Healthy individuals that control a latent infection with Mycobacterium tuberculosis express high levels of Th1 cytokines and the IL-4 antagonist IL-4delta2

The majority of healthy individuals exposed to Mycobacterium tuberculosis will not develop disease and identifying what constitutes "protective immunity" is one of the holy grails of M. tuberculosis immunology. It is known that IFN-gamma is essential for protection, but it is also apparent that IFN-gamma levels alone do not explain the immunity/susceptibility dichotomy. The controversy regarding correlates of immunity persists because identifying infected but healthy individuals (those who are immune) has been problematic. We have therefore used recognition of the M. tuberculosis virulence factor early secretory antigenic target 6 to identify healthy, but infected individuals from tuberculosis (TB)-endemic and nonendemic regions (Ethiopia and Denmark) and have compared signals for cytokines expressed directly ex vivo with the pattern found in TB patients. We find that TB patients are characterized by decreased levels of Th1 cytokines and increased levels of IL-10 compared with the healthy infected and noninfected community controls. Interestingly, the healthy infected subjects exhibited a selective increase of message for the IL-4 antagonist, IL-4delta2, compared with both TB patients or noninfected individuals. These data suggest that long-term control of M. tuberculosis infection is associated not just with elevated Th1 responses but also with inhibition of the Th2 response.     

Increased specific T cell cytokine responses in patients with active pulmonary tuberculosis from Central Africa

An understanding of T cell responses that are crucial for control of Mycobacterium tuberculosis (MTB) has major implications for the development of immune-based interventions. We studied the frequency of purified protein derivative (PPD)-specific CD3) cells expressing interleukin-2 (IL)-2, gamma interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha and IL-10 in HIV-negative pulmonary tuberculosis patients (TB, n=30) as well as in healthy individuals (controls, n=21) from Central Africa. Increased frequencies of PPD-stimulated CD3+ cells expressing IL-2, IFN-gamma, and TNF-alpha in TB were seen when compared with frequencies of controls. The presence of type 1 cytokine biased responses in TB patients was supported by a shift in the distribution pattern of cytokine expression from exclusively IL-2 or TNF-alpha expression seen in controls towards an increased frequency of IFN-gamma/IL-2 or IFN-gamma/TNF-alpha co-expression in TB. Higher levels of PPD-induced IFN-gamma in the supernatants from TB patients than from controls were found, which correlated with its intracellular expression. PPD was a weak inducer of IL-10 in T cells and insufficient in promoting cytokine production in TCRgammadelta+CD3+ cells. Non-specific stimulation with PMA and ionomycin revealed increased frequencies of CD4+ cells expressing IFN-gamma in controls, while expression of IL-2, IL-4, IL-10, IL-13, and TNF-alpha was not different. Non-specific cytokine responses of TCRgammadelta+CD3+ cells were similar in all groups. Pulmonary TB in Central Africa is associated with enhanced expression and secretion of specifically induced cytokines that are frequently implicated in host defense against MTB.     

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.     

Pulmonary tuberculosis: evaluation of interferon-gamma levels as an immunological healing marker based on the response to the Bacillus Calmette-Guerin

Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis whose interaction with the host may lead to a cell-mediated protective immune response. The presence of interferon gamma (IFN-gamma) is related to this response. With the purpose of understanding the immunological mechanisms involved in this protection, the lymphoproliferative response, IFN-gamma and other cytokines like interleukin (IL-5, IL-10), and tumor necrosis factor alpha (TNF-alpha) were evaluated before and after the use of anti-TB drugs on 30 patients with active TB disease, 24 healthy household contacts of active TB patients, with positive purified protein derivative (PPD) skin tests (induration > 10 mm), and 34 asymptomatic individuals with negative PPD skin test results (induration < 5 mm). The positive lymphoproliferative response among peripheral blood mononuclear cells of patients showed high levels of IFN-gamma, TNF-alpha, and IL-10. No significant levels of IL-5 were detected. After treatment with rifampicina, isoniazida, and pirazinamida, only the levels of IFN-gamma increased significantly (p < 0.01). These results highlight the need for further evaluation of IFN-gamma production as a healing prognostic of patients treated.     

