A Spontaneous Mouse Model of Lupus: Physiology and Therapy

Spontaneous models of lupus were recognized four decades ago beginning in the early 1960s with the NZB/NZW F1 (NZB/W F1) mouse, an F1 hybrid between the New Zealand Black (NZB) and New Zealand White (NZW) mice. Although the parental strains display limited autoimmunity, the NZB/W F1 develops severe lupus-like features similar to that of human lupus patients. Here, we will address the genetic characteristics of the model and discuss its main characteristics such as the presence of serum antinuclear autoantibodies (ANA) including anti-dsDNA, mild vasculitis, and the development of immune complex-mediated glomerulonephritis. Similar to human lupus, the disease develops primarily in female mice after six months of age, with a lesser percentage and severity in male mice. The relation of this phenomenon will be examined in the context of estrogen levels. The participation of both innate and adaptive immunity will be addressed as well as the contribution of both T and B cells in the development of the clinical aspects of the disease. We will focus on the use of the model as a valuable tool for elucidating the pathogenic mechanisms of the disease, as well as its use as preclinical testing of therapeutic for human use

Cells from discarded dressings differentiate chronic from acute wounds in patients with Epidermolysis Bullosa

Impaired wound healing complicates a wide range of diseases and represents a major cost to healthcare systems. Here we describe the use of discarded wound dressings as a novel, cost effective, accessible, and non-invasive method of isolating viable human cells present at the site of skin wounds. By analyzing 133 discarded wound dressings from 51 patients with the inherited skin-blistering disease epidermolysis bullosa (EB), we show that large numbers of cells, often in excess of 100 million per day, continually infiltrate wound dressings. We show, that the method is able to differentiate chronic from acute wounds, identifying significant increases in granulocytes in chronic wounds, and we show that patients with the junctional form of EB have significantly more cells infiltrating their wounds compared with patients with recessive dystrophic EB. Finally, we identify subsets of granulocytes and T lymphocytes present in all wounds paving the way for single cell profiling of innate and adaptive immune cells with relevance to wound pathologies. In summary, our study delineates findings in EB that have potential relevance for all chronic wounds, and presents a method of cellular isolation that has wide reaching clinical application

Alloreactive Regulatory T Cells Allow the Generation of Mixed Chimerism and Transplant Tolerance

Artículo de publicación ISIThe induction of donor-specific transplant tolerance is one of the main goals of modern immunology. Establishment of a mixed chimerism state in the transplant recipient has proven to be a suitable strategy for the induction of long-term allograft tolerance; however, current experimental recipient preconditioning protocols have many side effects, and are not feasible for use in future therapies. In order to improve the current mixed chimerism induction protocols, we developed a non-myeloablative bone-marrow transplant (NM-BMT) protocol using retinoic acid (RA)-induced alloantigen-specific Tregs, clinically available immunosuppressive drugs, and lower doses of irradiation. We demonstrate that RA-induced alloantigen-specific Tregs in addition to a NM-BMT protocol generates stable mixed chimerism and induces tolerance to allogeneic secondary skin allografts in mice. Therefore, the establishment of mixed chimerism through the use of donor-specific Tregs rather than non-specific immunosuppression could have a potential use in organ transplantation.FONDECY

Adoptive transfer of autoimmune splenic dendritic cells to lupus-prone mice triggers a B lymphocyte humoral response

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by increased autoantibody production that leads to multiple tissue injuries. Dendritic cells (DCs) are important orchestrators of immune responses and key components in fine-tuning the balance between tolerance and immunity. However, their role in autoimmune disorders such as SLE remains uncertain. We analyzed the contribution of DCs in triggering SLE by adoptively transferring splenic DCs from aged autoimmune [NZBxNZW]F1 (BWF1) mice to young healthy BWF1 mice. We observed that the transfer of DCs from autoimmune mice to pre-autoimmune mice induced high autoantibody titers in the serum of recipient mice. Moreover, autoimmune DCs from aged BWF1 mice were crucial for the expansion and differentiation of plasmablasts and CD5(+) B cells or B1-like cells in the peripheral blood, and spleen of recipient BWF1 mice, a phenomenon that is observed in autoimmune BWF1 mice. On the other hand, DCs from aged BWF1 mice participated in the expansion and differentiation of DCs and IFN-gamma-producing T cells. These results reveal that DCs from autoimmune BWF1 mice exhibit functional and phenotypic characteristics that allow them to trigger B cell hyperactivation, as well as DC and T cell expansion and differentiation, thereby promoting an exacerbated humoral response in lupus-prone mice.FONDECYT 1140431 1121478 PAI 791100009 CONICYT PFB-16 FONDEQUIP/EQM11413

