RESUMO
PURPOSE OF REVIEW: Inflammasomes are multimeric protein structures with crucial roles in host responses against infections and injuries. The importance of inflammasome activation goes beyond host defense as a dysregulated inflammasome and subsequent secretion of IL-1 family members is believed to be involved in the pathogenesis of various diseases, some of which also produce skeletal manifestations. The purpose of this review is to summarize recent developments in the understanding of inflammasome regulation and IL-1 family members in bone physiology and pathology and current therapeutics will be discussed. RECENT FINDINGS: Small animal models have been vital to help understand how the inflammasome regulates bone dynamics. Animal models with gain or loss of function in various inflammasome components or IL-1 family signaling have illustrated how these systems can impact numerous bone pathologies and have been utilized to test new inflammasome therapeutics. It is increasingly clear that a tightly regulated inflammasome is required not only for host defense but for skeletal homeostasis, as a dysregulated inflammasome is linked to diseases of pathological bone accrual and loss. Given the complexities of inflammasome activation and redundancies in IL-1 activation and secretion, targeting these pathways is at times challenging. Ongoing research into inflammasome-mediated mechanisms will allow the development of new therapeutics for inflammasome/IL-1 diseases.
Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Homeostase , Humanos , Inflamassomos/metabolismo , Interleucina-1beta , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Transdução de SinaisRESUMO
PURPOSE OF REVIEW: Neurogenic heterotopic ossification (NHO) is the abnormal formation of extra-skeletal bones in periarticular muscles after damage to the central nervous system (CNS) such as spinal cord injury (SCI), traumatic brain injury (TBI), stroke, or cerebral anoxia. The purpose of this review is to summarize recent developments in the understanding of NHO pathophysiology and pathogenesis. Recent animal models of NHO and recent findings investigating the communication between CNS injury, tissue inflammation, and upcoming NHO therapeutics are discussed. RECENT FINDINGS: Animal models of NHO following TBI or SCI have shown that NHO requires the combined effects of a severe CNS injury and soft tissue damage, in particular muscular inflammation and the infiltration of macrophages into damaged muscles plays a key role. In the context of a CNS injury, the inflammatory response to soft tissue damage is exaggerated and persistent with excessive signaling via substance P-, oncostatin M-, and TGF-ß1-mediated pathways. This review provides an overview of the known animal models and mechanisms of NHO and current therapeutic interventions for NHO patients. While some of the inflammatory mechanisms leading to NHO are common with other forms of traumatic and genetic heterotopic ossifications (HO), NHOs uniquely involve systemic changes in response to CNS injury. Future research into these CNS-mediated mechanisms is likely to reveal new targetable pathways to prevent NHO development in patients.
Assuntos
Sistema Nervoso Central/lesões , Ossificação Heterotópica/etiologia , Ossificação Heterotópica/fisiopatologia , Animais , Modelos Animais de Doenças , Humanos , Ossificação Heterotópica/terapiaRESUMO
Allogeneic hematopoietic stem cell transplantation is hampered by chronic graft-versus-host disease (cGVHD), resulting in multiorgan fibrosis and diminished function. Fibrosis in lung and skin leads to progressive bronchiolitis obliterans (BO) and scleroderma, respectively, for which new treatments are needed. We evaluated pirfenidone, a Food and Drug Administration (FDA)-approved drug for idiopathic pulmonary fibrosis, for its therapeutic effect in cGVHD mouse models with distinct pathophysiology. In a full major histocompatibility complex (MHC)-mismatched, multiorgan system model with BO, donor T-cell responses that support pathogenic antibody production are required for cGVHD development. Pirfenidone treatment beginning one month post-transplant restored pulmonary function and reversed lung fibrosis, which was associated with reduced macrophage infiltration and transforming growth factor-ß production. Pirfenidone dampened splenic germinal center B-cell and T-follicular helper cell frequencies that collaborate to produce antibody. In both a minor histocompatibility antigen-mismatched as well as a MHC-haploidentical model of sclerodermatous cGVHD, pirfenidone significantly reduced macrophages in the skin, although clinical improvement of scleroderma was only seen in one model. In vitro chemotaxis assays demonstrated that pirfenidone impaired macrophage migration to monocyte chemoattractant protein-1 (MCP-1) as well as IL-17A, which has been linked to cGVHD generation. Taken together, our data suggest that pirfenidone is a potential therapeutic agent to ameliorate fibrosis in cGVHD.
