Immune Responses to ESAT-6 and CFP-10 by FASCIA and Multiplex Technology for Diagnosis of M. tuberculosis Infection; IP-10 Is a Promising Marker

Background

There is a need for reliable markers to diagnose active and latent tuberculosis (TB). The interferon gamma release assays (IGRAs) are compared to the tuberculin skin test (TST) more specific, but cannot discriminate between recent or remote TB infection. Here the Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood (FASCIA), which quantifies expanded T-lymphoblasts by flow-cytometric analysis after long-term antigen stimulation of whole blood, is combined with cytokine/chemokine analysis in the supernatant by multiplex technology for diagnosis of Mycobacterium tuberculosis (Mtb) infection.

Methods and Findings

Consecutive patients with suspected TB (n = 85), with microbiologically verified active pulmonary TB (n = 33), extra pulmonary TB (n = 21), clinical TB (n = 11), presumed latent TB infection (LTBI) (n = 23), patients negative for TB (n = 8) and 21 healthy controls were studied. Blood samples were analyzed with FASCIA and multiplex technology to determine and correlate proliferative responses and the value of 14 cytokines for diagnosis of Mtb infection: IFN- γ, IL-2, TNF-α, IP-10, IL-12, IL-6, IL-4, IL-5, IL-13, IL-17, MIP-1β, GM-CSF, IFN-α2 and IL-10. Cytokine levels for IFN-γ, IP-10, MIP-1β, IL-2, TNF-α, IL-6, IL-10, IL-13 and GM-CSF were significantly higher after stimulation with the Mtb specific antigens ESAT-6 and CFP-10 in patients with active TB compared to healthy controls (p<0.05) and correlated with proliferative responses. IP-10 was positive in all patients with verified TB, if using a combination of ESAT-6 and CFP-10 and was the only marker significantly more sensitive in detecting active TB then IFN-γ (p = 0.012). Cytokine responses in patients with active TB were more frequent and detected at higher levels than in patients with LTBI.

Conclusions

IP-10 seems to be an important marker for diagnosis of active and latent TB. Patients with active TB and LTBI responded with similar cytokine profiles against TB antigens but proliferative and cytokine responses were generally higher in patients with active TB.     

Evaluation of the Diagnostic Potential of IP-10 and IL-2 as Biomarkers for the Diagnosis of Active and Latent Tuberculosis in a BCG-Vaccinated Population

Background

The Mycobacterium tuberculosis (Mtb)-specific T-cell interferon gamma release assays (IGRAs) are useful in detecting Mtb infection but perform poorly at distinguishing active tuberculosis disease (ATB) and latent tuberculosis infection (LTBI). This study is aimed at evaluating additional cytokines as biomarkers besides interferon-gamma (IFN-γ) to improve the identification of ATB and LTBI.

Methodology/Principal Findings

Sixty-six patients with ATB, 73 household contacts (HHC) of ATB patients and 76 healthy controls (HC) were recruited to undergo QuantiFERON TB GOLD in-tube assay (QFT) and the enzyme-linked immunosorbent assay (ELISA) where the release of IFN-γ, IFN-γ inducible protein 10 (IP-10), Interleukin 2 (IL-2) and Tumor Necrosis Factor-α (TNF-α) was determined in the whole blood with or without antigen-stimulation. The positive rates of the QFT, IP-10 and IL-2 tests were 86.4%, 89.4% and 86.4% for the ATB group with no difference between them (p>0.05). However, QFT in combination with IP-10 and IL-2 significantly increased the detection rate to 95.5% in the ATB group (p = 0.03) and the indeterminate rate of all samples decreased from 2.3% (5/215) to 0.4% (1/215). The un-stimulated level of IP-10 was significantly higher in the HHC than the ATB and HC groups. The IP-10 responses were strongly associated with extended Mtb exposure time and the degree of smear-positivity of the index cases. The IL-2/IFN-γ ratio in the antigen-stimulated plasma could discriminate LTBI from ATB with a sensitivity of 77.2% and a specificity of 87.2%.

Conclusion

The increased Mtb-specific antigen-stimulated expression of IP-10 and IL-2 may be useful for detecting both ATB and LTBI. Combining the QFT with IP-10 and IL-2 could increase the detection accuracy of active TB over the QFT alone.     

