γδ T Cells Mediate Protection Against Neutrophil-associated Lung Inflammation in Pulmonary Melioidosis.

Pulmonary melioidosis is a severe tropical infection caused by Burkholderia pseudomallei and is associated with high mortality despite early antibiotic treatment. γδ T cells have been increasingly implicated as drivers of the host neutrophil response during bacterial pneumonia, but their role in pulmonary melioidosis is unknown. Here, we report that in patients with melioidosis, a lower peripheral blood γδ T cell concentration is associated with higher mortality even when adjusting for severity of illness. γδ T cells were also enriched in the lung and protected against mortality in a mouse model of pulmonary melioidosis. γδ T cell deficiency in infected mice induced an early recruitment of neutrophils to the lung, independent of bacterial burden. Subsequently, γδ T cell deficiency resulted in increased neutrophil-associated inflammation in the lung as well as impaired bacterial clearance. Additionally, γδ T cells influenced neutrophil function and subset diversity in the lung after infection. Our results indicate that γδ T cells serve a novel protective role in the lung during a severe bacterial pneumonia by regulating excessive neutrophil-associated inflammation.

Flagellin-modulated inflammasome pathways characterize the human alveolar macrophage response to Burkholderia pseudomallei, a lung-tropic pathogen.

Melioidosis is an emerging tropical infection caused by inhalation, inoculation, or ingestion of the flagellated, facultatively intracellular pathogen Burkholderia pseudomallei. The melioidosis case fatality rate is often high, and pneumonia, the most common presentation, doubles the risk of death. The alveolar macrophage is a sentinel pulmonary host defense cell, but the human alveolar macrophage in B. pseudomallei infection has never been studied. The objective of this study was to investigate the host-pathogen interaction of B. pseudomallei infection with the human alveolar macrophage and to determine the role of flagellin in modulating inflammasome-mediated pathways. We found that B. pseudomallei infects primary human alveolar macrophages but is gradually restricted in the setting of concurrent cell death. Electron microscopy revealed cytosolic bacteria undergoing division, indicating that B. pseudomallei likely escapes the alveolar macrophage phagosome and may replicate in the cytosol, where it triggers immune responses. In paired human blood monocytes, uptake and intracellular restriction of B. pseudomallei are similar to those observed in alveolar macrophages, but cell death is reduced. The alveolar macrophage cytokine response to B. pseudomallei is characterized by marked interleukin (IL)-18 secretion compared to monocytes. Both cytotoxicity and IL-18 secretion in alveolar macrophages are partially flagellin dependent. However, the proportion of IL-18 release that is driven by flagellin is greater in alveolar macrophages than in monocytes. These findings suggest differential flagellin-mediated inflammasome pathway activation in the human alveolar macrophage response to B. pseudomallei infection and expand our understanding of intracellular pathogen recognition by this unique innate immune lung cell.

Discovery of a glutathione utilization pathway in Francisella that shows functional divergence between environmental and pathogenic species.

Glutathione (GSH) is an abundant metabolite within eukaryotic cells that can act as a signal, a nutrient source, or serve in a redox capacity for intracellular bacterial pathogens. For Francisella, GSH is thought to be a critical in vivo source of cysteine; however, the cellular pathways permitting GSH utilization by Francisella differ between strains and have remained poorly understood. Using genetic screening, we discovered a unique pathway for GSH utilization in Francisella. Whereas prior work suggested GSH catabolism initiates in the periplasm, the pathway we define consists of a major facilitator superfamily (MFS) member that transports intact GSH and a previously unrecognized bacterial cytoplasmic enzyme that catalyzes the first step of GSH degradation. Interestingly, we find that the transporter gene for this pathway is pseudogenized in pathogenic Francisella, explaining phenotypic discrepancies in GSH utilization among Francisella spp. and revealing a critical role for GSH in the environmental niche of these bacteria.

IL-1R2-based biomarker models predict melioidosis mortality independent of clinical data.

