Communication, construction, and fluid control: lymphoid organ fibroblastic reticular cell and conduit networks.

Fibroblastic reticular cells (FRCs) are a crucial part of the stromal cell infrastructure of secondary lymphoid organs (SLOs). Lymphoid organ fibroblasts form specialized niches for immune cell interactions and thereby govern lymphocyte activation and differentiation. Moreover, FRCs produce and ensheath a network of extracellular matrix (ECM) microfibers called the conduit system. FRC-generated conduits contribute to fluid and immune cell control by funneling fluids containing antigens and inflammatory mediators through the SLOs. We review recent progress in FRC biology that has advanced our understanding of immune cell functions and interactions. We discuss the intricate relationships between the cellular FRC and the fibrillar conduit networks, which together form the basis for efficient communication between immune cells and the tissues they survey.

Fibroblastic reticular cell response to dendritic cells requires coordinated activity of podoplanin, CD44 and CD9.

In adaptive immunity, CLEC-2+ dendritic cells (DCs) contact fibroblastic reticular cells (FRCs) inhibiting podoplanin-dependent actomyosin contractility, permitting FRC spreading and lymph node expansion. The molecular mechanisms controlling lymph node remodelling are incompletely understood. We asked how podoplanin is regulated on FRCs in the early phase of lymph node expansion, and which other proteins are required for the FRC response to DCs. We find that podoplanin and its partner proteins CD44 and CD9 are differentially expressed by specific lymph node stromal populations in vivo, and their expression in FRCs is coregulated by CLEC-2 (encoded by CLEC1B). Both CD44 and CD9 suppress podoplanin-dependent contractility. We find that beyond contractility, podoplanin is required for FRC polarity and alignment. Independently of podoplanin, CD44 and CD9 affect FRC-FRC interactions. Furthermore, our data show that remodelling of the FRC cytoskeleton in response to DCs is a two-step process requiring podoplanin partner proteins CD44 and CD9. Firstly, CLEC-2 and podoplanin binding inhibits FRC contractility, and, secondly, FRCs form protrusions and spread, which requires both CD44 and CD9. Together, we show a multi-faceted FRC response to DCs, which requires CD44 and CD9 in addition to podoplanin.

Characterization of human fibroblastic reticular cells as potential immunotherapeutic tools.

Fibroblastic reticular cells (FRCs) are essential players during adaptive immune responses not only as a structural support for the encounter of antigen-presenting cells and naive T lymphocytes but also as a source of modulatory signals. However, little is known about this cell population in humans. To address the phenotypical and functional analysis of human FRCs here we established splenic (SP) and mesenteric lymph node (LN) CD45-CD31-CD90+podoplanin+ myofibroblastic cell cultures. They shared the phenotypical characteristics distinctive of FRCs, including the expression of immunomodulatory factors and peripheral tissue antigens. Nevertheless, human FRCs also showed particular features, some differing from mouse FRCs, like the lack of nitric oxide synthase (NOS2) expression after interferon (IFN)γstimulation. Interestingly, SP-FRCs expressed higher levels of interleukin (IL)-6, BMP4, CCL2, CXCL12 and Notch molecules, and strongly adapted their functional profile to lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (Poly I:C) and IFNγ stimulation. In contrast, we found higher expression of transforming growth factor (TGF)ß and Activin A in LN-FRCs that barely responded via Toll-Like Receptor (TLR)3 and constitutively expressed retinaldehyde dehydrogenase 1 enzyme, absent in SP-FRCs. This study reveals human FRCs can be valuable models to increase our knowledge about the physiology of human secondary lymphoid organs in health and disease and to explore the therapeutic options of FRCs.

Autocrine activation of canonical BMP signaling regulates PD-L1 and PD-L2 expression in human dendritic cells.

