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Immunobiology journal homepage: www.elsevier.com/locate/imbio

Macrophages from a type 1 diabetes mouse model present dysregulated Pl3K/AKT, ERK 1/2 and SAPK/JNK levels Fernando Henrique Galvão Tessaro1, Thais Soprani Ayala1, Leonardo Mendes Bella, Joilson Oliveira Martins* Laboratory of Immunoendocrinology, Department of Clinical and Toxicological Analysis, School of Pharmaceutical Sciences - University of Sao Paulo (FCF/USP), São Paulo, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Diabetes Alloxan Macrophage PI3K ERK 1/2 TNF-alpha

Diabetes causes dysregulation in signal transduction in immune cells leading to an impaired response to pathogens. Herein, we investigated the impact of type 1 diabetes (T1D) in bone marrow-derived macrophages (BMDM), using male non-diabetic and diabetic C57BL/6 mice (alloxan 60 mg/kg, i.v., CEUA/FCF/USP - 467). Diabetic BMDM expressed impaired phosphoinositide 3-kinase (PI3K), being lower p-PI3K p55 levels and higher levels of PI3K p110 alpha, whereas protein kinase B (PKB/Akt) (Ser-473 and Thr-308), extracellular signalregulated kinases (ERK 1/2), and stress-activated protein kinase (SAPK/JNK) were enhanced compared to nondiabetic BMDM. Further evaluation of the responsiveness to lipopolysaccharide (LPS; 0.1 and 1 ug/mL), diabetic BMDM and peritoneal macrophage secreted dysregulated levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 levels. In 24 h, diabetic BMDM stimulated by LPS presented lower metabolic activity, with no differences in cell surveillance. Therefore, LPS re-stimulation (0.1 ug/mL) in diabetic BMDM resulted in higher secretion of TNF-α compared to non-diabetic BMDM. However, diabetic peritoneal macrophages secreted similar IL-6 levels in the first and additional 24 h of LPS stimulation. In general, our results demonstrated that diabetes exerts an impact in both BMDM and peritoneal macrophages ability to secrete cytokine under LPS stimulation.

1. Introduction Type 1 diabetes (T1D) is a metabolic disorder resulted from beta cell destruction by immune cells, reducing the levels of insulin in the blood (Atkinson et al., 2014). Insulin secretion at basal or stimulating conditions drives metabolism to tight control in different compartments in order to avoid organ dysfunction (American Diabetes Association, 2016). Without insulin treatment, T1D patients show a poor glycemic control by the organism having a range of complications that affects kidney, eyes, nerves, and cardiovascular system (American Diabetes Association, 2016; Atkinson et al., 2014). T1D patients present high mortality and morbidity rates in part due to an increase in the susceptibility to infections (American Diabetes Association, 2016; Casqueiro et al., 2012). Disruption in innate

immunity has been reported in diabetes mostly due to the difficulty in controlling glucose levels by the organism (Casqueiro et al., 2012). Environmental signals generated by diabetes promote modifications in bone marrow microenvironment and progenitor cells, which can contribute to alterations in myeloid cell maturation (Ratter et al., 2018; Bannon et al., 2013). Long-term inflammation perturbs hematopoiesis by increasing myeloid cell turnover at inflammatory sites due to elevated demand, generating abnormalities in monocytes and tissue-specific macrophages from several compartments of the body (Griseri et al., 2012). In general, T1D patients present a major number of myeloid cells (Ratter et al., 2018) and circulating mononuclear cells which express higher levels of Toll-like receptor (TLR)4, and its adapter molecules Myeloid differentiation primary response 88 (MyD88), and TIR-domain-containing adapter-inducing interferon-β (TRIF)

Abbreviations: BMDM, bone marrow-derived macrophages; ERK1/2, extracellular signal-regulated kinases; IL, Interleukin; LPS, lipopolysaccharide; MAPK, mitogenactivated protein kinases; MTT, 3-(4, 5-dimethylthiazol-2-yl,)-2, 5-diphenyltetrazolium bromide; MyD88, myeloid differentiation primary response 88; PI3K, phosphoinositide 3-kinase; PKB/Akt, protein kinase B; SAPK/JNK, stress-activated protein kinase; T1D, type 1 diabetes; TLR, toll-like receptor; TNF, tumor necrosis factor; TRIF, TIR-domain-containing adapter-inducing interferon-β ⁎ Corresponding author at: Laboratory of Immunoendocrinology, Department of Clinical and Toxicological Analysis, School of Pharmaceutical Sciences, University of São Paulo, Av. Prof. Lineu Prestes 580 - Bloco 17, 05508-000, São Paulo, SP, Brazil. E-mail address: [email protected] (J.O. Martins). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.imbio.2019.11.014 Received 15 January 2019; Accepted 26 November 2019 0171-2985/ © 2019 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