The T cell-specific CXC chemokines IP-10, Mig, and I-TAC are expressed by activated human bronchial epithelial cells

Recruitment of activated T cells to mucosal surfaces, such as the airway epithelium, is important in host defense and for the development of inflammatory diseases at these sites. We therefore asked whether the CXC chemokines IFN-induced protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC), which specifically chemoattract activated T cells by signaling through the chemokine receptor CXCR3, were inducible in respiratory epithelial cells. The effects of proinflammatory cytokines, including IFN-gamma (Th1-type cytokine), Th2-type cytokines (IL-4, IL-10, and IL-13), and dexamethasone were studied in normal human bronchial epithelial cells (NHBEC) and in two human respiratory epithelial cell lines, A549 and BEAS-2B. We found that IFN-gamma, but not TNF-alpha or IL-1 beta, strongly induced IP-10, Mig, and I-TAC mRNA accumulation mainly in NHBEC and that TNF-alpha and IL-1 beta synergized with IFN-gamma induction in all three cell types. High levels of IP-10 protein (> 800 ng/ml) were detected in supernatants of IFN-gamma/TNF-alpha-stimulated NHBEC. Neither dexamethasone nor Th2 cytokines modulated IP-10, Mig, or I-TAC expression. Since IFN-gamma is up-regulated in tuberculosis (TB), using in situ hybridization we studied the expression of IP-10 in the airways of TB patients and found that IP-10 mRNA was expressed in the bronchial epithelium. In addition, IP-10-positive cells obtained by bronchoalveolar lavage were significantly increased in TB patients compared with normal controls. These results show that activated bronchial epithelium is an important source of IP-10, Mig, and I-TAC, which may, in pulmonary diseases such as TB (in which IFN-gamma is highly expressed) play an important role in the recruitment of activated T cells.     

The impact of asymptomatic helminth co-infection in patients with newly diagnosed tuberculosis in north-west ethiopia

Background

Areas endemic of helminth infection, tuberculosis (TB) and HIV are to a large extent overlapping. The aim of this study was to assess the impact of asymptomatic helminth infection on the immunological response among TB patients with and without HIV, their house hold contacts and community controls.

Methodology

Consecutive smear positive TB patients (n = 112), their household contacts (n = 71) and community controls (n = 112) were recruited in Gondar town, Ethiopia. Stool microscopy, HIV serology, serum IgE level, eosinophil and CD4 counts were performed and tuberculosis patients were followed up for 3 months after initiation of anti-TB treatment.

Results

Helminth co-infection rate was 29% in TB patients and 21% in both community control and household contacts (p = 0.3) where Ascaris lumbricoides was the most prevalent parasite. In TB patients the seroprevalence of HIV was 47% (53/112). Eosinophilia and elevated IgE level were significantly associated with asymptomatic helminth infection. During TB treatment, the worm infection rate of HIV+/TB patients declined from 31% (10/32) at week 0 to 9% (3/32) at week 2 of TB treatment, whereas HIV−/TB patients showed no change from baseline to week 2, 29% (13/45) vs. 22.2% (10/45). This trend was stable at week 8 and 12 as well.

Conclusion

One third of smear positive TB patients were infected with helminths. Eosinophilia and elevated IgE level correlated with asymptomatic worm infection, indicating an effect on host immunity. The rate of worm infection declined during TB treatment in HIV+/TB co-infected patients whereas no decline was seen in HIV−/TB group.     

Th2 cytokines in susceptibility to tuberculosis

We need to understand what is different about susceptibility to tuberculosis (TB) in developing countries where most TB occurs, and where the current vaccine, Bacillus Calmette et Guérin (BCG) usually fails to protect. The presence of a background mixed IFN-gamma and Th2 response to mycobacterial antigens before infection with M. tuberculosis (Mtb), and the development of a large IL-4 response during progressive TB, are characteristics of individuals in the locations where BCG fails, which are also seen in animal models in the same countries. Recent data suggest that the background Th1 component in developing countries protects from low dose challenge with Mtb in mouse and man, but that following high dose challenge the pre-existing IL-4 component increases and blocks immunity unless the individual's immune system releases IL-4delta2, an antagonist of IL-4, which is raised in the blood of donors with stable latent TB. We outline how IL-4 (and IL-13) can undermine Th1-mediated immunity and drive inappropriate alternative activation of macrophages. The mechanisms of the effects of IL-4 include impaired antimicrobial activity due to reduced TNF-alpha-mediated apoptosis of infected cells, reduced activity of iNOS, increased availability of iron to intracellular Mtb, and increased proliferation of antigen-specific FOXP-3+ regulatory T cells. IL-4 also increases the toxicity of TNF-alpha and drives pulmonary fibrosis, thus enhancing immunopathology. The conclusion is that a vaccine that will work in developing countries might need to do more than enhance the existing Th1 response. In these environments it might be more important to block the Th2 component.     

