Kerryn Matthews

Hypercytokinaemia accompanies HIV-tuberculosis immune reconstitution inflammatory syndrome

Increased access to combination antiretroviral therapy in areas co-endemic for tuberculosis (TB) and HIV-1 infection is associated with an increased incidence of immune reconstitution inflammatory syndrome (TB-IRIS) whose cause is poorly understood. A case-control analysis of pro- and anti-inflammatory cytokines in TB-IRIS patients sampled at clinical presentation, and similar control patients with HIV-TB prescribed combined antiretroviral therapy who did not develop TB-IRIS. Peripheral blood mononuclear cells were cultured in the presence or absence of heat-killed Mycobacterium tuberculosis for 6 and 24 h. Stimulation with M. tuberculosis increased the abundance of many cytokine transcripts with interleukin (IL)-1&bgr;, IL-5, IL-6, IL-10, IL-13, IL-17A, interferon (IFN)-&ggr;, granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor (TNF) being greater in stimulated TB-IRIS cultures. Analysis of the corresponding proteins in culture supernatants, revealed increased IL-1&bgr;, IL-2, IL-6, IL-8, IL-10, IL-12p40, IFN-&ggr;, GM-CSF and TNF in TB-IRIS cultures. In serum, higher concentrations of TNF, IL-6, and IFN-&ggr; were observed in TB-IRIS patients. Serum IL-6 and TNF decreased during prednisone therapy in TB-IRIS patients. These data suggest that cytokine release contributes to pathology in TB-IRIS. IL-6 and TNF were consistently elevated and decreased in serum during corticosteroid therapy. Specific blockade of these cytokines may be rational approach to immunomodulation in TB-IRIS.

Corticosteroid-modulated immune activation in the tuberculosis immune reconstitution inflammatory syndrome.

RATIONALE HIV-tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an immunopathological reaction to mycobacterial antigens induced by antiretroviral therapy. Prednisone reduces morbidity in TB-IRIS, but the mechanisms are unclear. OBJECTIVES To determine the effect of prednisone on the inflammatory response in TB-IRIS (antigen-specific effector T cells, cytokines, and chemokines). METHODS Blood was taken from participants in a randomized placebo-controlled trial of prednisone for TB-IRIS, at 0, 2, and 4 weeks. Participants received prednisone at a dosage of 1.5 mg/kg/day for 2 weeks followed by 0.75 mg/kg/day for 2 weeks, or placebo at identical dosages. MEASUREMENTS AND MAIN RESULTS Analyses included IFN-γ enzyme-linked immunospot (ELISPOT), reverse transcription-polymerase chain reaction on peripheral blood mononuclear cells after restimulation with heat-killed Mycobacterium tuberculosis, Luminex multiplex cytokine analysis of corresponding tissue culture supernatants, and Luminex multiplex cytokine analysis of serum. Fifty-eight participants with TB-IRIS (31 receiving prednisone, 27 receiving placebo) were included. In serum, significant decreases in IL-6, IL-10, IL-12 p40, tumor necrosis factor-α, IFN-γ, and IFN-γ-induced protein-10 concentrations during prednisone, but not placebo, treatment were observed. No differences in ELISPOT responses comparing prednisone and placebo groups were shown in response to ESAT-6 (early secreted antigen target-6), Acr1, Acr2, 38-kD antigen, or heat-killed H37Rv M. tuberculosis. Purified protein derivative ELISPOT responses increased over 4 weeks in the prednisone group and decreased in the placebo group (P = 0.007). CONCLUSIONS The beneficial effects of prednisone in TB-IRIS appear to be mediated via suppression of predominantly proinflammatory cytokine responses of innate immune origin, not via a reduction of the numbers of antigen-specific T cells in peripheral blood.

