Granulysin produced by cytolytic T cells directly contributes to immune defense against tuberculosis (TB). We investigated granulysin as a candidate immune marker for childhood and adolescent TB.

Methods

Peripheral blood mononuclear cells (PBMC) from children and adolescents (1–17 years) with active TB, latent TB infection (LTBI), nontuberculous mycobacteria (NTM) infection and from uninfected controls were isolated and restimulated in a 7-day restimulation assay. Intracellular staining was then performed to analyze antigen-specific induction of activation markers and cytotoxic proteins, notably, granulysin in CD4⁺ CD45RO⁺ memory T cells.

Results

CD4⁺ CD45RO⁺ T cells co-expressing granulysin with specificity for Mycobacterium tuberculosis (Mtb) were present in high frequency in TB-experienced children and adolescents. Proliferating memory T cells (CFSE_lowCD4⁺CD45RO⁺) were identified as main source of granulysin and these cells expressed both central and effector memory phenotype. PBMC from study participants after TB drug therapy revealed that granulysin-expressing CD4⁺ T cells are long-lived, and express several activation and cytotoxicity markers with a proportion of cells being interferon-gamma-positive. In addition, granulysin-expressing T cell lines showed cytolytic activity against Mtb-infected target cells.

Conclusions

Our data suggest granulysin expression by CD4⁺ memory T cells as candidate immune marker for TB infection, notably, in childhood and adolescence.     

Differential Immune Responses and Protective Effects in Avirulent Mycobacterial Strains Vaccinated BALB/c Mice

Screening live mycobacterial vaccine candidates is the important strategy to develop new vaccines against adult tuberculosis (TB). In this study, the immunogenicity and protective efficacy of several avirulent mycobacterial strains including Mycobacterium smegmatis, M. vaccae, M. terrae, M. phlei, M. trivial, and M. tuberculosis H37Ra were compared with M. bovis BCG in BALB/c mice. Our results demonstrated that differential immune responses were induced in different mycobacterial species vaccinated mice. As BCG-vaccinated mice did, M. terrae immunization resulted in Th1-type responses in the lung, as well as splenocytes secreting IFN-γ against a highly conserved mycobacterial antigen Ag85A. M. smegmatis also induced the same splenocytes secreting IFN-γ as BCG and M. terrae did. In addition, M. terrae and M. smegmatis-immunized mice predominantly increased expression of IL-10 and TGF-β in the lung. Most importantly, mice vaccinated with H37Ra and M. vaccae could provide the same protection in the lung against virulent M. tuberculosis challenge as BCG. The result may have important implications in developing adult TB vaccine.     

A multifaceted analysis of immune-endocrine-metabolic alterations in patients with pulmonary tuberculosis

Our study investigated the circulating levels of factors involved in immune-inflammatory-endocrine-metabolic responses in patients with tuberculosis with the aim of uncovering a relation between certain immune and hormonal patterns, their clinical status and in vitro immune response. The concentration of leptin, adiponectin, IL-6, IL-1β, ghrelin, C-reactive protein (CRP), cortisol and dehydroepiandrosterone (DHEA), and the in vitro immune response (lymphoproliferation and IFN-γ production) was evaluated in 53 patients with active untreated tuberculosis, 27 household contacts and 25 healthy controls, without significant age- or sex-related differences. Patients had a lower body mass index (BMI), reduced levels of leptin and DHEA, and increased concentrations of CRP, IL-6, cortisol, IL-1β and nearly significant adiponectin values than household contacts and controls. Within tuberculosis patients the BMI and leptin levels were positively correlated and decreased with increasing disease severity, whereas higher concentrations of IL-6, CRP, IL-1β, cortisol, and ghrelin were seen in cases with moderate to severe tuberculosis. Household contacts had lower DHEA and higher IL-6 levels than controls. Group classification by means of discriminant analysis and the k-nearest neighbor method showed that tuberculosis patients were clearly different from the other groups, having higher levels of CRP and lower DHEA concentration and BMI. Furthermore, plasma leptin levels were positively associated with the basal in vitro IFN-γ production and the ConA-driven proliferation of cells from tuberculosis patients. Present alterations in the communication between the neuro-endocrine and immune systems in tuberculosis may contribute to disease worsening.