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Type 1 diabetes mellitus (T1D) is a chronic autoimmune disease characterized by insulin-producing pancreatic β-cell destruction and hyperglycemia. While monocytes and NOD-like receptor family-pyrin domain containing 3 (NLRP3) are associated with T1D onset and development, the specific receptors and factors involved in NLRP3 inflammasome activation remain unknown. Herein, we evaluated the inflammatory state of resident peritoneal macrophages (PMs) from genetically modified non-obese diabetic (NOD), NLRP3-KO, wild-type (WT) mice and in peripheral blood mononuclear cells (PBMCs) from human T1D patients. We also assessed the effect of docosahexaenoic acid (DHA) on the inflammatory status. Macrophages from STZ-induced T1D mice exhibited increased inflammatory cytokine/chemokine levels, nitric oxide (NO) secretion, NLRP3 and iNOS protein levels, and augmented glycolytic activity compared to control animals. In PMs from NOD and STZ-induced T1D mice, DHA reduced NO production and attenuated the inflammatory state. Furthermore, iNOS and IL-1β protein expression levels and NO production were lower in the PMs from diabetic NLRP3-KO mice than from WT mice. We also observed increased IL-1β secretion in PBMCs from T1D patients and immortalized murine macrophages treated with advanced glycation end products and palmitic acid. The present study demonstrated that the resident PMs are in a proinflammatory state characterized by increased NLRP3/iNOS pathway-mediated NO production, up-regulated proinflammatory cytokine/chemokine receptor expression and altered glycolytic activity. Notably, ex vivo treatment with DHA reverted the diabetes-induced changes and attenuated the macrophage inflammatory state. It is plausible that DHA supplementation could be employed as adjuvant therapy for treating individuals with T1D.

Introduction: Short-Chain Fatty Acids (SCFA) are products of intestinal microbial metabolism that can reach the brain and alter microglia in health and disease contexts. However, data are conflicting on the effect of acetate, the most abundant SCFA in the blood, in these cells. Objective: The authors aimed to investigate acetate as a modulator of the inflammatory response in microglia stimulated with LPS. Method: The authors used an immortalized cell line, C8-B4, and primary cells for in vitro treatments with acetate and LPS. Cell viability was analyzed by MTT, cytokine by RT-PCR, ELISA, and flow cytometry. The authors also performed in vivo and in silico analyses to study the role of acetate and the TNF-α contribution to the development of Experimental Autoimmune Encephalomyelitis (EAE). Results: Acetate co-administered with LPS was able to exacerbate the production of pro-inflammatory cytokines at gene and protein levels in cell lines and primary culture of microglia. However, the same effects were not observed when acetate was administered alone or as pretreatment, prior to the LPS stimulus. Additionally, pharmacological inhibition of histone deacetylase concomitantly with acetate and LPS led to decreased TNF-α production. In silico analysis showed a crucial role of the TNF-α pathway in EAE development. Moreover, acetate administration in vivo during the initial phase of EAE led to a better disease outcome and reduced TNF-α production. Conclusion: Treatment with acetate was able to promote the production of TNF-α in a concomitant LPS stimulus of microglia. However, the immune modulation of microglia by acetate pretreatment may be a component in the generation of future therapies for neurodegenerative diseases. HIGHLIGHTS Acetate was able to exacerbate the production of TNF-α in microglia. Acetate administered as pre-treatment to LPS acts as an anti-inflammatory. Histone deacetylase decreased TNF-α production in Acetate- and LPS-treated cells. Depending on the time of administration, Acetate modulates microglia’s activation. Acetate may threaten neurodegenerative and neuropsychiatric diseases.

OBJECTIVES: This study aimed to evaluate the potential anti-inflammatory effects of vitamin D supplementation under uremic conditions, both in vivo and in vitro, and its effects on the parameters of mineral metabolism. METHODS: Thirty-two hemodialysis patients were randomly assigned to receive placebo (N=14) or cholecalciferol (N=18) for six months. Serum levels of calcium, phosphate, total alkaline phosphatase, intact parathyroid hormone (iPTH), and vitamin D were measured at baseline and after three and six months. The levels of fibroblast growth factor-23 (FGF-23), interleukin-1β (IL-1β), and high-sensitivity C-reactive protein (hs-CRP) were also measured at baseline and at six months. Human monocytes were used for in vitro experiments and treated with cholecalciferol (150 nM) and uremic serum. Cell viability, reactive oxygen species (ROS) production, and cathelicidin (CAMP) expression were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, dichloro-dihydro-fluorescein diacetate assay, and real time-quantitative polymerase chain reaction, respectively. RESULTS: Both patient groups were clinically and biochemically similar at baseline. After six months, the levels of vitamin D and iPTH were higher and lower, respectively, in the cholecalciferol group than in the placebo group (p

