Comparative evaluation of ten blood biomarkers of inflammation in regular heated tobacco users and non-smoking healthy males–a pilot study

Abstract

Heated tobacco products (HTPs) are novel tobacco products that are alternatives to cigarettes. The study aimed to investigate the effect of HTPs on blood biomarkers of inflammation as well as to provide a comparative evaluation between daily heated tobacco users and healthy men who do not use nicotine products. This case–control study was carried out among 92 healthy males in Poland (Lodz-Province) aged 20–56 years: 44 daily heated tobacco users (daily use in the past 90 days) and 48 controls who do not use nicotine products. The history of use of the nicotine-containing products was self-reported and verified using a saliva cotinine test. A 20 ml blood sample was collected and the levels of ten blood biomarkers were analyzed. Among all heated tobacco users (n = 44), only the levels of interleukin 8 (IL-8) were significantly higher when compared to controls: 6.86 vs. 3.95 (p = 0.01). Among exclusive heated tobacco users (n = 33), the levels of IL-8 were also significantly higher when compared to controls: 7.76 vs. 3.95 (p = 0.01). IL-8 level was positively correlated (r = 0.37; p = 0.01) with the daily number of heated tobacco sticks. Out of 10 different biomarkers of inflammation, only IL-8 levels were significantly elevated in heated tobacco use compared to controls.

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Comparative evaluation of ten
blood biomarkers of inammation
in regular heated tobacco users
and non‑smoking healthy males–a
pilot study
Beata Świątkowska
1*, Mateusz Jankowski
2 & Dorota Kaleta
1
Heated tobacco products (HTPs) are novel tobacco products that are alternatives to cigarettes. The
study aimed to investigate the eect of HTPs on blood biomarkers of inammation as well as to
provide a comparative evaluation between daily heated tobacco users and healthy men who do not
use nicotine products. This case–control study was carried out among 92 healthy males in Poland
(Lodz‑Province) aged 20–56 years: 44 daily heated tobacco users (daily use in the past 90 days) and
48 controls who do not use nicotine products. The history of use of the nicotine‑containing products
was self‑reported and veried using a saliva cotinine test. A 20 ml blood sample was collected and the
levels of ten blood biomarkers were analyzed. Among all heated tobacco users (n = 44), only the levels
of interleukin 8 (IL‑8) were signicantly higher when compared to controls: 6.86 vs. 3.95 (p = 0.01).
Among exclusive heated tobacco users (n = 33), the levels of IL‑8 were also signicantly higher when
compared to controls: 7.76 vs. 3.95 (p = 0.01). IL‑8 level was positively correlated (r = 0.37; p = 0.01)
with the daily number of heated tobacco sticks. Out of 10 dierent biomarkers of inammation, only
IL‑8 levels were signicantly elevated in heated tobacco use compared to controls.
Heated tobacco products (HTPs) are novel tobacco products that are alternatives to cigarettes and combustible
tobacco products1–3. HTPs operate based on an electronically controlled heating element that heats dedicated
tobacco-containing sticks up to 350°C and generates aerosol inhaled by the user3. Nicotine levels in aerosol
generated by HTPs are estimated at 70–80% as those generated by combustible cigarettes4. When compared to
electronic cigarettes (e-cigarettes), HTPs-dedicated sticks contain processed tobacco which is a source of nicotine,
instead of a dedicated nicotine-containing liquid like in e-cigarettes5.
HTPs have been marketed since 20143. e market is dominated by the heating tobacco devices of major
tobacco companies, including “IQOS” from Philip Morris International, “glo” from British American Tobacco,
and “Ploom TECH” from Japan Tobacco3. It is estimated that 5% of the global population has ever used HTPs
and 1.5% of the global population has been identied as current HTP users6. In recent years, the prevalence of
HTPs increased rapidly. In Japan, the prevalence of HTPs increased from 0.2% in 2015 to 11.3% in 20197. In
Poland, the prevalence of daily HTP use increased from 0.4% in 2019 to 4.0% in 20228. e global prevalence of
HTPs is estimated to rise in the coming years6.
