microfluidic-platform-for-measuring-neutrophil-chemotaxis-from

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

Massachusetts General Hospital

Humans and mice infected with different Plasmodium strains are known to produce microvesicles derived from the infected red blood cells (RBCs), denoted RMVs. Studies in mice have shown that RMVs are elevated during infection and have proinflammatory activity. Here we present a detailed characterization of RMV composition and function in the human malaria parasite Plasmodium falciparum. Proteomics profiling revealed the enrichment of multiple host and parasite proteins, in particular of parasite antigens associated with host cell membranes and proteins involved in parasite invasion into RBCs. RMVs are quantitatively released during the asexual parasite cycle prior to parasite egress. RMVs demonstrate potent immunomodulatory properties on human primary macrophages and neutrophils. Additionally, RMVs are internalized by infected red blood cells and stimulate production of transmission stage parasites in a dose-dependent manner. Thus, RMVs mediate cellular communication within the parasite population and with the host innate immune system.

Malaria-infected erythrocyte-derived microvesicles mediate cellular communication within the parasite population and with the host immune system.

The current paradigm of unidirectional migration of neutrophils from circulation to sites of injury in tissues has been recently challenged by observations in zebrafish showing that neutrophils can return from tissues back into the circulation. However, the relevance of these observations to human neutrophils remains unclear, the forward and reverse migration of neutrophils is difficult to quantify, and the precise conditions modulating the reverse migration cannot be isolated. Here, we designed a microfluidic platform inside which we observed human neutrophil migration in response to chemoattractant sources inside channels, simulating the biochemical and mechanical confinement conditions at sites of injury in tissues. We observed that, after initially following the direction of chemoattractant gradients, more than 90% of human neutrophils can reverse their direction and migrate persistently and for distances longer than one thousand micrometers away from chemoattractant sources (retrotaxis). Retrotaxis is enhanced in the presence of lipoxin A4 (LXA4), a well-established mediator of inflammation resolution, or Tempol, a standard antioxidant. Retrotaxis stops after neutrophils encounter targets which they phagocytise or on surfaces presenting high concentrations of fibronectin. Our microfluidic model suggests a new paradigm for neutrophil accumulation at sites of inflammation, which depends on the balance of three simultaneous processes: chemotaxis along diffusion gradients, retrotaxis following mechanical guides, and stopping triggered by phagocytosis.

Retrotaxis of human neutrophils during mechanical confinement inside microfluidic channels.

Neutrophils are the most abundant type of white blood cells in the circulation, protecting the body against pathogens and responding early to inflammation. Although we understand how neutrophils respond to individual stimuli, we know less about how they prioritize between competing signals or respond to combinational signals. This situation is due in part to the lack of adequate experimental systems to provide signals in controlled spatial and temporal fashion. To address these limitations, we designed a platform for generating on-demand, competing chemical gradients and for monitoring neutrophil migration. On this platform, we implemented forty-eight assays generating independent gradients and employed synchronized valves to control the timing of these gradients. We observed faster activation of neutrophils in response to fMLP than to LTB4 and unveiled for the first time a potentiating effect for fMLP during migration towards LTB4. Our observations, enabled by the new tools, challenge the current paradigm of inhibitory competition between distinct chemoattractant gradients and suggest that human neutrophils are capable of complex integration of chemical signals in their environment.

On-demand, competing gradient arrays for neutrophil chemotaxis.

Immune responses in the brain are thought to play a role in disorders of the central nervous system, but an understanding of the process underlying how immune cells get into the brain and their fate there remains unclear. In this study, we used a 2-photon microscopy to reveal that neutrophils infiltrate brain and migrate toward amyloid plaques in a mouse model of Alzheimer's disease. These findings suggest a new molecular process underlying the pathophysiology of Alzheimer's disease.

Migration of neutrophils targeting amyloid plaques in Alzheimer's disease mouse model.

Diseases of failed inflammation resolution are common and largely incurable. Therapeutic induction of inflammation resolution is an attractive strategy to bring about healing without increasing susceptibility to infection. However, therapeutic targeting of inflammation resolution has been hampered by a lack of understanding of the underlying molecular controls. To address this drug development challenge, we developed an in vivo screen for proresolution therapeutics in a transgenic zebrafish model. Inflammation induced by sterile tissue injury was assessed for accelerated resolution in the presence of a library of known compounds. Of the molecules with proresolution activity, tanshinone IIA, derived from a Chinese medicinal herb, potently induced inflammation resolution in vivo both by induction of neutrophil apoptosis and by promoting reverse migration of neutrophils. Tanshinone IIA blocked proinflammatory signals in vivo, and its effects are conserved in human neutrophils, supporting a potential role in treating human inflammation and providing compelling evidence of the translational potential of this screening strategy.

A zebrafish compound screen reveals modulation of neutrophil reverse migration as an anti-inflammatory mechanism.

Neutrophil chemotaxis is critical for defense against infections and its alterations could lead to chronic inflammation and tissue injury. The central role that transient alterations of neutrophil chemotaxis could have on patient outcomes calls for its quantification in the clinic. However, current methods for measuring neutrophil chemotaxis require large volumes of blood and are time consuming. To address the need for rapid and robust assays, we designed a microfluidic device that measures neutrophil chemotaxis directly from a single droplet of blood. We validated the assay by comparing neutrophil chemotaxis from finger prick, venous blood and purified neutrophil samples. We found consistent average velocity of (19 ± 6 μm/min) and directionality (91.1%) between the three sources. We quantified the variability in neutrophil chemotaxis between healthy donors and found no significant changes over time. We also validated the device in the clinic and documented temporary chemotaxis deficiencies after burn injuries.

Article	Year
Malaria-infected erythrocyte-derived microvesicles mediate cellular communication within the parasite population and with the host immune system. Cell host & microbe\| PubMed ID: 23684304	2013
Retrotaxis of human neutrophils during mechanical confinement inside microfluidic channels. Integrative biology : quantitative biosciences from nano to macro\| PubMed ID: 24419464	2014
On-demand, competing gradient arrays for neutrophil chemotaxis. Lab on a chip\| PubMed ID: 24430002	2014
Migration of neutrophils targeting amyloid plaques in Alzheimer's disease mouse model. Neurobiology of aging\| PubMed ID: 24485508	2014
A zebrafish compound screen reveals modulation of neutrophil reverse migration as an anti-inflammatory mechanism. Science translational medicine\| PubMed ID: 24574340	2014
Measuring neutrophil speed and directionality during chemotaxis, directly from a droplet of whole blood. Technology\| PubMed ID: 24809064	2013

Bashar Hamza

Biography

JoVE Journal Publications

Other Publications