Optimizing abemaciclib-induced diarrhea management in patients with breast cancer: a pragmatic 2-group study using a postbiotic microbiota stabilizer.

BACKGROUND: Abemaciclib-induced diarrhea is a relevant concern in clinical practice. Postbiotics have emerged as a promising option for managing it. MATERIALS AND METHODS: We conducted a retrospective-prospective, 2-group, observational study to assess the impact of the postbiotic PostbiotiX-Restore, derived by Lactobacillus paracasei CNCM I-5220, on abemaciclib-induced diarrhea in patients with hormone receptor-positive HER2-negative breast cancer. The prospective population (Postbio group) received postbiotic during the first cycle of abemaciclib, while the retrospective one received standard care (Standard group). Diarrhea grading was defined according to the National Cancer Institute's Common Terminology Criteria for Adverse Events. RESULTS: During the first cycle, diarrhea occurred in 78.9% of patients in the Standard cohort and 97.1% in the Postbio one, with most cases being G1-G2. Severe (G3) diarrhea was significantly less frequent in the Postbio group (0%) compared to the Standard one (7.9%; P = .029). Over the entire study period, while the grading difference was not statistically significant, G3 events were less frequent in the Postbio population (5.9%) than the Standard one (15.4%). Moreover, Postbio patients required fewer dose reductions due to diarrhea compared to the Standard group (P = .002). Notably, in the Postbio population, G1 and G2 events had short median durations (3 and 1 days, respectively) and, for the 2 patients experiencing G3 events during the second abemaciclib cycle (off postbiotic), diarrhea lasted only 1 day. CONCLUSIONS: Our study demonstrates the effect of PostbiotiX-Restore in mitigating abemaciclib-induced diarrhea, resulting in reduced severity, fewer dose reductions, and shorter duration. Further exploration and validation in larger cohorts are needed.

BALB/c and C57BL/6 Mice Differ in Polyreactive IgA Abundance, which Impacts the Generation of Antigen-Specific IgA and Microbiota Diversity.

The interrelationship between IgAs and microbiota diversity is still unclear. Here we show that BALB/c mice had higher abundance and diversity of IgAs than C57BL/6 mice and that this correlated with increased microbiota diversity. We show that polyreactive IgAs mediated the entrance of non-invasive bacteria to Peyer's patches, independently of CX3CR1(+) phagocytes. This allowed the induction of bacteria-specific IgA and the establishment of a positive feedback loop of IgA production. Cohousing of mice or fecal transplantation had little or no influence on IgA production and had only partial impact on microbiota composition. Germ-free BALB/c, but not C57BL/6, mice already had polyreactive IgAs that influenced microbiota diversity and selection after colonization. Together, these data suggest that genetic predisposition to produce polyreactive IgAs has a strong impact on the generation of antigen-specific IgAs and the selection and maintenance of microbiota diversity.

Oral tolerance can be established via gap junction transfer of fed antigens from CX3CR1⁺ macrophages to CD103⁺ dendritic cells.

Antigen-presenting cells (APCs) in the gut are apt at oral tolerance establishment at steady state and immunity after infection; complex tasks in an environment exposed to the inflammatory burden of the microbiota. Here we show an unanticipated division of labor among APCs for the establishment of oral tolerance. Chemokine receptor CX3CR1(+) macrophages were found to take up soluble fed antigens and quickly transfer them to CD103(+) dendritic cells (DCs). Antigen transfer occurred via a mechanism that was Connexin 43-dependent and required membrane transfer, indicating a physiological role of gap junctions in antigen presentation. Deletion of Connexin 43 in APCs affected antigen transfer and resulted in the inability of CD103(+) DCs to acquire and present antigens in vivo, to drive T regulatory cell differentiation and to induce tolerance to food antigens. This functional cooperation between intestinal phagocytes might be a mechanism to avoid the exposure of tolerogenic DCs to the intestinal microbiota.

Microbiota-driven gut vascular barrier disruption is a prerequisite for non-alcoholic steatohepatitis development.