Please cite this article as: Fernando Henrique Galvão Tessaro, et al., Immunobiology, https://doi.org/10.1016/j.imbio.2019.11.014

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containing 30 % of L929 cell-conditioned medium, and 20 % of fetal bovine serum (Sigma Chemical Co, St. Louis, Mo). After 4 days, the cells received fresh supplemented medium in the same proportion and were incubated for three additional days. Then, after 7 days, cells were harvested using cold phosphate buffered saline (pH 7.4), seeded, and kept in RPMI 1640 containing 10 % of fetal bovine serum and 5 % of L929 cell-conditioned medium for 16 h at 37 °C with 5 % CO2 before the experiments. Peritoneal macrophages were obtained by peritoneal lavage, using sterilized cold phosphate buffer saline (pH 7.4). Cells were allowed to adhere for 2 h (37 °C, 5 % CO2), then cultured overnight in RPMI-1640 containing 10 % fetal bovine serum. BMDM and peritoneal macrophages were stimulated with or without 0.1 and 1 μg/mL of LPS from Escherichia coli (serotype 055:B5) (Sigma Chemical Co, St. Louis, Mo, USA) dissolved in RPMI 1640 for different time points. After 24 h on LPS-stimulation, macrophages were restimulated using 0.1 μg/mL of LPS for the next 24 h (Androulidaki et al., 2009).

(Filgueiras et al., 2015; Devaraj et al., 2008). Modification in macrophage development may contribute to the failure in infectious resolution (Sunahara and Martins, 2012; Lachmands et al., 2018). Macrophages are essential immune cells for cytokine secretion, phagocytosis, and tissue remodeling, displaying remarkable plasticity (Murray and Wynn, 2011). Diabetes contributes to disruptions in macrophage metabolism, therefore increasing the chances of an uncontrolled immune response (Ayala et al., 2019; Casqueiro et al., 2012; Esper et al., 2008). High glucose levels prime innate immune cells and change their epigenetic profile to a predisposed inflammatory state (van Diepen et al., 2016). In fact, TLR4 expression on mononuclear cells is driven by an increase in circulating glucose or exogenous insulin in T1D patients (Dandona et al., 2018). In addition, hyperglycemia conducts to enhance MyD88 levels in BMDM, peritoneal, and alveolar macrophages coming from diabetic mice (Filgueiras et al., 2015). Macrophages recognize LPS primarily by TLR4/MD2 subsequently via MyD88-dependent occurs the activation of mitogen-activated protein kinases (MAPK) — SAPK/JNK, p38 and ERK 1/2 — and phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB/Akt), culminating in synthesis and secretion of a range of inflammatory mediators (Liu et al., 2007; Koyasu, 2003). After being activated by LPS, macrophages reduce the expression of TLR4 on cell surface through a cellular mechanism of tolerance (Rajaiah et al., 2015). Alterations in TLR4/MD2 components might result in an impaired macrophage activation affecting its responsiveness to LPS (Diapen et al., 2016; Filgueiras et al., 2015; Casqueiro et al., 2012). Recently, authors have showed growing evidence for cell reprogramming in diabetes (Ratter et al., 2018; Diapen et al., 2016). Failing in the activation by LPS due to abnormal levels of TLR4/MD2 pathway molecules can result in a non-appropriate macrophage response in the course of inflammation (Filgueiras et al., 2015; Bannon et al., 2013; Casqueiro et al., 2012). Our aim was to investigate the impact of diabetes in key proteins involve to the LPS signal transduction in macrophages. For this purpose we used BMDM, and then we evaluate the responsiveness of both BMDM and tissue-specific peritoneal macrophages to the first and second stimuli of LPS.