Assuntos
Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas , Macrófagos/imunologia , Piridonas/farmacologia , Dermatopatias/prevenção & controle , Fator de Crescimento Transformador beta/imunologia , Aloenxertos , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Bronquiolite Obliterante/genética , Bronquiolite Obliterante/imunologia , Bronquiolite Obliterante/patologia , Bronquiolite Obliterante/prevenção & controle , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Interleucina-17/genética , Interleucina-17/imunologia , Macrófagos/patologia , Camundongos , Camundongos Mutantes , Fibrose Pulmonar/genética , Fibrose Pulmonar/imunologia , Fibrose Pulmonar/patologia , Fibrose Pulmonar/prevenção & controle , Dermatopatias/genética , Dermatopatias/imunologia , Dermatopatias/patologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/patologia , Fator de Crescimento Transformador beta/genéticaRESUMO
Chronic graft-versus-host disease (cGVHD) is a major cause of late mortality following allogeneic bone marrow transplantation (BMT) and is characterized by tissue fibrosis manifesting as scleroderma and bronchiolitis obliterans. The development of acute GVHD (aGVHD) is a powerful clinical predictor of subsequent cGVHD, suggesting that aGVHD may invoke the immunologic pathways responsible for cGVHD. In preclinical models in which sclerodermatous cGVHD develops after a preceding period of mild aGVHD, we show that antigen presentation within major histocompatibility complex (MHC) class II of donor dendritic cells (DCs) is markedly impaired early after BMT. This is associated with a failure of regulatory T-cell (Treg) homeostasis and cGVHD. Donor DC-restricted deletion of MHC class II phenocopied this Treg deficiency and cGVHD. Moreover, specific depletion of donor Tregs after BMT also induced cGVHD, whereas adoptive transfer of Tregs ameliorated it. These data demonstrate that the defect in Treg homeostasis seen in cGVHD is a causative lesion and is downstream of defective antigen presentation within MHC class II that is induced by aGVHD.
Assuntos
Apresentação de Antígeno , Transplante de Medula Óssea/efeitos adversos , Células Dendríticas/patologia , Doença Enxerto-Hospedeiro/patologia , Linfócitos T Reguladores/patologia , Doença Aguda , Transferência Adotiva , Animais , Doença Crônica , Células Dendríticas/imunologia , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/terapia , Antígenos de Histocompatibilidade Classe II/imunologia , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/transplanteRESUMO
The interplay between the inflammatory infiltrate and tissue resident cell populations invokes fibrogenesis. However, the temporal and mechanistic contributions of these cells to fibrosis are obscure. To address this issue, liver inflammation, ductular reaction (DR), and fibrosis were induced in C57BL/6 mice by thioacetamide administration for up to 12 weeks. Thioacetamide treatment induced two phases of liver fibrosis. A rapid pericentral inflammatory infiltrate enriched in F4/80(+) monocytes co-localized with SMA(+) myofibroblasts resulted in early collagen deposition, marking the start of an initial fibrotic phase (1 to 6 weeks). An expansion of bone marrow-derived macrophages preceded a second phase, characterized by accelerated progression of fibrosis (>6 weeks) after DR migration from the portal tracts to the centrilobular site of injury, in association with an increase in DR/macrophage interactions. Although chemokine (C-C motif) ligand 2 (CCL2) mRNA was induced rapidly in response to thioacetamide, CCL2 deficiency only partially abrogated fibrosis. In contrast, colony-stimulating factor 1 receptor blockade diminished C-C chemokine receptor type 2 [CCR2(neg) (Ly6C(lo))] monocytes, attenuated the DR, and significantly reduced fibrosis, illustrating the critical role of colony-stimulating factor 1-dependent monocyte/macrophage differentiation and linking the two phases of injury. In response to liver injury, colony-stimulating factor 1 drives early monocyte-mediated myofibroblast activation and collagen deposition, subsequent macrophage differentiation, and their association with the advancing DR, the formation of fibrotic septa, and the progression of liver fibrosis to cirrhosis.
