RESUMEN
Burns are a leading cause of morbidity and mortality worldwide with the most common cause of death resulting from sepsis, often from Pseudomonas aeruginosa. We previously reported that a non-lethal flame burn induced an altered host immune response. Using this model, gene expression in both the murine host and P. aeruginosa was measured using a NanoString custom probe panel. We observed differing patterns of gene expression in both host and P. aeruginosa in the skin, blood, liver, and spleen of mice that were burned and/or infected, compared to mice that were neither burned nor infected (i.e., Sham). In mice that were both burned and infected (B/I), we observed changes in gene expression in both the host and P. aeruginosa that were distinct from all other treatment conditions. These data suggest that the combination of the burned state and superimposed infection affects both host and pathogen gene expression to increase infection propensity. Gene transcription significantly changed from 6 to 24 h post-B/I in each tissue. Finally, inhibiting IL-10 signaling or co-administering arginine at the time of P. aeruginosa infection prolonged or restored survival in an otherwise 100% fatal burn and infection model. These findings suggest that disease states such as burns may differentially alter innate immune response gene expression in both a host- and pathogen-specific manner.IMPORTANCEThe interaction between an underlying disease process and a specific pathogen may lead to the unique expression of genes that affect bacterial pathogenesis. These genes may not be observed during infection in the absence of, or with a different underlying process or infection during the underlying process with a different pathogen. To test this hypothesis, we used Nanostring technology to compare gene transcription in a murine-burned wound infected with P. aeruginosa. The Nanostring probeset allowed the simultaneous direct comparison of immune response gene expression in both multiple host tissues and P. aeruginosa in conditions of burn alone, infection alone, and burn with infection. While RNA-Seq is used to discover novel transcripts, NanoString could be a technique to monitor specific changes in transcriptomes between samples and bypass the additional adjustments for multispecies sample processing or the need for the additional steps of alignment and assembly required for RNASeq. Using Nanostring, we identified arginine and IL-10 as important contributors to the lethal outcome of burned mice infected with P. aeruginosa. While other examples of altered gene transcription are in the literature, our study suggests that a more systematic comparison of gene expression in various underlying diseases during infection with specific bacterial pathogens may lead to the identification of unique host-pathogen interactions and result in more precise therapeutic interventions.
RESUMEN
Background: Streptococcus pneumoniae (Spn) is typically an asymptomatic colonizer of the nasopharynx but it also causes pneumonia and disseminated disease affecting various host anatomical sites. Transition from colonization to invasive disease is not well understood. Studies have shown that such a transition can occur as result of influenza A virus coinfection. Methods: We investigated the pneumococcal (serotype 19F, strain EF3030) and host transcriptomes with and without influenza A virus (A/California/07 2009 pH1N1) infection at this transition. This was done using primary, differentiated Human Bronchial Epithelial Cells (nHBEC) in a transwell monolayer model at an Air-Liquid Interface (ALI), with multispecies deep RNA-seq. Results: Distinct pneumococcal gene expression profiles were observed in the presence and absence of influenza. Influenza coinfection allowed for significantly greater pneumococcal growth and triggered the differential expression of bacterial genes corresponding to multiple metabolic pathways; in totality suggesting a fundamentally altered bacterial metabolic state and greater nutrient availability when coinfecting with influenza. Surprisingly, nHBEC transcriptomes were only modestly perturbed by infection with EF3030 alone in comparison to that resulting from Influenza A infection or coinfection, which had drastic alterations in thousands of genes. Influenza infected host transcriptomes suggest significant loss of ciliary function in host nHBEC cells. Conclusions: Influenza A virus infection of nHBEC promotes pneumococcal infection. One reason for this is an altered metabolic state by the bacterium, presumably due to host components made available as result of viral infection. Influenza infection had a far greater impact on the host response than did bacterial infection alone, and this included down regulation of genes involved in expressing cilia. We conclude that influenza infection promotes a pneumococcal metabolic shift allowing for transition from colonization to disseminated disease.
RESUMEN
Lymph node (LN) fibroblastic reticular cells (FRCs) define LN niches and regulate lymphocyte homeostasis through producing diverse extracellular matrix (ECM) components. We examined the role of ECM laminin α4 (Lama4) using FRC-Lama4 conditional KO Pdgfrb-Cre-/- × Lama4fl/fl mice. Single-cell RNA-sequencing (scRNA-Seq) data showed the promoter gene Pdgfrb was exclusively expressed in FRCs. Depleting FRC-Lama4 reduced Tregs and dendritic cells, decreased high endothelial venules, impaired the conduit system, and downregulated T cell survival factors in LNs. FRC-Lama4 depletion impaired the homing of lymphocytes to LNs in homeostasis and after allografting. Alloantigen-specific T cells proliferated, were activated to greater degrees in LNs lacking FRC-Lama4, and were more prone to differentiate into effector phenotypes relative to the Treg phenotype. In murine cardiac transplantation, tolerogenic immunosuppression was not effective in FRC-Lama4 recipients, which produced more alloantibodies than WT. After lung transplantation, FRC-Lama4-KO mice had more severe graft rejection with fewer Tregs in their LNs. Overall, FRC-Lama4 critically contributes to a tolerogenic LN niche by supporting T cell migration, constraining T cell activation and proliferation, and promoting Treg differentiation. Hence, it serves as a therapeutic target for immunoengineering.
