Evaluation of the TLR4 activation and its possible correlation with the endoplasmatic reticulum stress in neutrophils in Diabetes mellitus type 2.

Os receptores toll-like (TLR) reconhecem agentes invasores ou moléculas indicativas de injúria tecidual. Neutrófilos expressam a maioria dos receptores TLR e quando ativados desencadeiam a produção de citocinas e inicia-se o processo inflamatório. A ativação da via de TLR4 promove um aumento do estresse de retículo endoplasmático devido a alta demanda na produção de proteínas, principalmente citocinas e quimiocinas. O objetivo desse trabalho foi avaliar a resposta neutrofílica ao LPS e a interação das vias de TLR4 e UPR frente a duas condições: a obesidade e o diabetes tipo 2 (GK). Wistar alimentados com dieta hiperlipídica apresentaram um quadro de obesidade com diminuição da sensibilidade a insulina, enquanto animais GK apresentaram todo o fenótipo diabético tipo 2. Neutrófilos de animais GK e HFD produziram menos citocinas e migraram menos para o sítio de inflamação por mecanismos distintos. Por fim, neutrófilos dos grupos GK e HFD mostraram-se resistentes ao LPS por deficiência na via do TLR4.Toll-like receptors (TLRs) recognize invading agents or molecules indicative of tissue injury. Neutrophils express most TLR receptors and when activated trigger the production of cytokines and initiate the inflammatory process. Activation of the TLR4 pathway promotes an increase in endoplasmic reticulum stress due to high demand in the production of proteins, mainly cytokines and chemokines. The aim of this study was to evaluate the neutrophilic response to LPS and the interaction of the TLR4 and UPR pathways in two different conditions: obesity and type 2 diabetes (GK). HFD fed Wistar rats had a decrease in insulin sensitivity, whereas GK animals had the full type 2 diabetic phenotype. Neutrophils from GK and HFD produced lower cytokines and migrated less to the site of inflammation by different mechanisms. Finally, neutrophils from the GK and HFD groups were resistant to LPS because of deficiency in the TLR4 pathway

The role of the endoplasmatic reticulum in the process of death of neutrophils from diabetic rats.

O retículo endoplasmático (RE) vem ganhando evidência quando se trata de morte celular. O acúmulo de proteínas mal formadas inicia a ativação da Unfolded Protein Response (UPR), com a finalidade de manter a homeostasia do RE. Porém, quando o estímulo do estresse perdura por muito tempo e não é resolvido, a UPR pode ativar genes que conduzem à morte celular. Hiperglicemia, presente no diabetes mellitus, induz estresse de RE em vários tipos celulares. Assim, este estudo teve como objetivo investigar o possível envolvimento do estresse do retículo no processo de morte celular em neutrófilos de ratos diabéticos. Nosso estudo revelou que os neutrófilos de ratos diabéticos quando estimulados com PMA apresentam uma maior suscetibilidade à morte devido à ativação de IRE1a e subsequente fosforilação de JNK, redução na interação mitocôndria-RE na MAM e aumento da atividade da caspase-3. Dentre os resultados apresentados, neutrófilos provenientes de animais controle parecem estar protegidos do estresse de RE por apresentar maior expressão de GRP78 e das proteínas da MAM.Endoplasmic reticulum (ER) has been gaining evidence when it comes to cell death. The accumulation of unfolded proteins initiates the activation of the Unfolded Protein Response (UPR). The UPR may resolve the ER stress by upregulating genes responsible to maintain the ER homeostasis; or it can activate genes that lead to the cell death when the ER disbalance is not solved. Hyperglycemia, one of many symptoms observed is Diabetes, may cause ER stress in various types of cells. Thus, this study aims to investigate the possible involvement of the ER stress in the process of cell death in neutrophils from diabetic rats. In summary, our study found that neutrophils from diabetic rats when stimulated with PMA exhibit greater susceptibility to death due to activation of IRE1a and subsequent phosphorylation of JNK, reduced safety in mitochondria-ER interaction in the MAM compartment and increased caspase-3 activation. Control group seems to be protect against the ER stress by ROS production by higher expression of GRP78 and MAM proteins

Evaluation of the TLR4 activation and its possible correlation with the endoplasmatic reticulum stress in neutrophils in Diabetes mellitus type 2.

Os receptores toll-like (TLR) reconhecem agentes invasores ou moléculas indicativas de injúria tecidual. Neutrófilos expressam a maioria dos receptores TLR e quando ativados desencadeiam a produção de citocinas e inicia-se o processo inflamatório. A ativação da via de TLR4 promove um aumento do estresse de retículo endoplasmático devido a alta demanda na produção de proteínas, principalmente citocinas e quimiocinas. O objetivo desse trabalho foi avaliar a resposta neutrofílica ao LPS e a interação das vias de TLR4 e UPR frente a duas condições: a obesidade e o diabetes tipo 2 (GK). Wistar alimentados com dieta hiperlipídica apresentaram um quadro de obesidade com diminuição da sensibilidade a insulina, enquanto animais GK apresentaram todo o fenótipo diabético tipo 2. Neutrófilos de animais GK e HFD produziram menos citocinas e migraram menos para o sítio de inflamação por mecanismos distintos. Por fim, neutrófilos dos grupos GK e HFD mostraram-se resistentes ao LPS por deficiência na via do TLR4.Toll-like receptors (TLRs) recognize invading agents or molecules indicative of tissue injury. Neutrophils express most TLR receptors and when activated trigger the production of cytokines and initiate the inflammatory process. Activation of the TLR4 pathway promotes an increase in endoplasmic reticulum stress due to high demand in the production of proteins, mainly cytokines and chemokines. The aim of this study was to evaluate the neutrophilic response to LPS and the interaction of the TLR4 and UPR pathways in two different conditions: obesity and type 2 diabetes (GK). HFD fed Wistar rats had a decrease in insulin sensitivity, whereas GK animals had the full type 2 diabetic phenotype. Neutrophils from GK and HFD produced lower cytokines and migrated less to the site of inflammation by different mechanisms. Finally, neutrophils from the GK and HFD groups were resistant to LPS because of deficiency in the TLR4 pathway

