Immune cells and soluble immune markers in different stages of tuberculosis. Potential biomarkers for diagnosis and treatment efficacy

Tuberculosis (TB) is a major global health problem, especially in the developing world. In order to end the TB epidemic, reliable and rapid diagnostic tools that can identify and discriminate between latent and active TB are required. In addition, the emergence of multi- and extensively drug resistant strains of Mycobacterium tuberculosis (Mtb) highlights the need of improved treatment regimens and tools for monitoring the effect of treatment. It has been suggested that adjunct treatment that targets the host response to infection has the potential to facilitate eradication of Mtb, reduce tissue inflammation and shorten the treatment duration. The main aim of this thesis was to characterise immune cells and soluble immune markers in different stages of TB infection with focus on identifying potential biomarkers that may improve TB diagnostics and monitoring of treatment efficacy. The secondary aim was to explore the in vitro effects of the potential adjunct treatment option cyclooxygenase (COX)-inhibition on Mtb specific T cell responses. Peripheral blood mononuclear cells, plasma and supernatants from the QuantiFERON-TB Gold (QFT) test were obtained from individuals with active and latent TB before and during TB treatment and from QFT-negative controls. T cell subsets were studied by flow cytometry and potential immune modulating effects of the COX-inhibitor indomethacin on Mtb specific T cell responses were examined in vitro. Multiple soluble markers were measured in plasma and QFT supernatants by multiplex and enzyme immunoassays. In paper I, we found that the level of regulatory T cells (Treg) was higher in both the active and latent TB group compared with controls. The results of paper IV indicate that the COX-inhibitor indomethacin may be used to modulate the immune response in active TB by reducing the number of Mtb specific Treg. In paper II, we report that the plasma level of interferon gamma inducible protein 10 (IP-10), although not specific for TB, may differentiate between active and latent TB irrespective of human immunodeficiency virus (HIV) infection and may also be used to monitor the effect of treatment. In paper III, we did not find any marker with potential to differentiate between active and latent TB infection when Mtb specific marker levels were analysed in QFT supernatants. However, Mtb specific interleukin (IL)-1ra, IL-2 and IP-10 levels distinguished individuals with borderline QFT test results from QFT negative controls and these markers may improve the differentiation between latent TB and non-TB infected individuals. In conclusion, the results support that Treg may be a target for adjunct host directed therapy in TB, and that Mtb specific Treg can be reduced by COX-inhibitors which are well known drugs approved for other clinical conditions. Potential biomarkers for TB diagnosis and treatment efficacy have been identified. However, further studies are needed to examine whether it is possible to establish sufficient sensitive and specific test cut-offs for use in clinical practice

Cytokine Patterns in Tuberculosis Infection; IL-1ra, IL-2 and IP-10 Differentiate Borderline QuantiFERON-TB Samples from Uninfected Controls.

Interferon gamma release assays (IGRAs) do not discriminate between active tuberculosis (TB) and latent TB infection (LTBI), which limit their use in TB endemic areas. Subjects with QuantiFERON-TB (QFT) results around the diagnostic cut-off more likely show inconsistent results on serial testing which makes the interpretation of the assay difficult. We have studied potential biomarkers in patients with various stages of TB infection and with borderline QFT tests compared to those with higher values.27 soluble biomarkers were analysed in QFT supernatants from patients with active TB (n = 18), individuals with LTBI (n = 48) and from QFT negative controls (n = 16) by the Multiplex bead assay. The LTBI group was classified into two groups according to QFT IFN-γ levels; QFT borderline (0.35-0.70 IU/mL, n = 11) or QFT high (>0.70 IU/mL, n = 36).The levels of IL-1ra, IL-2, IL-13, IL-15, IFN-γ, IP-10 and MCP-1 in background corrected TB antigen stimulated supernatants (TBAg-Nil) significantly distinguished both active TB and LTBI QFT high groups from the QFT negative controls (p≤0.004). In addition, IL-1ra, IL-2 and IP-10 significantly differentiated the QFT borderline group from the controls (p≤0.001). Still, in the QFT borderline group the IL-1ra and IP-10 levels were not significant different from neither the QFT high nor the active TB group, whereas the IL-2 levels were lower (p≤0.003). The level of IP-10 showed the best separation between the QFT borderline group and the QFT negative controls (AUC 0.92) and offered 100% sensitivity for active TB.IL-1ra, IL-2 and IP-10 differentiate QFT borderline samples from uninfected controls and the majority of QFT borderline subjects were classified as LTBI by these markers. Still, inconsistency was seen, and further studies are needed to examine the performance of alternative markers before concluded if they could be used as diagnostics tools