Expression of CXC and CC type of chemokines and its receptors in tuberculous and non-tuberculous effusions

Chemokines mediate their biological functions by transmigration of various immune cells to the site of infection. Tuberculous pleurisy provides an effective model to study the role of chemokines in the recruitment of immune cells to the pleura. Our aim was to understand the cumulative effect of chemokines (IP-10, MIG, IL-8, MCP-1, MIP-1α and RANTES) and its receptors (CXCR2, CXCR3, CCR1, CCR2, CCR5 and CCR7) in the recruitment of CD4⁺ T cells obtained from blood (BL) and pleural fluid (PF) of tuberculous (TB) and non-tuberculous (NTB) patients. We observed significant increase in CD4⁺ T cells in TB PF indicating lymphocytic rich effusion. All chemokines except RANTES were significantly high in PF compared to BL in TB group, whereas IL-8 and MCP-1 showed significant increase only in NTB PF. The significantly high levels of IFN-γ and ΤΝF-α in TB PF and their positive correlation with IP-10 and MIP-1α indicated their synergistic action to elicit a strong protective Th1 response. In spite of high levels of Th1 cytokines and chemokines in TB PF, significantly lower levels of RANTES indicated its limited role at the site. The CXC receptors in PF of both the groups and CC receptors except CCR5 in TB PF were significantly high compared to BL. Only CXCR2, CCR5 and CCR7 showed significant increase in TB compared to NTB. Thus a selective concentration of chemokines, cytokines and abundant expression of chemokine receptors confirm the accumulation of activated and memory T cells at the site of infection and help in polarizing Th1 immune response.     

Increased Cytokines Response in Patients with Tuberculosis Complicated with Chronic Obstructive Pulmonary Disease

Objectives

To explore the change and its significance of cytokines in patients with pulmonary tuberculosis complicated with COPD.

Methods

The immune function of 152 cases of pulmonary tuberculosis with COPD was detected to compare with 150 cases of patients with pulmonary tuberculosis, 157 cases of patients with COPD and 50 cases of healthy volunteers who were in the hospital during the same period. T lymphocyte cell population in peripheral blood was detected by flow cytometry. The serum levels of sIL-2R, IL-6, IFN-γ, TNF-α were measured using ELISA.

Results

The percentage of CD4+ T cells in TB patients with or without COPD and COPD patients without TB was significantly lower than that in control group. The percentage of CD4+ T cells in patients with TB and COPD was significantly lower than that in the non-COPD TB patients. The percentage of CD8+ T cells was higher in the TB patients group than that in control group. The CD4+/CD8+ ratio in the TB patients group was significantly lower than that in control group. The concentrations of sIL-2R, IL-6, TNF-α, IFN-γ in TB patients with or without COPD and COPD patients without TB were significantly higher than those in control group. In addition, sIL-2R, IL-6, TNF-α concentrations in the patients with TB and COPD were higher than those in the non-COPD TB patients. The concentrations of sIL-2R, IL-6, TNF-α, IFN-γ in COPD patients with TB were significantly higher than those in COPD patients without TB. There was a significant negative correlation between serum levels of TNF-α, IL-6 and FEV1 (%, predicted) in COPD without TB group.

Conclusions

The patients with pulmonary tuberculosis complicated with COPD were impaired in cellular immunity, and its extent of immune impairment is more serious than those of the patients with pulmonary tuberculosis and the patients with COPD.     

Different Patterns of Cytokines and Chemokines Combined with IFN-γ Production Reflect Mycobacterium tuberculosis Infection and Disease

Background

IFN-γ is presently the only soluble immunological marker used to help diagnose latent Mycobacterium tuberculosis (M.tb) infection. However, IFN-γ is not available to distinguish latent from active TB infection. Moreover, extrapulmonary tuberculosis, such as tuberculous pleurisy, cannot be properly diagnosed by IFN-γ release assay. As a result, other disease- or infection-related immunological biomarkers that would be more effective need to be screened and identified.

