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Adipose-derived mesenchymal stem cells (ADMSCs) display immunosuppressive properties, suggesting a promising therapeutic application in several autoimmune diseases, but their role in type 1 diabetes (T1D) remains largely unexplored. The aim of this study was to investigate the immune regulatory properties of allogeneic ADMSC therapy in T cell-mediated autoimmune diabetes in NOD mice. ADMSC treatment reversed the hyperglycemia of early-onset diabetes in 78% of diabetic NOD mice, and this effect was associated with higher serum insulin, amylin, and glucagon-like peptide 1 levels compared with untreated controls. This improved outcome was associated with downregulation of the CD4(+) Th1-biased immune response and expansion of regulatory T cells (Tregs) in the pancreatic lymph nodes. Within the pancreas, inflammatory cell infiltration and interferon-γ levels were reduced, while insulin, pancreatic duodenal homeobox-1, and active transforming growth factor-β1 expression were increased. In vitro, ADMSCs induced the expansion/proliferation of Tregs in a cell contact-dependent manner mediated by programmed death ligand 1. In summary, ADMSC therapy efficiently ameliorates autoimmune diabetes pathogenesis in diabetic NOD mice by attenuating the Th1 immune response concomitant with the expansion/proliferation of Tregs, thereby contributing to the maintenance of functional β-cells. Thus, this study may provide a new perspective for the development of ADMSC-based cellular therapies for T1D. [ABSTRACT FROM AUTHOR]

Sirtuin 1 (SIRT1) is a class III histone deacetylase (HDAC3) that plays a crucial role in regulating the activation and differentiation of dendritic cells (DCs) as well as controlling the polarization and activation of T cells. Obesity, a chronic inflammatory condition, is characterized by the activation of immune cells in various tissues. We hypothesized that SIRT1 might influence the phenotype and functions of DCs through the Ido1 pathway, ultimately leading to the polarization towards pro-inflammatory T cells in obesity. In our study, we observed that SIRT1 activity was reduced in bone marrow-derived DCs (BMDCs) from obese animals. These BMDCs exhibited elevated oxidative phosphorylation (OXPHOS) and increased extracellular acidification rates (ECAR), along with enhanced expression of class II MHC, CD86, and CD40, and elevated secretion of IL-12p40, while the production of TGF-β was reduced. The kynurenine pathway activity was decreased in BMDCs from obese animals, particularly under SIRT1 inhibition. SIRT1 positively regulated the expression of Ido1 in DCs in a PPARγ-dependent manner. To support these findings, ATAC-seq analysis revealed that BMDCs from obese mice had differentially regulated open chromatin regions compared to those from lean mice, with reduced chromatin accessibility at the Sirt1 genomic locus in BMDCs from obese WT mice. Gene Ontology (GO) enrichment analysis indicated that BMDCs from obese animals had disrupted metabolic pathways, including those related to GTPase activity and insulin response. Differential expression analysis showed reduced levels of Pparg and Sirt1 in BMDCs from obese mice, which was challenged and confirmed using BMDCs from mice with conditional knockout of Sirt1 in dendritic cells (SIRT1∆). This study highlights that SIRT1 controls the metabolism and functions of DCs through modulation of the kynurenine pathway, with significant implications for obesity-related inflammation., (© 2024. The Author(s).)

Purpose: To analyze and compare the tear immunological profile in ocular GVHD (oGVHD) patients with that in non-oGVHD patients and to correlate them with ocular surface parameters based on the International Chronic Ocular GVHD Consensus Group (ICCGVHD) diagnostic criteria., Methods: Tear samples from 20 individuals who underwent allo-hematopoietic stem cell transplantation and were grouped according the presence or absence of oGVHD were analyzed using Bio-Plex assay., Results: IL-8 and MIP-1α levels were significantly higher in tears from oGVHD patients compared with those in tears from non-oGVHD patients (p<0.001 and p=0.001, respectively). Tear IL-8 levels correlated significantly with OSDI criteria (ρ=0.5159, p=0.001), ocular hyperemia (ρ=0.469, p=0.002), and corneal staining (ρ=0.339, p=0.032), whereas tear Mip-1α levels correlated with OSDI score (ρ=0.358, p=0.023)., Conclusion: We demonstrated higher tear levels of IL-8 and MIP-1α in oGVHD patients and significant correlations between theses cytokines and ocular surface parameters based on the ICCGVHDCG criteria.

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., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (Copyright © 2022 HCFMUSP. Published by Elsevier España, S.L.U. All rights reserved.)