Assuntos
Hepatite Animal/patologia , Cirrose Hepática Experimental/patologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/metabolismo , Animais , Quimiocinas/genética , Quimiocinas/metabolismo , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hepatite Animal/genética , Hepatite Animal/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/metabolismo , Fator Estimulador de Colônias de Macrófagos/genética , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , TioacetamidaRESUMO
Idiopathic pneumonia syndrome (IPS) is a relatively common, frequently fatal clinical entity, characterized by noninfectious acute lung inflammation following allogeneic stem cell transplantation (SCT), the mechanisms of which are unclear. In this study, we demonstrate that immune suppression with cyclosporin after SCT limits T-helper cell (Th) 1 differentiation and interferon-γ secretion by donor T cells, which is critical for inhibiting interleukin (IL)-6 generation from lung parenchyma during an alloimmune response. Thereafter, local IL-6 secretion induces donor alloantigen-specific Th17 cells to preferentially expand within the lung, and blockade of IL-17A or transplantation of grafts lacking the IL-17 receptor prevents disease. Studies using IL-6(-/-) recipients or IL-6 blockade demonstrate that IL-6 is the critical driver of donor Th17 differentiation within the lung. Importantly, IL-6 is also dysregulated in patients undergoing clinical SCT and is present at very high levels in the plasma of patients with IPS compared with SCT recipients without complications. Furthermore, at the time of diagnosis, plasma IL-6 levels were higher in a subset of IPS patients who were nonresponsive to steroids and anti-tumor necrosis factor therapy. In sum, pulmonary-derived IL-6 promotes IPS via the induction of Th17 differentiation, and strategies that target these cytokines represent logical therapeutic approaches for IPS.
Assuntos
Lesão Pulmonar Aguda/etiologia , Ciclosporina/efeitos adversos , Imunossupressores/efeitos adversos , Interleucina-17/imunologia , Interleucina-6/imunologia , Pulmão/patologia , Transplante de Células-Tronco/efeitos adversos , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/patologia , Animais , Ciclosporina/uso terapêutico , Feminino , Imunossupressores/uso terapêutico , Interferon gama/imunologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Transplante HomólogoRESUMO
Novel therapies for chronic graft-versus-host disease (cGVHD) are needed. Aberrant B-cell activation has been demonstrated in mice and humans with cGVHD. Having previously found that human cGVHD B cells are activated and primed for survival, we sought to further evaluate the role of the spleen tyrosine kinase (Syk) in cGVHD in multiple murine models and human peripheral blood cells. In a murine model of multiorgan system, nonsclerodermatous disease with bronchiolitis obliterans where cGVHD is dependent on antibody and germinal center (GC) B cells, we found that activation of Syk was necessary in donor B cells, but not T cells, for disease progression. Bone marrow-specific Syk deletion in vivo was effective in treating established cGVHD, as was a small-molecule inhibitor of Syk, fostamatinib, which normalized GC formation and decreased activated CD80/86(+) dendritic cells. In multiple distinct models of sclerodermatous cGVHD, clinical and pathological disease manifestations were not eliminated when mice were therapeutically treated with fostamatinib, though both clinical and immunologic effects could be observed in one of these scleroderma models. We further demonstrated that Syk inhibition was effective at inducing apoptosis of human cGVHD B cells. Together, these data demonstrate a therapeutic potential of targeting B-cell Syk signaling in cGVHD.
