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Researchers gain new insights into immune response during pediatric respiratory infections using the ImageXpress Pico system

“The ImageXpress Pico system enabled our team to accurately and efficiently assess the binding characteristics of mast cells to RSV-infected HLFs and acquire a better understanding of the inflammatory response to RSV.”

Seattle Children’s Hospital, Seattle Children’s Research Institute, University of Washington, Benaroya Research Institute

Lead Author: Dr. Stephen R. Reeves, Principal Investigator at Seattle Children’s Research Institute and Attending Physician in the Division of Pulmonary and Sleep Medicine department at Seattle Children’s Hospital

ImageXpress Pico Automated Cell Imaging System

The challenge

Understand the binding characteristics of mast cells and hyaluronan in the extracellular matrix of RSV-infected human lung fibroblasts

Respiratory syncytial virus (RSV) is one of the most common viruses infecting children worldwide. Mild cases of the virus are limited to upper respiratory tract (nose and throat) inflammation; more serious cases extend down to the lower respiratory tract (bronchial tubes and lungs) and are accompanied by coughing, low-grade fever, and a loss in appetite.

The team

More serious RSV infections can progress to pneumonia, respiratory failure, and/or death. RSV is highly contagious and predominantly affects children. However, adults and those with weakened immune systems are also susceptible. The symptoms and transmission of RSV is similar to other viruses that affect the upper and lower respiratory tract, including influenza A and B, rhinovirus, adenovirus, and the coronavirus strain, COVID-19.

Understanding more about the underlying mechanisms of these viruses could shed light on potential treatment options. Researchers at Seattle Children’s Research Institute, University of Washington and Benaroya Research Institute set out to study one of these potential mechanisms for RSV.

Dr. Stephen Reeves and his collaborators designed several experiments to determine the downstream inflammatory effects of interactions between mast cells and hyaluronan (HA) in RSV-infected human lung fibroblasts (HLFs). Both live and fixed-cell fluorescence imaging were needed to test a range of conditions, including the location and mechanism of action of HA within the extracellular matrix (ECM), mast cell protein expression, and molecular interactions between HA, mast cells, and the ECM. Traditional fluorescence microscopy required the use of glass cover slips for fixed cell imaging, which resulted in additional tissue handling and processing time. Dr. Reeves and his team were in need of an advanced system that could capture high quality images of both live cells and fixed tissue in a more efficient manner.

The solution

High-resolution live and fixed cell fluorescence imaging and analysis

The research team chose the ImageXpress® Pico Automated Cell Imaging System for its ease-of-use, flexibility, and robust analytic capabilities.

Assay protocols
To help the team get their experiments underway quickly, our Field Applications Scientists and Technical Support Specialists provided on-site training and support for the different assays. This included the development of assay protocols and on-the-fly analyses so that images could be simultaneously collected and analyzed.

Image acquisition
The research team performed live cell imaging and then subsequently fixed tissue to stain for the extracellular matrix components, which are not readily visualized using live cell techniques. The ImageXpress Pico system enabled the team to perform all of the imaging using multi-well plates thus significantly reducing the time associated with sample handling and processing. As part of the image acquisition, stitching of large areas of interest at higher magnification objectives was performed. This eliminated the need for additional replicates to be examined by traditional epiflourescence microscopy and yielded higher quality images.

Analysis
Dr. Reeves and his collaborators were able to perform a quantitative analysis of the interaction between mast cells (i.e., LUVA cells) and the ECM of human lung fibroblasts (HLFs) that were infected with respiratory syncytial virus (RSV). See images and analysis data below.

LUVA cells

The ImageXpress Pico system was also used to analyze the effects of RSV-infected HLFs on TSG-6 expression and mast cell (i.e., LUVA cell) adhesion over a 48-hour time period. See images and analysis data below. (Note: TSG-6 is a protein associated with inflammation and is shown to be upregulated in RSV-infected cells).

RSV-Infected Cells

The result

High quality data enables scientific breakthroughs sooner

Using the ImageXpress Pico system, Dr. Reeves’ team is the first to demonstrate that:

  • In pediatric donor-derived HLFs that are infected with RSV, there is greater HA synthesis, which results in a HA-enriched extracellular matrix (ECM)
  • An ECM enriched with HA promotes stronger adhesion of mast cells and increases the release of mast cell proteases that contribute to an inflammatory response (e.g., airway constriction, mucous production, cough)
  • RSV-infected HLFs exhibit increased expression of inflammatory mediators by mast cells, as well as increased mast cell adhesion

Findings from the study indicate that the formation of an HA-enriched ECM promotes a proinflammatory environment during acute respiratory viral infections. This mechanism could have far reaching implications for the therapeutic intervention for RSV and other respiratory viruses.

Infection of HLFs with RSV

Summary of findings: The study demonstrated that infection of HLFs with RSV induces upregulation of enzymes that synthesize HA (HAS 2&3) while downregulating enzymes that breakdown HA (HYAL 2), which leads to an increased accumulation of HA in the ECM and greater adherence of mast cells. Furthermore, RSV infected HLFs display increased expression of TSG-6, which enhances the binding of mast cells to the ECM. Mast cells bound to the RSV-induced ECM upregulate the expression of mast cell proteases contributing to the proinflammatory environment.

References

Find out more at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997473/

“The ImageXpress Pico system is constantly evolving to meet our ever-changing needs and is opening the door for further discoveries. We’re excited to see what comes next!”

Dr. Reeves and other corresponding authors are a part of Dr. Jason Debley’s lab. The Debley Lab utilizes epidemiological, clinical, and molecular methods to understand the evolution of asthma in early childhood and investigate the role of airway epithelium in childhood asthma.

Researchers at the Center for Immunity and Immunotherapies at Seattle Children’s Research Institute investigate some of the most challenging childhood diseases that affect the human immune system. Ultimately, the goal is to utilize the therapeutic power of immunology to devise novel disease treatments.