TITLE: The association of vitamin D with inflammatory cytokines in diabetic
peripheral neuropathy

  ABSTRACT:
[Purpose] The effects of vitamin D on the circulating levels of IL-17 and IL-13 were
investigated in patients with diabetic peripheral neuropathy, patients with diabetes
mellitus type 2 without neuropathy, and healthy controls. [Subjects and Methods] A
single-blind controlled clinical study was performed, including70 type 2 diabetic patients
with or without diabetic peripheral neuropathy and 33 healthy volunteer controls. The
25(OH)D levels were evaluated using ultra-performance liquid chromatography, and IL-17 and
IL-13 levels were assessed using enzyme-linked immunosorbent assays. [Results] The 25(OH)
vitamin D concentration was lower in diabetic peripheral neuropathy patients than in
diabetes mellitus patients without neuropathy and healthy controls. Similarly, 25(OH)D
levels were lower in diabetes mellitus patients than healthy controls. IL-17 and IL-13
levels were higher in diabetes mellitus patients than in controls. Additionally, IL-13
levels were higher in diabetic peripheral neuropathy patients than in diabetes mellitus
patients without neuropathy. These differences were statistically significant. There was a
significant positive correlation between 25(OH)D and IL-13,and a negative correlation
between 25(OH)D andIL-17 in the diabetic and diabetic neuropathy groups. [Conclusion]
Vitamin D is a potential modifiable risk factor for diabetic peripheral neuropathy and may
regulate inflammatory mediators, e.g., IL-17 and IL-13.

BODY.INTRODUCTION:
The global epidemic of diabetes mellitus (DM) and related complications are increasing
worldwide. Diabetic peripheral neuropathy (DPN) is a microvascular complication that affects
up to 50% of these diabetic patients and is a major cause of mortality and morbidity in this
population. The complex etiology of DPN is still not clear1, 2). However, hyperglycemia,
decreased blood flow, hypoxia, hypoxia-induced proangiogenesis, and pro-inflammatory
responses may play a role in the pathogenesis. Moreover, proinflammatory cytokines, such as
interleukins (IL), affect neurons and glial cells and are involved in the pathology of
diabetic neuropathy3).  Vitamin D deficiency is a suspected risk factor for DPN because it is related to
inflammation and hyperglycemia. Accordingly, deficiencies in vitamin D are associated with
an altered incidence of infections. Several studies have evaluated this relationship4, 5).  IL-13 is an immunoregulatory cytokine secreted mainly by activated Th (T helper)2 cells.
Additionally, IL-13 suppresses the production of pro-inflammatory cytokines and
prostaglandins by monocytes and macrophages6). IL-17 is a proinflammatory cytokine produced by activated Th17;it
has an important role in the regulation of immune responses. Moreover, the pathogenic
effects of Th17 cells are regulated by Th2 cytokines, such as IL-4, IL-5, IL-10, and
IL-137).  Proinflammatory cytokines and vitamin D deficiencies are thought to play a role in DPN
pathogenesis. The identification of the pathogenesis of DPN and its relationships with
modifiable risk factors may facilitate the development of novel therapies. Since vitamin D
deficiency is a potential modifiable risk factor for DPN, the aim of the present study was
to investigate the effects of vitamin D on the circulating levels of IL-17 and IL-13 in
patients with DPN, DM Type 2 patients without DPN, and healthy control individuals.