BACKGROUND & AIMS: Fatty liver disease, including non-alcoholic fatty liver (NAFLD) and steatohepatitis (NASH), has been associated with increased intestinal barrier permeability and translocation of bacteria or bacterial products into the blood circulation. In this study, we aimed to unravel the role of both intestinal barrier integrity and microbiota in NAFLD/NASH development. METHODS: C57BL/6J mice were fed with high-fat diet (HFD) or methionine-choline-deficient diet for 1 week or longer to recapitulate aspects of NASH (steatosis, inflammation, insulin resistance). Genetic and pharmacological strategies were then used to modulate intestinal barrier integrity. RESULTS: We show that disruption of the intestinal epithelial barrier and gut vascular barrier (GVB) are early events in NASH pathogenesis. Mice fed HFD for only 1 week undergo a diet-induced dysbiosis that drives GVB damage and bacterial translocation into the liver. Fecal microbiota transplantation from HFD-fed mice into specific pathogen-free recipients induces GVB damage and epididymal adipose tissue enlargement. GVB disruption depends on interference with the WNT/ß-catenin signaling pathway, as shown by genetic intervention driving ß-catenin activation only in endothelial cells, preventing GVB disruption and NASH development. The bile acid analogue and farnesoid X receptor agonist obeticholic acid (OCA) drives ß-catenin activation in endothelial cells. Accordingly, pharmacologic intervention with OCA protects against GVB disruption, both as a preventive and therapeutic agent. Importantly, we found upregulation of the GVB leakage marker in the colon of patients with NASH. CONCLUSIONS: We have identified a new player in NASH development, the GVB, whose damage leads to bacteria or bacterial product translocation into the blood circulation. Treatment aimed at restoring ß-catenin activation in endothelial cells, such as administration of OCA, protects against GVB damage and NASH development. LAY SUMMARY: The incidence of fatty liver disease is reaching epidemic levels in the USA, with more than 30% of adults having NAFLD (non-alcoholic fatty liver disease), which can progress to more severe non-alcoholic steatohepatitis (NASH). Herein, we show that disruption of the intestinal epithelial barrier and gut vascular barrier are early events in the development of NASH. We show that the drug obeticholic acid protects against barrier disruption and thereby prevents the development of NASH, providing further evidence for its use in the prevention or treatment of NASH.

Dichotomy of short and long thymic stromal lymphopoietin isoforms in inflammatory disorders of the bowel and skin.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) is a cytokine with pleiotropic functions in the immune system. It has been associated with allergic reactions in the skin and lungs but also homeostatic tolerogenic responses in the thymus and gut. OBJECTIVE: In human subjects TSLP is present in 2 isoforms, short and long. Here we wanted to investigate the differential expression of the TSLP isoforms and discern their biological implications under homeostatic or inflammatory conditions. METHODS: We evaluated the expression of TSLPs in tissues from healthy subjects, patients with ulcerative colitis, patients with celiac disease, and patients with atopic dermatitis and on epithelial cells and keratinocytes under steady-state conditions or after stimulation. We then tested the immune activity of TSLP isoforms both in vitro and in vivo. RESULTS: We showed that TSLP isoforms are responsible for 2 opposite immune functions. The short isoform is expressed under steady-state conditions and exerts anti-inflammatory activities by affecting the capacity of PBMCs and dendritic cells to produce inflammatory cytokines. Moreover, the short isoform TSLP ameliorates experimental colitis in mice and prevents endotoxin shock. The long isoform of TSLP is proinflammatory and is only expressed during inflammation. The isoforms are differentially regulated by pathogenic bacteria, such as Salmonella species and adhesive-invasive Escherichia coli. CONCLUSIONS: We have solved the dilemma of TSLP being both homeostatic and inflammatory. The TSLP isoform ratio is altered during several inflammatory disorders, with strong implications in disease treatment and prevention. Indeed, targeting of the long isoform of TSLP at the C-terminal portion, which is common to both isoforms, might lead to unwanted side effects caused by neutralization of the homeostatic short isoform.

Probiotic and postbiotic activity in health and disease: comparison on a novel polarised ex-vivo organ culture model.