Methodology

A panel of 41 soluble immunological molecules (17 cytokines and 24 chemokines) was tested using Luminex liquid array-based multiplexed immunoassays. Samples, including plasma and pleural effusions, from healthy donors (HD, n = 12) or patients with latent tuberculosis infection (LTBI, n = 20), pulmonary tuberculosis (TB, n = 12), tuberculous pleurisy (TP, n = 15) or lung cancer (LC, n = 15) were collected and screened for soluble markers. Peripheral blood mononuclear cells (PBMCs) and pleural fluid mononuclear cells (PFMCs) were also isolated to investigate antigen-specific immune factors.

Principal Findings

For the 41 examined factors, our results indicated that three patterns were closely associated with infection and disease. (1) Significantly elevated plasma levels of IL-2, IP-10, CXCL11 and CXCL12 were present in both patients with tuberculosis and in a sub-group participant with latent tuberculosis infection who showed a higher level of IFN-γ producing cells by ELISPOT assay compared with other latently infected individuals. (2) IL-6 and IL-9 were only significantly increased in plasma from active TB patients, and the two factors were consistently highly secreted after M.tb antigen stimulation. (3) When patients developed tuberculous pleurisy, CCL1, CCL21 and IL-6 were specifically increased in the pleural effusions. In particular, these three factors were consistently highly secreted by pleural fluid mononuclear cells following M.tb-specific antigen stimulation. In conclusion, our data imply that the specific secretion of soluble immunological factors, in addition to IFN-γ, may be used to evaluate M.tb infection and tuberculosis disease.     

Cigarette smoke attenuates the production of cytokines by human plasmacytoid dendritic cells and enhances the release of IL-8 in response to TLR-9 stimulation

Myeloid and plasmacytoid dendritic cells (mDCs, pDC) are crucial to the immune system, detecting microorganisms and linking the innate and adaptive immunity. pDC are present in small quantities in tissues that are in contact with the external environment; mainly the skin, the inner lining of the nose, lungs, stomach and intestines. They produce large amounts of IFN-α after stimulation and are pivotal for the induction of antiviral responses. Chronic obstructive pulmonary disease (COPD) patients are known to be more susceptible to viral infections. We have demonstrated that exposure of mDC to cigarette smoke extract (CSE) leads to the release of chemokines, however, not much is known about the role of pDC in COPD. In this study, we addressed several key questions with respect to the mechanism of action of CSE on human pDC in an in vitro model. Human pDCs were isolated from normal healthy volunteers and subjected to fresh CSE and the levels of IL-8, TNF-α, IP-10, IL-6, IL-1, IL-12 and IL-10 and IFN-α were studied by both ELISA and real time PCR methods. We observed that CSE augmented the production of IL-8 and suppressed the release of TNF-α, IL-6 and IFN-α. Moreover, CSE suppressed PI3K/Akt signalling in pDC. In conclusion, our data indicate that CSE has both the potential to diminish anti-viral immunity by downregulating the release of IFN-α and other pro-inflammatory cytokines while, at the same time, augmenting the pathogenesis of COPD via an IL-8 induced recruitment of neutrophils.     

Pro- and Anti-Inflammatory Cytokines against Rv2031 Are Elevated during Latent Tuberculosis: A Study in Cohorts of Tuberculosis Patients,Household Contacts and Community Controls in an Endemic Setting

Tuberculosis (TB) is among the leading causes of morbidity and mortality. The causative agent, Mycobacterium tuberculosis (Mtb), has evolved virulent factors for entry, survival, multiplication and immune evasion. Rv2031 (also called alpha crystallin, hspX, 16-kDa antigen), one of the most immunogenic latency antigens, is believed to play a key role in long-term viability of Mtb. Here, we report the dynamics of pro-inflammatory (IFN-γ, TNF-α) and anti-inflammatory (IL-10) cytokines against Rv2031 using whole blood assay in human cohorts in a TB endemic setting. Cytokine responses to ESAT-6-CFP-10 were also measured for comparison. Blood samples were collected from smear positive pulmonary TB patients and their contacts at baseline, 6 and 12 months, and from community controls at entry. At baseline, 54.4% of controls and 73.2% of contacts were QFT-GIT test positive. Baseline IFN-γ, TNF-α and IL-10 responses to Rv2031 were significantly higher in controls compared to contacts and untreated patients (p<0.001). Furthermore, untreated patients had significantly higher TNF-α and IL-10 responses to Rv2031 compared to contacts (p<0.001). In contacts and treated patients, IFN-γ, TNF-α and IL-10 responses to Rv2031 significantly increased over 12 months (p<0.0001) and became comparable with the corresponding levels in controls. There was a positive and significant correlation between Rv2031 and ESAT-6-CFP-10 specific cytokine responses in each study group. The fact that the levels of IFN-γ, TNF-α and IL-10 against Rv2031 were highest during latent TB infection may indicate their potential as markers of protection against TB. Taken together, the findings of this study suggest the potential of IFN-γ, TNF-α and IL-10 against Rv2031 as biomarkers of the host response to Mtb during convalescence from, and the absence of, active tuberculosis.     

