O diabetes mellitus do tipo 1 (T1D) é uma doença autoimune mediada por células T e caracterizada pela destruição seletiva das células ? pancreáticas produtoras de insulina. Células estromais mesenquimais (MSCs) e células tronco hematopoéticas (HSCs) são os principais componentes do nicho hematopoético na medula óssea. Estas células vêm sendo utilizadas nos últimos anos em transplantes autólogos para tratamento do T1D. O objetivo geral do presente trabalho foi avaliar o perfil de expressão gênica global de MSCs e HSCs de pacientes com T1D e compará-lo com células isoladas de indivíduos saudáveis através da técnica de microarray e programas específicos de bioinformática. As MSCs e HSCs foram isoladas da medula óssea de pacientes com T1D antes e após o tratamento com imunossupressão em altas doses seguida pelo transplante autólogo de células tronco hematopoéticas (AHSCT). As MSCs apresentaram valor elevado de expressão absoluta de diversas moléculas potencialmente relacionadas com suas funções de suporte à hematopoese. MSCs de pacientes diabéticos apresentaram perfil de expressão gênica global distinto das isoladas de indivíduos saudáveis, com hiper-regulação da sinalização via proteína G e hiporregulação da atividade transcricional. O receptor ?3 adrenérgico, assim como a sinalização simpática, foram hiper-expressos nas células dos pacientes. Genes que codificam moléculas que suportam a hematopoese e regulados pelo sistema nervoso simpático, VCAM1 e CXCL12, foram hiporregulados em nossa análise. Após o AHSCT, houve atenuação do perfil de expressão diferencial das MSCs dos pacientes, entretanto elas permaneceram com hiperatividade da sinalização via proteína G e déficit da atividade transcricional. As HSCs apresentaram altos níveis de expressão absoluta de diversas integrinas e receptores de citocinas e fatores de crescimento, potencialmente relacionados com funções na hematopoese. HSCs de pacientes com T1D apresentaram perfil de expressão gênica global distinto das de indivíduos saudáveis, com hiper-regulação de genes associados com a atividade transcricional. Os fatores de transcrição TCFL2 e p53, que têm papel fundamental na regulação do ciclo celular das HSCs, foram diferencialmente expressos entre as HSCs de pacientes diabéticos e controles. Assim, nossos resultados de expressão gênica global apontaram alterações intrínsecas nas HSCs e MSCs de pacientes diabéticos que podem estar relacionadas com a falha terapêutica dos transplantes autólogos. A implicação dessas alterações no desenvolvimento e patogênese do T1D permanece desconhecida e a realização de ensaios funcionais poderá esclarecer o significado biológico das mesmas. / Type 1 diabetes mellitus (T1D) is a T cell-mediated autoimmune disease, characterized by selective destruction of insulin-producing pancreatic ? cells. Mesenchymal stromal cells (MSCs) and hematopoietic stem cells (HSCs) are the main components of hematopoietic niches. In the last years, these cells are being used in autologous transplantation settings for T1D treatment. The main goal of this study was to evaluate the global gene expression profile of MSCs and HSCs from T1D patients, by using microarrays and bioinformatics specific programs. MSCs and HSCs were isolated from bone marrow of T1D patients before and after treatment with high dose immunossupression followed by hematopoietic stem cell transplantation. MSCs showed high absolute expression values of several molecules potentially related to their function of hematopoiesis support. MSCs from T1D patients exhibited distinct gene expression profile from control MSCs and presented up-regulation of the G protein-coupled receptor signaling pathway and down-regulation of transcriptional activity. The ?3 adrenergic receptor, as well the sympathetic nervous system signaling were up-regulated on patient´s cells. Genes that codify molecules which support hematopoeisis and are regulated by the symphatic nervous system, VCAM1 and CXCL12, were downregulated on our analysis. After AHSCT, there was an attenuation of the differential expression profile of MSCs from T1D patients, however they remained with G proteincoupled receptor signaling pathway hyperactivity and transcriptional activity deficit. HSCs exhibited high absolute expression values of integrins, cytokine receptors and growth factors, molecules potencially related to hematopoietic functions. HSCs from T1D patients showed distinct expression profile from control HSCs and demonstrated up-regulation of genes related to transcriptional activity. The transcription factors TCFL2 and p53, which have important role in regulating HSC cycle, were differentially expressed between HSCs from T1D patients and controls. Thus, our global gene expression analysis has revealed intrinsic alterations on MSCs and HSCs from T1D patients that could be related to the autologous transplant therapeutic failures. The implications of these alterations on the development and pathogenesis of T1D remain unknown and functional assays could unravel their biological meaning.