Objective: Astrocytes play a significant role in the pathology of multiple sclerosis (MS). Nevertheless, for ethical reasons, most studies in these cells were performed using the Experimental Autoimmune Encephalomyelitis model. As there are significant differences between human and mouse cells, we aimed here to better characterize astrocytes from patients with MS (PwMS), focusing mainly on mitochondrial function and cell metabolism., Methods: We obtained and characterized induced pluripotent stem cell (iPSC)-derived astrocytes from three PwMS and three unaffected controls, and performed electron microscopy, flow cytometry, cytokine and glutamate measurements, gene expression, in situ respiration, and metabolomics. We validated our findings using a single-nuclei RNA sequencing dataset., Results: We detected several differences in MS astrocytes including: (i) enrichment of genes associated with neurodegeneration, (ii) increased mitochondrial fission, (iii) increased production of superoxide and MS-related proinflammatory chemokines, (iv) impaired uptake and enhanced release of glutamate, (v) increased electron transport capacity and proton leak, in line with the increased oxidative stress, and (vi) a distinct metabolic profile, with a deficiency in amino acid catabolism and increased sphingolipid metabolism, which have already been linked to MS., Interpretation: Here we describe the metabolic profile of iPSC-derived astrocytes from PwMS and validate this model as a very powerful tool to study disease mechanisms and to perform non-invasive drug targeting assays in vitro. Our findings recapitulate several disease features described in patients and provide new mechanistic insights into the metabolic rewiring of astrocytes in MS, which could be targeted in future therapeutic studies. ANN NEUROL 2022;91:652-669., (© 2022 The Authors. Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Little is known about the diversity in immune profile of the different wild type strains of zebrafish ( Danio rerio ), despite its growing popularity as an animal model to study human diseases and drug testing. In the case of data resulting from modeling human diseases, differences in the background Danio fishes have rarely been taken into consideration when interpreting results and this is potentially problematic, as many studies not even mention the source and strain of the animals. In this study, we hypothesized that different wild type zebrafish strains could present distinct immune traits. To address the differences in immune responses between two commonly used wild type strains of zebrafish, AB and Tübingen (TU), we used an intestinal inflammation model induced by 2,4,6-Trinitrobenzenesulfonic acid (TNBS) and characterized the susceptibility and immune profile in these two strains. Our data demonstrates significant differences in survival between AB and TU strains when exposed to TNBS, suggesting important physiological differences in how these strains respond to inflammatory challenges. We observed that the AB strain presented increased mortality, higher neutrophilic intestinal infiltration, decreased goblet cell numbers and decreased IL-10 expression when exposed to TNBS, compared to the TU strain. In summary, our study demonstrates strain-specific immunological responses in AB and TU animals. Finally, the significant variations in strain-related susceptibility to inflammation and the differences in the immune profile shown here, highlight that the background of each strain need to be considered when utilizing zebrafish to model diseases and for drug screening purposes, thus better immune characterization of the diverse wild type strains of zebrafish is imperative., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 Published by Elsevier B.V.)

Purpose: This study aimed to characterize the tear film immunologic profile in keratoconus (KC) patients compared with healthy individuals (control group) and to investigate the correlation between the tear film immunologic profile and atopy, disease severity, and disease status over time., Methods: The study involved 30 KC patients and 18 healthy individuals. Tear collection was obtained using microcapillary tubes. Tear film levels of fractalkine, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-γ, interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17A, IL-21, IL-23, interferon-inducible T-cell alpha chemoattractant (ITAC), macrophage inflammatory protein-1 alpha (MIP-1α), MIP-1β, MIP-3α, and tumor necrosis factor (TNF)-α were detected. Keratometric measurements and topographic patterns were used to diagnose and define disease progression. Tear immunologic profiles were compared, emphasizing the presence or absence of ocular allergy. Correlations between the cytokine profile, disease severity, and disease status were also analyzed longitudinally in the KC patients., Results: Lacrimal cytokine concentrations were higher in the KC patients than they were in the controls in 14 of 21 cytokines analyzed. IL-6 was the most relevant cytokine found in KC patients, especially when associated with ocular allergy. There was no correlation between KC progression and the level of inflammatory cytokines when analyzed longitudinally. KC severity correlated with IL-6 concentration, where the more severe KC presented a higher IL-6 concentration in tears., Conclusions: Inflammatory activity seems to be involved in the pathogenesis of KC. Out of 21 cytokines, 14 were more concentrated in the tears of KC patients than healthy subjects. IL-6 was significantly higher in KC patients' tears and was related to disease severity. Disease progression did not correlate with cytokine levels when analyzed longitudinally., (Copyright © 2021 Molecular Vision.)

Plasmodium malariae has a wide geographic distribution, but mainly at very low parasitemias and in co-infections, leading to an underestimated prevalence of this species. Studies for the detection of antibodies against Plasmodium recombinant proteins are increasingly used to map geographical distributions, seroprevalence and transmission intensities of malaria infection. However, no seroepidemiological survey using recombinant P. malariae proteins has been conducted in Brazil. This work evaluated the antibody response in serum samples of individuals from endemic regions of Brazil (the Amazon region and Atlantic Forest) against five recombinant proteins of P. malariae merozoite surface protein 1 (MSP1), and the MSP1 C-terminal portions of P. vivax and P. falciparum , in a multiplex assay. The positivity was 69.5% of samples recognizing at least one MSP1 recombinant protein. The mean of the Reactivity Index for the C-terminal portion of the P. falciparum was significantly higher compared to the other recombinant proteins, followed by the C-terminal of P. vivax and the N-terminal of P. malariae . Among the recombinant P. malariae proteins, the N-terminal of P. malariae showed the highest Reactivity Index alone. This study validates the use of the multiplex assay to measure naturally acquired IgG antibodies against Plasmodium MSP1 proteins and demonstrate that these proteins are important tools for seroepidemiological surveys and could be used in malaria surveillance.

Sepsis is a complex infectious syndrome in which neutrophil participation is crucial for patient survival. Neutrophils quickly sense and eliminate the pathogen by using different effector mechanisms controlled by metabolic processes. The mammalian target of rapamycin (mTOR) pathway is an important route for metabolic regulation, and its role in neutrophil metabolism has not been fully understood yet, especially the importance of mTOR complex 2 (mTORC2) in the neutrophil effector functions. In this study, we observed that the loss of Rictor (mTORC2 scaffold protein) in primary mouse-derived neutrophils affects their chemotaxis by fMLF and their microbial killing capacity, but not the phagocytic capacity. We found that the microbicidal capacity was impaired in Rictor-deleted neutrophils because of an improper fusion of granules, reducing the hypochlorous acid production. The loss of Rictor also led to metabolic alterations in isolated neutrophils, increasing aerobic glycolysis. Finally, myeloid-Rictor-deleted mice (LysMRic Δ/Δ) also showed an impairment of the microbicidal capacity, increasing the bacterial burden in the Escherichia coli sepsis model. Overall, our results highlight the importance of proper mTORC2 activation for neutrophil effector functions and metabolism during sepsis., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

Uric acid (UA), a product of purine nucleotide degradation able to initiate an immune response, represents a breakpoint in the evolutionary history of humans, when uricase, the enzyme required for UA cleavage, was lost. Despite being inert in human cells, UA in its soluble form (sUA) can increase the level of interleukin-1β (IL-1β) in murine macrophages. We, therefore, hypothesized that the recognition of sUA is achieved by the Naip1-Nlrp3 inflammasome platform. Through structural modelling predictions and transcriptome and functional analyses, we found that murine Naip1 expression in human macrophages induces IL-1β expression, fatty acid production and an inflammation-related response upon sUA stimulation, a process reversed by the pharmacological and genetic inhibition of Nlrp3. Moreover, molecular interaction experiments showed that Naip1 directly recognizes sUA. Accordingly, Naip may be the sUA receptor lost through the human evolutionary process, and a better understanding of its recognition may lead to novel anti-hyperuricaemia therapies.