Production of TNF-α, IL-12(p40) and IL-17 Can Discriminate between Active TB Disease and Latent Infection in a West African Cohort

Background

Mycobacterium tuberculosis (MTb) infects approximately 2 billion people world-wide resulting in almost 2 million deaths per year. Determining biomarkers that distinguish different stages of tuberculosis (TB) infection and disease will provide tools for more effective diagnosis and ultimately aid in the development of new vaccine candidates. The current diagnostic kits utilising production of IFN-γ in response to TB antigens can detect MTb infection but are unable to distinguish between infection and disease. The aim of this study was to assess if the use of a longer term assay and the analysis of multiple cytokines would enhance diagnosis of active TB in a TB-endemic population.

Methods

We compared production of multiple cytokines (TNF-α, IFN-γ, IL-10, IL-12(p40), IL-13, IL-17 and IL-18) following long-term (7 days) stimulation of whole-blood with TB antigens (ESAT-6/CFP-10 (EC), PPD or TB10.4) from TB cases (n = 36) and their Mycobacterium-infected (TST+; n = 20) or uninfected (TST−; n = 19) household contacts (HHC).

Results and Conclusions

We found that TNF-α production following EC stimulation and TNF-α and IL-12(p40) following TB10.4 stimulation were significantly higher from TB cases compared to TST+ HHC, while production of IFN-γ and IL-13 were significantly higher from TST+ compared to TST- HHC following PPD or EC stimulation. Combined analysis of TNF-α, IL-12(p40) and IL-17 following TB10.4 stimulation resulted in 85% correct classification into TB cases or TST+ HHC. 74% correct classification into TST+ or TST− HHC was achieved with IFN-γ alone following TB10.4 stimulation (69% following EC) and little enhancement was seen with additional cytokines. We also saw a tendency for TB cases infected with M. africanum to have increased TNF-α and IL-10 production compared to those infected with M. tuberculosis. Our results provide further insight into the pathogenesis of tuberculosis and may enhance the specificity of the currently available diagnostic tests, particularly for diagnosis of active TB.     

Impaired M. tuberculosis Antigen-Specific IFN-γ Response without IL-17 Enhancement in Patients with Severe Cavitary Pulmonary Tuberculosis

BackgroundTh1 cells play an essential role in immune protection against tuberculosis. Th17 cells might be involved with immune pathology in active human tuberculosis (TB). The balance between Th1 and Th17 cells in patients with cavitary tuberculosis needs to be clarified which might help understanding the immunological basis of pathologic pathogenesis in TB.MethodInitially treated pulmonary TB (PTB) patients with or without cavities were recruited before chemotherapy. We isolated peripheral blood mononuclear cells, stimulated with phytohemagglutinin (PHA), PPD, or ESAT-6 antigens, and assayed supernatant IFN-γ and IL-17 by ELISA after 24 or 72 hours incubation, respectively. Cells were also stained with antibodies to CD3, CD4, CD8, IFN-γ or IL-17 and the proportion of stained cells was measured by flow cytometry.ResultsWe found wide variation of IFN-γ response in active PTB patients, but less subject-to-subject variation of IL-17 was observed as we previously reported. There were no significant differences in IFN-γ and IL-17 between cavitary and non-cavitary PTB; however, we found decreased IFN-γ secretion in severe cavitary PTB compared to mild lesion non-cavitary PTB (p < 0.05). We also found a decrease in the proportion of CD3+CD4+ T cells in the blood of severe cavitary PTB patients (p < 0.05).ConclusionsIL-17 seemed to have no association with the formation of cavities in active PTB from the study of PBMC. Impaired IFN-γ without IL-17 enhancement occurs in peripheral blood during severe cavitary PTB. Our results demonstrate that M. tuberculosis antigen-specific Th1 response is decreased when PTB lesions develop to severe cavities.     

