MICa/b-dependent activation of natural killer cells by CD64+ inflammatory type 2 dendritic cells contributes to autoimmunity.

Primary Sjögren's syndrome (pSS) is an inflammatory autoimmune disorder largely mediated by type I and II interferon (IFN). The potential contribution of innate immune cells, such as natural killer (NK) cells and dendritic cells (DC), to the pSS pathology remains understudied. Here, we identified an enriched CD16+ CD56hi NK cell subset associated with higher cytotoxic function, as well as elevated proportions of inflammatory CD64+ conventional dendritic cell (cDC2) subtype that expresses increased levels of MICa/b, the ligand for the activating receptor NKG2D, in pSS individuals. Circulating cDC2 from pSS patients efficiently induced activation of cytotoxic NK cells ex vivo and were found in proximity to CD56+ NK cells in salivary glands (SG) from pSS patients. Interestingly, transcriptional activation of IFN signatures associated with the RIG-I/DDX60 pathway, IFN I receptor, and its target genes regulate the expression of NKG2D ligands on cDC2 from pSS patients. Finally, increased proportions of CD64hi RAE-1+ cDC2 and NKG2D+ CD11b+ CD27+ NK cells were present in vivo in the SG after poly I:C injection. Our study provides novel insight into the contribution and interplay of NK and cDC2 in pSS pathology and identifies new potential therapy targets.

Immune synapse formation promotes lipid peroxidation and MHC-I upregulation in licensed dendritic cells for efficient priming of CD8+ T cells.

Antigen cognate dendritic cell (DC)-T cell synaptic interactions drive activation of T cells and instruct DCs. Upon receiving CD4+ T cell help, post-synaptic DCs (psDCs) are licensed to generate CD8+ T cell responses. However, the cellular and molecular mechanisms that enable psDCs licensing remain unclear. Here, we describe that antigen presentation induces an upregulation of MHC-I protein molecules and increased lipid peroxidation on psDCs in vitro and in vivo. We also show that these events mediate DC licensing. In addition, psDC adoptive transfer enhances pathogen-specific CD8+ T responses and protects mice from infection in a CD8+ T cell-dependent manner. Conversely, depletion of psDCs in vivo abrogates antigen-specific CD8+ T cell responses during immunization. Together, our data show that psDCs enable CD8+ T cell responses in vivo during vaccination and reveal crucial molecular events underlying psDC licensing.

ISG20L2: an RNA nuclease regulating T cell activation.

ISG20L2, a 3' to 5' exoribonuclease previously associated with ribosome biogenesis, is identified here in activated T cells as an enzyme with a preferential affinity for uridylated miRNA substrates. This enzyme is upregulated in T lymphocytes upon TCR and IFN type I stimulation and appears to be involved in regulating T cell function. ISG20L2 silencing leads to an increased basal expression of CD69 and induces greater IL2 secretion. However, ISG20L2 absence impairs CD25 upregulation, CD3 synaptic accumulation and MTOC translocation towards the antigen-presenting cell during immune synapsis. Remarkably, ISG20L2 controls the expression of immunoregulatory molecules, such as AHR, NKG2D, CTLA-4, CD137, TIM-3, PD-L1 or PD-1, which show increased levels in ISG20L2 knockout T cells. The dysregulation observed in these key molecules for T cell responses support a role for this exonuclease as a novel RNA-based regulator of T cell function.

Extracellular vesicles from Listeria monocytogenes-infected dendritic cells alert the innate immune response.

Communication through cell-cell contacts and extracellular vesicles (EVs) enables immune cells to coordinate their responses against diverse types of pathogens. The function exerted by EVs in this context depends on the proteins and nucleic acids loaded into EVs, which elicit specific responses involved in the resolution of infection. Several mechanisms control protein and nucleic acid loading into EVs; in this regard, acetylation has been described as a mechanism of cellular retention during protein sorting to exosomes. HDAC6 is a deacetylase involved in the control of cytoskeleton trafficking, organelle polarity and cell migration, defense against Listeria monocytogenes (Lm) infection and other immune related functions. Here, we show that the protein content of dendritic cells (DCs) and their secreted EVs (DEVs) vary during Lm infection, is enriched in proteins related to antiviral functions compared to non-infected cells and depends on HDAC6 expression. Analyses of the post-translational modifications revealed an alteration of the acetylation and ubiquitination profiles upon Lm infection both in DC lysates and DEVs. Functionally, EVs derived from infected DCs upregulate anti-pathogenic genes (e.g. inflammatory cytokines) in recipient immature DCs, which translated into protection from subsequent infection with vaccinia virus. Interestingly, absence of Listeriolysin O in Lm prevents DEVs from inducing this anti-viral state. In summary, these data underscore a new mechanism of communication between bacteria-infected DC during infection as they alert neighboring, uninfected DCs to promote antiviral responses.