There is a strong correlation between obesity and cancer. Here, we investigated the influence of IL-6 and gut microbiota of obese mice in melanoma development. We first evaluated B16F10 melanoma growth in preclinical models for obesity: mice deficient for leptin (ob/ob ) or adiponectin (AdpKO) and in wild-type mice (WT, C57BL/6J) fed a high-fat diet (HFD; 60% kcal from fat) for 12 weeks. The survival rates of ob/ob and HFD-fed mice were lower than those of their respective controls. AdpKO mice also died earlier than WT control mice. We then verified the involvement of IL-6 signaling in obese mice that were inoculated with melanoma cells. Both ob/ob and AdpKO mice had higher circulating IL-6 levels than wild-type mice. Melanoma tumor volumes in IL-6 KO mice fed an HFD were reduced compared to those of WT mice subjected to the same diet. Also evaluated the effect of microbiota in tumor development. Cohousing and fecal matter transfer experiments revealed that microbiota from ob/ob mice can stimulate tumor development in lean WT mice. Taken together, our data show that in some conditions IL-6 and the gut microbiota are key mediators that link obesity and melanoma.

Background: Host-microbiota interactions shape T-cell differentiation and promote tumour immunity. Although IL-9-producing T cells have been described as potent antitumour effectors, their role in microbiota-mediated tumour control remains unclear., Methods: We analysed the impact of the intestinal microbiota on the differentiation of colonic lamina propria IL-9-producing T cells in germ-free and dysbiotic mice. Systemic effects of the intestinal microbiota on IL-9-producing T cells and the antitumour role of IL-9 were analysed in a model of melanoma-challenged dysbiotic mice., Results: We show that germ-free mice have lower frequency of colonic lamina propria IL-9-producing T cells when compared with conventional mice, and that intestinal microbiota reconstitution restores cell frequencies. Long-term antibiotic treatment promotes host dysbiosis, diminishes intestinal IL-4 and TGF-β gene expression, decreases the frequency of colonic lamina propria IL-9-producing T cells, increases the susceptibility to tumour development and reduces the frequency of IL-9-producing T cells in the tumour microenvironment. Faecal transplant restores intestinal microbiota diversity, and the frequency of IL-9-producing T cells in the lungs of dysbiotic animals, restraining tumour burden. Finally, recombinant IL-9 injection enhances tumour control in dysbiotic mice., Conclusions: Host-microbiota interactions are required for adequate differentiation and antitumour function of IL-9-producing 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-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., (© 2019 Crohn’s & Colitis Foundation. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 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., Mariño, 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.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., Mariño, 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.

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 MOG 35-55 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., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Th9 cells orchestrate allergic lung inflammation by promoting recruitment and activation of eosinophils and mast cells, and by stimulating epithelial mucus production, which is known to be mainly dependent on IL-9. These cells share developmental pathways with induced regulatory T cells that may determine the generation of one over the other subset. In fact, the FOXP3 transcription factor has been shown to bind il9 locus and repress IL-9 production. The microbiota-derived short-chain fatty acids (SCFAs) butyrate and propionate have been described as FOXP3 inducers and are known to have anti-inflammatory properties. While SCFAs attenuate lung inflammation by inducing regulatory T cells and suppressing Th2 responses, their effects on Th9 cells have not been addressed yet. Therefore, we hypothesized that SCFAs would have a protective role in lung inflammation by negatively modulating differentiation and function of Th9 cells. Our results demonstrated that butyrate is more effective than propionate in promoting FOXP3 expression and IL-9 repression. In addition, propionate was found to negatively impact in vitro differentiation of IL-13-expressing T cells. Butyrate treatment attenuated lung inflammation and mucus production in OVA-challenged mice, which presented lower frequency of lung-infiltrated Th9 cells and eosinophils. Both Th9 cell adoptive transfer and IL-9 treatment restored lung inflammation in butyrate-treated OVA-challenged mice, indicating that the anti-inflammatory effects of butyrate may rely on suppressing Th9-mediated immune responses.

