Lack of mTORC2 signalling in CD11c+ myeloid cells inhibits their migration and ameliorates experimental colitis.

The Mammalian Target of Rapamycin (mTOR) pathway plays a key role in determining immune cells function through modulation of their metabolic status. By specific deletion of Rictor in CD11c+ myeloid cells (referred to here as CD11cRicΔ/Δ), this study investigated the role of mTOR complex 2 (mTORC2) signalling in dendritic cells (DCs) function in mice. We showed that upon DSS-induced colitis, lack of mTORC2 signalling CD11c+ cells diminishes colitis score, and abrogates dendritic cell (DC) migration to the mesenteric lymph nodes (MLN), thereby diminishing the infiltration of T helper (Th) 17 cells in the lamina propria (LP) and subsequent inflammation. These findings corroborate with abrogation of cytoskeleton organization and decreased activation of Rac1 and Cdc42 GTPases observed in CD11c+-mTORC2-deficient cells. Meta-analysis on colonic samples from ulcerative colitis (UC) patients revealed increased gene expression of pro-inflammatory cytokines which coincided with augmented expression of mTOR pathway, positive correlation between the DC marker ITGAX and IL-6, the expression of RICTOR, and CDC42. Together, this work proposes that targeting mTORC2 on DCs offers a key to hamper inflammatory responses and this way, ameliorates the progression and severity of intestinal inflammatory diseases.

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.

Tissue-specific metabolic profile drives iNKT cell function during obesity and liver injury.

Invariant natural killer T (iNKT) cells are a distinct population of lymphocytes characterized by their reactivity to glycolipids presented by CD1d. iNKT cells are found throughout the body, and little is known about their tissue-specific metabolic regulation. Here, we show that splenic and hepatic iNKT cells are metabolically comparable and rely on glycolytic metabolism to support their activation. Deletion of the pyruvate kinase M2 (Pkm2) gene in splenic and hepatic iNKT cells impairs their response to specific stimulation and their ability to mitigate acute liver injury. In contrast, adipose tissue (AT) iNKT cells exhibit a distinctive immunometabolic profile, with AMP-activated protein kinase (AMPK) being necessary for their function. AMPK deficiency impairs AT-iNKT physiology, blocking their capacity to maintain AT homeostasis and their ability to regulate AT inflammation during obesity. Our work deepens our understanding on the tissue-specific immunometabolic regulation of iNKT cells, which directly impacts the course of liver injury and obesity-induced inflammation.

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.

Protective role of NKT cells and macrophage M2-driven phenotype in bleomycin-induced pulmonary fibrosis.

Pulmonary fibrosis is a result of an abnormal wound healing in lung tissue triggered by an excessive accumulation of extracellular matrix proteins, loss of tissue elasticity, and debit of ventilatory function. NKT cells are a major source of Th1 and Th2 cytokines and may be crucial in the polarization of M1/M2 macrophages in pulmonary fibrogenesis. Although there appears to be constant scientific progress in that field, pulmonary fibrosis still exhibits no current cure. From these facts, we hypothesized that NKT cells could influence the development of pulmonary fibrosis via modulation of macrophage activation. Wild type (WT) and NKT type I cell-deficient mice (Jα18-/-) were subjected to the protocol of bleomycin-induced pulmonary fibrosis with or without treatment with NKT cell agonists α-galactosylceramide and sulfatide. The participation of different cell populations, collagen deposition, and protein levels of different cytokines involved in inflammation and fibrosis was evaluated. The results indicate a benign role of NKT cells in Jα18-/- mice and in wild-type α-galactosylceramide-sulfatide-treated groups. These animals presented lower levels of collagen deposition, fibrogenic molecules such as TGF-ß and vimentin and improved survival rates. In contrast, WT mice developed a Th2-driven response augmenting IL-4, 5, and 13 protein synthesis and increased collagen deposition. Furthermore, the arginase-1 metabolic pathway was downregulated in wild-type NKT-activated and knockout mice indicating lower activity of M2 macrophages in lung tissue. Hence, our data suggest that NKT cells play a protective role in this experimental model by down modulating the Th2 milieu, inhibiting M2 polarization and finally preventing fibrosis.

