Leukocyte dysfunction and reduced CTLA-4 expression are associated with perianal Crohn's disease.

Although perianal Crohn's disease (PCD) is highly associated with the exacerbated inflammation, the molecular basis and immunological signature that distinguish patients who present a history of perianal lesions are still unclear. This paper aims to define immunological characteristics related to PCD. In this cross-sectional observational study, we enrolled 20 healthy controls and 39 CD patients. Blood samples were obtained for the detection of plasma cytokines and lipopolysaccharides (LPS). Peripheral blood mononuclear cells (PBMCs) were phenotyped by flow cytometry. Leukocytes were stimulated with LPS or anti-CD3/anti-CD28 antibodies. Our results show that CD patients had augmented plasma interleukin (IL)-6 and LPS. However, their PBMC was characterized by decreased IL-6 production, while patients with a history of PCD produced higher IL-6, IL-8, and interferon-γ, along with decreased tumor necrosis factor alpha (TNF). CD patients had augmented FoxP3 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) regulatory markers, though the PCD subjects presented a significant reduction in CTLA-4 expression. CTLA-4 as well as IL-6 and TNF responses were able to distinguish the PCD patients from those who did not present perianal complications. In conclusion, IL-6, TNF, and CTLA-4 exhibit a distinct expression pattern in CD patients with a history of PCD, regardless of disease activity. These findings clarify some mechanisms involved in the development of the perianal manifestations and may have a great impact on the disease management.

The Microbiota-Dependent Worsening Effects of Melatonin on Gut Inflammation.

Dysbiosis and disturbances in gut homeostasis may result in dysregulated responses, which are common in inflammatory bowel diseases (IBD). These conditions may be refractory to the usual treatments and novel therapies are still necessary to reach a more successful regulation of intestinal immunity. The hormone melatonin (MLT) has been raised as a therapeutic alternative because of its known interactions with immune responses and gut microbiota. Hence, we evaluated the effects of MLT in experimental colitis that evolves with intestinal dysbiosis, inflammation and bacterial translocation. C57BL/6 mice were exposed to dextran sulfate sodium and treated with MLT. In acute colitis, the hormone led to increased clinical, systemic and intestinal inflammatory parameters. During remission, continued MLT administration delayed recovery, increased TNF, memory effector lymphocytes and diminished spleen regulatory cells. MLT treatment reduced Bacteroidetes and augmented Actinobacteria and Verrucomicrobia phyla in mice feces. Microbiota depletion resulted in a remarkable reversion of the colitis phenotype after MLT administration, including a counter-regulatory immune response, reduction in TNF and colon macrophages. There was a decrease in Actinobacteria, Firmicutes and, most strikingly, Verrucomicrobia phylum in recovering mice. Finally, these results pointed to a gut-microbiota-dependent effect of MLT in the potentiation of intestinal inflammation.

Peroxisome Proliferator-Activated Receptor Alpha Mediates the Beneficial Effects of Atorvastatin in Experimental Colitis.

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.

Microbiota Modulation of the Gut-Lung Axis in COVID-19.

COVID-19 is an infectious disease caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), and according to the World Health Organization (WHO), to date, SARS-CoV-2 has already infected more than 91.8 million people worldwide with 1,986,871 deaths. This virus affects mainly the respiratory system, but the gastrointestinal tract (GIT) is also a target, meanwhile SARS-CoV-2 was already detected in oesophagus, stomach, duodenum, rectum, and in fecal samples from COVID-19 patients. Prolonged GIT manifestations in COVID-19, mainly the diarrhea, were correlated with decreased richness and diversity of the gut microbiota, immune deregulation and delayed SARS-CoV-2 clearance. So, the bidirectional interactions between the respiratory mucosa and the gut microbiota, known as gut-lung axis, are supposed to be involved in the healthy or pathologic immune responses to SARS-CoV-2. In accordance, the intestinal dysbiosis is associated with increased mortality in other respiratory infections, due to an exacerbated inflammation and decreased regulatory or anti-inflammatory mechanisms in the lungs and in the gut, pointing to this important relationship between both mucosal compartments. Therefore, since the mucous membranes from the respiratory and gastrointestinal tracts are affected, in addition to dysbiosis and inflammation, it is plausible to assume that adjunctive therapies based on the modulation of the gut microbiota and re-establishment of eubiosis conditions could be an important therapeutic approach for constraining the harmful consequences of COVID-19. Then, in this review, we summarized studies showing the persistence of SARS-CoV-2 in the gastrointestinal system and the related digestive COVID-19 manifestations, in addition to the literature demonstrating nasopharyngeal, pulmonary and intestinal dysbiosis in COVID-19 patients. Lastly, we showed the potential beneficial role of probiotic administration in other respiratory infections, and discuss the possible role of probiotics as an adjunctive therapy in SARS-CoV-2 infection.