NLRP3 inflammasome [NLR (nucleotide-binding domain, leucine-rich repeat containing protein) Pyrin-domain-containing 3 ] functions as an innate sensor of several PAMPs and DAMPs (pathogen- and damage-associated molecular patterns). It has been also reported as a transcription factor related to Th2 pattern, although its role in the adaptive immunity has been controversial, mainly because the studies were performed using gene deletion approaches. In the present study, we have investigated the NLRP3 gain-of-function in the context of encephalomyelitis autoimmune disease (EAE), considered to be a Th1- and Th17-mediated disease. We took advantage of an animal model with NLRP3 gain-of-function exclusively to T CD4+ lymphocytes (CD4CreNLRP3fl/fl). These mice presented reduced clinical score, accompanied by less infiltrating T CD4+ cells expressing both IFN-γ and IL-17 at the central nervous system (CNS) during the peak of the disease. However, besides NLRP3 gain-of-function in lymphocytes, these mice lack NLRP3 expression in non-T CD4+ cells. Therefore, in order to circumvent this deficiency, we transferred naive CD4+ T cells from WT, NLRP3−/− or CD4CreNLRP3fl/fl into Rag-1−/− mice and immunized them with MOG35–55. Likewise, the animals repopulated with CD4CreNLRP3fl/fl T CD4+ cells presented reduced clinical score and decreased IFN-γ production at the peak of the disease. Additionally, primary effector CD4+ T cells derived from these mice presented reduced glycolytic profile, a metabolic profile compatible with Th2 cells. Finally, naive CD4+ T cells from CD4CreNLRP3fl/fl mice under a Th2-related cytokine milieu cocktail exhibited in vitro an increased IL-4 and IL-13 production. Conversely, naive CD4+ T cells from CD4CreNLRP3fl/fl mice under Th1 differentiation produced less IFN-γ and T-bet. Altogether, our data evidence that the NLRP3 gain-of-function promotes a Th2-related response, a pathway that could be better explored in the treatment of multiple sclerosis.

In contrast to mammals, zebrafish (Danio rerio) has the ability to regenerate injured sites such as different tissues present in the fin. It is known that cells of the innate immune system play essential roles in regeneration; however, some aspects of the molecular mechanisms by which these cells orchestrate regeneration remain unknown. This study aimed to evaluate the infiltration dynamics of neutrophils and macrophages in the regenerative process of fin fold in regard to the influence of the redox environment and oxidative pathways. Fin fold amputation was performed on transgenic larvae for macrophage-expressed gene 1 (mpeg1), lysozyme (lyz), myeloperoxidase (mpo) and tumour necrosis factor alpha (TNFα) at 3 days post-fertilization, followed by confocal microscopy imaging and measurement of the activities of oxidant and antioxidant enzymes. We observed initially an increase in the number of neutrophils (lyz:DsRed+/mpx:GFP+) and then macrophages (mpeg1+) in the injury site followed by a decrease in neutrophils at 7 days post-amputation (dpa). Moreover, macrophages switch from a pro-inflammatory to an anti-inflammatory profile throughout the process, while the activity of superoxide dismutase (SOD) increased at 1 dpa and catalase (CAT) at 5 dpa. Higher levels of lipid peroxidation were also detected during regeneration. Despite oxidative stress, there is, therefore, an antioxidant response throughout the regeneration of the caudal fin. The present work can contribute to future studies on the development of cell therapies, achieving greater effectiveness in the treatment of diseases related to the formation of fibrotic tissue.

The immune system can recognize microbes and sterile tissue damage. Among the damage-associated molecular patterns (DAMPs), uric acid is considered a major component which can trigger inflammation. It represents a breakpoint in the evolutionary history of humans as our ancestors lost the uricase gene, the enzyme responsible for its cleavage. High soluble uric acid (sUA) concentration is able to increase IL-1β in murine, but not human macrophages. We observed that sUA increased the mRNA expression of Naip1 in murine macrophages, and, therefore, we hypothesized that the recognition of sUA can be made by a Naip1-Nlrp3 inflammasome platform. Additionally, we used genome-wide transcriptome analysis, functional analyses and structural modeling predictions and observed that virus-transduction of murine Naip1 into human macrophages induced IL-1β after sUA stimulus, besides leading to fatty acid production and an inflammation-related response. Moreover, pharmacologic inhibition and genetic loss of Nlrp3 led to decreased IL-1β production upon sUA stimulus. Surface plasmon resonance and quartz crystal microbalance showed that sUA is able to interact with Naip1. Naip could be a lost receptor for sUA in the evolutionary process and a better understanding of the immune modulatory function of sUA could lead to design rational novel anti-hyperuricemic therapies.

Palmitoleic acid (POA, 16:1n-7) is a lipokine that has potential nutraceutical use to treat non-alcoholic fatty liver disease. We tested the effects of POA supplementation (daily oral gavage, 300 mg/Kg, 15 days) on murine liver inflammation induced by a high fat diet (HFD, 59% fat, 12 weeks). In HFD-fed mice, POA supplementation reduced serum insulin and improved insulin tolerance compared with oleic acid (OA, 300 mg/Kg). The livers of POA-treated mice exhibited less steatosis and inflammation than those of OA-treated mice with lower inflammatory cytokine levels and reduced toll-like receptor 4 protein content. The anti-inflammatory effects of POA in the liver were accompanied by a reduction in liver macrophages (LM, CD11c+; F4/80+; CD86+), an effect that could be triggered by peroxisome proliferator activated receptor (PPAR)-γ, a lipogenic transcription factor upregulated in livers of POA-treated mice. We also used HFD-fed mice with selective deletion of PPAR-γ in myeloid cells (PPAR-γ KOLyzCre+) to test whether the beneficial anti-inflammatory effects of POA are dependent on macrophages PPAR-γ. POA-mediated improvement of insulin tolerance was tightly dependent on myeloid PPAR-γ, while POA anti-inflammatory actions including the reduction in liver inflammatory cytokines were preserved in mice bearing myeloid cells deficient in PPAR-γ. This overlapped with increased CD206+ (M2a) cells and downregulation of CD86+ and CD11c+ liver macrophages. Moreover, POA supplementation increased hepatic AMPK activity and decreased expression of the fatty acid binding scavenger receptor, CD36. We conclude that POA controls liver inflammation triggered by fat accumulation through induction of M2a macrophages independently of myeloid cell PPAR-γ., Graphical abstract Unlabelled Image, Highlights • Palmitoleic acid (POA) supplementation reduced serum insulin and improved insulin tolerance; • Livers of POA-treated mice exhibited less steatosis and inflammation; • POA lowered the liver M1 macrophages population and the expression of inflammation-related immune-cell markers; • POA increased PPAR-γ, a transcription factor that regulates anti-inflammatory effects in macrophages; • However, POA reduced liver inflammation even in mice that lack PPAR-γ expression in myeloid cells; • POA controls liver inflammation through induction of M2a macrophages independently of PPAR-γ in myeloid cells.

