Pneumolysin suppresses the initial macrophage pro-inflammatory response to Streptococcus pneumoniae.

Published data for the Streptococcus pneumoniae virulence factor Pneumolysin (Ply) show contradictory effects on the host inflammatory response to infection. Ply has been shown to activate the inflammasome, but also can bind to MRC-1 resulting in suppression of dendritic cell inflammatory responses. We have used an in vitro infection model of human monocyte-derived macrophages (MDM), and a mouse model of pneumonia to clarify whether pro- or anti-inflammatory effects dominate the effects of Ply on the initial macrophage inflammatory response to S. pneumoniae, and the consequences during early lung infection. We found that infection with S. pneumoniae expressing Ply suppressed tumour necrosis factor (TNF) and interleukin-6 production by MDMs compared to cells infected with ply-deficient S. pneumoniae. This effect was independent of bacterial effects on cell death. Transcriptional analysis demonstrated S. pneumoniae expressing Ply caused a qualitatively similar but quantitatively lower MDM transcriptional response to S. pneumoniae compared to ply-deficient S. pneumoniae, with reduced expression of TNF and type I IFN inducible genes. Reduction of the MDM inflammatory response was prevented by inhibition of SOCS1. In the early lung infection mouse model, the TNF response to ply-deficient S. pneumoniae was enhanced and bacterial clearance increased compared to infection with wild-type S. pneumoniae. Overall, these data show Ply inhibits the initial macrophage inflammatory response to S. pneumoniae, probably mediated through SOCS1, and this was associated with improved immune evasion during early lung infection.

Exaggerated IL-17A activity in human in vivo recall responses discriminates active tuberculosis from latent infection and cured disease.

Host immune responses at the site of Mycobacterium tuberculosis infection can mediate pathogenesis of tuberculosis (TB) and onward transmission of infection. We hypothesized that pathological immune responses would be enriched at the site of host-pathogen interactions modeled by a standardized tuberculin skin test (TST) challenge in patients with active TB compared to those without disease, and interrogated immune responses by genome-wide transcriptional profiling. We show exaggerated interleukin-17A (IL-17A) and T helper 17 (TH17) responses among 48 individuals with active TB compared to 191 with latent TB infection, associated with increased neutrophil recruitment and matrix metalloproteinase-1 expression, both involved in TB pathogenesis. Curative antimicrobial treatment reversed these observed changes. Increased IL-1ß and IL-6 responses to mycobacterial stimulation were evident both in circulating monocytes and in molecular changes at the site of TST in individuals with active TB, supporting a model in which monocyte-derived IL-1ß and IL-6 promote TH17 differentiation within tissues. Modulation of these cytokine pathways may provide a rational strategy for host-directed therapy in active TB.

Anaerobe-enriched gut microbiota predicts pro-inflammatory responses in pulmonary tuberculosis.

BACKGROUND: The relationship between tuberculosis (TB), one of the leading infectious causes of death worldwide, and the microbiome, which is critical for health, is poorly understood. METHODS: To identify potential microbiome-host interactions, profiling of the oral, sputum and stool microbiota [n = 58 cases, n = 47 culture-negative symptomatic controls (SCs)] and whole blood transcriptome were done in pre-treatment presumptive pulmonary TB patients. This was a cross-sectional study. Microbiota were also characterised in close contacts of cases (CCCs, n = 73) and close contacts of SCs (CCSCs, n = 82) without active TB. FINDINGS: Cases and SCs each had similar α- and ß-diversities in oral washes and sputum, however, ß-diversity differed in stool (PERMANOVA p = 0•035). Cases were enriched with anaerobes in oral washes, sputum (Paludibacter, Lautropia in both) and stool (Erysipelotrichaceae, Blautia, Anaerostipes) and their stools enriched in microbial genes annotated as amino acid and carbohydrate metabolic pathways. In pairwise comparisons with their CCCs, cases had Megasphaera-enriched oral and sputum microbiota and Bifidobacterium-, Roseburia-, and Dorea-depleted stools. Compared to their CCSCs, SCs had reduced α-diversities and many differential taxa per specimen type. Cases differed transcriptionally from SCs in peripheral blood (PERMANOVA p = 0•001). A co-occurrence network analysis showed stool taxa, Erysipelotrichaceae and Blautia, to negatively co-correlate with enriched "death receptor" and "EIF2 signalling" pathways whereas Anaerostipes positively correlated with enriched "interferon signalling", "Nur77 signalling" and "inflammasome" pathways; all of which are host pathways associated with disease severity. In contrast, none of the taxa enriched in SCs correlated with host pathways. INTERPRETATION: TB-specific microbial relationships were identified in oral washes, induced sputum, and stool from cases before the confounding effects of antibiotics. Specific anaerobes in cases' stool predict upregulation of pro-inflammatory immunological pathways, supporting the gut microbiota's role in TB. FUNDING: European & Developing Countries Clinical Trials Partnership, South African-Medical Research Council, National Institute of Allergy and Infectious Diseases.

