|Year : 2022 | Volume
| Issue : 1 | Page : 9-12
Association of vitamin D with macrophage migration inhibitory factor and interleukin-8 in diabetic foot infection
Shalbha Tiwari1, Daliparthy D Pratyush2, Sanjeev K Gupta3, Surya K Singh4
1 Department of Endocrinology and Metabolism, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh; Department of Research, Chellaram Diabetes Institute, Pune, Maharashtra, India
2 Department of Endocrinology and Metabolism, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh; Division of ECD, Indian Council of Medical Research DHR, New Delhi, India
3 Department of General Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
4 Department of Endocrinology and Metabolism, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
|Date of Submission||20-Oct-2021|
|Date of Decision||01-Nov-2021|
|Date of Acceptance||02-Nov-2021|
|Date of Web Publication||07-Jan-2022|
Surya K Singh
Department of Endocrinology and Metabolism, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221 005, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: Diabetic foot wound is an important health challenge throughout the world. Interleukin 8 (IL8) and macrophage-migration inhibitory factor (MIF) play a significant role in host defense and in wound healing. Immune perturbation and Vitamin D deficiency also contribute to nonhealing of diabetic foot wounds. We aimed to evaluate the serum concentration of IL8 and MIF in diabetic patients with and without foot infection and its association with Vitamin D status. Material and Methods: Serum concentrations of 25 dihydroxyvitamin Vitamin D and cytokines (IL8 and MIF) were measured by radioimmunoassay and enzyme-linked immunosorbent assay, respectively, from 100 subjects with diabetes and foot infection as cases and 73 subjects with diabetes without foot infection as controls. Data were presented as mean (± standard error of mean [SE]) unless otherwise indicated and were analyzed by SPSS 16.0. Results: There was no significant difference in the mean (±SE) of age, duration of diabetes, hemoglobin A1C, and body mass index between the two groups. The mean (±SE) concentration of IL8 was significantly higher and MIF was significantly lower in cases than controls. Vitamin D correlated negatively with IL8 (r = −0.191) and positively (r = 0.1) with MIF. However, only the former correlation was statistically significant (P = 0.01). IL8 was also significantly high in patients with severe Vitamin D deficiency (<10 ng/ml) compared to patients with Vitamin D more than 10 ng/ml. Conclusion: Serum concentration of IL8 is significantly higher in diabetic foot infections compared to controls. Severe Vitamin D deficiency was associated with IL8 concentration in patients with diabetes.
Keywords: Cytokines, diabetic foot, interleukin 8, macrophage-migration inhibitory factor, Vitamin D
|How to cite this article:|
Tiwari S, Pratyush DD, Gupta SK, Singh SK. Association of vitamin D with macrophage migration inhibitory factor and interleukin-8 in diabetic foot infection. Chron Diabetes Res Pract 2022;1:9-12
|How to cite this URL:|
Tiwari S, Pratyush DD, Gupta SK, Singh SK. Association of vitamin D with macrophage migration inhibitory factor and interleukin-8 in diabetic foot infection. Chron Diabetes Res Pract [serial online] 2022 [cited 2023 Mar 29];1:9-12. Available from: https://cdrpj.org//text.asp?2022/1/1/9/335258
| Introduction|| |
Diabetic foot problems pose a huge economic burden on individuals., Complexity of diabetic foot wounds makes the treatment difficult and expensive. Although several studies have shown risk factors associated with it, still there is limited understanding about diabetic foot wound. In addition to several endogenous factors leading to causation and progression of diabetic foot wounds, “infection” is one of the exogenous factors that lead to the worsening of diabetic foot wounds. Nonhealing diabetic foot wounds symbolize hyper-inflammatory state and restoration of immune homeostasis in wound microenvironment is essential for effective wound healing.
Macrophages and related cytokines are one of the key components of host defense that not only help in infection control but also promote wound healing. Expression of macrophage migration inhibitory factor (MIF) at sites of inflammation, suggests its role in regulating the functions of macrophages in host defense. Interleukin 8 is a chemokine produced by stimulated monocytes/macrophages which primarily mediate activation and migration of neutrophils from peripheral blood to tissue site, a crucial step for infection control. Thus, it is important to understand the profile of these two important immune parameters in relation to diabetic foot infections.
The inflammatory cytokine concentrations in diabetic foot wounds are also found to be associated with Vitamin D deficiency. In the present study, we aimed to evaluate the serum level of MIF and interleukin 8 (IL8) in type 2 diabetes subjects with foot infection and without foot infection. and its correlation with Vitamin D status of the host.
| Material and Methods|| |
The study was conducted at endocrine and general surgery department of a medical college and university hospital in North India. Written informed consent was obtained. The study was approved by Institutional Ethics Committee.