With increasing public awareness of HTPs and the growing number of users, questions about the health
eects of HTP use are increasing2,9,10. HTPs are being marketed as reduced exposure alternatives to combustible
cigarettes3,9,11. Most of the scientic evidence on the health eects of HTP use is based on laboratory evaluation
of aerosol generated by HTP use as well as experimental studies on animal and cellular models, with limited
data from human-based studies3,9. Both industry-funded studies and independent research conrmed that the
concentration of chemical compounds in aerosol generated during HTP use is lower than during smoking
combustible cigarettes3,9,12,13. In general, toxic compounds related to combustion process were signicantly
reduced, but heated tobacco products are not risk-free3,9,12. Findings from the experimental animal and cellular
studies showed that aerosol from HTPs has lower toxicity than cigarette smoke3,9. ere are concerns that while
OPEN
1Department of Hygiene and Epidemiology, Medical University of Lodz, Żeligowskiego 7/9 Street, 90-752 Łódź,
Poland. 2Department of Population Health, School of Public Health, Centre of Postgraduate Medical Education,
Kleczewska 61/63 Street, 01-826 Warsaw, Poland. *email: beata.swiatkowska@umed.lodz.pl
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it may expose users to less of some toxins that are also found in traditional cigarettes, the use of heated tobacco
exposes users to other toxic substances whose toxicological proles of short- and long-term health eects are
unknown2,3,9,10. Nevertheless, HTP use may induce oxidative stress, and inammation and increase the risk of
respiratory tract infections9,14. While most research funded by the tobacco industry has demonstrated the health
advantages of switching from traditional cigarettes to HTP, certain independent studies point to some possible
negative eects of HTP aerosol exposure3,9,10.
ere is a limited number of human-based studies on the health eects of HTP use, mostly on short-term
health eects15–17. Majek etal. showed that the use of HTP for 5min evoked an increase in heart rate and blood
pressure as well as a decrease in fractional exhaled nitric oxide (FeNO) levels15. Goebel etal. reported small
airway obstruction and resistance as well as nicotine-related acute increase in arterial stiness and cardiovascular
stress aer HTP use16. Lyytinen etal. reported increased arterial stiness and platelet thrombus formation 5min
aer the HTP use in a group of healthy young adults17.
Smoking-induced oxidative stress, chronic inammation, and endothelial dysfunction are considered to play
major roles in the pathogenesis of tobacco-related diseases3,9,15–17. However, little is known about blood-based
biomarkers of inammation in regular heated tobacco users.
e study aimed to investigate the eect of heated tobacco products on blood biomarkers of inammation
as well as to provide a comparative evaluation between daily heated tobacco users and healthy men who do not
use nicotine products.
Material and methods
Study design and population
is case–control study was carried out among 92 healthy males in Poland (Lodz Province): 44 daily heated
tobacco users (past 90days) and 48 controls who do not use nicotine products. All procedures were carried
out in a medical facility and supervised by a physician. Subjects were recruited using an active recruitment
network, using the Medical University of Lodz and a network of private medical facilities operating in the Lodz
Province. Volunteers who declared a conscious willingness to participate in the study were screened to assess
the inclusion and exclusion criteria and qualications to participate in the study. Subjects were asked to undergo
the examination without a meal, and heated tobacco users were asked not to use nicotine products on the day of
the tests. A physical examination and a short interview were performed to assess the current health status. e
participants were then asked to ll out a set of questionnaires and blood samples were collected. All participants
provided written informed consent. e study protocol was reviewed and approved by the Ethics Committee of
the Medical University of Łódź (decision number: RNN/235/22/KE of 8/11/2022). All procedures performed
in this study were in accordance with the ethical standards of the institutional research committee and with the
Declaration of Helsinki.
Inclusion and exclusion criteria
Heated tobacco users
Males aged 20–56,without chronic diseases, who declare regular use of HTPs (IQOS or glo) – at least one
heated tobacco stick daily in the last 90days. Among heated tobacco users, subjects who use concurrently use
e-cigarettes regularly (at least 15days/month) or concurrently use combustible cigarettes/factory made cigarettes
were classied into a subgroup of dual users (subgroup 1: HTPs + e-cigarette; subgroup 2: HTPs + cigarette).