Acute kidney injury (AKI) and chronic kidney disease (CKD) are major concerns in worldwide public health, and their pathophysiology involves immune cells activation, being macrophages one of the main players of both processes. It is suggested that metabolic pathways could contribute to macrophage modulation and phosphatidylinositol‑3 kinase (PI3K) pathway was shown to be activated in kidneys subjected to ischemia and reperfusion as well as unilateral ureteral obstruction (UUO). Although PI3K inhibition is mostly associated with anti-inflammatory response, its use in kidney injuries has been shown controversial results, which indicates the need for further studies. Our aim was to unveil the role of PI3Kγ in macrophage polarization and in kidney diseases development. We analyzed bone-marrow macrophages polarization from wild-type (WT) and PI3Kγ knockout (PI3K KO) animals. We observed increased expression of M1 (CD86, CCR7, iNOS, TNF, CXCL9, CXCL10, IL-12 and IL-23) and decreased of M2 (CD206, Arg-1, FIZZ1 and YM1) markers in the lack of PI3Kγ. And this modulation was accompanied by higher levels of inflammatory cytokines in PI3K KO M1 cells. PI3K KO mice had increased M1 in steady state kidneys, and no protection was observed in these mice after acute and chronic kidney insults. On the contrary, they presented higher levels of protein-to-creatinine ratio and Kim-1 expression and increased tubular injury. In conclusion, our findings demonstrated that the lack of PI3Kγ favors M1 macrophages polarization providing an inflammatory-prone environment, which does not prevent kidney diseases progression., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cisplatin is a chemotherapy used to treat different types of cancer, such as testicular, bladder and head and neck. Physical exercise has been shown to improve cancer therapy and recently, it was demonstrated to be able to diminish side effects such as acute kidney injury (AKI), a common side effect in cisplatin treatment. In both cases, the modulation of inflammatory cytokines seems to be one of the mechanisms, but little is known about the immune cells in this process. Here, we investigated the role of CD4 + T cells in the AKI protection by physical exercise. We subjected C57Bl6 mice to long-term physical exercise (EX) before cisplatin treatment. Sedentary groups were used as control (CT). We confirmed that physical exercise decreased AKI by evaluating creatinine and Kim-1 levels, in the serum and kidney respectively. Analyzing the organs weight, we noticed a decrease in sedentary (CIS) and exercised (CIS-EX) cisplatin treated groups. Epididymal and brown adipose tissue weight were decreased in cisplatin treated subjects in comparison to untreated groups, as well as liver and spleen. We then investigated the profile of CD4 + T cells in the spleen and we observed increased levels of Tregs and CD4+CD25+ cells in CIS group, while CIS-EX presented similar amounts as control groups. Analyzing the kidney lymph nodes, we noticed a decrease of CD4+ cells in both CIS and CIS-EX group. However, a more activated phenotype (CD69+ and CD25+) was observed in CIS groups in comparison to CIS-EX group, as well as the presence of Tregs. We then investigated the production of cytokines by these cells and no difference among the groups was observed in cytokines production in splenic CD4 + T cells. However, a clear increase in TNF and IL-10 production was observed in CD4 + T cells from lymph nodes, while CIS-EX group presented similar levels as the control groups. We confirmed that physical exercise was able to diminish cisplatin-induced AKI with concomitant decrease in CD4 + T cell activation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Acute kidney injury (AKI) is considered an inflammatory disease in which toll-like receptors (TLRs) signaling pathways play an important role. The activation of TLRs results in production of several inflammatory cytokines leading to further renal damage. In contrast, TLRs are key players on autophagy induction, which is associated with a protective function on cisplatin-induced AKI. Hence, the present study aimed to evaluate the specific participation of TLR2 and TLR4 molecules on the development of cisplatin-induced AKI. Complementarily, we also investigated the link between TLRs and heme oxygenase-1 (HO-1), a promisor cytoprotective molecule. First, we observed that only the absence of TLR2 but not TLR4 in mice exacerbated the renal dysfunction, tissue injury and mortality rate, even under an immunologically privileged microenvironment. Second, we demonstrated that TLR2 knockout (KO) mice presented lower expression of autophagy-associated markers when compared with TLR4 KO animals. Similar parameter was confirmed in vitro , using tubular epithelial cells derived from both KO mice. To test the cross-talking between HO-1 and TLRs, hemin (an HO-1 internal inducer) was administrated in cisplatin-treated TLR2 and TLR4 KO mice and it was detected an improvement in the global renal tissue parameters. However, this protection was less evident at TLR2 KO mice. In summary, we documented that TLR2 plays a protective role in cisplatin-induced AKI progression, in part, by a mechanism associated with autophagy up-regulation, considering that its interplay with HO-1 can promote renal tissue recover., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

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, CCR2 rfp/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.

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., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The antineoplastic drug cisplatin promotes renal injury, which limits its use. Protocols that reduce renal cisplatin toxicity will allow higher doses to be used in cisplatin treatment. Here, we compare physical exercise and caloric restriction (CR) as protocols to reduce cisplatin renal injury in mice. Male C57BL/6 were divided into four groups: Control, cisplatin, exercise + cisplatin, and 30% CR + cisplatin. Animals were injected with a single dose of cisplatin (20 mg/kg i.p.) and sacrificed 96 h after injection. Quantitative real time PCR, histological analyses, immunohistochemistry, and biochemical measurements were performed to investigate renal injury, necrosis, apoptosis, and inflammatory mechanisms. Both protocols protected against cisplatin renal injury, but CR was more effective in reducing uraemia and renal necrosis. The CR + Cisplatin group exhibited reduced serum IL-1β and TNF-α levels. No differences were noted in the renal mRNA expression of cytokines. Both interventions reduced apoptosis, but only the CR + Cisplatin group decreased TNFR2 protein expression. PPAR-α was activated in mice after CR. An antagonist of PPAR-α blocked the protective effect of CR. Both interventions attenuated the nephrotoxicity caused by cisplatin injection, but CR + Cisplatin showed a better response by modulating TNFR2. Moreover, part of the CR benefit depends on PPAR-α activation.

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.

Mesenchymal stromal cells (MSCs) orchestrate tissue repair by releasing cell-derived microvesicles (MVs), which, presumably by small RNA species, modulate global gene expression. The knowledge of miRNA/mRNA signatures linked to a reparative status may elucidate some of the molecular events associated with MSC protection. Here, we used a model of cisplatin-induced kidney injury (acute kidney injury) to assess how MSCs or MVs could restore tissue function. MSCs and MVs presented similar protective effects, which were evidenced in vivo and in vitro by modulating apoptosis, inflammation, oxidative stress, and a set of prosurvival molecules. In addition, we observed that miRNAs (i.e., miR-880, miR-141, miR-377, and miR-21) were modulated, thereby showing active participation on regenerative process. Subsequently, we identified that MSC regulates a particular miRNA subset which mRNA targets are associated with Wnt/TGF-β, fibrosis, and epithelial-mesenchymal transition signaling pathways. Our results suggest that MSCs release MVs that transcriptionally reprogram injured cells, thereby modulating a specific miRNA-mRNA network.