The antitumor effect of metformin has been demonstrated in several types of cancer; however, the mechanisms involved are incompletely understood. In this study, we showed that metformin acts directly on melanoma cells as well as on the tumor microenvironment, particularly in the context of the immune response. In vitro, metformin induces a complex interplay between apoptosis and autophagy in melanoma cells. The anti-metastatic activity of metformin in vivo was assessed in several mouse models challenged with B16F10 cells. Metformin's activity was, in part, immune system-dependent, whereas its antitumor properties were abrogated in immunodeficient (NSG) mice. Metformin treatment increased the number of lung CD8-effector-memory T and CD4+Foxp3+IL-10+ T cells in B16F10-transplanted mice. It also decreased the levels of Gr-1+CD11b+ and RORγ+ IL17+CD4+ cells in B16F10-injected mice and the anti-metastatic effect was impaired in RAG-1-/- mice challenged with B16F10 cells, suggesting an important role for T cells in the protection induced by metformin. Finally, metformin in combination with the clinical metabolic agents rapamycin and sitagliptin showed a higher antitumor effect. The metformin/sitagliptin combination was effective in a BRAFV600E/PTEN tamoxifen-inducible murine melanoma model. Taken together, these results suggest that metformin has a pronounced effect on melanoma cells, including the induction of a strong protective immune response in the tumor microenvironment, leading to tumor growth control, and the combination with other metabolic agents may increase this effect.

Caloric restriction (CR) is the most effective intervention known to enhance lifespan, but its effect on the skin is poorly understood. Here, we show that CR mice display fur coat remodeling associated with an expansion of the hair follicle stem cell (HFSC) pool. We also find that the dermal adipocyte depot (dWAT) is underdeveloped in CR animals. The dermal/vennule annulus vasculature is enlarged, and a vascular endothelial growth factor (VEGF) switch and metabolic reprogramming in both the dermis and the epidermis are observed. When the fur coat is removed, CR mice display increased energy expenditure associated with lean weight loss and locomotion impairment. Our findings indicate that CR promotes extensive skin and fur remodeling. These changes are necessary for thermal homeostasis and metabolic fitness under conditions of limited energy intake, suggesting a potential adaptive mechanism.

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-β1 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.

Background: Inflammation is a key feature of aldosterone-induced vascular damage and dysfunction, but molecular mechanisms by which aldosterone triggers inflammation remain unclear. The NLRP3 inflammasome is a pivotal immune sensor that recognizes endogenous danger signals triggering sterile inflammation. Methods: We analyzed vascular function and inflammatory profile of wild-type (WT), NLRP3 knockout ( NLRP3 −/− ), caspase-1 knockout ( Casp-1 −/− ), and interleukin-1 receptor knockout ( IL-1R −/− ) mice treated with vehicle or aldosterone (600 µg·kg −1 ·d −1 for 14 days through osmotic mini-pump) while receiving 1% saline to drink. Results: Here, we show that NLRP3 inflammasome plays a central role in aldosterone-induced vascular dysfunction. Long-term infusion of aldosterone in mice resulted in elevation of plasma interleukin-1β levels and vascular abnormalities. Mice lacking the IL-1R or the inflammasome components NLRP3 and caspase-1 were protected from aldosterone-induced vascular damage. In vitro, aldosterone stimulated NLRP3-dependent interleukin-1β secretion by bone marrow–derived macrophages by activating nuclear factor-κB signaling and reactive oxygen species generation. Moreover, chimeric mice reconstituted with NLRP3-deficient hematopoietic cells showed that NLRP3 in immune cells mediates aldosterone-induced vascular damage. In addition, aldosterone increased the expression of NLRP3, active caspase-1, and mature interleukin-1β in human peripheral blood mononuclear cells. Hypertensive patients with hyperaldosteronism or normal levels of aldosterone exhibited increased activity of NLRP3 inflammasome, suggesting that the effect of hyperaldosteronism on the inflammasome may be mediated through high blood pressure. Conclusions: Together, these data demonstrate that NLRP3 inflammasome, through activation of IL-1R, is critically involved in the deleterious vascular effects of aldosterone, placing NLRP3 as a potential target for therapeutic interventions in conditions with high aldosterone levels.

