Influence of Lifestyle Factors on Neutrophil Migration Pilot Study

This study will investigate how external factors implicated in immunity such as exercise, caffeine ingestion, ethanol, glucose, and glucose and caffeine ingestion influence neutrophil migration.

Neutrophils are the most prominent immune cell in human blood and are involved in a complex equilibrium of immune protection and autoimmune damage. Their recruitment to an inflammatory or wounding site is controlled by the sensing and directed migration to a concentration gradient of attractant molecules, a process called chemotaxis. Immune cells are also implicated in many diseases including cancer. The ability to measure the amplitude of a response over time for a specific patient, and the variation of this response when the patient engages in certain activities or consumes certain substances will improve understanding of how certain lifestyle factors impact the immune response. Traditional assays require large volumes of blood and a long purification process, which may affect neutrophil function and strictly limits the number of draws possible from a single patient. The novel microfluidic assay proposed limits these drawbacks as it has the capability to purify neutrophils from a 3 µL drop of blood in less than 5 minutes and measure their chemotaxis in a gradient of chemokines. Critically, the proposed studies will begin to fill a gap in current understanding of immune response as previous studies focused on single endpoints likely missing early events in the response to external stimulus. Understanding this temporal response may have implications in the development of new treatments as well as improvements in diagnosis of improper immune response.

The KOALA (Kit On A Lid Assay) approach was developed in Professor Dave Beebe's lab and has been validated in a mouse model and in human asthmatic patients. In a collaboration with Dr. Anna Huttenlocher, it has been shown that, in contrast to traditional neutrophil purification, KOALA can be performed with small volumes of blood, and a much quicker purification time.

Due to its unique qualities, KOALA allows for repeated evaluation of neutrophil adhesion and chemotaxis properties, thus making it an attractive method for studying dynamic neutrophil changes that may occur as a result of an external factor.

Using traditional macrobiology tools, researchers have identified several factors that may play important roles in reducing neutrophil responsiveness and migration ability. Lifestyle and diet factors, amongst others, have been shown to impact neutrophil count, migration and biochemical function. For example, sleep deprivation has been linked with higher neutrophil count, despite a known immuno-depressive effect. Physical exercise results in increased neutrophil counts and increased neutrophil degranulation. Dietary factors, such as caffeine and ethanol have been shown to impact immune function.

Blood glucose will be tested at each time point using a blood glucose monitor to determine whether the lifestyle factor is affecting blood glucose (and consequently, neutrophil function). Alcohol consumption can cause both high and low blood sugar. Specifically, while low doses of alcohol may have a protective effect against risk of diabetes and metabolic syndrome, heavy drinking has been associated with higher glucose levels, resulting in increased risk of both diabetes and metabolic syndrome. Similarly, acute ingestion of caffeine has been shown to increase blood glucose levels, but several epidemiological studies have shown regular coffee consumption in associated with a lower risk of type 2 diabetes. Measuring blood glucose on subjects in the glucose study may show dose dependent effects on neutrophils.

To build on this research, the investigators intend to probe the role of these lifestyle factors on neutrophil migration over much shorter time scales than has been previously studied. The investigators propose to examine the effects of exercise, ethanol ingestion and caffeine consumption on neutrophil behavior.

Whole blood obtained from subjects by finger stick will be used in KOALA to isolate neutrophils. Neutrophil function will be assessed by measuring absolute migration speed, chemotactic index and chemotaxis velocity (directional velocity toward the formation of the gradient of chemoattractant).

The primary outcome of this study will be to determine whether exercise, caffeine consumption, ethanol, glucose and combined glucose and caffeine ingestion affect neutrophil chemotactic velocity. The secondary outcomes are to determine whether exercise, caffeine consumption, ethanol, glucose and combined glucose and caffeine ingestion affect the absolute speed and chemotactic index of neutrophils. The investigators will also assess whether cytokine profiles in the blood change as a result of engaging int he specified lifestyle factors.

Inclusion criteria

• Capacity to provide informed consent and ability to speak and read English.
• Male or female with no chronic or acute health concerns that might affect subject safety during the study or interfere with the study results
• No intake of medication that the researchers believe will significantly influence immune function in the 48 hours proceeding the lancet puncture (examples given in the section entitled "Exclusion Criteria")
• In good physical health
• Regularly exercise at least 30 minutes 3 times per week (exercise cohort)

Exclusion criteria

• Currently participating in another clinical trial
• History of significant systemic disease (eg. cancer, infection, hematological, renal, hepatic, coronary artery disease or other cardiovascular disease, endocrinologic, neurologic, rheumatologic, or gastrointestinal disease)
• Use of beta blockers or corticosteroids
• Currently taking medications that are not recommended to be taken in conjunction with alcohol
• Acute illness or evidence of clinically significant active infection
• Currently receiving immunotherapy
• Pregnant women
• Ingested medication (e.g. systemic corticosteroids) within 48 hours preceding the draw that the researchers believe may have an effect on immune response or the immune system
• Performed any activity that conflicts (eg. drinking any alcohol prior to the study), in the judgment of the investigator, with the external factor to be tested in the study (if any)
• Alcoholic or other health conditions for which alcohol consumption is contraindicated
• Consume more than 7 drinks per week (women alcohol cohort)
• Consume more than 14 drinks per week (men alcohol cohort)
• Consume more than three (8 oz.) servings of coffee, caffeinated soft drinks/tea (12 oz.) per day (caffeine cohort)