Background: The effect of the interaction between type 2 diabetes and dyslipidemia on inflammation and lipid peroxidation (LPO) has not been assessed., Aim: To investigate whether diabetes coupled with dyslipidemia alters oxidative metabolism leading to increased LPO products and inflammatory status., Methods: 100 patients were divided into four groups based upon diabetic and dyslipidemic status: poorly controlled diabetes with dyslipidemia (DM-PC/D), well-controlled diabetes with dyslipidemia (DM-WC/D), normoglycemic individuals with dyslipidemia (NG/D), and normoglycemic individuals without dyslipidemia (NG/ND). Plasma was evaluated for an LPO product (MDA), antioxidant levels and inflammatory cytokines., Results: Diabetics presented significantly higher levels of LPO (p<0.05) and the DM-PC/D had higher levels of proinflammatory cytokines and MDA in the plasma in comparison with normoglycemics (p<0.05). Interestingly IL1-β, IL-6, and TNF-α in DM-WC/D were not statistically different from those in DM-PC/D. Normoglycemic individuals with dyslipidemia presented significantly increased levels of IL-6 and TNF-α when compared to normoglycemic without dyslipidemia (p<0.05). MDA levels were also positively correlated with the presence of DM complications (r=0.42, p<0.01)., Conclusions: These findings show that dyslipidemia is associated with an increased inflammatory status, even in well-controlled diabetics and in normoglycemics. Our results suggest that lipid metabolism and peroxidation are important for the development of inflammation, which is elevated in several complications associated with diabetes., Competing Interests: There is no conflict of interest., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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 TH1/TH17 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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

Background and Purpose: Asthma is a disease of high prevalence and morbidity that generates high costs in hospitalization and treatment. Although the airway is involved in the physiopathology of asthma, there is also evidence of the importance of vascular and lung parenchyma inflammation and remodeling, which can contribute to the functional pulmonary alterations observed in asthmatic patients. Our aim was to evaluate treatment using sakuranetin, a flavone isolated from the twigs of Baccharis retusa (Asteraceae), on vascular and lung parenchyma alterations in an experimental murine model of asthma., Methods: Male BALB/c mice were subjected to a sensitization protocol with ovalbumin for 30days and were treated with or without sakuranetin (20mg/kg/mice) or dexamethasone (5mg/kg/mice); then, the lungs were collected for histopathological analysis. We evaluated extracellular matrix remodeling (collagen and elastic fibers), inflammation (eosinophils and NF-kB) and oxidative stress (8-isoprostane) in the pulmonary vessels and lung parenchyma. The thickness of the vascular wall was quantified, as well as the vascular endothelial growth factor (VEGF) levels., Results: We demonstrated that sakuranetin reduced the number of eosinophils and elastic fibers in both the pulmonary vessels and the lung parenchyma, probably due to a reduction of oxidative stress and of the transcription factor NF-kB and VEGF levels in the lung. In addition, it reduced the thickness of the pulmonary vascular wall. The treatment had no effect on the collagen fibers. In most of the parameters, the effect of sakuranetin was similar to the dexamethasone effect., Conclusions and Implications: Sakuranetin had anti-inflammatory and antioxidant effects, preventing vascular and distal parenchyma changes in this experimental model of asthma., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Background: Physical exercise induces positive alterations in gene expression involved in the metabolism of obesity. Maternal exercise provokes adaptations soon after birth in the offspring. Here, we investigated whether adult mouse offspring of swim-trained mothers is protected against the development of the deleterious effects of high fat diet (HFD)., Methods: Our study comprises two parts. First, female C57BL/6 mice were divided into one sedentary and one swim-trained group (before and during pregnancy, n = 18). In the second part, adult offspring (n = 12) of trained and sedentary mothers was challenged to HFD for 16 weeks. Notably, most of the analysis was done in male offspring., Results: Our results demonstrate that maternal exercise has several beneficial effects on the mouse offspring and protects them from the deleterious effects of HFD in the adult. Specifically, swimming during pregnancy leads to lower birth weight in offspring through 2 months of age. When subjected to HFD for 4 month in the adulthood, our study presents novel data on the male offspring's metabolism of trained mothers. The offspring gained less weight, which was accompanied by less body fat, and they used more calories during daytime compared with offspring of sedentary mothers. Furthermore, we observed increased adiponectin expression in skeletal muscle, which was accompanied by decreased leptin levels and increased insulin sensitivity. Decreased interleukin-6 expression and increased peptide PYY levels were observed in sera of adult offspring of mothers that swam during pregnancy., Conclusions: Our results point to the conclusion that maternal exercise is beneficial to protect the offspring from developing obesity, which could be important for succeeding generations as well.

Background: Severe scorpion envenomation can evolve to lung injury and, in some cases, death. The lung injury could be attributed to acute left ventricular failure and increased pulmonary vascular permeability secondary to the release of inflammatory mediators. In clinical practice, corticosteroids have been administered to reduce the early side effects of the anti-venom. We propose to study the effects of Tityus serrulatus venom and dexamethasone on pulmonary expression of sodium and water transporters, as well as on the inflammatory response., Methods: Wistar rats were injected intraperitoneally with saline (control group), dexamethasone, and saline (2.0 mg/kg body weight-60 min before saline injection; dexamethasone + saline group), venom (T. serrulatus venom-3.8 mg/kg body weight), or dexamethasone and venom (2.0 mg/kg body weight-60 min before venom injection; dexamethasone + venom group). At 60 min after venom/saline injection, experiments were performed in ventilated and non-ventilated animals. We analyzed sodium transporters, water transporters, and Toll-like receptor 4 (TLR4) by Western blotting, macrophage infiltration by immunohistochemistry, and serum interleukin (IL) by cytokine assay., Results: In the lung tissue of non-ventilated envenomed animals, protein expression of the epithelial sodium channel alpha subunit (α-ENaC) and aquaporin 5 (AQP5) were markedly downregulated whereas that of the Na-K-2Cl cotransporter (NKCC1) and TLR4 was elevated although expression of the Na,K-ATPase alpha 1 subunit was unaffected. Dexamethasone protected protein expression of α-ENaC, NKCC1, and TLR4 but not that of AQP5. We found that IL-6, IL-10, and tumor necrosis factor alpha were elevated in the venom and dexamethasone + venom groups although CD68 expression in lung tissue was elevated only in the venom group. Among the ventilated animals, both envenomed groups presented hypotension at 50 min after injection, and the arterial oxygen tension/fraction of inspired oxygen ratio was lower at 60 min than at baseline., Conclusions: Our results suggest that T. serrulatus venom and dexamethasone both regulate sodium transport in the lung and that T serrulatus venom regulates sodium transport via the TLR4 pathway.