Natural killer (NK) cell-derived extracellular-vesicle shuttled microRNAs control T cell responses.

Natural killer (NK) cells recognize and kill target cells undergoing different types of stress. NK cells are also capable of modulating immune responses. In particular, they regulate T cell functions. Small RNA next-generation sequencing of resting and activated human NK cells and their secreted extracellular vesicles (EVs) led to the identification of a specific repertoire of NK-EV-associated microRNAs and their post-transcriptional modifications signature. Several microRNAs of NK-EVs, namely miR-10b-5p, miR-92a-3p, and miR-155-5p, specifically target molecules involved in Th1 responses. NK-EVs promote the downregulation of GATA3 mRNA in CD4+ T cells and subsequent TBX21 de-repression that leads to Th1 polarization and IFN-γ and IL-2 production. NK-EVs also have an effect on monocyte and moDCs (monocyte-derived dendritic cells) function, driving their activation and increased presentation and costimulatory functions. Nanoparticle-delivered NK-EV microRNAs partially recapitulate NK-EV effects in mice. Our results provide new insights on the immunomodulatory roles of NK-EVs that may help to improve their use as immunotherapeutic tools.

Galectin-1 Expression in CD8+ T Lymphocytes Controls Inflammation in Contact Hypersensitivity.

Allergic contact dermatitis, also known as contact hypersensitivity, is a frequent T-cell‒mediated inflammatory skin disease characterized by red, itchy, swollen, and cracked skin. It is caused by the direct contact with an allergen and/or irritant hapten. Galectin-1 (Gal-1) is a ß-galactoside‒binding lectin, which is highly expressed in several types of immune cells. The role of endogenous Gal-1 in contact hypersensitivity is not known. We found that Gal-1‒deficient mice display more sustained and prolonged skin inflammation than wild-type mice after oxazolone treatment. Gal-1‒deficient mice have increased CD8+ T cells and neutrophilic infiltration in the skin. After the sensitization phase, Gal-1‒depleted mice showed an increased frequency of central memory CD8+ T cells and IFN-γ secretion by CD8+ T cells. The absence of Gal-1 does not affect the migration of transferred CD4+ and CD8+ T cells from the blood to the lymph nodes or to the skin. The depletion of CD4+ T lymphocytes as well as adoptive transfer experiments demonstrated that endogenous expression of Gal-1 on CD8+ T lymphocytes exerts a major role in the control of contact hypersensitivity model. These data underscore the protective role of endogenous Gal-1 in CD8+ but not CD4+ T cells in the development of allergic contact dermatitis.

Targeting L-type amino acid transporter 1 in innate and adaptive T cells efficiently controls skin inflammation.

BACKGROUND: Psoriasis is a frequent inflammatory skin disease that is mainly mediated by IL-23, IL-1ß, and IL-17 cytokines. Although psoriasis is a hyperproliferative skin disorder, the possible role of amino acid transporters has remained unexplored. OBJECTIVE: We sought to investigate the role of the essential amino acid transporter L-type amino acid transporter (LAT) 1 (SLC7A5) in psoriasis. METHODS: LAT1 floxed mice were crossed to Cre-expressing mouse strains under the control of keratin 5, CD4, and retinoic acid receptor-related orphan receptor γ. We produced models of skin inflammation induced by imiquimod (IMQ) and IL-23 and tested the effect of inhibiting LAT1 (JPH203) and mammalian target of rapamycin (mTOR [rapamycin]). RESULTS: LAT1 expression is increased in keratinocytes and skin-infiltrating lymphocytes of psoriatic lesions in human subjects and mice. LAT1 deletion in keratinocytes does not dampen the inflammatory response or their proliferation, which could be maintained by increased expression of the alternative amino acid transporters LAT2 and LAT3. Specific deletion of LAT1 in γδ and CD4 T cells controls the inflammatory response induced by IMQ. LAT1 deletion or inhibition blocks expansion of IL-17-secreting γ4+δ4+ and CD4 T cells and dampens the release of IL-1ß, IL-17, and IL-22 in the IMQ-induced model. Moreover, inhibition of LAT1 blocks expansion of human γδ T cells and IL-17 secretion by human CD4 T cells. IL-23 and IL-1ß stimulation upregulates LAT1 expression and induces mTOR activation in IL-17+ γδ and TH17 cells. Deletion or inhibition of LAT1 efficiently controls IL-23- and IL-1ß-induced phosphatidylinositol 3-kinase/AKT/mTOR activation independent of T-cell receptor signaling. CONCLUSION: Targeting LAT1-mediated amino acid uptake is a potentially useful immunosuppressive strategy to control skin inflammation mediated by the IL-23/IL-1ß/IL-17 axis.