Messenger RNA expression of IL-8, FOXP3, and IL-12beta differentiates latent tuberculosis infection from disease

Differentiation of active from latent tuberculosis (TB) is a major challenge in the control of TB. In this study, PBMC from latent TB-infected subjects, TB patients, and tuberculin skin test-negative donors stimulated with the Mycobacterium tuberculosis (Mtb)-specific Ag, early secretory antigenic target 6, and mRNA for 45 immune-related genes was measured by quantitative real-time PCR. Univariate analysis showed significant differences in the expression of 10 genes (IFN-gamma, FOXP3, IL-1alpha, IL-1beta, IL-2, IL-6, IL-8, IL-12alpha, IL-12beta, and IL-24) in PBMC from TB patients vs latent TB-infected subjects (p < 0.01). Multivariate logistic regression and classification and regression tree analyses revealed that expression of three genes, IL-8, FOXP3, and IL-12beta, is predictive for TB vs latent Mtb infection. Thus, measurement of Ag-specific expression of these three genes may offer a specific and noninvasive means of differentiating between latent Mtb infection and TB.     

Hepatitis C virus genotype and HIV coinfection affect cytokine mRNA levels in unstimulated PBMC but do not shift the T1/T2 balance

Rapid progression of hepatitis C virus (HCV) disease in patients with HIV/HCV may reflect different cytokine responses and be influenced by HCV genotype. This is addressed by a study of patients with HIV/HCV coinfection and infection with HCV genotype 2 or 3 (2/3). They are compared with coinfected patients infected with genotype 1 and HCV monoinfected patients matched for HCV genotype. IFN-gamma, IL-10, IL-4 and IL-4delta2 mRNA were quantified by real-time PCR in unstimulated PBMC and after in vitro stimulation with HCV core or nonstructural 3/4A antigen. In unstimulated PBMC, levels of IFN-gamma and IL-4 mRNA were lowest in HIV/HCV genotype 1 patients, intermediate in HIV/HCV genotype 2/3 patients and highest in HCV genotype 2/3 patients. Neither HCV genotype nor HIV affected levels of IL-10 mRNA in unstimulated PBMC or IFN-gamma, IL-4 and IL-10 mRNA in PBMC stimulated with HCV antigens. Levels of IL-4 and IL-4delta2 mRNA correlated in mitogen-stimulated PBMC from all patient groups but both were low in HIV/HCV genotype 1 patients. Serum soluble CD30 levels (a putative marker of a T2 cytokine environment) did not differ between patient groups. The data do not suggest a shift in the T1/T2 balance driven by HIV coinfection or HCV genotype but either may affect IL-4 bioavailability.     

Cytokine milieu of atopic dermatitis,as compared to psoriasis,skin prevents induction of innate immune response genes

Atopic dermatitis (AD) and psoriasis are the two most common chronic skin diseases. However patients with AD, but not psoriasis, suffer from frequent skin infections. To understand the molecular basis for this phenomenon, skin biopsies from AD and psoriasis patients were analyzed using GeneChip microarrays. The expression of innate immune response genes, human beta defensin (HBD)-2, IL-8, and inducible NO synthetase (iNOS) was found to be decreased in AD, as compared with psoriasis, skin (HBD-2, p = 0.00021; IL-8, p = 0.044; iNOS, p = 0.016). Decreased expression of the novel antimicrobial peptide, HBD-3, was demonstrated at the mRNA level by real-time PCR (p = 0.0002) and at the protein level by immunohistochemistry (p = 0.0005). By real-time PCR, our data confirmed that AD, as compared with psoriasis, is associated with elevated skin production of Th2 cytokines and low levels of proinflammatory cytokines such as TNF-alpha, IFN-gamma, and IL-1beta. Because HBD-2, IL-8, and iNOS are known to be inhibited by Th2 cytokines, we examined the effects of IL-4 and IL-13 on HBD-3 expression in keratinocyte culture in vitro. We found that IL-13 and IL-4 inhibited TNF-alpha- and IFN-gamma-induced HBD-3 production. These studies indicate that decreased expression of a constellation of antimicrobial genes occurs as the result of local up-regulation of Th2 cytokines and the lack of elevated amounts of TNF-alpha and IFN-gamma under inflammatory conditions in AD skin. These observations could explain the increased susceptibility of AD skin to microorganisms, and suggest a new fundamental rule that may explain the mechanism for frequent infection in other Th2 cytokine-mediated diseases.     