Assuntos
Linfócitos B/enzimologia , Doença Enxerto-Hospedeiro/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ativação Linfocitária/imunologia , Proteínas Tirosina Quinases/metabolismo , Aminopiridinas , Animais , Linfócitos B/imunologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Citometria de Fluxo , Imunofluorescência , Doença Enxerto-Hospedeiro/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Morfolinas , Oxazinas/farmacologia , Piridinas/farmacologia , Pirimidinas , Quinase SykRESUMO
The distribution, phenotype, and requirement of macrophages for fracture-associated inflammation and/or early anabolic progression during endochondral callus formation were investigated. A murine femoral fracture model [internally fixed using a flexible plate (MouseFix)] was used to facilitate reproducible fracture reduction. IHC demonstrated that inflammatory macrophages (F4/80(+)Mac-2(+)) were localized with initiating chondrification centers and persisted within granulation tissue at the expanding soft callus front. They were also associated with key events during soft-to-hard callus transition. Resident macrophages (F4/80(+)Mac-2(neg)), including osteal macrophages, predominated in the maturing hard callus. Macrophage Fas-induced apoptosis transgenic mice were used to induce macrophage depletion in vivo in the femoral fracture model. Callus formation was completely abolished when macrophage depletion was initiated at the time of surgery and was significantly reduced when depletion was delayed to coincide with initiation of early anabolic phase. Treatment initiating 5 days after fracture with the pro-macrophage cytokine colony stimulating factor-1 significantly enhanced soft callus formation. The data support that inflammatory macrophages were required for initiation of fracture repair, whereas both inflammatory and resident macrophages promoted anabolic mechanisms during endochondral callus formation. Overall, macrophages make substantive and prolonged contributions to fracture healing and can be targeted as a therapeutic approach for enhancing repair mechanisms. Thus, macrophages represent a viable target for the development of pro-anabolic fracture treatments with a potentially broad therapeutic window.
Assuntos
Fraturas do Fêmur/fisiopatologia , Consolidação da Fratura , Macrófagos/metabolismo , Osteogênese/fisiologia , Periósteo/metabolismo , Animais , Apoptose , Diferenciação Celular , Proliferação de Células , Citocinas/metabolismo , Progressão da Doença , Citometria de Fluxo , Fixação de Fratura , Imuno-Histoquímica , Inflamação , Fixadores Internos , Fator Estimulador de Colônias de Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Monócitos/citologia , FenótipoRESUMO
Donor T cells play pivotal roles in graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effects following bone marrow transplantation (BMT). DNAX accessory molecule 1 (DNAM-1) is a costimulatory and adhesion molecule, expressed mainly by natural killer cells and CD8(+) T cells at steady state to promote adhesion to ligand-expressing targets and enhance cytolysis. We have analyzed the role of this pathway in GVHD and GVL. The absence of DNAM-1 on the donor graft attenuated GVHD in major histocompatibility complex (MHC)-mismatched and MHC-matched BMT following conditioning with lethal and sublethal irradiation. In contrast, DNAM-1 was not critical for GVL effects against ligand (CD155) expressing and nonexpressing leukemia. The effects on GVHD following myeloablative conditioning were independent of CD8(+) T cells and dependent on CD4(+) T cells, and specifically donor FoxP3(+) regulatory T cells (Treg). The absence of DNAM-1 promoted the expansion and suppressive function of Treg after BMT. These findings provide support for therapeutic DNAM-1 inhibition to promote tolerance in relevant inflammatory-based diseases characterized by T-cell activation.
Assuntos
Antígenos de Diferenciação de Linfócitos T/fisiologia , Transplante de Medula Óssea , Doença Enxerto-Hospedeiro/prevenção & controle , Efeito Enxerto vs Leucemia/imunologia , Leucemia Experimental/prevenção & controle , Linfócitos T Reguladores/imunologia , Animais , Antígenos de Diferenciação de Linfócitos T/química , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/metabolismo , Feminino , Fatores de Transcrição Forkhead/metabolismo , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/imunologia , Leucemia Experimental/etiologia , Leucemia Experimental/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Condicionamento Pré-Transplante , Células Tumorais Cultivadas , Irradiação Corporal TotalRESUMO
Previous studies have generated conflicting results regarding the contribution of B cells to bone formation during physiology and repair. Here, we have investigated the role of B cells in osteoblast-mediated intramembranous anabolic bone modeling. Immunohistochemistry for CD45 receptor expression indicated that B cells had no propensity or aversion for endosteal regions or sites of bone modeling and/or remodeling in wild-type mice. In the endocortical diaphyseal region, quantitative immunohistology demonstrated that young wild-type and B-cell deficient mice had similar amounts of osteocalcin(+) osteoblast bone modeling surface. The degree of osteoblast-associated osteomac canopy was also comparable in these mice inferring that bone modeling cellular units were preserved in the absence of B cells. In a tibial injury model, only rare CD45 receptor positive B cells were located within areas of high anabolic activity, including minimal association with osterix(+) osteoblast-lineage committed mesenchymal cells in wild-type mice. Quantitative immunohistology demonstrated that collagen type I matrix deposition and macrophage and osteoclast distribution within the injury site were not compromised by the absence of B cells. Overall, osteoblast distribution during normal growth and bone healing via intramembranous ossification proceeded normally in the absence of B cells. These observations support that in vivo, these lymphoid cells have minimal influence, or at most, make redundant contributions to osteoblast function during anabolic bone modeling via intramembranous mechanisms.