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.

Dectin-1 Activation Exacerbates Obesity and Insulin Resistance in the Absence of MyD88.

The underlying mechanism by which MyD88 regulates the development of obesity, metainflammation, and insulin resistance (IR) remains unknown. Global deletion of MyD88 in high-fat diet (HFD)-fed mice resulted in increased weight gain, impaired glucose homeostasis, elevated Dectin-1 expression in adipose tissue (AT), and proinflammatory CD11c+ AT macrophages (ATMs). Dectin-1 KO mice were protected from diet-induced obesity (DIO) and IR and had reduced CD11c+ AT macrophages. Dectin-1 antagonist improved glucose homeostasis and decreased CD11c+ AT macrophages in chow- and HFD-fed MyD88 KO mice. Dectin-1 agonist worsened glucose homeostasis in MyD88 KO mice. Dectin-1 expression is increased in AT from obese individuals. Together, our data indicate that Dectin-1 regulates AT inflammation by promoting CD11c+ AT macrophages in the absence of MyD88 and identify a role for Dectin-1 in chronic inflammatory states, such as obesity. This suggests that Dectin-1 may have therapeutic implications as a biomarker for metabolic dysregulation in humans.

Dectin-1 activation exacerbates obesity and insulin resistance in the absence of myD88

The underlying mechanism by which MyD88 regulates the development of obesity, metainflammation, and insulin resistance (IR) remains unknown. Global deletion of MyD88 in high-fat diet (HFD)fed mice resulted in increased weight gain, impaired glucose homeostasis, elevated Dectin-1 expression in adipose tissue (AT), and proinflammatory CD11c+ AT macrophages (ATMs). Dectin-1 KO mice were protected from diet-induced obesity (DIO) and IR and had reduced CD11c+ AT macrophages. Dectin-1 antagonist improved glucose homeostasis and decreased CD11c+ AT macrophages in chow-and HFD-fed MyD88 KO mice. Dectin-1 agonist worsened glucose homeostasis in MyD88 KO mice. Dectin-1 expression is increased in AT from obese individuals. Together, our data indicate that Dectin-1 regulates AT inflammation by promoting CD11c+ AT macrophages in the absence of MyD88 and identify a role for Dectin-1 in chronic inflammatory states, such as obesity. This suggests that Dectin-1 may have ther-apeutic implications as a biomarker for metabolic dysregulation in humans.

Administration of α-Galactosylceramide Improves Adenine-Induced Renal Injury.

Natural killer T (NKT) cells are a subset of lymphocytes that reacts to glycolipids presented by CD1d. Invariant NKT cells (iNKT) correspond to >90% of the total population of NKTs and reacts to α-galactosylceramide (αGalCer). αGalCer promotes a complex mixture of Th1 and Th2 cytokines, as interferon (IFN)-γ and interleukin (IL)-4. NKT cells and IFN-γ are known to participate in some models of renal diseases, but further studies are still necessary to elucidate their mechanisms. The aim of our study was to analyze the participation of iNKT cells in an experimental model of tubule-interstitial nephritis. We used 8-wk-old C57BL/6j, Jα18KO and IFN-γKO mice. They were fed a 0.25% adenine diet for 10 d. Both adenine-fed wild-type (WT) and Jα18KO mice exhibited renal dysfunction, but adenine-fed Jα18KO mice presented higher expression of kidney injury molecule-1 (KIM-1), tumor necrosis factor (TNF)-α and type I collagen. To analyze the role of activated iNKT cells in our model, we administered αGalCer in WT mice during adenine ingestion. After αGalCer injection, we observed a significant reduction in serum creatinine, proinflammatory cytokines and renal fibrosis. However, this improvement in renal function was not observed in IFN-γKO mice after αGalCer treatment and adenine feeding, illustrating that this cytokine plays a role in our model. Our findings may suggest that IFN-γ production is one of the factors contributing to improved renal function after αGalCer administration.