Aim: Experimental autoimmune encephalomyelitis (EAE) is a CD4(+)-mediated autoimmune pathology of the central nervous system (CNS) that is used as a model for the study of the human neuroinflammatory disease, multiple sclerosis. During the development of EAE, auto-reactive Th1 and Th17 CD4(+) T cells infiltrate the CNS promoting inflammatory cells recruitment, focal inflammation and tissue destruction. In this sense, statins, agents used to lower lipid levels, have recently shown to exert interesting immunomodulatory function. In fact, statins promote a bias towards a Th2 response, which ameliorates the clinical outcome of EAE. Additionally, simvastatin can inhibit Th17 differentiation. However, many other effects exerted on the immune system by statins have yet to be clarified, in particular during neuroinflammation. Thus, the aim of this study was to investigate the effects of simvastatin on the development of experimental autoimmune encephalomyelitis., Methods: Mice were immunized with MOG(35-55) and EAE severity was assessed daily and scored using a clinical scale. Cytokine secretion by mononuclear cells infiltrating the CNS was evaluated by flow cytometry., Results: Simvastatin (5 mg/kg/day) improved clinical outcome, induced an increase in TGF-β mRNA expression and inhibited IL-6, IL-12p40, IL-12p70, RANTES and MIP-1β secretion (p < 0.05). This was accompanied by a significant decrease in CNS inflammatory mononuclear cell infiltration, with reduced frequencies of both Th1 and Th17 cells. Simvastatin inhibited the proliferation of T lymphocytes co-cultured with primary microglial cells., Conclusions: Simvastatin treatment promotes EAE clinical amelioration by inhibiting T cell proliferation and CNS infiltration by pathogenic Th1 and Th17 cells.

Acute and chronic kidney injuries (AKI and CKI) constitute syndromes responsible for a large part of renal failures, and are today still associated with high mortality rates. Given the lack of more effective therapies, there has been intense focus on the use stem cells for organ protective and regenerative effects. Mesenchymal stem cells (MSCs) have shown great potential in the treatment of various diseases of immune character, although there is still debate on its mechanism of action. Thus, for a greater understanding of the role of MSCs, we evaluated the effect of adipose tissue-derived stem cells (AdSCs) in an experimental model of nephrotoxicity induced by folic acid (FA) in FVB mice. AdSC-treated animals displayed kidney functional improvement 24h after therapy, represented by reduced serum urea after FA. These data correlated with cell cycle regulation and immune response modulation via reduced chemokine expression and reduced neutrophil infiltrate. Long-term analyses, 4 weeks after FA, indicated that AdSC treatment reduced kidney fibrosis and chronic inflammation. These were demonstrated by reduced interstitial collagen deposition and tissue chemokine and cytokine expression. Thus, we concluded that AdSC treatment played a protective role in the framework of nephrotoxic injury via modulation of inflammation and cell cycle regulation, resulting in reduced kidney damage and functional improvement, inhibiting organ fibrosis and providing long-term immune regulation.

Short-chain fatty acids (SCFAs) are fermentation end products produced by the intestinal microbiota and have anti-inflammatory and histone deacetylase-inhibiting properties. Recently, a dual relationship between the intestine and kidneys has been unraveled. Therefore, we evaluated the role of SCFA in an AKI model in which the inflammatory process has a detrimental role. We observed that therapy with the three main SCFAs (acetate, propionate, and butyrate) improved renal dysfunction caused by injury. This protection was associated with low levels of local and systemic inflammation, oxidative cellular stress, cell infiltration/activation, and apoptosis. However, it was also associated with an increase in autophagy. Moreover, SCFAs inhibited histone deacetylase activity and modulated the expression levels of enzymes involved in chromatin modification. In vitro analyses showed that SCFAs modulated the inflammatory process, decreasing the maturation of dendritic cells and inhibiting the capacity of these cells to induce CD4(+) and CD8(+) T cell proliferation. Furthermore, SCFAs ameliorated the effects of hypoxia in kidney epithelial cells by improving mitochondrial biogenesis. Notably, mice treated with acetate-producing bacteria also had better outcomes after AKI. Thus, we demonstrate that SCFAs improve organ function and viability after an injury through modulation of the inflammatory process, most likely via epigenetic modification., (Copyright © 2015 by the American Society of Nephrology.)

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.

Nephrotoxicity is substantial side effect for 30% of patients undergoing cancer therapy with cisplatin and may force them to change or even abandon the treatment. Studies regarding aerobic exercise have shown its efficacy for the treatment of many types of diseases and its capacity to reduce tumors. However, little is known about the impact of physical exercise on cisplatin-induced acute kidney injury (AKI). In the present study, our aim was to investigate the role of physical exercise in AKI induced by cisplatin. We submitted C57Bl6 male mice to seven weeks of chronic exercise on a training treadmill and treated them with single i.p. injection of cisplatin (20 mg/kg) in the last week. Exercise efficacy was confirmed by an increased capillary-to-fiber ratio in the gastrocnemius muscle of exercised groups (EX and CIS-EX). The group submitted to exercise before cisplatin administration (CIS-EX) exhibited less weight loss and decreased serum urea levels compared to the cisplatin group (CIS). Exercise also showed a protective role against cisplatin-induced cell death in the kidney. The CIS-EX group showed a lower inflammatory response, with less TNF and IL-10 expression in the kidney and serum. In the same group, we observed an increase of IL-6 and HO-1 expression in the kidney. Taken together, our results indicate that chronic aerobic exercise is able to attenuate AKI by inducing IL-6 and HO-1 production, which results in lower inflammatory and apoptotic profiles in the kidney.