Bone morphogenetic proteins (BMPs) are multifunctional growth factors regulating differentiation and proliferation in numerous systems including the immune system. Previously, we described that the BMP signaling pathway is functional in human monocyte-derived dendritic cells (MoDCs), which were found to express both the specific receptors and the Smad proteins required for signal transduction. In this study, we provide evidence that human MoDCs produce BMP-4 and that this production is increased over the maturation process as is BMP signal transduction. When DCs are matured in the presence of an inhibitor of the BMP pathway, the expression of the maturation markers PD-L1 and PD-L2 is reduced, while cytokine production is not affected. As a result, these mature DCs present an augmented ability to stimulate both T cells and NK cells. Eventually, the inhibition of BMP signaling during maturation causes a reduced expression of IRF-1, a transcription factor that positively regulates the expression of PD-L1 and PD-L2. The present study indicates that the BMP signaling pathway regulates PD-L1 and PD-L2 expression in human MoDCs during the maturation process, probably through the IRF-1 transcription factor, and also points out that the manipulation of BMP signaling might considerably improve the immunogenicity of MoDCs used in immunotherapy.

A discrete population of IFN λ-expressing BDCA3hi dendritic cells is present in human thymus.

Human thymus contains two major subpopulations of dendritic cells (DCs), conventional DCs (cDCs) and plasmacytoid DCs (pDCs), which are mainly involved in central tolerance and also in protecting the thymus against infections. In blood and peripheral organs cDCs include the subpopulation of BDCA3(hi) DCs, considered as equivalents to mouse CD8α(+) DCs. In this study we describe in human thymus the presence of a discrete population of BDCA3(hi) DCs that, like their peripheral counterparts, express CD13, low-intermediate levels of CD11c, CLEC9A, high levels of XCR1, IRF8 and TLR3, and mostly lack the expression of CD11b, CD14 and TLR7. Thymic BDCA3(hi) DCs display immature features with a low expression of costimulatory molecules and HLA-DR, and a low allostimulatory capacity. Also, BDCA3(hi) DCs exhibit a strong response to TLR3 stimulation, producing high levels of interferon (IFN)-λ1 and CXCL10, which indicates that, similarly to thymic pDCs, BDCA3(hi) DCs can have an important role in thymus protection against viral infections.

Expression of BMPRIA on human thymic NK cell precursors: role of BMP signaling in intrathymic NK cell development.

The bone morphogenetic protein (BMP) signaling pathway regulates survival, proliferation, and differentiation of several cell types in multiple tissues, including the thymus. Previous reports have shown that BMP signaling negatively regulates T-cell development. Here, we study the subpopulation of early human intrathymic progenitors expressing the type IA BMP receptor (BMPRIA) and provide evidence that CD34(+)CD1a(-)BMPRIA(+) precursor cells mostly express surface cell markers and transcription factors typically associated with NK cell lineage. These CD34(+) cells mostly differentiate into functional CD56(+) natural killer (NK) cells when they are cocultured with thymic stromal cells in chimeric human-mouse fetal thymic organ cultures and also in the presence of SCF and IL-15. Moreover, autocrine BMP signaling can promote the differentiation of thymic NK cells by regulating the expression of key transcription factors required for NK cell lineage (eg, Id3 and Nfil3) as well as one of the components of IL-15 receptor, CD122. Subsequently, the resulting population of IL-15-responsive NK cell precursors can be expanded by IL-15, whose action is mediated by BMP signaling during the last steps of thymic NK cell differentiation. Our results strongly suggest that BMPRIA expression identifies human thymic NK cell precursors and that BMP signaling is relevant for NK cell differentiation in the human thymus.

Modulation of tumor microenvironment by targeting histone acetylation in bladder cancer.