Nonalcoholic steatohepatitis (NASH), especially as part of the metabolic syndrome (MS), is an increasing burden in Western countries. Statins are already used in MS and seem to be beneficial in liver diseases. The aim of this study was to investigate the molecular mechanisms underlying pleiotropic effects on small GTPases of statins in NASH. NASH within MS was induced in 12-wk-old apoE−/− mice after 7 wk of Western diet (NASH mice). Small GTPases were inhibited by activated simvastatin (SMV), NSC23766 (NSC), or Clostridium sordellii lethal toxin (LT) by using subcutaneous osmotic minipumps. Hepatic steatosis, inflammation, and fibrosis were assessed by histology, Western blot, and RT-PCR measurements of cholesterol and hydroxyproline content. SMV treatment significantly decreased hepatic inflammation and fibrosis, but had no significant effect on steatosis and hepatic cholesterol content in NASH. SMV blunted fibrosis due to inhibition of both RhoA/Rho kinase and Ras/ERK pathways. Interestingly, inhibition of RAC1 and Ras (by LT) failed to decrease fibrosis to the same extent. Inhibition of RAC1 (by NSC) showed no significant effect at all. Inhibition of RhoA and Ras downstream signaling by statins is responsible for the beneficial hepatic effects in NASH.

Chronic kidney disease (CKD) is associated with several other long-lasting conditions such as diabetes and cardiovascular diseases and it is a significant contributor to mortality worldwide. Obstructive kidney disease is one of the leading causes of CKD in children and may result from a wide variety of pathologic processes. Recent studies have shown that α7 nicotinic acetylcholine receptor (α7 nAChR) activation in the cholinergic anti-inflammatory pathway reduces production of inflammatory mediators and consequently prevents tissue injury and death. Here, we examined the role of endogenous release of acetylcholine on the development of fibrosis in renal tissue using a model of unilateral ureter obstruction (UUO)-induced CKD, in which obstruction promotes inflammation-mediated kidney damages. To interfere with acetylcholine secretion, we used mice in which the vesicular acetylcholine transporter is genetically reduced (VAChT KD hom mice). We observed a higher renal damage in VAChT mutant mice when compared to wild type controls, exemplified by higher proteinuria and increased amount of type 1 collagen in the kidney tissue, indicating accentuated fibrogenesis. These results were accompanied by enhanced localized kidney inflammation, with increased T H 1/T H 17 profile response. Administration of PNU-282987, a selective agonist of α7 nAChR, significantly attenuated kidney injury after UUO in VAChT KD hom mice, indicating that the lack of acetylcholine release decrease the action of the cholinergic anti-inflammatory pathway, promoting an up-regulation of pro-inflammatory and pro-fibrotic pathways. These results suggest that physiological activation of the cholinergic anti-inflammatory pathway regulates inflammatory responses in the kidney suggesting a new therapeutic approach for kidney disease.

Butyrate is a short-chain fatty acid derived from the metabolism of indigestible carbohydrates by the gut microbiota. Butyrate contributes to gut homeostasis, but it may also control inflammatory responses and host physiology in other tissues. Butyrate inhibits histone deacetylases, thereby affecting gene transcription, and also signals through the metabolite-sensing G protein receptor (GPR)109a. We produced an mAb to mouse GPR109a and found high expression on podocytes in the kidney. Wild-type and Gpr109a-/- mice were induced to develop nephropathy by a single injection of Adriamycin and treated with sodium butyrate or high butyrate-releasing high-amylose maize starch diet. Butyrate improved proteinuria by preserving podocyte at glomerular basement membrane and attenuated glomerulosclerosis and tissue inflammation. This protective phenotype was associated with increased podocyte-related proteins and a normalized pattern of acetylation and methylation at promoter sites of genes essential for podocyte function. We found that GPR109a is expressed by podocytes, and the use of Gpr109a-/- mice showed that the protective effects of butyrate depended on GPR109a expression. A prebiotic diet that releases high amounts of butyrate also proved highly effective for protection against kidney disease. Butyrate and GPR109a play a role in the pathogenesis of kidney disease and provide one of the important molecular connections between diet, the gut microbiota, and kidney disease.-Felizardo, R. J. F., de Almeida, D. C., Pereira, R. L., Watanabe, I. K. M., Doimo, N. T. S., Ribeiro, W. R., Cenedeze, M. A., Hiyane, M. I., Amano, M. T., Braga, T. T., Ferreira, C. M., Parmigiani, R. B., Andrade-Oliveira, V., Volpini, R. A., Vinolo, M. A. R., Marino, E., Robert, R., Mackay, C. R., Camara, N. O. S. Gut microbial metabolite butyrate protects against proteinuric kidney disease through epigenetic- and GPR109a-mediated mechanisms.

SUMMARY The evolutionarily conserved miR-302 family of microRNAs is expressed during early mammalian embryonic development. Here, we report that deletion of miR-302a-d in mice results in a fully penetrant late embryonic lethal phenotype. Knockout embryos have an anterior neural tube closure defect associated with a thickened neuroepithelium. The neuroepithelium shows increased progenitor proliferation, decreased cell death, and precocious neuronal differentiation. mRNA profiling at multiple time points during neurulation uncovers a complex pattern of changing targets over time. Overexpression of one of these targets, Fgf15, in the neuroepithelium of the chick embryo induces precocious neuronal differentiation. Compound mutants between mir-302 and the related mir-290 locus have a synthetic lethal phenotype prior to neurulation. Our results show that mir-302 helps regulate neurulation by suppressing neural progenitor expansion and precocious differentiation. Furthermore, these results uncover redundant roles for mir-290 and mir-302 early in development., Graphical Abstract

