Humanized Mouse Models of Rheumatoid Arthritis for Studies on Immunopathogenesis and Preclinical Testing of Cell-Based Therapies

Rodent models of rheumatoid arthritis (RA) have been used over decades to study the immunopathogenesis of the disease and to explore intervention strategies. Nevertheless, mouse models of RA reach their limit when it comes to testing of new therapeutic approaches such as cell-based therapies. Differences between the human and the murine immune system make it difficult to draw reliable conclusions about the success of immunotherapies. To overcome this issue, humanized mouse models have been established that mimic components of the human immune system in mice. Two main strategies have been pursued for humanization: the introduction of human transgenes such as human leukocyte antigen molecules or specific T cell receptors, and the generation of mouse/human chimera by transferring human cells or tissues into immunodeficient mice. Recently, both approaches have been combined to achieve more sophisticated humanized models of autoimmune diseases. This review discusses limitations of conventional mouse models of RA-like disease and provides a closer look into studies in humanized mice exploring their usefulness and necessity as preclinical models for testing of cell-based therapies in autoimmune diseases such as RA

Regulation of Tolerogenic Features on Dexamethasone-Modulated MPLA-Activated Dendritic Cells by MYC

The potential of tolerogenic dendritic cells (tolDCs) to shape immune responses and restore tolerance has turn them into a promising therapeutic tool for cellular therapies directed toward immune regulation in autoimmunity. Although the cellular mechanisms by which these cells can exert their regulatory function are well-known, the mechanisms driving their differentiation and function are still poorly known, and the variety of stimuli and protocols applied to differentiate DCs toward a tolerogenic phenotype makes it even more complex to underpin the molecular features involved in their function. Through transcriptional profiling analysis of monocyte-derived tolDCs modulated with dexamethasone (Dex) and activated with monophosphoryl lipid A (MPLA), known as DM-DCs, we were able to identify MYC as one of the transcriptional regulators of several genes differentially expressed on DM-DCs compared to MPLA-matured DCs (M-DCs) and untreated/immature DCs (DCs) as revealed by Ingenuity Pathway Analysis (IPA) upstream regulators evaluation. Additionally, MYC was also amidst the most upregulated genes in DM-DCs, finding that was confirmed at a transcriptional as well as at a protein level. Blockade of transactivation of MYC target genes led to the downregulation of tolerance-related markers IDO1 and JAG1. MYC blockade also led to downregulation of PLZF and STAT3, transcription factors associated with immune regulation and inhibition of DC maturation, further supporting a role of MYC as an upstream regulator contributing to the regulatory phenotype of DM-DCs. On the other hand, we had previously shown that fatty acid oxidation, oxidative metabolism and zinc homeostasis are amongst the main biological functions represented in DM-DCs, and here we show that DM-DCs exhibit higher intracellular expression of ROS and Zinc compared to mature M-DCs and DCs. Taken together, these findings suggest that the regulatory profile of DM-DCs is partly shaped by the effect of the transcriptional regulation of tolerance-inducing genes by MYC and the modulation of oxidative metabolic processes and signaling mediators such as Zinc and ROS

The role of dendritic cells and T cell regulation in the pathogenesis of Whipple's disease