Combination of Cytokine Responses Indicative of Latent TB and Active TB in Malawian Adults

Background

An IFN-γ response to M. tuberculosis-specific antigens is an effective biomarker for M. tuberculosis infection but it cannot discriminate between latent TB infection and active TB disease. Combining a number of cytokine/chemokine responses to M. tuberculosis antigens may enable differentiation of latent TB from active disease.

Methods

Asymptomatic recently-exposed individuals (spouses of TB patients) were recruited and tuberculin skin tested, bled and followed-up for two years. Culture supernatants, from a six-day culture of diluted whole blood samples stimulated with M. tuberculosis-derived PPD or ESAT-6, were measured for IFN-γ, IL-10, IL-13, IL-17, TNF-α and CXCL10 using cytokine ELISAs. In addition, 15 patients with sputum smear-positive pulmonary TB were recruited and tested.

Results

Spouses with positive IFN-γ responses to M. tuberculosis ESAT-6 (>62.5 pg/mL) and TB patients showed high production of IL-17, CXCL10 and TNF-α. Higher production of IL-10 and IL-17 in response to ESAT-6 was observed in the spouses compared with TB patients while the ratios of IFN-γ/IL-10 and IFN-γ/IL-17 in response to M. tuberculosis-derived PPD were significantly higher in TB patients compared with the spouses. Tuberculin skin test results did not correlate with cytokine responses.

Conclusions

CXCL10 and TNF-α may be used as adjunct markers alongside an IFN-γ release assay to diagnose M. tuberculosis infection, and IL-17 and IL-10 production may differentiate individuals with LTBI from active TB.     

Dysregulated Immune Activation in Second-Line HAART HIV+ Patients Is Similar to That of Untreated Patients

Background

Successful highly active antiretroviral therapy (HAART) has changed the outcome of AIDS patients worldwide because the complete suppression of viremia improves health and prolongs life expectancy of HIV-1+ patients. However, little attention has been given to the immunological profile of patients under distinct HAART regimens. This work aimed to investigate the differences in the immunological pattern of HIV-1+ patients under the first- or second-line HAART in Brazil.

Methods

CD4+ T cell counts, Viral load, and plasma concentration of sCD14, sCD163, MCP-1, RANTES, IP-10, IL-1β, IL-6, TNF-α, IL-12, IFN-α, IFN-γ, IL-4, IL-5, and IL-10 were assessed for immunological characterization of the following clinical groups: Non-infected individuals (NI; n = 66), HIV-1+ untreated (HIV; n = 46), HIV-1+ treated with first-line HAART (HAART 1; n = 15); and HIV-1+ treated with second-line HAART (HAART 2; n = 15).

Results

We found that the immunological biosignature pattern of HAART 1 is similar to that of NI individuals, especially in patients presenting slow progression of the disease, while patients under HAART 2 remain in a moderate inflammatory state, which is similar to that of untreated HIV patients pattern. Network correlations revealed that differences in IP-10, TNF-α, IL-6, IFN-α, and IL-10 interactions were primordial in HIV disease and treatment. Heat map and decision tree analysis identified that IP-10>TNF-α>IFN-α were the best respective HAART segregation biomarkers.

Conclusion

HIV patients in different HAART regimens develop distinct immunological biosignature, introducing a novel perspective into disease outcome and potential new therapies that consider HAART patients as a heterogeneous group.     

Cytokines in Acute Chikungunya

Introduction

Acute chikungunya (CHIKV) is predominantly an acute onset of excruciatingly painful, self-limiting musculoskeletal (MSK) arbovirus illness and this was further reported by us during the 2006 Indian epidemic [Chopra et al. Epidemiol Infect 2012]. Selected serum cytokines profile in subjects within one month of onset of illness is being presented.

