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

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.

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.

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.

Inflammation Biomarkers of Advanced Disease in Nongingival Tissues of Chronic Periodontitis Patients.

Chronic periodontitis is a multifactorial inflammatory disease that affects supporting structures of the teeth. Although the gingival response is largely described, little is known about the immune changes in the alveolar bone and neighboring tissues that could indicate periodontal disease (PD) activity. Then, in this study we identified the ongoing inflammatory changes and novel biomarkers for periodontitis in the tissues directly affected by the destructive disease in PD patients. Samples were collected by osteotomy in 17 control subjects during extraction of third molars and 18 patients with advanced PD, in which alveoloplasty was necessary after extraction of teeth with previous extensive periodontal damage. Patients presented mononuclear cells infiltration in the connective tissue next to the bone and higher fibrosis area, along with increased accumulation of IL-17(+) and TRAP(+) cells. The levels of TNF-α and MMP-2 mRNA were also elevated compared to controls and a positive and significant correlation was observed between TNF-α and MMP-2 mRNA expression, considering all samples evaluated. In conclusion, nongingival tissues neighboring large periodontal pockets present inflammatory markers that could predict ongoing bone resorption and disease spreading. Therefore, we suggested that the detailed evaluation of these regions could be of great importance to the assessment of disease progression.

Association of matrix metalloproteinase inducer (EMMPRIN) with the expression of matrix metalloproteinases-1, -2 and -9 during periapical lesion development.

OBJECTIVE: To evaluate the expression of matrix metalloproteinase inducer (EMMPRIN) and its correlation with the expression of matrix metalloproteinases (MMPs)-1, -2 and -9 during the development of periapical lesion in mice. METHODS: Periapical lesions were induced in the lower first molars of mice and after 7, 14, 21 and 42 days the mandibles were removed. The periapical lesions were measured by micro-computed tomography. The expression of EMMPRIN, MMPs-1, -2, and -9 genes were determined by real-time RT-PCR. The location and expression of EMMPRIN and MMPs were evaluated by immunohistochemistry. RESULTS: At 14 days, the periapical lesion area was higher than at 7 days. At 21 and 42 days no statistically significant bone loss was observed in comparison to 14 days. The control group showed discrete and occasional EMMPRIM, MMP-1, -2 and -9 immunostaining in the periodontal ligament fibroblasts. At 7, 14, 21 and 42 days intense immunoexpression was observed for EMMPRIN, MMPs-1, -2 and -9 in the region adjacent to the apical foramen. The EMMPRIN immunoexpression was higher at 7, 14, 21 and 42 days compared with the control. There was a positive correlation between gene expression of EMMPRIN and MMPs in the active phase of periapical lesion development. CONCLUSION: There is a high expression of EMMPRIM mainly by the inflammatory infiltrate in the region adjacent to the apical foramen during periapical lesion development. Furthermore, the positive correlation with MMP-1, -2, and -9 during the first days after periapical lesion induction indicates that EMMPRIM may be involved in the active phase of periapical lesions development.

Dynamic changes of the Th17/Tc17 and regulatory T cell populations interfere in the experimental autoimmune diabetes pathogenesis.

A balance between proinflammatory (Th17 and Tc17) and anti-inflammatory (regulatory T cells) subsets of T cells is essential to maintain immunological tolerance and prevent the onset of several autoimmune diseases, including type 1 diabetes. However, the kinetics of these subsets and disease severity during the streptozotocin (STZ)-induced diabetes course has not been determined. Thus, susceptible C57BL/6 mice were administrated with multiple low doses of STZ and we evaluated the frequency/absolute number of these T cell subsets in the pancreatic lymph nodes (PLNs) and spleen and Th1, Th17, Treg cytokine production in the pancreatic tissue. At different time points of the disease progression (6, 11, 18 and 25 days after the last STZ administration), the histopathological alterations were also evaluated by H&E and immunohistochemistry staining. During the initial phase of diabetes development (day 6), we noted increased numbers of CD4(+) and CD8(+) T cells in spleen and PLNs. At the same time, the frequencies of Th17 and Tc17 cells in PLNs were also enhanced. In addition, the early augment of interferon gamma (IFN-γ), tumoral necrosis factor (TNF-α), IL-6 and IL-17 levels in pancreatic tissue correlated with pancreatic islet inflammation and mild ß-cell damage. Notably, the absolute number of Treg cells increased in PLNs during over time when compared to control group. Interestingly, increased IL-10 levels were associated with control of the inflammatory process during the late phase of the type 1 diabetes (day 25). In agreement, mice lacking the expression of IL-17 receptor (Il17r) showed impairment in STZ-induced diabetes progression, reduced peri-insulitis and beta cells preservation when compared with wild-type mice. Our findings suggest that dynamic changes of pathogenic Th17/Tc17 and regulatory T cell subsets numbers is associated with early strong inflammation in the pancreatic islets followed by late regulatory profile during the experimental STZ-induced diabetes course.