HDAC6 controls innate immune and autophagy responses to TLR-mediated signalling by the intracellular bacteria Listeria monocytogenes.

Recent evidence on HDAC6 function underlines its role as a key protein in the innate immune response to viral infection. However, whether HDAC6 regulates innate immunity during bacterial infection remains unexplored. To assess the role of HDAC6 in the regulation of defence mechanisms against intracellular bacteria, we used the Listeria monocytogenes (Lm) infection model. Our data show that Hdac6-/- bone marrow-derived dendritic cells (BMDCs) have a higher bacterial load than Hdac6+/+ cells, correlating with weaker induction of IFN-related genes, pro-inflammatory cytokines and nitrite production after bacterial infection. Hdac6-/- BMDCs have a weakened phosphorylation of MAPK signalling in response to Lm infection, suggesting altered Toll-like receptor signalling (TLR). Compared with Hdac6+/+ counterparts, Hdac6-/- GM-CSF-derived and FLT3L-derived dendritic cells show weaker pro-inflammatory cytokine secretion in response to various TLR agonists. Moreover, HDAC6 associates with the TLR-adaptor molecule Myeloid differentiation primary response gene 88 (MyD88), and the absence of HDAC6 seems to diminish the NF-κB induction after TLR stimuli. Hdac6-/- mice display low serum levels of inflammatory cytokine IL-6 and correspondingly an increased survival to a systemic infection with Lm. The impaired bacterial clearance in the absence of HDAC6 appears to be caused by a defect in autophagy. Hence, Hdac6-/- BMDCs accumulate higher levels of the autophagy marker p62 and show defective phagosome-lysosome fusion. These data underline the important function of HDAC6 in dendritic cells not only in bacterial autophagy, but also in the proper activation of TLR signalling. These results thus demonstrate an important regulatory role for HDAC6 in the innate immune response to intracellular bacterial infection.

miRNA profiling during antigen-dependent T cell activation: A role for miR-132-3p.

microRNAs (miRNAs) are tightly regulated during T lymphocyte activation to enable the establishment of precise immune responses. Here, we analyzed the changes of the miRNA profiles of T cells in response to activation by cognate interaction with dendritic cells. We also studied mRNA targets common to miRNAs regulated in T cell activation. pik3r1 gene, which encodes the regulatory subunits of PI3K p50, p55 and p85, was identified as target of miRNAs upregulated after T cell activation. Using 3'UTR luciferase reporter-based and biochemical assays, we showed the inhibitory relationship between miR-132-3p upregulation and expression of the pik3r1 gene. Our results indicate that specific miRNAs whose expression is modulated during T cell activation might regulate PI3K signaling in T cells.

CD9 Regulates Major Histocompatibility Complex Class II Trafficking in Monocyte-Derived Dendritic Cells.

