Macrophages play crucial roles in many human disease processes. However, obtaining large numbers of primary cells for study is often difficult. We describe 2D and 3D methods for directing human ...induced pluripotent stem cells (hiPSCs) into macrophages (iMACs). iMACs generated in 2D culture showed functional similarities to human primary monocyte-derived M2-like macrophages, and could be successfully polarized into a M1-like phenotype. Both M1- and M2-like iMACs showed phagocytic activity and reactivity to endogenous or exogenous stimuli. In contrast, iMACs generated by a 3D culture system showed mixed M1- and M2-like functional characteristics. 2D-iMACs from patients with fibrodysplasia ossificans progressiva (FOP), an inherited disease with progressive heterotopic ossification driven by inflammation, showed prolonged inflammatory cytokine production and higher Activin A production after M1-like polarization, resulting in dampened responses to additional LPS stimulation. These results demonstrate a simple and robust way of creating hiPSC-derived M1- and M2-like macrophage lineages, while identifying macrophages as a source of Activin A that may drive heterotopic ossification in FOP.
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•Macrophage-like cells can be created from human induced pluripotent stem cells (iMACs).•These iMACs can be differentiated into M1-like or M2-like states.•iMACs show cytokine responses when stimulated with LPS, HMGB1, or S100A8/A9.•iMACs carrying the ACVR1 R206H mutation show increased macrophage responses.•This reveals new inflammatory roles in fibrodysplasia ossificans progressiva (FOP).
To present an updated summary of the relationship between inflammation and localized and generalized bone loss in the rheumatic diseases.
In addition to the well established role of inflammatory ...cytokines in promoting enhanced osteoclast function and bone loss, recent work has discovered the cytokine milieu may also inhibit osteoblast function and bone repair. The WNT and bone morphogenetic protein pathways provide molecular links between inflammation and altered bone homeostasis in chronic inflammatory states. These pathways and others have been the targets of emerging therapies for the management of inflammatory bone loss.
Inflammation and bone loss are linked through a number of molecular pathways. Both of these processes need to be addressed when designing an effective treatment strategy for the rheumatic diseases.
Inflammation helps regulate normal growth and tissue repair. Although bone morphogenetic proteins (BMPs) and inflammation are known contributors to abnormal bone formation, how these pathways ...interact in ossification remains unclear.
We examined this potential link in patients with fibrodysplasia ossificans progressiva (FOP), a genetic condition of progressive heterotopic ossification caused by activating mutations in the Activin A type I receptor (ACVR1/ALK2). FOP patients show exquisite sensitivity to trauma, suggesting that BMP pathway activation may alter immune responses. We studied primary blood, monocyte, and macrophage samples from control and FOP subjects using multiplex cytokine, gene expression, and protein analyses; examined CD14+ primary monocyte and macrophage responses to TLR ligands; and assayed BMP, TGF-β activated kinase 1 (TAK1), and NF-κB pathways.
FOP subjects at baseline without clinically evident heterotopic ossification showed increased serum IL-3, IL-7, IL-8, and IL-10. CD14+ primary monocytes treated with the TLR4 activator LPS showed increased CCL5, CCR7, and CXCL10; abnormal cytokine/chemokine secretion; and prolonged activation of the NF-κB pathway. FOP macrophages derived from primary monocytes also showed abnormal cytokine/chemokine secretion, increased TGF-β production, and p38MAPK activation. Surprisingly, SMAD phosphorylation was not significantly changed in the FOP monocytes/macrophages.
Abnormal ACVR1 activity causes a proinflammatory state via increased NF-κB and p38MAPK activity. Similar changes may contribute to other types of heterotopic ossification, such as in scleroderma and dermatomyositis; after trauma; or with recombinant BMP-induced bone fusion. Our findings suggest that chronic antiinflammatory treatment may be useful for heterotopic ossification.
Post-transplant cyclophosphamide (PTCy) combined with tacrolimus (TAC) as graft-versus-host disease (GvHD) prophylaxis post-hematopoietic cell transplantation (HCT) is safe and effective. Optimal ...serum levels of TAC in this combination remain undetermined. We hypothesized that TAC at initial steady state (TISS) of <10 ng/mL could promote optimal transplant outcomes and prevent TAC-associated toxicities. We retrospectively analyzed a consecutive case series of 210 patients who received PTCy/TAC-based prophylaxis post-HCT from 1/2013-6/2018. Patients received HCT from haploidentical (n = 172) or mismatched donors (n = 38), and flat dose (FD) or weight-based dose (WBD) TAC. Twenty-four-month overall survival (OS), disease free survival (DFS), and relapse rate (RR) were 61%, 56%, and 22%, respectively, in TISS < 10 ng/mL cohort (n = 176), and 50%, 43%, and 35%, respectively, in TISS ≥ 10 ng/mL cohort (n = 34) (OS, P = 0.71; DFS, P = 0.097; RR, P = 0.031). OS, DFS, RR, non-relapse mortality, acute GvHD grade II-IV, grade III-IV or chronic GvHD by TISS were similar in multivariable analysis. TISS ≥ 10 ng/mL conferred increased risk of viral infection (P = 0.003). More patients receiving FD vs. WBD had TISS < 10 ng/mL (P = 0.001). Overall, TISS < 10 ng/mL early post HCT conferred similar survival outcomes and lowered risk of viral infection and toxicities compared to TISS ≥ 10 ng/mL.