Controls
Males aged , 20–56,without chronic diseases, who do not use nicotine products and did not smokers or have
smoked less than 100 cigarettes in their life who have not used nicotine replacement therapy in the last 90days,
and who have used an e-cigarette no more than once in their entire life and e-cigarette use has occurred more
than 90days before the examination, and have used HTP no more than once in their entire life and HTP use
has occurred more than 90days before the examination. e control group was recruited in such a way that it
corresponded to the demographic characteristics of the group of heated tobacco users.
Smoking status
e history of use of the nicotine-containing products was based on self-reported and veried using a saliva
cotinine test (Salimetrics 1-2002, Stratech Scientic Ltd, UK; cotinine concentration in ng per ml). e mean
cotinine concentration was 0.55ng/ml among control, which is below the cut-of value for non-smokers published
on the website18 of the test manufacturer (5ng/ml), comparted to 360.9ng/ml (p < 0.001) among HTPs (with
cut-o value for smokers 100ng/ml)18.
Participants were asked about ever and past 90days use of heated tobacco use, e-cigarette use, and cigarette
smoking. ose who declared the use of nicotine-containing products (current or in the past) were asked about
the characteristics of nicotine product use (over 20 questions on each type of nicotine-containing product). A
total of 88 questions on nicotine-containing products were addressed. Heated tobacco users were asked about
the frequency of use, the number of heated tobacco sticks consumed per day, the type of heated tobacco device
used, heated tobacco stick, motivation for HTPs use, indoor use of HTPs, harm perception of HTPs, and self-
reported health eects of HTPs use, as well as the addictive potential of HTPs. Questions on the history of HTPs
use were based on the Global Adults Tobacco Survey (GATS) and adapted to HTPs use19.
Measures
A 20ml blood sample was obtained from each subject. Levels of the following blood-based biomarkers of
inammation20 in blood serum were analyzed:
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• Proinammatory cytokine levels: interleukin (IL) IL-1β, IL-6, IL-8, IL-10, IL-12p70, tumor necrosis factor
alpha (TNF-α) (Magnetic Luminex® performance assay, FCSTM09, Luminex Corp, Austin, TX, US). Cytokine
levels were presented in picograms per milliliter [pg/ml].
• c-reactive protein (CRP) and brinogen levels (MILLIPLEX® human cardiovascular disease (CVD) magnetic
bead panel, HCVD3MAG-67K, Merck, Darmstadt Germany). CRP levels were presented in milligrams per
liter [mg/l] and brinogen levels were presented in milligrams per deciliter [mg/dl].
• Adhesion molecules: vascular cell adhesion molecule (VCAM-1) and intercellular adhesion molecule (ICAM-
1) (Human Luminex® Discovery Assay, LXSAHM-08, Luminex Corp, Austin, TX, US). Adhesion molecule
levels were presented in nanograms per milliliter [ng/ml].
All laboratory tests were carried out in the Central Scientic Laboratory of the Medical University of Lodz
(CoreLab). All diagnostics procedures were performed by qualied medical personnel with experience in
medical diagnostics following the laboratory in accordance with the recommendations of the diagnostic test
manufacturers.
Statistical analysis
Data analysis was performed using procedures available in IBM SPSS Statistics 29 (IBM, Armonk, NY, USA). e
normality of distributions was tested using the Shapiro–Wilk test. Dierences in the distribution of quantitative
variables between the HTPs users and control group were analyzed using Student’s t-test or non-parametric
tests U Mann–Whitney test. Spearman’s rank correlation was used to analyze the correlation between the blood
biomarker levels and the daily number of heated tobacco sticks used in the last 90days. Data were presented
separately for all heated tobacco users (only HTPs or dual use HPTs and cigarette or e-cigarette) and exclusive
heated tobacco users (only HTPs in the last 90days). Statistical signicance was assessed at p < 0.05.