Methods

Out of 509 clinical CHIKV cases (43% population) identified during a rural population survey, 225 subjects consented blood investigations. 132 examined within 30 days of febrile onset are the study cohort. Anti-CHIKV IgM and IgG antibodies tested by immunochromatography and indirect immunofluorescence respectively. Interferons (IFN)-α, -β and -γ, Interferon Gamma-Induced Protein-10 (CXCL-10/IP-10), Tumor Necrosis Factor-α (TNF-α), Interleukin-1β (IL-1β), Interleukin-6 (IL-6), Interleukin-13 (IL-13), Monocyte Chemoattractant Protein-1 (MCP-1), Interleukin–4 (IL-4) and Interleukin–10 (IL-10) performed by ELISA. Samples collected from neighboring community a year prior to the epidemic used as healthy controls.

Results

Seropositivity for anti-CHIKV IgM and IgG was 65% and 52% respectively. IFN-α, IFN-β, IFN-γ, CXCL10/IP-10 and IL-1β showed intense response in early acute phase. Cytokines (particularly TNF-α, MCP-1, IL-4, IL-6 and IL-10) was maximum in extended symptomatic phase and remained elevated in recovered subjects. Higher (p<0.05) IFN and IL-4 seen in patients seropositive for anti-CHIKV IgG. Elderly cases (≥65 years) showed elevated cytokines (except IFN) and anti-CHIKV antibodies near similar to younger subjects. Significant correlations (p<0.05) found between cytokines and clinical features (fatigue, low back ache, myalgia) and anti-CHIKV antibodies.

Conclusion

An intense cytokine milieu was evident in the early and immediate persistent symptomatic phase and in recovered subjects. Early persistent IgM and lower IgG to anti-CHKV and intense Th2 cytokine phenotype seem to be associated with delay in resolution of MSK symptoms. Intriguingly, maximum TNF-α, IL-6 and IL-13 with low anti-CHIKV IgM response found in subjects recovered from CHIKV within one month of illness.     

Early Specific Host Response Associated with Starting Effective Tuberculosis Treatment in an Infection Controlled Placebo Controlled Mouse Study

Recently we proposed exploring the potential of treatment stimulated testing as diagnostic method for tuberculosis (TB). An infection controlled placebo controlled mouse study was performed to investigate whether serum cytokine levels changed measurably during the early phase of TB chemotherapy. Serum was collected prior to and during the first 3 weeks of isoniazid (INH) and rifampicin (RIF) chemotherapy, and levels of 23 selected cytokines/chemokines were measured using a liquid bead array. The serum levels of IFNγ, IP-10, MIG, MCP-1, IL-17 and IL-6 were elevated in the TB infected mice compared to non-infected mice at least at 1 time point measured. In infected mice, IFNγ, IP-10, MIG and MCP-1 levels decreased within 7 days of treatment with RIF+INH compared to placebo. Treatment of non-infected mice in the absence of tuberculosis infection had no effect on these cytokines. IL-17 and IL-6 had decreased to baseline in all infected mice prior to the initiation of treatment. This study demonstrates that systemic levels of some cytokines, more specifically IFNγ, IP-10, MIG and MCP-1, rapidly and specifically change upon starting TB chemotherapy only in the presence of infection in a mouse model. Thus, IFNγ, IP-10, MIG and MCP-1 are promising ‘Treat-to-Test’ targets for the diagnosis of TB and deserve further investigation in a study on human TB suspects.     

Pleural Tuberculosis in Patients with Early HIV Infection Is Associated with Increased TNF-Alpha Expression and Necrosis in Granulomas

Although granulomas may be an essential host response against persistent antigens, they are also associated with immunopathology. We investigated whether HIV co-infection affects histopathological appearance and cytokine profiles of pleural granulomas in patients with active pleural tuberculosis (TB). Granulomas were investigated in pleural biopsies from HIV positive and negative TB pleuritis patients. Granulomas were characterised as necrotic or non-necrotic, graded histologically and investigated for the mRNA expression of IL-12, IFN-γ, TNF-α and IL-4 by in situ hybridisation. In all TB patients a mixed Th1/Th2 profile was noted. Necrotic granulomas were more evident in HIV positive patients with a clear association between TNF-α and necrosis. This study demonstrates immune dysregulation which may include TNF-α-mediated immunopathology at the site of disease in HIV infected pleural TB patients.     