Bone and the Innate Immune System Charles, Julia F.; Nakamura, Mary C.
Current osteoporosis reports,
03/2014, Letnik:
12, Številka:
1
Journal Article
Recenzirano
Odprti dostop
The immune system and bone are intimately linked with significant physical and functionally related interactions. The innate immune system functions as an immediate response system to initiate ...protections against local challenges such as pathogens and cellular damage. Bone is a very specific microenvironment, in which infectious attack is less common but repair and regeneration are ongoing and important functions. Thus, in the bone the primary goal of innate immune and bone interactions is to maintain tissue integrity. Innate immune signals are critical for removal of damaged and apoptotic cells and to stimulate normal tissue repair and regeneration. In this review we focus on the innate immune mechanisms that function to regulate bone homeostasis.
Missense mutations that reduce or abrogate myeloid cell expression of the F-BAR domain protein, proline serine threonine phosphatase-interacting protein 2 (PSTPIP2), lead to autoinflammatory disease ...involving extramedullary hematopoiesis, skin and bone lesions. However, little is known about how PSTPIP2 regulates osteoclast development. Here we examined how PSTPIP2 deficiency causes osteopenia and bone lesions, using the mouse PSTPIP2 mutations, cmo, which fails to express PSTPIP2 and Lupo, in which PSTPIP2 is dysfunctional. In both models, serum levels of the pro-osteoclastogenic factor, MIP-1α, were elevated and CSF-1 receptor (CSF-1R)–dependent production of MIP-1α by macrophages was increased. Treatment of cmo mice with a dual specificity CSF-1R and c-Kit inhibitor, PLX3397, decreased circulating MIP-1α and ameliorated the extramedullary hematopoiesis, inflammation, and osteopenia, demonstrating that aberrant myelopoiesis drives disease. Purified osteoclast precursors from PSTPIP2-deficient mice exhibit increased osteoclastogenesis in vitro and were used to probe the structural requirements for PSTPIP2 suppression of osteoclast development. PSTPIP2 tyrosine phosphorylation and a functional F-BAR domain were essential for PSTPIP2 inhibition of TRAP expression and osteoclast precursor fusion, whereas interaction with PEST-type phosphatases was only required for suppression of TRAP expression. Thus, PSTPIP2 acts as a negative feedback regulator of CSF-1R signaling to suppress inflammation and osteoclastogenesis.
Cbl is an adaptor protein and an E3 ligase that plays both positive and negative roles in several signaling pathways that affect various cellular functions. Tyrosine 737 is unique to Cbl and is ...phosphorylated by Syk and Src family kinases. Phosphorylated Cbl Tyr737 creates a binding site for the p85 regulatory subunit of PI3K, which also plays an important role in the regulation of bone resorption by osteoclasts. To investigate the role of Cbl-PI3K interaction in bone homeostasis, we examined the knock-in mice (CblYF/YF) in which the PI3K binding site in Cbl is ablated due to the mutation in the regulatory tyrosine. We report that in CblYF/YF mice, despite increased numbers of osteoclasts, bone volume is increased due to defective osteoclast function. Additionally, in ex vivo cultures, mature CblYF/YF osteoclasts showed an increased ability to survive in the presence of RANKL due to delayed onset of apoptosis. RANKL-mediated signaling is perturbed in CblYF/YF osteoclasts, and most interestingly, AKT phosphorylation is up-regulated, suggesting that the lack of PI3K sequestration by Cbl results in increased survival and decreased bone resorption. Cumulatively, these in vivo and in vitro results show that, on one hand, binding of Cbl to PI3K negatively regulates osteoclast differentiation, survival, and signaling events (e.g. AKT phosphorylation), whereas on the other hand it positively influences osteoclast function.
Deficiency of the signaling adapter protein DAP12 is associated with bony abnormalities in both mice and humans. We identify specific DAP12‐associated receptors expressed by osteoclasts and examine ...function of DAP12 in murine osteoclasts in vivo and in vitro. These data show a new role for DAP12 signaling in regulating formation of multinucleated osteoclasts.