Results
Characteristics of the study population
A total of 92 healthy males were recruited. e mean age of HTP users was 33.6years and control 33.4years
(Table1). Most of the subjects had higher education: (56.8% of HTP users and 60.4% of controls) and lived in
cities above 500,000 residents (61.4% of HTP users and 83.3% of controls). Most of the subjects had full-time
jobs (59.1% of HTPs users and 64.6% of controls). Among heated tobacco users, 95.5% had ever tried a cigarette
and 84.1% had ever tried an e-cigarette (Table1). Among controls, 18.8% had ever tried a cigarette, 20.8% had
ever tried an e-cigarette and 12.5% had ever tried heated tobacco products. Detailed characteristics is presented
in Table1.
Patterns of heated tobacco product use
Out of all heated tobacco product users (n = 44), 11.4% were daily smokers and 15.9% declared daily use of
e-cigarettes. In total, there were 10 dual users (HTPs and cigarette or e-cigarette) and 1 triple user (HTPs,
cigarette, e-cigarette). Most of the HTP users consumed from 11 to 20 heated tobacco sticks per day (Table2).
e most popular heated tobacco device was IQOS, used by 52.3% of heated tobacco users, 40.9% used glo and
6.8% used lil SOLID 2.0. Mint or menthol avors were the most common ones, 13.6% used blueberry avor
and only one-half of HTP users used tobacco avor sticks. Most of the HTPs users used heated tobacco devices
indoors (93.2%) and 54.5% used HTPs in places where smoking is prohibited (Table2). Detailed characteristics
of the heated tobacco users is presented in Table2. e average number of heated tobacco stick packs per week
was 5.1 ± 2.9, median of 5 packs per week. Most of the participants used HTP for 3years (29.5%), one-h used
HTP for 2years, 15.9% used HTP for less than 1year, 13.3% for 4years and 11.4% of HTP users used heated
tobacco products for 5years (Table2).
Blood biomarkers of inammation levels
A comparative evaluation of blood biomarkers in regular heated tobacco users and healthy males who do not
use nicotine products is presented in Table3. Among all heated tobacco users (n = 44), the levels of interleukin 8
were signicantly higher when compared to controls: 6.86 vs. 3.95 (p = 0.01) (Table3). Among exclusive heated
tobacco users (n = 33), the levels of interleukin 8 were also signicantly higher when compared to controls: 7.76
vs. 3.95 (p = 0.01) (Table4). Among all heated tobacco users (n = 44), interleukin 8 level was positively correlated
(r = 0.37; p = 0.01) with the daily number of heated tobacco sticks (Table5). is correlation was not statistically
signicant in subanalysis for exclusive heated tobacco users (Table6). ere were no statistically signicant
dierences (p > 0.05) in the levels of 9 other blood biomarkers of inammation, including IL-1β, IL-6, IL-10,
IL-12p70, TNF-α, CRP, brinogen and adhesion molecules (VCAM-1 and ICAM-1) between heated tobacco
users and healthy males who do not use nicotine products (Tables3, 4, 5, 6).
Discussion
is is one of the rst human-based case–control studies on the blood biomarkers of inammation of regular
(past 90days) heated tobacco use. Out of 10 blood biomarkers analyzed in this study, only interleukin 8 levels
were signicantly (p < 0.05) higher among heated tobacco users compared to healthy males who do not use
nicotine products. ese dierences were observed both among all heated tobacco users (exclusive and dual
users with cigarettes or e-cigarettes) and exclusive heated tobacco users. e level of IL-8 raised with the daily
number of heated tobacco sticks consumed by HTP users.
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Most of the currently available data on the health eects of heated tobacco use are limited to laboratory-based
experimental studies or studies on short-term health eects of HTP use among humans3,9,10,14–17. Previously
published human-based studies assessed the levels of dierent biomarkers immediately (mostly within 5min)
aer the use of heated tobacco sticks15–17. Small airway obstruction and resistance as well as a decrease in
fractional exhaled nitric oxide were reported as the immediate health eects of HTP use15,16. Moreover, an
increase in heart rate and blood pressure, increased arterial stiness, and platelet thrombus formation were
described as cardiovascular eects of heated tobacco use17. Findings from experimental studies on cell lines
and animal models suggest that HTP use may evoke chronic inammation14,21–23. Based on mice models, Sawa
etal. reported that short-term exposure to HTP aerosols may increase oxidative stress and induce the secretion
of inammatory cytokines: IL-6 and GM-CSF14. Using mice models, Gu etal. reported that chronic exposure
to aerosol generated by HTP use resulted in impaired pulmonary function and lung tissue damage21. Bhat etal.
reported that HTP use induces inammatory immune-cell accumulation in the lungs as well as augments the
levels of proinammatory cytokines and chemokines in the BAL uid22. Yamamoto etal. reported that HTP use
induced IL-8 overexpression with an increase in human monocyte THP-1 cell lines23.