O diabetes mellitus do tipo 1 (T1D) é uma doença autoimune mediada por células T e caracterizada pela destruição seletiva das células ? pancreáticas produtoras de insulina. Células estromais mesenquimais (MSCs) e células tronco hematopoéticas (HSCs) são os principais componentes do nicho hematopoético na medula óssea. Estas células vêm sendo utilizadas nos últimos anos em transplantes autólogos para tratamento do T1D. O objetivo geral do presente trabalho foi avaliar o perfil de expressão gênica global de MSCs e HSCs de pacientes com T1D e compará-lo com células isoladas de indivíduos saudáveis através da técnica de microarray e programas específicos de bioinformática. As MSCs e HSCs foram isoladas da medula óssea de pacientes com T1D antes e após o tratamento com imunossupressão em altas doses seguida pelo transplante autólogo de células tronco hematopoéticas (AHSCT). As MSCs apresentaram valor elevado de expressão absoluta de diversas moléculas potencialmente relacionadas com suas funções de suporte à hematopoese. MSCs de pacientes diabéticos apresentaram perfil de expressão gênica global distinto das isoladas de indivíduos saudáveis, com hiper-regulação da sinalização via proteína G e hiporregulação da atividade transcricional. O receptor ?3 adrenérgico, assim como a sinalização simpática, foram hiper-expressos nas células dos pacientes. Genes que codificam moléculas que suportam a hematopoese e regulados pelo sistema nervoso simpático, VCAM1 e CXCL12, foram hiporregulados em nossa análise. Após o AHSCT, houve atenuação do perfil de expressão diferencial das MSCs dos pacientes, entretanto elas permaneceram com hiperatividade da sinalização via proteína G e déficit da atividade transcricional. As HSCs apresentaram altos níveis de expressão absoluta de diversas integrinas e receptores de citocinas e fatores de crescimento, potencialmente relacionados com funções na hematopoese. HSCs de pacientes com T1D apresentaram perfil de expressão gênica global distinto das de indivíduos saudáveis, com hiper-regulação de genes associados com a atividade transcricional. Os fatores de transcrição TCFL2 e p53, que têm papel fundamental na regulação do ciclo celular das HSCs, foram diferencialmente expressos entre as HSCs de pacientes diabéticos e controles. Assim, nossos resultados de expressão gênica global apontaram alterações intrínsecas nas HSCs e MSCs de pacientes diabéticos que podem estar relacionadas com a falha terapêutica dos transplantes autólogos. A implicação dessas alterações no desenvolvimento e patogênese do T1D permanece desconhecida e a realização de ensaios funcionais poderá esclarecer o significado biológico das mesmas. / Type 1 diabetes mellitus (T1D) is a T cell-mediated autoimmune disease, characterized by selective destruction of insulin-producing pancreatic ? cells. Mesenchymal stromal cells (MSCs) and hematopoietic stem cells (HSCs) are the main components of hematopoietic niches. In the last years, these cells are being used in autologous transplantation settings for T1D treatment. The main goal of this study was to evaluate the global gene expression profile of MSCs and HSCs from T1D patients, by using microarrays and bioinformatics specific programs. MSCs and HSCs were isolated from bone marrow of T1D patients before and after treatment with high dose immunossupression followed by hematopoietic stem cell transplantation. MSCs showed high absolute expression values of several molecules potentially related to their function of hematopoiesis support. MSCs from T1D patients exhibited distinct gene expression profile from control MSCs and presented up-regulation of the G protein-coupled receptor signaling pathway and down-regulation of transcriptional activity. The ?3 adrenergic receptor, as well the sympathetic nervous system signaling were up-regulated on patient´s cells. Genes that codify molecules which support hematopoeisis and are regulated by the symphatic nervous system, VCAM1 and CXCL12, were downregulated on our analysis. After AHSCT, there was an attenuation of the differential expression profile of MSCs from T1D patients, however they remained with G proteincoupled receptor signaling pathway hyperactivity and transcriptional activity deficit. HSCs exhibited high absolute expression values of integrins, cytokine receptors and growth factors, molecules potencially related to hematopoietic functions. HSCs from T1D patients showed distinct expression profile from control HSCs and demonstrated up-regulation of genes related to transcriptional activity. The transcription factors TCFL2 and p53, which have important role in regulating HSC cycle, were differentially expressed between HSCs from T1D patients and controls. Thus, our global gene expression analysis has revealed intrinsic alterations on MSCs and HSCs from T1D patients that could be related to the autologous transplant therapeutic failures. The implications of these alterations on the development and pathogenesis of T1D remain unknown and functional assays could unravel their biological meaning.