Persistent T Cell Repertoire Perturbation and T Cell Activation in HIV After Long Term Treatment.

Objective: In people living with HIV (PLHIV), we sought to test the hypothesis that long term anti-retroviral therapy restores the normal T cell repertoire, and investigate the functional relationship of residual repertoire abnormalities to persistent immune system dysregulation. Methods: We conducted a case-control study in PLHIV and HIV-negative volunteers, of circulating T cell receptor repertoires and whole blood transcriptomes by RNA sequencing, complemented by metadata from routinely collected health care records. Results: T cell receptor sequencing revealed persistent abnormalities in the clonal T cell repertoire of PLHIV, characterized by reduced repertoire diversity and oligoclonal T cell expansion correlated with elevated CD8 T cell counts. We found no evidence that these expansions were driven by cytomegalovirus or another common antigen. Increased frequency of long CDR3 sequences and reduced frequency of public sequences among the expanded clones implicated abnormal thymic selection as a contributing factor. These abnormalities in the repertoire correlated with systems level evidence of persistent T cell activation in genome-wide blood transcriptomes. Conclusions: The diversity of T cell receptor repertoires in PLHIV on long term anti-retroviral therapy remains significantly depleted, and skewed by idiosyncratic clones, partly attributable to altered thymic output and associated with T cell mediated chronic immune activation. Further investigation of thymic function and the antigenic drivers of T cell clonal selection in PLHIV are critical to efforts to fully re-establish normal immune function.

Vitamin D3 replacement enhances antigen-specific immunity in older adults.

Introduction: Ageing is associated with increased number of infections, decreased vaccine efficacy and increased systemic inflammation termed inflammageing. These changes are reflected by reduced recall responses to varicella zoster virus (VZV) challenge in the skin of older adults. Vitamin D deficiency is more common in the old and has been associated with frailty and increased inflammation. In addition, vitamin D increases immunoregulatory mechanisms and therefore has the potential to inhibit inflammageing. Objectives: We investigated the use of vitamin D3 replacement to enhance cutaneous antigen-specific immunity in older adults (≥65 years). Methods: Vitamin D insufficient older adults (n = 18) were administered 6400IU of vitamin D3/day orally for 14 weeks. Antigen-specific immunity to VZV was assessed by clinical score assessment of the injection site and transcriptional analysis of skin biopsies collected from challenged injection sites pre- and post-vitamin D3 replacement. Results: We showed that older adults had reduced VZV-specific cutaneous immune response and increased non-specific inflammation as compared to young. Increased non-specific inflammation observed in the skin of older adults negatively correlated with vitamin D sufficiency. We showed that vitamin D3 supplementation significantly increased the response to cutaneous VZV antigen challenge in older adults. This enhancement was associated with a reduction in inflammatory monocyte infiltration with a concomitant enhancement of T cell recruitment to the site of antigen challenge in the skin. Conclusion: Vitamin D3 replacement can boost antigen-specific immunity in older adults with sub-optimal vitamin D status.

Blood transcriptional biomarkers for active pulmonary tuberculosis in a high-burden setting: a prospective, observational, diagnostic accuracy study.

