Armin Deffur

HIV-tuberculosis-associated immune reconstitution inflammatory syndrome is characterized by Toll-like receptor and inflammasome signalling.

Patients with HIV-associated tuberculosis (TB) initiating antiretroviral therapy (ART) may develop immune reconstitution inflammatory syndrome (TB-IRIS). No biomarkers for TB-IRIS have been identified and the underlying mechanisms are unclear. Here we perform transcriptomic profiling of the blood samples of patients with HIV-associated TB. We identify differentially abundant transcripts as early as week 0.5 post ART initiation that predict downstream activation of proinflammatory cytokines in patients who progress to TB-IRIS. At the characteristic time of TB-IRIS onset (week 2), the signature is characterized by over-representation of innate immune mediators including TLR signalling and TREM-1 activation of the inflammasome. In keeping with the transcriptional data, concentrations of plasma cytokines and caspase-1/5 are elevated in TB-IRIS. Inhibition of MyD88 adaptor and group 1 caspases reduces secretion of cytokines including IL-1 in TB-IRIS patients. These data provide insight on the pathogenesis of TB-IRIS and may assist the development of specific therapies.

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.

Neutrophil-Associated Central Nervous System Inflammation in Tuberculous Meningitis Immune Reconstitution Inflammatory Syndrome

Tuberculous meningitis immune reconstitution inflammatory syndrome (TBM-IRIS) is characterized by severe, compartmentalized cerebral inflammation, involving mediators of innate and adaptive immune responses. A high baseline cerebrospinal fluid bacillary load predisposes to recurrent inflammation during antiretroviral therapy, manifesting as TBM-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.

Co-infection with Mycobacterium tuberculosis and human immunodeficiency virus: an overview and motivation for systems approaches

Tuberculosis is a devastating disease that accounts for a high proportion of infectious disease morbidity and mortality worldwide. HIV-1 co-infection exacerbates tuberculosis. Enhanced understanding of the host-pathogen relationship in HIV-1 and Mycobacterium tuberculosis co-infection is required. While reductionist approaches have yielded many valuable insights into disease pathogenesis, systems approaches are required that develop data-driven models able to predict emergent properties of this complex co-infection system in order to develop novel therapeutic approaches and to improve diagnostics. Here, we provide a pathogenesis-focused overview of HIV-TB co-infection followed by an introduction to systems approaches and concrete examples of how such approaches are useful.

Tricks to translating TB transcriptomics

Transcriptomics and other high-throughput methods are increasingly applied to questions relating to tuberculosis (TB) pathogenesis. Whole blood transcriptomics has repeatedly been applied to define correlates of TB risk and has produced new insight into the late stage of disease pathogenesis. In a novel approach, authors of a recently published study in Science Translational Medicine applied complex data analysis of existing TB transcriptomic datasets, and in vitro models, in an attempt to identify correlates of protection in TB, which are crucially required for the development of novel TB diagnostics and therapeutics to halt this global epidemic. Utilizing latent TB infection (LTBI) as a surrogate of protection, they identified IL-32 as a mediator of interferon gamma (IFNγ)-vitamin D dependent antimicrobial immunity and a marker of LTBI. Here, we provide a review of all TB whole-blood transcriptomic studies to date in the context of identifying correlates of protection, discuss potential pitfalls of combining complex analyses originating from such studies, the importance of detailed metadata to interpret differential patient classification algorithms, the effect of differing circulating cell populations between patient groups on the interpretation of resulting biomarkers and we decipher weighted gene co-expression network analysis (WGCNA), a recently developed systems biology tool which holds promise of identifying novel pathway interactions in disease pathogenesis. In conclusion, we propose the development of an integrated OMICS platform and open access to detailed metadata, in order for the TB research community to leverage the vast array of OMICS data being generated with the aim of unraveling the holy grail of TB research: correlates of protection.