Predominance of interleukin-22 over interleukin-17 at the site of disease in human tuberculosis

Summary The inflammatory response to Mycobacterium tuberculosis (M.tb) at the site of disease is Th1 driven. Whether the Th17 cytokines, IL-17 and IL-22, contribute to this response in humans is unknown. We hypothesized that IL-17 and IL-22 contribute to the inflammatory response in pleural and pericardial disease sites of human tuberculosis (TB). We studied pleural and pericardial effusions, established TB disease sites, from HIV-uninfected TB patients. Levels of soluble cytokines were measured by ELISA and MMP-9 by luminex. Bronchoalveolar lavage or pericardial mycobacteria-specific T cell cytokine expression was analyzed by intracellular cytokine staining. IL-17 was not abundant in pleural or pericardial fluid. IL-17 expression by mycobacteria-specific disease site T cells was not detected in healthy, M.tb-infected persons, or patients with TB pericarditis. These data do not support a major role for IL-17 at established TB disease sites in humans. IL-22 was readily detected in fluid from both disease sites. These IL-22 levels exceeded matching peripheral blood levels. Further, IL-22 levels in pericardial fluid correlated positively with MMP-9, an enzyme known to degrade the pulmonary extracellular matrix. We propose that our findings support a role for IL-22 in TB-induced pathology or the resulting repair process.

HIV-1 infection alters CD4+ memory T-cell phenotype at the site of disease in extrapulmonary tuberculosis

HIV‐1‐infected people have an increased risk of developing extrapulmonary tuberculosis (TB), the immunopathogenesis of which is poorly understood. Here, we conducted a detailed immunological analysis of human pericardial TB, to determine the effect of HIV‐1 co‐infection on the phenotype of Mycobacterium tuberculosis (MTB)‐specific memory T cells and the role of polyfunctional T cells at the disease site, using cells from pericardial fluid and blood of 74 patients with (n=50) and without (n=24) HIV‐1 co‐infection. The MTB antigen‐induced IFN‐γ response was elevated at the disease site, irrespective of HIV‐1 status or antigenic stimulant. However, the IFN‐γ ELISpot showed no clear evidence of increased numbers of antigen‐specific cells at the disease site except for ESAT‐6 in HIV‐1 uninfected individuals (p=0.009). Flow cytometric analysis showed that CD4+ memory T cells in the pericardial fluid of HIV‐1‐infected patients were of a less differentiated phenotype, with the presence of polyfunctional CD4+ T cells expressing TNF, IL‐2 and IFN‐γ. These results indicate that HIV‐1 infection results in altered phenotype and function of MTB‐specific CD4+ T cells at the disease site, which may contribute to the increased risk of developing TB at all stages of HIV‐1 infection.

Role of the Interleukin 10 Family of Cytokines in Patients With Immune Reconstitution Inflammatory Syndrome Associated With HIV Infection and Tuberculosis

Background.  The interleukin 10 (IL-10) family comprises cytokines structurally related to IL-10 that share signaling receptors that have conserved signaling cascades. The immunopathogenesis of immune reconstitution inflammatory syndrome (IRIS) in patients with human immunodeficiency virus (HIV) infection and tuberculosis remains incompletely understood. We hypothesized that a deficiency of IL-10 and its homologs may contribute to the immunopathology of IRIS in these patients. Methods. We performed a case-control analysis involving patients with HIV infection and tuberculosis who had IRIS at clinical presentation (tuberculosis-IRIS) and similar patients with HIV infection and tuberculosis who did not develop tuberculosis-IRIS (non-IRIS). Peripheral blood mononuclear cells (PBMCs) were cultured in the presence or absence of heat-killed Mycobacterium tuberculosis for 6 and 24 hours. Messenger RNA was analyzed by quantitative reverse transcription polymerase chain reaction analysis. Cytokine concentrations in serum were also determined. Results. Cultures of PBMCs stimulated with M. tuberculosis for 24 hours yielded higher IL-10 and interleukin 22 (IL-22) transcript levels for tuberculosis-IRIS patients, compared with non-IRIS patients. Analysis of corresponding serum samples showed significantly higher concentrations of IL-10 and IL-22 in tuberculosis-IRIS patients, compared with non-IRIS patients. Conclusions. IL-10 and IL-22 were differentially induced in PBMCs from tuberculosis-IRIS patients after in vitro stimulation, and higher concentrations of their corresponding proteins were detected in serum (in vivo). The higher levels of IL-10 observed in this study may represent a compensatory antiinflammatory response during tuberculosis-IRIS. The elevated levels of IL-22 suggest an association between this cytokine and immunopathology during tuberculosis-IRIS.

