Low density neutrophils (LDNs) are described in a number of inflammatory conditions, cancers and infections and associated with immunopathology, and a mechanistic role in disease. The role of LDNs at homeostasis in healthy individuals has not been investigated. We have developed an isolation protocol that generates high purity LDNs from healthy donors. Healthy LDNs were identical to healthy normal density neutrophils (NDNs), aside from reduced neutrophil extracellular trap formation. CD66b, CD16, CD15, CD10, CD54, CD62L, CXCR2, CD47 and CD11b were expressed at equivalent levels in healthy LDNs and NDNs and underwent apoptosis and ROS production interchangeably. Healthy LDNs had no differential effect on CD4+ or CD8+ T cell proliferation or IFNγ production compared with NDNs. LDNs were generated from healthy NDNs in vitro by activation with TNF, LPS or fMLF, suggesting a mechanism of LDN generation in disease however, we show neutrophilia in people with Cystic Fibrosis (CF) was not due to increased LDNs. LDNs are present in the neutrophil pool at homeostasis and have limited functional differences to NDNs. We conclude that increased LDN numbers in disease reflect the specific pathology or inflammatory environment and that neutrophil density alone is inadequate to classify discrete functional populations of neutrophils.

Low density neutrophils (LDNs) are described in a number of inflammatory conditions, cancers and infections and associated with immunopathology, and a mechanistic role in disease. The role of LDNs at homeostasis in healthy individuals has not been investigated. We have developed an isolation protocol that generates high purity LDNs from healthy donors. Healthy LDNs were identical to healthy NDNs, aside from reduced neutrophil extracellular trap formation. CD66b, CD16, CD15, CD10, CD54, CD62L, CXCR2, CD47 and CD11b were expressed at equivalent levels in LDNs and normal density neutrophils (NDNs) and underwent apoptosis and ROS production interchangeably. Healthy LDNs had no differential effect on CD4 + or CD8 + T cell proliferation or IFNγ production compared with NDNs.LDNs were generated from healthy NDNs in vitro by activation with TNF, LPS or fMLF, suggesting a mechanism of LDN generation in disease however, we show neutrophilia in people with Cystic Fibrosis (CF) was not due to increased LDNs. LDNs are present in the neutrophil pool at homeostasis and have limited functional differences to NDNs. We conclude that increased LDN numbers in disease reflect the specific pathology or inflammatory environment and that neutrophil density alone is inadequate to classify discrete functional populations of neutrophils.

Gut microbiota are fundamental for healthy animal function, but the evidence that host function can be predicted from microbiota taxonomy remains equivocal, and natural populations remain understudied compared to laboratory animals. Paired analyses of covariation in microbiota and host parameters are powerful approaches to characterise host-microbiome relationships mechanistically, especially in wild populations of animals that are also lab models, enabling insight into the ecological basis of host function at a molecular and cellular level. The fruitfly Drosophila melanogaster is a preeminent model organism, amenable to field investigation by 'omic analyses. Here we present an analysis of wild male D. melanogaster, with paired measurements of (A) bacterial diversity and abundance, measured by 16S amplicon sequencing; and (B) the host gut transcriptome. We found orthogonal axes of microbial genera, which correspond to differential expression of host genes. The differentially-expressed gene sets were enriched in functions including protein translation, mitochondrial respiration, immunity and reproduction. Each gene set had a distinct functional signature, suggesting that wild flies exhibit a range of distinct axes of functional variation, which correspond to orthogonal axes of microbiome variation. These findings strengthen the bridge between the wild ecology and functional genetics of a leading host-microbiome model.