Substance P augments chemokine production by Staphylococcus aureus infected murine osteoclasts

Staphylococcal osteomyelitis is a serious infection of the bone and joints characterized by progressive inflammatory tissue damage and leukocyte recruitment leading to net bone loss. Resident bone cells are capable of recognizing Staphylococcus aureus and initiating an inflammatory immune response that recruits leukocytes and alters bone homeostasis. Importantly, bone tissue is richly innervated with substance P containing nerve fibers and we have previously shown that this neuropeptide can augment the inflammatory responses of both osteoblasts and osteoclasts to S. aureus infection via neurokinin-1 receptors (NK-1R). Here, we have extended these studies by demonstrating that pharmacological inhibition of NK-1R ameliorates disease severity in a mouse model of staphylococcal osteomyelitis. This effect was associated with the abolition of infection-induced increases in neutrophil-attracting chemokine production, and reduced local levels of osteoclast and neutrophil activity. We then assessed the effect of S. aureus infection on bone-marrow derived osteoclast gene expression in the absence or presence of substance P. We determined that infection upregulates osteoclast expression of mRNAs encoding inflammatory mediators that include the neutrophil-attracting chemokines identified in vivo. Importantly, we found that, while substance P has no effect on chemokine mRNA expression in infected cells, this neuropeptide significantly increases the release of these chemokines by S. aureus challenged osteoclasts but not osteoblasts. Together, these data further support the ability of substance P to exacerbate inflammatory damage in staphylococcal osteomyelitis and indicate that this effect may be due, in part, to an augmentation of the leukocyte-recruiting immune functions of osteoclasts. Primary murine osteoclasts were infected with S. aureus at a bacteria-to-osteoclast ratio of 75:1. At 4 hours post-infection, RNA was isolated with TRIzol and purified using a GeneJET RNA Cleanup and Concentration Micro Kit according to the manufacturer’s guidelines (ThermoFisher Scientific). The Genomic Sequencing and Analysis Facility at the University of Texas at Austin prepared Tag-Seq libraries using an established method and they were sequenced using an NovaSeq S1 to generate 100bp single end reads for 3’ end sequencing (35). The adapters of the sequenced reads were adapter cut using cutadapt v. 2.6 (36) and the reads were quality checked using fastqc v0.11.9. The GRCm39 RefSeq transcriptome (GCF_000001635.27) was used as a reference to map the quality controlled reads using Bowtie2 v 2.4.1 (37). Differential gene expression analysis was performed with the R package DESeq2 version 1.40.2 (38). Significant changes in gene expression were characterized as a log2 fold change greater than two, and an adjusted p-value of less than 0.05 (Wald Statistic, Benjamini & Hochberg correction). Gene ontology terms for the transcriptome were annotated using Interproscan 5.60 - 92.0 (39). Heatmaps were generated using the ComplexHeatmap v2.16.0 package (40). ShinyGO v0.80 was used to determine pathway enrichment where the top 20 pathways with a False Discovery Rate (FDR) less than 0.05 were sorted by fold enrichment (41). The original datasets are available in the BioProject publicly accessible repository under the identifier PRJNA1168989.