Chemokines are a large family of proteins that, once associated to its receptor on leukocytes, stimulate their movement and migration from blood to tissues. Once in the tissue, immune cells trigger inflammation that, when uncontrolled, leads to fibrosis development. Among the immune cells, macrophages take a special role in fibrosis formation, since macrophage depletion reflects less collagen deposition. The majority of tissue macrophages is derived from monocytes, especially monocytes expressing the chemokine receptor CCR2. Here, we investigated the role of infiltrating CCR2+ cells in the development of fibrosis, and specifically, the dynamic of infiltration of these cells into kidneys under chronic obstructive lesion. Using liposome-encapsulated clodronate, we observed that macrophage depletion culminated in less collagen deposition and reduced chemokines milieu that were released in the damaged kidney after obstructive nephropathy. We also obstructed the kidneys of CCL3-/-, CCR2-/-, CCR4-/-, CCR5-/-, and C57BL/6 mice and we found that among all animals, CCR2-/- mice demonstrated the more robust protection, reflected by less inflammatory and Th17-related cytokines and less collagen formation. Next we evaluated the dynamic of CCR2+/rfp cell infiltration and we observed that they adhere onto the vessels at early stages of disease, culminating in increased recruitment of CCR2+/rfp cells at later stages. On the other hand, CCR2rfp/rfp animals exhibited less fibrosis formation and reduced numbers of recruited cells at later stages. We have experimentally demonstrated that inflammatory CCR2+ cells that reach the injured kidney at initial stages after tissue damage are responsible for the fibrotic pattern observed at later time points in the context of UUO.

Assembly of a relatively large protein aggregate or "speck" formed by the adaptor protein ASC is a common downstream step in the activation of most inflammasomes. This unique feature of ASC allows its visualization by several imaging techniques and constitutes a reliable and feasible readout for inflammasome activation in cells and tissues. We have previously described step-by-step protocols to generate immortalized cell lines stably expressing ASC fused to a fluorescent protein for measuring inflammasome activation by confocal microscopy, and immunofluorescence of endogenous ASC in primary cells. Here, we present two more methods to detect ASC speck formation: (1) Assessment of ASC speck formation by flow cytometry; and (2) Chemical cross-linking of ASC followed by immunoblotting. These methods allow for the discrimination of inflammasome-activated versus non-activated cells, the identification of lineage-specific inflammasome activation in complex cell mixtures, and sorting of inflammasome-activated cells for further analysis.

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-β 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.

Uric acid is a damage-associated molecular pattern (DAMP), released from ischemic tissues and dying cells which, when crystalized, is able to activate the NLRP3 inflammasome. Soluble uric acid (sUA) is found in high concentrations in the serum of great apes, and even higher in some diseases, before the appearance of crystals. In the present study, we sought to investigate whether uric acid, in the soluble form, could also activate the NLRP3 inflammasome and induce the production of IL-1β. We monitored ROS, mitochondrial area and respiratory parameters from macrophages following sUA stimulus. We observed that sUA is released in a hypoxic environment and is able to induce IL-1β release. This process is followed by production of mitochondrial ROS, ASC speck formation and caspase-1 activation. Nlrp3−/− macrophages presented a protected redox state, increased maximum and reserve oxygen consumption ratio (OCR) and higher VDAC protein levels when compared to WT and Myd88−/− cells. Using a disease model characterized by increased sUA levels, we observed a correlation between sUA, inflammasome activation and fibrosis. These findings suggest sUA activates the NLRP3 inflammasome. We propose that future therapeutic strategies for renal fibrosis should include strategies that block sUA or inhibit its recognition by phagocytes. © The Author(s) 2017. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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.

Deficient wound healing is a common multifactorial complication in diabetic patients, but the cellular and molecular mechanisms involved are poorly defined. In the present study, we analyzed the effects of hyperglycemia on integrins expression in rat dermal fibroblasts and addressed its role in cell adhesion and migration. Diabetes Mellitus was induced in rats by streptozotocin injection and maintained for 30 days. Primary cultures of dermal fibroblasts from control and diabetic rats were maintained under low glucose (5 mM D-glucose) or high glucose (30 mM D-glucose) for 7 days. Cell adhesion and migration were studied by kymography, transwell, and time-lapse assays, and the expressions of integrin subunits αv and α5 were studied by immunocytochemistry and western blotting. Fibroblasts derived from diabetic rats confirmed a reduced migration speed and delayed spreading compared to fibroblasts derived from control rats. The membrane fraction of diabetic-derived fibroblasts showed a decrease of integrin subunits α5 and αv, which was confirmed by immunocytochemistry assays. A reduction in the pericellular fibronectin matrix was also observed. The exposure of diabetic-derived cells to a higher concentration of exogenous fibronectin improved migration velocity and the expression of αv but did not completely restore their migration capacity. In conclusion, the mechanisms involved in the deleterious effects of Diabetes Mellitus on wound healing include the ability of fibroblasts to secrete and to adhere to fibronectin.

The innate immune system plays an important role as a first response to tissue injury. This first response is carried out via germline-encoded receptors. They can recognize exogenous Pathogen-Associated Molecular Patterns and endogenous Dangers-Associated Molecular Patterns. The Toll-Like Receptor (TLR) family is well-studied, but more recently another family in the cytoplasmic compartment, called nod-like receptor (NLR), was discovered. In addition to being present in inflammatory cells, these receptors are widely distributed in various cell types, including renal tissue, where these receptors have an important role in triggering the inflammatory response during renal diseases. This review summarizes the present data regarding the role of TLRs and NLRs in the course and development of various kidney pathologies.