Indiana University-Purdue University Indianapolis (IUPUI) / Graft-versus-host disease (GVHD) remains the major complication after allogeneic hematopoietic stem cell transplantation (HSCT), resulting from immunological attack on target organs such as gastrointestinal (GI) tract, liver and skin from donor allogeneic T cells. The most common treatment for GVHD is immunosuppressive drugs such as corticosteroids, which may result in many side effects including the loss of the beneficial graft-versus-leukemia (GVL) effect and increased infection rates. However, GVHD-specific drugs have yet to be implemented. Here we show that by targeting on a novel pathogenic CD4+ T cell subpopulation that our lab previously found in patients with GI GVHD, we can develop new avenues to treat GVHD. This novel population is characterized as CD146+CCR5+ T cells, co-expressing IL-17A and IFN-γ. We found that the inducible T-cell costimulator (ICOS), which has been reported to be important for human Th17 differentiation in vitro, is critical for the development of this nonconventional T Helper 1 (Th1*)-polarized CD146+CCR5+ conventional T cells (Tconvs) population. Furthermore, we found that ICOS can induce the generation of Th1*-polarized CD146+CCR5+ regulatory T cells (Tregs) population, lowering the frequencies of phenotypic markers of functional Tregs. Our data also showed that inhibiting the major transcriptional factor of Th17, RAR-related orphan receptor gamma t (RORγt), could prevent the development of CD146+CCR5+ Tconvs in vitro. Our results demonstrate how pathogenic CD146+CCR5+ T cells are induced through ICOS or RORγt, suggesting new targets for GVHD treatment. We anticipate our assay to be a starting point for the development of novel GVHD-specific drugs. For example, the treatments that focus on inhibiting RORγ would have fewer side effects than general immunosuppressive drugs that GVHD patients use today and inhibit GVHD while sparing the GVL effect. Furthermore, we expect the CD146+CCR5+ Tconvs and/or Tregs can be used as GVHD biomarkers. These biomarkers may guide preemptive treatments such as RORγt inhibitor.

Indiana University-Purdue University Indianapolis (IUPUI) / More than 300 million people world-wide suffer from breathlessness, wheezing, chest tightness, and coughing characteristic of chronic bronchial asthma, the global incidence of which is on the rise. Allergen-sensitization and challenge elicits pulmonary expression of chemoattractants that promote a chronic eosinophil-rich infiltrate. Eosinophils are increasingly recognized as important myeloid effectors in chronic inflammation characteristic of asthma, although few eosinophil molecular signaling pathways have successfully been targeted in asthma therapy. p21 activated kinases (PAKs), members of the Ste-20 family of serine/threonine kinases, act as molecular switches in cytoskeletal-dependent processes involved in cellular motility. We hypothesized that PAK1 modulated eosinophil infiltration in an allergic airway disease (AAD) murine model. In this model, Pak1 deficient mice developed reduced inflammatory AAD responses in vivo with notable decreases in eosinophil infiltration in the lungs and broncho-alveolar lavage fluids (BALF). To test the importance of PAK1 in hematopoietic cells in AAD we used complementary bone marrow transplant experiments that demonstrated decreased eosinophil inflammation in hosts transplanted with Pak1 deficient bone marrow. In in vitro studies, we show that eotaxin-signaling through PAK1 facilitated eotaxin-mediated eosinophil migration. Ablating PAK1 expression by genetic deletion in hematopoietic progenitors or siRNA treatment in derived human eosinophils impaired eotaxin-mediated eosinophil migration, while ectopic PAK1 expression promoted this migration. Together these data suggest a key role for PAK1 in the development of atopic eosinophil inflammation and eotaxin-mediated eosinophil migration.

