Cytokine Profile Analysis of IL - and TNF-α in Diabetic Patients
Infected by C.albicans
Regina Purnama Dewi Iskandar
1
, Retno Pudji Rahayu
2
1
Graduate Student of Immunology, Postgraduate School, Universitas Airlangga;
2
Department of Oral and Maxillofacial Pathology, Faculty of Dental Medicine, Universitas Airlangga
Keywords: Diabetes mellitus, C.albicans, oral candidiasis, IL-1β, and TNF-α
Abstract: Background: The prevalence of Diabetes Mellitus (DM) in Indonesia in 2020 is expected to affect more
than 7 million people. The disease would lead to complication and oral manifestations. One of the oral
manifestations of DM is an oral infection caused by Candida albicans. The patients are often in an
immunocompromised state with an aberrant immune response. It was reported that diabetic patients have a
higher level of pro-inflammatory cytokines. Objectives: This study aims to analyze the cytokine profile of
diabetic patients infected with C.albicans. Methods: The subjects consist of 33 people with oral candidiasis,
who were grouped into regulated DM, unregulated DM, and non-DM individuals. HbA1C was measured to
classify the patients into the regulated DM or unregulated DM groups. Diagnosis of oral candidiasis was
based on the clinical appearance of oral mucosa and Papanicolaou staining. There was 7cc of peripheral
blood that was obtained from all subjects to analyze IL- and TNF-α levels, using an indirect ELISA
technique. The data were statistically analyzed using one-way ANOVA. Results: The level of IL-and
TNF-α were significantly different (α = 0.05) between each group. On the contrary, there was no significant
difference in IL-and TNF-α level between the regulated DM group and the control group. Conclusion:
The oral candidosis plays a role in altering pro-inflammatory cytokines levels in individuals with DM, in
both regulated and unregulated groups.
1 INTRODUCTION
Diabetes Mellitus (DM) is indicated with chronic
hyperglycemia due to impaired insulin secretion and
function (Bigna et al., 2018). As well as in
Indonesia, the global prevalence of DM is
continuously increasing each year. There were
approximately 415 million people suffering from
DM according to the International Diabetes
Foundation (Tankeu et al., 2016). It is estimated that
the world prevalence of DM will increase to 7.7%
(439 million) by 2030. An epidemiological study of
diabetic patients in Indonesia showed that the
prevalence of DM is 5.7% (Mihardja et al., 2014).
There were 8.5 million of adults with DM in
Indonesia in 2013, while in 2035 it is estimated there
will be 14.1 million of adults suffering from DM
based on the International Diabetes Foundation
(Forouhi and Wareham, 2014). The disease may
progressively develop complications with increased
morbidity, disability, and mortality that threatens life
(Papatheodorou et al., 2016).
The mechanism that underlies complications is
Advanced Glycation End (AGE) products. The
substance targets extracellular proteins to undergo
damaging crosslinks (Nass et al., 2007). The
accelerated AGE formation occurs in people with
DM as an effect of abundant concentration of
glucose, AGE precursors, and oxidative stress.
AGEs are known for their damaging effect of
inducing cell death, reducing cell adhesion and
migration, and interfering protein function
(Nowotny et al., 2015). Moreover, AGE deposition
causes complications and oral manifestations in
people with DM (Nass et al., 2007).
One of the oral manifestations in diabetic
patients is a candida infection. The frequency of oral
infection with candida is higher in diabetic patients
due to a high concentration of salivary glucose and
low salivary secretion that facilitates the adherence
of yeast in epithelial cells (Obradovet al., 2011).
Diabetic patients are prone to infection because of
Purnama Dewi Iskandar, R. and Pudji Rahayu, R.
Cytokine Profile Analysis of IL - 1ç and TNF-a in Diabetic Patients Infected by C.albicans.
DOI: 10.5220/0007541402810284
In Proceedings of the 2nd Inter national Conference Postgraduate School (ICPS 2018), pages 281-284
ISBN: 978-989-758-348-3
Copyright
c
2018 by SCITEPRESS – Science and Technology Publications, Lda. All rights reserved
281
their impaired immune system. Chronic
hyperglycemia provides a disadvantageous
environment that compromises the immune system,
which is known as the immunocompromised state.
