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The current therapeutic options for Inflammatory Bowel Diseases (IBD) are limited. Even using common anti-inflammatory, immunosuppressive or biological therapies, many patients become unresponsive to the treatments, immunosuppressed or unable to restrain secondary infections. Statins are cholesterol-lowering drugs with non-canonical anti-inflammatory properties, whose underlying mechanisms of action still remain poorly understood. Here, we described that in vitro atorvastatin (ATO) treatment was not toxic to splenocytes, constrained cell proliferation and modulated IL-6 and IL-10 production in a dose-dependent manner. Mice exposed to dextran sulfate sodium (DSS) for colitis induction and treated with ATO shifted their immune response from Th17 towards Th2, improved the clinical and histological aspects of intestinal inflammation and reduced the number of circulating leukocytes. Both experimental and in silico analyses revealed that PPAR-α expression is reduced in experimental colitis, which was reversed by ATO treatment. While IBD patients also downregulate PPAR-α expression, the responsiveness to biological therapy relied on the restoration of PPAR-α levels. Indeed, the in vitro and in vivo effects induced by ATO treatment were abrogated in Ppara-/- mice or leukocytes. In conclusion, the beneficial effects of ATO in colitis are dependent on PPAR-α, which could also be a potential predictive biomarker of therapy responsiveness in IBD.

Synthetic biology has been advancing cellular and molecular biology studies through the design of synthetic circuits capable to examine diverse endogenously or exogenously driven regulatory pathways. While early genetic devices were engineered to be insulated from intracellular crosstalk, more recently the need of achieving dynamic control of cellular behavior has led to the development of smart interfaces that connect signal information (sensor) to desired output activation (actuator). Sensor-actuator circuits can respond to diverse inputs, including small molecules, exogenous and endogenous mRNA, noncoding RNA (i.e., miRNA), and proteins to regulate downstream events, transcriptionally, posttranscriptionally, and translationally. These devices require attentive engineering to either create complex chimeric proteins or modify protein structures to be amenable to the specific circuits' architecture and/or purpose.In this chapter, we describe how to implement two different protein-based devices in mammalian cells: (1) a modular platform that sense and respond to disease-associated proteins and (2) a protein-based system that allows simultaneous regulation of RNA translation and protein activity, via RNA-protein and newly engineered protein-protein interactions.

Synthetic biology has been advancing cellular and molecular biology studies through the design of synthetic circuits capable to examine diverse endogenously or exogenously driven regulatory pathways. While early genetic devices were engineered to be insulated from intracellular crosstalk, more recently the need of achieving dynamic control of cellular behavior has led to the development of smart interfaces that connect signal information (sensor) to desired output activation (actuator). Sensor-actuator circuits can respond to diverse inputs, including small molecules, exogenous and endogenous mRNA, noncoding RNA (i.e., miRNA), and proteins to regulate downstream events, transcriptionally, posttranscriptionally, and translationally. These devices require attentive engineering to either create complex chimeric proteins or modify protein structures to be amenable to the specific circuits' architecture and/or purpose.In this chapter, we describe how to implement two different protein-based devices in mammalian cells: (1) a modular platform that sense and respond to disease-associated proteins and (2) a protein-based system that allows simultaneous regulation of RNA translation and protein activity, via RNA-protein and newly engineered protein-protein interactions.

Envenomation by the larval or pupal stages of moths occurs when the victim presses their hairs. They penetrate the subcutaneous tissue, releasing toxins such as proteolytic enzymes, histamine and other pro-inflammatory substances. Cutaneous reactions, including severe pain, oedema and erythema are frequent local manifestations of caterpillar envenomation, but, in some cases, the reactions can evolve into vesicles, bullae, erosions, petechiae, superficial skin necrosis and ulcerations. Alternatively, some individual can develop allergic reactions, renal failure, osteochondritis, deformity and immobilization of the affected joints and intracerebral bleeding. Caterpillars produce venom to protect themselves from predators; contact with humans is accidental and deserves close attention. Their venoms have not been well studied, except for toxins from some few species. The present review brings together data on venomous caterpillars of moths, primarily addressing the available literature on diversity among the different families that cause accident in humans, the structures used in their defense, venom composition and clinical aspects of the envenomations. Understanding the molecular mechanisms of action of caterpillars' toxins may lead to the development of more adequate treatments.