Prevalence, Hemodynamics, and Cytokine Profile of Effusive-Constrictive Pericarditis in Patients with Tuberculous Pericardial Effusion

Background Effusive constrictive pericarditis (ECP) is visceral constriction in conjunction with compressive pericardial effusion. The prevalence of proven tuberculous ECP is unknown. Whilst ECP is distinguished from effusive disease on hemodynamic grounds, it is unknown whether effusive-constrictive physiology has a distinct cytokine profile. We conducted a prospective study of prevalence and cytokine profile of effusive-constrictive disease in patients with tuberculous pericardial effusion. Methods From July 2006 through July 2009, the prevalence of ECP and serum and pericardial levels of inflammatory cytokines were determined in adults with tuberculous pericardial effusion. The diagnosis of ECP was made by combined pericardiocentesis and cardiac catheterization. Results Of 91 patients evaluated, 68 had tuberculous pericarditis. The 36/68 patients (52.9%; 95% confidence interval [CI]: 41.2-65.4) with ECP were younger (29 versus 37 years, P=0.02), had a higher pre-pericardiocentesis right atrial pressure (17.0 versus 10.0 mmHg, P<0.0001), serum concentration of interleukin-10 (IL-10) (38.5 versus 0.2 pg/ml, P<0.001) and transforming growth factor-beta (121.5 versus 29.1 pg/ml, P=0.02), pericardial concentration of IL-10 (84.7 versus 20.4 pg/ml, P=0.006) and interferon-gamma (2,568.0 versus 906.6 pg/ml, P=0.03) than effusive non-constrictive cases. In multivariable regression analysis, right atrial pressure > 15 mmHg (odds ratio [OR] = 48, 95%CI: 8.7-265; P<0.0001) and IL-10 > 200 pg/ml (OR=10, 95%CI: 1.1, 93; P=0.04) were independently associated with ECP. Conclusion Effusive-constrictive disease occurs in half of cases of tuberculous pericardial effusion, and is characterized by greater elevation in the pre-pericardiocentesis right atrial pressure and pericardial and serum IL-10 levels compared to patients with effusive non-constrictive tuberculous pericarditis.

Cytotoxic Mediators in Paradoxical HIV–Tuberculosis Immune Reconstitution Inflammatory Syndrome

Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) frequently complicates combined antiretroviral therapy and antituberculosis therapy in HIV-1–coinfected tuberculosis patients. The immunopathological mechanisms underlying TB-IRIS are incompletely defined, and improved understanding is required to derive new treatments and to reduce associated morbidity and mortality. We performed longitudinal and cross-sectional analyses of human PBMCs from paradoxical TB-IRIS patients and non-IRIS controls (HIV-TB–coinfected patients commencing antiretroviral therapy who did not develop TB-IRIS). Freshly isolated PBMC stimulated with heat-killed Mycobacterium tuberculosis H37Rv (hkH37Rv) were used for IFN-γ ELISPOT and RNA extraction. Stored RNA was used for microarray and RT-PCR, whereas corresponding stored culture supernatants were used for ELISA. Stored PBMC were used for perforin and granzyme B ELISPOT and flow cytometry. There were significantly increased IFN-γ responses to hkH37Rv in TB-IRIS, compared with non-IRIS PBMC (p = 0.035). Microarray analysis of hkH37Rv-stimulated PBMC indicated that perforin 1 was the most significantly upregulated gene, with granzyme B among the top five (log2 fold difference 3.587 and 2.828, respectively), in TB-IRIS. Downstream experiments using RT-PCR, ELISA, and ELISPOT confirmed the increased expression and secretion of perforin and granzyme B. Moreover, granzyme B secretion reduced in PBMC from TB-IRIS patients during corticosteroid treatment. Invariant NKT cell (CD3+Vα24+) proportions were higher in TB-IRIS patients (p = 0.004) and were a source of perforin. Our data implicate the granule exocytosis pathway in TB-IRIS pathophysiology. Further understanding of the immunopathogenesis of this condition will facilitate development of specific diagnostic and improved therapeutic options.