Indiana University-Purdue University Indianapolis (IUPUI) / Hematopoietic stem cell (HSC) transplantation is a lifesaving therapy for a number of hematological disorders. However, to be effective, transplanted HSCs must efficiently “home” to supportive niches within the bone marrow. Limited HSC number and poor function are complications of transplant in some circumstances, and can lead to delayed engraftment and immune reconstitution, or in some cases, bone marrow failure. Enhancing HSC homing is a strategy to improve stem cell transplantation efficiency. We have previously shown that ex vivo treatment of mouse or human HSCs with 16-16 dimethyl PGE2 (dmPGE2) increases their bone marrow homing efficiency and engraftment, resulting in part from upregulation of surface CXCR4 expression. We now show that pulse-treatment of mouse or human HSPCs with dmPGE2 stabilizes HIF1α in HSPCs, and that similar treatment with the hypoxia mimetic DMOG produces analogous effects to dmPGE2 on HSPC CXCR4 expression and homing. This suggests that HIF1α is responsible for PGE2’s enhancing effects on HSPCs. Pharmacological inhibition of HIF1α stabilization in vitro with Sodium Nitroprusside (SNP), confirms the requirement of HIF1α for dmPGE2-enhanced migration and CXCR4 upregulation. Additionally, we confirm the requirement for HIF1α in dmPGE2-enhanced in vivo homing using a conditional knockout mouse model of HIF1α gene deletion. Finally, we validate that the hypoxia response element located 1.3kb from the transcriptional start site within the CXCR4 promoter is required for enhanced CXCR4 expression after PGE2 treatment. Interestingly, we also observe an increase in the small GTPase Rac1 after dmPGE2 treatment, as well as a defect in PGE2-enhanced migration and CXCR4 expression in Rac1 knockout HSPCs. Using state-of-the-art imaging technology we, confirm an increase in Rac1 and CXCR4 colocalization after dmPGE2 treatment that likely explains enhanced sensitivity of PGE2-treated HSPCs to SDF-1. Taken together, these results define a precise mechanism through which ex vivo pulse treatment of HSPC with dmPGE2 enhances HSPC function through alterations in cell motility and homing, and describe a role for hypoxia and HIF1α in enhancement of hematopoietic transplantation.

Indiana University-Purdue University Indianapolis (IUPUI) / Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in malignant tumors, and its activation is associated with high histological grade and advanced cancer stage. STAT3 has been shown to play important roles in multiple aspects of cancer aggressiveness including proliferation, survival, self-renewal, migration, invasion, angiogenesis and immune response by regulating the expression of diverse downstream target genes. Thus, inhibiting STAT3 promises to be an attractive strategy for treatment of advanced tumors with metastatic potential. We firstly identified a STAT3 inhibitor, inS3-54, by targeting the DNA-binding site of STAT3 using an in-silico screening approach; however, inS3-54 was finally found not to be appropriate for further studies because of low specificity on STAT3 and poor absorption in mice. To develop an effective and specific STAT3 inhibitor, we identified 89 analogues for the structure-activity relationship analysis. By using hematopoietic progenitor cells isolated from wild-type and STAT3 conditional knockout mice, further studies showed that three analogues (A18, A26 and A69) only inhibited STAT3-dependent colony formation of hematopoietic progenitor cells, indicating a higher selectivity for STAT3 than their parental compound, inS3-54. These compounds were found to (1) inhibit STAT3-specific DNA binding activity; (2) bind to STAT3 protein; (3) suppress proliferation of cancer cells harboring aberrant STAT3 signaling; (4) inhibit migration and invasion of cancer cells and (5) inhibit STAT3-dependent expression of downstream targets by blocking the binding of STAT3 to the promoter regions of responsive genes in cells. In addition, A18 can reduce tumor growth in a mouse xenograft model of lung cancer with little effect on body weight. Taken together, we conclude that it is feasible to inhibit STAT3 by targeting its DNA-binding domain for discovery of anticancer therapeutics.

