Chronic hyperglycaemia drives functional impairment of lymphocytes in diabetic INSC94Y transgenic pigs

People with diabetes mellitus have an increased risk for infections, however, there is still a critical gap in precise knowledge about altered immune mechanisms in this disease. Since diabetic INSC94Y transgenic pigs exhibit elevated blood glucose and a stable diabetic phenotype soon after birth, they provide a favourable model to explore functional alterations of immune cells in an early stage of diabetes mellitus in vivo. Hence, we investigated peripheral blood mononuclear cells (PBMC) of these diabetic pigs compared to non-transgenic wild-type littermates. We found a 5-fold decreased proliferative response of T cells in INSC94Y tg pigs to polyclonal T cell mitogen phytohaemagglutinin (PHA). Using label-free LC-MS/MS, a total of 2,704 proteins were quantified, and distinct changes in protein abundances in CD4+ T cells of early-stage diabetic pigs were detectable. Additionally, we found significant increases in mitochondrial oxygen consumption rate (OCR) and higher basal glycolytic activity in PBMC of diabetic INSC94Y tg pigs, indicating an altered metabolic immune cell phenotype in diabetics. Thus, our study provides new insights into molecular mechanisms of dysregulated immune cells triggered by permanent hyperglycaemia.

People with diabetes mellitus have an increased risk for infections, however, there is still a critical gap 21 in precise knowledge about altered immune mechanisms in this disease. Since diabetic INS C94Y 22 transgenic pigs exhibit elevated blood glucose and a stable diabetic phenotype soon after birth, they 23 provide a favourable model to explore functional alterations of immune cells in an early stage of 24 diabetes mellitus in vivo. Hence, we investigated peripheral blood mononuclear cells (PBMC) of these 25 diabetic pigs compared to non-transgenic wild-type littermates. We found a 5-fold decreased 26 proliferative response of T cells in INS C94Y tg pigs to polyclonal T cell mitogen phytohaemagglutinin 27 (PHA). Using label-free LC-MS/MS, a total of 2,704 proteins were quantified, and distinct changes in 28 protein abundances in CD4 + T cells of early-stage diabetic pigs were detectable. Additionally, we found 29 significant increases in mitochondrial oxygen consumption rate (OCR) and higher basal glycolytic 30 activity in PBMC of diabetic INS C94Y tg pigs, indicating an altered metabolic immune cell phenotype 31 in diabetics. Thus, our study provides new insights into molecular mechanisms of dysregulated immune 32 cells triggered by permanent hyperglycaemia. 33

Introduction 34
Diabetes mellitus is a major risk factor regarding the outcome of bacterial and viral infections (Harding,35 Pavkov, Magliano, Shaw, & Gregg, 2019). Among diabetic patients, increased numbers of highly 36 susceptible individuals suffering from tuberculosis (Kumar et al., 2019) or influenza (Marshall et al.,37 2020) are common. Recently, meta-analyses revealed an enhanced severity and mortality of  19 infections in people with diabetes mellitus (Y. Hu et al., 2020;Zhou et al., 2020). Undoubtedly,39 there is a link between the disease and an impaired immune system, but the molecular mechanisms 40 involved in altered immune cell function in diabetic patients are still unknown to date. Dysfunctional 41 T cells may play a pivotal role in immunological impairment in diabetes mellitus (Nicholas et   Germany). 109

Magnetic activated cell sorting (MACS) for CD4 + T cells 110
Briefly, a total of 6 x 10 7 cells was incubated in staining buffer with mouse anti-pig CD4 alpha (clone 111 MIL17, Bio-Rad AbD Serotec, Puchheim, Germany, 1:50) at 4°C for 20 min. Staining buffer contained 112 phosphate-buffered saline (pH 7.2) and was supplemented with 2 mM EDTA and 0.5% bovine serum 113 albumin (BSA). In the next step, cells were resuspended in 480 μL staining buffer before adding 120 114 μL anti-mouse IgG2a/b MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) for an incubation 115 period of 15 min. In further steps, BSA in staining buffer was omitted to prevent interference with mass 116 spectrometry. Magnetic separation was performed using LS columns (Miltenyi Biotec, Bergisch 117 Gladbach, Germany). Magnetically-labelled CD4 + T cells were retained in the magnetic field, while 118 unwanted cells were eliminated by three washing steps. Positive CD4 + T cell fraction was eluted by 119 removing the column from magnetic field and flushing with staining buffer. 6 x 10 5 positive selected 120 cells were pelleted and stored at −20 °C until filter-aided sample preparation (FASP). The isolation of 121 porcine CD4 + T cell routinely achieved >90% purity, confirmed by flow cytometry. 122