Selection of subjects
Type 2 diabetes subjects with foot infection admitted to the endocrine and surgery unit wards of the university hospital were recruited in the study as cases and subjects with diabetes without foot infection were recruited as study controls. Subjects on immunosuppressants and having a compromised blood supply of the limbs were excluded from the study.
Blood samples were collected after an overnight fast in a plain vial and in ethylenediamine tetraacetic acid vial for serum biomolecular profiles (biochemical and immunological) and hemoglobin A1C (HbA1c), respectively. Aliquoted serum samples were stored at −80° C till the assay period.
Biochemical and immunological analysis
HbA1c was measured routinely in the endocrine laboratory using Drew Scientific DS5 HbA1c analyzer. Serum 25 hydroxy Vitamin D concentration was measured by radioimmunoassay (RIA) using commercially available kit (Diasorin, Italy). The intra and inter-assay variations coefficients of variation (% CV) were 11.7 and 12.5, respectively. MIF (RayBiotech, Inc. Norcross GA) and IL8 (Diaclone, France) concentrations were measured by enzyme-linked immunosorbent assay as per manufacturer's protocol. The minimum detectable dose (sensitivity) of MIF was <6 pg/ml. The intra and interassay CV were <10 and <12, respectively. The minimum detectable dose (sensitivity) of IL8 was 29 pg/ml. Intra and interassay %CV were 3.1 and 9.7, respectively.
Data were analyzed by using SPSS 16.0 (SPSS Inc., Chicago, US) and Graph Pad 3.0 (GraphPad Software, Inc. San Diego, US) software. Unpaired t-test was used to compare the clinical characteristics and the cytokine levels of cases and controls. Correlation analysis was used to assess the association between Vitamin D and cytokine level in the study population. Independent “t”-test was used to compare the cytokine level in subjects with severe Vitamin D deficiency from that of remaining subjects.
| Results|| |
Cytokine concentrations in cases and controls
The age, duration of diabetes, HbA1c, Vitamin D, and body mass index (BMI) of study participants of the two groups are summarized in [Table 1]. There was no significant difference in the mean (±Standard Error of Mean [SE]) age, duration of diabetes, HbA1c, and BMI between the two groups on statistical analysis. Although Vitamin D level was comparatively lower in cases than controls, the difference did not reach statistical significance. The mean (±SE) concentration of inflammatory cytokines IL8 was significantly (P = 0.009) higher and of MIF was significantly (P < 0.0001) lower in cases than controls.
|Table 1: Comparison of clinical parameters and cytokine profile of diabetic foot infection cases and controls with diabetes|
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Vitamin D and cytokines-correlations analysis
There was a significant negative correlation of Vitamin D with IL8 [Figure 1]. No significant correlation of Vitamin D with MIF [Figure 2] was observed. The correlation coefficient and P value for IL8 were r = −0.191; P = 0.01 and MIF were r = 0.1; P = 0.3.
|Figure 2: Correlation between Vitamin D and macrophage-migration inhibitory factor|
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Cytokine level in severe Vitamin D deficiency
Subjects with severe Vitamin D deficiency (25 dihydroxyvitamin [OH] Vitamin D <10 ng/ml) have significantly higher level of IL8 compared to those having 25 (OH) Vitamin D ≥10 ng/ml. There was no difference observed in MIF concentration between the two sub groups [Table 2]. The analysis did not show statistical difference in cytokine levels with Vitamin D concentration cutoff of 20 ng/ml.
|Table 2: Comparison of cytokines levels in diabetes with and without severe Vitamin D deficiency|
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| Discussion|| |
Increased IL-8 concentration in patients with diabetic foot infection in our study substantiated the findings of Hirao et al., who also found significantly higher level of IL-8 in patients with septic syndrome than healthy controls and suggested IL-8 as a valuable early indicator of bacterial infection. It has also been proposed that IL-8 in addition to tumor necrosis factor-alpha (TNF-α) and transforming growth factor beta 1 (TGF-β1) may participate in development and progression of the diabetic complications.
