B-cell intrinsic and extrinsic signals that regulate central tolerance of mouse and human B cells.

The random recombination of immunoglobulin V(D)J gene segments produces unique IgM antibodies that serve as the antigen receptor for each developing B cell. Hence, the newly formed B cell repertoire is comprised of a variety of specificities that display a range of reactivity with self-antigens. Newly generated IgM+ immature B cells that are non-autoreactive or that bind self-antigen with low avidity are licensed to leave the bone marrow with their intact antigen receptor and to travel via the blood to the peripheral lymphoid tissue for further selection and maturation. In contrast, clones with medium to high avidity for self-antigen remain within the marrow and undergo central tolerance, a process that revises their antigen receptor or eliminates the autoreactive B cell altogether. Thus, central B cell tolerance is critical for reducing the autoreactive capacity and avidity for self-antigen of our circulating B cell repertoire. Bone marrow cultures and mouse models have been instrumental for understanding the mechanisms that regulate the selection of bone marrow B cells. Here, we review recent studies that have shed new light on the contribution of the ERK, PI3K, and CXCR4 signaling pathways in the selection of mouse and human immature B cells that either bind or do not bind self-antigen.

Central human B cell tolerance manifests with a distinctive cell phenotype and is enforced via CXCR4 signaling in hu-mice.

Central B cell tolerance, the process restricting the development of many newly generated autoreactive B cells, has been intensely investigated in mouse cells while studies in humans have been hampered by the inability to phenotypically distinguish autoreactive and nonautoreactive immature B cell clones and the difficulty in accessing fresh human bone marrow samples. Using a human immune system mouse model in which all human Igκ+ B cells undergo central tolerance, we discovered that human autoreactive immature B cells exhibit a distinctive phenotype that includes lower activation of ERK and differential expression of CD69, CD81, CXCR4, and other glycoproteins. Human B cells exhibiting these characteristics were observed in fresh human bone marrow tissue biopsy specimens, although differences in marker expression were smaller than in the humanized mouse model. Furthermore, the expression of these markers was slightly altered in autoreactive B cells of humanized mice engrafted with some human immune systems genetically predisposed to autoimmunity. Finally, by treating mice and human immune system mice with a pharmacologic antagonist, we show that signaling by CXCR4 is necessary to prevent both human and mouse autoreactive B cell clones from egressing the bone marrow, indicating that CXCR4 functionally contributes to central B cell tolerance.

The development of human immune system mice and their use to study tolerance and autoimmunity.

Autoimmune diseases evolve from complex interactions between the immune system and self-antigens and involve several genetic attributes, environmental triggers and diverse cell types. Research using experimental mouse models has contributed key knowledge on the mechanisms that underlie these diseases in humans, but differences between the mouse and human immune systems can and, at times, do undermine the translational significance of these findings. The use of human immune system (HIS) mice enables the utility of mouse models with greater relevance for human diseases. As the name conveys, these mice are reconstituted with mature human immune cells transferred directly from peripheral blood or via transplantation of human hematopoietic stem cells that nucleate the generation of a complex human immune system. The function of the human immune system in HIS mice has improved over the years with the stepwise development of better models. HIS mice exhibit key benefits of the murine animal model, such as small size, robust and rapid reproduction and ease of experimental manipulation. Importantly, HIS mice also provide an applicable in vivo setting that permit the investigation of the physiological and pathological functions of the human immune system and its response to novel treatments. With the gaining popularity of HIS mice in the last decade, the potential of this model has been exploited for research in basic science, infectious diseases, cancer, and autoimmunity. In this review we focus on the use of HIS mice in autoimmune studies to stimulate further development of these valuable models.

Correction: Severe Changes in Thymic Microenvironment in a Chronic Experimental Model of Paracoccidioidomycosis.

[This corrects the article DOI: 10.1371/journal.pone.0164745.].

Severe Changes in Thymic Microenvironment in a Chronic Experimental Model of Paracoccidioidomycosis.