In this study, the past 90days of use of heated tobacco products evoked a signicant increase in IL-8 levels,
when compared to healthy adults who do not use nicotine products. e levels of IL-8 in blood samples collected
from HTP users were elevated regardless of the pattern of heated tobacco use – exclusive HTP use or dual-use
(HTPs and cigarettes or e-cigarettes). Interleukin 8 (IL-8) is a major mediator of inammation and acts as a
chemoattractant for neutrophils, basophils, and T cells24,25. Previous studies showed that cigarette smoke induces
IL-8 release from macrophages. IL-8 is a key chemokine during the initiation and progression of tobacco-related
lung inammation as well as the development of chronic obstructive pulmonary disease (COPD)26,27. Findings
from cancer research25 also showed that elevated levels of IL-8 correspond to increased severity of numerous
cancers, including melanoma28. breast29, renal30, prostate31 gastric32, and colorectal cancers33. Our ndings suggest
that chronic use of heated tobacco products may lead to the development of lung diseases (especially COPD)
and cancer progression.
It is estimated that over half of HTP users use heated tobacco with other nicotine products, mostly cigarettes
or e-cigarettes (so-called dual users)34. In this study, elevated levels of IL-8 were observed also among exclusive
Table 1. Characteristics of the study population.
HTPs users (n = 44) Control group (n = 48)
n (%) n (%)
Gender
Male 44 (100.0) 48 (100.0)
Age
Mean ± SD 33.6 ± 7.27 33.4 ± 10.20
Min–max 21–46 20–56
Having higher education
No 19 (43.2) 19 (39.6)
Yes 25 (56.8) 29 (60.4)
Place of residence
City up to 10,000 residents 6 (13.6) 1 (2.1)
City from 10,000 to 20,000 residents 1 (2.3) 3 (6.3)
City from 20,000 to 50,000 residents 7 (15.9) 2 (4.2)
City from 50,000 to 100,000 residents 2 (4.5) 2 (4.2)
City from 100,000 to 200,000 residents 1 (2.3) 0 (0.0)
City from 200,000 to 500,000 residents 0 (0.0) 0 (0.0)
City above 500,000 residents 27 (61.4) 40 (83.3)
Occupational status
Full-time job 26 (59.1) 31 (64.6)
Part-time job 5 (11.4) 6 (12.5)
Self-employment 13 (29.5) 7 (14.6)
Student 3 (6.8) 12 (25.0)
Unemployed 1 (2.3) 1 (2.1)
Marital status
Single 13 (29.5) 14 (29.2)
Married 23 (52.3) 20 (41.7)
Informal relationship 8 (18.2) 14 (29.2)
Ever cigarette smoking 42 (95.5) 9 (18.8)
Ever e-cigarette use 37 (84.1) 10 (20.8)
Ever heated tobacco use 44 (100.0) 6 (12.5)
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heated tobacco users, which suggests that heated tobacco use is the sucient factor that increases the IL-8 levels.
Further studies should analyze dierences in blood biomarkers of inammation between exclusive heated tobacco
users and dual users.
Among all heated tobacco users, interleukin 8 level was positively correlated with the daily number of heated
tobacco sticks, which suggests a dose-dependent reaction. is observation is in line with the previous ndings
from tobacco research which proved that the risk of diseases is dose-dependent and varies on the number of
cigarettes smoked per day35,36.
Heated tobacco products are marketed as less harmful alternatives to cigarettes3,11. Findings from the
toxicological studies suggest that HTPs compared to cigarettes may be products with a reduced risk of respiratory
diseases, cardiovascular diseases, and cancer2,9. Out of 10 dierent biomarkers analyzed in this study, the level
Table 2. Characteristics of the heated tobacco users (n = 44).
