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Although some glycolytic intermediates have been shown to modulate several cell type formation and activation, the functional role of fructose 1,6-bisphosphate (FBP) on osteoclastogenesis is still unknown. Osteoclastogenesis was evaluated on bone marrow preosteoclasts cultured with M-CSF − 30 ng/ml, RANKL − 10 ng/ml, and two concentrations of FBP (100 and 300 µM). TRAP-positive stained cells were counted, and osteoclastogenic marker genes expression were evaluated by qPCR. Osteoclasts resorption capacity was evaluated by the expression of specific enzymes and capacity to resorb a mineralized matrix. The NF-κB activation was detected using RAW 264.7, stably expressing luciferase on the NF-κB responsive promoter. We show that FBP, the product of the first stage of glycolysis, inhibited RANKL-induced osteoclasts differentiation and TRAP activity. The treatment of preosteoclasts with FBP attenuated osteoclast fusion and formation, without affecting cell viability. Moreover, the inhibition of several osteoclastogenic marker genes expression (TRAP, OSCAR, DC-STAMP, Integrin αv, NFATc1) by FBP correlates with a reduction of mineralized matrix resorption capacity. The mechanism underlying FBP-inhibition of osteoclastogenesis involves NF-κB/NFATc1 signaling pathway inhibition. Altogether these data show a protective role of a natural glycolytic intermediate in bone homeostasis that may have therapeutic benefit for osteolytic diseases.