2.4. Immunoblotting Cell extracts were lysed using RIPA buffer containing protease and phosphatase inhibitors (Cell Signaling Technology), and protein concentrations were determined by a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Inc., Rockford, IL). Lysates were separated by 10 % SDS-PAGE, transferred with the Amersham TE 70 PWR Semi-Dry Transfer System (Amersham Biosciences Corp., Piscataway, NJ, USA) onto a nitrocellulose membrane, and blotted with primary and secondary antibodies (Abs). Finally, proteins were visualized by enhanced chemiluminescence (GE Healthcare), and images were acquired by Amersham™ Imager 600 (GE Healthcare, Chicago, Illinois, EUA). For immunoblotting, nitrocellulose membranes were incubated in Trisbuffered saline-Tween buffer (150 mM NaCl, 20 mM Tris, 1 % Tween 20, pH 7.4) containing 5 % nonfat dried milk for 1 h. Afterwards, the blots were washed with Tris-buffered saline-Tween buffer three times for 5 min each and then probed for 12−14 h with antibodies (1:1000 dilution) against phospho-PI3 kinase p85 (Tyr458)/p55 (Tyr199), PI3 kinase p110α, PI3 kinase p110β, PI3 kinase p85, phospho-PI3 kinase p85 (Tyr458)/p55 (Tyr199), phospho-ERK 1/2 MAP kinase (Thr183/ Tyr185), phospho-p38 MAP kinase (Thr180/Tyr182), phospho-Akt (308-Thr), and phospho-Akt (473-Ser) diluted in 5 % bovine serum albumin at 4 °C. The antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The blots were then incubated with anti-rabbit secondary antibody (1:10,000) for 1 h (Abcam), developed using enhanced chemiluminescence detection and exposed over the nitrocellulose membrane. The band densities were determined by densitometric analysis using Image Studio Lite Version 5.2. The density values of the bands were normalized in each lane to β-actin or GAPDH (Sigma Chemical Co, St. Louis, Mo, USA).

2. Material and methods 2.1. Animals and ethics statement We used male C57BL/6 mice (8–12 weeks old, wild-type phenotype, and weighing 25 ± 2 g at baseline). The animals were maintained at 23 ± 2 °C under a 12-h light:dark cycle. Food and water were provided ad libitum before and during the experimental period. This study was performed in accordance with the principles and guidelines of the National Council for the Control of Animal Experimentation and approved by the Ethics Committee on Animal Use at the School of Pharmaceutical Sciences, University of São Paulo, Brazil (protocol number: 467). Cellular content was collected under ketamine/xylazine anesthesia, and all efforts were made to minimize animal suffering.

2.5. Enzyme-linked immunosorbent assay Cells were cultured at different time points, and supernatants were collected, centrifuged, and analyzed by enzyme-linked immunosorbent assay using cytokine-specific kits (R&D Systems, Minneapolis, MN, USA).

2.2. Induction of T1D T1D was induced via intravenous alloxan monohydrate injection (60 mg/kg) (Sigma Chemical Co, St. Louis, Mo) dissolved in saline (0.9 % NaCl), as previously described (Tessaro et al., 2017). After 10 days, the glycemia of the animals was measured using Accu-Chek Advantage II (Roche Diagnostics, São Paulo, SP, Brazil). Only animals with glycemia levels above 300 mg/dL were considered diabetic for this study.

2.6. Cell counting and viability Cell counting was performed by trypan blue exclusion (Thermo Fisher Scientific Inc., Rockford, IL). The percentage of viable cells was determined using a gate strategy, forward scatter and side scatter, to select macrophages that were negative for propidium iodide (PI) as viable cells. Briefly, BMDM were seeded at a concentration of 2 × 106 cells in 60 mm dishes in 2 ml RPMI 1640, 10 % of fetal bovine serum, and 5 % L929 cell-conditioned medium by overnight. After 24 h of treatment, cells were harvested using cold phosphate buffer saline containing fetal bovine serum at 5 % (pH 7.4), and stained with PI

2.3. Cell culture and LPS stimulation Bone marrow cells were isolated from the femurs of 8- to 12-wk-old mice, as described elsewhere (Tessaro et al., 2017). Cells were seeded in Petri dishes (10 cm) in RPMI 1640 (Sigma Chemical Co, St. Louis, Mo) 2

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3.2. PI3K/Akt is dysregulated in macrophages from diabetic mice

(1:100) for 30 min at 4 °C protected from light. Cell viability was analyzed by flow cytometry in a FACScan flow cytometer (BD Biosciences). To evaluate cell viability, we performed a second assay using as parameter of evaluation the metabolic activity of BMDM. Briefly, BMDM were seeded at a concentration of 2 × 105 cells/well in 96-well plates in 200 μl of mix containing 85 % of RPMI 1640, 10 %, of fetal bovine serum, and 5 % of L929 cell-conditioned medium overnight. Cells were incubated under the indicated experimental conditions. At the 24 -h time point, the medium was removed, and 100 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma Chemical Co, St. Louis, Mo, USA) solution (0.5 mg/ml in RPMI 1640) was added to each well. After 30 min at 37 °C, the medium was removed, and 100 μL of dimethyl sulfoxide was added (Lombardo et al., 2007). Spectrophotometric measurements at 510 nm were performed using a 96-well plate reader (Epoch Microplate Spectrophotometer, Biotek).