Formaldehyde is ubiquitous pollutant that induces oxidative stress in the lung. Several lung diseases have been associated with oxidative stress and their control is necessary. Photobiomodulation therapy (PBMT) has been highlighted as a promissory treatment, but its mechanisms need to be better investigated. Our objective was to evaluate the effects of PBMT on the oxidative stress generated by FA exposure. Male Wistar rats were submitted to FA exposure of 1% or vehicle (3 days) and treated or not with PBMT (1 and 5 h after each FA exposure). Rats treated only with laser were used as control. Twenty-four hours after the last FA exposure, we analyzed the effects of PBMT on the generation of nitrites and hydrogen peroxide, oxidative burst, glutathione reductase, peroxidase, S-transferase enzyme activities, the gene expression of nitric oxide, cyclooxygenase, superoxide dismutase, the catalase enzyme, and heme oxygenase-1. PBMT reduced the generation of nitrites and hydrogen peroxide and increased oxidative burst in the lung cells. A decreased level of oxidant enzymes was observed which were concomitantly related to an increased level of antioxidants. This study provides new information about the antioxidant mechanisms of PBMT in the lung and might constitute an important tool for lung disease treatment.

Introduction Macrophages are heterogeneous and thus can be correlated with distinct tissue outcomes after injury. Conflicting data have indicated that the M2-related phenotype directly triggers fibrosis. Conversely, we hypothesize here that the inflammatory milieu provided by early infiltration of pro-inflammatory macrophages dictates tissue scarring after injury. Methods and results We first determined that tissue-localized macrophages exhibit a pro-inflammatory phenotype (p40IL12(+)CCR7(+)CD11b(+)) during the early phase of a chronic injury model, in contrast to a pro-resolving phenotype (Arg1(+)IL10(+)CD206(+)CD11b(+)) at a later stage. Then, we evaluated the effects of injecting macrophages differentiated in vitro in the presence of IFNγ + LPS or IL4 + IL13 or non-differentiated macrophages (hereafter, M0) on promoting inflammation and progression of chronic injury in macrophage-depleted mice. In addition to enhancing the expression of pro-inflammatory cytokines, the injection of M (IFNγ + LPS), but not M (IL4 + IL13) or M0, accentuated fibrosis while augmenting levels of anti-inflammatory molecules, increasing collagen deposition and impairing organ function. We observed a similar profile after injection of sorted CCR7(+)CD11b(+) cells and a more pronounced effect of M (IFNγ + LPS) cells originated from Stat6(-/-) mice. The injection of M (IFNγ + LPS) cells was associated with the up-regulation of inflammation- and fibrosis-related proteins (Thbs1, Mmp7, Mmp8, and Mmp13). Conclusions Our results suggest that pro-inflammatory macrophages promote microenvironmental changes that may lead to fibrogenesis by inducing an inflammatory milieu that alters a network of extracellular-related genes, culminating in tissue fibrosis.

Inhalation of formaldehyde (FA) during the pregnancy induces oxidative stress in the uterus, and here we hypothesized that this mechanism may be responsible for the impaired immune response detected in the offspring. In order to investigate the protective effects of Vitamin C on the oxidative stress induced by FA in the uterine microenvironment, pregnant Wistar rats were treated with vitamin C (150mg/kg, gavage) or vehicle (distilled water, gavage) 1h before FA exposure (0.92mg/m(3), 1h/day, 5days/week), for 21days, and the 30days old offspring were submitted to LPS injection (Salmonella abortus equi, 5mg/kg, i.p.). The enhanced gene expression of iNOS, COX-1 and COX-2 and decreased gene expression of SOD-2 in the uterus of FA exposed mothers was rescued by Vit C treatment. Moreover, vitamin C rescued the impaired immune response elicited by LPS in the offspring from FA exposed mothers, by increasing the number of blood and bone marrow leukocytes, and augmenting gene expression of IL-6 and reducing mRNA levels of IL-10 and IFN in the lungs. Vitamin C treatment did not rescue the impaired TLR4-NF-kB pathway in the lung of the offspring, suggesting that FA-induced uterine oxidative stress affects other inflammatory pathways activated by LPS in the offspring. Together, data obtained here confirm our hypothesis that FA-induced oxidative stress in the uterine microenvironment modifies the programming mechanisms of the immune defenses of offspring, leading to an impaired host defense.

Lung diseases constitute an important public health problem and its growing level of concern has led to efforts for the development of new therapies, particularly for the control of lung inflammation. Low Level Laser Therapy (LLLT) has been highlighted as a non-invasive therapy with few side effects, but its mechanisms need to be better understood and explored. Considering that pollution causes several harmful effects on human health, including lung inflammation, in this study, we have used formaldehyde (FA), an environmental and occupational pollutant, for the induction of neutrophilic lung inflammation. Our objective was to investigate the local and systemic effects of LLLT after FA exposure. Male Wistar rats were exposed to FA (1%) or vehicle (distillated water) during 3 consecutive days and treated or not with LLLT (1 and 5 hours after each FA exposure). Non-manipulated rats were used as control. 24 h after the last FA exposure, we analyzed the local and systemic effects of LLLT. The treatment with LLLT reduced the development of neutrophilic lung inflammation induced by FA, as observed by the reduced number of leukocytes, mast cells degranulated, and a decreased myeloperoxidase activity in the lung. Moreover, LLLT also reduced the microvascular lung permeability in the parenchyma and the intrapulmonary bronchi. Alterations on the profile of inflammatory cytokines were evidenced by the reduced levels of IL-6 and TNF-α and the elevated levels of IL-10 in the lung. Together, our results showed that LLLT abolishes FA-induced neutrophilic lung inflammation by a reduction of the inflammatory cytokines and mast cell degranulation. This study may provide important information about the mechanisms of LLLT in lung inflammation induced by a pollutant.

