Gillian S. Tomlinson

Cerebrospinal fluid cytokine profiles predict risk of early mortality and immune reconstitution inflammatory syndrome in HIV-associated cryptococcal meningitis.

Understanding the host immune response during cryptococcal meningitis (CM) is of critical importance for the development of immunomodulatory therapies. We profiled the cerebrospinal fluid (CSF) immune-response in ninety patients with HIV-associated CM, and examined associations between immune phenotype and clinical outcome. CSF cytokine, chemokine, and macrophage activation marker concentrations were assayed at disease presentation, and associations between these parameters and microbiological and clinical outcomes were examined using principal component analysis (PCA). PCA demonstrated a co-correlated CSF cytokine and chemokine response consisting primarily of Th1, Th2, and Th17-type cytokines. The presence of this CSF cytokine response was associated with evidence of increased macrophage activation, more rapid clearance of Cryptococci from CSF, and survival at 2 weeks. The key components of this protective immune-response were interleukin (IL)-6 and interferon-γ, IL-4, IL-10 and IL-17 levels also made a modest positive contribution to the PC1 score. A second component of co-correlated chemokines was identified by PCA, consisting primarily of monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α). High CSF chemokine concentrations were associated with low peripheral CD4 cell counts and CSF lymphocyte counts and were predictive of immune reconstitution inflammatory syndrome (IRIS). In conclusion CSF cytokine and chemokine profiles predict risk of early mortality and IRIS in HIV-associated CM. We speculate that the presence of even minimal Cryptococcus-specific Th1-type CD4+ T-cell responses lead to increased recruitment of circulating lymphocytes and monocytes into the central nervous system (CNS), more effective activation of CNS macrophages and microglial cells, and faster organism clearance; while high CNS chemokine levels may predispose to over recruitment or inappropriate recruitment of immune cells to the CNS and IRIS following peripheral immune reconstitution with ART. These results provide a rational basis for future studies of immune modulation in CM, and demonstrate the potential of baseline immune profiling to identify CM patients most at risk of mortality and subsequent IRIS.

TLR-mediated inflammatory responses to Streptococcus pneumoniae are highly dependent on surface expression of bacterial lipoproteins.

Streptococcus pneumoniae infections induce inflammatory responses that contribute toward both disease pathogenesis and immunity, but the host–pathogen interactions that mediate these effects are poorly defined. We used the surface lipoprotein-deficient ∆lgt pneumococcal mutant strain to test the hypothesis that lipoproteins are key determinants of TLR-mediated immune responses to S. pneumoniae. We show using reporter assays that TLR2 signaling is dependent on pneumococcal lipoproteins, and that macrophage NF-κB activation and TNF-α release were reduced in response to the ∆lgt strain. Differences in TNF-α responses between Δlgt and wild-type bacteria were abrogated for macrophages from TLR2- but not TLR4-deficient mice. Transcriptional profiling of human macrophages revealed attenuated TLR2-associated responses to ∆lgt S. pneumoniae, comprising many NF-κB–regulated proinflammatory cytokine and chemokine genes. Importantly, non-TLR2–associated responses were preserved. Experiments using leukocytes from IL-1R–associated kinase-4–deficient patients and a mouse pneumonia model confirmed that proinflammatory responses were lipoprotein dependent. Our data suggest that leukocyte responses to bacterial lipoproteins are required for TLR2- and IL-1R–associated kinase-4–mediated inflammatory responses to S. pneumoniae.

Adherent Human Alveolar Macrophages Exhibit a Transient Pro-Inflammatory Profile That Confounds Responses to Innate Immune Stimulation