MIF is a pleiotropic cytokine that functions as a critical mediator of innate immunity and promotes several pathophysiological processes including autoimmune diabetes and type 2 diabetes. We observed low circulating level of MIF in subjects with diabetic foot infection compared to subjects with diabetes without foot infection. Our findings were contrary to the previous study where a blood level of MIF was markedly elevated in association with fatal outcome in sepsis.,,
Our findings indicated dysregulation in the inflammatory cytokine milieu in subjects with diabetes similar to the earlier reports. Researchers have shown that subjects with diabetic foot ulcers exhibit specific and nonrandom upregulation of several acute-phase proteins, cytokines, and chemokines but their study has limitation of confounding variables including age and HbA1c that vary significantly in the subjects with diabetes with and without foot ulcer.
A potential physiological role for Vitamin D in regulating innate and adaptive component of immunity was documented previously. A negative correlation of Vitamin D with IL8 in the study participants strongly supported its anti-inflammatory role. Similar were the findings from another study where it was shown that 1α, 25 OH D3 downregulated the expression of inflammatory cytokines TNF-α, IL-6, and IL-1 β and suppressed interferon gamma “mediated macrophage activation., Zhang et al. have shown that Vitamin D inhibits monocyte/macrophage pro-inflammatory cytokine production.
Our observation was further strengthened by another important finding of this study. A significant difference in the level of IL-8 was present in subjects with severe vitamin D deficiency, i.e., serum level <10 ng/ml in comparison to subjects with Vitamin D ≥10 ng/ml. This substantiated the previous observation where Vitamin D <10 ng/ml was proposed as a risk factor for unfavorable immune alteration in diabetic foot infection. Preliminary study showed elevated cytokine response in diabetic foot infection consequent upon Vitamin D deficiency. Previously, Khoo et al. had also shown that elevated Vitamin D3 level in vivo was associated with downregulation of cytokine response in healthy subjects.
Studies have also shown the effect of Vitamin D on glucose homeostasis, insulin resistance, and β cell dysfunction in subjects at risk of type 2 diabetes and on markers of inflammation in nondiabetic adults. Experimental studies support the involvement of Vitamin D in modulating the inflammatory response but human studies are scarce investigating inflammatory biomarkers specifically in subjects with or at high risk of type 2 diabetes and its complications., Our study reported the influence of Vitamin D status on markers of inflammation in subjects with diabetes and diabetic foot infection. This could possibly have a clinical implication on the use of Vitamin D supplementation in diabetic foot.
The underlying complications and insulin status of the two groups were not evaluated before comparing the cytokine data which is a limitation of the present study. Furthermore, the cytokine status of nondiabetic subjects with soft tissue infection was not included in this study as our intention was to identify the immune changes in subjects with diabetes in presence and absence of foot infection.
| Conclusion|| |
IL 8 was higher in patients with diabetic foot infection than in patients with diabetes. IL 8 was negatively correlated with serum vitamin D level in the study subjects of both the groups. Thus, the present study provides a valuable information about the role of vitamin D and its serum level to influence the marker of inflammation in patients with diabetes and diabetic foot infection.
The authors wish to thank Dr T. B Singh, Professor-Biostatistics at Institute of Medical Sciences, Banaras Hindu University for providing support in the statistical analysis. The work was funded by the Indian Council of Medical Research, New Delhi, India, in the form of a grant (IRIS ID no. ICMR/RHN/2008-04670) to SKS. ST and DDP thank the University Grant Commission, New Delhi, India, for financial assistance as research fellowships. Part of the study has been presented in Desert Foot 2011 conference at, Phoenix, Arizona, USA by S. T and been awarded with third prize.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Cavanagh P, Attinger C, Abbas Z, Bal A, Rojas N, Xu ZR. Cost of treating diabetic foot ulcers in five different countries. Diabetes Metab Res Rev 2012;28 Suppl 1:107-11.
Lu Q, Wang J, Wei X, Wang G, Xu Y, Lu Z, et al
. Cost of diabetic foot ulcer management in China: A 7-year single-center retrospective review. Diabetes Metab Syndr Obes 2020;13:4249-60.
Trengove NJ, Bielefeldt-Ohmann H, Stacey MC. Mitogenic activity and cytokine levels in non-healing and healing chronic leg ulcers. Wound Repair Regen 2000;8:13-25.
Khanna S, Biswas S, Shang Y, Collard E, Azad A, Kauh C, et al.
Macrophage dysfunction impairs resolution of inflammation in the wounds of diabetic mice. PLoS One 2010;5:e9539.
Farr L, Ghosh S, Moonah S. Role of MIF cytokine/CD74 receptor pathway in protecting against injury and promoting repair. Front Immunol 2020;11:1273.