Mutations in NOD2 predisposes to Inflammatory Bowel Diseases. Therefore, we evaluated the role of this innate receptor in the modulation of immunity in face of host microbiota changes. NOD2-/- mice presented higher susceptibility to experimental colitis than WT, with increased CD4 and CD8 T lymphocytes in the spleen. NOD2 deficiency also led to reduced Th17-related cytokines in the colon, with overall augmented IFN-γ in the gut and spleen. Nonetheless, there was increased frequency of CD4+IL-4+ cells in the mesenteric lymph nodes besides elevated CTLA-4 and FoxP3 regulatory markers in the spleen of NOD2-/- mice, although it did not result in more efficient control of gut inflammation. Indeed, these animals also had augmented IL-1β and IL-5 in the peritoneum, indicating that this receptor may be important to control bacteria translocation too. Microbiota exchanging between cohoused WT and NOD2-/- mice led to colitis worsening in the absence of the receptor, while antibiotic therapy in WT mice abrogated this effect. Then, not only the genetic mutation confers increased susceptibility to inflammation, but it is also influenced by the microbiota harbored by the host. Finally, NOD2-/- mice are more prone to intestinal inflammation due to deregulated immune response and increased susceptibility to colitogenic bacteria.

Sepsis is a severe syndrome that arises when the host response to an insult is exacerbated, leading to organ failure and frequently to death. How a chronic infection that causes a prolonged Th1 expansion affects the course of sepsis is unknown. In this study, we showed that mice chronically infected with Toxoplasma gondii were more susceptible to sepsis induced by cecal ligation and puncture (CLP). Although T. gondii-infected mice exhibited efficient control of the bacterial burden, they showed increased mortality compared to the control groups. Mechanistically, chronic T. gondii infection induces the suppression of Th2 lymphocytes via Gata3-repressive methylation and simultaneously induces long-lived IFN-γ-producing CD4+ T lymphocytes, which promotes systemic inflammation that is harmful during CLP. Chronic T. gondii infection intensifies local and systemic Th1 cytokines as well as nitric oxide production, which reduces systolic and diastolic arterial blood pressures after sepsis induction, thus predisposing the host to septic shock. Blockade of IFN-γ prevented arterial hypotension and prolonged the host lifespan by reducing the cytokine storm. Interestingly, these data mirrored our observation in septic patients, in which sepsis severity was positively correlated to increased levels of IFN-γ in patients who were serologically positive for T. gondii. Collectively, these data demonstrated that chronic infection with T. gondii is a critical factor for sepsis severity that needs to be considered when designing strategies to prevent and control the outcome of this devastating disease.

Crohn's disease (CD) and ulcerative colitis (UC) are intestinal disorders that comprise the inflammatory bowel diseases (IBD). These disorders have a significant effect on the quality of life of affected patients and the increasing number of IBD cases worldwide is a growing concern. Because of the overall burden of IBD and its multifactorial etiology, efforts have been made to improve the medical management of these inflammatory conditions. The classical therapeutic strategies aim to control the exacerbated host immune response with aminosalicylates, antibiotics, corticosteroids, thiopurines, methotrexate and anti-tumor necrosis factor (TNF) biological agents. Although successful in the treatment of several CD or UC conditions, these drugs have limited effectiveness, and variable responses may culminate in unpredictable outcomes. The ideal therapy should reduce inflammation without inducing immunosuppression, and remains a challenge to health care personnel. Recently, a number of additional approaches to IBD therapy, such as new target molecules for biological agents and cellular therapy, have shown promising results. A deeper understanding of IBD pathogenesis and the availability of novel therapies are needed to improve therapeutic success. This review describes the overall key features of therapies currently employed in clinical practice as well as novel and future alternative IBD treatment methods.

