Exposure to Mycobacterium remodels alveolar macrophages and the early innate response to Mycobacterium tuberculosis infection.

Alveolar macrophages (AMs) play a critical role during Mycobacterium tuberculosis (Mtb) infection as the first cells in the lung to encounter bacteria. We previously showed that AMs initially respond to Mtb in vivo by mounting a cell-protective, rather than pro-inflammatory response. However, the plasticity of the initial AM response was unknown. Here, we characterize how previous exposure to Mycobacterium, either through subcutaneous vaccination with Mycobacterium bovis (scBCG) or through a contained Mtb infection (coMtb) that mimics aspects of concomitant immunity, impacts the initial response by AMs. We find that both scBCG and coMtb accelerate early innate cell activation and recruitment and generate a stronger pro-inflammatory response to Mtb in vivo by AMs. Within the lung environment, AMs from scBCG vaccinated mice mount a robust interferon-associated response, while AMs from coMtb mice produce a broader inflammatory response that is not dominated by Interferon Stimulated Genes. Using scRNAseq, we identify changes to the frequency and phenotype of airway-resident macrophages following Mycobacterium exposure, with enrichment for both interferon-associated and pro-inflammatory populations of AMs. In contrast, minimal changes were found for airway-resident T cells and dendritic cells after exposures. Ex vivo stimulation of AMs with Pam3Cys, LPS and Mtb reveal that scBCG and coMtb exposures generate stronger interferon-associated responses to LPS and Mtb that are cell-intrinsic changes. However, AM profiles that were unique to each exposure modality following Mtb infection in vivo are dependent on the lung environment and do not emerge following ex vivo stimulation. Overall, our studies reveal significant and durable remodeling of AMs following exposure to Mycobacterium, with evidence for both AM-intrinsic changes and contributions from the altered lung microenvironments. Comparisons between the scBCG and coMtb models highlight the plasticity of AMs in the airway and opportunities to target their function through vaccination or host-directed therapies.

Type I interferon decreases macrophage energy metabolism during mycobacterial infection.

Metabolic reprogramming powers and polarizes macrophage functions, but the nature and regulation of this response during infection with pathogens remain controversial. In this study, we characterize the metabolic and transcriptional responses of murine macrophages to Mycobacterium tuberculosis (Mtb) in order to disentangle the underlying mechanisms. We find that type I interferon (IFN) signaling correlates with the decreased glycolysis and mitochondrial damage that is induced by live, but not killed, Mtb. Macrophages lacking the type I IFN receptor (IFNAR) maintain glycolytic flux and mitochondrial function during Mtb infection in vitro and in vivo. IFNß itself restrains the glycolytic shift of inflammatory macrophages and initiates mitochondrial stress. We confirm that type I IFN acts upstream of mitochondrial damage using macrophages lacking the protein STING. We suggest that a type I IFN-mitochondrial feedback loop controls macrophage responses to mycobacteria and that this could contribute to pathogenesis across a range of diseases.

Designing monitoring protocols to measure population trends of threatened insects: A case study of the cryptic, flightless grasshopper Brachaspis robustus.

Statistically robust monitoring of threatened populations is essential for effective conservation management because the population trend data that monitoring generates is often used to make decisions about when and how to take action. Despite representing the highest proportion of threatened animals globally, the development of best practice methods for monitoring populations of threatened insects is relatively uncommon. Traditionally, population trend data for the Nationally Endangered New Zealand grasshopper Brachaspis robustus has been determined by counting all adults and nymphs seen on a single ~1.5 km transect searched once annually. This method lacks spatial and temporal replication, both of which are essential to overcome detection errors in highly cryptic species like B. robustus. It also provides no information about changes in the grasshopper's distribution throughout its range. Here, we design and test new population density and site occupancy monitoring protocols by comparing a) comprehensive plot and transect searches at one site and b) transect searches at two sites representing two different habitats (gravel road and natural riverbed) occupied by the species across its remaining range. Using power analyses, we determined a) the number of transects, b) the number of repeated visits and c) the grasshopper demographic to count to accurately detect long term change in relative population density. To inform a monitoring protocol design to track trends in grasshopper distribution, we estimated the probability of detecting an individual with respect to a) search area, b) weather and c) the grasshopper demographic counted at each of the two sites. Density estimates from plots and transects did not differ significantly. Population density monitoring was found to be most informative when large adult females present in early summer were used to index population size. To detect a significant change in relative density with power > 0.8 at the gravel road habitat, at least seventeen spatial replicates (transects) and four temporal replicates (visits) were required. Density estimates at the natural braided river site performed poorly and likely require a much higher survey effort. Detection of grasshopper presence was highest (pg > 0.6) using a 100 m x 1 m transect at both sites in February under optimal (no cloud) conditions. At least three visits to a transect should be conducted per season for distribution monitoring. Monitoring protocols that inform the management of threatened species are crucial for better understanding and mitigation of the current global trends of insect decline. This study provides an exemplar of how appropriate monitoring protocols can be developed for threatened insect species.