Mass spectrometry and data analysis 123
Purified CD4 + T cells of four diabetic INS C94Y tg pigs and five littermate wildtypes were analyzed. 124 CD4 + T cell pellets were lysed in urea buffer (8M in 0.1M Tris/HCl pH 8.5) and 10 μg total protein of 125 each sample was proteolysed with LysC and trypsin by a modified filter-aided sample preparation 126 (FASP) as described (Grosche et al., 2016). Acidified eluted peptides were analyzed in the data-127 independent acquisition mode on a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific, 128 Waltham, MA, USA) online coupled to an ultra-high-performance liquid chromatography (UHPLC) 129 system (Ultimate 3000, Thermo Fisher Scientific Karlsruhe, Germany, 1:100) for 30 min at RT. 5 x 10 4 cells were transferred to microscope slides and 153 centrifugated (300 x g, 10 min) before coverslip using mounting medium (Serva, Rosenheim, 154 Germany). Visualization of stained targets was performed using a Leica Dmi8 microscope with 155 associated LAS-X-software (Leica, Wetzlar, Germany). 156

Measurement of OCR and ECAR by Seahorse XFe Analyzer 157
Metabolic phenotypes of porcine PBMC were determined using a Seahorse XFe Analyzer (Agilent 158 Technologies, Waldbronn, Germany) measuring oxygen consumption rate (OCR), which is attributed  littermates. Interestingly, while no differences could be observed between the two groups after 182 stimulation with PWM (Fig 1a) and ConA (Fig 1b), lymphocytes of diabetic pigs (n=11) revealed a 183 significantly 5-fold decreased proliferation rate in response to PHA compared to wildtypes (n=13) ( Fig  184  1c; ***p≤0.001). 185 Lymphocyte subpopulation ratio did not differ between wildtypes and diabetic pigs 186 Next, we examined whether the decreased ability to proliferate correlated with altered lymphocyte 187 subpopulations in diabetic INS C94Y tg pigs. There were no significant differences in the percentage of 188 B or T cell population between wildtypes and diabetic pigs ( Supplementary Fig 1a, b). Moreover, 189 measurements of T cell subsets CD4 + (Supplementary Fig 1c), CD8α + (Supplementary Fig 1d), 190 CD4 + CD8α + αβT cells (Supplementary Fig 1e) and SWC5 + γδT cells ( Supplementary Fig 1f) revealed 191 no differences between both groups. 192

Proteomic profiling identified disease-associated protein ANXA1 in CD4 + T cells of diabetic 193 INS C94Y transgenic pigs 194
We characterized the proteomes of porcine CD4 + T cells, which we hypothesized to be the key drivers 195 of impaired proliferation response. Purified CD4 + T cells were examined with differential proteome 196 analyses, using data-independent acquisition LC−MS/MS. A high-resolution proteome with 2,704 197 identified proteins was obtained (quantified with at least two unique peptides; Supplementary Table  198 1), with 80 proteins significantly different in abundance (*p < 0.05; Fig 2a). Two of these proteins had 199 a ≥ 2-fold difference in abundance, named MYL4 and ANXA1 (Fig 2b). In the whole sample set, 200 ANXA1 was highlighted with highest statistical significance in protein abundance differences between 201 wildtypes and diabetic pigs (***p < 0.001). Interestingly, higher levels of ANXA1 were also already 202 described in serum and plasma of type  Fig 3 a-c). 208 Furthermore, we analysed ANXA1 subcellular localization in purified CD4 + T cells with 209 immunocytology and flow cytometry. While intracellular ANXA1 levels were equivalent between both 210 groups (Fig 3 d- CD4 + T cells of wildtypes were "Lagging strand synthesis", "Gap-filling DNA repair synthesis and 217 ligation in GG-NER" and "Polymerase switching" (Table 1). In contrast, proteins with significantly 218 higher abundances in CD4 + T cells of INS C94Y tg pigs were associated with the pathways "Downstream 219 signal transduction", "RUNX1 regulates transcription of genes involved in differentiation of myeloid 220 cells" and "Lipophagy" (Table 2)

Increased basal glycolytic activity and mitochondrial respiration in PBMC of diabetic pigs 225
Interestingly, the basal glycolytic rate of PBMC from diabetic pigs was significantly higher compared 226 to wild-type PBMC (Fig 4a; *p<0.05), indicating increased lactate production and glycolytic activity 227 of diabetic PBMC. Furthermore, analyses of OCR revealed a significant increase in oxygen 228 consumption at all measured time points in diabetic pigs. Compared to wild-type PBMC, a rise of 229 basal, ATP-linked and maximal mitochondrial respiration (**p<0.01) as well as spare respiratory 230 capacity (*p<0.05) was present in PBMC from diabetic pigs (Fig 4b). These differences indicated a 231 fundamentally different metabolic phenotype of PBMC from wildtypes (n=15) and diabetic INS C94Y tg 232 pigs (n=10). 233