Der Morbus Whipple (MW) ist eine chronische systemische Infektion mit dem ubiquitär vorkommenden Bakterium Tropheryma (T.) whipplei, welche mit subtilen Immundefekten assoziiert zu sein scheint. Kennzeichnend für MW-Patienten ist die massive Infiltration der duodenalen Mukosa mit T. whipplei-gefüllten Makrophagen und das Fehlen einer T. whipplei-spezifischen T-Helferzell Typ 1 (Th1)-Antwort. Obwohl dendritische Zellen (DC) für die Initiierung von T-Zell- Antworten zur Abwehr intrazellulärer Bakterien essenziell sind, war bislang nichts über ihre Rolle in der Pathogenese des MW bekannt. Ebenfalls unklar war, ob regulatorische T-Zellen (Treg) bei MW-Patienten an der Suppression von T-Zell Antworten beteiligt sind. Daher war es das Ziel dieser Arbeit, DC von MW Patienten hinsichtlich der Verteilung in Blut und Geweben, der Funktionalität und deren Beeinflussung durch T. whipplei zu untersuchen sowie bei MW-Patienten Veränderungen der CD4+ T-Zellpopulation und suppressive Effekte durch Treg zu analysieren. In der duodenalen Mukosa und in Lymphknoten von MW-Patienten war die Anzahl der DC im Vergleich zu Kontrollgewebe nicht signifikant verändert. Im Blut von unbehandelten MW-Patienten waren myeloide DC im Vergleich zu Kontrollpersonen jedoch reduziert und zeigten eine verringerte Ausreifung und Interleukin (IL)-12-Produktion in Reaktion auf bakterielle Stimuli. Aus Monozyten in vitro differenzierte DC von MW-Patienten unterschieden sich nicht hinsichtlich Phänotyp, Endozytosekapazität und Fähigkeit zur Aktivierung Antigen-spezifischer CD4+ T-Zellen von DC der Kontrollpersonen. Allerdings zeigten die DC aus MW Patienten in vitro eine defekte IL-12-Sekretion und waren in der Kokultur mit autologen T-Zellen nicht in der Lage, eine T. whipplei-spezifische Th1-Reaktion zu induzieren. Sowohl in vitro als auch in situ wurde T. whipplei von unreifen DC aufgenommen, lieferte jedoch nur schwache Stimuli für die Aktivierung der DC. Lymphknoten mit einer T. whipplei-Besiedlung zeigten strukturelle Veränderungen, welche von einer signifikanten Reduktion der Lymphozyten-Proliferation begleitet wurden. Dies spiegelte sich auch in der verringerten Anzahl der CD4+ T-Zellen im Blut von unbehandelten MW-Patienten wider. Zudem waren bei MW-Patienten unabhängig vom Therapiestatus sowohl der Anteil der zirkulierenden CD4+ T-Zellen mit Darmtropismus als auch die Anzahl der CD4+ und CD45RO+ (Effektor-/Gedächtnis-) T-Zellen in der duodenalen Mukosa reduziert. Dagegen zeigten unbehandelte MW-Patienten im Vergleich zu Kontrollpersonen eine lokal erhöhte Anzahl an Treg im Duodenum und eine erhöhtes suppressives Potenzial der Treg gegenüber Th1- und Th17-Zellen im Blut. Aktivierte T-Zellen nahmen im Blut von unbehandelten MW-Patienten im Vergleich zu Kontrollpersonen zwar einen höheren Anteil innerhalb der CD4+ T-Zellen ein, zeigten aber einen anergen Phänotyp und eine reduzierte Reaktivität gegenüber Recall-Antigenen und polyklonaler Stimulation. Während die polyklonale und Antigen-spezifische T-Zell-Reaktivität nach der Behandlung wieder das Normalniveau erreichte, blieb die fehlende T. whipplei-spezifische Th1-Antwort der MW Patienten bestehen. Die Ergebnisse dieser Arbeit verdeutlichen, dass die reduzierte IL-12-Produktion der DC, deren unzureichende Aktivierung durch T. whipplei und die Treg-vermittelte Suppression von T-Zell-Antworten an der Immunpathogenese des MW beteiligt sind.Whipple’s disease (WD) is a chronic systemic infection with the ubiquitous bacterium Tropheryma (T.) whipplei, which seems to be associated with subtile immunodeficiencies. The hallmark of WD patients is the massive infiltration of the duodenal mucosa with T. whipplei-stuffed macrophages and the lack of a T. whipplei-specific T helper cell type 1 (Th1) response. Although dendritic cells (DC) are essential for the initiation of T cell responses to ward off intracellular bacteria, nothing was known so far about their role in the pathogenesis of WD. It was also unclear, if regulatory T cells (Treg) were involved in the suppression of T cell responses in WD patients. Therefore the aim of this work was to examine DC from WD patients in terms of distribution in blood and tissues, functionality and the influence of T. whipplei, as well as to analyze alterations within the CD4+ T cell population and suppressive effects of Treg in WD patients. Within the duodenal mucosa and lymph nodes from WD patients the number of DC was not significantly altered as compared to control tissue. But myeloid DC were reduced in the blood of untreated WD patients in comparison to control subjects, and showed a reduced maturation and production of interleukin (IL)-12 in response to bacterial stimuli. In vitro, monocyte-derived DC of WD patients did not differ in phenotype, endocytotic capacity, and the ability to activate antigen-specific CD4+ T cells from DC of control subjects. However, DC from WD patients exhibited a defective IL-12 secretion in vitro, and were not able to induce a T. whipplei- specific Th1 reaction when co-cultured with autologous T cells. T. whipplei was ingested by immature DC, both in vitro and in situ, but provided only weak stimuli for the activation of DC. Lymph nodes colonized by T. whipplei showed structural changes accompanied by a significant reduction of lymphocyte proliferation. This was also reflected by the reduced number of CD4+ T cells in the peripheral blood of untreated WD patients. Moreover, the proportion of circulating CD4+ T cells with gut tropism as well as the number of CD4+ and CD45RO+ (effector/memory) T cells in the duodenal mucosa were reduced in WD patients, irrespective of their treatment status. In contrast, untreated WD patients showed a locally increased number of Treg in the duodenum and an enhanced suppressive potential of Treg towards Th1 and Th17 cells in the blood as compared to control subjects. Activated T cells comprised a higher proportion within the CD4+ T cells in the blood of untreated WD patients than of control subjects, but showed an anergic phenotype and a reduced reactivity towards recall antigens and polyclonal stimulation. While the polyclonal and antigen-specific T cell reactivity reached normal levels after treatment, the lack of a T. whipplei-specific Th1 response remained in WD patients. The results of this work indicate that the reduced IL-12 production by DC, their insufficient activation by T. whipplei, and the Treg-mediated suppression of T cell responses are involved in the immunopathogenesis of WD