Antigen presentation by dendritic cells (DCs) stimulates naive CD4+ T cells, triggering T cell activation and the adaptive arm of the immune response. Newly synthesized major histocompatibility complex class II (MHC-II) molecules accumulate at MHC-II-enriched endosomal compartments and are transported to the plasma membrane of DCs after binding to antigenic peptides to enable antigen presentation. In DCs, MHC-II molecules are included in tetraspanin-enriched microdomains (TEMs). However, the role of tetraspanin CD9 in these processes remains largely undefined. Here, we show that CD9 regulates the T cell-stimulatory capacity of granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent bone marrow-derived DCs (BMDCs), without affecting antigen presentation by fms-like tyrosine kinase 3 ligand (Flt3L)-dependent BMDCs. CD9 knockout (KO) GM-CSF-dependent BMDCs, which resemble monocyte-derived DCs (MoDCs), induce lower levels of T cell activation than wild-type DCs, and this effect is related to a reduction in MHC-II surface expression in CD9-deficient MoDCs. Importantly, MHC-II targeting to the plasma membrane is largely impaired in immature CD9 KO MoDCs, in which MHC-II remains arrested in acidic intracellular compartments enriched in LAMP-1 (lysosome-associated membrane protein 1), and MHC-II internalization is also blocked. Moreover, CD9 participates in MHC-II trafficking in mature MoDCs, regulating its endocytosis and recycling. Our results demonstrate that the tetraspanin CD9 specifically regulates antigenic presentation in MoDCs through the regulation of MHC-II intracellular trafficking.

Tetraspanin CD9 Limits Mucosal Healing in Experimental Colitis.

Tetraspanins are a family of proteins with four transmembrane domains that associate between themselves and cluster with other partner proteins, conforming a distinct class of membrane domains, the tetraspanin-enriched microdomains (TEMs). These TEMs constitute macromolecular signaling platforms that regulate key processes in several cellular settings controlling signaling thresholds and avidity of receptors. In this study, we investigated the role of CD9, a tetraspanin that regulates major biological processes such as cell migration and immunological responses, in two mouse models of colitis that have been used to study the pathogenesis of inflammatory bowel disease (IBD). Previous in vitro studies revealed an important role in the interaction of leukocytes with inflamed endothelium, but in vivo evidence of the involvement of CD9 in inflammatory diseases is scarce. Here, we studied the role of CD9 in the pathogenesis of colitis in vivo. Colitis was induced by administration of dextran sodium sulfate (DSS), a chemical colitogen that causes epithelial disruption and intestinal inflammation. CD9-/- mice showed less severe colitis than wild-type counterparts upon exposure to DSS (2% solution) and enhanced survival in response to a lethal DSS dose (4%). Decreased neutrophil and macrophage cell infiltration was observed in colonic tissue from CD9-/- animals, in accordance with their lower serum levels of TNF-α, IL-6, and other proinflammatory cytokines in the colon. The specific role of CD9 in IBD was further dissected by transfer of CD4+ CD45RBhi naive T cells into the Rag1-/- mouse colitis model. However, no significant differences were observed in these settings between both groups, ruling out a role for CD9 in IBD in the lymphoid compartment. Experiments with bone marrow chimeras revealed that CD9 in the non-hematopoietic compartment is involved in colon injury and limits the proliferation of epithelial cells. Our data indicate that CD9 in non-hematopoietic cells plays an important role in colitis by limiting epithelial cell proliferation. Future strategies to repress CD9 expression may be of therapeutic benefit in the treatment of IBD.

CD81 controls immunity to Listeria infection through rac-dependent inhibition of proinflammatory mediator release and activation of cytotoxic T cells.

Despite recent evidence on the involvement of CD81 in pathogen binding and Ag presentation by dendritic cells (DCs), the molecular mechanism of how CD81 regulates immunity during infection remains to be elucidated. To investigate the role of CD81 in the regulation of defense mechanisms against microbial infections, we have used the Listeria monocytogenes infection model to explore the impact of CD81 deficiency in the innate and adaptive immune response against this pathogenic bacteria. We show that CD81(-/-) mice are less susceptible than wild-type mice to systemic Listeria infection, which correlates with increased numbers of inflammatory monocytes and DCs in CD81(-/-) spleens, the main subsets controlling early bacterial burden. Additionally, our data reveal that CD81 inhibits Rac/STAT-1 activation, leading to a negative regulation of the production of TNF-α and NO by inflammatory DCs and the activation of cytotoxic T cells by splenic CD8α(+) DCs. In conclusion, this study demonstrates that CD81-Rac interaction exerts an important regulatory role on the innate and adaptive immunity against bacterial infection and suggests a role for CD81 in the development of novel therapeutic targets during infectious diseases.