Dysregulation of proinflammatory and regulatory cytokines in HIV infected persons with active tuberculosis

Proinflammatory and regulatory cytokines have been implicated to play important role in immunopathology of HIV and tuberculosis (TB) infection. Capacity of unstimulated and mitogen-stimulated peripheral blood mononuclear cells (PBMCs) to secrete cytokines (interleukin (IL)-2, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), IL-4, IL-10 and IL-6) was estimated for 15 HIV-TB coinfected patients, 22 HIV seropositives without TB, 32 HIV negative TB patients, and 36 healthy subjects. Dually infected patients had suppression of both Th1 and Th2 cytokine secretion as evidenced by significantly lower production of IL-2, IFN-gamma and TNF-alpha as well as IL-4 and IL-10. Production of IL-2 and TNF-alpha was significantly decreased only in case of HIV infection. Significantly higher IL-6 secretion was found in unstimulated cultures in dually infected patients. The mitogen induced cytokine secretion was generally lower in HIV-TB coinfected patients indicating profound perturbation of both Th1 and Th2 responses.     

IL-13-mediated worm expulsion is B7 independent and IFN-gamma sensitive

B7 costimulation is a required component of many type 2 immune responses, including allergy and protective immunity to many nematode parasites. This response includes elevations in Th2 cytokines and associated effector functions including elevations in serum IgG1 and IgE and parasite expulsion. In studies of mice infected with Trichuris muris, blocking B7 ligand interactions inhibited protective immunity, suppressed IL-4 production, and enhanced IFN-gamma production, but unexpectedly did not inhibit production of the Th2 cytokine, IL-13. Blocking both IFN-gamma and B7 restored protective immunity, which was IL-13 dependent, but did not restore IL-4 or associated IgE responses. Although IL-13 was required for worm expulsion in mice in which both IFN-gamma and B7 were blocked, IL-4 could mediate expulsion in the absence of both IL-13 and IFN-gamma. These studies demonstrate that 1) B7 costimulation is required to induce IL-4, but not IL-13 responses; 2) IL-13 is elevated in association with the IFN-gamma response that occurs following inhibition of B7 interactions, but can only mediate IL-4-independent protection when IFN-gamma is also inhibited; and 3) increased IL-13 production, in the absence of increased IL-4 production, is not associated with an IgE response, even in the absence of IFN-gamma.     

Kinetics of T cell cytokine gene expression in gerbils after a primary subcutaneous Brugia pahangi infection

The majority of patients infected with lymphatic filariae are microfilaremic but tend to manifest little obvious pathology because of the infections. Data collected from the Mongolian gerbil-Brugia spp. model for human lymphatic filariasis suggest this experimental animal model system most closely represents this patient group and will be useful in studying immunological parameters associated with chronic infections. This article reports the quantitation of interleukin (IL)-4, IL-5, IL-10, IL-13, and interferon (IFN)-gamma messenger RNA (mRNA) in gerbils after a primary subcutaneous infection with Brugia pahangi. Chronically infected gerbils showed elevated IL-4 in all tissues, compared with earlier time points, linking this Th2 cytokine to the downregulation of responsiveness, which develops in gerbils and humans. Both IL-5 and IL-13 mRNA expression were transient in all tissues. The peak in IL-5 at 14-28 days postinfection reflects the peak of peripheral eosinophilia observed in B. pahangi-infected gerbils. Little IFN-gamma mRNA was reported from chronically infected gerbils. The data collected thus far suggest that the expression profile of many of the measured cytokines in B. pahangi-infected gerbils reflects what is seen in an important subset of humans infected with lymphatic filariae, the microfilaremic, asymptomatic patient.     