T cell maturation takes place within the thymus, a primary lymphoid organ that is commonly targeted during infections. Previous studies showed that acute infection with Paracoccidioides brasiliensis (Pb), the causative agent of paracoccidioidomycosis (PCM), promotes thymic atrophy that is associated with the presence of yeast cells in the organ. However, as human PCM is a chronic infection, it is imperative to investigate the consequences of Pb infection over the thymic structure and function in chronic infection. In this sense, we developed a new experimental model where Pb yeast cells are injected through the intraperitoneal route and mice are evaluated over 120 days of infection. Thymuses were analyzed in chronically infected mice and we found that the thymus underwent extensive morphological alterations and severe infiltration of P. brasiliensis yeast cells. Further analyses showed an altered phenotype and function of thymocytes that are commonly found in peripheral mature T lymphocytes. We also observed activation of the NLRP3 inflammasome in the thymus. Our data provide new information on the severe changes observed in the thymic microenvironment in a model of PCM that more closely mimics the human infection.

Artesunate Ameliorates Experimental Autoimmune Encephalomyelitis by Inhibiting Leukocyte Migration to the Central Nervous System.

BACKGROUND AND AIMS: Experimental autoimmune encephalomyelitis (EAE) is T-cell-dependent disease of the central nervous system (CNS) of mice. This model resembles multiple sclerosis (MS) in many aspects. Therapies that focus in the modulation of the immune response and cellular infiltration in the CNS present best effects in the clinics. Artesunate (Art) is a semi-synthetic sesquiterpene derivative from artemisinin and has been shown to reduce the clinical signs of autoimmune disease models through mechanisms not yet understood. In this study, we aimed to evaluate whether administration of Art would ameliorate EAE. METHODS AND RESULTS: C57BL6 mice were immunized with MOG35-55 peptide to induce EAE. At the same time, Art treatment started (3 mg/kg/day via i.p.) for five consecutive days. We found that Art treatment reduced the clinical signs of EAE and that correlated with a reduced infiltration of cells in the CNS. Disease amelioration did not correlate with immunomodulation as recall responses, leukocyte subpopulations, and gene expression analysis were similar among treated and untreated mice. Ultimately, further analysis provided data indicating that a possible mechanism of action for Art is dependent on the cellular migration to the CNS. CONCLUSIONS: Artesunate reduces the severity of EAE by inhibiting migration of pathogenic T cells to the CNS.

Paracoccidioides brasiliensis infection promotes thymic disarrangement and premature egress of mature lymphocytes expressing prohibitive TCRs.

BACKGROUND: Paracoccidioidomycosis, a chronic granulomatous fungal disease caused by Paracoccidioides brasiliensis yeast cells affects mainly rural workers, albeit recently cases in immunosuppressed individuals has been reported. Protective immune response against P. brasiliensis is dependent on the activity of helper T cells especially IFN-γ-producing Th1 cells. It has been proposed that Paracoccidioides brasiliensis is able to modulate the immune response towards a permissive state and that the thymus plays a major role in it. METHODS: In this paper, we show that acute infection of BALB/c mice with P. brasiliensis virulent isolate (Pb18) might cause alterations in the thymic environment as well as the prohibitive TCR-expressing T cells in the spleens. RESULTS: After seven days of infection, we found yeast cells on the thymic stroma, the thymic epithelial cells (TEC) were altered regarding their spatial-orientation and inflammatory mediators gene expression was increased. Likewise, thymocytes (differentiating T cells) presented higher migratory ability in ex vivo experiments. Notwithstanding, P. brasiliensis-infected mice showed an increased frequency of prohibitive TCR-expressing T cells in the spleens, suggesting that the selection processes that occur in the thymus may be compromised during the acute infection. CONCLUSION: In this paper, for the first time, we show that acute infection with Paracoccidioides brasiliensis yeast cells promotes thymic alterations leading to a defective repertoire of peripheral T cells. The data presented here may represent new mechanisms by which P. brasiliensis subverts the immune response towards the chronic infection observed in humans.

Differential Response of Human Hepatocyte Chromatin to HDAC Inhibitors as a Function of Microenvironmental Glucose Level.