To induce macrophage differentiation, cells remained in conditioned medium for 7 days, as described before. Then, after counting the cells, we seeded BMDM with fresh medium in 6-well plates staying incubated overnight. Initially, we evaluated PI3K and Akt (Fig. 2) activation in non-diabetic and diabetic BMDM cell lysate. Our findings showed that BMDM from diabetic animals had higher expression levels of PI3K p110 alpha catalytic subunit (Fig. 2B), whereas PI3K p110β levels were similar in BMDM from diabetic and non-diabetic mice (Fig. 2C). PI3K p85 regulatory subunit levels in their total (Fig. 2D) or phosphorylated form (Fig. 2E; Fig. 2F) presented similar levels in both diabetic and non-diabetic BMDM. Thus, phosphorylation levels of the regulatory PI3K p55 subunit, which is an isoform of PI3K 85 normalized by β-actin (Fig. 2G), showed no difference between diabetic and non-diabetic BMDM; however, when we normalized the total levels of PI3K p85, the subunit showed fewer levels of phosphorylation in BMDM from diabetic animals compared to BMDM from non-diabetic mice (Fig. 2H). Our analysis of Akt showed that diabetic BMDM presented a higher phosphorylation at both threonine (Fig. 2I) and serine residues (Fig. 2J) in comparison to the non-diabetic BMDM.

2.7. Statistical analysis The data are presented as the mean ± SEM from at least three independent experiments. Statistical analyses were performed using GraphPad software (San Diego, CA, USA), and Student's t-test and analysis of variance followed by the Tukey-Kramer test were used to perform comparisons. A p-value lower than 0.05 was considered statistically significant.

3.3. ERK 1/2 and SAPK/JNK present enhanced phosphorylation in macrophages from diabetic mice MAPK phosphorylation levels (Fig. 3A) were also evaluated in BMDM cell lysate. We found that the levels of p38 phosphorylation (Fig. 3B) were similar in diabetic and non-diabetic animals BMDM. However, BMDM from diabetic animals showed higher phosphorylated levels of both p42 (Fig. 3C), and p44 MAPK (Fig. 3D), also known as ERK 1/2. For the SAPK/JNK phosphorylation, our result showed that diabetic BMDM had enhanced activation of both p46 (Fig. 3E), and p54 (Fig. 3F).

2.8. Data availability The values behind the means, standard deviations and other measures reported in the data supporting findings of this study can be obtained from the corresponding author upon reasonable request (Dr. Joilson de Oliveira Martins, [email protected]).

3. Results

3.4. BMDM from T1D animals showed impaired cytokine secretion after LPS stimulation

3.1. Type 1 diabetes experimental model Our results regarding TLR4 signaling proteins showed that diabetic BMDM had abnormal activation in PI3K/Akt, ERK 1/2 MAPK, and SAPK/JNK proteins. Then, we challenged both diabetic and non-diabetic BMDM with LPS. Our results showed that BMDM stimulated by 0.1 or 1 μg/mL of LPS secreted higher levels of TNF-α (Fig. 4A, B), and IL-6 (Fig. 4C, D) compared to the non-stimulated BMDM at 6 and 24 h.

C57BL/6 mice received an injection of alloxan. After 10 days, diabetic mice exhibited hyperglycemia (Fig. 1A), reduction in body weight gain (Fig. 1B), and low levels of circulating insulin (Fig. 1C) in comparison to the corresponding values found in mice that received the vehicle (saline).

Fig. 1. Type 1 diabetes mouse model. Alloxan was administered to C57BL/6 mice (i.v., 60 mg/kg), and (A) blood glucose (mg/dL), (B) body weight gain (g) and (C) insulin levels (ng/mL) were measured at different time points. The results are expressed as the mean ± SEM of at least four independent experiments. * p < 0.05 vs. Vehicle. 3

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Fig. 2. Expression of PI3K p110alpha and Akt in BMDM are enhanced in BMDM from type 1 diabetes mouse model. PI3K protein and phosphorylation levels and Akt phosphorylation were determined from cell lysates by Western blot. (A) Protein expression of PI3K p110alpha (110 kDa), PI3K p110beta (110 kDa), PI3K p85 (85 kDa), pPI3K p85 (85 kDa), pPI3K p55 (60 kDa), pAkt (Thr308) pAkt (Ser473) (60 kDa) and β-actin (42 kDa) (B) PI3K p110alpha and β-actin ratio; (C) PI3K p110beta and β-actin ratio; (D) PI3K p85 and β-actin ratio; (E) Relationship pPI3K p85 and PI3K p85; (F) pPI3K p85 and β-actin ratio; (G) pPI3K p55 and β-actin ratio; (H) pPI3K p55 and PI3K p85 ratio; (I) pAkt (Thr 308)/β-actin ratio; and (J) pAkt (Ser 473)/β-actin ratio. Results expressed as the mean ± SEM of at least 3 independent experiments. * p < 0.05 vs. Vehicle.