The influence of dehydroepiandrosterone on effector functions of neutrophils

Dehydroepiandrosterone (DHEA) is a steroid hormone secreted by the adrenal glands, gonads and brain. It is a precursor to sex hormones and also is known to have immune modulatory activity. However, little is known about the relationship between DHEA and neutrophils and thus our study evaluates the influence of DHEA in the effector functions of neutrophils. Human neutrophils were treated in vitro with DHEA and further infected with Salmonella enterica serovar Typhimurium. The treatment of neutrophils with 0.01 µM of DHEA increased the phagocytosis of Salmonella independent of TLR4 as the treatment did not modulate the TLR4 expression. Additionally, DHEA caused a decrease in ROS (reactive oxygen species) production and did not influence the formation of the neutrophil extracellular trap (NET). Steroid treated neutrophils, infected or stimulated with LPS (lipopolysaccharide), showed reduced production of IL-8, compared to untreated cells. Also, the protein levels of p-NFκB were decreased in neutrophils treated with DHEA, and this reduction could explain the reduced levels of IL-8. These results led us to conclude that the steroid hormone DHEA has important modulatory functions in neutrophils

The Aryl Hydrocarbon Receptor (AHR) as a Potential Target for the Control of Intestinal Inflammation: Insights from an Immune and Bacteria Sensor Receptor.

The aryl hydrocarbon receptor (AHR) is widely expressed in immune and non-immune cells of the gut and its activation has been correlated to the outcome of inflammatory bowel diseases (IBD). In ulcerative colitis and Crohn's disease, there is an excessive chronic inflammation with massive accumulation of leukocytes in the gut, in an attempt to constrain the invasion of pathogenic microorganisms on the damaged organ. Accordingly, it is known that dietary components, xenobiotics, and some chemicals or metabolites can activate AHR and induce the modulation of inflammatory responses. In fact, the AHR triggering by specific ligands during inflammatory conditions results in decreased IFNγ, IL-6, IL-12, TNF, IL-7, and IL-17, along with reduced microbial translocation and fibrosis in the gut. Moreover, upon AHR activation, there are increased regulatory mechanisms such as IL-10, IL-22, prostaglandin E2, and Foxp3, besides the production of anti-microbial peptides and epithelial repair. Most interestingly, commensal bacteria or their metabolites may also activate this receptor, thus contributing to the restoration of gut normobiosis and homeostasis. In line with that, Lactobacillus reuteri, Lactobacillus bulgaricus, or microbial products such as tryptophan metabolites, indole-3-pyruvic acid, urolithin A, short-chain fatty acids, dihydroxyquinoline, and others may regulate the inflammation by mechanisms dependent on AHR activation. Hence, here we discussed the potential modulatory role of AHR on intestinal inflammation, focused on the reestablishment of homeostasis through the receptor triggering by microbial metabolites. Finally, the development of AHR-based therapies derived from bacteria products could represent an important future alternative for controlling IBD.

COVID-19: Integrating the Complexity of Systemic and Pulmonary Immunopathology to Identify Biomarkers for Different Outcomes.