Assuntos
Linfócitos B/patologia , Depleção Linfocítica , Osteogênese , Tíbia/lesões , Tíbia/patologia , Cicatrização , Animais , Medula Óssea/patologia , Remodelação Óssea , Microambiente Celular , Diáfises/patologia , Modelos Animais de Doenças , Antígenos Comuns de Leucócito/metabolismo , Membranas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Ossificação Heterotópica/patologia , Ossificação Heterotópica/fisiopatologia , Tíbia/fisiopatologiaRESUMO
Hematopoietic stem cells (HSCs) interact with osteoblastic, stromal, and vascular components of the BM hematopoietic microenvironment (HM) that are required for the maintenance of long-term self-renewal in vivo. Osteoblasts have been reported to be a critical cell type making up the HSC niche in vivo. Rac1 GTPase has been implicated in adhesion, spreading, and differentiation of osteoblast cell lines and is critical for HSC engraftment and retention. Recent data suggest a differential role of GTPases in endosteal/osteoblastic versus perivascular niche function. However, whether Rac signaling pathways are also necessary in the cell-extrinsic control of HSC function within the HM has not been examined. In the present study, genetic and inducible models of Rac deletion were used to demonstrate that Rac depletion causes impaired proliferation and induction of apoptosis in the OP9 cell line and in primary BM stromal cells. Deletion of Rac proteins caused reduced trabecular and cortical long bone growth in vivo. Surprisingly, HSC function and maintenance of hematopoiesis in vivo was preserved despite these substantial cell-extrinsic changes. These data have implications for therapeutic strategies to target Rac signaling in HSC mobilization and in the treatment of leukemia and provide clarification to our evolving concepts of HSC-HM interactions.
Assuntos
Desenvolvimento Ósseo/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Osteoblastos/metabolismo , Transdução de Sinais , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Apoptose , Western Blotting , Células da Medula Óssea , Comunicação Celular , Diferenciação Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Citometria de Fluxo , Hematopoese , Técnicas Imunoenzimáticas , Camundongos , Camundongos Knockout , Neuropeptídeos/fisiologia , Osteoblastos/citologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Células Estromais , Microtomografia por Raio-X , Proteínas rac de Ligação ao GTP/antagonistas & inibidores , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/fisiologia , Proteínas rac1 de Ligação ao GTP , Proteína RAC2 de Ligação ao GTPRESUMO
FoxP3(+) confers suppressive properties and is confined to regulatory T cells (T(reg)) that potently inhibit autoreactive immune responses. In the transplant setting, natural CD4(+) T(reg) are critical in controlling alloreactivity and the establishment of tolerance. We now identify an important CD8(+) population of FoxP3(+) T(reg) that convert from CD8(+) conventional donor T cells after allogeneic but not syngeneic bone marrow transplantation. These CD8(+) T(reg) undergo conversion in the mesenteric lymph nodes under the influence of recipient dendritic cells and TGF-ß. Importantly, this population is as important for protection from GVHD as the well-studied natural CD4(+)FoxP3(+) population and is more potent in exerting class I-restricted and antigen-specific suppression in vitro and in vivo. Critically, CD8(+)FoxP3(+) T(reg) are exquisitely sensitive to inhibition by cyclosporine but can be massively and specifically expanded in vivo to prevent GVHD by coadministering rapamycin and IL-2 antibody complexes. CD8(+)FoxP3(+) T(reg) thus represent a new regulatory population with considerable potential to preferentially subvert MHC class I-restricted T-cell responses after bone marrow transplantation.