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.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Instituto Nacional de Ciencia e Tecnologia de Fluidos Complexos (INCT Complex Fluids) Macrophages play a special role in the onset of several diseases, including acute and chronic kidney injuries. in this sense, tubule interstitial nephritis (TIN) represents an underestimated insult, which can be triggered by different stimuli and, in the absence of a proper regulation, can lead to fibrosis deposition. Based on this perception, we evaluated the participation of macrophage recruitment in the development of TIN. Initially, we provided adenine-enriched food to WT and searched for macrophage presence and action in the kidney. Also, a group of animals were depleted of macrophages with the clodronate liposome while receiving adenine-enriched diet. We collected blood and renal tissue from these animals and renal function, inflammation, and fibrosis were evaluated. We observed higher expression of chemokines in the kidneys of adenine-fed mice and a substantial protection when macrophages were depleted. Then, we specifically investigated the role of some key chemokines, CCR5 and CCL3, in this TIN experimental model. Interestingly, CCR5 KO and CCL3 KO animals showed less renal dysfunction and a decreased proinflammatory profile. Furthermore, in those animals, there was less profibrotic signaling. in conclusion, we can suggest that macrophage infiltration is important for the onset of renal injury in the adenine-induced TIN. Univ São Paulo, Inst Biomed Sci, Dept Immunol, Lab Transplantat Immunobiol, BR-05508900 São Paulo, Brazil Universidade Federal de São Paulo, Div Nephrol, Lab Clin & Expt Immunol, BR-04023900 São Paulo, Brazil Universidade Federal de São Paulo, Dept Biophys, BR-04023062 São Paulo, Brazil Univ São Paulo, Inst Chem, Dept Biochem, BR-05508000 São Paulo, Brazil Univ São Paulo, Sch Med Ribeirao Preto, Dept Biochem & Immunol, BR-14049900 Ribeirao Preto, SP, Brazil Universidade Federal de São Paulo, Div Nephrol, Lab Clin & Expt Immunol, BR-04023900 São Paulo, Brazil Universidade Federal de São Paulo, Dept Biophys, BR-04023062 São Paulo, Brazil FAPESP: 2009/54474-8 FAPESP: 2012/02270-2 FAPESP: 2013/25010-9 Web of Science

Platelet-activating factor (PAF) is a lipid mediator with important pro-inflammatory effects, being synthesized by several cell types including kidney cells. Although there is evidence of its involvement in acute renal dysfunction, its role in progressive kidney injury is not completely known. In the present study, we investigated the role of PAF receptor (PAFR) in an experimental model of chronic renal disease. Wild-type (WT) and PAFR knockout (KO) mice underwent unilateral ureter obstruction (UUO), and at kill time, urine and kidney tissue was collected. PAFR KO animals compared with WT mice present: (a) less renal dysfunction, evaluated by urine protein/creatinine ratio; (b) less fibrosis evaluated by collagen deposition, type I collagen, Lysyl Oxidase-1 (LOX-1) and transforming growth factor β (TGF-β) gene expression, and higher expression of bone morphogenetic protein 7 (BMP-7) (3.3-fold lower TGF-β/BMP-7 ratio); (c) downregulation of extracellular matrix (ECM) and adhesion molecule-related machinery genes; and (d) lower levels of pro-inflammatory cytokines. These indicate that PAFR engagement by PAF or PAF-like molecules generated during UUO potentiates renal dysfunction and fibrosis and might promote epithelial-to-mesenchymal transition (EMT). Also, early blockade of PAFR after UUO leads to a protective effect, with less fibrosis deposition. In conclusion, PAFR signaling contributes to a pro-inflammatory environment in the model of obstructive nephropathy, favoring the fibrotic process, which lately will generate renal dysfunction and progressive organ failure.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) National Institute of Science and Technology (INCT) Inflammation contributes to the pathogenesis of chronic kidney disease (CKD). Molecules released by the inflamed injured tissue can activate toll-like receptors (TLRs), thereby modulating macrophage and CD4(+) T-cell activity. We propose that in renal fibrogenesis. M2 macrophages are recruited and activated in a T helper subset 2 cell (T(H)2)-prone inflammatory milieu in a MyD88-dependent manner. Mice submitted to unilateral ureteral ligation (UUO) demonstrated an increase in macrophage infiltration with collagen deposition after 7 d. Conversely, TLR2, TLR4 and MyD88 knockout (KO) mice had an improved renal function together with diminished TH2 cytokine production and decreased fibrosis formation. Moreover, TLR2, TLR4 and MyD88 KO animals exhibited less M2 macrophage infiltration, namely interleukin (IL)-10(+) and CD206(+). CD11b(high) cells, at 7 d after surgery. We evaluated the role of a T(H)2 cytokine in this context, and observed that the absence of IL-4 was associated with better renal function, decreased IL-13 and TGF-beta levels, reduced arginase activity and a decrease in fibrosis formation when compared with IL-12 KO and wild-type (WT) animals. Indeed, the better renal outcomes and the decreased fibrosis formation were restricted to the deficiency of IL-4 in the hematopoietic compartment. Finally, macrophage depletion, rather than the absence of T cells, led to reduced lesions of the glomerular filtration barrier and decreased collagen deposition. These results provide evidence that future therapeutic strategies against renal fibrosis should be accompanied by the modulation of the M1 :M2 and T(H)1:T(H)2 balance, as T(H)2 and M2 cells are predictive of fibrosis toward mechanisms that are sensed by innate immune response and triggered in a MyD88-dependent pathway. Online address: http://www.molmed.org doi: 10.2119/molmed.2012.00131 Univ São Paulo, Lab Transplantat Immunobiol, Dept Immunol, Inst Biomed Sci 4, BR-05508900 São Paulo, Brazil Universidade Federal de São Paulo, Lab Clin & Expt Immunol, Div Nephrol, São Paulo, Brazil Univ São Paulo, Dept Pharmacol, Inst Biomed Sci, BR-05508900 São Paulo, Brazil Univ São Paulo, Dept Biochem, Inst Chem, BR-05508900 São Paulo, Brazil Universidade Federal de São Paulo, Lab Clin & Expt Immunol, Div Nephrol, São Paulo, Brazil FAPESP: 07/07139-3 FAPESP: 10/52180-4 Web of Science