ROC curve analyses for differentiation between the QFT borderline group and QFT negative controls.

<p>ROC curve analyses for differentiation between the QFT borderline group and QFT negative controls.</p

Clinical characteristics of the study participants.

<p>Clinical characteristics of the study participants.</p

Markers differentiating TB infection from QFT negative controls.

<p>TB antigen stimulated background corrected (TBAg-Nil) levels of IL-1ra, IL-2, IP-10, IFN-γ, IL-13, IL-15, IL-17a and MCP-1 in patients with active TB (ATB), latent TB infected subjects with QFT result >0.70 IU/mL (LTBI QFT>0.7), subjects with QFT result in the borderline zone 0.35–0.70 IU/mL (LTBI QFT<0.7) and in QFT negative controls (QFT negative). The horizontal lines show the median values. Mann-Whitney U test was used for comparison between groups. The TBAg-Nil level of IP-10 and MCP-1 were excluded from statistical analyses when the LTBI and ATB groups were compared because high proportions of study subjects in both groups had levels above the UDL of the assay. Brackets represents statistically significant differences (p<0.005).</p

Number of subjects classified as active TB and LTBI by IL-1ra, IL-2 and IP-10.

<p>Number of subjects classified as active TB and LTBI by IL-1ra, IL-2 and IP-10.</p

Markers differentiating between active and latent TB infection.

<p>Nil levels (pg/mL) of IL-1β, IL-1ra, IL-9 and IL-17a in patients with active TB (ATB), latent TB infection (LTBI) and in QFT negative controls (QFT negative). The horizontal lines show the median values. Mann-Whitney U test was used for comparison between groups. Brackets represents statistically significant differences (p≤0.004).</p

The COX- inhibitor indomethacin reduces Th1 effector and T regulatory cells in vitro in Mycobacterium tuberculosis infection

Abstract Background Tuberculosis (TB) causes a major burden on global health with long and cumbersome TB treatment regimens. Host-directed immune modulating therapies have been suggested as adjunctive treatment to TB antibiotics. Upregulated cyclooxygenase-2 (COX-2)-prostaglandin E2 (PGE2) signaling pathway may cause a dysfunctional immune response that favors survival and replication of Mycobacterium tuberculosis (Mtb). Methods Blood samples were obtained from patients with latent TB (n = 9) and active TB (n = 33) before initiation of anti-TB chemotherapy. COX-2 expression in monocytes and ESAT-6 and Ag85 specific T cell cytokine responses (TNF-α, IFN-γ, IL-2), proliferation (carboxyfluorescein succinimidyl ester staining) and regulation (FOXP3+ T regulatory cells) were analysed by flow cytometry and the in vitro effects of the COX-1/2 inhibitor indomethacin were measured. Results We demonstrate that indomethacin significantly down-regulates the fraction of Mtb specific FOXP3+ T regulatory cells (ESAT-6; p = 0.004 and Ag85; p < 0.001) with a concomitant reduction of Mtb specific cytokine responses and T cell proliferation in active TB. Although active TB tend to have higher levels, there are no significant differences in COX-2 expression between unstimulated monocytes from patients with active TB compared to latent infection. Monocytes in both TB groups respond with a significant upregulation of COX-2 after in vitro stimulation. Conclusions Taken together, our in vitro data indicate a modulation of the Th1 effector and T regulatory cells in Mtb infection in response to the COX-1/2 inhibitor indomethacin. The potential role as adjunctive host-directed therapy in TB disease should be further evaluated in both animal studies and in human clinical trials