BACKGROUND AND AIMS: Probiotics and their metabolic products, here called postbiotics, have been proposed as food supplements for a healthier intestinal homeostasis, but also as therapeutic aids in inflammatory bowel disease (IBD) with, however, very little clinical benefit. This may be due to the lack of reliable preclinical models for testing the efficacy of different strains. METHODS: The activity of three probiotic strains of Lactobacillus (or a postbiotic) was analysed and compared with a pathogenic strain of Salmonella on a novel organ culture system of human healthy and IBD intestinal mucosa developed in our laboratory. The system maintains an apical to basolateral polarity during stimulation due to the presence of a glued cave cylinder. The cylinder is detached at the end of the experiment and the tissue is processed for histology and immunohistochemistry. Cytokines released from the basolateral side are analysed. RESULTS: The model system provides several physiological characteristics typical of a mucosal microenvironment including the presence of an organised mucus layer and an apical to basolateral polarity. Polarised administration of bacteria is critical to control the ensuing immune response as it mimics the physiological entrance of bacteria. The authors show that probiotics are not always beneficial for the healthy host and can also be detrimental in inflamed IBD. This study shows that a potent postbiotic can protect against the inflammatory properties of invasive Salmonella on healthy tissue and also downregulate ongoing inflammatory processes in IBD tissue. CONCLUSIONS: Probiotics can have inflammatory activities in both healthy and IBD tissue. Valid preclinical data on proper model systems should therefore be obtained before specific probiotic strains enter the clinics, especially if administered during acute inflammatory responses. Postbiotics may be a safe alternative for the treatment of patients with IBD in the acute inflammatory phase.

Lactobacillus paracasei CNCM I-5220-derived postbiotic protects from the leaky-gut.

The maintenance of intestinal barrier function is essential for preventing different pathologies, such as the leaky gut syndrome (LGS), which is characterized by the passage of harmful agents, like bacteria, toxins, and viruses, into the bloodstream. Intestinal barrier integrity is controlled by several players, including the gut microbiota. Various molecules, called postbiotics, are released during the natural metabolic activity of the microbiota. Postbiotics can regulate host-microbe interactions, epithelial homeostasis, and have overall benefits for our health. In this work, we used in vitro and in vivo systems to demonstrate the role of Lactobacillus paracasei CNCM I-5220-derived postbiotic (LP-PBF) in preserving intestinal barrier integrity. We demonstrated in vitro that LP-PBF restored the morphology of tight junctions (TJs) that were altered upon Salmonella typhimurium exposure. In vivo, LP-PBF protected the gut vascular barrier and blocked S. typhimurium dissemination into the bloodstream. Interestingly, we found that LP-PBF interacts not only with the host cells, but also directly with S. typhimurium blocking its biofilm formation, partially due to the presence of biosurfactants. This study highlights that LP-PBF is beneficial in maintaining gut homeostasis due to the synergistic effect of its different components. These results suggest that LP-PBF could be utilized in managing several pathologies displaying an impaired intestinal barrier function.

Sensitizing cancer cells to immune checkpoint inhibitors by microbiota-mediated upregulation of HLA class I.

Recent data have shown that gut microbiota has a major impact on the clinical response to immune checkpoint inhibitors (ICIs) in the context of solid tumors. ICI-based therapy acts by unlocking cognate cytotoxic T lymphocyte (CTL) effector responses, and increased sensitivity to ICIs is due to an enhancement of patients' tumor antigen (TA)-specific CTL responses. Cancer clearance by TA-specific CTL requires expression of relevant TAs on cancer cells' HLA class I molecules, and reduced HLA class I expression is a common mechanism used by cancer cells to evade the immune system. Here, we show that metabolites released by bacteria, in particular, phytosphingosine, can upregulate HLA class I expression on cancer cells, sensitizing them to TA-specific CTL lysis in vitro and in vivo, in combination with immunotherapy. This effect is mediated by postbiotic-induced upregulation of NLRC5 in response to upstream MYD88-NF-κB activation, thus significantly controlling tumor growth.

Biomimetic superabsorbent hydrogel acts as a gut protective dynamic exoskeleton improving metabolic parameters and expanding A. muciniphila.