Introduction: Osteoclasts are bone‐resorbing cells derived from hematopoietic precursors in the myeloid lineage. In other myeloid cell types, the signaling adapter protein DAP12 transmits activating signals on ligation of a DAP12‐associated receptor (DAR). The aim of this study was to clarify the role of DAP12 signaling during osteoclast development.
Materials and Methods: Osteoclasts from DAP12−/− or control mice were analyzed in vitro for morphology, function, and for osteoclast markers. DARs were identified in osteoclast cultures through reverse transcriptase‐polymerase chain reaction (RT‐PCR). Bone density of DAP12−/− and control mice were analyzed by microcomputed tomography. DAP12−/− osteoclasts were retrovirally reconstituted with DAP12. RAW264.7 cells were transfected with FLAG‐tagged DAP12 or TREM2 and stimulated by anti‐FLAG antibody during in vitro osteoclastogenesis.
Results: C57BL/6 DAP12‐deficient mice have higher bone mass than C57BL/6 wildtype controls. We verified the presence of DAP12 in pre‐osteoclasts and osteoclasts derived from C57BL/6 or the pre‐osteoclast line RAW 264.7 and identified the DARs expressed. DAP12−/− osteoclasts developed in vitro with macrophage colony‐stimulating factor (M‐CSF) and RANKL formed only intensely TRACP+ mononuclear cells and failed to generate multinuclear osteoclasts. These mononuclear cells are functional osteoclast‐like cells because, by RT‐PCR, they express other osteoclast markers and generate resorption pits on dentine slices, although quantitative assessment of bone resorption shows decreased resorption by DAP12−/− osteoclasts compared with C57BL/6 osteoclasts. Restoration of DAP12 expression by retroviral transduction of DAP12−/− osteoclast precursors rescued in vitro osteoclast multinucleation. Direct stimulation of DAP12 expressed in RAW264.7 during in vitro osteoclastogenesis led to a marked increase in the number of TRACP+ multinucleated osteoclast‐like cells formed.
Conclusion: Our studies indicate that stimulation of the DAP12 adapter protein plays a significant role in formation of multinuclear osteoclasts and that DAP12 and DARs likely participate in the regulation of bony remodeling.
BackgroundAdoptive transfer of CD19-specific chimeric antigen receptor (CD19CAR) T cells can induce dramatic disease regression in patients with B cell malignancies. CD19CAR T cell therapy may be ...limited by insufficient engraftment and persistence, resulting in tumor relapse. We previously demonstrated a proof of principle that cytomegalovirus (CMV)-specific T cells can be isolated and enriched prior to CD19CAR transduction to produce CMV-CD19CAR T cells, and that these CMV-CD19CAR T cells can be expanded in vivo through CMV vaccination, resulting in better tumor control in a murine model. Here we developed a clinical platform for generating CMV-CD19CAR T cells.MethodsPeripheral blood mononuclear cells (PBMCs) collected from CMV-seropositive healthy donors were stimulated with a good manufacturing practices-grade PepTivator overlapping CMVpp65 peptide pool and enriched for CMV-responsive interferon γ (IFNγ)+T cells using IFNγ Catchmatrix, within the CliniMACS Prodigy Cytokine Capture System (Miltenyi Biotec). Resulting CMV-specific T cells were transduced with a lentiviral vector encoding a second generation CD19R:CD28:ζ/EGFRt CAR and expanded with interleukin 2 (IL-2) and IL-15 for 15 days before characterization.ResultsCMV-specific T cells were enriched from 0.8%±0.5 of input PBMC to 76.3%±11.6 in nine full-scale qualification runs (absolute yield of 4.2±3.3×106 IFNγ+T cells from an input of 1×109 PBMCs). Average CD19CAR transduction efficiency of CMV-specific T cells was 27.0%±14.2 in the final products, which underwent rapid expansion, resulting in a total cell dose of 6.2±0.9 × 106 CD19CAR-tranduced T cells with CMV specificity (ie, functionally bispecific). CMV-CD19CAR T cells were polyclonal, expressed memory markers but had low expression of exhaustion markers, responded to both CD19 and CMVpp65 stimulation with rapid proliferation and exhibited antigen-specific effector functions against both CD19-expressing tumors and CMVpp65 antigen. The final products passed release criteria for clinical use.ConclusionsWe demonstrated the feasibility of our large-scale platform for generating CMV-CD19CAR T cells for clinical application. We plan to initiate a clinical trial at City of Hope using CMV-CD19CAR T cells for patients with intermediate/high-grade B cell non-Hodgkin’s lymphoma immediately after autologous hematopoietic cell transplantation followed by vaccination with a novel CMV vaccine based on Modified Vaccinia Ankara (Triplex) 28 days and 56 days post-T cell infusion.
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