Poor Penetration of Antibiotics Into Pericardium in Pericardial Tuberculosis

Pericardial tuberculosis (TB) is associated with high therapy failure and high mortality rates. Antibiotics have to penetrate to site of infection at sufficient non-protein bound concentrations, and then enter bacteria to inhibit intracellular biochemical processes. The antibiotic concentrations achieved in pericardial fluid in TB pericarditis have never been measured before. We recruited two cohorts of patients with TB pericarditis, and left a pigtail catheter in-situ for serial drug concentration measurements over 24 h. Altogether, 704 drug concentrations were comodeled for pharmacokinetic analyses. The drug concentrations achieved in pericardial fluid were compared to the minimum inhibitory concentrations (MICs) of clinical Mycobacterium tuberculosis isolates. The total rifampicin concentration pericardial-to-serum ratios in 16 paired samples were 0.19 ± 0.33. The protein concentrations of the pericardial fluid in TB pericarditis were observed to be as high as in plasma. The non-protein bound rifampicin concentrations in pericardial fluid were 4-fold lower than rifampicin MICs in the pilot study, and the peak concentration was 0.125 versus 0.208 mg/L in the second (p = 0.001). The rifampicin clearance from pericardial fluid was 9.45 L/h versus 7.82 L/h in plasma (p = 0.002). Ethambutol peak concentrations had a pericardial-to-plasma ratio of 0.55 ± 0.22; free ethambutol peak concentrations were 2.30-lower than MICs (p < 0·001). The pericardial fluid pH was 7.34. The median pyrazinamide peak concentrations were 42.93 mg/L versus a median MIC of 800 mg/L at pH 7.34 (p < 0.0001). There was no significant difference between isoniazid pericardial fluid and plasma concentrations, and isoniazid peak concentrations were above MIC. This is the first study to measure anti-TB drug concentrations, pH and protein in the pericardial TB fluid. Pericardial concentrations of the key sterilizing drugs for TB were below MIC, which could contribute to poor outcomes. A new regimen that overcomes these limitations might need to be crafted.

Six host-range restricted poxviruses from three genera induce distinct gene expression profiles in an in vivo mouse model.

BackgroundHost-range restricted poxviruses make promising vaccine vectors due to their safety profile and immunogenicity. An understanding of the host innate immune responses produced by different poxvirus vectors would aid in the assessment, selection and rational design of improved vaccines for human and veterinary applications. Novel avipoxviruses are being assessed to determine if they are different from other poxvirus vectors. Analysis of the transcriptome induced in a mouse model would aid in determining if there were significant differences between different poxvirus vectors which may reflect different adjuvant potential as well as establish if they should be further evaluated as vaccine vectors.ResultsWe compared host transcript abundance in the spleens of BALB/c mice twenty four hours after intravenous infection (105 pfu/mouse) with six host-restricted poxvirus species from three genera, namely Lumpy Skin Disease virus (LSDV), Canarypox virus (CNPV), Fowlpox virus (FWPV), modified vaccinia Ankara (MVA) and two novel South African avipoxviruses, Feral Pigeonpox virus (FeP2) and Penguinpox virus (PEPV). These six viruses produced qualitatively and quantitatively distinct host responses with LSDV, followed by MVA, inducing the greatest interferon (IFN) response. FeP2 and PEPV caused very little change to host transcript abundance compared to the other 4 viruses tested. CNPV and FWPV induced the up regulation of two immunoglobulin genes (Ighg and Ighg3 (IgG3)) with CNPV inducing a third, Ighm (IgM). HIV-1–specific IgG3 antibodies have been correlated with decreased risk of HIV-1 infection in the RV144 trial, which included a CNPV-based vector (Yates et al. (Sci Transl Med, 6(228) p228, 2014). Up regulation of IgG3 by CNPV and FWPV but not the other poxviruses tested in vivo, implies that these two avipoxvirus-vector backbones may be involved in stimulation of the clinically important IgG3 antibody subclass. Differential transcript abundance associated with the different poxviruses is further discussed with particular emphasis on responses related to immune responses.ConclusionSix, genetically diverse host-restricted poxviruses produce different responses in a mouse model early after infection. These differences may affect the immune response induced to vaccine antigen in vectors based on these viruses. The two novel avipoxviruses were clearly distinguishable from the other viruses.

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.