Discussion 234
To assess the impact of permanent hyperglycaemia on immune cell function in vivo, we characterised 235 lymphocytes of a transgenic pig model for permanent neonatal diabetes mellitus. Significant 236 differences in proliferative response, CD4 + T cell proteomes and the metabolic phenotypes were 237 already detectable in these early diabetic pigs. Our findings point to main distinct changes in the T cell 238 compartment triggered by early-life high blood glucose and indicate that several immune alterations 239 already occur in the early stage of diabetes mellitus. 240 We observed no differences of lymphocyte proliferation between controls and diabetic pigs after 241 polyclonal stimulation with B and T cell mitogen PWM and T cell mitogen ConA. However, after 242 polyclonal stimulation with T cell mitogen PHA, we detected a distinct decreased proliferation of 243 lymphocytes from diabetic pigs, which points to an impaired immune response selectively induced by To gain deeper insights into hyperglycaemia-associated molecular changes of CD4 + T cells, we 275 analysed the proteome of these cells from diabetic pigs and wildtypes as a hypothesis-generating 276 approach for the dysregulated immune responses. Notably, we obtained a comprehensive proteomic 277 data set for porcine CD4 + T cells with a high resolution of 2,704 identified proteins. Among these, we 278 found a total of 3% (80 proteins) with significantly different protein abundances between both groups, 279 indicating distinct proteome differences in CD4 + T cells from early stage diabetic pigs, functionally 280 associated to DNA damage responses and cell protection, actin cytoskeleton organization and 281 regulation of cellular energy metabolism. While many studies of immune cells from diabetic type 2 282 patients were performed by a 2D-gelelectrophoresis approach, only significantly altered spots were 283 analysed subsequently with mass spectrometry ( diabetic PBMC in this study revealed an increase in mitochondrial respiration and basal glycolytic 320 activity. We found significant increases in mitochondrial oxygen consumption rate (OCR) in PBMC 321 of diabetic pigs. Interestingly, in line with our findings, increased oxygen consumption was also 322 demonstrated in PBMC of type 2 diabetes patients (Hartman et al., 2014). Previously, this group also 323 measured a higher production of reactive oxygen species (ROS) in these cells and hypothesised 324 mitochondrial damage in diabetic PBMC caused by enhanced oxidative stress (Widlansky et al., 2010). 325 Since ROS synthesis is essential for physiological immune activation of lymphocytes but lead to 326 cellular damage if concentration surpasses (Alfatni et al., 2020), this parameter should be determined 327 in PBMC of diabetic pigs, in order to evaluate whether immune cells are impaired by oxidative cell 328 stress. On the contrary, mitochondrial respiration with increased oxygen consumption is used by 329 immune cells to supplement increased glycolysis, which is the main metabolic pathway fueling effector 330 function upon T cell activation (Gaber, Chen, Krauss, & Buttgereit, 2019). Besides increased oxygen 331 consumption rates, we also found a higher basal glycolytic activity in early diabetic pigs, suggesting 332 that hyperglycaemia induced metabolic adaption to local conditions of immune cells and promoted a 333 more activated status in T cells (Bonacina, Baragetti, Catapano, & Norata, 2019). A rapid increase in 334 glycolysis is an important determinant in cellular energy production and required for effective cell 335 proliferation, size expansion, and differentiation (Gaber et al., 2019). Hence, it is also possible that 336 PBMC in young diabetic pigs are highly metabolically active to challenge hyperglycaemic conditions. 337 Our findings indicate that metabolic disturbances occurring at early stage diabetes mellitus, as observed 338 in INS C94Y tg pigs, likely cause metabolic reprogramming in PBMC and may also influence their 339 activation status. Since further studies are absolutely required for a better understanding of bioenergetic 340 profiles of PBMC in diabetes mellitus, diabetic pigs enable to define mitochondrial properties and 341 cellular energy production in these cells at initial stage of disease. 342 Taken together, our findings point to an early, fundamental dysregulation of immune cells when 343 exposed to prolonged high blood glucose. Chronic hyperglycaemia leads to profound molecular and 344 functional changes of T cells reasoned by proteomic and metabolic alterations, and, moreover, affects 345 T cell immune responses in vitro. the manuscript and approved the final version to be published. CD is the guarantor of this work and, 368 as such, had full access to all the data in the study and takes responsibility for the integrity of the data 369 and the accuracy of the data analysis. 370

Data availability 371
The data are available on request from the authors. 372  proliferation rate (***p≤0.001) compared to non-transgenic wild-type littermates (n=13). Data are 535 represented as means ± SEM; horizontal bars indicate group means. 536 FCCP, rotenone and antimycin A. Compared to PBMC from wild-types (wt, n=15), mitochondrial 561 respiration of PBMC from diabetic pigs (n=10) yielded a significantly increased basal, ATP-linked and 562 maximal respiration (**p<0.01) as well as a significantly increased spare respiratory capacity 563 (*p<0.05). Data are represented as means ± SEM. 564