Circulating Leukocyte Alterations and the Development/Progression of Diabetic Retinopathy in Type 1 Diabetic Patients - A Pilot Study

ABSTRACT

Background/Aims

The aim of this study was to investigate the relationship between alterations in circulating leukocytes and the initiation and progression of DR in people with type 1 diabetes (T1D).

Methods

Forty-one patients with T1D [13 mild non-proliferative DR (mNPDR), 14 active proliferative DR (aPDR) and 14 inactive PDR (iPDR)], and 13 age- and gender-matched healthy controls were recruited prospectively. Circulating leukocytes, including CD4⁺ and CD8⁺ T-cells, CD14⁺CD16⁻, CD14⁻CD16⁺ and CD14⁺CD16⁺ monocytes; CD16⁺HLA-DR⁻ neutrophils, CD19⁺ B-cells and CD56⁺ natural killer cells and their cell surface adhesion molecules and chemokine receptors (HLA-DR, CD62L, CCR2, CCR5, CD66a, CD157 and CD305) were examined by flow cytometry.

Results

In DR patients, compared to healthy controls, increased proportions of neutrophils (p = .0152); reduced proportions of lymphocytes (p = .0002), HLA-DR⁺ leukocytes (p = .0406) and non-classical monocytes (p = .0204); and reduced expression of CD66a (p = .0048) and CD157 (p = .0007) on CD4⁺ T cells were observed. Compared to healthy controls, CD19⁺ B cells were reduced at the mNPDR but not aPDR patients. Total lymphocytes, CD4⁺ T cells and CD8⁺ T cells progressively decreased whereas neutrophils, the neutrophil/lymphocyte ratio and the neutrophil/CD4⁺ ratio progressively increased from early to late stages of DR, reaching statistical significance at the aPDR stage. Longer diabetes duration was associated with a reduced proportion of CD8⁺ T cells (p = .002) and increased neutrophil/CD8⁺ ratio (p = .033).

Conclusions

In this pilot study, DR is associated with increased innate cellular immunity especially neutrophils and reduced adaptive cellular immunity particularly lymphocytes. Impaired B-cell immunity may play a role in the initiation of DR; whereas impaired T-cell immunity with increased neutrophil response may contribute to progression of DR from non-proliferative to proliferative stages in T1D patients. Large multicenter studies are needed to further understand the immune dysregulation in DR initiation and progression.

KEYWORDS:

• Type 1 diabetes
• diabetic retinopathy
• neutrophils
• lymphocytes
• flow cytometry

Abbreviations

AGEs	=	advanced glycation end-products
ANOVA	=	analysis of variance
APCs	=	antigen-presenting cells
aPDR	=	active proliferative diabetic retinopathy
BMI	=	body mass index
BP	=	blood pressure
BRB	=	blood-retina-barrier
DR	=	diabetic retinopathy
EC	=	endothelial cell
EDTA	=	ethylenediaminetetraacetic acid
G-CSF	=	granulocyte colony-stimulating factor
iPDR	=	inactive proliferative diabetic retinopathy
LMR	=	lymphocyte-monocyte ratio
MHC-II	=	major histocompatibility complex, class II
mNPDR	=	mild non-proliferative diabetic retinopathy
NLR	=	neutrophil-lymphocyte ratio;
NPDR	=	non-proliferative diabetic retinopathy
PDR	=	proliferative diabetic retinopathy
PRP	=	pan-retinal photocoagulation
SEM	=	standard error of the mean
SPSS	=	statistical package for the social sciences
T1D	=	type 1 diabetes
TCR	=	T-cell receptor
VEGF	=	vascular endothelial growth factor

Acknowledgments

We thank the patients who participated in this study.

Authors’ contributions

HX, NL and MC conceived the study. GO, HX and NL designed the experiments. GO conducted the experiments and acquired data. GO and DMW analysed the results. GO, HX, NL, MC, AL and JRH discussed and interpreted the data. NL, DA and NJ recruited patients. GO and HX drafted the manuscript and all authors reviewed the manuscript.

Availability of data and materials

All data generated or analysed during this study are included in this published article.

Competing interests

The authors declare that they have no competing interests.

Declarations

• The study was approved by the Office for Research Ethics Committees Northern Ireland (ORECNI, Ref: 14/NI/0084) and procedures were performed in accordance with the tenets of the Declaration of Helsinki on research into human volunteers. Written informed consent was obtained from all participants prior to their enrolment in this study.

• Some data have been presented in abstract/poster format at the Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO) 2017, 6-11 May, Baltimore, MD, USA (Poster A0040).

Additional information

Funding

This work was supported by funding from Juvenile Diabetes Research Foundation (JDRF; Ref: 2-SRA-2014-141-Q-R) and through the generous support of Miss Elizabeth Sloan.