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., (© 2014. Published by The Company of Biologists Ltd.)

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.

Background/aims: We investigated the effects of leptin in the development of lipopolysaccharide (LPS)-induced acute lung inflammation (ALI) in lean mice., Methods: Mice were administered leptin (1.0µg/g) or leptin (1.0µg/g) followed by LPS (1.5µg/g) intranasally. Additionally, some animals were given LPS (1.5µg/g) or saline intranasally alone, as a control. Tissue samples and fluids were collected six hours after instillation., Results: We demonstrated that leptin alone did not induce any injury. Local LPS exposure resulted in significant acute lung inflammation, characterized by a substantial increase in total cells, mainly neutrophils, in bronchoalveolar lavages (BAL). We also observed a significant lymphocyte influx into the lungs associated with enhanced lung expression of chemokines and cytokines (KC, RANTES, TNF-α, IFN-γ, GM-CSF and VEGF). LPS-induced ALI was characterized by the enhanced expression of ICAM-1 and iNOS in the lungs. Mice that received LPS showed an increase in insulin levels. Leptin, when administered prior to LPS instillation, abolished all of these effects. LPS induced an increase in corticosterone levels, and leptin potentiated this event., Conclusion: These data suggest that exogenous leptin may promote protection during sepsis, and downregulation of the insulin levels and upregulation of corticosterone may be important mechanisms in the amelioration of LPS-induced ALI., (© 2014 S. Karger AG, Basel.)

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.

Plasmodium chabaudi infection induces a rapid and intense splenic CD4(+) T cell response that contributes to both disease pathogenesis and the control of acute parasitemia. The subsequent development of clinical immunity to disease occurs concomitantly with the persistence of low levels of chronic parasitemia. The suppressive activity of regulatory T (T(reg)) cells has been implicated in both development of clinical immunity and parasite persistence. To evaluate whether IL-2 is required to induce and to sustain the suppressive activity of T(reg) cells in malaria, we examined in detail the effects of anti-IL-2 treatment with JES6-1 monoclonal antibody (mAb) on the splenic CD4(+) T cell response during acute and chronic P. chabaudi AS infection in C57BL/6 mice. JES6-1 treatment on days 0, 2 and 4 of infection partially inhibits the expansion of the CD4(+)CD25(+)Foxp3(+) cell population during acute malaria. Despite the concomitant secretion of IL-2 and expression of high affinity IL-2 receptor by large CD4(+) T cells, JES6-1 treatment does not impair effector CD4(+) T cell activation and IFN-γ production. However, at the chronic phase of the disease, an enhancement of cellular and humoral responses occurs in JES6-1-treated mice, with increased production of TNF-α and parasite-specific IgG2a antibodies. Furthermore, JES6-1 mAb completely blocked the in vitro proliferation of CD4(+) T cells from non-treated chronic mice, while it further increased the response of CD4(+) T cells from JES6-1-treated chronic mice. We conclude that JES6-1 treatment impairs the expansion of T(reg) cell population during early P. chabaudi malaria and enhances the Th1 cell response in the late phase of the disease.

The Th1/Th2 balance represents an important factor in the pathogenesis of renal ischemia-reperfusion injury (IRI). In addition, IRI causes a systemic inflammation that can affect other tissues, such as the lungs. To investigate the ability of renal IRI to modulate pulmonary function in a specific model of allergic inflammation, C57Bl/6 mice were immunized with ovalbumin/albumen on days 0 and 7 and challenged with an ovalbumin (OA) aerosol on days 14 and 21. After 24 h of the second antigen challenge, the animals were subjected to 45 minutes of ischemia. After 24 h of reperfusion, the bronchoalveolar lavage (BAL) fluid, blood and lung tissue were collected for analysis. Serum creatinine levels increased in both allergic and non-immunized animals subjected to IRI. However, BAL analysis showed a reduction in the total cells (46%) and neutrophils (58%) compared with control allergic animals not submitted to IRI. In addition, OA challenge induced the phosphorylation of ERK and Akt and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lung homogenates. After renal IRI, the phosphorylation of ERK and expression of COX-2 and iNOS were markedly reduced; however, there was no difference in the phosphorylation of Akt between sham and ischemic OA-challenged animals. Mucus production was also reduced in allergic mice after renal IRI. IL-4, IL-5 and IL-13 were markedly down-regulated in immunized/challenged mice subjected to IRI. These results suggest that renal IRI can modulate lung allergic inflammation, probably by altering the Th1/Th2 balance and, at least in part, by changing cellular signal transduction factors., (Copyright © 2012 S. Karger AG, Basel.)

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.

It has been well-documented that leukotrienes (LTs) are released in allergic lung inflammation and that they participate in the physiopathology of asthma. A role for LTs in innate immunity has recently emerged: Cys-LTs were shown to enhance FcgammaR-mediated phagocytosis by alveolar macrophages (AMs). Thus, using a rat model of asthma, we evaluated FcgammaR-mediated phagocytosis and killing of Klebsiella pneumoniae by AMs. The effect of treatment with a cys-LT antagonist (montelukast) on macrophage function was also investigated. Male Wistar rats were immunized twice with OVA/alumen intraperitoneally and challenged with OVA aerosol. After 24 h, the animals were killed, and the AMs were obtained by bronchoalveolar lavage. Macrophages were cultured with IgG-opsonized red blood cells (50:1) or IgG-opsonized K. pneumoniae (30:1), and phagocytosis or killing was evaluated. Leukotriene C(4) and nitric oxide were quantified by the EIA and Griess methods, respectively. The results showed that AMs from sensitized and challenged rats presented a markedly increased phagocytic capacity via FcgammaR (10X compared to controls) and enhanced killing of K. pneumoniae (4X higher than controls). The increased phagocytosis was inhibited 15X and killing 3X by treatment of the rats with montelukast, as compared to the non-treated group. cys-LT addition increased phagocytosis in control AMs but had no effect on macrophages from allergic lungs. Montelukast reduced nitric oxide (39%) and LTC(4) (73%). These results suggest that LTs produced during allergic lung inflammation potentiate the capacity of AMs to phagocytose and kill K. pneumonia via FcgammaR., (Copyright 2010 S. Karger AG, Basel.)