Altered Ratio of IFN-γ/IL-10 in Patients with Drug Resistant Mycobacterium tuberculosis and HIV- Tuberculosis Immune Reconstitution Inflammatory Syndrome

We have described a clinical relationship between HIV-Tuberculosis Immune Reconstitution Inflammatory Syndrome (TB-IRIS) and anti-tubercular drug resistance. Here we studied the immune response of TB-IRIS patients from whom a drug-resistant (n = 11) or drug-susceptible (n = 25) Mycobacterium tuberculosis (MTB) strain was isolated after presenting with TB-IRIS. ELISpot analysis and multiplex cytokine analysis of the supernatant collected from peripheral blood mononuclear cells stimulated overnight with the heat-killed H37Rv MTB laboratory strain was used. Although there was no statistical difference in IFN-gamma ELISpot responses between the two groups, the results point towards higher bacterial load in the drug-resistant patients, possibly due to failed therapy. The ratio between secreted IFN-gamma/IL-10 and IL-2/IL-10 was significantly lower in TB-IRIS patients in whom the cause of TB was a drug-resistant strain compared to those with a fully sensitive strain (p = 0.02). Since host immune responses are dependent on the bacterial load, we hypothesise that the impaired cytokine balance is likely to be caused by the poorly controlled bacterial growth in these patients.

Scientific letter: Ac-SDKP (N-acetyl-seryl-aspartyl-lysyl-proline) and Galectin-3 levels in tuberculous pericardial effusion: implications for pathogenesis and prevention of pericardial constriction

To the Editor: The incidence of constrictive pericarditis in HIV uninfected patients with pericardial tuberculosis is very high (31.65 cases per 1000 person-years) despite modern rifampicin-based antituberculosis treatment.1 The cellular mediators and molecular mechanisms of post-tuberculous pericardial fibrosis are unknown. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a ubiquitous tetrapeptide with important antifibrotic properties and galectin-3 is an activator of myofibroblasts, promoter of collagen and extracellular matrix deposition and is associated with organ fibrosis.2 ,3 Ac-SDKP, which is inactivated by ACE, exerts part of its antifibrotic effect by inhibiting galectin-3 (figure 1).4 Currently, it is not known whether endogenous Ac-SDKP and galectin-3 are present in normal pericardial effusion, and whether there are any changes in the context of pericarditis. Figure 1 Hypothesised mechanism by which the tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) inhibits fibrosis Ac-SDKP is generated from the G-actin binding peptide, thymosin β4, by …

A Compartmentalized Profibrotic Immune Response Characterizes Pericardial Tuberculosis, Irrespective of HIV-1 Infection