Alterations in the epigenetic machinery in both tumor and immune cells contribute to bladder cancer (BC) development, constituting a promising target as an alternative therapeutic option. Here, we have explored the effects of a novel histone deacetylase (HDAC) inhibitor CM-1758, alone or in combination with immune checkpoint inhibitors (ICI) in BC. We determined the antitumor effects of CM-1758 in various BC cell lines together with the induction of broad transcriptional changes, with focus on the epigenetic regulation of PD-L1. Using an immunocompetent syngeneic mouse model of metastatic BC, we studied the effects of CM-1758 alone or in combination with anti-PD-L1 not only on tumor cells, but also in the tumor microenvironment. In vitro, we found that CM-1758 has cytotoxic and cytostatic effects either by inducing apoptosis or cell cycle arrest in BC cells at low micromolar levels. PD-L1 is epigenetically regulated by histone acetylation marks and is induced after treatment with CM-1758. We also observed that treatment with CM-1758 led to an important delay in tumor growth and a higher CD8 + T cell tumor infiltration. Moreover, anti-PD-L1 alone or in combination with CM-1758 reprogramed macrophage differentiation towards a M1-like polarization state and increased of pro-inflammatory cytokines systemically, yielding potential further antitumor effects. Our results suggest the possibility of combining HDAC inhibitors with immunotherapies for the management of advanced metastatic BC.

Wnt5a skews dendritic cell differentiation to an unconventional phenotype with tolerogenic features.

Dendritic cells (DCs) are critical regulators of immune responses that integrate signals from the innate and adaptive immune system and orchestrate T cell responses toward either immunity or tolerance. Growing evidence points to the Wnt signaling pathway as a pivotal piece in the immune balance and focuses on DCs as a direct target for their immunoregulatory role. Our results show that the increase in Wnt5a signaling during the differentiation of human DCs from monocytes alters their phenotype and compromises their subsequent capacity to mature in response to TLR-dependent stimuli. These Wnt5a-DCs produce scant amounts of IL-12p70 and TNF-α but increased levels of IL-10. Consequently, these Wnt5a-DCs have a reduced capacity to induce Th1 responses that promote IL-10 secretion by CD4 T cells. Changes in the transcriptional profile of Wnt5a-DCs correlate with their unconventional phenotype caused presumably by increased IL-6/IL-10 signaling during the process of DC differentiation. The effect of Wnt5a is not a consequence of ß-catenin accumulation but is dependent on noncanonical Ca(2+)/calmodulin-dependent protein kinase II/NF-κB signaling. Our results therefore suggest that under high levels of Wnt5a, typical of the inflammatory state and sepsis, monocytes could differentiate into unconventional DCs with tolerogenic features.

Toward Tumor Fight and Tumor Microenvironment Remodeling: PBA Induces Cell Cycle Arrest and Reduces Tumor Hybrid Cells' Pluripotency in Bladder Cancer.

Bladder cancer (BC) is the second most frequent cancer of the genitourinary system. The most successful therapy since the 1970s has consisted of intravesical instillations of Bacillus Calmette-Guérin (BCG) in which the tumor microenvironment (TME), including macrophages, plays an important role. However, some patients cannot be treated with this therapy due to comorbidities and severe inflammatory side effects. The overexpression of histone deacetylases (HDACs) in BC has been correlated with macrophage polarization together with higher tumor grades and poor prognosis. Herein we demonstrated that phenylbutyrate acid (PBA), a HDAC inhibitor, acts as an antitumoral compound and immunomodulator. In BC cell lines, PBA induced significant cell cycle arrest in G1, reduced stemness markers and increased PD-L1 expression with a corresponding reduction in histone 3 and 4 acetylation patterns. Concerning its role as an immunomodulator, we found that PBA reduced macrophage IL-6 and IL-10 production as well as CD14 downregulation and the upregulation of both PD-L1 and IL-1ß. Along this line, PBA showed a reduction in IL-4-induced M2 polarization in human macrophages. In co-cultures of BC cell lines with human macrophages, a double-positive myeloid-tumoral hybrid population (CD11b+EPCAM+) was detected after 48 h, which indicates BC cell-macrophage fusions known as tumor hybrid cells (THC). These THC were characterized by high PD-L1 and stemness markers (SOX2, NANOG, miR-302) as compared with non-fused (CD11b-EPCAM+) cancer cells. Eventually, PBA reduced stemness markers along with BMP4 and IL-10. Our data indicate that PBA could have beneficial properties for BC management, affecting not only tumor cells but also the TME.

