Chronic Cannabis Smoking, Oxidative Stress and the Pulmonary Innate Immune Response

I. Hypotheses and Specific Aims:

With changing legislation, the landscape of cannabis use is shifting; data demonstrates that
public perception of marijuana's "safety" has contributed to increasing usage most commonly
via the inhaled route (smoking). The effects of habitual cannabis smoking on lung health,
however, remains unclear. Spirometric data has been inconclusive regarding risk of airflow
limitation and development of chronic obstructive pulmonary disease, a finding that may be
attributable to inaccurate reporting due to its former illegal nature. It seems clear,
however, that these patients suffer from an increased incidence of bronchitis symptoms from
an uncertain pathophysiologic mechanism. Animal data from the 1990s demonstrates that
cannabinoid exposure (of which Tetrahydrocannabinol (THC) is an example) results in increased
intra-pulmonary oxidative stress indices and immunomodulatory effects resulting in abnormal
macrophage function; a finding that may be related the aforementioned symptoms. However, this
data is likely now outmoded given the markedly increased (THC) content of today's cannabis
for sale (12 percent Tetrahydrocannabinol (THC) compared to 3 percent in the 1990s).
Furthermore, most of these findings have not been validated in human subjects. Thus, the
investigators hypothesize that habitually smoked cannabis increases intrapulmonary oxidative
stress resulting in impaired alveolar macrophage (AM) phagocytosis, elicits an attenuated AM
response to pathogen-associated molecular patterns (PAMPs) and promotes AM apoptosis thereby
increasing risk of upper and lower airway infections.

II. Background and Significance:

Cannabis use in the United States is rising since legalization for medical and, more
recently, recreational purposes. The United Nations Office on Drugs and Crime estimates that
in 2012, when cannabis was legal for medical purposes in 17 states, but illegal for
recreational consumption nationally, cannabis use among the American population increased
from 11.5% to 12.1%. Over the past 2 years, an additional seven states have legalized medical
cannabis, and Colorado and Washington have recently legalized its sale for recreational use.
Since January 2014, publicly available Colorado data indicate increasing tax revenue from
cannabis sales (both medical and recreational), suggesting a rise in state-wide consumption
in a relatively short period of time.

Effects of modern inhaled cannabis on lung health are not established. Published
epidemiologic data collected prior to the widespread legalization of cannabis and
commercialization of the cannabis industry demonstrate consistent associations between
"regular" (e.g. near daily) and "heavy" (e.g. for multiple years) cannabis use with poorer
lung health. Clinically, regular inhaled cannabis users complain of increased chronic
bronchitis symptoms (eg. wheeze, chronic cough) compared to non-smokers; an increased use of
medical services for respiratory infections due to immunosuppressive effects of the drug has
also been reported. However, characterization of cannabis use in these investigations varies
widely, ranging from 7 joint-years (1 joint per day for 1 year) 12 to 117 joint-years in
cohorts examined. These studies were also potentially confounded by under-reporting of
cannabis use due to the drug's illegal status, and concomitant use of inhaled tobacco in some
study populations. Moreover, the quantity of THC in modern cannabis products has been
increasing over the past two decades. Therefore, it remains unclear what use patterns of
modern inhaled cannabis are harmful to lung health, although more cannabis is being consumed
now than ever before, both for medical and recreational purposes. Today, due to the
decriminalization of cannabis sale and purchase, the accuracy of self-reported cannabis use
is likely much greater than in prior investigations, providing a novel opportunity to more
accurately establish patterns of use associated with ill effects on health. Importantly, no
study to date has utilized time series analyses, such as the time line follow-back (TLFB), to
quantitate the effects of regular cannabis consumption in association with lung health. TLFB
techniques are used extensively to quantitate substance use among chronic users of alcohol,
tobacco, and more recently, cannabis. TLFB are calendar-assisted, structured interviews that
cue memory to enhance accurate recall, and have been determined to be both reliable and valid
in quantitating current substance use in detail. TLFB interviews to characterize past 30 day
cannabis use have been routinely conducted in research subjects by Dr. Corsi's (co-mentor to
Dr. Biehl) research group since 2010, and longer term joint-year data has been collected as
well.

Inhaled cannabis use adversely affects function of alveolar macrophages (AMs), critical
pulmonary innate immune effectors. AMs express cannabinoid (CB) receptors, primarily CB2, on
their surface, whose ligand is THC15 (a cannabinoid). AMs represent the first line of defense
against invading pathogens in the lower airways, and function to keep the lungs sterile.
Published investigations using AMs isolated via bronchoalveolar lavage (BAL) from small
numbers (n<20) of habitual inhaled cannabis users have reported an inappropriately diminished
response to S. aureus, an important lung pathogen. When AMs were exposed to S. aureus, they
exhibited decreased bacterial phagocytosis and killing; hampered production of the
pro-inflammatory cytokines tumor necrosis factor-α, IL-6, and granulocyte monocyte colony
stimulating factor; and decreased production of nitric oxide. Preclinical data has suggested
that AM viability and apoptosis may also be adversely influenced by THC in a dose- and
time-dependent manner through the CB2 receptor, further impairing the AM's ability to respond
to pathogens. As mentioned, cannabis use is increasing on a per capita basis, and continues
to be used via an inhaled route, while its THC quantity is increasing. Collectively, this
suggests the possibility that individuals who regularly use small doses of inhaled cannabis,
or have used cannabis for a shorter length of time, may still be at risk for AM dysfunction,
clinically manifested by increased respiratory symptoms and an increased risk for pulmonary
infections, particularly since pre-clinical data has consistently reported adverse effects of
cannabis exposure on infectious disease resistance.