To the Editor: Tuberculosis (TB) remains a major threat to health in developing countries and in HIV-1–infected persons (1). In sub-Saharan Africa, the most common etiology of pericardial effusions in HIV-1–infected persons is TB (2). Mortality in patients with tuberculous pericarditis coinfected with HIV-1 can reach 40% in the absence of antiretroviral treatment, and neither antimicrobial therapy of TB alone nor the addition of corticosteroids to chemotherapy resulted in a clinically satisfactory mortality reduction, although corticosteroids significantly reduced hospitalization and incidence of constrictive pericarditis, regardless of HIV status (3). Improved understanding of immunological mechanisms at the disease site is required for the development of more effective host-directed therapies. Because HIV-1 coinfection alters the memory phenotype of CD4+ T cells in the pericardium (4), we hypothesized that HIV infection would also affect transcript abundance of key immune mediators in pericardial TB at the disease site. Based on known transcriptional perturbation in extrapulmonary TB (5–9), we selected 42 analytes and performed differential transcriptomic analysis by quantitative reverse transcription polymerase chain reaction in paired blood and pericardial fluid from 27 patients (15 definite and 12 probable patients with tuberculous pericarditis, and 17 patients coinfected with HIV-1). We report a detailed immunopathological characterization of pericardial TB, with 21 analytes also confirmed at the protein level. Methods, patient characteristics (see Tables E1 and E2 in the online supplement), and raw data for all genes in blood and pericardial fluid (Tables E3 and E4) are detailed in the online supplement. Preliminary data have been reported in the form of an abstract (10). A rigorous data analysis was conducted (Figure 1A). Briefly, pairwise fluid versus blood comparisons of individual genes yielded 21 differentially expressed genes in pericardial fluid compared with blood after application of Benjamini-Hochberg multiple testing correction (Tables E4). Next, cluster analysis was employed, with the resulting heat map (Figure E1) showing that strikingly, most blood and pericardial fluid samples from individual patients clustered together. This appeared to be driven by highly correlated gene expression patterns between the two compartments, as further shown in the correlation matrices of blood and pericardial fluid samples (Figure E2). Gene coexpression patterns in blood differed from pericardial fluid, in which pronounced coexpression of fibrosis-associated and neutrophil-associated genes were evident, as well as coexpression of some pro- and anti-inflammatory genes. Given the very low levels of transcript for some genes, a nonspecific filter was applied, which removed transcripts with a delta cycle threshold value higher than 38 in 5% or more of the samples. This step left 22 genes that separated blood and pericardial fluid, with five samples not assigned to either of the main blood or pericardial fluid clusters (Figure E3). This result indicates that overall gene expression in the two compartments is very different and confirmed our previous impression that genes with low levels of expression drive the patient-specific clustering. Figure 1. (A) Outline showing the flow of data in the analytic pipeline. (B) Clustered heat map of 17 selected genes, showing blood and pericardial fluid samples grouped into two separate compartments with prominence of fibrosis-associated genes in pericardial ... Next, computation of differential transcript abundance with multiple testing correction using a linear models approach identified 17 transcripts to be differentially abundant between the blood and pericardial fluid compartments (Table 1), which collectively clustered the samples into two separate compartments of blood and pericardial fluid (Figure 1B). A correlation matrix of the 17 selected genes pointed to the prominent coexpression of fibrosis-associated genes in pericardial fluid (Figure E4). Principal component analysis of the data using the three gene sets (“all genes,” showing no differential gene expression between the compartments; 22 “filtered genes,” which indicated a difference between the two compartments; and 17 “selected genes”) showed that the first principal component of the 17 differentially expressed genes explained 60% of the observed variance (Figure E5). Table 1. Differential Abundance of Gene Products by Disease Site, as Measured by Either Reverse Transcription Polymerase Chain Reaction (RNA) or ELISA/Luminex (Protein) To assess whether differential transcript abundance between blood and pericardial fluid was modulated by factors that plausibly reflect differential immune status or modulation thereof, we implemented an analysis in limma (Linear Models for Microarray Data), based on a factorial design, in which we compared the transcript abundance levels between compartments, taking into account the following interaction terms: HIV-1 coinfection status, CD4 count below 200 cells/μl, concurrent corticosteroid use, and pericardial fluid Mycobacterium tuberculosis culture result. None of these factors had a significant effect on differential transcript abundance (Figure 1B and Figure E6). The immunophenotype of the host response at the disease site was next assessed at the protein level (Table E4); results of fold changes between compartments are summarized in Table 1. In general, protein levels and patterns of over- or underabundance mirrored those of mRNA transcripts. Two exceptions were IFN-γ and IL-1β, both of which were significantly more abundant as proteins in pericardial fluid, but the corresponding mRNA transcript abundance was either no different or significantly lower than in blood (Table 1). We hypothesized that the difference could be a result of antigen-specific T cells that enter the pericardium, release IFN-γ, and die, potentially activating the inflammasome pathway, resulting in pyroptosis and release of IL-1β protein (11). Therefore, we assessed cell death in the pericardial compartment by comparing the cell death enrichment factors in pericardial fluid from cases of tuberculous pericarditis (n = 24) and those from asymptomatic controls undergoing cardiac surgery (n = 28) and found that cell death significantly increased in tuberculous pericarditis cases (P = 2.4 × 10−7) compared with controls (Figure E7), with no difference observed between HIV-infected and uninfected samples. In summary, we report a strong profibrotic response of gene expression in pericardial fluid, with a differentially stronger pro-inflammatory response also confirmed at the protein level. We show a transcriptomic gene expression signature of 17 genes that differentiate blood from pericardial fluid in patients with pericardial TB, and these transcripts were associated with fibrosis and regulators of fibrosis, as well as matrix metalloproteinases and tissue inhibitors of metalloproteinases. Also, unexpectedly and contrary to the effect on T-cell phenotype, HIV-1 infection does not affect the expression profile of key immune mediators at the pericardial TB disease site. Although we recognize the bias and the limitation introduced by the selection of 42 very specific genes, our data serve to justify a full transcriptional analysis at the microarray level. Further studies including more patients and an unbiased selection of genes will provide more detailed insight into molecular mechanisms and immunopathology during TB infection of the pericardium, and thereby lead to improved host-directed therapy. Future therapeutic approaches could target regulators of fibrosis and apoptosis, with the aim of preventing fibrosis, morbidity, and mortality.