Role of TNF α, IL-6 and CXCL10 in dengue disease severity

Background and objective: Dengue virus infections (Dengue) have become increasingly common in Pakistan and can result in case fatalities if not managed appropriately. Patients with Dengue virus infection may be asymptomatic or present with Dengue fever (DF), Dengue with warning signs (DWS) or severe Dengue (SD). Severity in Dengue is coincident with an exacerbated production of lymphocyte-induced cytokines and chemokines which are associated with plasma leakage. We investigated the association of circulating levels of cytokines such as Interleukin (IL)-6, tumor necrosis factor (TNF)-alpha and CXCL-10 in Dengue patients with differing severity of disease.Material and methods: Dengue infection was confirmed by testing for human IgM to the Dengue virus. Dengue patients (n=58) and healthy controls (n=33) were recruited. Dengue patients were grouped into those with DF (n=39), DWS (n=15) and SD (n=4). Serum IL-6, TNFα and CXCL10 levels were tested by ELISA. The Mann Whitney U test was used for statistical analysis.Result: Circulating levels of TNFα (p≤0.001) and CXCL10 (p≤0.001) levels were increased in Dengue patients as compared with controls. When patients were stratified for disease severity, it was observed that CXCL10 was increased in DWS as compared to DF (p=0.046). IL-6 levels were increased in patients with SD as compared to those with DWS (p=0.044). TNFα levels were not found to differ between different groups of Dengue patients.Conclusion: Raised CXCL10 and TNFα levels were associated with increased clinical severity of Dengue infection and probably increased disease progression due to excessive inflammation and increased vascular changes in the patients

ESAT6-Induced IFNγ and CXCL9 Can Differentiate Severity of Tuberculosis

BACKGROUND: Protective responses against Mycobacterium tuberculosis are dependent on appropriate T cell and macrophage activation. Mycobacterial antigen six kDa early secreted antigenic target (ESAT6) and culture filtrate protein 10 (CFP10) can detect M. tuberculosis specific IFNgamma responses. However, most studies have been performed in non-endemic regions and to study pulmonary tuberculosis (PTB). We have studied ESAT6 and CFP10 induced cytokine and chemokines responses in PTB and extrapulmonary (EPul) TB. METHODOLOGY: IFNgamma, IL10, CXCL9 and CCL2 responses were determined using an ex vivo whole blood assay system in PTB (n = 30) and EPulTB patients with limited (LNTB, n = 24) or severe (SevTB, n = 22) disease, and in healthy endemic controls (ECs). Responses to bacterial LPS were also determined. PRINCIPAL FINDINGS: ESAT6- and CFP10-induced IFNgamma was comparable between ECs and TB patients. Both ESAT6- and CFP10-induced IFNgamma secretion was greater in LNTB than PTB. ESAT6-induced CXCL9 was greater in EPulTB as compared with PTB, with an increase in SevTB as compared with LNTB. CFP10-induced CCL2 was higher in PTB than LNTB patients. LPS-stimulated CXCL9 was greatest in SevTB and LPS-induced CCL2 was increased in PTB as compared with LNTB patients. A positive correlation between ESAT6-induced IFNgamma and CXCL9 was present in all TB patients, but IFNgamma and CCL2 was only correlated in LNTB. ESAT-induced CCL2 and CXCL9 were significantly associated in LNTB while correlation in response to LPS was only present in SevTB. CONCLUSIONS: ESAT6 induced IFNgamma and CXCL9 can differentiate between limited and severe TB infections

Expression of M. tuberculosis-induced suppressor of cytokine signaling (SOCS) 1, SOCS3, FoxP3 and secretion of IL-6 associates with differing clinical severity of tuberculosis