The impaired immune system is characterized by
suppressed neutrophil function, antioxidant system,
cellular and humoral immunity (Casqueiro et al.,
2012). Diabetic patients have a 21% higher risk of
infection than non-diabetic people, and it affects all
organs and systems, as well as oral cavities (Duka et
al., 2017). DM is reported to elevate production and
function of Interleukin 1-beta (IL-) and Tumor
necrosis factor-alpha (TNF-α) to facilitate systemic
and tissue inflammation, as well as contribute to
insulin resistance (Cardoso et al., 2017; Mohammadi
et al., 2017; Peiró et al., 2017). Based on the pivotal
roles of IL- and TNF-α in DM and oral
candidiasis, the present study aims to analyze the
cytokine profile of IL- and TNF-α in people
diagnosed with DM and Oral candidiasis.
2 MATERIALS AND METHODS
The subjects consist of 33 people with oral
candidiasis who were grouped into regulated DM,
unregulated DM, and non-diabetic. The
measurement of HbA1C was performed to classify
the patients into regulated DM or unregulated DM
groups. Subjects with HbA1C higher than eight were
considered as unregulated DM, subjects with
HbA1C ranges from 6.5 to 8 were considered as
regulated DM, while subjects with HbA1C lower
than 6.5 were considered as a non-diabetic
population or control. The subjects were diagnosed
from suffering oral candidiasis based on the clinical
appearance of oral mucosa and laboratory
examination. There were 7 ccs of peripheral blood
obtained from all subjects to analyze the level of
cytokines IL-and TNF-α using an indirect ELISA
technique. Scrubbing of oral mucosa was performed
to obtain C.albicans and was cultured in Saboroud
Dextrose Agar medium (Difco) afterwards to
conduct a gram staining and carbohydrate
fermentation test. The obtained C.albicans were also
stained using Papanicolaou staining. The data were
statistically analyzed using a one-way ANOVA test.
3 RESULTS
Indirect Enzyme Linked Immuno Assay (ELISA)
was performed to analyze IL- and TNF-α level
from the blood serum of all subjects. The ELISA is
an effective method to identify the
immunocompromised status of subjects with
regulated DM, unregulated DM, and non-diabetic
control affected with oral candidiasis. According to
the one-way ANOVA analysis, it was shown that p
< 0,012 = 0,05). In accordance with IL-1β, the
result for TNF-α is p < 0,007 = 0,05), then it was
followed by the Tukey HSD test.
Figure 1 : The level of IL- (pg/ml) in non-diabetic
subjects, subjects with DM, and subjects with unregulated
DM.
Figure 2 : The level of TNF-α (pg/ml) in non-diabetic
subjects, subjects with DM, and subjects with
unregulated DM.
Statistical analysis showed there were significant
differences = 0,05) for IL- and TNF-α analysis
between unregulated diabetic patients groups and
non-diabetic control populations infected with
C.albicans. The level of IL- in unregulated
diabetic patients showed no insignificant difference
compared to non-diabetic group (Figure 1). There
was a significant difference in TNF-α level between
unregulated diabetic patients compared to the non-
diabetic group, while the level of TNF-α in regulated
diabetic patients group and the non-diabetic group
was not significant (Figure 2). The aberrant cytokine
profile obtained from the present study was due to
the impaired immune response in unregulated
diabetic patients that induced AGEs product to
stimulate a higher level of IL-1β and TNF-α.