Evidências crescentes de que os processos metabólicos desempenham um papel no controle das funções efetoras imunes, nos fazem compreender melhor os papéis e funções das vias metabólicas nas respostas imunes, assim possibilitar o surgimento de uma oportunidade terapêutica baseada no imunometabolismo. De forma interessante, a frutose 1,6-bifosfato (FBP) é um intermediário endógeno da via glicolítica que tem efeitos anti-inflamatórios quando administrado em modelo de isquemia ou artrite. Embora o mecanismo não seja totalmente claro, esse efeito protetor já foi descrito por aumentar os níveis de adenosina (ADO) como resultado de uma produção elevada de ATP proveniente da glicólise. Além disso, o aumento da glicólise é um mecanismo chave nas células ativadas por LPS, pode ocorrer pela indução da isoenzima piruvato quinase M2 (PKM2), regulando o fluxo glicolítico. Entretanto, não foi demonstrado como o PKM2 participa dessa reprogramação metabólica e modula a resposta inflamatória de macrófagos. Nossa hipótese é que a reprogramação metabólica por FBP ou ativação alostérica da PKM2 pode modular a resposta inflamatória de macrófagos. Demonstramos em várias condições experimentais que a FBP induziu uma resposta anti-inflamatória mediada por IL-10, através do aumento do metabolismo glicolítico em macrófagos. Confirmamos por metaboloma e outros ensaios que o tratamento com FBP aumenta o metabolismo celular, aumentando a produção e liberação de ATP que será transformado em adenosina pela via de CD39/CD73. Usando macrófagos deficientes para o receptor de adenosina (A2A KO), confirmamos que a produção de IL-10 induzida por FBP ocorre via sinalização de adenosina/A2A. Além disso, o efeito antiinflamatório gerado pela FBP foi confirmado em modelos experimentais de peritonite e colite. No entanto, ao conhecer os efeitos da FBP e sabendo que ela é o ativador alostérico natural da PKM2, avaliamos se o aumento da produção de IL-10 pela FBP ocorre de forma dependente de PKM2. Porém, demonstramos que os efeitos da FBP não eram dependentes da presença de PKM2. Mesmo assim, avaliamos se a ativação alostérica de PKM2 por TEPP-46 geraria uma resposta anti-inflamatória. Observamos em BMDM que o tratamento com TEPP-46 também foi capaz de aumentar a produção de IL-10, e essa resposta foi gerada por aumentar o metabolismo celular, elevando a produção de ATP glicolítico e assim, os níveis de adenosina pela ação de CD39/CD73 na via adenosinérgica. Em modelo experimental de peritonite, observamos que o tratamento com o TEPP-46 reduz parâmetros inflamatório dependentes da sinalização de adenosina pelo A2A. Juntos, esses dados demostraram que a via glicolítica é essencial para o efeito anti-inflamatório da FBP e do TEPP-46, levando ao surgimento de novas estratégias terapêuticas baseadas na modulação do imunometabolismo. Growing evidence that metabolic processes play a role in control of immune effector functions leads to emergence of a therapeutic opportunity based on immunometabolism, when understand roles and functions of metabolic pathways in immune responses. Of interest, fructose 1,6-bisphosphate (FBP) is an endogenous intermediate of glycolytic pathway that has anti-inflammatory effects when administrated in ischemic or arthritis model. Although the mechanism is not fully understood, this protective effect is attributed to increased adenosine (ADO) levels as a result of enhance in ATP production by glycolysis. Besides that, increased glycolysis is key mechanism in LPS-activated cells, which may occur by inducing isoenzyme pyruvate kinase M2 (PKM2), regulating the glycolytic flux. How ever, has not been demonstrated how PKM2 can participate that metabolic reprogramming modulate the macrophage inflammatory response. We hypothesized that metabolic reprogramming by FBP or PKM2 allosteric activation could modulate the macrophage inflammatory response. Here we demonstrated in several experimental conditions that FBP induces an anti-inflammatory response mediated by IL-10 via increase of the glycolytic metabolism in macrophages. We confirmed by metabolomics and others assays that FBP treatment enhances the cellular metabolism, increasing the production and release of ATP that will be turned into adenosine by CD39/CD73 pathway. Using macrophages knockout to adenosine receptor (A2A KO) we confirmed that the IL-10 production induced by FBP occur via adenosine/A2A signaling. Furthermore, the regulatory immunometabolic effect of FBP was confirmed in the peritonitis and colitis model. Knowing the FBP effects and that it is the natural allosteric activator of PKM2, we evaluated if increased of IL-10 production by FBP occurs by PKM2-depend. Nonetheless, we demonstrated that FBP effects were not dependent the PKM2 presence. Even so, we evaluated whether the allosteric activation of PKM2 by TEPP-46 generated an antiinflammatory response. We observed in BMDM that TEPP-46 was also able to increase the production of IL-10, and this response was generated by increased cellular metabolism, enhance glycolytic ATP production and adenosine level by CD39/CD73 action in adenosinergic pathway, reducing the inflammatory response in peritonitis level. Together, these data showed that the IL-10-dependent glycolytic pathway is essential for the antiinflammatory effect of FBP and allosteric activation of PKM2 by TEPP-46, leading to the emergence of new therapeutic strategies based on immunometabolism modulation.

Sepsis results in elevated adenosine in circulation. Extracellular adenosine triggers immunosuppressive signaling via the A2a receptor (A2aR). Sepsis survivors develop persistent immunosuppression with increased risk of recurrent infections. We utilized the cecal ligation and puncture (CLP) model of sepsis and subsequent infection to assess the role of adenosine in post-sepsis immune suppression. A2aR-deficient mice showed improved resistance to post-sepsis infections. Sepsis expanded a subset of CD39hi B cells and elevated extracellular adenosine, which was absent in mice lacking CD39-expressing B cells. Sepsis-surviving B cell-deficient mice were more resistant to secondary infections. Mechanistically, metabolic reprogramming of septic B cells increased production of ATP, which was converted into adenosine by CD39 on plasmablasts. Adenosine signaling via A2aR impaired macrophage bactericidal activity and enhanced interleukin-10 production. Septic individuals exhibited expanded CD39hi plasmablasts and adenosine accumulation. Our study reveals CD39hi plasmablasts and adenosine as important drivers of sepsis-induced immunosuppression with relevance in human disease.