Background Appropriate immune activation of T cells and macrophages is central for the control of Mycobacterium tuberculosis infections. IFN-γ stimulated responses are lowered in tuberculosis (TB), while expression of Suppressor of Cytokine Signaling (SOCS) molecules – 1 and 3 and CD4+CD25+FoxP3+T regulatory cells is increased. Here we investigated the association of these molecules in regard to clinical severity of TB. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from patients with pulmonary TB (PTB, n = 33), extra-pulmonary TB (ETB, n = 33) and healthy endemic controls (EC, n = 15). Cases were classified as moderately advanced or far advanced PTB, and less severe or severe disseminated ETB. M. tuberculosis -stimulated IFN-γ, SOCS1, SOCS3 and FoxP3 gene expression and secretion of Th1 and Th2 cytokines was measured. Statistical analysis was performed using Mann–Whitney U, Wilcoxon Rank and Kruskal Wallis non-parametric tests. Results In un-stimulated PBMCs, IL-6 (p = 0.018) and IL-10 (p = 0.013) secretion levels were increased in PTB while IL-10 was also increased in ETB (p = 0.003), all in comparison with EC. M. tuberculosis-stimulated IL-6 (p = 0.003) was lowered in ETB as compared with EC. SOCS1 mRNA expression in M. tuberculosis stimulated PBMCs levels in moderately advanced PTB (p = 0.022), far advanced (p = 0.014) PTB, and severe ETB (p = 0.009) were raised as compared with EC. On the other hand, SOCS1 mRNA titers were reduced in less severe ETB, in comparison with severe ETB (p = 0.027) and far advanced PTB (p = 0.016). SOCS3 mRNA accumulation was reduced in far advanced PTB (p = 0.007) and FoxP3 mRNA expression was increased in less severe ETB as compared with EC (p = 0.017). Conclusions The lowered SOCS1 mRNA levels in patients with less severe extra-pulmonary TB as compared to those with more severe ETB and PTB may lead to elevated IFN-γ pathway gene expression in the latter group. As localized ETB has shown to be associated with more effective Th1 immunity and adaptive responses, this suggests a role for SOCS1 in determining disease outcome in extra-pulmonary TB

MAD 20 alleles of merozoite surface protein-1 (msp-1) are associated with severe Plasmodium falciparum malaria in Pakistan

Background: Various factors determine the outcome of Plasmodium falciparum infection such as parasite load, sequestration, adhesion molecules, and immune mediators. P. falciparum merozoite surface protein-1 (msp-1) and msp-2 genotypes were also found associated with severe disease. We investigated the association between msp-1 and msp-2 genotypes in patients with uncomplicated malaria (UM) and severe malaria (SM).Methods: Twenty-two malaria patients with microscopy-confirmed P. falciparum infection and eight healthy endemic controls were selected for analysis. Nested polymerase chain reaction (PCR) was used to identify P. falciparum genotypes. The plasma concentration of cytokines [tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interferon-gamma (IFN-gamma)] and chemokines [chemokine (C-X-C motif) ligand 9 (CXCL9) and CXCL10] were evaluated using enzyme-linked immunosorbent assay (ELISA).Results: TNF-alpha levels were significantly higher in both UM (389 pg/mL, p = 0.020) and SM (771 pg/mL, p = 0.004) compared with healthy controls, while they were greater in SM (p = 0.012) as compared to UM. CXCL9 levels were significantly raised in SM as compared to UM and negative controls (NCs). CXCL10 levels were raised in UM (550 pg/mL, p = 0.001) and SM (1480 pg/mL, p = 0.01) as compared with NCs. Increased levels of IL-6 were found in patients carrying the FC27 allelic type of msp-2. A higher prevalence of MAD 20 and K1 msp-1 alleles was observed in the SM group compared to UM.CONCLUSION: Overall, a greater prevalence of MAD 20 alleles and increased serum TNF-alpha and CXCL9 levels were associated with severe outcome in malaria. Understanding the diversity of malaria genotypes is important for predicting disease-related outcomes of P. falciparum infection in endemic areas

Measurement of ESAT6-induced IFNγ responses adjunct with CXCL9 increases the rate of diagnosis of active tuberculosis in an endemic region

Due to difficulties in direct diagnosis of Mycobacterium tuberculosis infection where site-specific specimens are not available, indirect methods of testing for infection are required. M. tuberculosisearly secreted antigen target-6 (ESAT6) induced IFN-γ responses are specific, but do not differentiate between latent and active TB. The use of adjunct biomarkers for TB diagnosis has been proposed, such as the chemokines: CXCL9, CXCL10 and CCL2. ESAT6-induced IFN-γ CXCL9, CXCL10 and CCL2 was measured in whole blood cell supernatants of patients with pulmonary tuberculosis (PTB, n = 36) and extrapulmonary TB (ETB, n = 31) and compared with healthy endemic controls (EC, n = 33). ESAT6-induced IFN-γ responses were positive in 32% of TB cases as compared with 15% of EC cases (p = 0.048). ESAT6-induced CXCL9 responses were positive in 42% of TB cases and 15% of EC cases (p = 0.006). ESAT6-induced-CXCL10 and -CCL2 responses did not discriminate between TB and EC groups. Measurement of IFN-γ or CXCL9 together diagnosed TB (53%) cases and was significant as compared with EC (p = 0.014) cases. IFN-γ and CXCL10 together did not increase the number of TB cases diagnosed. Within TB groups, ESAT6-IFN-γ/CXCL9-based detection increased to 53% in PTB (p = 0.031) and 54% in ETB (p = 0.021), with comparable diagnosis in less severe extrapulmonary TB (L-ETB, 55%) and severe disseminated extrapulmonary TB (D-ETB, 50%). Given that 47% of TB cases remained undetected, this study shown that ESAT6-induced IFNγ and CXCL9 can support diagnosis, but must be supported by clinical correlation and other relevant investigations