Obesity in kidney disease: A heavyweight opponent.

Obesity is an important worldwide challenge that must be faced in most developed and developing countries because of unhealthy nutritional habits. The consequences of obesity and being overweight are observed in different organs, but the kidney is one of the most affected. Excess adipose tissue causes hemodynamic alterations in the kidney that can result in renal disease. However, obesity is also commonly associated with other comorbidities such as chronic inflammation, hypertension and diabetes. This association of several aggravating factors is still a matter of concern in clinical and basic research because the pathophysiologic mechanisms surrounding chronic kidney disease development in obese patients remain unclear. This review will discuss the consequences of obesity in the context of renal injury.

Balance between the two kinin receptors in the progression of experimental focal and segmental glomerulosclerosis in mice.

Focal and segmental glomerulosclerosis (FSGS) is one of the most important renal diseases related to end-stage renal failure. Bradykinin has been implicated in the pathogenesis of renal inflammation, whereas the role of its receptor 2 (B2RBK; also known as BDKRB2) in FSGS has not been studied. FSGS was induced in wild-type and B2RBK-knockout mice by a single intravenous injection of Adriamycin (ADM). In order to further modulate the kinin receptors, the animals were also treated with the B2RBK antagonist HOE-140 and the B1RBK antagonist DALBK. Here, we show that the blockage of B2RBK with HOE-140 protects mice from the development of FSGS, including podocyte foot process effacement and the re-establishment of slit-diaphragm-related proteins. However, B2RBK-knockout mice were not protected from FSGS. These opposite results were due to B1RBK expression. B1RBK was upregulated after the injection of ADM and this upregulation was exacerbated in B2RBK-knockout animals. Furthermore, treatment with HOE-140 downregulated the B1RBK receptor. The blockage of B1RBK in B2RBK-knockout animals promoted FSGS regression, with a less-inflammatory phenotype. These results indicate a deleterious role of both kinin receptors in an FSGS model and suggest a possible cross-talk between them in the progression of disease.

TLR2, TLR4 and the MYD88 signaling pathway are crucial for neutrophil migration in acute kidney injury induced by sepsis.

The aim of this study was to investigate the role of TLR2, TLR4 and MyD88 in sepsis-induced AKI. C57BL/6 TLR2(-/-), TLR4(-/-) and MyD88(-/-) male mice were subjected to sepsis by cecal ligation and puncture (CLP). Twenty four hours later, kidney tissue and blood samples were collected for analysis. The TLR2(-/-), TLR4(-/-) and MyD88(-/-) mice that were subjected to CLP had preserved renal morphology, and fewer areas of hypoxia and apoptosis compared with the wild-type C57BL/6 mice (WT). MyD88(-/-) mice were completely protected compared with the WT mice. We also observed reduced expression of proinflammatory cytokines in the kidneys of the knockout mice compared with those of the WT mice and subsequent inhibition of increased vascular permeability in the kidneys of the knockout mice. The WT mice had increased GR1(+low) cells migration compared with the knockout mice and decreased in GR1(+high) cells migration into the peritoneal cavity. The TLR2(-/-), TLR4(-/-), and MyD88(-/-) mice had lower neutrophil infiltration in the kidneys. Depletion of neutrophils in the WT mice led to protection of renal function and less inflammation in the kidneys of these mice. Innate immunity participates in polymicrobial sepsis-induced AKI, mainly through the MyD88 pathway, by leading to an increased migration of neutrophils to the kidney, increased production of proinflammatory cytokines, vascular permeability, hypoxia and apoptosis of tubular cells.