Human Rhinovirus Induced Cytokine/Chemokine Responses in Human Airway Epithelial and Immune Cells

Infections with human rhinovirus (HRV) are commonly associated with acute upper and lower respiratory tract disease and asthma exacerbations. The role that HRVs play in these diseases suggests it is important to understand host-specific or virus-specific factors that contribute to pathogenesis. Since species A HRVs are often associated with more serious HRV disease than species B HRVs, differences in immune responses they induce should inform disease pathogenesis. To identify species differences in induced responses, we evaluated 3 species A viruses, HRV 25, 31 and 36 and 3 species B viruses, HRV 4, 35 and 48 by exposing human PBMCs to HRV infected Calu-3 cells. To evaluate the potential effect of memory induced by previous HRV infection on study responses, we tested cord blood mononuclear cells that should be HRV naïve. There were HRV-associated increases (significant increase compared to mock-infected cells) for one or more HRVs for IP-10 and IL-15 that was unaffected by addition of PBMCs, for MIP-1α, MIP-1β, IFN-α, and HGF only with addition of PBMCs, and for ENA-78 only without addition of PBMCs. All three species B HRVs induced higher levels, compared to A HRVs, of MIP-1α and MIP-1β with PBMCs and ENA-78 without PBMCs. In contrast, addition of CBMCs had less effect and did not induce MIP-1α, MIP-1β, or IFN-α nor block ENA-78 production. Addition of CBMCs did, however, increase IP-10 levels for HRV 35 and HRV 36 infection. The presence of an effect with PBMCs and no effect with CBMCs for some responses suggest differences between the two types of cells possibly because of the presence of HRV memory responses in PBMCs and not CBMCs or limited response capacity for the immature CBMCs relative to PBMCs. Thus, our results indicate that different HRV strains can induce different patterns of cytokines and chemokines; some of these differences may be due to differences in memory responses induced by past HRV infections, and other differences related to virus factors that can inform disease pathogenesis.     

Discordance in CD4+T-Cell Levels and Viral Loads with Co-Occurrence of Elevated Peripheral TNF-α and IL-4 in Newly Diagnosed HIV-TB Co-Infected Cases

Background

Cytokines are the hallmark of immune response to different pathogens and often dictate the disease outcome. HIV infection and tuberculosis (TB) are more destructive when confronted together than either alone. Clinical data related to the immune status of HIV-TB patients before the initiation of any drug therapy is not well documented. This study aimed to collect the baseline information pertaining to the immune status of HIV-TB co-infected patients and correlate the same with CD4+T cell levels and viral loads at the time of diagnosis prior to any drug therapy.

Methodology/Principal Findings

We analyzed the cytokines, CD4+T cell levels and viral loads to determine the immune environment in HIV-TB co-infection. The study involved four categories namely, Healthy controls (n = 57), TB infected (n = 57), HIV infected (n = 59) and HIV-TB co-infected (n = 57) patients. The multi-partite comparison and correlation between cytokines, CD4+T-cell levels and viral loads prior to drug therapy, showed an altered TH1 and TH2 response, as indicated by the cytokine profiles and skewed IFN-γ/IL-10 ratio. Inadequate CD4+T cell counts in HIV-TB patients did not correlate with high viral loads and vice-versa. When compared to HIV category, 34% of HIV-TB patients had concurrent high plasma levels of IL-4 and TNF-α at the time of diagnosis. TB relapse was observed in 5 of these HIV-TB co-infected patients who also displayed high IFN-γ/IL-10 ratio.

Conclusion/Significance

With these studies, we infer (i) CD4+T-cell levels as baseline criteria to report the disease progression in terms of viral load in HIV-TB co-infected patients can be misleading and (ii) co-occurrence of high TNF-α and IL-4 levels along with a high ratio of IFN-γ/IL-10, prior to drug therapy, may increase the susceptibility of HIV-TB co-infected patients to hyper-inflammation and TB relapse.     