Fig. 3. Phosphorylation levels of ERK 1/2 MAPK and SAPK/JNK are enhanced in BMDM from type 1 diabetes mouse model. MAPK phosphorylation levels were determined from cell lysates by Western blot. (A) Protein phosphorylation of p-p38, p-ERK 1/2, p-SAPK/JNK, and protein expression of GAPDH. Levels of the ratio between (B) p-p38/GAPDH, (C) ERK pp42/GAPDH, (D) ERK p-p44/GAPDH, (E) SAPK/JNK p-p46/GAPDH, and (F) SAPK/JNK p-p54/GAPDH. The results are expressed as the mean ± SEM of at least 4 independent experiments. *p < 0.05 vs. Vehicle.

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Fig. 4. Secretion of pro- and anti-inflammatory cytokines after stimulation with LPS is dysregulated by BMDM from type 1 diabetes mouse model. BMDM were stimulated with 0.1 or 1 μg/mL LPS, and cytokine levels were determined in the cell culture supernatant by ELISA. Secretion of TNF-α at (A) 6 and (B) 24 h; secretion of IL-6 in.(C) 6 and (D) 24 h; secretion of IL-10 in.(E) 6 and (F) 24 h. (G) Metabolic activity was determined after 24 h of treatment by MTT assay and (H) PI levels were determined by flow cytometry in BMDM CD11b+. Values represent the mean ± SEM of four independent experiments. *p < 0.05 vs. Ctrl (Vehicle); **p < 0.05 vs. Ctrl (Alloxan); +p < 0.05 vs. 0.1 μg/mL (Vehicle); ++p vs. 1 μg / mL (Vehicle).

Meanwhile, non-diabetic BMDM stimulated by 1 μg/mL of LPS for 24 h secreted higher levels of IL-10 (Fig. 4E, F) than BMDM in LPS unstimulated cells. Compared to non-diabetic BMDM, diabetic BMDM stimulated by LPS secreted higher levels of TNF-α at 24 h of incubation. Meanwhile, diabetic BMDM secreted lower levels of both IL-6 and IL-10 compared to non-diabetic mice; the values cited were visualized at 6 h to IL-6, and at 24 h to IL-10. Regarded to IL-10, the reduction in secreted levels was observed when BMDM was stimulated only by 1 μg/ mL of LPS.

BMDM than non-diabetic BMDM. 3.7. Peritoneal macrophages from T1D animals presented dysregulated cytokine secretion after LPS stimulation Herein, we evaluate the responsiveness to LPS by tissue-specific macrophages using the same protocol previously described to BMDM. Peritoneal macrophages secreted higher levels of cytokine with 0.1 or 1 μg/mL of LPS (Fig. 6) compared to the unstimulated peritoneal macrophages in 6 and 24 h of incubation. After, we challenged both diabetic and non-diabetic peritoneal macrophages with 0.1 or 1 μg/mL of LPS. Our results showed that diabetic peritoneal macrophages secreted lower levels of TNF-α in 6 h when stimulated by 1 μg/mL of LPS (Fig. 6A); after 24 h of incubation, diabetic peritoneal macrophages stimulated by 0.1 and 1 μg/mL of LPS secreted lesser levels of TNF-α compared to non-diabetic peritoneal macrophages (Fig. 6B). Regarded to IL-6 secretion, diabetic peritoneal macrophages secreted higher levels at 6 h under stimulation by 0.1 μg/mL of LPS (Fig. 6C); however, secreted minor levels when stimulated by 0.1 and 1 μg/mL of LPS in 24 h of incubation in comparison to non-diabetic peritoneal macrophages (Fig. 6D). IL-10 secreted levels by diabetic peritoneal macrophages were lower after stimulation by 0.1 μg/mL of LPS in comparison to non-diabetic macrophages (Fig. 6E). Peritoneal macrophages were maintained in RPMI-1640, or LPS (0.1 or 1 μg/mL) for 24 h, and cells received a LPS re-stimulation (0.1 μg/ mL) for more additional 24 h. Then our results showed that diabetic peritoneal macrophages secreted higher levels of TNF-α (Fig. 6F) and IL-6 (Fig. 6G) compared to non-diabetic peritoneal macrophages. Both non-diabetic and diabetic peritoneal macrophages that received a second LPS stimulation (0.1 μg/mL) secreted lesser levels of TNF-α