31
33.62
35.79
28
29
30
31
32
33
34
35
36
37
Kontrol Regulasi Baik Regulasi Jelek
Kadar IL1-
(pg/ml)
Control
Regulated DM
Level of IL
-1Zβ
(pg/ml)
201.13
215.43
233.25
180
190
200
210
220
230
240
Kontrol Regulasi Baik Regulasi Jelek
Kadar TNF-
(pg/ml)
Control
Regulated DM
Unregulated
DM
Level of TNF
-α
(pg/ml)
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4 DISCUSSION
An indirect ELISA (ELISA Bendermed system Kit)
was used in the present study to analyze the
immunocompromised state with oral candidiasis in
unregulated diabetic patients, regulated diabetic
patients, and non-diabetic control populations. Oral
infection by C.albicans stimulated pro-inflammatory
cytokines. IL-and TNF-α level were investigated
in the present study as they are major cytokines
involved in inflammation both in diabetic and non-
diabetic populations (Peiró et al., 2017).
The level of IL- and TNF-α in the present
study was not measured in local tissue, but
systemically through plasma obtained from the
whole blood. This consideration is due to the fact
that there is no significant difference in the oral
mucosal immune response in C.albicans infection
between the regulated diabetic, unregulated diabetic,
and non-diabetic populations. According to
statistical analysis, there was a significant difference
= 0,05) of IL-level in the unregulated diabetic
group compared to the IL-level in the regulated
diabetic and non-diabetic populations (Figure 1).
The level of TNF-α in regulated diabetic and non-
diabetic groups was significant = 0,05; Figure 2).
On the contrary, the level of IL-and TNF-α in the
regulated diabetic group were not significantly
different to their level in a non-diabetic population
(Figures 1 and 2). The data obtained from the study
reveals that the immune response in regulated
diabetic patients is relatively impaired and could
generate an immune response against C.albicans. It
is confirmed through ELISA analysis that pro-
inflammatory cytokine levels in regulated diabetic
patients were not significantly different from the
non-diabetic population. Whereas the pro-
inflammatory cytokine levels were exacerbated in
unregulated diabetic patients, represented by
cytokine IL-and TNF-α.
Prolonged hyperglycemia initiates a non-
enzymatic glycosylation process that alters the
structure and function of proteins and biologic
molecules (Negre-Salvayre et al., 2009). AGEs were
formed through prolonged glycation induced by
chronic hyperglycemia. AGEs stimulate
macrophages to continuously release pro-
inflammatory cytokines, particularly IL- and
TNF-α (Byun et al., 2017). The literature is coherent
to results in the present study, which stated that the
levels of IL-and TNF-α in unregulated diabetics
were significantly higher = 0,012 for IL- and α
= 0,05 for TNF-α) than in unregulated diabetics and
the control group. The underlying mechanism that
makes unregulated diabetic patients prone to
infection is the high concentration of pro-
inflammatory cytokines detected in unregulated
diabetic patients impaired immune response, in
addition to AGE (Cardoso et al., 2017; Mohammadi
et al., 2017; Peiró et al., 2017).
The immunocompromised condition that
commonly affects unregulated diabetic patients
suppresses antigen recognition activity, interferes
with phagocytosis, and intracellular killing that
would lead to immune defects (Gordon, 2016).
Moreover, reduced cytokines levels also occur in
diabetic patients due to the impaired function of
polymorphonuclear (PMN) and macrophages. T
lymphocyte deficiency and neutrophil dysfunction
contribute to the pathogenesis of oral candidiasis.
Cytokines mainly regulate the activity of PMN.
However, the antifungal activity of PMN is initiated
by Large Granular Lymphocytes (LGLs) that secrete
interferon-γ (IFN-γ), interferon-α (IFN-α), TNF-α,
and IL-1. Thus, lymphocyte deficiency may reduce
cytokine levels secreted by Th1 and Th2
(Samaranayake et al., 1990).
5 CONCLUSION
The oral candidosis plays a role in altering pro-
inflammatory cytokine levels in individuals with
DM, in both the regulated and unregulated groups.
Unregulated diabetic patients in this study had
significantly greater IL- and TNF-α levels
compared to regulated diabetic patients and non-
diabetic controls. Moreover, the levels of pro-
inflammatory cytokines could be used to determine
the severity of diabetic patients and their risk of
suffering from oral candidiasis.
REFERENCES
Bigna JJ, Nansseu JR, Katte J-C, Noubiap JJ. 2018.