Sepsis results in elevated adenosine in circulation. Extracellular adenosine triggers immunosuppressive signaling via the A2a receptor (A2aR). Sepsis survivors develop persistent immunosuppression with increased risk of recurrent infections. We utilized the cecal ligation and puncture (CLP) model of sepsis and subsequent infection to assess the role of adenosine in post-sepsis immune suppression. A2aR-deficient mice showed improved resistance to post-sepsis infections. Sepsis expanded a subset of CD39

BackgroundPrevious data have reported that the growth of established tumors may be facilitated by postsepsis disorder through changes in the microenvironment and immune dysfunction. However, the influence of postsepsis disorder in initial carcinogenesis remains elusive.MethodsIn the present work, the effect of postsepsis on inflammation-induced early carcinogenesis was evaluated in an experimental model of colitis-associated colorectal cancer (CAC). We also analyzed the frequency and role of intestinal T regulatory cells (Treg) in CAC carcinogenesis.ResultsThe colitis grade and the tumor development rate were evaluated postmortem or in vivo through serial colonoscopies. Sepsis-surviving mice (SSM) presented with a lower colonic DNA damage, polyp incidence, reduced tumor load, and milder colitis than their sham-operated counterparts. Ablating Treg led to restoration of the ability to develop colitis and tumor polyps in the SSM, in a similar fashion to that in the sham-operated mice. On the other hand, the growth of subcutaneously inoculated MC38luc colorectal cancer cells or previously established chemical CAC tumors was increased in SSM.ConclusionOur results provide evidence that postsepsis disorder has a dual effect in cancer development, inhibiting inflammation-induced early carcinogenesis in a Treg-dependent manner, while increasing the growth of previously established tumors.

International audience; Macrophages play a critical role in the regulation of immune responses. They are highly plastic cells, responding to diverse environmental stimuli to acquire different functional phenotypes. Intracellular signaling through mitogen-activated protein kinases (MAPKs) has been reported to regulate the differentiation of macrophages, but the role of the atypical MAPK ERK5 signaling in IL-4-mediated M2 macrophage differentiation is still unclear. Here, we showed that the ERK5 signaling pathway plays a critical role in IL-4-induced M2 macrophage differentiation. We found that pharmacological inhibition of MEK5, an immediate upstream activator of ERK5, with BIX markedly reduced the expression of classical M2 markers, such as Arg-1, Ym-1, and Fizz-1, as well as the production of M2-related chemokines and cytokines, CCL22, CCL17 and IGF-1 in IL-4-stimulated macrophages. Moreover, pharmacological inhibition of ERK5 with XMD8-92 also decreased the expression of several M2 markers induced by IL-4. In accordance, myeloid cell-specific Erk5 depletion (ERK5 ∆mye), using LysM cre /Erk5 f/f mice, confirmed the involvement of ERK5 signaling in IL-4-induced M2 polarization. Mechanistically, we found that pharmacological inhibition of ERK5 did not affect STAT6 phosphorylation, suggesting that ERK5 signaling regulates M2 differentiation in a STAT6-independent manner. However, we found that genetic deficiency or pharmacological inhibition of the MEK5/ERK5 pathway blocked the expression of c-Myc in IL-4-activated macrophages, which is a critical transcription factor involved in M2 differentiation. Taken together, our results reveal that activation of the MEK5/ERK5 signaling pathway is crucial in IL-4-induced M2 macrophage differentiation through the induction of c-Myc expression.