The rising prevalence of obesity and metabolic disorders worldwide highlights the urgent need to find new long-term and clinically meaningful weight-loss therapies. Here, we evaluate the therapeutic potential and the mechanism of action of a biomimetic cellulose-based oral superabsorbent hydrogel (OSH). Treatment with OSH exerts effects on intestinal tissue and gut microbiota composition, functioning like a protective dynamic exoskeleton. It protects from gut barrier permeability disruption and induces rapid and consistent changes in the gut microbiota composition, specifically fostering Akkermansia muciniphila expansion. The mechanobiological, physical, and chemical structures of the gel are required for A. muciniphila growth. OSH treatment induces weight loss and reduces fat accumulation, in both preventative and therapeutic settings. OSH usage also prevents liver steatosis, immune infiltration, and fibrosis, limiting the progression of non-alcoholic fatty liver disease. Our work shows the potential of using OSH as a non-systemic mechanobiological approach to treat metabolic syndrome and its comorbidities.

Farnesoid X receptor activation inhibits inflammation and preserves the intestinal barrier in inflammatory bowel disease.

BACKGROUND & AIMS: Inflammatory bowel disease (IBD) is characterised by chronic intestinal inflammation, resulting from dysregulation of the mucosal immune system and compromised intestinal epithelial barrier function. The bile salt, nuclear farnesoid X receptor (FXR), was recently implicated in intestinal antibacterial defence and barrier function. The aim of this study was to investigate the therapeutic potential of FXR agonists in the treatment of intestinal inflammation in complementary in vivo and in vitro models. METHODS: Colitis was induced in wild-type (WT) and Fxr-null mice using dextran sodium sulfate, and in WT mice using trinitrobenzenesulfonic acid. Mice were treated with vehicle or the FXR agonist INT-747, and colitis symptoms were assessed daily. Epithelial permeability assays and cytokine expression analysis were conducted in mouse colon and enterocyte-like cells (Caco-2/HT29) treated with medium or INT-747. Inflammatory cytokine secretion was determined by ELISA in various human immune cell types. RESULTS: INT-747-treated WT mice are protected from DSS- and TNBS-induced colitis, as shown by significant reduction of body weight loss, epithelial permeability, rectal bleeding, colonic shortening, ulceration, inflammatory cell infiltration and goblet cell loss. Furthermore, Fxr activation in intestines of WT mice and differentiated enterocyte-like cells downregulates expression of key proinflammatory cytokines and preserves epithelial barrier function. INT-747 significantly decreases tumour necrosis factor α secretion in activated human peripheral blood mononuclear cells, purified CD14 monocytes and dendritic cells, as well as in lamina propria mononuclear cells from patients with IBD. CONCLUSIONS: FXR activation prevents chemically induced intestinal inflammation, with improvement of colitis symptoms, inhibition of epithelial permeability, and reduced goblet cell loss. Furthermore, FXR activation inhibits proinflammatory cytokine production in vivo in the mouse colonic mucosa, and ex vivo in different immune cell populations. The findings provide a rationale to explore FXR agonists as a novel therapeutic strategy for IBD.

A 'Multiomic' Approach of Saliva Metabolomics, Microbiota, and Serum Biomarkers to Assess the Need of Hospitalization in Coronavirus Disease 2019.

BACKGROUND AND AIMS: The SARS-CoV-2 pandemic has overwhelmed the treatment capacity of the health care systems during the highest viral diffusion rate. Patients reaching the emergency department had to be either hospitalized (inpatients) or discharged (outpatients). Still, the decision was taken based on the individual assessment of the actual clinical condition, without specific biomarkers to predict future improvement or deterioration, and discharged patients often returned to the hospital for aggravation of their condition. Here, we have developed a new combined approach of omics to identify factors that could distinguish coronavirus disease 19 (COVID-19) inpatients from outpatients. METHODS: Saliva and blood samples were collected over the course of two observational cohort studies. By using machine learning approaches, we compared salivary metabolome of 50 COVID-19 patients with that of 270 healthy individuals having previously been exposed or not to SARS-CoV-2. We then correlated the salivary metabolites that allowed separating COVID-19 inpatients from outpatients with serum biomarkers and salivary microbiota taxa differentially represented in the two groups of patients. RESULTS: We identified nine salivary metabolites that allowed assessing the need of hospitalization. When combined with serum biomarkers, just two salivary metabolites (myo-inositol and 2-pyrrolidineacetic acid) and one serum protein, chitinase 3-like-1 (CHI3L1), were sufficient to separate inpatients from outpatients completely and correlated with modulated microbiota taxa. In particular, we found Corynebacterium 1 to be overrepresented in inpatients, whereas Actinomycetaceae F0332, Candidatus Saccharimonas, and Haemophilus were all underrepresented in the hospitalized population. CONCLUSION: This is a proof of concept that a combined omic analysis can be used to stratify patients independently from COVID-19.