Vitamin A Impairs the Reprogramming of Tregs into IL-17-Producing Cells during Intestinal Inflammation

Maintaining the identity of Foxp3+ regulatory T cells (Tregs) is critical for controlling immune responses in the gut, where an imbalance between Tregs and T effector cells has been linked to inflammatory bowel disease. Accumulating evidence suggests that Tregs can convert into Th17 cells and acquire an inflammatory phenotype. In this study, we used an adoptive transfer model of Ag-specific T cells to study the contribution of different factors to the reprogramming of in vitro-generated Treg cells (iTreg) into IL-17-producing cells in a mouse model of gut inflammation in vivo. Our results show that intestinal inflammation induces the reprogramming of iTreg cells into IL-17-producing cells and that vitamin A restrains reprogramming in the gut. We also demonstrate that the presence of IL-2 during the in vitro generation of iTreg cells confers resistance to Th17 conversion but that IL-2 and retinoic acid (RA) cooperate to maintain Foxp3 expression following stimulation under Th17-polarizing conditions. Additionally, although IL-2 and RA differentially regulate the expression of different Treg cell suppressive markers, Treg cells generated under different polarizing conditions present similar suppressive capacity

Purinergic Signaling as a Regulator of Th17 Cell Plasticity

T helper type 17 (Th17) lymphocytes, characterized by the production of interleukin-17 and other pro-inflammatory cytokines, are present in intestinal lamina propria and have been described as important players driving intestinal inflammation. Recent evidence, supporting the notion of a functional and phenotypic instability of Th17 cells, has shown that Th17 differentiate into type 1 regulatory (Tr1) T cells during the resolution of intestinal inflammation. Moreover, it has been suggested that the expression of CD39 ectonucleotidase endows Th17 cells with immunosuppressive properties. However, the exact role of CD39 ectonucleotidase in Th17 cells has not been studied in the context of intestinal inflammation. Here we show that Th17 cells expressing CD39 ectonucleotidase can hydrolyze ATP and survive to ATP-induced cell death. Moreover, in vitro-generated Th17 cells expressing the CD39 ectonucleotidase produce IL-10 and are less pathogenic than CD39 negative Th17 cells in a model of experimental colitis in Rag-/- mice. Remarkably, we show that CD39 activity regulates the conversion of Th17 cells to IL-10-producing cells in vitro, which is abrogated in the presence of ATP and the CD39-specific inhibitor ARL67156. All these data suggest that CD39 expression by Th17 cells allows the depletion of ATP and is crucial for IL-10 production and survival during the resolution of intestinal inflammation

DataSheet1_Safety and efficacy of clinical-grade, cryopreserved menstrual blood mesenchymal stromal cells in experimental acute respiratory distress syndrome.pdf

Background: Treatment for critical care conditions, such as acute respiratory distress syndrome (ARDS), requires ready-to-administer injectable mesenchymal stromal cells (MSCs). A validated cryopreserved therapy based on MSCs derived from menstrual blood (MenSCs) is an attractive option that offers advantages over freshly cultured cells and allows its use as an off-the-shelf therapy in acute clinical conditions. The main goal of this study is to provide evidence on the impact of cryopreservation on different biological functions of MenSCs and to determine the optimal therapeutic dose, safety, and efficacy profile of clinical-grade, cryopreserved (cryo)-MenSCs in experimental ARDS.Methods: Biological functions of fresh versus cryo-MenSCs were compared in vitro. The effects of cryo-MenSCs therapy were evaluated in vivo in ARDS-induced (Escherichia coli lipopolysaccharide) C57BL/6 mice. After 24 h, the animals were treated with five doses ranging from 0.25×105 to 1.25×106 cells/animal. At 2 and 7 days after induction of ARDS, safety and efficacy were evaluated.Results: Clinical-grade cryo-MenSCs injections improved lung mechanics and reduced alveolar collapse, tissue cellularity, and remodelling, decreasing elastic and collagen fiber content in alveolar septa. In addition, administration of these cells modulated inflammatory mediators and promoted pro-angiogenic and anti-apoptotic effects in lung-injured animals. More beneficial effects were observed with an optimal dose of 4×106 cells/Kg than with higher or lower doses.Conclusion: From a translational perspective, the results showed that clinical-grade cryopreserved MenSCs retain their biological properties and exert a therapeutic effect in mild to moderate experimental ARDS. The optimal therapeutic dose was well-tolerated, safe, and effective, favouring improved lung function. These findings support the potential value of an off-the-shelf MenSCs-based product as a promising therapeutic strategy for treating ARDS.</p