Paclitaxel is an antineoplastic agent widely used to treat several solid tumor types. The primary mechanism of action of paclitaxel is based on microtubule stabilization inducing cell-cycle arrest. Here, we use several tumor models to show that paclitaxel not only induces tumor cell-cycle arrest, but also promotes antitumor immunity. In vitro, paclitaxel reprogrammed M2-polarized macrophages to the M1-like phenotype in a TLR4-dependent manner, similarly to LPS. Paclitaxel also modulated the tumor-associated macrophage (TAM) profile in mouse models of breast and melanoma tumors; gene expression analysis showed that paclitaxel altered the M2-like signature of TAMs toward an M1-like profile. In mice selectively lacking TLR4 on myeloid cells, for example, macrophages (LysM-Cre+/−/TLR4fl/fl), the antitumor effect of paclitaxel was attenuated. Gene expression analysis of tumor samples from patients with ovarian cancer before and after treatment with paclitaxel detected an enrichment of genes linked to the M1 macrophage activation profile (IFNγ-stimulated macrophages). These findings indicate that paclitaxel skews TAMs toward an immunocompetent profile via TLR4, which might contribute to the antitumor effect of paclitaxel and provide a rationale for new combination regimens comprising paclitaxel and immunotherapies as an anticancer treatment. Significance: This study provides new evidence that the antitumor effect of paclitaxel occurs in part via reactivation of the immune response against cancer, guiding tumor-associated macrophages toward the M1-like antitumor phenotype. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/20/5891/F1.large.jpg. Cancer Res; 78(20); 5891–900. ©2018 AACR. See related commentary by Garassino et al., p. 5729

The mechanisms of muscle wasting and decreased mobility have a major functional effect in rheumatoid arthritis, but they have been poorly studied. The objective of our study is to describe muscular involvement and the pathways in an experimental model of arthritis compared to the pathways in disuse atrophy. Female Wistar rats were separated into three groups: control (CO), collagen-induced arthritis (CIA), and immobilized (IM). Spontaneous locomotion and weight were evaluated weekly. The gastrocnemius muscle was evaluated by histology and immunoblotting to measure the expression of myostatin (a negative regulator), LC3 (autophagy), MuRF-1 (proteasome-mediated proteolysis), MyoD, and myogenin (satellite-cell activation). The significance level was set at P

Background Rheumatoid arthritis (RA) is an inflammatory autoimmune disease of unknown etiology, affecting mainly the joint but also other tissues. RA patients usually present weakness and muscle atrophy, nonarticular manifestations of the disease. Although causing great impact, the understanding of muscle atrophy, its development, and the mechanisms involved is still very limited. The objective of this study is to evaluate the development of muscle atrophy in skeletal muscle of a murine model of arthritis. Methods The experimental murine model of collagen-induced arthritis (CIA) was used. DBA/1J mice were randomly divided into three groups: control (CO, n = 25), sham arthritis (SA, n = 25), and arthritis (CIA, n = 28), analyzed in different time points: 25, 35, and 45 days after the induction of arthritis. The arthritis development was followed by clinical scores and hind paw edema three times a week. The spontaneous exploratory locomotion and weight were evaluated weekly. In all time points, serum was collected before the death of the animals for cytokine analysis, and myofiber cross-sectional areas (CSA) of gastrocnemius (GA) and tibialis anterior (TA) skeletal muscles were evaluated. Results The clinical parameters of arthritis progressively increased in CIA in all experimental times, demonstrating the greatest difference from other groups at 45 days after induction (clinical score: CO, 00 ± 00; SA, 1.00 ± 0.14; CIA, 3.28 ± 0.41 p > 0.05). The CIA animals had lower weights during all the experimentation periods with a difference of 6 % from CO at 45 days (p > 0.05). CIA animals also demonstrated progressive decrease in distance walked, with a reduction of 54 % in 35 and 74 % at 45 days. Cytokine analysis identified significant increase in IL-6 serum levels in CIA than CO and SA in all experimental times. CSA of the myofiber of GA and TA was decreased 26 and 31 % (p > 0.05) in CIA in 45 days after the induction of disease, respectively. There was significant and inverse correlation between the disease clinical score and myofiber CSA in 45 days (GA: r = −0.71; p = 0.021). Conclusion Our results point to a progressive development of muscle wasting, with premature onset arthritis. These observations are relevant to understand the development of muscle loss, as well as for the design of future studies trying to understand the mechanisms involved in muscle wasting. As far as we are concerned, this is the first study to evaluate the relation between disease score and muscle atrophy in a model of arthritis. Electronic supplementary material The online version of this article (doi:10.1007/s13539-013-0102-1) contains supplementary material, which is available to authorized users.