Human benign prostatic hyperplasia stromal cells as inducers and targets of chronic immuno-mediated inflammation.

Benign prostatic hyperplasia (BPH), a highly prevalent prostatic condition, could involve an inflammatory component in disease pathogenesis. In this study, we show that human stromal prostate cells obtained from BPH tissue can actively contribute to the inflammatory process by secreting proinflammatory cytokines as well as chemokines able to recruit lymphomonuclear cells and by acting as APCs. BPH cells express all of the TLRs and their ligation leads to the secretion of CXCL8/IL-8, CXCL10, and IL-6. In addition, BPH cells express costimulatory as well as class I and class II MHC molecules, which activate alloreactive CD4(+) cells that in turn markedly up-regulate IL-12/IL-23p40 and IL-12p75 secretion by BPH cells. Alloreactive CD4(+) cells activated by BPH cells secrete IFN-gamma and IL-17. These cytokines up-regulate IL-6, IL-8, and CXCL10 production by BPH cells, creating a positive feedback loop that can amplify inflammation. IL-8 induces autocrine/paracrine proliferation of BPH cells, indicating also a growth-promoting activity of this chemokine in disease pathogenesis. These results show that human BPH cells represent nonprofessional APCs able to induce and sustain chronic inflammatory processes, supporting the relevance of inflammation in BPH pathogenesis.

Functional characterization and immunomodulatory properties of Lactobacillus helveticus strains isolated from Italian hard cheeses.

Lactobacillus helveticus carries many properties such as the ability to survive gastrointestinal transit, modulate the host immune response, accumulate biopeptides in milk, and adhere to the epithelial cells that could contribute to improving host health. In this study, the applicability as functional cultures of four L. helveticus strains isolated from Italian hard cheeses was investigated. A preliminary strain characterization showed that the ability to produce folate was generally low while antioxidant, proteolytic, peptidase, and ß-galactosidase activities resulted high, although very variable, between strains. When stimulated moDCs were incubated in the presence of live cells, a dose-dependent release of both the pro-inflammatory cytokine IL-12p70 and the anti-inflammatory cytokine IL-10, was shown for all the four strains. In the presence of cell-free culture supernatants (postbiotics), a dose-dependent, decrease of IL-12p70 and an increase of IL-10 was generally observed. The immunomodulatory effect took place also in Caciotta-like cheese made with strains SIM12 and SIS16 as bifunctional (i.e., immunomodulant and acidifying) starter cultures, thus confirming tests in culture media. Given that the growth of bacteria in the cheese was not necessary (they were killed by pasteurization), the results indicated that some constituents of non-viable bacteria had immunomodulatory properties. This study adds additional evidence for the positive role of L. helveticus on human health and suggests cheese as a suitable food for delivering candidate strains and modulating their anti-inflammatory properties.

Identification of a class of non-conventional ER-stress-response-derived immunogenic peptides.

Efforts to overcome resistance to immune checkpoint blockade therapy have focused on vaccination strategies using neoepitopes, although they cannot be applied on a large scale due to the "private" nature of cancer mutations. Here, we show that infection of tumor cells with Salmonella induces the opening of membrane hemichannels and the extracellular release of proteasome-generated peptides by the exacerbation of endoplasmic reticulum (ER) stress. Peptides released by cancer cells foster an antitumor response in vivo, both in mice bearing B16F10 melanomas and in dogs suffering from osteosarcoma. Mass spectrometry analysis on the supernatant of human melanoma cells revealed 12 peptides capable of priming healthy-donor CD8+ T cells that recognize and kill human melanoma cells in vitro and when xenotransplanted in vivo. Hence, we identified a class of shared tumor antigens that are generated in ER-stressed cells, such as tumor cells, that do not induce tolerance and are not presented by healthy cells.

Identification of a choroid plexus vascular barrier closing during intestinal inflammation.

