Cytokine Profile Analysis of IL - 1β and TNF-α in Diabetic Patients

Infected by C.albicans

Regina Purnama Dewi Iskandar

, Retno Pudji Rahayu

Graduate Student of Immunology, Postgraduate School, Universitas Airlangga;

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-1β and TNF-α levels, using an indirect ELISA

technique. The data were statistically analyzed using one-way ANOVA. Results: The level of IL-1β and

TNF-α were significantly different (α = 0.05) between each group. On the contrary, there was no significant

difference in IL-1β 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 (Obradović et al., 2011).

Diabetic patients are prone to infection because of

Purnama Dewi Iskandar, R. and Pudji Rahayu, R.

Cytokine Proﬁle Analysis of IL - 1Ã§ 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

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-1β) 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-1β and TNF-α in DM and oral

candidiasis, the present study aims to analyze the

cytokine profile of IL-1β 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-1β 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-1β 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 (α = 0,05). In accordance with IL-1β, the

result for TNF-α is p < 0,007 (α = 0,05), then it was

followed by the Tukey HSD test.

Figure 1 : The level of IL-1β (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 (α = 0,05) for IL-1β and TNF-α analysis

between unregulated diabetic patients groups and

non-diabetic control populations infected with

C.albicans. The level of IL-1β 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.

33.62

35.79

Kontrol Regulasi Baik Regulasi Jelek

Kadar IL1-

(pg/ml)

Control

Regulated DM

Unregulated

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

Level of TNF

-α

(pg/ml)

ICPS 2018 - 2nd International Conference Postgraduate School

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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-1β 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-1β 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

(α = 0,05) of IL-1β level in the unregulated diabetic

group compared to the IL-1β 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 (α = 0,05; Figure 2).

On the contrary, the level of IL-1β 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-1β 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-1β and

TNF-α (Byun et al., 2017). The literature is coherent

to results in the present study, which stated that the

levels of IL-1β and TNF-α in unregulated diabetics

were significantly higher (α = 0,012 for IL-1β 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-1β 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.

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