In the last few months, the coronavirus disease 2019 (COVID-19) pandemic has affected millions of people worldwide and has provoked an exceptional effort from the scientific community to understand the disease. Clinical evidence suggests that severe COVID-19 is associated with both dysregulation of damage tolerance caused by pulmonary immunopathology and high viral load. In this review article, we describe and discuss clinical studies that show advances in the understanding of mild and severe illness and we highlight major points that are critical for improving the comprehension of different clinical outcomes. The understanding of pulmonary immunopathology will contribute to the identification of biomarkers in an attempt to classify mild, moderate, severe and critical COVID-19 illness. The interface of pulmonary immunopathology and the identification of biomarkers are critical for the development of new therapeutic strategies aimed to reduce the systemic and pulmonary hyperinflammation in severe COVID-19.

Reappraisal of antibodies against Saccharomyces cerevisiae (ASCA) as persistent biomarkers in quiescent Crohn's disease.

A clear correlation exists between microbiota and the dysregulation of the immune response in Inflammatory Bowel Diseases (IBD), which comprise Crohn's disease (CD) and ulcerative colitis (UC). These unbalanced reactions also involve humoral responses, with antibodies against Saccharomyces cerevisiae. Thus, here we aimed to quantify IgA and IgG specific to S. cerevisiae (ASCA) in quiescent CD and UC, to correlate the production of these antibodies with patient's inflammatory response and disease clinical presentation. Twenty-nine subjects (16 CD and 13 UC) and 45 healthy controls were enrolled in this study and had plasma samples tested for ASCA and cytokines (IL-2, IL-4, IL-6, IL-10, IFN-γ, TNF-α), besides clinical evaluation. IBD patients had increase IgA and IgG ASCA, especially those with colonic (L2) and fistulizing (B3) CD. Similarly, patients who dropped out the treatment had augmented ASCA, while IgG was reduced in those receiving sulfasalazine treatment. Furthermore, the quiescent CD patients had elevated IL-6 on plasma, especially in the absence of treatment, together with increased counter regulatory response of IL-10. There was a positive correlation between IgA and IgG on CD but not UC, as well as between IgA and TNF in total IBD patients. In addition, the levels of IgG x TNF, IgA x IL-10 and IgG x IL-10 were also correlated in CD, indicating that ASCA production may be influenced by the inflammatory response. Finally, we concluded that ASCA could be pointed as relevant biomarker of CD presentation and residual inflammation, even in clinical remission patients.

Absence of NOD2 receptor predisposes to intestinal inflammation by a deregulation in the immune response in hosts that are unable to control gut dysbiosis.

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-1ß 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.

Inducible nitric oxide synthase controls experimental Strongyloides infection.

Infection with Strongyloides sp. induces a host immune response, predominantly the Th2 type, that is able to eliminate the parasite. However, little is known about the role of the nitric oxide (NO) mediator, induced by the enzyme nitric oxide synthase (NOS), in strongyloidiasis. Therefore, in this study, we investigated the immune response of mice genetically deficient in the enzyme inducible nitric oxide synthase (iNOS-/- ), infected with Strongyloides venezuelensis. C57BL/6 wild-type (WT) and iNOS-/- mice were individually inoculated by subcutaneous injection of 3000 S. venezuelensis L3 larvae. In the absence of iNOS, mice were more susceptible to the infection than WT animals, in which the parasite was completely eliminated. The overall production of cytokines and specific IgG, IgG1 or IgE antibodies against the parasite was significantly lowered in infected iNOS-/- mice. The expression of iNOS was observed in the intestine of WT hosts but mainly in the wall of the parasite, despite the presence of iNOS in mice. Altogether, we concluded that iNOS expression may play an important role in the control of S. venezuelensis infection.

The inhibition of 5-Lipoxygenase (5-LO) products leukotriene B4 (LTB4) and cysteinyl leukotrienes (cysLTs) modulates the inflammatory response and improves cutaneous wound healing.