Up to 40% of patients with inflammatory bowel disease present with psychosocial disturbances. We previously identified a gut vascular barrier that controls the dissemination of bacteria from the intestine to the liver. Here, we describe a vascular barrier in the brain choroid plexus (PVB) that is modulated in response to intestinal inflammation through bacteria-derived lipopolysaccharide. The inflammatory response induces PVB closure after gut vascular barrier opening by the up-regulation of the wingless-type, catenin-beta 1 (Wnt/ß-catenin) signaling pathway, rendering it inaccessible to large molecules. In a model of genetically driven closure of choroid plexus endothelial cells, we observed a deficit in short-term memory and anxiety-like behavior, suggesting that PVB closure may correlate with mental deficits. Inflammatory bowel disease­related mental symptoms may thus be the consequence of a deregulated gut­brain vascular axis.

Paralysis of the cytotoxic granule machinery is a new cancer immune evasion mechanism mediated by chitinase 3-like-1.

BACKGROUND: Natural killer (NK) cells require a functional lytic granule machinery to mediate effective antitumor responses. Evading the lytic cargo deployed at the immune synapse (IS) could be a critical step for cancer progression through yet unidentified mechanisms. METHODS: NK cell antibody-dependent cellular cytotoxicity (ADCC) is a major determinant of the clinical efficacy of some therapeutic antibodies including the anti-HER2 Trastuzumab. Thus, we screened sera of Trastuzumab-resistant HER2 +patients with breast cancer for molecules that could inhibit NK cell ADCC. We validated our findings in vitro using cytotoxicity assays and confocal imaging of the lytic granule machinery and in vivo using syngeneic and xenograft murine models. RESULTS: We found that sera from Trastuzumab-refractory patients could inhibit healthy NK cell ADCC in vitro. These sera contained high levels of the inflammatory protein chitinase 3-like 1 (CHI3L1) compared with sera from responders and healthy controls. We demonstrate that recombinant CHI3L1 inhibits both ADCC and innate NK cell cytotoxicity. Mechanistically, CHI3L1 prevents the correct polarization of the microtubule-organizing center along with the lytic granules to the IS by hindering the receptor of advanced glycation end-products and its downstream JNK signaling. In vivo, CHI3L1 administration drastically impairs the control of NK cell-sensitive tumors, while CHI3L1 blockade synergizes with ADCC to cure mice with HER2 +xenografts. CONCLUSION: Our work highlights a new paradigm of tumor immune escape mediated by CHI3L1 which acts on the cytotoxic machinery and prevents granule polarization. Targeting CHI3L1 could mitigate immune escape and potentiate antibody and cell-based immunotherapies.

Gut vascular barrier impairment leads to intestinal bacteria dissemination and colorectal cancer metastasis to liver.

Metastasis is facilitated by the formation of a "premetastatic niche," which is fostered by primary tumor-derived factors. Colorectal cancer (CRC) metastasizes mainly to the liver. We show that the premetastatic niche in the liver is induced by bacteria dissemination from primary CRC. We report that tumor-resident bacteria Escherichia coli disrupt the gut vascular barrier (GVB), an anatomical structure controlling bacterial dissemination along the gut-liver axis, depending on the virulence regulator VirF. Upon GVB impairment, bacteria disseminate to the liver, boost the formation of a premetastatic niche, and favor the recruitment of metastatic cells. In training and validation cohorts of CRC patients, we find that the increased levels of PV-1, a marker of impaired GVB, is associated with liver bacteria dissemination and metachronous distant metastases. Thus, PV-1 is a prognostic marker for CRC distant recurrence and vascular impairment, leading to liver metastases.

Endogenous murine microbiota member Faecalibaculum rodentium and its human homologue protect from intestinal tumour growth.

The microbiota has been shown to promote intestinal tumourigenesis, but a possible anti-tumourigenic effect has also been postulated. Here, we demonstrate that changes in the microbiota and mucus composition are concomitant with tumourigenesis. We identified two anti-tumourigenic strains of the microbiota-Faecalibaculum rodentium and its human homologue, Holdemanella biformis-that are strongly under-represented during tumourigenesis. Reconstitution of ApcMin/+ or azoxymethane- and dextran sulfate sodium-treated mice with an isolate of F. rodentium (F. PB1) or its metabolic products reduced tumour growth. Both F. PB1 and H. biformis produced short-chain fatty acids that contributed to control protein acetylation and tumour cell proliferation by inhibiting calcineurin and NFATc3 activation in mouse and human settings. We have thus identified endogenous anti-tumourigenic bacterial strains with strong diagnostic, therapeutic and translational potential.