The leukocyte activation receptor CD69 controls T cell differentiation through its interaction with galectin-1.

CD69 is involved in immune cell homeostasis, regulating the T cell-mediated immune response through the control of Th17 cell differentiation. However, natural ligands for CD69 have not yet been described. Using recombinant fusion proteins containing the extracellular domain of CD69, we have detected the presence of a ligand(s) for CD69 on human dendritic cells (DCs). Pulldown followed by mass spectrometry analyses of CD69-binding moieties on DCs identified galectin-1 as a CD69 counterreceptor. Surface plasmon resonance and anti-CD69 blocking analyses demonstrated a direct and specific interaction between CD69 and galectin-1 that was carbohydrate dependent. Functional assays with both human and mouse T cells demonstrated the role of CD69 in the negative effect of galectin-1 on Th17 differentiation. Our findings identify CD69 and galectin-1 to be a novel regulatory receptor-ligand pair that modulates Th17 effector cell differentiation and function.

CD69 modulates sphingosine-1-phosphate-induced migration of skin dendritic cells.

In this study, we have investigated the role of CD69, an early inducible leukocyte activation receptor, in murine dendritic cell (DC) differentiation, maturation, and migration. Skin DCs and DC subsets present in mouse lymphoid organs express CD69 in response to maturation stimuli. Using a contact sensitization model, we show that skin DCs migrated more efficiently to draining lymph nodes (LNs) in the absence of CD69. This was confirmed by subcutaneous transfer of CD69-/- DCs, which presented an increased migration to peripheral LNs. Two-photon microscopy analysis showed that once DCs reached the LNs, CD69 deficiency did not alter DC interstitial motility in the LNs. Chemotaxis to sphingosine-1-phosphate (S1P) was enhanced in CD69-/- DCs compared with wild-type DCs. Accordingly, we detected a higher expression of S1P receptor type-1 (S1P(1)) by CD69-/- DCs, whereas S1P(3) expression levels were similar in wild-type and CD69-/- DCs. Moreover, in vivo treatment with S1P analogs SEW2871 and FTY720 during skin sensitization reduced skin DC migration to peripheral LNs. These results suggest that CD69 regulates S1P-induced skin DC migration by modulating S1P(1) function. Together, our findings increase our knowledge on DC trafficking patterns in the skin, enabling the development of new directed therapies using DCs for antigen (Ag) delivery.

CD69 limits the severity of cardiomyopathy after autoimmune myocarditis.

BACKGROUND: Experimental autoimmune myocarditis (EAM), a mouse model of post-infectious cardiomyopathy, reflects mechanisms of inflammatory cardiomyopathy in humans. EAM is characterized by an infiltration of inflammatory cells into the myocardium that can be followed by myocyte fibrosis, edema, and necrosis, leading to ventricular wall dysfunction and heart failure. Different data indicate that CD69 exerts an important immunoregulatory effect in vivo. However, the possible role of CD69 in autoimmune myocarditis has not been studied. METHODS AND RESULTS: We have explored the role of the leukocyte regulatory molecule CD69 in the inflammation that leads to cardiac dysfunction after myocardial injury in EAM. We have found that after induction of EAM, the draining lymph nodes from CD69-deficient mice developed an exacerbated Th17 inflammatory response, resulting in increases in the numbers of infiltrating leukocytes in the myocardium. In the chronic phase of EAM, transthoracic echocardiography revealed a significantly reduced left ventricular fractional shortening and a decreased ejection fraction in CD69-deficient mice, indicative of an impaired cardiac contractility. This condition was accompanied by a greater extent of myocardial fibrosis, an elevated number of sinus pauses on ECG, and an enhanced ratio of heart weight to body weight in CD69-/- mice. Moreover, both bone marrow transplantation and adoptive transfer of Th17 cells isolated from immunized CD69-/- mice with EAM into naive wild-type recipients reproduced the severity of the disease, demonstrating that CD69 exerts its function within the lymphocyte compartment. CONCLUSION: Our findings indicate that CD69 negatively regulates heart-specific Th17 responses, cardiac inflammation, and heart failure progression in EAM.

CD69 association with Jak3/Stat5 proteins regulates Th17 cell differentiation.