To analyze the participation of the enzyme 5-lipoxygenase (5-LO) in skin repair, WT wounds were compared to those in 5-LO deficient mice (5-LO-/-), which presented faster closure and reduced inflammatory infiltrate in the skin, together with increased CD4 regulatory T cells markers in the draining lymph nodes. The 5-LO-/- wounds also had diminished TNF-α, CCL11, CCL7, CCL2, CXCL9, CCR1 and CXCR2 mRNA expression in the lesions, besides differential extracellular matrix remodeling. Furthermore, when cysteinyl leukotriene (cysLT) and leukotriene (LTB4) receptors were antagonized in WT mice, there was a remarkable reduction in TNF-α expression and faster skin healing, similarly to the findings in 5-LO-/- animals. Finally, our results suggested that 5-LO products, in special cysLT and LTB4, underline skin inflammation that follows skin injury and their neutralization may be an important strategy to improve cutaneous healing.

New Horizons on Molecular Pharmacology Applied to Drug Discovery: When Resonance Overcomes Radioligand Binding.

One of the cornerstones of rational drug development is the measurement of molecular parameters derived from ligand-receptor interaction, which guides therapeutic windows definition. Over the last decades, radioligand binding has provided valuable contributions in this field as key method for such purposes. However, its limitations spurred the development of more exquisite techniques for determining such parameters. For instance, safety risks related to radioactivity waste, expensive and controlled disposal of radioisotopes, radiotracer separation-dependence for affinity analysis, and one-site mathematical models-based fitting of data make radioligand binding a suboptimal approach in providing measures of actual affinity conformations from ligands and G proteincoupled receptors (GPCR). Current advances on high-throughput screening (HTS) assays have markedly extended the options of sparing sensitive ways for monitoring ligand affinity. The advent of the novel bioluminescent donor NanoLuc luciferase (Nluc), engineered from Oplophorus gracilirostris luciferase, allowed fitting bioluminescence resonance energy transfer (BRET) for monitoring ligand binding. Such novel approach named Nluc-based BRET (NanoBRET) binding assay consists of a real-time homogeneous proximity assay that overcomes radioligand binding limitations but ensures the quality in affinity measurements. Here, we cover the main advantages of NanoBRET protocol and the undesirable drawbacks of radioligand binding as molecular methods that span pharmacological toolbox applied to Drug Discovery. Also, we provide a novel perspective for the application of NanoBRET technology in affinity assays for multiple-state binding mechanisms involving oligomerization and/or functional biased selectivity. This new angle was proposed based on specific biophysical criteria required for the real-time homogeneity assigned to the proximity NanoBRET protocol.

The impact of intestinal resection on the immune function of short bowel syndrome patients.

Short bowel syndrome (SBS) is characterized by a massive intestinal loss after surgery resection. Likewise, disturbances involving the intestine, which represents a complex immune environment, may result in breakdown of homeostasis and altered responses, thus leading to unpredictable clinical outcomes. However, the consequences of bowel resection were poorly investigated until now. Therefore, this study aimed to evaluate the immunological status of SBS-patients. For this purpose, ten subjects and nine healthy controls were evaluated. Along with some metabolic disturbances, the main results showed higher levels of the inflammatory cytokine IL-6 in plasma among SBS-patients. However, there were no differences in the frequency of CD3+, CD3+CD4+ or CD3+CD8+ T lymphocytes. An augmented frequency in CD4+ and CD8+ cells producing IFN-γ was also observed in peripheral blood mononuclear cells (PBMC), together with elevated percentage of CD4+ cells producing IL-10. No differences were observed in the frequency of total CD4+CD25-, CD4+CD25+ lymphocytes nor in the expression of FoxP3 or GITR. Nevertheless, SBS-patients showed higher frequency of the regulatory T cell population CD4+CD25+CD39+ cells in PBMC. In conclusion, these data pointed to SBS as an important disturbance that compromises not only the intestinal environment but also negatively influences systemic immune components.

Adrenal-Derived Hormones Differentially Modulate Intestinal Immunity in Experimental Colitis.