The canonical BMP signaling pathway is involved in human monocyte-derived dendritic cell maturation.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-ß superfamily, are multifunctional polypeptides regulating a broad spectrum of functions in embryonic and adult tissues. Recent reports have demonstrated that BMPs regulate the survival, proliferation and differentiation of several cell types in the immune system. In this study, we investigate the effects of BMP signaling activation on the capacity of human dendritic cells (DCs) to stimulate immune responses. Human DCs express type I and type II BMP receptors (BMPRIA, BMPRIB, type IA activin receptor, BMPRII) and BMP signal transduction molecules (Smad1, 5, and 8, as well as Smad4). On BMP stimulation, Id1-3 (inhibitor of differentiation 1-3/DNA binding) mRNA expression is upregulated and this effect can be blocked with the inhibitor dorsomorphin, showing that the canonical BMP signal transduction pathway is functionally active in DCs. BMP signaling activation promotes the phenotypic maturation of human DCs by increasing the expression of co-stimulatory molecules and also CD83, programmed cell death ligand 1 (PD-L1) and PD-L2, and stimulates cytokine secretion, mainly interleukin-8 and tumor necrosis factor-α. Accordingly, BMP-treated DCs exhibit an enhanced T-cell stimulatory capacity. BMP signaling also enhances the survival of human DCs increasing the Bcl-2/Bax ratio. Finally, the expression of Runx transcription factors is increased in mature DCs, and the mRNA levels of Runx1-3 are upregulated in response to BMP stimulation, indicating that Runx transcription factor family may mediate the effects of BMP signaling in human DC maturation.

Podoplanin drives dedifferentiation and amoeboid invasion of melanoma.

Melanoma is an aggressive skin cancer developing from melanocytes, frequently resulting in metastatic disease. Melanoma cells utilize amoeboid migration as mode of local invasion. Amoeboid invasion is characterized by rounded cell morphology and high actomyosin contractility driven by Rho GTPase signalling. Migrastatic drugs targeting actin polymerization and contractility are therefore a promising treatment option for metastatic melanoma. To predict amoeboid invasion and metastatic potential, biomarkers functionally linked to contractility pathways are needed. The glycoprotein podoplanin drives actomyosin contractility in lymphoid fibroblasts and is overexpressed in many cancers. We show that podoplanin enhances amoeboid invasion in melanoma. Podoplanin expression in murine melanoma drives rounded cell morphology, increasing motility, and invasion in vivo. Podoplanin expression is increased in a subset of dedifferentiated human melanoma, and in vitro is sufficient to upregulate melanoma-associated marker Pou3f2/Brn2. Together, our data define podoplanin as a functional biomarker for dedifferentiated invasive melanoma and a promising migrastatic therapeutic target.

The CXCL12/CXCR4 pair in aged human thymus.

CXCL12 is an important CXC chemokine involved in numerous biological processes. We had previously demonstrated the synergistic participation of CXCL12 and IL-7 in the control of both survival and proliferation of CD34(+) human thymic lymphoid progenitors. On this basis, we hypothesize a presumptive role for CXCL12 and its receptor, CXCR4, in the thymus involution. In this respect, in the current report we describe the expression of both molecules in the human thymus during aging. Our results demonstrate that, despite the profound alterations observed in the thymic epithelial microenvironment of aged thymuses, the proportions of different CD4/CD8 thymocyte subsets do not undergo significant variations. Remarkably, a strong CXCL12 expression was found in older thymuses, which appeared in the same locations as in younger thymuses: the subcapsulary and medullary areas. The proportions of CXCR4(+) cells, most of them belonging to the CD3(-) compartment, showed no important variations in the older thymuses. However, within the CD34(+) cell population, a significant reduction in the expression of CXCR4 molecules was observed.

Survival and function of human thymic dendritic cells are dependent on autocrine Hedgehog signaling.