Introduction: Inflammation influences colorectal carcinogenesis and patients with inflammatory bowel disease are more prone to develop colorectal cancer (CRC). Varipous evidence points towards sepsis-surviving individuals experiencing a prolonged state of immunosuppression. The aim of this study was to evaluate if colitis-induced CRC initiation and progression was affected in sepsis-surviving mice. Experimental Procedures: C57Bl6 mice were submitted to cecal ligation and puncture (CLP) model, and after 15 days, sepsis-surviving mice and one control group received azoxymethane (AOM, 10 mg/kg, ip) and dextran sodium sulfate (DSS 2%, ad libitum, 3 cycles of 5 days) on days 20-25, 40-45, and 60-65 post-CLP. Colitis and tumor development were evaluated by serial colonoscopies. Cytokines (IFN-γ, IL-1β, TNF, IL-6, IL-17, TGF), chemokines (KC and MIG), and GM-CSF were quantified on days 0, 12, 52, and 65 by Milliplex and Treg cells on days 12 and 65 by flow cytometry. Two groups of C57 animals received cyclophosphamide (CYP) (100 mg/kg, ip), and two groups of animals expressing diphtheria toxin (DTX) receptor under-regulation of Foxp3 received DTX (0.5 mg/animal, ip) before the AOM/DSS protocol. Another animal cohort was submitted to CLP after receiving AOM/DSS, and two other groups were injected with murine CRC cells (MC38luc) 105 cells s.c. or 106 cells intra-splenically 15 days post-CLP. Results: Sepsis-surviving animals showed reduced colitis and lower incidence, number of tumors, and tumor load than control animals. Lower levels of IFN-γ in the colon on day 12, and of IL-1β, TNF, IL-6, IL-17, KC, and MIG on days 12 and 65, in addition to IL-33, TG,F and GM-CSF on day 65, were detected in sepsis-surviving mice. Tregs accumulated in a more pronounced fashion in sepsis-surviving mice on D15. Treg depletion through CYP or DTX restored colitis and CRC incidence in post-sepsis mice. Conversely, mice submitted to sepsis after AOM/DSS showed an increase in tumor load 30 days after CLP when compared to controls. Furthermore, in the subcutaneous and intrasplenic colorectal cancer model, increased tumor growth was observed in post-sepsis animals when compared to the controls. Conclusion: Colitis-dependent CRC carcinogenesis was reduced by post-sepsis state in mice in a Treg-dependent manner. Citation Format: Caio Abner Vitorino Goncalves Leite, Jose Mauricio Segundo Correia Mota, Kalil Alves Lima, Leticia Almeida Nascimento, Marcela Davoli Ferreira, Paula Ramos Viacava, Maria Dirlei Begnami, Jose Carlos Alves Filho, Roberto Cesar Pereira Lima Jr., Vladmir Claudio Cordeiro Lima, Fernando Queiroz Cunha. Colorectal carcinogenesis is precluded by intestinal Treg cell accumulation in post-sepsis state [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A70.

Reproduction alters the male physiology. We performed a comprehensive study to examine oxidative stress in the brains of male rats with (experienced) or without (naive) reproductive activity during aging. Oxidative stress was assessed by measuring the activity of catalase, glutathione peroxidase, superoxide dismutase, glutathione S-transferase, aconitase, and aconitase reactivated, and by measuring lipid peroxidation, protein carbonylation, nitrite and nitrate levels, vitamin C levels, and glutathione (total, reduced, oxidized forms) levels in brain tissue, as well as testosterone and estradiol levels in serum. Reproductively active animals exhibited increased testosterone levels and aconitase activity, suggesting an increased metabolism. Increased antioxidant enzyme activities and increased levels of antioxidant compounds were observed, yet damage to biomolecules was also observed in experienced rats. During aging changes in oxidative stress were observed. We found higher activities of antioxidant enzymes, higher amounts of antioxidants, and more damage at six months of age among experienced animals than among naive animals. Similar antioxidant activities and levels, and damage were found between the groups at twenty-four months of age. These results add comprehensive data regarding changes in oxidative stress during aging, and suggest an explanation for the costs of reproduction.