Alveolar macrophages (AM) are thought to have a key role in the immunopathogenesis of respiratory diseases. We sought to test the hypothesis that human AM exhibit an anti-inflammatory bias by making genome-wide comparisons with monocyte derived macrophages (MDM). Adherent AM obtained by bronchoalveolar lavage of patients under investigation for haemoptysis, but found to have no respiratory pathology, were compared to MDM from healthy volunteers by whole genome transcriptional profiling before and after innate immune stimulation. We found that freshly isolated AM exhibited a marked pro-inflammatory transcriptional signature. High levels of basal pro-inflammatory gene expression gave the impression of attenuated responses to lipopolysaccharide (LPS) and the RNA analogue, poly IC, but in rested cells pro-inflammatory gene expression declined and transcriptional responsiveness to these stimuli was restored. In comparison to MDM, both freshly isolated and rested AM showed upregulation of MHC class II molecules. In most experimental paradigms ex vivo adherent AM are used immediately after isolation. Therefore, the confounding effects of their pro-inflammatory profile at baseline need careful consideration. Moreover, despite the prevailing view that AM have an anti-inflammatory bias, our data clearly show that they can adopt a striking pro-inflammatory phenotype, and may have greater capacity for presentation of exogenous antigens than MDM.

HIV-1 Infection of Macrophages Dysregulates Innate Immune Responses to Mycobacterium tuberculosis by Inhibition of Interleukin-10

Human immunodeficiency virus (HIV)-1 and Mycobacterium tuberculosis (M. tuberculosis) both target macrophages, which are key cells in inflammatory responses and their resolution. Therefore, we tested the hypothesis that HIV-1 may modulate macrophage responses to coinfection with M. tuberculosis. HIV-1 caused exaggerated proinflammatory responses to M. tuberculosis that supported enhanced virus replication, and were associated with deficient stimulus-specific induction of anti-inflammatory interleukin (IL)-10 and attenuation of mitogen-activated kinase signaling downstream of Toll-like receptor 2 and dectin-1 stimulation. Our in vitro data were mirrored by lower IL-10 and higher proinflammatory IL-1β in airway samples from HIV-1-infected patients with pulmonary tuberculosis compared with those with non-tuberculous respiratory tract infections. Single-round infection of macrophages with HIV-1 was sufficient to attenuate IL-10 responses, and antiretroviral treatment of replicative virus did not affect this phenotype. We propose that deficient homeostatic IL-10 responses may contribute to the immunopathogenesis of active tuberculosis and propagation of virus infection in HIV-1/M. tuberculosis coinfection.

Transcriptional profiling of innate and adaptive human immune responses to mycobacteria in the tuberculin skin test

The tuberculin skin test (TST) is a model of integrated innate and adaptive human immune responses to Mycobacterium tuberculosis, but the component processes that are involved in this model have not previously been defined in vivo. We used transcriptional profiling to study these responses within the TST at molecular and system levels. Skin biopsies from TST injection sites were examined in subjects classified as TST+ or TST− by clinical and histological criteria. Genome‐wide expression arrays showed evolution of immune responses reflecting T‐cell activation and recruitment with uniquely Th1‐polarized responses and cytotoxic T cells (CTLs). In addition, distinct innate immune and IFN‐γ‐stimulated gene expression signatures were identified, under the regulation of NF‐κB and STAT1 transcriptional control. These were highly enriched for chemokines and MHC class II molecules providing a potential mechanism for paracrine amplification of inflammatory responses in the TST, by supporting cellular recruitment and enhancing antigen presentation. The same repertoire of innate and adaptive immune responses was evident in TST+ and TST− subjects alike, clinically positive TSTs being distinguished only by quantitatively greater differences. These data provide new insights into complex multifaceted responses within the TST, with much greater sensitivity than previous clinical or histological assessments.