Lactate: an alternative pathway for the immunosuppressive properties of mesenchymal stem/stromal cells

International audienceAbstract Background The metabolic reprogramming of mesenchymal stem/stromal cells (MSC) favoring glycolysis has recently emerged as a new approach to improve their immunotherapeutic abilities. This strategy is associated with greater lactate release, and interestingly, recent studies have proposed lactate as a functional suppressive molecule, changing the old paradigm of lactate as a waste product. Therefore, we evaluated the role of lactate as an alternative mediator of MSC immunosuppressive properties and its contribution to the enhanced immunoregulatory activity of glycolytic MSCs. Materials and methods Murine CD4 + T cells from C57BL/6 male mice were differentiated into proinflammatory Th1 or Th17 cells and cultured with either L-lactate, MSCs pretreated or not with the glycolytic inductor, oligomycin, and MSCs pretreated or not with a chemical inhibitor of lactate dehydrogenase A (LDHA), galloflavin or LDH siRNA to prevent lactate production. Additionally, we validated our results using human umbilical cord-derived MSCs (UC-MSCs) in a murine model of delayed type 1 hypersensitivity (DTH). Results Our results showed that 50 mM of exogenous L-lactate inhibited the proliferation rate and phenotype of CD4 + T cell-derived Th1 or Th17 by 40% and 60%, respectively. Moreover, the suppressive activity of both glycolytic and basal MSCs was impaired when LDH activity was reduced. Likewise, in the DTH inflammation model, lactate production was required for MSC anti-inflammatory activity. This lactate dependent-immunosuppressive mechanism was confirmed in UC-MSCs through the inhibition of LDH, which significantly decreased their capacity to control proliferation of activated CD4 + and CD8 + human T cells by 30%. Conclusion These findings identify a new MSC immunosuppressive pathway that is independent of the classical suppressive mechanism and demonstrated that the enhanced suppressive and therapeutic abilities of glycolytic MSCs depend at least in part on lactate production

Th17_TGF-β1 cells present a regulatory phenotype.

<p>(A) IL-17-GFP+ Th17_TGF-β1 and Th17_IL-23 cells were sorted and then analyzed by real-time PCR for mRNA expression of several transcription factors and cytokines (n = 3). (B) IL-17-GFP+ Th17_TGF-β1 and Th17_IL-23 cells were sorted and then reactivated for 4 hrs with PMA plus ionomycin to assess cytokine production by CBA or (C) in the presence of PMA, ionomycin and brefeldin A to analyze GM-CSF production by FACS (n = 5). (D) Percentage of GM-CSF+ cells within IL-17-GFP+ Th17_TGF-β1 and Th17_IL-23 cells (n = 5). Data are presented as mean ± S.E.M. *p<0.05, **p<0.01 and ***p<0.001 determined by t-test (A) or Mann-Whitney test (B and D).</p

ATP hydrolysis by CD39 on Th17_TGF-β1 cells promotes their conversion into IL-10-producing cells.

<p>(A) IL-10 production by IL-17-GFP+ Th17_TGF-β1 and Th17_IL-23 cells restimulated for 3 days with anti-CD3 and anti-CD28 antibodies in the presence and absence of Tr1 polarizing cytokines (TGF-β1, IL-21 and IL-27), 50 μM ATP and 250 μM ARL67156. IL-10 production was analyzed by CBA (n = 4). (B) Th17_IL-23 cells from wild-type and P2X7R knockout mice were restimulated for 3 days with anti-CD3 and anti-CD28 antibodies and IL-10 production was analyzed by CBA (n = 3). Data are presented as mean ± S.E.M. *p<0.05; **p<0.01 determined by repeated measures analysis of variance.</p