BACKGROUND: Blood transcriptional signatures are candidates for non-sputum triage or confirmatory tests of tuberculosis. Prospective head-to-head comparisons of their diagnostic accuracy in real-world settings are necessary to assess their clinical use. We aimed to compare the diagnostic accuracy of candidate transcriptional signatures identified by systematic review, in a setting with a high burden of tuberculosis and HIV. METHODS: We did a prospective observational study nested within a diagnostic accuracy study of sputum Xpert MTB/RIF (Xpert) and Xpert MTB/RIF Ultra (Ultra) tests for pulmonary tuberculosis. We recruited consecutive symptomatic adults aged 18 years or older self-presenting to a tuberculosis clinic in Cape Town, South Africa. Participants provided blood for RNA sequencing, and sputum samples for liquid culture and molecular testing using Xpert and Ultra. We assessed the diagnostic accuracy of candidate blood transcriptional signatures for active tuberculosis (including those intended to distinguish active tuberculosis from other diseases) identified by systematic review, compared with culture or Xpert MTB/RIF positivity as the standard reference. In our primary analysis, patients with tuberculosis were defined as those with either a positive liquid culture or Xpert result. Patients with missing blood RNA or sputum results were excluded. Our primary objective was to benchmark the diagnostic accuracy of candidate transcriptional signatures against the WHO target product profile (TPP) for a tuberculosis triage test. FINDINGS: Between Feb 12, 2016, and July 18, 2017, we obtained paired sputum and RNA sequencing data from 181 participants, 54 (30%) of whom had confirmed pulmonary tuberculosis. Of 27 eligible signatures identified by systematic review, four achieved the highest diagnostic accuracy with similar area under the receiver operating characteristic curves (Sweeney3: 90·6% [95% CI 85·6-95·6]; Kaforou25: 86·9% [80·9-92·9]; Roe3: 86·9% [80·3-93·5]; and BATF2: 86·8% [80·6-93·1]), independent of age, sex, HIV status, previous tuberculosis, or sputum smear result. At test thresholds that gave 70% specificity (the minimum WHO TPP specificity for a triage test), these four signatures achieved sensitivities between 83·3% (95% CI 71·3-91·0) and 90·7% (80·1-96·0). No signature met the optimum criteria, of 95% sensitivity and 80% specificity proposed by WHO for a triage test, or the minimum criteria (of 65% sensitivity and 98% specificity) for a confirmatory test, but all four correctly identified Ultra-positive, culture-negative patients. INTERPRETATION: Selected blood transcriptional signatures met the minimum WHO benchmarks for a tuberculosis triage test but not for a confirmatory test. Further development of the signatures is warranted to investigate their possible effects on clinical and health economic outcomes as part of a triage strategy, or when used as add-on confirmatory test in conjunction with the highly sensitive Ultra test for Mycobacterium tuberculosis DNA. FUNDING: Royal Society Newton Advanced Fellowship, Wellcome Trust, National Institute of Health Research, and UK Medical Research Council.

Concise whole blood transcriptional signatures for incipient tuberculosis: a systematic review and patient-level pooled meta-analysis.

BACKGROUND: Multiple blood transcriptional signatures have been proposed for identification of active and incipient tuberculosis. We aimed to compare the performance of systematically identified candidate signatures for incipient tuberculosis and to benchmark these against WHO targets. METHODS: We did a systematic review and individual participant data meta-analysis. We searched Medline and Embase for candidate whole blood mRNA signatures discovered with the primary objective of diagnosis of active or incipient tuberculosis, compared with controls who were healthy or had latent tuberculosis infection. We tested the performance of eligible signatures in whole blood transcriptomic datasets, in which sampling before tuberculosis diagnosis was done and time to disease was available. Culture-confirmed and clinically or radiologically diagnosed pulmonary or extrapulmonary tuberculosis cases were included. Non-progressor (individuals who remained tuberculosis-free during follow-up) samples with less than 6 months of follow-up from the date of sample collection were excluded, as were participants with prevalent tuberculosis and those who received preventive therapy. Scores were calculated for candidate signatures for each participant in the pooled dataset. Receiver operating characteristic curves, sensitivities, and specificities were examined using prespecified intervals to tuberculosis (<3 months, <6 months, <1 year, and <2 years) from sample collection. This study is registered with PROSPERO, number CRD42019135618. RESULTS: We tested 17 candidate mRNA signatures in a pooled dataset from four eligible studies comprising 1126 samples. This dataset included 183 samples from 127 incipient tuberculosis cases in South Africa, Ethiopia, The Gambia, and the UK. Eight signatures (comprising 1-25 transcripts) that predominantly reflect interferon and tumour necrosis factor-inducible gene expression, had equivalent diagnostic accuracy for incipient tuberculosis over a 2-year period with areas under the receiver operating characteristic curves ranging from 0·70 (95% CI 0·64-0·76) to 0·77 (0·71-0·82). The sensitivity of all eight signatures declined with increasing disease-free time interval. Using a threshold derived from two SDs above the mean of uninfected controls to prioritise specificity and positive-predictive value, the eight signatures achieved sensitivities of 24·7-39·9% over 24 months and of 47·1-81·0% over 3 months, with corresponding specificities of more than 90%. Based on pre-test probability of 2%, the eight signatures achieved positive-predictive values ranging from 6·8-9·4% over 24 months and 11·2-14·4% over 3 months. When using biomarker thresholds maximising sensitivity and specificity with equal weighting to both, no signature met the minimum WHO target product profile parameters for incipient tuberculosis biomarkers over a 2-year period. INTERPRETATION: Blood transcriptional biomarkers reflect short-term risk of tuberculosis and only exceed WHO benchmarks if applied to 3-6-month intervals. Serial testing among carefully selected target groups might be required for optimal implementation of these biomarkers. FUNDING: Wellcome Trust and National Institute for Health Research.

