Elsevier

Journal of Proteomics

Volume 224, 30 July 2020, 103843
Journal of Proteomics

Proteome profile of neutrophils from a transgenic diabetic pig model shows distinct changes

https://doi.org/10.1016/j.jprot.2020.103843Get rights and content

Highlights

  • Diabetic INSC94Y pig neutrophils show changed proteome compared to wild type.

  • Higher abundant neutrophil proteins in diabetes associate to cytoskeletal dynamics.

  • Myosin regulatory light chain 9 abundance is 5-fold higher in INSC94Y neutrophils.

  • Altered motility of neutrophils might affect sufficient immune response in diabetes.

Abstract

INSC94Y transgenic pigs develop a stable diabetic phenotype early after birth and therefore allow studying the influence of hyperglycemia on primary immune cells in an early stage of diabetes mellitus in vivo. Since immune response is altered in diabetes mellitus, with deviant neutrophil function discussed as one of the possible causes in humans and mouse models, we investigated these immune cells in INSC94Y transgenic pigs and wild type controls at protein level. A total of 2371 proteins were quantified by label-free LC-MS/MS. Subsequent differential proteome analysis of transgenic animals and controls revealed clear differences in protein abundances, indicating a deviant behavior of granulocytes in the diabetic state. Interestingly, abundance of myosin regulatory light chain 9 (MLC-2C) was increased 5-fold in cells of diabetic pigs. MLC-2C directly affects cell contractility by regulating myosin ATPase activity, can act as transcription factor and was also associated with inflammation. It might contribute to impaired neutrophil cell adhesion, migration and phagocytosis.

Our study provides novel insights into proteome changes in neutrophils from a large animal model for permanent neonatal diabetes mellitus and points to dysregulation of neutrophil function even in an early stage of this disease.

Data are available via ProteomeXchange with identifier PXD017274.

Significance

Our studies provide novel basic information about the neutrophil proteome of pigs and contribute to a better understanding of molecular mechanisms involved in altered immune cell function in an early stage diabetes. We demonstrate proteins that are dysregulated in neutrophils from a transgenic diabetic pig and have not been described in this context so far. The data presented here are highly relevant for veterinary medicine and have translational quality for diabetes in humans.

Introduction

With increasing prevalence in both developed and developing countries, diabetes mellitus has become the most important metabolic disease in humans today [1,2]. A link between diabetes and increased susceptibility for infections was proven by numerous studies [3]. In addition to this, severe courses of disease like bacteremia are seen more often in diabetic patients than in people with no disease history [4]. While clinical importance of these findings is beyond questioning, the underlying mechanisms responsible for the impaired immune function in the diabetic condition, poorly understood so far, are unknown to date.

As first line effector cells of the innate immune system, neutrophils are able to react in multiple ways to potential threats, namely through phagocytosis, degranulation, ROS release and formation of DNA-based extracellular traps (NETs) [5,6]. Besides this essential defensive function of neutrophils, their role as regulatory elements in the healthy organism becomes more and more obvious [7]. Lately neutrophil granulocytes, formerly considered to depend mainly on glycolysis, were proven to adapt their metabolic pathways via transcriptional regulation to a changed environment [8]. During pathological condition of diabetes these adaptions include deficiency in ROS production, chemotaxis, adhesion and phagocytosis [8]. By which regulatory pathways these effects are transduced inside the cell is not fully understood to date [9]. However, recent studies showed that insulin is not only required for maintaining glucose homeostasis but also acts as an immunomodulatory hormone [9]. It can affect cytokine release and expression of adhesion molecules in leukocytes [9]. Whether these observations of insulin impacting the immune system may be mediated solely by an increased blood glucose level is currently under discussion [10].