In Vivo Molecular Dissection of the Effects of HIV-1 in Active Tuberculosis

Increased risk of tuberculosis (TB) associated with HIV-1 infection is primarily attributed to deficient T helper (Th)1 immune responses, but most people with active TB have robust Th1 responses, indicating that these are not sufficient to protect against disease. Recent findings suggest that favourable outcomes following Mycobacterium tuberculosis infection arise from finely balanced inflammatory and regulatory pathways, achieving pathogen control without immunopathology. We hypothesised that HIV-1 and antiretroviral therapy (ART) exert widespread changes to cell mediated immunity, which may compromise the optimal host protective response to TB and provide novel insights into the correlates of immune protection and pathogenesis. We sought to define these effects in patients with active TB by transcriptional profiling of tuberculin skin tests (TST) to make comprehensive molecular level assessments of in vivo human immune responses at the site of a standardised mycobacterial challenge. We showed that the TST transcriptome accurately reflects the molecular pathology at the site of human pulmonary TB, and used this approach to investigate immune dysregulation in HIV-1/TB co-infected patients with distinct clinical phenotypes associated with TST reactivity or anergy and unmasking TB immune reconstitution inflammatory syndrome (IRIS) after initiation of ART. HIV-1 infected patients with positive TSTs exhibited preserved Th1 responses but deficient immunoregulatory IL10-inducible responses. Those with clinically negative TSTs revealed profound anergy of innate as well as adaptive immune responses, except for preservation of type 1 interferon activity, implicated in impaired anti-mycobacterial immunity. Patients with unmasking TB IRIS showed recovery of Th1 immunity to normal levels, but exaggerated Th2-associated responses specifically. These mechanisms of immune dysregulation were localised to the tissue microenvironment and not evident in peripheral blood. TST molecular profiling categorised different mechanisms of immunological dysfunction in HIV-1 infection beyond the effects on CD4 T cells, each associated with increased risk of TB disease and amenable to host-directed therapies.

Transcriptional Profiling of Endobronchial Ultrasound-Guided Lymph Node Samples Aids Diagnosis of Mediastinal Lymphadenopathy

Background Endobronchial ultrasound (EBUS)-guided biopsy is the mainstay for investigation of mediastinal lymphadenopathy for laboratory diagnosis of malignancy, sarcoidosis, or TB. However, improved methods for discriminating between TB and sarcoidosis and excluding malignancy are still needed. We sought to evaluate the role of genomewide transcriptional profiling to aid diagnostic processes in this setting. Methods Mediastinal lymph node samples from 88 individuals were obtained by EBUS-guided aspiration for investigation of mediastinal lymphadenopathy and subjected to transcriptional profiling in addition to conventional laboratory assessments. Computational strategies were used to evaluate the potential for using the transcriptome to distinguish between diagnostic categories. Results Molecular signatures associated with granulomas or neoplastic and metastatic processes were clearly discernible in granulomatous and malignant lymph node samples, respectively. Support vector machine (SVM) learning using differentially expressed genes showed excellent sensitivity and specificity profiles in receiver operating characteristic curve analysis with area under curve values > 0.9 for discriminating between granulomatous and nongranulomatous disease, TB and sarcoidosis, and between cancer and reactive lymphadenopathy. A two-step decision tree using SVM to distinguish granulomatous and nongranulomatous disease, then between TB and sarcoidosis in granulomatous cases, and between cancer and reactive lymphadenopathy in nongranulomatous cases, achieved > 90% specificity for each diagnosis and afforded greater sensitivity than existing tests to detect TB and cancer. In some diagnostically ambiguous cases, computational classification predicted granulomatous disease or cancer before pathologic abnormalities were evident. Conclusions Machine learning analysis of transcriptional profiling in mediastinal lymphadenopathy may significantly improve the clinical utility of EBUS-guided biopsies.

S78 Determinants of macrophage responses to Streptococcus pneumoniae

Introduction and Objectives Streptococcus pneumoniae is the commonest cause of pneumonia and associated with marked inflammatory responses that underpin its immunopathogenesis. Surprisingly little is known about the molecular determinants of host–pathogen interactions that mediate these responses. We have studied the role of the pneumococcal capsule and surface lipoproteins in innate immune responses by macrophages that comprise the first line of cellular immune defence within the lung. Methods Human macrophage responses to wild type S pneumoniae (TIGR4) and isogenic mutant strains deficient in capsule (P1672) or surface lipoproteins (Δlgt) were investigated at the level of intracellular signalling, genome-wide transcriptional profiling and at protein level by cytokine ELISA. Results Unencapsulated bacteria invoked greater activation of the classical NFkB pathway, suggesting that the capsule may serve to inhibit some innate immune host pathogen interactions. In contrast, the Δlgt strain showed attenuated activation of NFkB, suggesting that lipoproteins are important ligands for innate immune recognition of pneumococci. Transcriptional responses to both unencapsulated and Δlgt strains showed marked differences to wild type pneumococci. However, quantitatively, major gene expression changes were preserved in the mutant strains. Despite the divergent effects on NFkB activation, both unencapsulated and Δlgt strains showed attenuated responses amongst these genes, although some key responses such as upregulation of TNFα were equivalent in all strains. Transcription factor enrichment analysis was conducted for the list of genes up-regulated by each strain to obtain new insight into the different pathways by which pneumococci may activate inflammatory responses. As expected, genes up-regulated by the wild type strain were enriched only for the NFkB family. In keeping with the signalling data, the Δlgt strain was not enriched for NFkB but only the PPARγ-RXR transactivator, and the unencapsulated strain was highly enriched for NFkB and a raft of other transcription factors. Conclusion Pneumococcal capsule and lipoproteins are important determinants of inflammatory responses to pneumococci. Our data suggest that the capsule inhibits multiple innate immune signalling pathways and that lipoproteins are critically important for activation of the canonical NFkB pathway. TNFα responses are independent of capsule and lipoproteins.