The adrenal glands are able to modulate immune responses through neuroimmunoendocrine interactions and cortisol secretion that could suppress exacerbated inflammation such as in inflammatory bowel disease (IBD). Therefore, here we evaluated the role of these glands in experimental colitis induced by 3% dextran sulfate sodium (DSS) in C57BL/6 mice subjected to adrenalectomy, with or without glucocorticoid (GC) replacement. Mice succumbed to colitis without adrenals with a higher clinical score and augmented systemic levels of IL-6 and lower LPS. Furthermore, adrenalectomy negatively modulated systemic regulatory markers. The absence of adrenals resulted in augmented tolerogenic lamina propria dendritic cells but no compensatory local production of corticosterone and decreased mucosal inflammation associated with increased IFN-γ and FasL in the intestine. To clarify the importance of GC in this scenario, GC replacement in adrenalectomized mice restored different markers to the same degree of that observed in DSS group. Finally, this is the first time that adrenal-derived hormones, especially GC, were associated with the differential local modulation of the gut infiltrate, also pointing to a relationship between adrenalectomy and the modulation of systemic regulatory markers. These findings may elucidate some neuroimmunoendocrine mechanisms that dictate colitis outcome.

Dehydroepiandrosterone (DHEA) restrains intestinal inflammation by rendering leukocytes hyporesponsive and balancing colitogenic inflammatory responses.

Dehydroepiandrosterone (DHEA) is a hormone that plays an important role in the modulation of inflammatory responses. However, the precise mechanisms that link the actions of this androgen with protection or susceptibility to inflammatory bowel diseases (IBD) remain uknown. Here we showed that low dose DHEA inhibited proliferation of spleen cells and IFN-у production. The hormone was not toxic to myeloid lineage cells, although it caused necrosis of spleen cells at the intermediate and highest doses in vitro (50 and 100µM). The treatment of C57BL/6 mice with DHEA during colitis induction by dextran sodium sulfate (DSS) led to a reduction in weight loss and clinical signs of disease. There were decreased peripheral blood monocytes on day 6 of DSS exposure and treatment, besides increase in circulating neutrophils in the tissue repair phase. DHEA also led to reduced lamina propria cellularity and restoration of normal colon length. These results were accompanied by decreased expression of IL-6 and TGF-ß mRNA, while IL-13 was augmented in the colon on day 6, which was probably related to attenuation of inflammation. There was retention of CD4(+) cells in the spleen after use of DHEA, along with augmented frequency of CD4(+)IL-4(+) cells, decreased CD4(+)IFN-É£(+) in spleen and constrained CD4(+)IL-17(+) population in the mesenteric lymph nodes. Moreover, splenocytes of mice treated with DHEA became hyporesponsive, as observed by reduced proliferation after re-stimulation ex-vivo. In conclusion, DHEA modifyies leukocyte activity and balances the exacerbated immune responses which drive local and systemic damages in IBD.

Decreased Toll-Like Receptor 2 and Toll-Like Receptor 7/8-Induced Cytokines in Parkinson's Disease Patients.

OBJECTIVES: Toll-like receptors (TLRs) are expressed in several immune cells including blood monocytes and resident macrophages, such as microglia in the central nervous system. TLRs recognize pathogen- or damage-associated molecular patterns, leading to the release of inflammatory and toxic molecules, which can contribute to neuroinflammation associated with Parkinson's disease (PD). The aim of this study was to compare the potential of peripheral blood cells from PD patients or healthy subjects to produce cytokines after exposure to TLR agonists, and to investigate TLR2 and TLR4 expression on monocyte subsets. METHODS: Twenty-one patients and 21 healthy controls were recruited. Patients were evaluated according to the Unified Parkinson's Disease Rating Scale, and Hoehn and Yahr stage. Cytokines were measured in supernatants of whole blood cultures after incubation with TLR2, TLR4, or TLR7/8 agonists, by cytometric bead array. Expression of CD14, CD16, TLR2, and TLR4 was analyzed by cytometry. RESULTS: Patient blood cells produced lower levels of cytokines in response to TLR2 and also after TLR7/8/R848 activation than controls. Percentages of CD14+CD16+ or CD14+CD16- monocytes and TLR2 and TLR4 expression were similar between patients and controls. CONCLUSIONS: Blood leukocyte TLR2 and TLR7/8 responses are impaired in PD. This was neither associated with imbalance in monocyte subsets nor with TLR2/TLR4 expression on these cells. The association between a decreased TLR response in periphery and damage of brain in PD must be further investigated.