Mycobacterial antigens in pleural fluid mononuclear cells to diagnose pleural tuberculosis in HIV co-infected patients

Background Extra pulmonary manifestation of tuberculosis (TB) accounts for approximately one-half of TB cases in HIV-infected individuals with pleural TB as the second most common location. Even though mycobacteria are cleared, mycobacterial antigens may persist in infected tissues, causing sustained inflammation and chronicity of the disease. The aim of this study was to explore various mycobacterial antigens in pleural effusions, the impact of HIV infection and CD4+ T-cell depletion on the presence of antigens, and the diagnostic potential of antigens for improved and rapid diagnosis of pleural TB. Methods Pleural fluid specimens were collected from patients presenting with clinically suspected pleural TB, and processed routinely for culture, cytology, and adenosine deaminase activity analysis. HIV status and CD4+ T-cell counts were recorded. Pleural fluid mononuclear cells (PFMC) were isolated, and cell smears were stained with acid-fast staining and immunocytochemistry for various mycobacterial antigens. Real-time and nested-PCR were performed. Patients were categorized as pleural TB or non-TB cases using a composite reference standard. Performance of the mycobacterial antigens as diagnostic test was assessed. Results A total of 41 patients were enrolled, of which 32 were classified as pleural TB and 9 as non-TB. Thirteen patients had culture confirmed pleural TB, 26 (81%) were HIV-TB co-infected, and 64% had < 100 CD4+ T-cells/microL. Both secreted and cell-wall mycobacterial antigens were detected in PFMC. Lipoarabinomannan (LAM) was the most frequently detected antigen. There was no direct correlation between positive culture and antigens. Cases with low CD4+ T-cell counts had higher bacterial and antigen burden. By combining detection of secreted antigen or LAM, the sensitivity and specificity to diagnose pleural TB was 56 and 78%, respectively, as compared to 41 and 100% for culture, 53 and 89% for nested PCR, and 6 and 100% for real-time PCR. Conclusion Mycobacterial antigens were detectable in PFMC from tuberculous pleural effusions, even in cases where viable mycobacteria or bacterial DNA were not always detected. Thus, a combination of secreted antigen and LAM detection by immunocytochemistry may be a complement to acid-fast staining and contribute to rapid and accurate diagnosis of pleural TB

IP-10 differentiates between active and latent tuberculosis irrespective of HIV status and declines during therapy

Objectives: Biomarkers for diagnosis and therapy efficacy in tuberculosis (TB) are requested. We have studied biomarkers that may differentiate between active and latent TB infection (LTBI), the influence of HIV infection and changes during anti-TB chemotherapy. Methods: Thirty-eight plasma cytokines, assessed by multiplex and enzyme immunoassays, were analyzed in patients with active TB before and during 24 weeks of anti-TB chemotherapy (n = 65), from individuals with LTBI (n = 34) and from QuantiFERON-TB (QFT) negative controls (n = 65). The study participants were grouped according to HIV status. Results: Plasma levels of the CXC chemokine IP-10 and soluble TNF receptor type 2 (sTNFr2) significantly differentiated active TB from the LTBI group, irrespective of HIV status. In the HIV-infected group the sensitivity and specificity was 100% for IP-10 with a cut-off of 2547 pg/mL. Plasma IP-10 declined gradually during anti-TB chemotherapy (12–24 weeks, p = 0.002) to a level comparable to LTBI and QFT negative control groups. sTNFr2 fluctuated throughout therapy, but was decreased after 12–24 weeks (p = 0.006). Conclusions: IP-10 distinguished with high accuracy active TB from LTBI irrespective of HIV infection and declined during anti-TB chemotherapy. Plasma IP-10 may serve as a diagnostic biomarker to differentiate between the stages of TB infection and for monitoring therapy efficacy