Gene Expression Profiling of Human Monocyte-derived Dendritic Cells – Searching for Molecular Regulators of Tolerogenicity

Artículo de publicación ISIThe ability of dendritic cells (DCs) to initiate and modulate antigen-specific immune responses has made them attractive targets for immunotherapy. Since DC research in humans is limited by the scarcity of DC populations in the blood circulation, most of our knowledge about DC biology and function has been obtained in vitro from monocyte-derived DCs (moDCs), which can be readily generated in sufficient numbers and are able to differentiate into distinct functional subsets depending on the nature of stimulus. In particular, moDCs with tolerogenic properties (tolDCs) possess great therapeutic potential for the treatment of autoimmune diseases. Several protocols have been developed to generate tolDCs in vitro, able to reinstruct auto-reactive T cells and to promote regulatory cells. While ligands and soluble mediators, by which DCs shape immune responses, have been vastly studied, the intracellular pathways and transcriptional regulators that govern tolDC differentiation and function are poorly understood. Whole-genome microarrays and proteomics provide useful strategies to dissect the complex molecular processes that promote tolerogenicity. Only few attempts have been made to understand tolDC biology through a global view on "omics" profiles. So far, the identification of a common regulator of tolerogenicity has been hampered by the fact that each protocol, used for tolDC generation, targets distinct signaling pathways. Here, we review the progress in understanding the transcriptional regulation of moDC differentiation, with a special focus on tolDCs, and highlight candidate molecules that might be associated with DC tolerogenicity.FONDECYT-Chile, Millennium Institute on Immunology and Immunotherapy, Fundacion Ciencia Translacional from Chil

Dexamethasone and monophosphoryl lipid A-modulated dendritic cells promote antigen-specific tolerogenic properties on naïve and memory CD4+ T cells