Indiana University-Purdue University Indianapolis (IUPUI) / The potential of hematopoietic stem cells (HSC) to home and to anchor within the bone marrow (BM) microenvironment controls the ability of transplanted HSCs to establish normal hematopoiesis. Activated Leukocyte Cell Adhesion Molecule (ALCAM; also identified as CD166), which participates in homophilic interactions, is expressed on a group of osteoblasts in the hematopoietic niche capable of sustaining functional HSC in vitro. Since we could also detect ALCAM expression on HSC, we suspect that ALCAM may play a role in anchoring primitive hematopoietic cells to ALCAM expressing components of the hematopoietic niche via dimerization. We investigated the role of ALCAM on the homing abilities of hematopoietic stem and progenitor cells (HSPC) by calculating recovery frequency of Sca-1+ALCAM+ cells in an in vivo murine bone marrow transplantation model. Our data supports the notion that ALCAM promotes improved homing potential of hematopoietic Sca-1+ cells. Recovery of BM-homed Sca-1+ cells from the endosteal region was 1.8-fold higher than that of total donor cells. However, a 3.0-fold higher number of Sca-1+ALCAM+ cells homed to the endosteal region compared to total donor cells. Similarly, homed Sca-1+ALCAM+ cells were recovered from the vascular region at 2.1-fold greater frequency than total homed donor cells from that region, compared to only a 1.3-fold increase in the recovery frequency of Sca-1+ cells. In vitro quantitation of clonogenic BM-homed hematopoietic progenitors corroborate the results from the homing assay. The frequency of in vitro clonogenic progenitors was significantly higher among endosteal-homed Sca-1+ALCAM+ cells compared to other fractions of donor cells. Collectively, these data demonstrate that engrafting HSC expressing ALCAM home more efficiently to the BM and within the BM microenvironment, these cells preferentially seed the endosteal niche.

Indiana University-Purdue University Indianapolis (IUPUI) / Immunotherapy for cancer has held much promise as a potent modality of cancer treatment. The ability to selectively destroy diseased cells and leave healthy cells unharmed has been the goal of cancer immunotherapy for the past thirty years. However, the full capabilities of cancer immunotherapies have been elusive. Cancer immunotherapies have been consistently hampered by limited immune reactivity, a diminishing immune response over time, and a failure to overcome self-tolerance. Many of these deficiencies have been borne-out by immunotherapies that have focused on the adoptive transfer of activated or genetically modified mature CD8+ T cells. The limitations inherent in therapies involving terminally differentiated mature lymphocytes include limited duration, lack of involvement of other components of the immune system, and limited clinical efficacy. We sought to overcome these limitations by altering and enhancing long-term host immunity by genetically modifying then transplanting HSCs. To study these questions and test the efficiency of gene transfer, we cloned a tumor reactive HLA-DR4-restricted CD4+ TCR specific for the melanocyte differentiation antigen TRP-1, then constructed both a high expression lentiviral delivery system and a TCR Tg expressing the same TCR genes. We demonstrate with both mouse and human HSCs durable, high-efficiency TCR gene transfer, following long-term transplantation. We demonstrate the induction of spontaneous autoimmune vitiligo and a TCR-specific TH1 polarized memory effector CD4+ T cell population. Most importantly, we demonstrate the destruction of subcutaneous melanoma without the aid of vaccination, immune modulation, or cytokine administration. Overall, these results demonstrate the creation of a novel translational model of durable lentiviral gene transfer, the induction of spontaneous CD4+ T cell immunity, the breaking of self-tolerance, and the induction of anti-tumor immunity.