HTPs users (n = 44)
n (%)
Current cigarette smoking 5 (11.4)
Current e-cigarette use 7 (15.9)
Number of heated tobacco sticks used per day
1 2 (4.5)
2–5 8 (18.2)
6–10 7 (15.9)
11–20 21 (47.7)
Over 20 6 (13.6)
How long do you use heated tobacco products?
Less than 1year 7 (15.9)
For 1year 4 (9.1)
For 2years 9 (20.5)
For 3years 13 (29.5)
For 4years 6 (13.6)
For 5years 5 (11.4)
Type of heated tobacco device
IQOS 23 (52.3)
Glo 18 (40.9)
lil SOLID 2.0 3 (6.8)
Most frequently used avors of tobacco sticks in the last 90days
Tobacco avor 11 (25.0)
Mint avor 13 (29.5)
Menthol avor 11 (25.0)
Blueberry avor 6 (13.6)
Do you use HTPs in places where smoking is prohibited?
Yes 24 (54.5)
No 20 (45.5)
Do you use HTPs indoors?
Yes 41 (93.2)
No 3 (6.8)
How soon aer waking up do you use heated tobacco?
Up to 5min 3 (6.8)
6–30min 13 (29.5)
31–60min 12 (27.3)
Over 60min 16 (36.4)
Do you have diculty refraining from using heated tobacco in places where it is prohibited?
Yes 7 (15.9)
No 37 (84.1)
Which heated tobacco stick is the most dicult for you to give up?
From rst in the morning 24 (54.5)
From any other 20 (45.5)
At what time of day do you use heated tobacco more oen?
Within the rst hours aer waking up 12 (27.3)
During the rest of the day 32 (72.7)
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Table 3. Blood biomarkers in regular heated tobacco users (all) and control group. SD standard deviation, SE
standard error, Min minimum, Max maximum, H heated tobacco users, C controls. *U Mann–Whitney test;
**Student’s t-test. Signicant values are in bold.
Study
group
Mean SD SE Median Min Max pn
IL-1β [pg/ml] H 44 1.91 1.02 0.15 1.60 0.59 5.43 0.9*
C 48 2.07 1.34 0.19 1.73 0.29 7.83
IL-6 [pg/ml] H 44 2.83 1.12 0.17 2.73 0.97 6.63 0.2*
C 48 3.57 3.03 0.44 3.11 1.03 21.9
IL-8 [pg/ml] H 44 6.86 16.5 2.49 4.37 1.97 113.76 0.01*
C 48 3.95 0.90 0.13 3.82 2.28 6.66
IL-10 [pg/ml] H 44 2.04 0.89 0.13 1.97 0.66 4.52 0.5*
C 48 2.29 1.21 0.17 1.91 0.66 5.20
IL-12p70 [pg/ml] H 44 17.47 11.42 1.72 14.42 0.00 49.76 0.1*
C 48 13.41 7.72 1.11 12.53 0.55 33.93
TNF-α [pg/ml] H 44 13.45 2.98 0.44 13.37 7.73 18.43 0.8*
C 48 13.57 3.84 0.55 13.23 6.79 26.91
CRP [mg/l] H 44 8.26 15.67 2.36 2.20 0.00 89.00 0.2*
C 48 17.77 59.19 8.54 4.21 0.00 404.03
Fibrinogen [mg/dl] H 44 162.86 88.40 13.33 125.20 83.47 358.51 0.2*
C 48 195.64 99.65 14.38 137.28 86.69 387.80
VCAM-1 [ng/ml] H 44 967.43 254.34 38.34 954.85 496.65 1685.09 0.9**
C 48 959.28 265.51 38.32 920.52 460.61 1482.51
ICAM-1 [ng/ml] H 44 394.54 448.81 67.66 250.27 98.25 2458.535 0.7*
C 48 424.29 485.77 70.11 236.11 27.48 1999.79
Table 4. Blood biomarkers in exclusive regular heated tobacco users and control group. SD standard
deviation, SE standard error, Min minimum, Max maximum, He exclusive heated tobacco users, C controls. *U
Mann–Whitney test; **Student’s t-test. Signicant values are in bold.