Spatial Network Mapping of Pulmonary Multidrug-Resistant Tuberculosis Cavities Using RNA Sequencing.

Rationale: There is poor understanding about protective immunity and the pathogenesis of cavitation in patients with tuberculosis.Objectives: To map pathophysiological pathways at anatomically distinct positions within the human tuberculosis cavity.Methods: Biopsies were obtained from eight predetermined locations within lung cavities of patients with multidrug-resistant tuberculosis undergoing therapeutic surgical resection (n = 14) and healthy lung tissue from control subjects without tuberculosis (n = 10). RNA sequencing, immunohistochemistry, and bacterial load determination were performed at each cavity position. Differentially expressed genes were normalized to control subjects without tuberculosis, and ontologically mapped to identify a spatially compartmentalized pathophysiological map of the cavity. In silico perturbation using a novel distance-dependent dynamical sink model was used to investigate interactions between immune networks and bacterial burden, and to integrate these identified pathways.Measurements and Main Results: The median (range) lung cavity volume on positron emission tomography/computed tomography scans was 50 cm3 (15-389 cm3). RNA sequence reads (31% splice variants) mapped to 19,049 annotated human genes. Multiple proinflammatory pathways were upregulated in the cavity wall, whereas a downregulation "sink" in the central caseum-fluid interface characterized 53% of pathways including neuroendocrine signaling, calcium signaling, triggering receptor expressed on myeloid cells-1, reactive oxygen and nitrogen species production, retinoic acid-mediated apoptosis, and RIG-I-like receptor signaling. The mathematical model demonstrated that neuroendocrine, protein kinase C-θ, and triggering receptor expressed on myeloid cells-1 pathways, and macrophage and neutrophil numbers, had the highest correlation with bacterial burden (r > 0.6), whereas T-helper effector systems did not.Conclusions: These data provide novel insights into host immunity to Mycobacterium tuberculosis-related cavitation. The pathways defined may serve as useful targets for the design of host-directed therapies, and transmission prevention interventions.

Enhancement of cutaneous immunity during aging by blocking p38 mitogen-activated protein (MAP) kinase-induced inflammation.