T-cell differentiation involves the early decision to commit to a particular pattern of response to an antigen. Here, we show that the leukocyte activation antigen CD69 limits differentiation into proinflammatory helper T cells (Th17 cells). Upon antigen stimulation in vitro, CD4(+) T cells from CD69-deficient mice generate an expansion of Th17 cells and the induction of greater mRNA expression of interleukin 17 (IL-17), IL 23 receptor (IL-23R), and the nuclear receptor retinoic acid-related orphan receptor γt (RORγt). In vivo studies with CD69-deficient mice bearing OTII T-cell receptors (TCRs) specific for OVA peptide showed a high proportion of antigen-specific Th17 subpopulation in the draining lymph nodes, as well as in CD69-deficient mice immunized with type II collagen. Biochemical analysis demonstrated that the CD69 cytoplasmic tail associates with the Jak3/Stat5 signaling pathway, which regulates the transcription of RORγt and, consequently, differentiation toward the Th17 lineage. Functional experiments in Th17 cultures demonstrated that the selective inhibition of Jak3 activation enhanced the transcription of RORγt. Moreover, the addition of exogenous IL-2 restored Stat5 phosphorylation and inhibited the enhanced Th17 differentiation in CD69-deficient cells. These results support the early activation receptor CD69 as an intrinsic modulator of the T-cell differentiation program that conditions immune inflammatory processes.

The leukocyte activation antigen CD69 limits allergic asthma and skin contact hypersensitivity.

BACKGROUND: Allergic diseases have a major health care impact in industrialized countries. The development of these diseases is influenced by exposure to allergen and to immunological and genetic factors. However, the molecular mechanisms underlying the inflammatory response that triggers allergy are not well defined. OBJECTIVE: We have investigated the role of the leukocyte activation antigen CD69 in the regulation of two allergic diseases, asthma and contact dermatitis. METHODS: Analysis of two models of allergic diseases in CD69 knockout and wild-type mice: ovalbumin-induced allergic airway inflammation (BALB/c genetic background) and contact hypersensitivity to oxazolone (C57BL/6J genetic background). RESULTS: CD69 deficiency dramatically enhanced the inflammatory response in the ovalbumin-induced asthma model of antigen-induced airway allergy. CD69 knockout mice showed exacerbated pulmonary eosinophil recruitment, high vascular cell adhesion molecule 1 expression levels in lung vasculature, and enhanced T(H)2 and T(H)17 cytokines in the bronchoalveolar space and lung tissue. In the hapten-induced cutaneous contact hypersensitivity model, both CD69 deficiency and treatment with anti-CD69 mAb increased inflammation. Treatment with contact allergens induced enhanced T(H)1 and T(H)17 responses in CD69 deficient mice, and neutralizing anti-IL-17 antibodies reduced skin inflammation. In both experimental systems, adoptive transfer of lymph node cells from CD69 knockout mice increased the inflammatory response in recipient mice. CONCLUSION: These results demonstrate that the early activation receptor CD69 is an intrinsic modulator of immune allergic processes through the negative regulation of allergen-induced T-cell effector responses.

BMP-7 blocks mesenchymal conversion of mesothelial cells and prevents peritoneal damage induced by dialysis fluid exposure.

BACKGROUND: During peritoneal dialysis (PD), mesothelial cells (MC) undergo an epithelial-to-mesenchymal transition (EMT), and this process is associated with peritoneal membrane (PM) damage. Bone morphogenic protein-7 (BMP-7) antagonizes transforming growth factor (TGF)-beta1, modulates EMT and protects against fibrosis. Herein, we analysed the modulating role of BMP-7 on EMT of MC in vitro and its protective effects in a rat PD model. METHODS: Epitheliod or non-epitheliod MC were analysed for the expression of BMP-7, TGF-beta1, activated Smads, epithelial cadherin (E-cadherin), collagen I, alpha smooth muscle cell actin (alpha-SMA) and vascular endothelial growth factor (VEGF) using standard procedures. Rats were daily instilled with PD fluid with or without BMP-7 during 5 weeks. Histological analyses were carried out in parietal peritoneum. Fibrosis was quantified with van Gieson or Masson's trichrome staining. Vasculature, activated macrophages and invading MC were quantified by immunofluorescence analysis. Quantification of infiltrating leukocytes and MC density in liver imprints was performed by May-Grünwald-Giemsa staining. Hyaluronic acid levels were determined by ELISA. RESULTS: MC constitutively expressed BMP-7, and its expression was downregulated during EMT. Treatment with recombinant BMP-7 resulted in blockade of TGF-beta1-induced EMT of MC. We provide evidence of a Smad-dependent mechanism for the blockade of EMT. Exposure of rat peritoneum to PD fluid resulted in inflammatory and regenerative responses, invasion of the compact zone by MC, fibrosis and angiogenesis. Administration of BMP-7 decreased the number of invading MC and reduced fibrosis and angiogenesis. In contrast, BMP-7 had no effect on inflammatory and regenerative responses, suggesting that these are EMT-independent, and probably upstream, processes. CONCLUSIONS: Data point to a balance between BMP-7 and TGF-beta1 in the control of EMT and indicate that blockade of EMT may be a therapeutic approach to ameliorate peritoneal membrane damage during PD.