Prevalence of Prediabetes and Diabetes Mellitus
Among Adults Residing in Cameroon: A
Systemic Review and Meta-Analysis. Diabetes
Research and Clinical Practice; 137: 109-18.
Byun K, Yoo YC, Son M, Lee J, Jeong G-B, Park
YM, Salekdeh GH, Lee B. 2017. Advanced
Glycation End-Products Produced Systemically
And By Macrophages: A Common Contributor
To Inflammation And Degenerative Diseases.
Pharmacology & Therapeutics; 177: 44-55.
Cytokine Profile Analysis of IL - 1ç and TNF-a in Diabetic Patients Infected by C.albicans
283
Cardoso JF, Gomes KB, Fernandes AP, Domingueti
CP. 2017. Evaluation of Cytokines Type 1
Diabetes Patients with And Without
Retinopathy. J Bras Patol Med Lab; 53(1): 31-7.
Casqueiro J, Casqueiro J, Alves C. 2012. Infections
in Patients With Diabetes Mellitus: A Review of
Pathogenesis. Indian J Endocrinol Metab; 16(1):
S27-36.
Duka E, Puca E, Çomo N, Pipero P, Harxhi A,
Akshija I, Resuli M, Kraja D. 2017. Infections in
Immunocompromised Patients from Diabetes
Mellitus. International Journal of Healthcare
Sciences; 5(1): 583-7.
Forouhi NG, Wareham NJ. 2014. Epidemiology of
Diabetes. Medicine; 42(12): 698 702.
Gordon S. 2016. Phagocytosis: An Immunobiologic
Process. Immunity; 44: 463-75.
Mihardja L, Soetrisno U, Soegondo S. 2014.
Prevalence and Clinical Profile of Diabetes
Mellitus in Productive Aged Urban Indonesians.
J Diabetes Invest; 5: 507-12.
Mohammadi M, Gozashti MH, Aghadavood M,
Mejdizadeh MR, Hayatbakhsh MM. 2017.
Clinical significance of Serum IL-6 and TNF-α
Levels in Patient With Metabolic Syndrome.
Reports of Biochemistry and Molecular Biology;
6(1): 74-9.
Nass N, Bartling B, Santor AN, Scheubel RJ,
Börgermann J, Silber RE, Simm A. 2007.
Advanced Glycation End Products, Diabetes
And Ageing. Zeitschriftr Gerontologie und
Geriatrie; 40(5): 349-56.
Negre-Salvayre A, Salvayre R, Augé N, Pampiona
R, Portero-Otín M. 2009. Hyperclicemia and
Glycation in Diabetic Complications.
Antioxidant & Redox Signaling; 11(12): 3071-
109.
Nowotny K, Jung T, hn A, Weber D, Grune T.
2015.Advanced Glycation End Products and
Oxidative Stress in Type 2 Diabetes Mellitus.
Biomolecules; 5: 194-222.
Obradović RR, Kesić LG, PejčAN, PetrovMS,
Živkov ND, Živković DM. 2011. Diabetes
Mellitus and Oral Candidiasis. Acta
Stomatologica Naissi; 27(63): 1025-34.
Papatheodorou K, Papanas N, Banach M,
Papazoglou D, Edmonds M. 2016.
Complications of Diavetes 2016. Journal of
Diabetes Research; vol. 2016; article ID
6989453.
Peiró C, Lorenzo Ó, Carraro R, nchez-Ferrer CF.
2017. IL- Inihibtion in Caridovascular
Complications Associated to Diabetes Mellitus.
Front. Pharmacol; 8: 363.
Samaranayake L P and Macfarlane TW. 1990. Oral
Candidosis. First Ed. Butterworth and co Ltd
Publishing London Boston Singapore: 16-132
Tankeu AT, Bigna JJ, Nansseu JR, Endomba FTA,
Wafeu GS, Kaze AD, Noubiap JJ. 2017. Global
prevalence of diabetes mellitus in patients with
tuberculosis: a systematic review and meta-
analysis protocol. BMJ;7:e015170.
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