Cannabis exposure in vitro leads to oxidative stress that in turn affects pulmonary cellular
viability. Murine lung epithelial cells exposed to cannabis smoke extract in vitro
demonstrate a dose-related increase in oxidative stress, while human lung BEAS-2B cells
exposed to cannabis smoke extract in vitro display an increase in reactive oxygen species
production. Enhanced oxidative stress in each investigation was further associated with
evidence of cytotoxicity, characterized by cellular apoptosis and DNA damage. It seems
possible, then, that enhanced oxidative stress due to habitual inhaled cannabis may
additionally influence the viability and function of AMs in the lower airways. However, the
effect of inhaled cannabis on oxidative stress and its relationship to AMs has never been
specifically explored with primary cells from human subjects.

In summary, our collaboration will utilize validated methods to characterize regular and
chronic cannabis use patterns among exclusive medical and/or recreational cannabis users, in
order to examine their impact on cells critical to the maintenance of lung health.
Information the investigators derive may be used to counsel active and contemplating cannabis
users, and inform medical providers and researchers.

Inclusion Criteria:

INCLUSION CRITERIA for CANNABIS USING patient: Subjects will be eligible if they meet ALL
of the following criteria:

1. Daily or near daily cannabis use (inhaled via joint/cigarette or pipe) equivalent to
AT LEAST 20 "joint years" (number of joints/cigarettes per day multiplied by number of
years during which cannabis was smoked) by self-report and validated through urine
drug screen.

2. Never or limited exposure to inhaled tobacco products (equivalent to LESS THAN one
"pack year" in a lifetime) by selfreport and validated through negative urine cotinine
screen.

3. Capacity to answer screening questions and provide informed consent at time of
interview, along with contact information

INCLUSION CRITERIA for CONTROL patient: Subjects will be eligible if they meet all of the
following:

1. No cannabis use ever by self-report, and validated through urine drug screen.

2. Never users of tobacco/cigarettes

3. Capacity to answer screening questions and provide informed consent at time of
interview, along with contact information.

Exclusion Criteria:

EXCLUSION CRITERIA for CANNABIS USING patient: Subjects will be ineligible if they meet ANY
of the following criteria:

1. LESS THAN near daily cannabis use (inhaled via joint/cigarette or pipe) equivalent to
LESS THAN 20 "joint years" (number of joints/cigarettes per day multiplied by number
of years during which cannabis was smoked) or a negative urine drug screen (for
cannabis)

2. Inhaled tobacco product exposure EXCEEDING one "pack year" or a positive urine
cotinine screen

3. Elevated AUDIT-C score: A 3 item questionnaire to identify subjects with alcohol use
disorders.

4. Prior medical history of liver disease: cirrhosis, total bilirubin > 2.0 mg/dL or
albumin <3

5. Prior medical history of myocardial infarction or congestive heart failure

6. Prior medical history of end-stage renal disease or serum creatinine >3 mg/dL

7. Prior history of or current use of illicit drug use defined as a positive toxicology
screen for opiates or cocaine

8. Prior history of diabetes mellitus

9. Prior history of chronic obstructive pulmonary disease (COPD) or asthma that is not
clinically controlled (have not required systemic corticosteroids in the past month)

10. Prior history of HIV, not controlled or on medication

11. Peripheral white blood cell count of less than 3000

12. Acute worsening (<7 days) in respiratory symptoms (such as change in cough frequency
or sputum production, fever, dyspnea, abnormal chest radiograph), or room air pulse
oximetry of < 92% at rest or spirometry of < 50% predicted for FEV1 and FVC

13. Use of systemic antibiotics for any reason in the past month (4 weeks)

14. Failure of a subject or the subject's substance abuse counselor to provide assent

15. Nutritional risk index of less than 95

16. Age < 21 or > 55 (using an age of 55 limits the likelihood of comorbid conditions that
may increase the risk of adverse events with bronchoscopy)

17. Pregnancy

18. Decisionally challenged

19. Prisoners.

EXCLUSION CRITERIA for CONTROL patient: Subjects will be ineligible to participate if they
meet ANY of the following criteria:

1. History of inhaled cannabis exposure EXCEEDING one "joint year" or positive urine
toxicology screen (for cannabis)

2. History of inhaled tobacco product exposure EXCEEDING one "pack year" or a positive
urine cotinine screen

3. Elevated AUDIT-C score: A 3 item questionnaire to identify subjects with alcohol use
disorders.

4. Prior medical history of liver disease: cirrhosis, total bilirubin > 2.0 mg/dL or
albumin <3

5. Prior medical history of myocardial infarction or congestive heart failure

6. Prior medical history of end-stage renal disease or serum creatinine >3 mg/dL

7. Prior history of or current use of illicit drug use defined as a positive toxicology
screen for opiates or cocaine

8. Prior history of diabetes mellitus

9. Prior history of chronic obstructive pulmonary disease (COPD) or asthma that is not
clinically controlled (have not required systemic corticosteroids in the past month)

10. Prior history of HIV, not controlled or on medication

11. Peripheral white blood cell count of less than 3000

12. Acute worsening (<7 days) in respiratory symptoms (such as change in cough frequency
or sputum production, fever, dyspnea, abnormal chest radiograph), or room air pulse
oximetry of < 92% at rest or spirometry of < 50% predicted for FEV1 and FVC

13. Use of systemic antibiotics in the past month (4 weeks)

14. Failure of a subject or the subject's substance abuse counselor to provide assent

15. Nutritional risk index of less than 95

16. Age < 21 or > 55

17. Pregnancy

18. Decisionally challenged

19. Prisoners.