Inflammatory bowel disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC), is a chronic disorder that affects thousands of people around the world. These diseases are characterized by exacerbated uncontrolled intestinal inflammation that leads to poor quality of life in affected patients. Although the exact cause of IBD still remains unknown, compelling evidence suggests that the interplay among immune deregulation, environmental factors, and genetic polymorphisms contributes to the multifactorial nature of the disease. Therefore, in this review we present classical and novel findings regarding IBD etiopathogenesis. Considering the genetic causes of the diseases, alterations in about 100 genes or allelic variants, most of them in components of the immune system, have been related to IBD susceptibility. Dysbiosis of the intestinal microbiota also plays a role in the initiation or perpetuation of gut inflammation, which develops under altered or impaired immune responses. In this context, unbalanced innate and especially adaptive immunity has been considered one of the major contributing factors to IBD development, with the involvement of the Th1, Th2, and Th17 effector population in addition to impaired regulatory responses in CD or UC. Finally, an understanding of the interplay among pathogenic triggers of IBD will improve knowledge about the immunological mechanisms of gut inflammation, thus providing novel tools for IBD control.

Heme oxygenase-1 (HO-1) is an enzyme that catabolizes free heme, which induces an intense inflammatory response. The expression of HO-1 is induced by different stimuli, triggering an anti-inflammatory response during biological stress. It was previously verified that HO-1 is able to induce indoleamine 2,3-dioxygenase (IDO), an enzyme that is induced by IFN-γ in Toxoplasma gondii infection. To verify the role of HO-1 during in vivo T. gondii infection, BALB/c and C57BL/6 mice were infected with the ME49 strain and treated with zinc protoporphyrin IX (ZnPPIX) or hemin, which inhibit or induce HO-1 activity, respectively. The results show that T. gondii infection induced high levels of HO-1 expression in the lung of BALB/c and C57BL6 mice. The animals treated with ZnPPIX presented higher parasitism in the lungs of both lineages of mice, whereas hemin treatment decreased the parasite replication in this organ and in the small intestine of infected C57BL/6 mice. Furthermore, C57BL/6 mice infected with T. gondii and treated with hemin showed higher levels of IDO expression in the lungs and small intestine than uninfected mice. In conclusion, our data suggest that HO-1 activity is involved in the control of T. gondii in the lungs of both mouse lineages, whereas the hemin, a HO-1 inducer, seems to be involved in the control of parasitism in the small intestine of C57BL/6 mice.