Malaria associated-acute kidney injury (AKI) is associated with 45% of mortality in adult patients hospitalized with severe form of the disease. However, the causes that lead to a framework of malaria-associated AKI are still poorly characterized. Some clinical studies speculate that oxidative stress products, a characteristic of Plasmodium infection, as well as proinflammatory response induced by the parasite are involved in its pathophysiology. Therefore, we aimed to investigate the development of malaria-associated AKI during infection by P. berghei ANKA, with special attention to the role played by the inflammatory response and the involvement of oxidative stress. For that, we took advantage of an experimental model of severe malaria that showed significant changes in the renal pathophysiology to investigate the role of malaria infection in the renal microvascular permeability and tissue injury. Therefore, BALB/c mice were infected with P. berghei ANKA. To assess renal function, creatinine, blood urea nitrogen, and ratio of proteinuria and creatininuria were evaluated. The products of oxidative stress, as well as cytokine profile were quantified in plasma and renal tissue. The change of renal microvascular permeability, tissue hypoxia and cellular apoptosis were also evaluated. Parasite infection resulted in renal dysfunction. Furthermore, we observed increased expression of adhesion molecule, proinflammatory cytokines and products of oxidative stress, associated with a decrease mRNA expression of HO-1 in kidney tissue of infected mice. The measurement of lipoprotein oxidizability also showed a significant increase in plasma of infected animals. Together, our findings support the idea that products of oxidative stress, as well as the immune response against the parasite are crucial to changes in kidney architecture and microvascular endothelial permeability of BALB/c mice infected with P. berghei ANKA.

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.

Focal and segmental glomerulosclerosis (FSGS) is one of the most important causes of end-stage renal failure. The bradykinin B1 receptor has been associated with tissue inflammation and renal fibrosis. To test for a role of the bradykinin B1 receptor in podocyte injury, we pharmacologically modulated its activity at different time points in an adriamycin-induced mouse model of FSGS. Estimated albuminuria and urinary protein to creatinine ratios correlated with podocytopathy. Adriamycin injection led to loss of body weight, proteinuria, and upregulation of B1 receptor mRNA. Early treatment with a B1 antagonist reduced albuminuria and glomerulosclerosis, and inhibited the adriamycin-induced downregulation of podocin, nephrin, and α-actinin-4 expression. Moreover, delayed treatment with antagonist also induced podocyte protection. Conversely, a B1 agonist aggravated renal dysfunction and even further suppressed the levels of podocyte-related molecules. Thus, we propose that kinin has a crucial role in the pathogenesis of FSGS operating through bradykinin B1 receptor signaling.

Tubule-interstitial nephritis (TIN) results in decreased renal function and interstitial inflammation, which ultimately leads to fibrosis. Excessive adenine intake can cause TIN because xanthine dehydrogenase (XDH) can convert this purine into an insoluble compound, which precipitates in the tubuli. Innate immune sensors, such as Toll-like receptors (TLR) and inflammasome complex, play a crucial role in the initiation of inflammation. The aim of this study was to evaluate the roles of TLR-2 and -4, Myd88 and inflammasome complex in an experimental model of TIN. Here, we show that wild-type (WT) mice fed adenine-enriched food exhibited significant renal dysfunction and enhanced cellular infiltration accompanied by collagen deposition. They also presented higher gene and protein expression of pro-inflammatory cytokines. In contrast, TLR-2, -4, MyD88, ASC and Caspase-1 KO mice showed renoprotection associated with expression of inflammatory molecules at levels comparable to controls. Furthermore, treatment of WT animals with allopurinol, an XDH inhibitor, led to reduced levels of uric acid, oxidative stress, collagen deposition and a downregulation of the NF-kB signaling pathway. We concluded that MyD88 signaling and inflammasome participate in the development of TIN. Furthermore, inhibition of XDH seems to be a promising way to therapeutically target the developing inflammatory process.