The Hedgehog (Hh) family of signaling molecules functions in the development of numerous tissues during embryogenesis and has also been involved in adult self-renewing tissues. Recent results have demonstrated that the different components of the Hh signaling pathway are expressed in the human thymus. In this study, we investigate whether thymic dendritic cells (DCs) are cell targets for Hh signaling. Both components of the Hh receptor, Patched and Smoothened, as well as other Hh-binding proteins with modulating functions, are expressed by human thymic DCs. The expression of Gli1, Gli2, and Gli3 transcription factors suggests that the Hh signaling pathway is active in thymic DCs, and approximately one-half of thymic DCs produces Sonic Hh (Shh). The culture of thymic DCs with Shh protects them from apoptosis [similarly to CD40 ligand (CD40L)], and these antiapoptotic effects are related to an up-regulation of Bcl-2 and Bcl-X(L) protein expression. The addition of the Hh pathway inhibitor, cyclopamine, decreases DC viability and impairs their allostimulatory function in vitro. In addition, the blockade of the Hh signaling pathway by cyclopamine treatment abrogates the up-regulation of HLA-DR, CD86, CD80, and CD83 expression induced by CD40L on thymic DCs. Finally, we also show that after activation with CD40L thymic DCs down-regulate the expression of Hh receptor components as well as Shh production. Taken together, these results suggest that the survival and function of thymic DCs are regulated by an autocrine Hh signaling.

Immunotherapy: breaching the barriers for cancer treatment.

The great ambition to treat cancer through harnessing a patient's own immune responses has started to become reality. Clinical trials have shown impressive results and some patients reaching the end of existing treatment options have achieved full remission. Yet the response rate even within the most promising trials remain at just 30-40% of patients. To date, the focus of immunotherapy research has been to identify tumour antigens, and to enhance activation of effector lymphocytes. Yet this is only the first step to effective immunotherapy for a broader range of patients. Activated cytotoxic T cells can only act on their tumour cell targets if they have free and easy access to all tumour regions. Solid tumours are complex, heterogeneous environments which vary greatly in their physical properties. We must now focus our efforts on understanding how factors such as the composition, density and geometry of tumour extracellular matrix acts to impede or promote immune cell infiltration and activation, and work to design novel pharmacological interventions which restore and enhance leucocyte trafficking within solid tumours. This article is part of a discussion meeting issue 'Forces in cancer: interdisciplinary approaches in tumour mechanobiology'.

Epigenetics of Bladder Cancer: Where Biomarkers and Therapeutic Targets Meet.

Bladder cancer (BC) is the most common neoplasia of the urothelial tract. Due to its high incidence, prevalence, recurrence and mortality, it remains an unsolved clinical and social problem. The treatment of BC is challenging and, although immunotherapies have revealed potential benefit in a percentage of patients, it remains mostly an incurable disease at its advanced state. Epigenetic alterations, including aberrant DNA methylation, altered chromatin remodeling and deregulated expression of non-coding RNAs are common events in BC and can be driver events in BC pathogenesis. Accordingly, these epigenetic alterations are now being used as potential biomarkers for these disorders and are being envisioned as potential therapeutic targets for the future management of BC. In this review, we summarize the recent findings in these emerging and exciting new aspects paving the way for future clinical treatment of this disease.

Fibroblastic Reticular Cells Control Conduit Matrix Deposition during Lymph Node Expansion.

Lymph nodes (LNs) act as filters, constantly sampling peripheral cues. This is facilitated by the conduit network, a tubular structure of aligned extracellular matrix (ECM) fibrils ensheathed by fibroblastic reticular cells (FRCs). LNs undergo rapid 3- to 5-fold expansion during adaptive immune responses, but these ECM-rich structures are not permanently damaged. Whether conduit flow or filtering function is affected during LN expansion is unknown. Here, we show that conduits are partially disrupted during acute LN expansion, but FRC-FRC contacts remain connected. We reveal that polarized FRCs deposit ECM basolaterally using LL5-ß and that ECM production is regulated at transcriptional and secretory levels by the C-type lectin CLEC-2, expressed by dendritic cells. Inflamed LNs maintain conduit size exclusion, and flow is disrupted but persists, indicating the robustness of this structure despite rapid tissue expansion. We show how dynamic communication between peripheral tissues and LNs provides a mechanism to prevent inflammation-induced fibrosis in lymphoid tissue.