Assuntos
Transplante de Medula Óssea , Linfócitos T CD8-Positivos/citologia , Fatores de Transcrição Forkhead/metabolismo , Tolerância Imunológica/imunologia , Linfócitos T Reguladores/citologia , Animais , Anticorpos/administração & dosagem , Anticorpos/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Proliferação de Células/efeitos dos fármacos , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Epitopos/imunologia , Feminino , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Tolerância Imunológica/efeitos dos fármacos , Interleucina-2/imunologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Sirolimo/administração & dosagem , Sirolimo/farmacologia , Análise de Sobrevida , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta/farmacologia , Transplante HomólogoRESUMO
Neurogenic heterotopic ossifications (NHO) are heterotopic bones that develop in periarticular muscles after severe central nervous system (CNS) injuries. Several retrospective studies have shown that NHO prevalence is higher in patients who suffer concomitant infections. However, it is unclear whether these infections directly contribute to NHO development or reflect the immunodepression observed in patients with CNS injury. Using our mouse model of NHO induced by spinal cord injury (SCI) between vertebrae T11 to T13 , we demonstrate that lipopolysaccharides (LPS) from gram-negative bacteria exacerbate NHO development in a toll-like receptor-4 (TLR4)-dependent manner, signaling through the TIR-domain-containing adapter-inducing interferon-ß (TRIF/TICAM1) adaptor rather than the myeloid differentiation primary response-88 (MYD88) adaptor. We find that T11 to T13 SCI did not significantly alter intestinal integrity nor cause intestinal bacteria translocation or endotoxemia, suggesting that NHO development is not driven by endotoxins from the gut in this model of SCI-induced NHO. Relevant to the human pathology, LPS increased expression of osteoblast markers in cultures of human fibro-adipogenic progenitors isolated from muscles surrounding NHO biopsies. In a case-control retrospective study in patients with traumatic brain injuries, infections with gram-negative Pseudomonas species were significantly associated with NHO development. Together these data suggest a functional association between gram-negative bacterial infections and NHO development and highlights infection management as a key consideration to avoid NHO development in patients. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
Assuntos
Ossificação Heterotópica , Traumatismos da Medula Espinal , Camundongos , Animais , Humanos , Lipopolissacarídeos/farmacologia , Estudos Retrospectivos , Traumatismos da Medula Espinal/complicações , Ossificação Heterotópica/patologia , Bactérias , MineraisRESUMO
In the bone marrow, hematopoietic stem cells (HSCs) reside in specific niches near osteoblast-lineage cells at the endosteum. To investigate the regulation of these endosteal niches, we studied the mobilization of HSCs into the bloodstream in response to granulocyte colony-stimulating factor (G-CSF). We report that G-CSF mobilization rapidly depletes endosteal osteoblasts, leading to suppressed endosteal bone formation and decreased expression of factors required for HSC retention and self-renewal. Importantly, G-CSF administration also depleted a population of trophic endosteal macrophages (osteomacs) that support osteoblast function. Osteomac loss, osteoblast suppression, and HSC mobilization occurred concomitantly, suggesting that osteomac loss could disrupt endosteal niches. Indeed, in vivo depletion of macrophages, in either macrophage Fas-induced apoptosis (Mafia) transgenic mice or by administration of clodronate-loaded liposomes to wild-type mice, recapitulated the: (1) loss of endosteal osteoblasts and (2) marked reduction of HSC-trophic cytokines at the endosteum, with (3) HSC mobilization into the blood, as observed during G-CSF administration. Together, these results establish that bone marrow macrophages are pivotal to maintain the endosteal HSC niche and that the loss of such macrophages leads to the egress of HSCs into the blood.
