Gene Expression Profile Changes in Neutrophils - From Sterile Compartments into Sites of Inflammation

Neutrophils, key cells of the innate immune system, are responsible for preventing bacterial infections. They are rapidly recruited to sites of infection where they eliminate bacteria through killing methods that require reactive oxygen dependent processes. It has recently been established that neutrophils are capable of rapid and complex changes in gene expression during inflammatory responses. The concept that neutrophils only directly kill bacteria has been replaced by the concept that activated neutrophils can influence the immune response through the secretion of a variety of cytokines and by acting as antigen-presenting cell (APC) expressing MHC Class II, allowing for activation of T cells. Recent advances in neutrophil biology demonstrated that neutrophils also have an active regulatory role in angiogenesis and tumoral fate. It has been noted that a number of diseases including arthritis, periodontitis and acute respiratory distress syndrome (ARDS) are associated with neutrophil hyperactivity that results in significant tissue damage. Our group has previously shown that for some periodontal diseases, neutrophil hyperactivity is a key determinant of disease progression and severity. However, it remains unclear what factors are responsible for a patient developing a hyperactive neutrophil mediated disease. I hypothesize that local gene expression changes in neutrophils are responsible for the hyperactive behaviour of these cells during an inflammatory response. In order to assess this, I characterized the neutrophil gene expression profile in various compartments (bone marrow, blood and peritoneum in mice and blood and oral cavity in humans) and then characterized this genetic and phenotypic profile during an inflammatory response. I hypothesize that the neutrophil has a characteristic set of genes that are normally activated when it enters a site of inflammation from the circulation and that neutrophils can be polarized into a different functional subset under certain conditions that result in inflammation mediated diseases. To identify changes in neutrophil gene expression in the circulation and inflamed tissue I used recent advances in neutrophil isolation, RNA amplification, and microarray technologies to characterize the specific transcriptome associated with neutrophil site-specific responses.Ph

Identification of CD Marker Expression and Neutrophil Surface Marker Changes in Health and Disease using High-throughput Screening Flow Cytometry

Neutrophil hyperactivation can contribute to tissue damage in inflammatory diseases. Although many cell-surface proteins are known to be expressed on neutrophils there is no comprehensive study of the surface-markers that can be used to phenotype neutrophils. Neutrophils subpopulations isolated (blood and oral rinses) from healthy and chronic-periodontitis patients were screened against a panel of 374 known Cluster of Differentiation (CD) antibodies to identify cell-surface markers specific to neutrophils. This screen identified CD11b+, CD16b+, and CD66c+ as markers that are always expressed on neutrophils. Cell-sorting with an antibody against CD11b/CD16b/CD66c allowed for the enrichment of mature neutrophils, yielding populations of up to 99%, confirming the validity of these markers when isolating neutrophils. These findings provide a simple method for isolating neutrophils from humans, and thereby establish a validated method that allows for the accurate identification of neutrophils. This knowledge will be crucial for identifying neutrophil subtypes associated with neutrophil-mediated inflammatory diseases.M.Sc

Oral Neutrophil Transcriptome Changes Result in a Pro-Survival Phenotype in Periodontal Diseases

<div><p>Background</p><p>Periodontal diseases are inflammatory processes that occur following the influx of neutrophils into the periodontal tissues in response to the subgingival bacterial biofilm. Current literature suggests that while neutrophils are protective and prevent bacterial infections, they also appear to contribute to damage of the periodontal tissues. In the present study we compare the gene expression profile changes in neutrophils as they migrate from the circulation into the oral tissues in patients with chronic periodontits and matched healthy subjects. We hypothesized that oral neutrophils in periodontal disease patients will display a disease specific transcriptome that differs from the oral neutrophil of healthy subjects.</p><p>Methods</p><p>Venous blood and oral rinse samples were obtained from healthy subjects and chronic periodontitis patients for neutrophil isolation. mRNA was isolated from the neutrophils, and gene expression microarray analysis was completed. Results were confirmed for specific genes of interest by qRT-PCR and Western Blot analysis.</p><p>Results and Discussion</p><p>Chronic periodontitis patients presented with increased recruitment of neutrophils to the oral cavity. Gene expression analysis revealed differences in the expression levels of genes from several biological pathways. Using hierarchical clustering analysis, we found that the apoptosis network was significantly altered in patients with chronic inflammation in the oral cavity, with up-regulation of pro-survival members of the Bcl-2 family and down-regulation of pro-apoptosis members in the same compartment. Additional functional analysis confirmed that the percentages of viable neutrophils are significantly increased in the oral cavity of chronic periodontitis patients.</p><p>Conclusions</p><p>Oral neutrophils from patients with periodontal disease displayed an altered transcriptome following migration into the oral tissues. This resulted in a pro-survival neutrophil phenotype in chronic periodontitis patients when compared with healthy subjects, resulting in a longer-lived neutrophil. This is likely to impact the severity and length of the inflammatory response in this oral disease.</p></div

Demographics and clinical parameters of study subjects.

<p>ns - not significant;</p>*<p>p-value ≤0.05 vs. Healthy subjects.</p

Graphic representation of Death Receptor Pathway and Apoptosis Signaling Pathway in neutrophils demonstrates that they are altered in neutrophils in chronic inflammation.

<p>IPA canonical pathway analysis of Death Receptor Signaling Pathway in neutrophils from (A) healthy individuals and (B) chronic periodontitis patients. Red represents up-regulated genes, green are down-regulated genes and white symbols depict neighbouring genes in this analysis.</p

DAVID pathway analysis – Common pathways of Chronic Periodontitis and Healthy Subjects.

<p>Additional analysis can be found in File S3.</p

Oral chronic inflammation increases neutrophil recruitment to the site of infection.

<p>The proportion of neutrophils recruited to the oral cavity was counted in healthy patients, patients with chronic periodontits. * p ≤ 0.02; vs. Healthy. All data are mean ± SEM. Healthy subjects (n = 14); Generalized Periodontitis (n = 62).</p

DAVID pathway analysis –Pathways up-regulated in Chronic Periodontitis patients only.

<p>Additional analysis can be found in File S3.</p

The oral neutrophil in chronic periodontitis has high transcriptional activity compared to the oral neutrophil in a healthy patient.

<p>(A) Graphic representation of the number of genes that are either up regulated (red) or down regulated (green), when blood neutrophils (PMN-B) were compared with oral neutrophils (PMN-O) in healthy individuals and chronic periodontitis patients FC ≥2; p-value ≤0.05. (B) Heatmap of genes with FC ≥5. Genes shown in red are up-regulated and those shown in green are down-regulated in Oral samples of Chronic Periodontitis patients (n = 4). The complete list of genes can be found at the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068983#pone.0068983.s002" target="_blank">file S1</a>.</p