CXCL12/CXCR4 signaling promotes human thymic dendritic cell survival regulating the Bcl-2/Bax ratio.

CXCL12, a member of the chemokine CXC subfamily, and its physiologic receptor CXCR4 are essential for the development of various organs during embryonic development and are also involved in the control of cell survival, proliferation and migration in adult tissues. In the human thymus, CXCL12 is produced by epithelial cells located in the subcapsular and medullary regions and CXCR4 is expressed in different thymocyte subpopulations. Several results have demonstrated that CXCL12/CXCR4 signaling participates in different intrathymic processes including the control of human precursor cell survival and proliferation, and the exit of mature thymocytes to the periphery. In this study, we show that CXCL12 is also produced by human thymic dendritic cells (DCs), most of which express CXCR4 receptor. The addition of exogenous CXCL12 significantly inhibited the serum depletion-induced apoptosis in thymic DCs, and the treatment with neutralizing antibodies against CXCL12 or CXCR4 decreased their survival. The survival-promoting effect of CXCL12 was mediated by the up-regulation of Bcl-2 protein expression and the concomitant down-regulation of Bax protein expression. The higher viability of thymic DCs also enhanced their allostimulatory capacity. Taken together, the results suggest a new function of CXCL12 in the human thymus controlling the survival and functionality of thymic DCs.

Overexpression of hypoxia-inducible factor 1 alpha improves immunomodulation by dental mesenchymal stem cells.

BACKGROUND: Human dental mesenchymal stem cells (MSCs) are considered as highly accessible and attractive MSCs for use in regenerative medicine, yet some of their features are not as well characterized as other MSCs. Hypoxia-preconditioning and hypoxia-inducible factor 1 (HIF-1) alpha overexpression significantly improves MSC therapeutics, but the mechanisms involved are not fully understood. In the present study, we characterize immunomodulatory properties of dental MSCs and determine changes in their ability to modulate adaptive and innate immune populations after HIF-1 alpha overexpression. METHODS: Human dental MSCs were stably transduced with green fluorescent protein (GFP-MSCs) or GFP-HIF-1 alpha lentivirus vectors (HIF-MSCs). A hypoxic-like metabolic profile was confirmed by mitochondrial and glycolysis stress test. Capacity of HIF-MSCs to modulate T-cell activation, dendritic cell differentiation, monocyte migration, and polarizations towards macrophages and natural killer (NK) cell lytic activity was assessed by a number of functional assays in co-cultures. The expression of relevant factors were determined by polymerase chain reaction (PCR) analysis and enzyme-linked immunosorbent assay (ELISA). RESULTS: While HIF-1 alpha overexpression did not modify the inhibition of T-cell activation by MSCs, HIF-MSCs impaired dendritic cell differentiation more efficiently. In addition, HIF-MSCs showed a tendency to induce higher attraction of monocytes, which differentiate into suppressor macrophages, and exhibited enhanced resistance to NK cell-mediated lysis, which supports the improved therapeutic capacity of HIF-MSCs. HIF-MSCs also displayed a pro-angiogenic profile characterized by increased expression of CXCL12/SDF1 and CCL5/RANTES and complete loss of CXCL10/IP10 transcription. CONCLUSIONS: Immunomodulation and expression of trophic factors by dental MSCs make them perfect candidates for cell therapy. Overexpression of HIF-1 alpha enhances these features and increases their resistance to allogenic NK cell lysis and, hence, their potential in vivo lifespan. Our results further support the use of HIF-1 alpha-expressing dental MSCs for cell therapy in tissue injury and immune disorders.

BMP4 Induces M2 Macrophage Polarization and Favors Tumor Progression in Bladder Cancer.