Background Rheumatoid arthritis (RA) is an inflammatory autoimmune disease of unknown etiology, affecting the joint and also other tissues [Altindag, 2007]. RA patients usually present weakness and muscle atrophy, non-articular manifestation of the disease [Mirό, 1996]. Although causing great impact, the development of muscle atrophy and mechanisms involved is still very limited. Objectives To evaluate the development of muscle atrophy in skeletal muscle of murine model of arthritis. Methods We used the experimental murine model of collagen induced arthritis (CIA) conform Brand et al [2007]. Experimental animals were randomly divided into three groups (n=79): control (CO), sham arthritis (SA) and arthritis (CIA), analyzed in different time-points: 25, 35 and 45 days after induction of the arthritis. The arthritis development was followed by clinical scores three times a week and weight was evaluated weekly. Cytokine analysis was evaluated by CBA in serum collected before the death of the animals. Cross-sectional area of the myofiberof skeletal muscle (tibialis anterior - TA, gastrocnemius - GA) was measured. Significance was considered p

Background Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory disease of unknown etiology, affecting not only the joints but also other tissues such as skeletal muscle [1]. Muscle loss is a secondary manifestation that impairs the function and quality of life of patients with RA [1]. Physical exercise is associated with reduced morbidity and other beneficial effects inseveral diseases [2, 3]. Objectives To evaluate the effect of aerobic exercise on muscle loss of animals with collagen-induced arthritis (CIA). Methods CIA was inducedin male mice DBA/1J asdescribed by Brand, 2010 [4]. The animals were divided into two CIA groups: (i) animals with exercise (protocol: walking for 30 min, from 9 to 15 m.min -1 , three times a week for 4 weeks after the onset of the disease), (ii) animals without exercise. Clinical score and paw swelling were assessed daily during all the experimental period. The weight of the animals and spontaneous exploratory locomotion were evaluated weekly. After death, the myofiber area of the tibialis anterior and gastrocnemius muscles were evaluated as well as ankle joint histopathology. For statistical analysis, t-test were performed and statistical significance was considered for p values p Results No significant difference was observed in clinical parameters of disease activity (clinical score, paw swelling and animal weight) and spontaneous locomotion in the walking group compared with the group without exercise. Although not significant, exercise slightly decreased joint inflammation (1.6 ± 0.17 and 1.9 ± 0.20 arbitrary value, p=.16) and bone erosion (1.3 ± 0.26 and 1.8 ± 0.23 arbitrary value, p=.12), besides that, cartilage erosion were significantly reduced in exercised joint (1.3 ± 0.26 and 1.8 ± 0.23 arbitrary value, p 2 and 481 ± 63 μm 2 , respectively, p Conclusions This is the first intervention study with forced walk in the experimental model CIA. According to results, aerobic exercise applied from the first clinical signs showed benefit over cartilage erosion and muscle atrophy in an experimental model of arthritis, observed mainly by the reduction of myofiber area, but does not seem to impact in the process of arthritis. References McInnes IB, Schett G. New England Journal of Medicine 2011, 365 (23). Navarro F et al. Cell Biochemistry and Function 2010, 28 (4):266-273. Nader GA, Lundberg IE. Current Opinion in Rheumatology 2009, 21 (6):599-603. Brand DD et al. Nature Protocols 2007, 2 (5):1269-1275. Disclosure of Interest None Declared