Increased Inflammatory Response in Cytomegalovirus Seropositive Patients with Alzheimer’s Disease

Alzheimer’s disease (AD) has been associated with increased local inflammation in the affected brain regions, and in some studies also with elevated levels of proinflammatory cytokines in peripheral blood. Cytomegalovirus (CMV) is known to promote a more effector-oriented phenotype in the T-cell compartment, increasing with age. The aim of this study was to investigate the inflammatory response of peripheral blood mononuclear cells (PBMCs) from AD patients and non-demented (ND) controls. Using a multiplex Luminex xMAP assay targeting GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IP-10 and TNF-α, cytokine profiles from PBMCs were analysed after stimulation with anti-CD3/CD28 beads, CMV pp65 peptide mix or amyloid β (Aβ) protofibrils, respectively. CMV seropositive AD subjects presented with higher IFN-γ levels after anti-CD3/CD28 and CMV pp65 but not after Aβ stimulation, compared to CMV seropositive ND controls. When analysing IFN-γ response to anti-CD3/CD28 stimulation on a subgroup level, CMV seropositive AD subjects presented with higher levels compared to both CMV seronegative AD and CMV seropositive ND subjects. Taken together, our data from patients with clinically manifest AD suggest a possible role of CMV as an inflammatory promoter in AD immunology. Further studies of AD patients at earlier stages of disease, could provide better insight into the pathophysiology.     

ApoE Production in Human Monocytes and Its Regulation by Inflammatory Cytokines

The apoE production by tissue macrophages is crucial for the prevention of atherosclerosis and the aim of this study was to further elucidate how this apolipoprotein is regulated by cytokines present during inflammation. Here we studied apoE production in peripheral blood mononuclear cells (PBMC) and analysis was made with a newly developed apoE ELISpot assay. In PBMC, apoE secretion was restricted to monocytes with classical (CD14⁺⁺CD16⁻) and intermediate (CD14⁺CD16⁺) monocytes being the main producers. As earlier described for macrophages, production was strongly upregulated by TGF-β and downregulated by bacterial lipopolysaccharide (LPS) and the inflammatory cytokines IFN-γ, TNF-α and IL-1β. We could here show that a similar down-regulatory effect was also observed with the type I interferon, IFN-α, while IL-6, often regarded as one of the more prominent inflammatory cytokines, did not affect TGF-β-induced apoE production. The TNF-α inhibitor Enbrel could partly block the down-regulatory effect of IFN-γ, IFN-α and IL-1β, indicating that inhibition of apoE by these cytokines may be dependent on or synergize with TNF-α. Other cytokines tested, IL-2, IL-4, IL-12, IL-13, IL-17A and IL-23, had no inhibitory effect on apoE production. In contrast to the effect on monocytes, apoE production by primary hepatocytes and the hepatoma cell line HepG2 was more or less unaffected by treatment with cytokines or LPS.     

Coincident Pre-Diabetes Is Associated with Dysregulated Cytokine Responses in Pulmonary Tuberculosis

Background

Cytokines play an important role in the pathogenesis of pulmonary tuberculosis (PTB) - Type 2 diabetes mellitus co-morbidity. However, the cytokine interactions that characterize PTB coincident with pre-diabetes (PDM) are not known.

Methods

To identify the influence of coincident PDM on cytokine levels in PTB, we examined circulating levels of a panel of cytokines in the plasma of individuals with TB-PDM and compared them with those without PDM (TB-NDM).

Results

TB-PDM is characterized by elevated circulating levels of Type 1 (IFNγ, TNFα and IL-2), Type 17 (IL-17A and IL-17F) and other pro-inflammatory (IL-1β, IFNβ and GM-CSF) cytokines. TB-PDM is also characterized by increased systemic levels of Type 2 (IL-5) and regulatory (IL-10 and TGFβ) cytokines. Moreover, TB antigen stimulated whole blood also showed increased levels of pro-inflammatory (IFNγ, TNFα and IL-1β) cytokines as well. However, the cytokines did not exhibit any significant correlation with HbA1C levels or with bacterial burdens.

Conclusion

Our data reveal that pre-diabetes in PTB individuals is characterized by heightened cytokine responsiveness, indicating that a balanced pro and anti - inflammatory cytokine milieu is a feature of pre-diabetes - TB co-morbidity.