Tolerogenic dendritic cells (DCs) are a promising tool to control T cell-mediated autoimmunity. Here, we evaluate the ability of dexamethasone-modulated and monophosphoryl lipid A-activated DCs (MPLA-tDCs) to exert immunomodulatory effects on naïve and memory CD4+ T cells in an antigen-specific manner. For this purpose MPLA-tDCs were loaded with purified protein derivative (PPD) as antigen and co-cultured with autologous naïve or memory CD4+ T cells. Lymphocytes were re-challenged with autologous PPD-pulsed mature DCs (mDCs), evaluating proliferation and cytokine production by flow cytometry. On primed-naïve CD4+ T cells, the expression of regulatory T cell markers was evaluated and their suppressive ability was assessed in autologous co-cultures with CD4+ effector T cells and PPD-pulsed mDCs. We detected that memory CD4+ T cells primed by MPLA-tDCs presented reduced proliferation and pro-inflammatory cytokine expression in response to PPD and were refractory to subsequent stimulation. Naïve CD4+ T cells were instructed by MPLA-tDCs to be hyporesponsive to antigen-specific re-stimulation, and to suppress the induction of T helper cell type 1 and 17 responses. In conclusion MPLA-tDCs are able to modulate antigen-specific responses of both naïve and memory CD4+ T cells and might be a promising strategy to turn off self-reactive CD4+ effector T cells in autoimmunity

Humanized mouse models of rheumatoid arthritis for studies on immunopathogenesis and preclinical testing of cell-based therapies

Rodent models of rheumatoid arthritis (RA) have been used over decades to study the immunopathogenesis of the disease and to explore intervention strategies. Nevertheless, mouse models of RA reach their limit when it comes to testing of new therapeutic approaches such as cell-based therapies. Differences between the human and the murine immune system make it difficult to draw reliable conclusions about the success of immunotherapies. To overcome this issue, humanized mouse models have been established that mimic components of the human immune system in mice. Two main strategies have been pursued for humanization: the introduction of human transgenes such as human leukocyte antigen molecules or specific T cell receptors, and the generation of mouse/human chimera by transferring human cells or tissues into immunodeficient mice. Recently, both approaches have been combined to achieve more sophisticated humanized models of autoimmune diseases. This review discusses limitations of conventional mouse models of RA-like disease and provides a closer look into studies in humanized mice exploring their usefulness and necessity as preclinical models for testing of cell-based therapies in autoimmune diseases such as RA

The role of interleukin-6 signalling and its therapeutic blockage in skewing the T cell balance in rheumatoid arthritis

Therapeutic blockage of cytokine signalling in autoimmune diseases has improved our understanding of the role of these cytokines in triggering, shaping and perpetuating autoimmune responses. In rheumatoid arthritis (RA), immunopathology is driven by a predominance of arthritogenic T helper cells secreting interferon- [T helper type 1 (Th1)] and interleukin (IL)-17 (Th17) over regulatory T cells (T-reg). The pleiotropic cytokine IL-6 is crucial to the differentiation of Th17 cells and the balance between pathogenic Th17 and protective T-reg. Targeting the IL-6 receptor (IL-6R) by humanized antibodies improves signs and symptoms of RA, and has provided new insights into the mechanisms of inflammation and immune regulation. Here we review current evidence on the role of IL-6 in the pathogenesis of RA and the molecular consequences of IL-6R blockage in disease, with special focus on the Th17/T-reg balance and plasticity.FONDECYT, 1121100, 1112149

Tolerogenic dendritic cells for reprogramming of lymphocyte responses in autoimmune diseases