Cloning of the T-cell receptor genes is a critical step when generating T-cell receptor transgenic mice. Because T-cell receptor molecules are clonotypical, isolation of their genes requires reverse transcriptase-assisted PCR using primers specific for each different Valpha or Vbeta genes or by the screening of cDNA libraries generated from RNA obtained from each individual T-cell clone. Although feasible, these approaches are laborious and costly. The aim of the present study was to test the application of the non-palindromic adaptor-PCR method as an alternative to isolate the genes encoding the T-cell receptor of an antigen-specific T-cell hybridoma. For this purpose, we established hybridomas specific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen. These T-cell hybridomas were characterized with regard to their ability to secrete interferon-gamma, IL-4, and IL-10 after stimulation with the antigen. A CD3+, CD4+, CD8- interferon-gamma-producing hybridoma was selected for the identification of the variable regions of the T-cell receptor by the non-palindromic adaptor-PCR method. Using this methodology, we were able to rapidly and efficiently determine the variable regions of both T-cell receptor chains. The results obtained by the non-palindromic adaptor-PCR method were confirmed by the isolation and sequencing of the complete cDNA genes and by the recognition with a specific antibody against the T-cell receptor variable beta chain. We conclude that the non-palindromic adaptor-PCR method can be a valuable tool for the identification of the T-cell receptor transcripts of T-cell hybridomas and may facilitate the generation of T-cell receptor transgenic mice.

We previously described that DNA vaccination with the gene encoding amastigote surface protein 2 (ASP-2) protects approximately 65% of highly susceptible A/Sn mice against the lethal Trypanosoma cruzi infection. Here, we explored the possibility that bacterial recombinant proteins of ASP-2 could be used to improve the efficacy of vaccinations. Initially, we compared the protective efficacy of vaccination regimens using either a plasmid DNA, a recombinant protein, or both sequentially (DNA priming and protein boosting). Survival after the challenge was not statistically different among the three mouse groups and ranged from 53.5 to 75%. The fact that immunization with a recombinant protein alone induced protective immunity revealed the possibility that this strategy could be pursued for vaccination. We investigated this possibility by using six different recombinant proteins representing distinct portions of ASP-2. The vaccination of mice with glutathione S-transferase fusion proteins representing amino acids 261 to 500 or 261 to 380 of ASP-2 in the presence of the adjuvants alum and CpG oligodeoxynucleotide 1826 provided remarkable immunity, consistently protecting 100% of the A/Sn mice. Immunity was completely reversed by the in vivo depletion of CD8(+) T cells, but not CD4(+) T cells, and was associated with the presence of CD8(+) T cells specific for an epitope located between amino acids 320 and 327 of ASP-2. We concluded that a relatively simple formulation consisting of a recombinant protein with a selected portion of ASP-2, alum, and CpG oligodeoxynucleotide 1826 might be used to cross-prime strong CD8(+)-T-cell-dependent protective immunity against T. cruzi infection.

Protective immunity against lethal infection is developed when BALB/c or C57BL/6 mice are immunized with plasmids containing genes from the protozoan parasite Trypanosoma cruzi. However, genetic vaccination of the highly susceptible mouse strain A/Sn promoted limited survival after challenge. This observation questioned whether this type of vaccination would be appropriate for highly susceptible individuals. Here, we compared the protective efficacy and the immune response after individual or combined genetic vaccination of A/Sn mice with genes encoding trans-sialidase (TS) or the amastigote surface protein-2 (ASP-2). After challenge, a significant proportion of A/Sn mice immunized with either the asp-2 gene or simultaneously with asp-2 and ts genes, survived infection. In contrast, the vast majority of mice immunized with the ts gene or the vector alone died. Parasitological and histological studies performed in the surviving mice revealed that these mice harbored parasites; however, minimal inflammatory responses were seen in heart and striated muscle. We used this model to search for an in vitro correlation for protection. We found that protective immunity correlated with a higher secretion of interferon- by spleen cells on in vitro restimulation with ASP-2 and the presence of ASP-2-specific CD8 cells. Depletion of either CD4 or CD8 or both T-cell subpopulations prior to the challenge rendered the mice susceptible to infection demonstrating the critical contribution of both cell types in protective immunity. Our results reinforce the prophylactic potential of genetic vaccination with asp-2 and ts genes by describing protective immunity against lethal T. cruzi infection and chronic tissue pathology in a highly susceptible mouse strain.

Obligatory intracellular parasites such as Plasmodium sp, Trypanosoma cruzi, Toxoplasma gondii and Leishmania sp are responsible for the infection of hundreds of millions of individuals every year. These parasites can deliver antigens to the host cell cytoplasm that are presented through MHC class I molecules to protective CD8 T cells. The in vivo priming conditions of specific CD8 T cells during natural infection are largely unknown and remain as an area that has been poorly explored. The antiparasitic mechanisms mediated by CD8 T cells include both interferon-gamma-dependent and -independent pathways. The fact that CD8 T cells are potent inhibitors of parasitic development prompted many investigators to explore whether induction of these T cells can be a feasible strategy for the development of effective subunit vaccines against these parasitic diseases. Studies performed on experimental models supported the hypothesis that CD8 T cells induced by recombinant viral vectors or DNA vaccines could serve as the basis for human vaccination. Regimens of immunization consisting of two different vectors (heterologous prime-boost) are much more efficient in terms of expansion of protective CD8 T lymphocytes than immunization with a single vector. The results obtained using experimental models have led to clinical vaccination trials that are currently underway.