Pigs are used as large animal models in a wide field of biomedical research [11]. Their close similarity to humans in terms of size, anatomy, diet and metabolism offers some significant benefits for diabetes research [11]. Developing pathologic alterations like diabetic retinopathy, vasculopathy and nephropathy, diabetes in pigs mimics the human diabetic phenotype much better than the commonly used rodent models do [11]. Genetic engineering made several transgenic breeding lines available for different approaches within the field of diabetes research [12]. The INSC94Y transgenic pig model used in this study develops a stable diabetic phenotype within the first week of life, similar to permanent neonatal diabetes mellitus (PNDM) or mutant INS gene-induced diabetes of youth (MIDY) in humans [13]. Due to a point mutation within the insulin gene and consequential amino acid sequence (Csingle bondY), the secretion of the protein by beta cells is impaired in these pigs [13]. Therefore, fasting and postprandial hyperglycemia is observed, rendering the model suitable to detect hyperglycemia-associated pathophysiologies [13].

Abnormal behavior of primary polymorphonuclear leukocytes (PMN) in diabetic settings was reported in humans and in experiments in mice [14,15]. While studies in man can display differences between diabetes patients depending on their state of glycemic control, early stages of diabetes are difficult to observe due to the insidious disease process. Hence, we used PMN from young INSC94Y transgenic pigs and matched wild type controls with the aim to gain insight into changes in the neutrophil proteome that point to dysfunction occurring in the initial stage of diabetes.

Section snippets

Sample preparation

PMN from heparinized venous whole blood of six 12 week old INSC94Y transgenic (INSC94Y) pigs and six age-matched wild type (wt) littermates were used in this study. In detail, neutrophils of four INSC94Y and three wt animals underwent mass spectrometric analysis. Immunofluorescence staining was conducted at a later time with PMN of the other five individuals (two INSC94Y, three wt). Relative amount of leukocyte subsets was determined via DiffQuick stained blood smears for all pigs and

The porcine neutrophil proteome shows divergent protein abundances in INSC94Y transgenic pigs

We identified a total of 2371 proteins in the whole sample set of primary pig neutrophils (Supplementary Table 2). Of these, 1680 were identified with at least two unique peptides. The cell fraction analyzed here consisted of 98.4 ± 1.9% neutrophils with lymphocytes being the most frequent impurity (1.2 ± 1.2%, Supplementary Fig. 1C). Quantitative comparison of the protein repertoire revealed 51 proteins with significant differences in abundance between INSC94Y transgenic animals and control

Discussion

Using label-free LC-MS/MS, we obtained a proteomic data set for porcine neutrophils with an unprecedented high resolution of 2371 identified proteins. Hence our data provide a basis for further research regarding the innate immune system of pigs. Moreover, the aim of the present study was to characterize neutrophil granulocytes in INSC94Y transgenic pigs and gain deeper insight into proteomic changes related to the early stage of diabetes mellitus. 51 of all identified proteins differed

Conclusion

Our study provides novel information on the proteome of porcine neutrophil granulocytes in general and shows that significant changes can be detected in an initial stage of diabetes mellitus in a transgenic diabetic pig model. We gained insights into the differentiated reaction of neutrophil proteome to permanently elevated blood glucose levels. Further experiments are needed to elucidate the exact role of the different abundant candidates and especially myosin regulatory light chain 9 in the

Author contributions

C.D. conceived and designed the experiment; M.W., C.D., B.A. and S.M.H. performed the experiments and analyzed the data; S.R. and E.W. contributed reagents, materials and analysis tools; M.W., R.D. and C.D. wrote the manuscript. All authors critically read the manuscript and approved the final version to be published.

Funding

This work was supported by grants from the Deutsche Forschungsgemeinschaft SPP project 2127 DFG DE 719/7-1 (to C.D.) and HA 6014/5-1 (to S.M.H.).

Declaration of Competing Interest

The authors declare that they have no competing interests.

Acknowledgement

The authors would like to thank Carmen Wiedemann, Bernhard Hobmaier and Isabella Giese for critical discussions and for assistance in blood sampling.

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