NADPH Oxidase-Dependent Production of Reactive Oxygen Species Induces Endoplasmatic Reticulum Stress in Neutrophil-Like HL60 Cells

<div><p>Reactive oxygen species (ROS) primarily produced via NADPH oxidase play an important role for killing microorganisms in neutrophils. In this study we examined if ROS production in Human promyelocytic leukemia cells (HL60) differentiated into neutrophil-like cells (dHL60) induces ER stress and activates the unfolded protein response (UPR). To cause ROS production cells were treated with PMA or by chronic hyperglycemia. Chronic hyperglycemia failed to induce ROS production and did not cause activation of the UPR in dHL60 cells. PMA, a pharmacologic NADPH oxidase activator, induced ER stress in dHL60 cells as monitored by IRE-1 and PERK pathway activation, and this was independent of calcium signaling. The NADPH oxidase inhibitor, DPI, abolished both ROS production and UPR activation. These results show that ROS produced by NADPH oxidase induces ER stress and suggests a close association between the redox state of the cell and the activation of the UPR in neutrophil-like HL60 cells.</p></div

Effect of PMA, fMLP and hyperglycemia on calcium influx and ER calcium content in neutrophil-like HL60 cells (dHL60).

<p>PMA (1 μM) (A) did not alter calcium dynamics in dHL60 cells. fMLP (1 μM) (B-D) promoted calcium influx in neutrophil-like HL60 cells; and hyperglycemia (C) did not disturb the calcium intake by fMLP stimulation. ER calcium content in dHL60 was not altered by hyperglycemia (E; F). Calcium dynamics were monitored by Indo-1-AM (1 μM) using fluorometry. [NG] normoglycemic media (5.5 mM of glucose); [MN] Mannitol enriched media (5.5 mM of glucose + 19.5 mM of mannitol); [HG] Hyperglycemic media (25 mM of glucose). Graphs show fluorescence intensity during the time of analysis (A; B; C; E). Histograms show the mean intracellular calcium concentration ± S.E.M. (D) and the mean ± S.E.M. of ER calcium content (F). Results are from 4 independent experiments. (**) Indicate p <0.01 and (*) indicate p<0,05.</p

Effect of ROS production on UPR regulated proteins in neutrophil-like HL60 cells (dHL60).

<p><b> </b> PMA (1 μM) treatment for 1 and 4h increased phosphorylated eIF2α (A; C) and GRP78 protein levels (B; D). This was blocked by DPI (10 μM), a NADPH oxidase inhibitor (A; B; C; D). GADD34 and ATF4 protein content was not altered by PMA (E; F). Histograms (C; D; E; F) show the mean ± S.E.M. of the optical density (OD) of the protein bands. γ Tubulin was used as loading control. Results are from at least 3 independent experiments. (***) Indicates p <0.001 and (**) Indicates p <0.01.</p

Effect of ROS production on the expression of genes involved in the unfolded protein response (UPR).

<p>GAPDH and 18S were used as reference control genes. Results are from 6 independent experiments. Thapsigargin (1μM) 1h and Tunicamycin (2 μg/ml) 16h were used as positive controls. (***) Indicates p <0.001; (**) Indicates p <0.01 and (*) Indicates p <0.05; (###) indicates p<0.001 <i>vs</i> control group; (##) indicates p<0.01 <i>vs</i> control group; (#) indicates p<0.05 <i>vs</i> control group.</p

Effect of hyperglycemia and PMA on ROS production in neutrophil-like HL60 cells (dHL60) and non-differentiated HL60 cells.

<p>PMA (1μM) triggered the production of ROS only in dHL60 (A; C). Hyperglycemia did not affect the ROS production in neutrophil-like HL60 cells (B). DPI (10 μm) was used as NADPH oxidase inhibitor. [NG] normoglycemic media (5.5 mM of glucose); [MN] Mannitol enriched media (5.5 mM of glucose + 19.5 mM of mannitol); [HG] Hyperglycemic media (25 mM of glucose). DHR (10 μM) was used to monitor ROS production by flow cytometry. Graphs show median of fluorescence ± S.E.M. Results are from 6 independent experiments. (***) Indicate p <0.001.</p

Effect of hyperglycemia and PMA on eIF2α phosphorylation and splicing of XBP1 mRNA in neutrophil-like HL60 cells (dHL60).

<p>Hyperglycemia failed to modulate eIF2α phosphorylation (A; B) and splicing of XBP1 (C; D). Splicing of XBP1 mRNA was caused by PMA (1 μM) stimulus (1 and 4h) (C; D). [NG] normoglycemic media (5.5 mM of glucose); [MN] Mannitol enriched media (5.5 mM of glucose + 19.5 mM of mannitol); [HG] Hyperglycemic media (25 mM of glucose). Histogram (B) shows the mean ± S.E.M. optical density (OD) of the protein bands (A). γ Tubulin was used as loading control. (C) Complementary cDNA bands of unspliced XBP1 (uXBP1) (top band) and spliced (sXBP1) (bottom band). Results is representative of 3 independent experiments. [Tg] Positive control (1 μM Thapsigargin, 1h); [N1h] NG + 1h PMA; [M1h] MN + 1h PMA; [H1h] HG + 1h PMA; [N4h] NG + 4h PMA; [M4h] MN + 4h PMA; [H4h] HG + 4h PMA.</p