Introduction: Melioidosis is an often-fatal tropical infectious disease caused by the Gram-negative bacillus Burkholderia pseudomallei, but few studies have identified promising biomarker candidates to predict outcome. Methods: In 78 prospectively enrolled patients hospitalized with melioidosis, six candidate protein biomarkers, identified from the literature, were measured in plasma at enrollment. A multi-biomarker model was developed using least absolute shrinkage and selection operator (LASSO) regression, and mortality discrimination was compared to a clinical variable model by receiver operating characteristic curve analysis. Mortality prediction was confirmed in an external validation set of 191 prospectively enrolled patients hospitalized with melioidosis. Results: LASSO regression selected IL-1R2 and soluble triggering receptor on myeloid cells 1 (sTREM-1) for inclusion in the candidate biomarker model. The areas under the receiver operating characteristic curve (AUC) for mortality discrimination for the IL-1R2 + sTREM-1 model (AUC 0.81, 95% CI 0.72-0.91) as well as for an IL-1R2-only model (AUC 0.78, 95% CI 0.68-0.88) were higher than for a model based on a modified Sequential Organ Failure Assessment (SOFA) score (AUC 0.69, 95% CI 0.56-0.81, p < 0.01, p = 0.03, respectively). In the external validation set, the IL-1R2 + sTREM-1 model (AUC 0.86, 95% CI 0.81-0.92) had superior 28-day mortality discrimination compared to a modified SOFA model (AUC 0.80, 95% CI 0.74-0.86, p < 0.01) and was similar to a model containing IL-1R2 alone (AUC 0.82, 95% CI 0.76-0.88, p = 0.33). Conclusion: Biomarker models containing IL-1R2 had improved 28-day mortality prediction compared to clinical variable models in melioidosis and may be targets for future, rapid test development.

A macrophage-targeted platform for extending drug dosing with polymer prodrugs for pulmonary infection prophylaxis.

Pulmonary melioidosis is a bacterial disease with high morbidity and a mortality rate that can be as high as 40% in resource-poor regions of South Asia. This disease burden is linked to the pathogen's intrinsic antibiotic resistance and protected intracellular localization in alveolar macrophages. Current treatment regimens require several antibiotics with multi-month oral and intravenous administrations that are difficult to implement in under-resourced settings. Herein, we report that a macrophage-targeted polyciprofloxacin prodrug acts as a surprisingly effective pre-exposure prophylactic in highly lethal murine models of aerosolized human pulmonary melioidosis. A single dose of the polymeric prodrug maintained high lung drug levels and targeted an intracellular depot of ciprofloxacin to the alveolar macrophage compartment that was sustained over a period of 7 days above minimal inhibitory concentrations. This intracellular pharmacokinetic profile provided complete pre-exposure protection in a BSL-3 model with an aerosolized clinical isolate of Burkholderia pseudomallei from Thailand. This total protection was achieved despite the bacteria's relative resistance to ciprofloxacin and where an equivalent dose of pulmonary-administered ciprofloxacin was ineffective. For the first time, we demonstrate that targeting the intracellular macrophage compartment with extended antibiotic dosing can achieve pre-exposure prophylaxis in a model of pulmonary melioidosis. This fully synthetic and modular therapeutic platform could be an important therapeutic approach with new or re-purposed antibiotics for melioidosis prevention and treatment, especially as portable inhalation devices in high-risk, resource-poor settings.

Genome-Wide Approach to the CD4 T-Cell Response to Human Herpesvirus 6B.

Human herpesvirus 6 (HHV-6) and cytomegalovirus (CMV) are population-prevalent betaherpesviruses with intermittent lytic replication that can be pathogenic in immunocompromised hosts. Elucidation of the adaptive immune response is valuable for understanding pathogenesis and designing novel treatments. Knowledge of T-cell antigens has reached the genome-wide level for CMV and other human herpesviruses, but study of HHV-6 is at an earlier stage. Using rare-cell enrichment combined with an HLA-agnostic, proteome-wide approach, we queried HHV-6B-specific CD4 T cells from 18 healthy donors with each known HHV-6B protein. We detected a low abundance of HHV-6-specific CD4 T cells in blood; however, the within-person CD4 T-cell response is quite broad: the median number of open reading frame (ORF) products recognized was nine per person. Overall, the data expand the number of documented HHV-6B CD4 T-cell antigens from approximately 11 to 60. Epitopes in the proteins encoded by U14, U90, and U95 were mapped with synthetic peptides, and HLA restriction was defined for some responses. Intriguingly, CD4 T-cell antigens newly described in this report are among the most population prevalent, including U73, U72, U95, and U30. Our results indicate that selection of HHV-6B ORFs for immunotherapy should consider this expanded panel of HHV-6B antigens.IMPORTANCE Human herpesvirus 6 is highly prevalent and maintains chronic infection in immunocompetent individuals, with the potential to replicate widely in settings of immunosuppression, leading to clinical disease. Antiviral compounds may be ineffective and/or pose dose-limiting toxicity, and therefore, immune-based therapies have garnered increased interest in recent years. Attempts at addressing this unmet medical need begin with understanding the cellular response to HHV-6 at the individual and population levels. The present study provides a comprehensive assessment of HHV-6-specific T-cell responses that may inform the development of cell-based therapies directed at this virus.