Toxoplasma gondii é um protozoário intracelular obrigatório que causa a toxoplasmose. Em modelo experimental, camundongos C57BL/6 infectados por via oral com 100 cistos de T. gondii, cepa ME-49, desenvolvem sérias lesões intestinais similares as observadas em doenças inflamatórias intestinais. Ao invadir as células epiteliais intestinais, o parasito induz uma resposta inflamatória de padrão T helper (Th) 1 elevada, ativada pela produção de quimiocinas e citocinas envolvidas na migração e ativação celular. Para que ocorra essa migração celular para o sítio de infecção é necessário a presença de receptores de quimiocinas. O receptor de quimiocinas CCR5 é muito importante para o recrutamento celular em algumas infecções e está envolvido com a migração de vários subtipos celulares como células dendríticas, células T e, em particular, células T reguladoras. CCR5 pode estar relacionado também a mecanismos independentes da migração celular, no qual a sinalização intracelular e ativação de NF-B podem levar a intensificação da resposta imunológica. Ainda não está claro o papel do receptor CCR5 no modelo de infecção oral por T. gondii. Dessa forma, animais C57BL/6 e deficientes em CCR5 foram infectados por via oral com 5 cistos de T. gondii, cepa ME-49, e alguns parâmetros imunológicos e bioquímicos foram avaliados no 8º dia de infecção. Os resultados mostraram que animais CCR5-/- apresentaram alta suscetibilidade à infecção oral por T. gondii, exibindo um intenso infiltrado inflamatório no íleo e regiões de ulceração epitelial, quando comparados com animais C57BL/6. Independentemente de serem deficientes ou não de CCR5, os camundongos apresentaram focos inflamatórios dispersos pelo parênquima do fígado, entretanto camundongos CCR5-/- apresentaram uma extensiva vacuolização dos hepatócitos, com excessivo acúmulo de lipídeos no órgão e elevada concentração sérica de triglicérides e de transaminases. A carga parasitária foi significativamente mais elevada no intestino delgado e no fígado dos animais CCR5-/- em comparação com animais C57BL/6. Foi observada também uma menor migração de células NK no intestino delgado, bem como um aumento na frequência de células T CD4+ neste órgão e uma menor concentração de IFN- e IL-12p40 no macerado do fígado dos animais CCR5-/- em comparação com C57BL/6. Análise de expressão gênica no fígado revelou redução na formação de transcritos para PPAR nos animais deficientes em CCR5, e quando os camundongos foram tratados com Gemfibrozil, um agonista de PPAR, houve reversão na vacuolização hepática e na concentração de triglicérides no soro dos animais CCR5-/-. Estes dados sugerem que a migração celular dependente de CCR5 é essencial para a modulação da resposta inflamatória induzida por T. gondii no intestino delgado. Além do mais, a ausência de CCR5 compromete a integridade hepática durante a infecção oral por T. gondii e os mecanismos moleculares envolvidos podem estar relacionados à expressão de PPAR. T. gondii is an obligate intracellular protozoan parasite which is the causative agent of toxoplasmosis. In experimental model, C57BL/6 mice orally infected with a high parasitic load develop serious intestinal lesions, whose injuries are similar to those observed in Inflammatory Bowel Disease. This inflammation is caused due to parasite invasion of intestinal epithelial cells that elicit a robust Th1 type immune response. Moreover, chemokines produced by intestinal epithelial cells are involved in the migration and activation of inflammatory cells. In particular, the chemokine receptor CCR5 is important for cell recruitment in some infections and is involved with the migration of various cells subsets such as dendritic cells, T cells and, in particular regulatory T cells. CCR5 may also be related to mechanisms independent of cell migration, in which the intracellular signaling and activation of NF-B may lead to intensification of the immune response. The role of CCR5 has not been clear in the experimental oral T. gondii infection. Thus, wild type C57BL/6 mice and CCR5-/- littermates were infected with T. gondii by gavage and immune and biochemical parameters, were analyzed at day 8 after infection. The CCR5-/- mice showed to be highly susceptible to the parasite, with intense inflammatory infiltration in the ilea and regions of epithelial ulcerations in comparison with WT mice. Both strain of mice presented inflammatory foci scattered by parenchyma of the liver, however the CCR5-/- mice presented an extensive hepatocyte vacuolization with an excessive accumulation of lipids in the organ and elevated serum triglycerides and transaminases concentration. The parasite load was significantly higher on small intestine and liver samples of CCR5-/- in comparison with WT mice. There was also a minor migration of NK cells in the small intestine, as well as greater frequency of CD4+ T cells in this organ and a lower IFN- and IL-12p40 levels in liver homogenate samples in the CCR5-/- mice compared with WT mice. Gene expression analysis revealed a reduction in the formation of transcripts for PPAR in mice deficient in CCR5, and when the animals were treated with Gemfibrozil, a PPAR agonist, there was an improvement in the level of vacuolization and reduced triglycerides. These data suggest that a CCR5-dependent cell migration is essential for the modulation of T. gondii-induced inflammatory response in the small intestine. In addition, hepatic integrity during T. gondii oral infection is compromised in the absence of CCR5, and the molecular mechanisms involved can be related to PPAR expression.

CCR5, an important receptor related to cell recruitment and inflammation, is expressed during experimental Toxoplasma gondii infection. However, its role in the immunopathology of toxoplasmosis is not clearly defined yet. Thus, we inoculated WT and CCR5(-/-) mice with a sub lethal dose of the parasite by oral route. CCR5(-/-) mice were extremely susceptible to infection, presenting higher parasite load and lower tissue expression of IL-12p40, IFN-γ, TNF, IL-6, iNOS, Foxp3, T-bet, GATA-3 and PPARα. Although both groups presented inflammation in the liver with prominent neutrophil infiltration, CCR5(-/-) mice had extensive tissue damage with hepatocyte vacuolization, steatosis, elevated serum triglycerides and transaminases. PPARα agonist Gemfibrozil improved the vacuolization but did not rescue CCR5(-/-) infected mice from high serum triglycerides levels and enhanced mortality. We also found intense inflammation in the ileum of CCR5(-/-) infected mice, with epithelial ulceration, augmented CD4 and decreased frequency of NK cells in the gut lamina propria. Most interestingly, these findings were accompanied by an outstanding accumulation of neutrophils in the ileum, which seemed to be involved in the gut immunopathology, once the depletion of these cells was accompanied by reduced local damage. Altogether, these data demonstrated that CCR5 is essential to the control of T. gondii infection and to maintain the metabolic, hepatic and intestinal integrity. These findings add novel information on the disease pathogenesis and may be relevant for directing future approaches to the treatment of multi-deregulated diseases.