Study
group
Mean SD SE Median Min Max pn
IL-1β [pg/ml] He 33 1.95 1.11 0.19 1.56 0.59 5.43 0.9*
C 48 2.07 1.34 0.19 1.73 0.29 7.83
IL-6 [pg/ml] He 33 2.96 1.18 0.21 2.78 0.97 6.63 0.4*
C 48 3.57 3.03 0.44 3.11 1.03 21.9
IL-8 [pg/ml] He 33 7.76 19.06 3.32 4.39 2.33 113.76 0.01*
C 48 3.95 0.90 0.13 3.82 2.28 6.66
IL-10 [pg/ml] He 33 2.05 0.86 0.15 2.02 0.91 4.51 0.6*
C 48 2.29 1.21 0.17 1.91 0.66 5.20
IL-12p70 [pg/ml] He 33 17.77 12.5 2.18 14.53 0.00 49.76 0.2*
C 48 13.41 7.72 1.11 12.53 0.55 33.93
TNF-α [pg/ml] He 33 12.78 2.73 0.48 12.33 7.73 18.44 0.5*
C 48 13.57 3.84 0.55 13.23 6.79 26.91
CRP [mg/l] He 33 8.22 16.67 2.90 1.96 0.00 89.00 0.1*
C 48 17.77 59.19 8.54 4.21 0.00 404.03
Fibrinogen [mg/dl] He 33 161.80 88.97 15.49 124.89 83.47 358.51 0.2*
C 48 195.64 99.65 14.38 137.28 86.69 387.80
VCAM-1 [ng/ml] He 33 902.31 215.96 37.59 906.31 496.65 1384.20 0.3**
C 48 959.28 265.51 38.32 920.52 460.61 1482.51
ICAM-1 [ng/ml] He 33 366.67 358.52 62.41 248.84 98.25 1743.55 0.9*
C 48 424.29 485.77 70.11 236.11 27.48 1999.79
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of only one biomarker (IL-8) was signicantly higher among HTP users compared to controls. ere were no
dierences in the levels of important biomarkers like TNF-α, CRP, brinogen, and adhesion molecules as well
as other interleukins. In this study, exclusive cigarette smokers were not included as a separate group, and the
blood biomarkers of inammation were compared between heated tobacco users and nicotine-free controls. IL-6
is a predictor of the frequency of COPD exacerbation37 and is linked to chronic inammation-related cancers38.
However, in this study, there were no statistically signicant dierences in IL-6 levels between HTP users and
the control group. C-reactive protein levels are raised in stable COPD patients39. In this study, the mean age of
the subjects was 33years and participants diagnosed with COPD were excluded, so changes in CRP observed
usually in patients with COPD were not observed. ere were no signicant dierences in VCAM-1 and ICAM-
1, biomarkers associated with cardiovascular disease risk40, but due to the younger age of the study population,
this observation should be analyzed carefully as Lyytinen etal.17 revealed potential cardiovascular risk of HTP
use. e mode of operation of heated tobacco products diers from cigarette smoking, so the impact of regular
heated tobacco use on biomarkers of inammation may dier from those observed in cigarette smokers. However,
due to the limited sample size (preliminary results), our ndings should be interpreted carefully and point to the
need for further cohort studies and prospective analyses.
is study has practical implications for tobacco control and public health. Our ndings suggest that regular
heated tobacco use may lead to the development of chronic diseases (especially respiratory diseases) and the risk
is dose dependent. However, the risk of diseases may be lower than among cigarette smokers. Further cohort
studies are needed to assess the impact of regular heated tobacco use on the risk of chronic diseases, especially
those evoked by chronic inammation.
ere are several limitations of this study. is is a single-point case–control study. e history of heated
tobacco use was based on self-reported data but veried by the cotinine levels and set of questions on tobacco
use patterns. Findings are based on single-point observations, and follow-ups are needed to assess the impact
of heated tobacco use on the dynamics of blood biomarkers (especially proinammatory cytokines) changes.
is is a pilot study, so the number of participants is low. e follow-up study is planned for 2024 and the more
advanced analyses will be published aer the completion of the study group. In one subject, the IL-8 levels were
113.76pg/ml, however, even aer the exclusions of this subject from the analysis, dierences in IL-8 levels
between heated tobacco users and control were statistically signicant. is study aimed to assess the real-life
pattern of heated tobacco use, so exposure to HTPs diers between the participants. Nevertheless, this is one
Table 5. Correlation between the levels of blood biomarkers and the daily number of heated tobacco sticks
used in the last 90days among all heated tobacco users (n = 44). r the Spearman rank correlation coecient.