World TB Day 2018: The Challenge of Drug Resistant Tuberculosis

On 24th March, the world commemorates the day in 1882 when Dr Robert Koch announced his discovery of Mycobacterium tuberculosis (MTB). Over 130 years later, tuberculosis (TB) continues to affect individuals, communities, and entire health systems and economies. Koch unsuccessfully tried to ‘cure’ TB, and despite major advances in other areas of medicine, control of TB remains elusive- in 2016 TB was the leading infectious cause of death. The STOP TB partnership and World Health Organization (WHO) have announced their theme for World TB Day 2018 “Wanted: Leaders for a TB-Free World. You can make history. End TB.” This theme recognizes that TB is much larger than any one person, institute or discipline of research, and provides an opportunity for us to reflect on the major challenges and consider how we, as a scientific community, can work together and take the lead to address the global crisis of drug-resistant TB (DR-TB).

S36 DIFFERENTIATION OF TUBERCULOSIS AND SARCOIDOSIS BY TRANSCRIPTIONAL PROFILING OF IMMUNE RESPONSES IN MEDIASTINAL LYMPH NODE SAMPLES

Introduction and Objectives Differentiating tuberculosis and sarcoidosis can be difficult, particularly in the context of mediastinal lymphadenopathy, because both diseases are characterised by overlapping clinical phenotypes and histologically similar granulomatous inflammation. Currently, diagnosis relies heavily on microbiological confirmation of tuberculosis which is only available in <50% of cases. Therefore, novel diagnostic strategies are needed to prevent morbidity associated with delayed or inappropriate treatment. We tested the hypothesis that genome wide transcriptional profiling of mediastinal lymph node samples obtained by minimally invasive endobronchial ultrasound guidance could identify gene signatures that differentiate tuberculosis and sarcoidosis. Methods In vivo immune responses were compared in mediastinal lymph node biopsies obtained via endobronchial ultrasound guidance from patients with tuberculosis, sarcoidosis or non-granulomatous disease using genome-wide transcriptional profiling. Machine learning algorithms were used to test the discriminatory power of identified gene signatures which distinguished granulomatous from non-granulomatous disease or tuberculosis from sarcoidosis. Results Comparison of lymph node genome‑wide transcriptional profiles by principal component analysis revealed clear differences between granulomatous and non-granulomatous disease. Granulomatous profiles showed significant enrichment for genes involved in antigen presentation, inflammatory responses, innate immune responses and T cell activation, in keeping with the processes involved in granuloma generation. As expected, sarcoidosis and tuberculosis sample profiles were very similar, however, significant gene expression differences were still evident between these two groups. In particular, several genes related to development of granuloma architecture were more highly expressed in sarcoidosis samples. Next we used machine learning tools in order to test the discriminatory power of differentially expressed gene signatures and found that the support vector machines algorithm correctly classified up to 97% of granulomatous and non-granulomatous disease cases. Importantly, this technique successfully distinguished sarcoidosis from tuberculosis in up to 100% cases. Conclusions Transcriptomic analysis of lymph node samples from the site of disease identifies gene signatures that can reliably distinguish tuberculosis from sarcoidosis using computational classification tools. Our data highlight the superior discriminatory power of multiple gene expression differences over a single marker in complex disease and generate a pathway for biomarker discovery in the management of tuberculosis and sarcoidosis.