Introduction: Autoimmune diseases including type 1 diabetes (T1D) are thought to have a Th1/Th17 bias, but the underlying mechanisms driving the differentiation and activation of these pathogenic T cells has been investigated. Recently, the IL-1beta has been shown to regulate Th17 cell differentiation through of the expression of the transcription factor RORγt. Objective: For this reason, we addressed the respective IL-1 receptor (IL-1R) role in the TD1. Methods: IL-1R deficient mice and their wild-type (C57BL/6) were inoculated intraperitoneally with 40mg/Kg of streptozotocin for 5 consecutive days. Blood glucose levels and body weight were monitored weekly. The pancreatic lymph nodes (PLN) were removed to assess the Th17 and Th1 frequency by flow cytometry. Results: Diabetic IL-1R deficient mice developed lower hyperglycemia with only 50% of mice becoming diabetic. In agreement, mice lacking IL-1R have reduced inflammatory infiltrate (insulitis) and augmented insulin content into pancreatic islets by second week. In addition, the IL-1R deficiency caused a decrease in IL-17 and IFN-γ-producing CD4+ T cell population in pancreatic lymph nodes (PLN). The IL-17, IL-6, TNF-α, IFN-γ levels were not altered, but IL-10 and IL-4 levels were higher in pancreatic tissue of diabetic IL-1R deficient mice. Conclusion: These results suggest that IL-1receptor promotes the Th17/Th1 skewing in the PLN and possibly contributes to pancreatic islet damage during the T1D onset.

The Toxoplasma gondii infection induces intense intestinal inflammation and tissue damage in susceptible hosts. Here, we studied the regulation of immune response during intestinal mucosal inflammation induced by T. gondii, focusing on the role of Th17, Th22 and regulatory T cells (Tregs). C57BL/6-susceptible and BALB/c-resistant mice orally infected with T. gondii exhibited increased frequencies of Th17 and Th22 cells in Lamina Propria and Peyers patches during disease progression. However, whereas C57BL/6 mice exhibited higher frequency of Th17, BALB/c mice showed higher counts of Treg and Th22 cells. The infection also impaired the effector function of Treg cells in susceptible hosts, since Tregs obtained from C57BL/6-infected mice showed a reduced suppressive activity in vitro. The transfer of Foxp3+ cells sorted from non-infected mice, but not from infected mice, increased mice survival. In parallel, we evaluated the role of IL-17 and IL-22 in the disease and we observed that IL-17R-/- and IL-22-/- mice were more susceptible, presented higher parasite burden and intestinal inflammation and lower Treg frequency than C57BL/6. Moreover, IL-17R-/- showed a significant reduction of Th22 cells compared to C57BL/6 mice. Taken together, these findings suggest that IL-17 and IL-22 production are essential for resistance against T. gondii infection, by controlling parasite burden and along with IL-22, Tregs are important for controlling the intestinal inflammation.

Introduction: The majority of studies regarding host-pathogen interactions have focused on a single pathogen. However, host organisms in a natural environment are commonly exposed to multiple pathogens, such as T. gondii infection, which induces a strong immune response by Th1 pathway, that can persists throughout the life of the host. Therefore, we hypothesized that chronic infection with T. gondii could alter the course of host response against bacterial infection. Methodology: C57BL/6 mice were orally infected with 5 cysts of ME-49 strain of T. gondii and 40 days post infection, subjected infection by cecal ligation and puncture (CLP) sepsis model. Results: Our data showed that, 12 and 24 hours after of CLP, the group of mice infected with T. gondii exhibited decreased bacterial count, increased inflammation characterized by an increase in myeloperoxidase content in lung and increase in IFN-γ and TNF cytokines levels, as compared mice subjected to the same stimulus infectious without previous T. gondii infection. Moreover, after CLP, these animals showed a significantly increase in mortality rate, which was reduced in mice TLR-2 knockout or treated with low concentrations of anti-IFN-γ or anti-TNF antibodies. Conclusion: This study demonstrates that T. gondii infection potentiates the host response against polymicrobial infection, which can be harmful to the host. The mechanism by which this happens seems to be mediated via the overproduction of IFN-γ and TNF.