3.5. Evaluation of cell viability and metabolic activity of BMDM MTT assay is used to cell viability thought metabolic activity due to the ability of BMDM of reduce the tetrazolium dye MTT to its insoluble formazan (Lombardo et al., 2007). We found that without LPS stimulus, non-diabetic and diabetic BMDM presented similar levels of cell mitochondrial activity. When stimulated by 0.1 or 1 μg/mL of LPS, diabetic BMDM presented lower metabolic activity than non-diabetic BMDM (Fig. 4G). The presence or not of different concentrations of LPS in BMDM did not alter BMDM viability measured by PI (Fig. 4H). 3.6. Diabetic BMDM secrete higher levels of TNF-α under LPS re-stimulation BMDM from diabetic animals maintained in RPMI-1640 for 24 h, then stimulated by 0.1 μg/mL of LPS for 24 additional hours secreted higher levels of TNF-α (Fig. 5A), and IL-6 (Fig. 5B) compared to nondiabetic BMDM. Meanwhile, a second LPS stimulus with 0.1 μg/mL promoted a higher secretion of TNF-α levels, but not IL-6 and IL-10 levels (Fig. 5C). In addition, the ratio between TNF-α/IL-10 (Fig. 5D) and IL-6/10 (Fig. 5E) showed higher values coming from diabetic 5

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Fig. 5. LPS induced enhanced levels of secreted TNF-α by tolerized macrophages from type 1 diabetes mouse model. Secretion of pro- and anti-inflammatory cytokines after re-stimulation with LPS by BMDM from non-diabetic and diabetic animals. Cytokine levels were determined in the cell culture supernatant by ELISA. Secretion of the cytokines (A) TNF-α, (B) IL-6 and (C) IL-10 after re-stimulation with LPS (0.1 μg / ml) for 24 h present in the cell culture supernatant. Ratio of (D) TNF-α/IL-10 and (E) IL-6/IL-10 values. Values represent the mean ± EPM of four independent experiments. *p < 0.05 vs. Ctrl (Vehicle); **p < 0.05 vs. Ctrl (Alloxan); p < 0.05 vs. Ctrl (Vehicle); § p < 0.05 vs. 0.1 μg/mL (Vehicle); +p < 0.05 vs. 0.1 μg/mL (Vehicle); ++p vs. 1 μg/mL (Vehicle).

compared to the peritoneal macrophage that received the first LPS stimulus with no differences between in non-diabetic and diabetic peritoneal macrophages. By the same comparison, non-diabetic peritoneal macrophages secreted lower levels of IL-6 (Fig. 6G) in comparison to peritoneal macrophages first stimulated, meanwhile diabetic peritoneal macrophages secreted similar levels when stimulates or restimulated in the 24 additional hours. Regarded to IL-10, the levels in the supernatant were not detected by the technique used in this work.

results, we observed a different phenotype of BMDM activation from diabetic animals compared to BMDM of non-diabetic mice. Macrophages residing at T1D recipient present different process of activation (Diapen et al., 2016; Sunahara et al., 2014; Bannon et al., 2013; Sun et al., 2012) beyond signaling pathways disruption (Filgueiras et al., 2015; Sun et al., 2012), and consequently failing to secrete immune mediators in response to bacterial products (Lachmands et al., 2018; Filgueiras et al., 2015). In alloxan-treated animals, diabetes affects intracellular signaling pathways in 7-days differentiated macrophages coming from bone marrow cells. Resulting in an increase of PI3K p110 alpha, and a reduction in the activation of PI3K p55 (p85 subunit). Hyperglycemia from diabetes induces epigenetic changes reprograming the macrophage and modifying the response to subsequent cellular stimulus (Ratter et al., 2018; Sun et al., 2012). PI3K is formed by a catalytic subunit (p110), and regulatory subunits (p85 and p55), which controls the phosphorylation of phosphatidylinositol 4, 5-bisphosphate in phosphatidylinositol 3, 4, 5-bisphosphate (Jean and Kiger, 2014; Guillermet-Guibert et al., 2008). Several stimuli activate PI3K generating higher levels of phosphatidylinositol 3, 4, 5-bisphosphate that recruits both PDK1 and PKB/Akt directly to the cell membrane (Jean and Kiger, 2014). High glucose in vitro increases Akt phosphorylation in peritoneal macrophages (Sun et al., 2012). In addition, Akt represents a family of serine-threonine