Differential Live Mycobacterium tuberculosis-, M. bovis BCG-, Recombinant ESAT6-, and Culture Filtrate Protein 10-Induced Immunity in Tuberculosis▿

The high prevalence of Mycobacterium tuberculosis makes it imperative that immune responses to evaluate could be predictive of infection. We investigated live Mycobacterium- and recombinant antigen-induced cytokine and chemokine responses in patients with active tuberculosis (TB) compared with those of healthy controls from an area where TB is endemic (ECs). M. tuberculosis-, M. bovis BCG-, ESAT6-, and culture filtrate protein 10 (CFP10)-induced responses were determined in peripheral blood mononuclear cells from patients with pulmonary TB (n = 38) and ECs (n = 39). The levels of the cytokines gamma interferon (IFN-γ) and interleukin-10 (IL-10) and the chemokines CCL2, CCL3, and CXCL9 were measured. The levels of M. tuberculosis- and BCG-induced IFN-γ secretion were significantly reduced (P = 0.002 and P < 0.01, respectively), while the amount of IL-10 induced by both virulent (P < 0.01) and avirulent (P = 0.002) mycobacteria was increased in patients with TB. The ESAT6-induced IFN-γ responses were increased in the patients with TB (P = 0.013) compared with those in the EC group. When tuberculin skin test (TST)-negative (TST−; induration, <10 mm) and TST-positive (TST+) donors were studied separately, both TST− and TST+ individuals showed increased IFN-γ responses to M. tuberculosis compared with the responses of the patients with TB (P = 0.037 and P = 0.006, respectively). However, only TST+ ECs showed reduced IFN-γ responses to ESAT6 (P = 0.008) compared with the responses of the patients with TB. The levels of M. tuberculosis-induced CCL2 (P = 0.006) and CXCL9 (P = 0.017) were greater in the patients with TB. The levels of CCL3 secretion in response to Mycobacterium and antigen stimulation were comparable between the two groups. While the levels of ESAT6-induced chemokines did not differ between the patients with TB and the ECs, the levels of CFP10-induced CCL2 (P = 0.01) and CXCL9 (P = 0.001) were increased in the patients. These data indicate differential host IFN-γ, CXCL9, and CCL2 responses to live mycobacteria and mycobacterial antigens and have implications for the identification of potential biomarkers of infection which could be used for the diagnosis of TB

Differential LPS-induced CXCL9 and CCL2 in limited and severe TB.

<p>Whole blood cells were stimulated with LPS at 1 µg/ml and CXCL9 measured in cell supernatants harvested at 2 days post-stimulation. All other parameters as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005158#pone-0005158-g001" target="_blank">Fig. 1</a>. A. CXCL9 and B. CCL2.</p

Differential ESAT6-induced IFNγ and CXCL9 in pulmonary and extrapulmonary TB.

<p>Whole blood cells were stimulated with ESAT6 at 5 µg/ml, and IFNγ measured in cell supernatants harvested at 5 day post-stimulation, while CXCL9 was measured at 2 days post-stimulation. TB patient groups comprised those with pulmonary TB (PTB, n = 30), less severe EPulTB (LNTB, n = 24) and severe EPulTB (SevTB, n = 22). The box plots represent data from each group after cytokine secretion from unstimulated cells has been subtracted. The whiskers indicate the 25^th and 75^th quartile respectively, while the median line separates the two. ‘*’ denotes significant differences between groups, p<0.05; (*). A. IFNγ, B. CXCL9.</p

ESAT6- and CFP10-induced cytokine and chemokine responses.

<p>EC, healthy endemic controls; TB, tuberculosis patients.</p><p>‘δ’ denotes cytokine secretion after background subtraction in each case.</p>*<p>denotes significant difference (p<0.05) as compared with EC.</p><p>All statistical analyses performed using the Mann-Whitney U non-parametric test.</p