Signicant values are in bold.
r p
IL-1β & daily number of heated tobacco sticks − 0.11 0.5
IL-6 & daily number of heated tobacco sticks − 0.01 0.9
IL-8 & daily number of heated tobacco sticks 0.37 0.01
IL-10 & daily number of heated tobacco sticks − 0.02 0.9
IL-12p70 & daily number of heated tobacco sticks 0.11 0.46
TNF-α & daily number of heated tobacco sticks 0.03 0.83
CRP & daily number of heated tobacco sticks − 0.11 0.5
Fibrinogen & daily number of heated tobacco sticks − 0.09 0.5
VCAM-1 & daily number of heated tobacco sticks − 0.27 0.08
ICAM-1 & daily number of heated tobacco sticks − 0.20 0.2
Table 6. Correlation between the levels of blood biomarkers and the daily number of heated tobacco sticks
used in the last 90days among exclusive heated tobacco users (n = 33). r the Spearman rank correlation
coecient.
r p
IL-1β & daily number of heated tobacco sticks − 0.04 0.8
IL-6 & daily number of heated tobacco sticks − 0.10 0.6
IL-8 & daily number of heated tobacco sticks 0.08 0.6
IL-10 & daily number of heated tobacco sticks − 0.20 0.3
IL-12p70 & daily number of heated tobacco sticks 0.03 0.9
TNF-α & daily number of heated tobacco sticks − 0.06 0.8
CRP & daily number of heated tobacco sticks 0.01 0.9
Fibrinogen & daily number of heated tobacco sticks − 0.17 0.3
VCAM-1 & daily number of heated tobacco sticks − 0.18 0.3
ICAM-1 & daily number of heated tobacco sticks − 0.11 0.5
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of the rst case–control studies on the health eects of heated tobacco use, carried out in humans. Due to the
increasing prevalence of HTP use and limited scientic data on their health consequences, investigating cause-
and-eect relationships in this area is a new challenge for public health. e results of our study demonstrated
that only one of the ten blood-based biomarker of inammation (IL-8) was signicantly higher among the eect
use of heated tobacco on blood-based biomarkers of inammation and higher interleukin 8, one of the main
measures of inammation, in users of heated tobacco compared to non-smokers. Given the lack of signicant
changes in the other biomarkers of inammation, the relevance of increases in IL8 needs further investigations
to better understand the complex interplay among various interleukins.
In conclusion, daily use of heated tobacco during the last 90days was associated with an increased level of
IL-8 among heated tobacco users compared to healthy controls who do not use nicotine products. An increase
in IL-8 level was positively correlated with the number of heated tobacco sticks consumed per day. ere were
no dierences in 9 of 10 blood biomarkers of inammation between HTP users and controls, so heated tobacco
products may be a modied risk tobacco product compared to combustible cigarettes.
Data availability
e datasets generated during and analysed during the current study are available from the corresponding author
on reasonable request.
Received: 12 December 2023; Accepted: 9 April 2024
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Author contributions
BS, MJ, DK conceived of the study and participated in study design, data interpretation. BS and DK coordinated
the measurements and data collection. MJ was responsible for statistical analysis. DK was responsible for project
supervision. BS, MJ, DK worked on manuscript. All authors read and approved the nal manuscript.
Funding
is study was funded from the state budget under the program of the Polish Minister of Education and Science
called "Science for Society", project entitled: "Use of heated tobacco—health and social aspects" project no.
NdS/549728/2022/20222 of November 2, 2022 amount co-nancing PLN 1,013,150.00 total project value PLN
1,013,150.00, Polska.
Competing interests
e authors declare no competing interests.
Additional information
Correspondence and requests for materials should be addressed to B.Ś.
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