4. Discussion Reduced levels of insulin are seen in T1D patients, and it promotes a state of intense catabolism of carbohydrates, lipids, and amino acids (American Diabetes Association, 2016; Atkinson et al., 2014; Oliveira et al., 2013). Modification in host metabolism is accompanied by the progress of diabetes, which have been also associated with phenotype changes in macrophages (Bannon et al., 2013; Sun et al., 2012). Macrophages are virtually residing in all organs, contributing to maintain homeostasis by removing microorganisms and cell debris from different sites (Murray and Wynn, 2011). Dysregulated cytokine secretion generated by this environment contributes to increased susceptibility to infectious diseases in diabetic patients (Lachmands et al., 2018; Filgueiras et al., 2015; Casqueiro et al., 2012). Taken together our 6

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Fig. 6. Secretion of pro- and anti-inflammatory cytokines after the first and second stimulus with LPS is dysregulated by peritoneal macrophages from type 1 diabetes mouse model. Peritoneal macrophages were stimulated with 0.1 or 1 μg/mL of LPS, and then re-stimulated with LPS (0.1 μg/mL). Cytokine levels were determined in the cell culture supernatant by ELISA. Secretion of TNF-α at (A) 6 and (B) 24 h; secretion of IL-6 in (C) 6 and (D) 24 h; secretion of IL-10 in (E) 24 h. After additional 24 h of re-stimulation, levels of TNF-α (F) and IL-6 (G) were determined. *p < 0.05 vs. Ctrl (Vehicle); **p < 0.05 vs. Ctrl (Alloxan); p < 0.05 vs. Ctrl (Vehicle); § p < 0.05 vs. 0.1 μg/mL (Vehicle); +p < 0.05 vs. 0.1 μg/mL (Vehicle); ++p vs. 1 μg/mL (Vehicle).

LPS-activated macrophages through a cellular mechanism of tolerance reduce the expression of TLR4 on cell surface (Rajaiah et al., 2015). Akt has an important role in the mechanism of TLR4 tolerance, and LPS re-stimulation is known to promote hyporesponsiveness with a minor release of cytokines by macrophages (Androulidaki et al., 2009). Tolerance state needs to be tightly regulated by the cell since alterations in this mechanism can provoke exacerbated responses by macrophages (López-Collazo and Del Fresno, 2013, Androulidaki et al., 2009). We have demonstrated that diabetic BMDM present changes in the basal level of some important components of LPS signaling, which possibly is modifying the levels of cytokine secretion. Both acute and local production of TNF-α is beneficial in host defense against pathogens, meanwhile chronic exposure to high levels of TNF-α is detrimental (Tracey et al., 1986). Diabetic BMDM failed to respond adequately upon receiving the first and second LPS stimulation in the additionally 24 h secreting enhanced TNF-α levels even mantaing the tolerance state, visualized by IL-6 and IL-10 secreted levels. Meanwhile, after re-stimulation peritoneal macrophages secreted similar levels of TNF-α in 24 h showing the presence of a hyporesponsiveness state. However, diabetic peritoneal macrophages failed in secreting similar levels of IL-6 after being stimulated for the first and second in the additionally 24 h. Modifications in LPS response generated by the impact of diabetes in macrophages have changed the way these cells secrete cytokines in its own microenvironment. When occurs an impaired secretion of TNFα and IL-6 by macrophages, there is an increase of the risk in the development of inflammatory conditions, which together can activate immune cells and contribute to the failures in macrophage activation (Filgueiras et al., 2015; Awad et al., 2015; Kanter et al., 2000). The balance between TNF-α and IL-10 is the result of autocrine control in which TNF-α activates the synthesis and secretion of IL-10, while IL-10 negatively regulates the formation of TNF-α by negative feedback