BACKGROUND: Immunity decreases with age, which leads to reactivation of varicella zoster virus (VZV). In human subjects age-associated immune changes are usually measured in blood leukocytes; however, this might not reflect alterations in tissue-specific immunity. OBJECTIVES: We used a VZV antigen challenge system in the skin to investigate changes in tissue-specific mechanisms involved in the decreased response to this virus during aging. METHODS: We assessed cutaneous immunity based on the extent of erythema and induration after intradermal VZV antigen injection. We also performed immune histology and transcriptomic analyses on skin biopsy specimens taken from the challenge site in young (<40 years) and old (>65 years) subjects. RESULTS: Old human subjects exhibited decreased erythema and induration, CD4+ and CD8+ T-cell infiltration, and attenuated global gene activation at the site of cutaneous VZV antigen challenge compared with young subjects. This was associated with increased sterile inflammation in the skin in the same subjects related to p38 mitogen-activated protein kinase-related proinflammatory cytokine production (P < .0007). We inhibited systemic inflammation in old subjects by means of pretreatment with an oral small-molecule p38 mitogen-activated protein kinase inhibitor (Losmapimod; GlaxoSmithKline, Brentford, United Kingdom), which reduced both serum C-reactive protein levels and peripheral blood monocyte secretion of IL-6 and TNF-α. In contrast, cutaneous responses to VZV antigen challenge were increased significantly in the same subjects (P < .0003). CONCLUSION: Excessive inflammation in the skin early after antigen challenge retards antigen-specific immunity. However, this can be reversed by inhibition of inflammatory cytokine production that can be used to promote vaccine efficacy and the treatment of infections and malignancy during aging.

Quantitative Characterization of the T Cell Receptor Repertoire of Naïve and Memory Subsets Using an Integrated Experimental and Computational Pipeline Which Is Robust, Economical, and Versatile.

The T cell receptor (TCR) repertoire can provide a personalized biomarker for infectious and non-infectious diseases. We describe a protocol for amplifying, sequencing, and analyzing TCRs which is robust, sensitive, and versatile. The key experimental step is ligation of a single-stranded oligonucleotide to the 3' end of the TCR cDNA. This allows amplification of all possible rearrangements using a single set of primers per locus. It also introduces a unique molecular identifier to label each starting cDNA molecule. This molecular identifier is used to correct for sequence errors and for effects of differential PCR amplification efficiency, thus producing more accurate measures of the true TCR frequency within the sample. This integrated experimental and computational pipeline is applied to the analysis of human memory and naive subpopulations, and results in consistent measures of diversity and inequality. After error correction, the distribution of TCR sequence abundance in all subpopulations followed a power law over a wide range of values. The power law exponent differed between naïve and memory populations, but was consistent between individuals. The integrated experimental and analysis pipeline we describe is appropriate to studies of T cell responses in a broad range of physiological and pathological contexts.

Tumor Necrosis Factor (TNF) Bioactivity at the Site of an Acute Cell-Mediated Immune Response Is Preserved in Rheumatoid Arthritis Patients Responding to Anti-TNF Therapy.

The impact of anti-tumor necrosis factor (TNF) therapies on inducible TNF-dependent activity in humans has never been evaluated in vivo. We aimed to test the hypothesis that patients responding to anti-TNF treatments exhibit attenuated TNF-dependent immune responses at the site of an immune challenge. We developed and validated four context-specific TNF-inducible transcriptional signatures to quantify TNF bioactivity in transcriptomic data. In anti-TNF treated rheumatoid arthritis (RA) patients, we measured the expression of these biosignatures in blood, and in skin biopsies from the site of tuberculin skin tests (TSTs) as a human experimental model of multivariate cell-mediated immune responses. In blood, anti-TNF therapies attenuated TNF bioactivity following ex vivo stimulation. However, at the site of the TST, TNF-inducible gene expression and genome-wide transcriptional changes associated with cell-mediated immune responses were comparable to that of RA patients receiving methotrexate only. These data demonstrate that anti-TNF agents in RA patients do not inhibit inducible TNF activity at the site of an acute inflammatory challenge in vivo, as modeled by the TST. We hypothesize instead that their therapeutic effects are limited to regulating TNF activity in chronic inflammation or by alternative non-canonical pathways.

Validation of Immune Cell Modules in Multicellular Transcriptomic Data.

Numerous gene signatures, or modules have been described to evaluate the immune cell composition in transcriptomes of multicellular tissue samples. However, significant diversity in module gene content for specific cell types is associated with heterogeneity in their performance. In order to rank modules that best reflect their purported association, we have generated the modular discrimination index (MDI) score that assesses expression of each module in the target cell type relative to other cells. We demonstrate that MDI scores predict modules that best reflect independently validated differences in cellular composition, and correlate with the covariance between cell numbers and module expression in human blood and tissue samples. Our analyses demonstrate that MDI scores provide an ordinal summary statistic that reliably ranks the accuracy of gene expression modules for deconvolution of cell type abundance in transcriptional data.