Flaster H, Bernhagen J, Calandra T, Bucala R. The macrophage migration inhibitory factor-glucocorticoid dyad: Regulation of inflammation and immunity. Mol Endocrinol 2007;21:1267-80.
Meniailo ME, Malashchenko VV, Shmarov VA, Gazatova ND, Melashchenko OB, Goncharov AG, et al.
Interleukin-8 favors pro-inflammatory activity of human monocytes/macrophages. Int Immunopharmacol 2018;56:217-21.
Hirao Y, Kanda T, Aso Y, Mitsuhashi M, Kobayashi I. Interleukin 8 – An early marker for bacterial infection. Lab Med 2000;31:39-44.
Tiwari S, Pratyush DD, Gupta SK, Singh SK. Vitamin D deficiency is associated with inflammatory cytokine concentrations in patients with diabetic foot infection. Br J Nutr 2014;112:1938-43.
Huseynova GR, Azizova GI, Efendiyev AM. Quantitative changes in serum IL-8, TNF-α and TGF-β1 levels depending on compensation stage in type 2 diabetic patients. Int J Diabetes Metab 2009;17:59-62.
Cvetkovic I, Al-Abed Y, Miljkovic D, Maksimovic-Ivanic D, Roth J, Bacher M, et al.
Critical role of macrophage migration inhibitory factor activity in experimental autoimmune diabetes. Endocrinology 2005;146:2942-51.
Yabunaka N, Nishihira J, Mizue Y, Tsuji M, Kumagai M, Ohtsuka Y, et al.
Elevated serum content of macrophage migration inhibitory factor in patients with type 2 diabetes. Diabetes Care 2000;23:256-8.
Emonts M, Sweep FC, Grebenchtchikov N, Geurts-Moespot A, Knaup M, Chanson AL, et al.
Association between high levels of blood macrophage migration inhibitory factor, inappropriate adrenal response, and early death in patients with severe sepsis. Clin Infect Dis 2007;44:1321-8.
Bozza FA, Gomes RN, Japiassú AM, Soares M, Castro-Faria-Neto HC, Bozza PT, et al.
Macrophage migration inhibitory factor levels correlate with fatal outcome in sepsis. Shock 2004;22:309-13.
Weigelt C, Rose B, Poschen U, Ziegler D, Friese G, Kempf K, et al.
Immune mediators in patients with acute diabetic foot syndrome. Diabetes Care 2009;32:1491-6.
Hewison M. Vitamin D and the immune system: New perspectives on an old theme. Endocrinol Metab Clin North Am 2010;39:365-79.
Giulietti A, van Etten E, Overbergh L, Stoffels K, Bouillon R, Mathieu C. Monocytes from type 2 diabetic patients have a pro-inflammatory profile. 1, 25-Dihydroxyvitamin D3 works as anti-inflammatory. Diabetes Res Clin Pract 2007;77:47-57.
Helming L, Böse J, Ehrchen J, Schiebe S, Frahm T, Geffers R, et al.
1alpha, 25-Dihydroxyvitamin D3 is a potent suppressor of interferon gamma-mediated macrophage activation. Blood 2005;106:4351-8.
Zhang Y, Leung DY, Richers BN, Liu Y, Remigio LK, Riches DW, et al.
Vitamin D inhibits monocyte/macrophage proinflammatory cytokine production by targeting MAPK phosphatase-1. J Immunol 2012;188:2127-35.
Khoo AL, Chai LY, Koenen HJ, Sweep FC, Joosten I, Netea MG, et al.
Regulation of cytokine responses by seasonality of vitamin D status in healthy individuals. Clin Exp Immunol 2011;164:72-9.
Kayaniyil S, Vieth R, Retnakaran R, Knight JA, Qi Y, Gerstein HC, et al.
Association of vitamin D with insulin resistance and beta-cell dysfunction in subjects at risk for type 2 diabetes. Diabetes Care 2010;33:1379-81.
Pittas AG, Harris SS, Stark PC, Dawson-Hughes B. The effects of calcium and vitamin D supplementation on blood glucose and markers of inflammation in nondiabetic adults. Diabetes Care 2007;30:980-6.
Boucher BJ. Vitamin D insufficiency and diabetes risks. Curr Drug Targets 2011;12:61-87.
Chagas CE, Borges MC, Martini LA, Rogero MM. Focus on vitamin D, inflammation and type 2 diabetes. Nutrients 2012;4:52-67.
[Figure 1], [Figure 2]
[Table 1], [Table 2]