Cyclooxygenase-2 mediates dialysate-induced alterations of the peritoneal membrane.

During peritoneal dialysis (PD), exposure of the peritoneal membrane to nonphysiologic solutions causes inflammation, ultimately leading to altered structure and function. Myofibroblasts, one of the cell types that contribute to dysfunction of the peritoneal membrane, can originate from mesothelial cells (MCs) by epithelial-to-mesenchymal transition (EMT), a process that has been associated with an increased rate of peritoneal transport. Because cyclooxygenase-2 (COX-2) is induced by inflammation, we studied the role of COX-2 in the deterioration of the peritoneal membrane. We observed that nonepithelioid MCs found in peritoneal effluent expressed higher levels of COX-2 than epithelioid MCs. The mass transfer coefficient for creatinine correlated with MC phenotype and with COX-2 levels. Although COX-2 was upregulated during EMT of MCs in vitro, COX-2 inhibition did not prevent EMT. In a mouse model of PD, however, COX-2 inhibition with Celecoxib resulted in reduced fibrosis and in partial recovery of ultrafiltration, outcomes that were associated with a reduction of inflammatory cells. Furthermore, PD fluid with a low content of glucose degradation products did not induce EMT or COX-2; the peritoneal membranes of mice treated with this fluid showed less worsening than mice exposed to standard fluid. In conclusion, upregulation of COX-2 during EMT may mediate peritoneal inflammation, suggesting COX-2 inhibition as a potential strategy to ameliorate peritoneal deterioration in PD patients.

Ex vivo analysis of dialysis effluent-derived mesothelial cells as an approach to unveiling the mechanism of peritoneal membrane failure.

During peritoneal dialysis (PD), the peritoneum is exposed to bioincompatible dialysis fluids, which causes progressive fibrosis and angiogenesis and, ultimately, ultrafiltration failure. In addition, repeated episodes of peritonitis or hemoperitoneum may accelerate all these processes. Fibrosis has been classically considered the main cause of peritoneal membrane functional decline. However, in parallel with fibrosis, the peritoneum also displays increases in capillary number (angiogenesis) and vasculopathy in response to PD. Nowadays, there is emerging evidence pointing to peritoneal microvasculature as the main factor responsible for increased solute transport and ultrafiltration failure. However, the pathophysiologic mechanism(s) involved in starting and maintaining peritoneal fibrosis and angiogenesis remain(s) elusive. Peritoneal stromal fibroblasts have been considered (for many years) the cell type mainly involved in structural and functional alterations of the peritoneum; whereas mesothelial cells have been considered mere victims of peritoneal injury caused by PD. Recently, ex vivo cultures of effluent-derived mesothelial cells, in conjunction with immunohistochemical analysis of peritoneal biopsies from PD patients, have identified mesothelial cells as culprits, at least in part, in peritoneal membrane deterioration. This review discusses recent findings that suggest new peritoneal myofibroblastic cells may arise from local conversion of mesothelial cells by epithelial-to-mesenchymal transition during the repair responses that take place in PD. The transdifferentiated mesothelial cells may retain a permanent mesenchymal state, as long as initiating stimuli persist, and contribute to PD-induced fibrosis and angiogenesis, and hence to membrane failure. Future therapeutic interventions could be designated in order to prevent or reverse epithelial-to-mesenchymal transition of mesothelial cells, or its pernicious effects.