Insulin Influences Autophagy Response Distinctively in Macrophages of Different Compartments

Background/Aims: Diabetes mellitus (DM) is characterized by hyperglycemia, associated to a lack or inefficiency of the insulin to regulate glucose metabolism. DM is also marked by alterations in a diversity of cellular processes that need to be further unraveled. In this study, we examined the autophagy pathway in diabetic rat macrophages before and after treatment with insulin. Methods: Bone marrow-derived macrophages (BMM), bronchoalveolar lavage (BAL) and splenic tissue of diabetic male Wistar rats (alloxan, 42 mg/kg, i.v., 10 days) and control rats (physiological saline, i.v.). Some diabetic rats were given neutral protamine Hagedorn insulin (4 IU, s.c.) 8 h before experiments. For characterization of the model and evaluation of the effect of insulin on the autophagic process, the following analyzes were performed: (a) concentrations of cytokines: interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-4, IL-10, cytokine-induced neutrophil chemoattractant (CINC)-1 and CINC-2 in the BAL supernatant was measured by ELISA; (b) characterization of alveolar macrophage (AM) of the BAL as surface antigens (MHCII, pan-macrophage KiM2R, CD11b) and autophagic markers (protein microtubule-associated light chain (LC)3, autophagy protein (Atg)12 by flow cytometry and confocal microscopy (c) study of macrophages differentiated from the bone marrow by flow cytometry and confocal microscopy (d) histology of the spleen by immunohistochemistry associated with confocal microscopy. Results: Interestingly, insulin exerted antagonistic effects on macrophages from different tissues. Macrophages from bronchoalveolar lavage (BAL) enhanced their LC3 autophagosome bound content after treatment with insulin whereas splenic macrophages from red pulp in diabetic rats failed to enhance their Atg 12 levels compared to control animals. Insulin treatment in diabetic rats did not change LC3 content in bone marrow derived macrophages (BMM). M1 and M2 macrophages behaved accordingly to the host they were derived from. Diabetic M1 BMM had their LC3 vesicle-bound content diminished and M2 BMM enhanced their LC3 levels and insulin treatment failed to rescue autophagy to control levels. Insulin normalizes CINC-2 level but does not modulate autophagy markers. Conclusion: Taking these results together, diabetic macrophages derived from different compartments show different levels of autophagy markers compared to healthy animals, therefore, they suffer distinctively in the absence of insulin

Deletion of Wiskott–Aldrich syndrome protein triggers Rac2 activity and increased cross-presentation by dendritic cells

Wiskott–Aldrich syndrome (WAS) is caused by loss-of-function mutations in the WASp gene. Decreased cellular responses in WASp-deficient cells have been interpreted to mean that WASp directly regulates these responses in WASp-sufficient cells. Here, we identify an exception to this concept and show that WASp-deficient dendritic cells have increased activation of Rac2 that support cross-presentation to CD8+ T cells. Using two different skin pathology models, WASp-deficient mice show an accumulation of dendritic cells in the skin and increased expansion of IFNγ-producing CD8+ T cells in the draining lymph node and spleen. Specific deletion of WASp in dendritic cells leads to marked expansion of CD8+ T cells at the expense of CD4+ T cells. WASp-deficient dendritic cells induce increased cross-presentation to CD8+ T cells by activating Rac2 that maintains a near neutral pH of phagosomes. Our data reveals an intricate balance between activation of WASp and Rac2 signalling pathways in dendritic cells

Modification of Heterotrimeric G-Proteins in Swiss 3T3 Cells Stimulated with <em>Pasteurella multocida</em> Toxin

<div><p>Many bacterial toxins covalently modify components of eukaryotic signalling pathways in a highly specific manner, and can be used as powerful tools to decipher the function of their molecular target(s). The <em>Pasteurella multocida</em> toxin (PMT) mediates its cellular effects through the activation of members of three of the four heterotrimeric G-protein families, G_q, G₁₂ and G_i. PMT has been shown by others to lead to the deamidation of recombinant Gα_i at Gln-205 to inhibit its intrinsic GTPase activity. We have investigated modification of native Gα subunits mediated by PMT in Swiss 3T3 cells using 2-D gel electrophoresis and antibody detection. An acidic change in the isoelectric point was observed for the Gα subunit of the G_q and G_i families following PMT treatment of Swiss 3T3 cells, which is consistent with the deamidation of these Gα subunits. Surprisingly, PMT also induced a similar modification of Gα₁₁, a member of the G_q family of G-proteins that is not activated by PMT. Furthermore, an alkaline change in the isoelectric point of Gα₁₃ was observed following PMT treatment of cells, suggesting differential modification of this Gα subunit by PMT. G_s was not affected by PMT treatment. Prolonged treatment with PMT led to a reduction in membrane-associated Gα_i, but not Gα_q. We also show that PMT inhibits the GTPase activity of G_q.</p> </div

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field