Diabetes is a complex multifactorial disorder characterized by chronic hyperglycemia due to impaired insulin secretion. Recent observations suggest that the complexity of the disease cannot be entirely accounted for genetic predisposition and a compelling argument for an epigenetic component is rapidly emerging. The use of histone deacetylase inhibitor (HDACi) in clinical setting is an emerging area of investigation. In this study, we have aimed to understand and compare the response of hepatocyte chromatin to valproic acid (VPA) and trichostatin A (TSA) treatments under normoglycemic or hyperglycemic conditions to expand our knowledge about the consequences of HDACi treatment in a diabetes cell model. Under normoglycemic conditions, these treatments promoted chromatin remodeling, as assessed by image analysis and H3K9ac and H3K9me2 abundance. Simultaneously, H3K9ac marks shifted to the nuclear periphery accompanied by HP1 dissociation from the heterochromatin and a G1 cell cycle arrest. More striking changes in the cell cycle progression and mitotic ratios required drastic treatment. Under hyperglycemic conditions, high glucose per se promoted chromatin changes similar to those promoted by VPA and TSA. Nonetheless, these results were not intensified in cells treated with HDACis under hyperglycemic conditions. Despite the absence of morphological changes being promoted, HDACi treatment seems to confer a physiological meaning, ameliorating the cellular hyperglycemic state through reduction of glucose production. These observations allow us to conclude that the glucose level to which the hepatocytes are subjected affects how chromatin responds to HDACi and their action under high-glucose environment might not reflect on chromatin remodeling. J. Cell. Physiol. 231: 2257-2265, 2016. © 2016 Wiley Periodicals, Inc.

Violacein Treatment Modulates Acute and Chronic Inflammation through the Suppression of Cytokine Production and Induction of Regulatory T Cells.

Inflammation is a necessary process to control infection. However, exacerbated inflammation, acute or chronic, promotes deleterious effects in the organism. Violacein (viola), a quorum sensing metabolite from the Gram-negative bacterium Chromobacterium violaceum, has been shown to protect mice from malaria and to have beneficial effects on tumors. However, it is not known whether this drug possesses anti-inflammatory activity. In this study, we investigated whether viola administration is able to reduce acute and chronic autoimmune inflammation. For that purpose, C57BL/6 mice were intraperitoneally injected with 1 µg of LPS and were treated with viola (3.5mg/kg) via i.p. at the same time-point. Three hours later, the levels of inflammatory cytokines in the sera and phenotypical characterization of leukocytes were determined. Mice treated with viola presented a significant reduction in the production of inflammatory cytokines compared with untreated mice. Interestingly, although viola is a compound derived from bacteria, it did not induce inflammation upon administration to naïve mice. To test whether viola would protect mice from an autoimmune inflammation, Experimental Autoimmune Encephalomyelitis (EAE)-inflicted mice were given viola i.p. at disease onset, at the 10th day from immunization. Viola-treated mice developed mild EAE disease in contrast with placebo-treated mice. The frequencies of dendritic cells and macrophages were unaltered in EAE mice treated with viola. However, the sole administration of viola augmented the levels of splenic regulatory T cells (CD4+Foxp3+). We also found that adoptive transfer of viola-elicited regulatory T cells significantly reduced EAE. Our study shows, for the first time, that violacein is able to modulate acute and chronic inflammation. Amelioration relied in suppression of cytokine production (in acute inflammation) and stimulation of regulatory T cells (in chronic inflammation). New studies must be conducted in order to assess the possible use of viola in therapeutic approaches in human autoimmune diseases.

Dendritic cells treated with crude Plasmodium berghei extracts acquire immune-modulatory properties and suppress the development of autoimmune neuroinflammation.

Dendritic cells (DCs) are professional antigen-presenting cells specifically targeted during Plasmodium infection. Upon infection, DCs show impaired antigen presentation and T-cell activation abilities. In this study, we aimed to evaluate whether cellular extracts obtained from Plasmodium berghei-infected erythrocytes (PbX) modulate DCs phenotypically and functionally and the potential therapeutic usage of PbX-modulated DCs in the control of experimental autoimmune encephalomyelitis (EAE, the mouse model for human multiple sclerosis). We found that PbX-treated DCs have impaired maturation and stimulated the generation of regulatory T cells when cultured with naive T lymphocytes in vitro. When adoptively transferred to C57BL/6 mice the EAE severity was reduced. Disease amelioration correlated with a diminished infiltration of cytokine-producing T cells in the central nervous system as well as the suppression of encephalitogenic T cells. Our study shows that extracts obtained from P. berghei-infected erythrocytes modulate DCs towards an immunosuppressive phenotype. In addition, the adoptive transfer of PbX-modulated DCs was able to ameliorate EAE development through the suppression of specific cellular immune responses towards neuro-antigens. To our knowledge, this is the first study to present evidence that DCs treated with P. berghei extracts are able to control autoimmune neuroinflammation.