Murine B cells expressing Thy-1 after in vivo immunization selectively secrete IgE

IL-4 induces Thy-1 expression and IgE secretion by LPS--and T cell-stimulated murine B cells in vitro. IFN-gamma inhibits both of these IL-4-mediated effects. IL-4 and IFN-gamma are often exclusively produced by different CD4+ T cell subsets. Injection of mice with a polyclonal goat anti-mouse IgD antibody (GaM delta) stimulates large increases in the serum concentration of IgE through the production of IL-4. Neutralization of endogenous IFN-gamma production in GaM delta-injected mice leads to further increases in serum IgE levels. We show that IL-4 and IFN-gamma, produced after GaM delta injection, respectively, stimulate and inhibit the number of splenic B cells expressing Thy-1 in vivo. Increased numbers of Thy-1-expressing B cells are observed concomitantly with the onset of enhanced IgE secretion. These Thy-1-expressing B cells are highly and selectively enriched for IgE-secreting cells.     

Gamma delta T cell responses associated with the development of tuberculosis in health care workers

This study evaluated T cell immune responses to purified protein derivative (PPD) and Mycobacterium tuberculosis (Mtb) in health care workers who remained free of active tuberculosis (HCWs w/o TB), health care workers who went on to develop active TB (HCWs w/TB), non-health care workers who were TB free (Non-HCWs) and tuberculosis patients presenting with minimal (Min TB) or advanced (Adv TB) disease. Peripheral blood mononuclear cells (PBMC) were stimulated with Mtb and PPD and the expression of T cell activation markers CD25+ and HLA-DR+, intracellular IL-4 and IFN-gamma production and cytotoxic responses were evaluated. PBMC from HCWs who developed TB showed decreased percentages of cells expressing CD8+CD25+ in comparison to HCWs who remained healthy. HCWs who developed TB showed increased gammadelta TCR+ cell cytotoxicity and decreased CD3+gammadelta TCR- cell cytotoxicity in comparison to HCWs who remained healthy. PBMC from TB patients with advanced disease showed decreased percentages of CD25+CD4+ and CD25+CD8+ T cells that were associated with increased IL-4 production in CD8+ and gammadelta TCR+ phenotypes, in comparison with TB patients presenting minimal disease. TB patients with advanced disease showed increased gammadelta TCR+ cytotoxicity and reduced CD3+gammadelta TCR- cell cytotoxicity. Our results suggest that HCWs who developed TB show an early compensatory mechanism involving an increase in lytic responses of gammadelta TCR+ cells which did not prevent TB.     

Expression of IL-15 and IL-4 in IFN-gamma-independent control of experimental human Cryptosporidium parvum infection

We have previously demonstrated interferon gamma (IFN-gamma) in intestinal mucosa after experimental human Cryptosporidium parvum infection, but expression was limited to sensitized volunteers. To characterize IFN-gamma-independent mechanisms in control of infection, jejunal biopsies from immunocompetent volunteers experimentally challenged with C. parvum were examined by in situ hybridization for interleukin (IL-)15 and IL-4 mRNA with confirmation by immunohistochemistry. Cytokine expression was correlated with prechallenge anti- C. parvum IgG, symptoms, oocyst shedding, and prior IFN-gamma expression data. IL-15 expression was noted only in those without prior sensitization, who did not express IFN-gamma. By contrast, expression of IL-4 was associated with prior sensitization. IL-15 was only detected in those with symptoms (6/14 symptomatic vs 0/3 asymptomatic, P<0.05). Among 14 volunteers who did not express IFN-gamma, oocyst shedding was lower in those expressing IL-15. Overall, 14/15 volunteers who did not shed oocysts expressed either IFN-gamma or IL-15. There was no correlation between expression of IL-4 and symptoms or oocyst shedding. In conclusion, IL-15 expression was associated with control of oocyst shedding in those not expressing IFN-gamma. These data suggest that IL-15 is involved in IFN-gamma independent mechanisms of control of human cryptosporidiosis, perhaps via activation of the innate immune response.