Purpose: Bladder cancer is a current clinical and social problem. At diagnosis, most patients present with nonmuscle-invasive tumors, characterized by a high recurrence rate, which could progress to muscle-invasive disease and metastasis. Bone morphogenetic protein (BMP)-dependent signaling arising from stromal bladder tissue mediates urothelial homeostasis by promoting urothelial cell differentiation. However, the possible role of BMP ligands in bladder cancer is still unclear.Experimental Design: Tumor and normal tissue from 68 patients with urothelial cancer were prospectively collected and analyzed for expression of BMP and macrophage markers. The mechanism of action was assessed in vitro by experiments with bladder cancer cell lines and peripheral blood monocyte-derived macrophages.Results: We observed BMP4 expression is associated and favored type II macrophage differentiation. In vitro experiments showed that both recombinant BMP4 and BMP4-containing conditioned media from bladder cancer cell lines favored monocyte/macrophage polarization toward M2 phenotype macrophages, as shown by the expression and secretion of IL10. Using a series of human bladder cancer patient samples, we also observed increased expression of BMP4 in advanced and undifferentiated tumors in close correlation with epithelial-mesenchymal transition (EMT). However, the p-Smad 1,5,8 staining in tumors showing EMT signs was reduced, due to the increased miR-21 expression leading to reduced BMPR2 expression.Conclusions: These findings suggest that BMP4 secretion by bladder cancer cells provides the M2 signal necessary for a protumoral immune environment. In addition, the repression of BMPR2 by miR-21 makes the tumor cells refractory to the prodifferentiating actions mediated by BMP ligands, favoring tumor growth. Clin Cancer Res; 23(23); 7388-99. ©2017 AACR.

Blockade of bone morphogenetic protein signaling potentiates the pro-inflammatory phenotype induced by interleukin-17 and tumor necrosis factor-α combination in rheumatoid synoviocytes.

INTRODUCTION: Bone morphogenetic proteins (BMPs) are multifunctional secreted growth factors regulating a broad spectrum of functions in numerous systems. An increased expression and production of specific BMPs have been described in the rheumatoid arthritis (RA) synovium. The aim of this study was to analyze the involvement of the BMP signaling pathway in RA synoviocytes in response to interleukin-17 (IL-17) and tumor necrosis factor-alpha (TNF-α). METHODS: The expression of components of the BMP signaling pathway (BMP receptors, BMP ligands, BMP signal transducers, and BMP antagonists) was analyzed by quantitative polymerase chain reaction before and after treatment of RA synoviocytes with TNF-α or IL-17 or both. Regulation was studied in the presence of the specific BMP inhibitor DMH1 (dorsomorphin homologue 1) or an exogenous BMP ligand, BMP6. Expression and production of pro-inflammatory cytokines (IL-6 and granulocyte-macrophage colony-stimulating factor), chemokines (IL-8, CCL2, CCL5, and CXCL10), and matrix metalloproteinases (MMP-1, -2, -3, -9, and -13) were analyzed. RESULTS: RA synoviocytes express BMP receptors (mainly BMPRIA, ACTRIA, and BMPRII), signal transducers of the Smad family (Smad1 and 5 and co-Smad4), and different BMP antagonists. The modulation of the expression of the BMP target genes-Id (inhibitor of DNA-binding/differentiation) proteins and Runx (Runt-related transcription factor) transcription factors-after the addition of exogenous BMP shows that the BMP signaling pathway is active. RA synoviocytes also express BMP ligands (BMP2, BMP6, and BMP7) which are highly upregulated after activation with TNF-α and IL-17. Autocrine BMP signaling pathway can be blocked by treatment with the inhibitor DMH1, leading to an increase in the upregulated expression of pro-inflammatory cytokines, chemokines, and MMPs induced by the activation of RA synoviocytes with TNF-α and IL-17. Conversely, the additional stimulation of the BMP pathway with the exogenous addition of the BMP6 ligand decreases the expression of those pro-inflammatory and pro-destructive factors. CONCLUSION: The results indicate that the canonical BMP pathway is functionally active in human RA synoviocytes and that the inhibition of autocrine BMP signaling exacerbates the pro-inflammatory phenotype induced in RA synoviocytes by the stimulation with IL-17 and TNF-α.