The vitamin D receptor agonist elocalcitol inhibits IL-8-dependent benign prostatic hyperplasia stromal cell proliferation and inflammatory response by targeting the RhoA/Rho kinase and NF-kappaB pathways.

BACKGROUND: Benign prostatic hyperplasia (BPH) is characterized by an important inflammatory component. Stimulation of human prostate stromal cells from BPH tissues with proinflammatory cytokines leads to secretion of IL-8, a chemokine involved in BPH pathogenesis. The vitamin D receptor (VDR) agonist elocalcitol can arrest prostate growth in BPH patients, but its mechanism of action in this pathology is still incompletely understood. METHODS: IL-8 levels were measured by real-time RT-PCR and ELISA. NF-kappaB translocation and COX-2 expression were evaluated by confocal microscopy. RhoA and Rho-kinase (ROCK) gene expression and functional activity were studied by real-time RT-PCR, immuno-kinase assays, Western blot analysis, confocal microscopy, and cell invasion. RESULTS: Stimulation of BPH cells with IL-8 activates the calcium-sensitizing RhoA/ROCK pathway, as demonstrated by the increased membrane translocation of RhoA and by phosphorylation of the ROCK substrate myosin phosphatase target subunit 1 (MYPT-1). In agreement with these data, C3 exoenzyme, a selective RhoA inhibitor, inhibits IL-8-induced invasion of BPH cells. The VDR agonist elocalcitol significantly inhibits IL-8 production by BPH cells stimulated with inflammatory cytokines, and IL-8-induced proliferation of BPH cells. In addition, elocalcitol inhibits IL-8-induced membrane translocation of RhoA and MYPT-1 phosphorylation in BPH cells, and inhibits dose-dependently their IL-8-dependent invasion. The inhibition induced by elocalcitol of IL-8 production by BPH cells is accompanied by decreased COX-2 expression and PGE(2) production and by arrest of NF-kappaB p65 nuclear translocation, associated with inhibition of the RhoA/ROCK pathway. CONCLUSIONS: These data provide a mechanistic explanation for the anti-proliferative and anti-inflammatory properties of elocalcitol in BPH cells.

Induction of tolerogenic dendritic cells by vitamin D receptor agonists.

Dendritic cells induce and regulate T cell responses, and tolerogenic dendritic cells (DCs) can promote the development of regulatory T cells with suppressive activity. Thus, the possibility to manipulate DCs using different pharmacological or biological agents enables them to exert tolerogenic activities, could be exploited to better control a variety of chronic inflammatory conditions, from autoimmune diseases to allograft rejection. A variety of both biological and pharmacological agents can induce tolerogenic DCs, and several in vitro studies have demonstrated that human regulatory T cells can be induced by DCs manipulated to acquire and/or enhance tolerogenic properties, with in vivo data also accumulating. Within this context, we have explored the immunoregulatory activities of vitamin D receptor (VDR) agonists, secosteroid hormones able to induce tolerogenic DCs and regulatory T cells. Tolerogenic DCs induced by a short treatment with VDR agonists promote CD4(+) CD25(+) Foxp3(+) suppressor T cells that are able to mediate transplantation tolerance and to arrest the development of autoimmune diseases. VDR agonists not only favour the induction of CD4(+) CD25(+) regulatory T cells, but can also enhance their recruitment to inflammatory sites. VDR agonists have been proven effective and safe drugs in a variety of autoimmune disease and graft rejection models, highlighting their potential applicability to chronic inflammatory conditions sustained by autoreactive or alloreactive immune responses. In addition to the topical treatment of psoriasis, a Th1-mediated autoimmune disease of the skin where VDR agonists are the most used topical drugs; these agents might eventually find a broader application in the treatment of inflammatory conditions, where their modulatory effects on DCs enhancing T cells with regulatory functions could turn out to be quite beneficial.