Dendritic cells (DCs) control immune responses by driving potent inflammatory actions against external and internal threats while generating tolerance to self and harmless components. This duality and their potential to reprogram immune responses in an antigen-specific fashion have made them an interesting target for immunotherapeutic strategies to control autoimmune diseases. Several protocols have been described for in vitro generation of tolerogenic DCs (tolDCs) capable of modulating adaptive immune responses and restoring tolerance through different mechanisms that involve anergy, generation of regulatory lymphocyte populations, or deletion of potentially harmful inflammatory T cell subsets. Recently, the capacity of tolDCs to induce interleukin (IL-10)-secreting regulatory B cells has been demonstrated. In vitro assays and rodent models of autoimmune diseases provide insights to the molecular regulators and pathways enabling tolDCs to control immune responses. Here we review mechanisms through which tolDCs modulate adaptive immune responses, particularly focusing on their suitability for reprogramming autoreactive CD4(+) effector T cells. Furthermore, we discuss recent findings establishing that tolDCs also modulate B cell populations and discuss clinical trials applying tolDCs to patients with autoimmune diseases.Fondecyt-Chile 1140553 Millennium Institute on Immunology and Immunotherapy P09-016-F Fundacion Ciencia Translational from Chil

Specific and Nonspecific B-Cell Function in the Small Intestines of Patients with Whipple's Disease ▿

Whipple's disease is a chronic multisystemic infection caused by Tropheryma whipplei that is characterized by arthritis, weight loss, and diarrhea. The immunological defects in the duodenal mucosa, the site of major replication of the agent underlying the pathogenesis of Whipple's disease, are poorly understood. Mucosal immunoglobulins are essential for the defense against intestinal pathogens; therefore, we analyzed the B-cell response in duodenal specimens and sera of Whipple's disease patients. Whereas systemic immunoglobulin production was affected only marginally, duodenal biopsy specimens of Whipple's disease patients contained reduced numbers of immunoglobulin-positive plasma cells and secreted less immunoglobulin compared to healthy controls but showed a weak secretory IgA response toward T. whipplei. This T. whipplei-specific intestinal immune response was not observed in controls. Thus, we were able to demonstrate that general mucosal immunoglobulin production in Whipple's disease patients is impaired. However, this deficiency does not completely abolish T. whipplei-specific secretory IgA production that nonetheless does not protect from chronic infection

Dexamethasone and Monophosphoryl Lipid A Induce a Distinctive Profile on Monocyte-Derived Dendritic Cells through Transcriptional Modulation of Genes Associated With Essential Processes of the Immune Response

There is growing interest in the use of tolerogenic dendritic cells (tolDCs) as a potential target for immunotherapy. However, the molecular bases that drive the differentiation of monocyte-derived DCs (moDCs) toward a tolerogenic state are still poorly understood. Here, we studied the transcriptional profile of moDCs from healthy subjects, modulated with dexamethasone (Dex) and activated with monophosphoryl lipid A (MPLA), referred to as Dex-modulated and MPLA-activated DCs (DM-DCs), as an approach to identify molecular regulators and pathways associated with the induction of tolerogenic properties in tolDCs. We found that DM-DCs exhibit a distinctive transcriptional profile compared to untreated (DCs) and MPLA-matured DCs. Differentially expressed genes downregulated by DM included MMP12, CD1c, IL-1B, and FCER1A involved in DC maturation/inflammation and genes upregulated by DM included JAG1, MERTK, IL-10, and IDO1 involved in tolerance. Genes related to chemotactic responses, cell-to-cell signaling and interaction, fatty acid oxidation, metal homeostasis, and free radical scavenging were strongly enriched, predicting the activation of alternative metabolic processes than those driven by counterpart DCs. Furthermore, we identified a set of genes that were regulated exclusively by the combined action of Dex and MPLA, which are mainly involved in the control of zinc homeostasis and reactive oxygen species production. These data further support the important role of metabolic processes on the control of the DC-driven regulatory immune response. Thus, Dex and MPLA treatments modify gene expression in moDCs by inducing a particular transcriptional profile characterized by the activation of tolerance-associated genes and suppression of the expression of inflammatory genes, conferring the potential to exert regulatory functions and immune response modulation