Obesity increases blood-brain barrier permeability and aggravates the mouse model of multiple sclerosis.

Obesity-induced insulin resistance (OIR) has been associated with an increased prevalence of neurodegenerative disorders such as multiple sclerosis. Obesity results in increased blood-brain barrier (BBB) permeability, specifically in the hypothalamic regions associated with the control of caloric intake. In obesity, the chronic state of low-grade inflammation has been implicated in several chronic autoimmune inflammatory disorders. However, the mechanisms that connect the inflammatory profile of obesity with the severity of experimental autoimmune encephalomyelitis (EAE) are poorly defined. In this study, we show that obese mice are more susceptible to EAE, presenting a worse clinical score with more severe pathological changes in the spinal cord when compared with control mice. Analysis of immune infiltrates at the peak of the disease shows that high-fat diet (HFD)- and control (chow)-fed groups do not present any difference in innate or adaptive immune cell compartments, indicating the increased severity occurs prior to disease onset. In the setting of worsening EAE in HFD-fed mice, we observed spinal cord lesions in myelinated regions and (blood brain barrier) BBB disruption. We also found higher levels of pro-inflammatory monocytes, macrophages, and IFN-γ+CD4+ T cells in the HFD-fed group compared to chow-fed animals. Altogether, our results indicate that OIR promotes BBB disruption, allowing the infiltration of monocytes/macrophages and activation of resident microglia, ultimately promoting CNS inflammation and exacerbation of EAE.

iNKT Cel Transfer: The Use of Cell Sorting Combined with Flow Cytometry Validation Approach.

Natural killer T (NKT) cells are an innate-like T cell subset that recognize lipid antigens presented by CD1d-expressing antigen presenting cells (APCs), such as dendritic cells, macrophages, and B cells. They can be subdivided into two different subsets according to the variation in αß TCR chains: type I and type II NKT cells. Type I, also called invariant NKT cells (iNKT), express restricted TCRs with an invariant α-chain (Vα24-Jα18 in humans and Vα14-Jα18 in mice) and limited ß-chains. Here we have established a protocol in which iNKT cells are isolated from a donor wild-type mouse and transferred into iNKT KO (Jα18-/-) mouse. Below we will explore the methods for cell sorting of splenic iNKTs, iNKT cells transfer, and detection of transferred cells into the liver using flow cytometry technique.

Using flow cytometry for mitochondrial assays.

The understanding of how different cell types adapt their metabolism in the face of challenges has been attracting the attention of researchers for many years. Recently, immunologists also started to focus on how the metabolism of immune cells can impact the way that immunity drives its responses. The presence of a pathogen or damage in a tissue changes severely the way that the immune cells need to respond. When activated, immune cells usually shift their metabolism from a high energy demanding status using mitochondria respiration to a glycolytic based rapid ATP production. The diminished amount of respiration leads to changes in the mitochondrial membrane potential and, consequently, generation of reactive oxygen species. Here, we show how flow cytometry can be used to track changes in mitochondrial mass, membrane potential and superoxide (ROS) production in live immune cells. ● This protocol suggests a quick way of evaluating mitochondrial fitness using flow cytometry. We propose using the probes MitoTraker Green and MitoTracker Red/ MitoSOX at the same time. This way, it is possible to evaluate different parameters of mitochondrial biology in living cells. ● Flow cytometry is a highly used tool by immunologists. With the advances of studies focusing on the metabolism of immune cells, a simplified application of flow cytometry for mitochondrial studies and screenings is a helpful clarifying method for immunology.

M-CSF- and L929-derived macrophages present distinct metabolic profiles with similar inflammatory outcomes.

Macrophages are essential components of the immune system. Macrophages can be derived from the bone marrow of mice with either recombinant M-CSF or L929 supernatant. Recent literature considers recombinant M-CSF- and L929-derived macrophages as equals, even though L929-derived macrophages are exposed to other substances secreted in the L929 supernatant, and not only M-CSF. Thus, we decided to perform a comparative analysis of both inflammatory and metabolic profiles of macrophages differentiated under the aforementioned conditions, which is relevant for standardization and interpretation of in vitro studies. We observed that, when treated with LPS, L929macs secrete lower levels of proinflammatory cytokines (TNF-α, IL-6, IL12) and present higher glycolysis and oxygen consumption when compared with M-CSFmacs. L929macs also have increased mitochondrial mass, with higher percentage of dysfunctional mitochondria. This sort of information can help direct further studies towards a more specific approach for macrophage generation.

Fecal IgA Levels and Gut Microbiota Composition Are Regulated by Invariant Natural Killer T Cells.

BACKGROUND: The gut microbiota is a key element to support host homeostasis and the development of the immune system. The relationship between the microbiota and immunity is a 2-way road, in which the microbiota contributes to the development/function of immune cells and immunity can affect the composition of microbes. In this context, natural killer T cells (NKT cells) are distinct T lymphocytes that play a role in gut immunity and are influenced by gut microbes. In our work, we investigated the involvement of invariant NKT cells (iNKT) in intestinal homeostasis. RESULTS: We found that iNKT-deficient mice (iNKT-KO) had reduced levels of fecal IgA and an altered composition of the gut microbiota, with increased Bacteroidetes. The absence of iNKT cells also affected TGF-ß1 levels and plasma cells, which were significantly reduced in knockout (KO) mice. In addition, when submitted to dextran sodium sulfate colitis, iNKT-KO mice had worsening of colitis when compared with wild-type (WT) mice. To further address iNKT cell contribution to intestinal homeostasis, we adoptively transferred iNKT cells to KO mice, and they were submitted to colitis. Transfer of iNKT cells improved colitis and restored fecal IgA levels and gut microbiota. CONCLUSIONS: Our results indicate that intestinal NKT cells are important modulators of intestinal homeostasis and that gut microbiota composition may be a potential target in the management of inflammatory bowel diseases.

Mesenchymal stromal cells modulate gut inflammation in experimental colitis.

Inflammatory bowel diseases (IBDs) affect millions of people worldwide and their frequencies in developed countries have increased since the twentieth century. In this context, there is an intensive search for therapies that modulate inflammation and provide tissue regeneration in IBDs. Recently, the immunomodulatory activity of adipose tissue-derived mesenchymal stromal cells (ADMSCs) has been demonstrated to play an important role on several immune cells in different conditions of inflammatory and autoimmune diseases. In this study, we explored the immunomodulatory potential of ADMSC in a classical model of DSS-induced colitis. First, we found that treatment of mice with ADMSC ameliorated the severity of DSS-induced colitis, reducing colitis pathological score and preventing colon shortening. Moreover, a prominent reduction of pro-inflammatory cytokines levels (i.e., IFN-γ, TNF-α, IL-6 and MCP-1) was observed in the colon of animals treated with ADMSC. We also observed a significant reduction in the frequencies of macrophages (F4/80+CD11b+) and dendritic cells (CD11c+CD103+) in the intestinal lamina propria of ADMSC-treated mice. Finally, we detected the up-regulation of immunoregulatory-associated molecules in intestine of mice treated with ADMSCs (i.e., elevated arginase-1 and IL-10). Thus, this present study demonstrated that ADMSC modulates the overall gut inflammation (cell activation and recruitment) in experimental colitis, providing support to the further development of new strategies in the treatment of intestinal diseases.