Assuntos
Células da Medula Óssea/citologia , Células-Tronco Hematopoéticas/citologia , Macrófagos/citologia , Nicho de Células-Tronco/metabolismo , Animais , Células da Medula Óssea/metabolismo , Diferenciação Celular , Linhagem da Célula , Movimento Celular/fisiologia , Separação Celular , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos , Células-Tronco Hematopoéticas/metabolismo , Imuno-Histoquímica , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Neurogenic heterotopic ossifications (NHOs) are incapacitating complications of traumatic brain and spinal cord injuries (SCI) that manifest as abnormal bone formation in periarticular muscles. Using a unique model of NHO after SCI in genetically unmodified mice, we have previously established that the innate immune system plays a key driving role in NHO pathogenesis. The role of adaptive immune cells in NHO pathogenesis, however, remains unexplored in this model. Here we established that B lymphocytes were reduced in the spleen and blood after SCI and increased in muscles of mice in which NHO develops, whereas minimal changes in T cell frequencies were noted. Interestingly, Rag1 -/- mice lacking mature T and B lymphocytes, developed NHO, similar to wild-type mice. Finally, mice that underwent splenectomy before SCI and muscle damage also developed NHO to the same extent as non-splenectomized SCI controls. Overall, our findings show that functional T and B lymphocytes have minimal influence or dispensable contributions to NHO development after experimental SCI in mice.
RESUMO
We show that pro-inflammatory oncostatin M (OSM) is an important regulator of hematopoietic stem cell (HSC) niches in the bone marrow (BM). Treatment of healthy humans and mice with granulocyte colony-stimulating factor (G-CSF) dramatically increases OSM release in blood and BM. Using mice null for the OSM receptor (OSMR) gene, we demonstrate that OSM provides a negative feed-back acting as a brake on HSPC mobilization in response to clinically relevant mobilizing molecules G-CSF and CXCR4 antagonist. Likewise, injection of a recombinant OSM molecular trap made of OSMR complex extracellular domains enhances HSC mobilization in poor mobilizing C57BL/6 and NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice. Mechanistically, OSM attenuates HSC chemotactic response to CXCL12 and increases HSC homing to the BM signaling indirectly via BM endothelial and mesenchymal cells which are the only cells expressing OSMR in the BM. OSM up-regulates E-selectin expression on BM endothelial cells indirectly increasing HSC proliferation. RNA sequencing of HSCs from Osmr-/- and wild-type mice suggest that HSCs have altered cytoskeleton reorganization, energy usage and cycling in the absence of OSM signaling in niches. Therefore OSM is an important regulator of HSC niche function restraining HSC mobilization and anti-OSM therapy combined with current mobilizing regimens may improve HSPC mobilization for transplantation.
Assuntos
Medula Óssea/fisiologia , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Oncostatina M/metabolismo , Nicho de Células-Tronco , Animais , Medula Óssea/efeitos dos fármacos , Feminino , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NODRESUMO
Neurogenic heterotopic ossifications (NHOs) form in periarticular muscles after severe spinal cord (SCI) and traumatic brain injuries. The pathogenesis of NHO is poorly understood with no effective preventive treatment. The only curative treatment remains surgical resection of pathological NHOs. In a mouse model of SCI-induced NHO that involves a transection of the spinal cord combined with a muscle injury, a differential gene expression analysis revealed that genes involved in inflammation such as interleukin-1ß (IL-1ß) were overexpressed in muscles developing NHO. Using mice knocked-out for the gene encoding IL-1 receptor (IL1R1) and neutralizing antibodies for IL-1α and IL-1ß, we show that IL-1 signaling contributes to NHO development after SCI in mice. Interestingly, other proteins involved in inflammation that were also overexpressed in muscles developing NHO, such as colony-stimulating factor-1, tumor necrosis factor, or C-C chemokine ligand-2, did not promote NHO development. Finally, using NHO biopsies from SCI and TBI patients, we show that IL-1ß is expressed by CD68+ macrophages. IL-1α and IL-1ß produced by activated human monocytes promote calcium mineralization and RUNX2 expression in fibro-adipogenic progenitors isolated from muscles surrounding NHOs. Altogether, these data suggest that interleukin-1 promotes NHO development in both humans and mice. © 2021 American Society for Bone and Mineral Research (ASBMR).