BODY.SUBJECTS AND METHODS:
The procedures conformed to the ethical standards of the Responsible Committee on Human
Experimentation and with the Declaration of Helsinki. This study was approved by the local
ethics committee of Namik Kemal University. Informed consent was obtained from all
individuals before inclusion in the study.  Demographic data, HbA1c, fasting blood glucose, serum lipid profiles, arterial blood
pressure, and medical data were recorded for all participants.  The diagnosis of DPN included evaluations of the clinical symptom history, a neurological
examination, electrophysiological tests, quantitative sensory testing, and autonomic
function tests. The diagnoses were based on clinical symptoms and the results of the entire
electromyography8,9,10). Medical data were
documented. Patients with type 1 DM, clinical evidence of cardiovascular or cerebrovascular
disease, hepatic or renal failure, malignancy, autoimmune diseases, acute or chronic
infections, a history of trauma or surgery, or pregnancy were excluded.  Group I (n=33) was composed of type 2 DM patients without DPN and group II (n=37) was
composed of type 2 DM patients with DPN. Group III (n=33) was composed of healthy control
subjects who were age-, gender-, and body mass index-matched with patients in the DM
groups.  Blood samples were collected after a 30-minute resting period between 8:30 and 9:30 a.m.
Serum was centrifuged at 2,000 × g for 15 minutes at 4 °C. All samples were stored at −80 °C
until the analysis.  Fasting plasma glucose, total cholesterol, HDL-cholesterol, and triglycerides were analyzed
using an automated analyzer (Cobas e6000-e501; Roche Diagnostics, Tokyo, Japan).
LDL-cholesterol was calculated using the Friedewald formula11).  Serum 25(OH)D levels were evaluated using ultra-performance liquid chromatography with a
ClinRep commercial kit (Germany) and an UPLC analyzer (Thermo Scientific, Waltham, MA, USA).
The results are reported in ng/ml. Serum IL-17 concentrations were measured using the RayBio
Human IL-17 ELISA Kit (Atlanta, GA, USA).The sensitivity of the IL-17 commercial kits was
80 pg/ml, and the intra- and inter-assay coefficients of variation were <10% and <12%,
respectively. Serum IL-13 concentrations were measured using the RayBio Human IL-13 ELISA
Kit. The sensitivity of the IL-13 commercial kit was 0.15 pg/ml. The intra- and inter-assay
coefficients of variation were <10% and <12%, respectively.  Statistical analyses were performed with SPSS version 17 (Chicago, IL, USA). The
homogeneity of data within groups was analyzed with the Shapiro-Wilk test. Results are
expressed as means ± standard deviation or medians and ranges, depending on the distribution
of data. Normally distributed data were analyzed by independent t-tests and
non-normally distributed data were analyzed by Mann-Whitney U tests. In normally distributed
multiple group comparisons, one-way ANOVA with the Bonferroni correction was used. The
Pearson's test was used for the correlation analysis. The alpha significance level was set
at <0.05.

BODY.RESULTS:
DM patients without DPN (Group I: 15 males and 18 females), diabetic patients with DPN
(Group II: 17 males and 20 females), and healthy volunteers (Group III: 15 males and 18
females) were enrolled in the study. Demographic data for each group were recorded. No
significant difference was observed in terms of age, gender, and body mass index among
groups (p>0.05).  No statistically significant difference was found between Group I and Group II in terms of
height, weight, FBG, systolic and diastolic blood pressure, HbA1C, TC, TG, and HDL and LDL
levels.  The 25(OH)D levels of Group I and Group II were lower than those of Group III (p=0.036 and
p<0.001, respectively). The 25(OH)D levels of Group II were significantly lower than
those of Group I (p=0.005).  IL-17 levels of Group I and Group II were higher than those of Group III (p<0.001 and
p<0.001, respectively). The difference in IL-17 levels between Group II and Group I was
not statistically significant (p=0.897).  The IL-13 levels of Group I and Group II were higher than those of group III (p=0.038 and
p<0.001, respectively). The IL-13 levels of Group II were significantly higher than those
of Group I (p<0.001). Biochemical test results are described in Table 1Table 1.Demographic data and inter-group comparisons of biochemical parameters
.  A significant positive correlation was detected between IL-13 levels and 25(OH)D levels
(r=0.301, p=0.013). There was a significant negative correlation
betweenIL-17and 25(OH)D levels (r=−0.354, p=0.003).