protein kinases that regulate cell survival and TLR4 expression (Androulidaki et al., 2009). Diabetes affected BMDM driving to a higher phosphorylation in Akt levels at both threonine and serine residues. In addition, our findings demonstrated that diabetes itself enhanced the phosphorylation of JNK/SAPK, which their activation are in response to inflammatory diseases involved in the control of insulin sensitivity and metabolic regulation (Gary and Nakamura, 2007). Increased levels of ERK1/2 phosphorylation in macrophages and other cells mostly occur by exerting a protective effect to the chronic high glucose environment (Través et al., 2012). Both SAPK/JNK and ERK 1/2 presented major activation in BMDM coming from diabetic mice. Alterations in both PI3K/Akt and MAPK levels in diabetic BMDM did not promote differences in spontaneous release of TNF-α, IL-6, and IL-10 levels. When stimulated by LPS, BMDM recognizes LPS primarily by TLR4/MD2 via MyD88-dependent signal transduction through MAPK and PI3K/Akt promoting synthesis and secretion of inflammatory mediators (Liu et al., 2007; Murray and Wynn, 2011; Jean and Kiger, 2014). Together, diabetic BMDM presented a reduction in phosphorylation of PI3K p55, an increase in PI3K p110 alpha, and activation of Akt, ERK 1/2, and SAPK/JNK. Thus, these BMDM secreted higher levels of TNF-α (24 h), lower levels of IL-6 (6 h), and IL-10 (24 h) after LPS stimulation compared to the non-diabetic BMDM without altering cell surveillance. However, we found that diabetic BMDM stimulated by LPS showed a reduced metabolic activity in 24 h compared to non-diabetic BMDM showing modification in cell metabolism behavior, however cell viability evaluated by PI showed no injuries. In diabetes, impaired cytokine secretion contributes to the failure in the resolution of inflammatory process (Casqueiro et al., 2012), such as LPS response (Filgueiras et al., 2015), bacterial elimination (Filgueiras et al., 2015; Esper et al., 2008) and wound healing (Bannon et al., 2013).

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(Salez et al., 2000). Therefore, adequate levels of IL-10 are important because they directly modulate the functions of macrophages, which include the release of immune proinflammatory mediators, antigen presentation and phagocytosis (Sabat et al., 2010). Furthermore, IL-10 suppresses IL-6 synthesis, stimulating the production of the TNF-α receptor in its soluble form (Fullerton and GIROY, 2016; Sabat et al., 2010). Failures in IL-10 secretion drive an inflammatory macrophage profile due to an increase in the secretion of proinflammatory mediators. After LPS re-stimulation, the ratio of TNF-α and IL-6 versus IL-10 reflected a presence of an inflammatory microenvironment promoted by diabetic BMDM. Herein, we found that diabetes impairs PI3K/Akt, and MAPK signaling proteins in BMDM. Therefore, dysregulates TNF-α, IL-6, and IL10 secretion levels in both BMDM and peritoneal macrophages coming from diabetic mice. In 24 h, diabetic BMDM showed a decrease in the metabolism activity. Additionally, in a tolerant state, diabetic BMDMs secreted higher levels of TNF-α, but not IL-6 and IL-10; while diabetic peritoneal macrophages secreted enhanced levels of IL-6, but not of TNF-α.

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5. Conclusions Diabetic macrophages from different compartments have an impaired capacity of response and tolerance to LPS contributing to a poor inflammatory control. These results provide new insights into the diabetes field and generation of macrophages in a hyperglycemic environment. Declaration of Competing Interest The authors declare no competing financial interests. Author Contributions FHT, TSA and JOM conceived and designed the experiments. FHT, TSA, and LMB performed the experiments. FHT, TSA and JOM analyzed the data. JOM contributed reagents/materials/analysis tools. FHT, TSA and JOM wrote the paper with the assistance of all the authors. Acknowledgments The authors sincerely would like to thank Silene Migliorini and Renata Chaves Albuquerque for their expert technical help. The authors are supported by grant 2010/02272-0, 2014/05214-1, 2017/11540-7 and 2019/25999-7 from São Paulo Research Foundation (FAPESP), grant 470523/2013-1 and 301617/2016-3 from National Counsel of Technological and Scientific Development (CNPq, Projeto Universal 2013, PQ-1D), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil. References American Diabetes Association, 2016. Sec. 1. In Standards of Medical Care in Diabetes. 39. pp. S6–S12. Androulidaki, A., et al., 2009. Akt1 controls macrophage response to LPS by regulating microRNAs immunity. Immunity 31, 220–231. Atkinson, M.A., Eisenbarth, G.S., Michels, A.W., 2014. Type 1 diabetes. Lancet 383, 69–82. Awad, A.S., You, H., Gao, T., Cooper, T.K., Nedospasov, S.A., Vacher, J., Wilkinson, P.F., Farrell, F.X., Brian Reeves, W., 2015. Macrophage-derived tumor necrosis factor-α mediates diabetic renal injury. Kidney Int. 88, 722–733. Ayala, T.S., Tessaro, F.H.G., Jannuzzi, G.P., Bella, L.M., Ferreira, K.S., Martins, J.O., 2019. High glucose environments interfere with bone marrow-derived macrophage inflammatory mediator release, the TLR4 pathway and glucose metabolism. Sci. Rep. 9 (1), 11447. Bannon, P., Wood, S., Restivo, T., Campbell, L., Hardman, M.J., Mace, K.A., 2013.

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