Assuntos
Interleucina-1beta/metabolismo , Ossificação Heterotópica , Traumatismos da Medula Espinal , Animais , Humanos , Inflamação/complicações , Interleucina-1 , Camundongos , Músculos/patologia , Ossificação Heterotópica/patologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Traumatismos da Medula Espinal/complicaçõesRESUMO
Perforin is a key effector of lymphocyte-mediated cell death pathways and contributes to transplant rejection of immunologically mismatched grafts. We have developed a novel series of benzenesulfonamide (BZS) inhibitors of perforin that can mitigate graft rejection during allogeneic bone marrow/stem cell transplantation. Eight such perforin inhibitors were tested for their murine pharmacokinetics, plasma protein binding, and their ability to block perforin-mediated lysis in vitro and to block the rejection of major histocompatibility complex (MHC)-mismatched mouse bone marrow cells. All compounds showed >99% binding to plasma proteins and demonstrated perforin inhibitory activity in vitro and in vivo. A lead compound, compound 1, that showed significant increases in allogeneic bone marrow preservation was evaluated for its plasma pharmacokinetics and in vivo efficacy at multiple dosing regimens to establish a pharmacokinetic/pharmacodynamic (PK/PD) relationship. The strongest PK/PD correlation was observed between perforin inhibition in vivo and time that total plasma concentrations remained above 900 µM, which correlates to unbound concentrations similar to 3× the unbound in vitro IC90 of compound 1. This PK/PD relationship will inform future dosing strategies of BZS perforin inhibitors to maintain concentrations above 3× the unbound IC90 for as long as possible to maximize efficacy and enhance progression toward clinical evaluation.
RESUMO
The cells of origin of neurogenic heterotopic ossifications (NHOs), which develop frequently in the periarticular muscles following spinal cord injuries (SCIs) and traumatic brain injuries, remain unclear because skeletal muscle harbors two progenitor cell populations: satellite cells (SCs), which are myogenic, and fibroadipogenic progenitors (FAPs), which are mesenchymal. Lineage-tracing experiments using the Cre recombinase/LoxP system were performed in two mouse strains with the fluorescent protein ZsGreen specifically expressed in either SCs or FAPs in skeletal muscles under the control of the Pax7 or Prrx1 gene promoter, respectively. These experiments demonstrate that following muscle injury, SCI causes the upregulation of PDGFRα expression on FAPs but not SCs and the failure of SCs to regenerate myofibers in the injured muscle, with reduced apoptosis and continued proliferation of muscle resident FAPs enabling their osteogenic differentiation into NHOs. No cells expressing ZsGreen under the Prrx1 promoter were detected in the blood after injury, suggesting that the cells of origin of NHOs are locally derived from the injured muscle. We validated these findings using human NHO biopsies. PDGFRα+ mesenchymal cells isolated from the muscle surrounding NHO biopsies could develop ectopic human bones when transplanted into immunocompromised mice, whereas CD56+ myogenic cells had a much lower potential. Therefore, NHO is a pathology of the injured muscle in which SCI reprograms FAPs to undergo uncontrolled proliferation and differentiation into osteoblasts.
RESUMO
Hematopoiesis and bone interact in various developmental and pathological processes. Neurogenic heterotopic ossifications (NHO) are the formation of ectopic hematopoietic bones in peri-articular muscles that develop following severe lesions of the central nervous system such as traumatic cerebral or spinal injuries or strokes. This review will focus on the hematopoietic facet of NHO. The characterization of NHO demonstrates the presence of hematopoietic marrow in which quiescent hematopoietic stem cells (HSC) are maintained by a functional stromal microenvironment, thus documenting that NHOs are neo-formed ectopic HSC niches. Similarly to adult bone marrow, the NHO permissive environment supports HSC maintenance, proliferation and differentiation through bidirectional signaling with mesenchymal stromal cells and endothelial cells, involving cell adhesion molecules, membrane-bound growth factors, hormones, and secreted matrix proteins. The participation of the nervous system, macrophages and inflammatory cytokines including oncostatin M and transforming growth factor (TGF)-ß in this process, reveals how neural circuitry fine-tunes the inflammatory response to generate hematopoietic bones in injured muscles. The localization of NHOs in the peri-articular muscle environment also suggests a role of muscle mesenchymal cells and bone metabolism in development of hematopoiesis in adults. Little is known about the establishment of bone marrow niches and the regulation of HSC cycling during fetal development. Similarities between NHO and development of fetal bones make NHOs an interesting model to study the establishment of bone marrow hematopoiesis during development. Conversely, identification of stage-specific factors that specify HSC developmental state during fetal bone development will give more mechanistic insights into NHO.