BODY.DISCUSSION:
There is increasing evidence that nerve tissues in DM undergo a pro-inflammatory process
that causes symptoms and precipitates the development of neuropathy. Certainly, macrophages
and lymphocytes invade the diabetic nerves and release IL or TNF-α12,13,14).  Moreover, there is considerable evidence that pro-inflammatory cytokines play a role in the
pathogenesis of diabetic neuropathy. New data regarding the relationship between neuropathy
and inflammatory cytokines have resulted in new approaches to suppress neuropathy via
specific targets of cytokines15,16,17,18,19). Nevertheless,
very few studies have examined DPN and cytokines.  Doupis et al. evaluated the relationship between inflammation and microvascular reactivity
as well as the development of DPN. They suggested that DPN is associated with altered levels
of biochemical indicators of endothelial dysfunction andinflammation16).  Hang et al. evaluated the roles of plasma cytokines, including IL-13 and IL-17, in another
kind of microvascular complication of DM, diabetic retinopathy (DR). DPN and DR have a
similar pathogenetic basis. Hang et al. assessed cytokine levels and their relationship with
the severity of DR and found significantly elevated cytokine levels, including IL-13 and
IL-17, in patients with DM. TNF-α plasma levels were higher in proliferative-DR compared
with the levels in patients with non-proliferative DR and patients with no apparent DR20).  In this study, IL-13 and IL-17 levels of diabetes patients with and without DPN were higher
than the levels observed in the control group. While the IL-13 levels of DPN patients were
statistically significantly higher than those of diabetes patients without DPN, the
difference in IL-17 levels between the groups was not statistically significant. These
differences in cytokine levels might reflect known inflammatory processes in diabetes and
its complications. Moreover, IL-13 levels might indicate DPN development in type 2 DM.  Additionally, the status of vitamin D deficiency affects the immune system and tends to
increase infections4, 5). Since IL-13 is an immunoregulatory factor and IL-17 is a
proinflammatory cytokine, the effects of vitamin D levels on these cytokines may provide
insight into the pathogenesis of DPN. The role of vitamin D in the prevention and treatment
of various neurological diseases has been evaluated for several years. The effect of vitamin
D is confirmed by VDR and CYP27B1 expression in glial cells in the nervous system21). Despite many studies on the relationship
between diabetes and its complications and vitamin D deficiency, there is little data
concerning the association between neuropathy and vitamin D deficiency.  Several studies suggest a possible relationship between DPN and vitamin D deficiency in
type 2 DM patients, but the general pattern is in conclusive. Therefore, it is still unclear
whether serum 25(OH) D levels are related to DPN risk in type 2 DM patients22).  Shebab et al. evaluated the incidence of vitamin D deficiency in 210 type 2 DM patients
with and without DPN. In total, 81.5% of diabetic neuropathy patients had a vitamin D
deficiency, compared with 60.4% of patients without diabetic neuropathy. Accordingly, the
authors suggested that vitamin D deficiency is a risk factor of DPN5).  Ahmadieh et al. investigated 25(OH)D levels in diabetic neuropathy patients compared to
those without neuropathy. They found lower vitamin D levels in DPN patients and suggested
that 25(OH)D vitamin levels are significant predictors of diabetic neuropathy23).  Skalli et al. evaluated serum 25(OH)D levels in 111 DPN patients. They found significantly
lower 25(OH) D levels in the group with DPN than without DPN24).  The results of this study were consistent with the limited literature on this issue.
Vitamin D levels in diabetic patients were lower than those in the control group. Meanwhile,
vitamin D levels of the DPN group were lower than those of non-neuropathic diabetes
patients. Vitamin D levels had a positive correlation with IL-13 and a negative correlation
with IL-17. These results suggested that vitamin D impacts DPN development via inflammatory
pathogenesis.  Highlighting the pathogenesis of DPN and relationships with modifiable risk factors as well
as methods for the early diagnosis of DPN could help inhibit disease progression and
facilitate the development of new treatment strategies with a molecular basis. Cytokines
like IL-13 and IL-17 may reflect low-grade inflammation in DM. Moreover, IL-13 might
indicate DPN in type2 DM patients. Additionally, vitamin D deficiency seems to play a role
in the development of DPN, and it is highly likely to be a modifiable risk factor for DPN in
type 2 diabetes patients.  This study had a few limitations that should be considered. The number of patients included
was relatively small. The study design was cross-sectional, and it was a single-center
study. Additional studies with larger sample sizes and multi-center designs are needed to
confirm the results.

Question: Compared to diabetes mellitus patients without neuropathy (GI) what was the result of diabetic peripheral neuropathy patients (GII) on 25(OH) vitamin D concentration?
Evidence: The 25(OH)D levels of Group II were significantly lower than
those of Group I (p=0.005).  
Answer: significantly decreased