Quantitative analysis of immune cell motility and mechanics on hydrogel substrates

Cell migration is central to the development and regulation of immune function, yet the biophysical and molecular basis of motility and force generation in immune cells is not well understood. Previous studies examining immune cell behavior have either been conducted in vivo or in non-physiologic tissue culture systems such as glass or plastic. Few studies have been completed to date to investigate the behavior of immune cells on compliant hydrogel surfaces that more closely resemble in vivo environments. The use of hydrogel substrates offers a number of advantages in the investigation of immune cell behavior as the mechanical and chemical properties of hydrogels are easily controlled and quantitative information on cell behavior can be obtained. In this thesis, we develop a novel system to enable chemical gradients to be generated over hydrogel surfaces using microfluidic technology. Using this system, we investigate the motility and force generation of neutrophils, a key cellular component of the innate immune response. Through systematic studies of neutrophil motility on compliant hydrogel substrates, we develop a quantitative understanding of how neutrophils respond to chemical and mechanical cues. We find that neutrophil adhesion, migration and force generation are influenced by both chemical and mechanical properties of their surrounding matrix. In response to changes in substrate mechanics, neutrophils can rapidly modulate their adhesive area, directionality, and traction forces. We are the first to show that traction force generation can influence neutrophil polarity and the response to chemotactic signals. Lastly, using inhibitors and in vivo mouse models, we show that both integrin and RhoA signaling are implicated in force generation in neutrophils

Traction Forces of Neutrophils Migrating on Compliant Substrates

AbstractProper functioning of the innate immune response depends on migration of circulating neutrophils into tissues at sites of infection and inflammation. Migration of highly motile, amoeboid cells such as neutrophils has significant physiological relevance, yet the traction forces that drive neutrophil motion in response to chemical cues are not well characterized. To better understand the relationship between chemotactic signals and the organization of forces in motile neutrophils, force measurements were made on hydrogel surfaces under well-defined chemotactic gradients created with a microfluidic device. Two parameters, the mean chemoattractant concentration (CM) and the gradient magnitude (Δc/Δx) were varied. Cells experiencing a large gradient with CM near the chemotactic receptor KD displayed strong punctate centers of uropodial contractile force and strong directional motion on stiff (12 kPa) surfaces. Under conditions of ideal chemotaxis—cells in strong gradients with mean chemoattractant near the receptor KD and on stiffer substrates—there is a correlation between the magnitude of force generation and directional motion as measured by the chemotactic index. However, on soft materials or under weaker chemotactic conditions, directional motion is uncorrelated with the magnitude of traction force. Inhibition of either β2 integrins or Rho-associated kinase, a kinase downstream from RhoA, greatly reduced rearward traction forces and directional motion, although some vestigial lamellipodium-driven motility remained. In summary, neutrophils display a diverse repertoire of methods for organizing their internal machinery to generate directional motion

Development of Pandemic Vaccines: ERVEBO Case Study

Preventative vaccines are considered one of the most cost-effective and efficient means to contain outbreaks and prevent pandemics. However, the requirements to gain licensure and manufacture a vaccine for human use are complex, costly, and time-consuming. The 2013–2016 Ebola virus disease (EVD) outbreak was the largest EVD outbreak to date and the third Public Health Emergency of International Concern in history, so to prevent a pandemic, numerous partners from the public and private sectors combined efforts and resources to develop an investigational Zaire ebolavirus (EBOV) vaccine candidate (rVSVΔG-ZEBOV-GP) as quickly as possible. The rVSVΔG-ZEBOV-GP vaccine was approved as ERVEBOTM by the European Medicines Authority (EMA) and the United States Food and Drug Administration (FDA) in December 2019 after five years of development. This review describes the development program of this EBOV vaccine, summarizes what is known about safety, immunogenicity, and efficacy, describes ongoing work in the program, and highlights learnings applicable to the development of pandemic vaccines