Expression Of SIRPα Research Articles

Surfactant Protein D Inhibits Interleukin-12p40 Production by Macrophages Through the SIRPα/ROCK/ERK Signaling Pathway

ObjectiveInterleukin (IL)-12 has a pivotal profibrotic role in the development of idiopathic pulmonary fibrosis (IPF). Medical research trials based on IPF registry databases have actively recruited patients. Surfactant protein D (SP-D) is a useful biomarker in patients with IPF. SP-D binds to signal regulatory protein α (SIRPα), which acts as an inhibitory receptor, and this SP-D/SIRPα interaction may have an anti-inflammatory effect. Accordingly, the inhibitory effect of SP-D on IL-12p40 production by lipopolysaccharide (LPS)-stimulated macrophages was investigated. Materials and MethodsHuman granulocyte-macrophage colony-stimulating factor (GM-CSF)-stimulated macrophages (day 9 of culture) was used to investigate IL-12p40 production after stimulation with SP-D. ResultsGM-CSF was found to upregulate SIRPα expression by macrophages. PD98059 (an extracellular signal-regulated kinase [ERK] inhibitor) blunted induction of SIRPα expression by GM-CSF. SP-D significantly attenuated IL-12p40 production by macrophages after stimulation with LPS. Silencing of SIRPα/β/γ significantly reversed this inhibitory effect of SP-D. In contrast, neither SB023580 (a p38α/β MAPK inhibitor) nor BIRB796 (a p38γ/δ MAPK inhibitor) attenuated the inhibitory effect of SP-D on LPS-stimulated production of IL-12p40. Silencing of SHP also had no influence on this effect of SP-D. Interestingly, a Rho-associated protein kinase (ROCK) inhibitor (Y-27632) abolished the inhibition of LPS-stimulated IL-12p40 production by SP-D, whereas silencing of ERK 2 significantly blunted this effect of Y-27632. ConclusionsThese findings suggest that SP-D inhibits LPS-stimulated production of IL-12p40 via the SIRPα/ROCK/ERK signaling pathway.

Signal-Regulatory Protein-α (SIRP- α) Expression Delineates Distinct Subsets in Monocytes/Macrophages in Normal Tissue and in B-Cell Non-Hodgkin Lymphoma

BACKGROUNDMonocytes and macrophages (mo/mΦ) are a key part of the composition of peripheral blood (PB) and tissues and increased numbers of mo/mΦ have been associated with patient outcome in non-Hodgkin lymphoma (NHL). Because CD14 is abundantly expressed on the surface of human mo/mΦ, it is often used to identify or isolate human mo/mΦ, and immunosuppressive CD14+HLA-DRlow monocytes have been shown to be increased in the peripheral blood of NHL patients. However, we have previously shown that CD14 expression on mo/mΦ is substantially lower than CD68 expression suggesting that many CD68+ mo/mΦ are CD14 negative, especially in spleen and lymph node tissues. To characterize both CD14+ and CD14- mo/mΦ in PB and tissues, we isolated all mo/mΦ from B-cell NHL specimens and normal controls and assessed their phenotype and function.METHODSHuman mo/mΦ were isolated by negative selection from PB and tissue biopsy specimens (B-cell NHL and normal tissues) using the immunomagnetic isolation (monocyte enrichment kit). Morphological and immunophenotypic characteristics of isolated mo/mΦ were determined by Giemsa stain and flow cytometry. Phagocytosis and migration assays were used to determine the function of isolated mo/mΦ. T cells were co-cultured with mo/mΦ and T cell proliferation was evaluated by CFSE staining and detected by flow cytometry.RESULTSUsing a monocyte enrichment kit to isolate all mo/mΦ, the purity of isolated mo/mϕ was 85~99%, which was defined by the percentage of lineage-negative cells (i.e. cells without expression of CD3,CD19, CD20, and CD56). We found that these isolated mo/mΦ constituted 2 populations: a more frequent population of larger cells and a less common population of smaller cells. In contrast to PB, CD14 positive mo/mΦ constituted less than 40% of the tissue mo/mΦ from the isolated population. Furthermore, we found that the cell size from CD14+ mo/mΦ were larger than CD14- mo/mΦ. Using CD14 and SIRP-α, we could identify 3 populations of mo/mΦ: CD14+SIRP-αhigh, CD14-SIRP-αdim and CD14-SIRP-α- cells. CD14+SIRP-αhigh cells and CD14-SIRP-αdim cells typically constituted the population of larger cells, while CD14-SIRP-α- cells constituted the population of smaller cells. CD14-SIRP-α- cells lacked the typical phenotypic markers and had decreased phagocytic and migratory ability compared to CD14+SIRP-αhigh and CD14-SIRP-αdim cells. Furthermore, we found that these 3 populations of mo/mΦ had a differential effect on activated T-cells and that the CD14-SIRP-α- cells appeared increased number in biopsy specimens from NHL when compared to normal tissues.CONCLUSIONSWe have identified a unique population of small CD68+ mo/mΦ that lack expression of CD14, SIRP-α, and other FcγR markers. This subset of mo/mΦ is more prevalent in NHL tissues and has limited phagocytic and migratory functions. This CD14-SIRP-α- mo/mΦ subpopulation may play an inhibitory role in anti-cancer and inflammatory responses. DisclosuresNo relevant conflicts of interest to declare.

Signal regulatory protein α associated with the progression of oral leukoplakia and oral squamous cell carcinoma regulates phenotype switch of macrophages.

Signal regulatory protein α (SIRPα) is a cell-surface protein expressed on macrophages that are regarded as an important component of the tumor microenvironment. The expression of SIRPα in oral leukoplakia (OLK) and oral squamous cell carcinoma (OSCC), and further explored the role of SIRPα on the phenotype, phagocytosis ability, migration, and invasion of macrophages in OSCC were investigated. The expression of SIRPα in OLK was higher than in OSCC, correlating with the expression of CD68 and CD163 on macrophages. After cultured with the conditioned media of oral cancer cells, the expression of SIRPα on THP-1 cells was decreased gradually. In co-culture system, macrophages were induced into M2 phenotype by oral cancer cells. Blockade of SIRPα inhibited phagocytosis ability and IL-6, TNF-α productions of macrophages. In addition, the proliferation, migration, and IL-10, TGF-β productions of macrophages were upregulated after blockade of SIRPα. Macrophages upregulated the expression of SIRPα and phagocytosis ability, and inhibited the migration and invasion when the activation of NF-κB was inhibited by pyrrolidine dithiocarbamate ammonium (PDTC). Hence, SIRPα might play an important role in the progression of OLK and oral cancer, and could be a pivotal therapeutic target in OSCC by regulating the phenotype of macrophages via targeting NF-κB.

Silencing of CD47 and SIRPα by Polypurine reverse Hoogsteen hairpins to promote MCF-7 breast cancer cells death by PMA-differentiated THP-1 cells.

BackgroundIn the context of tumor immunology, tumor cells have been shown to overexpress CD47, an anti-phagocytic signal directed to macrophages to escape from phagocytosis by interacting with Signal Regulatory Protein α SIRPα.In the present work, we designed Polypurine reverse Hoogsteen hairpins, PPRHs, to silence the expression of CD47 in tumor cells and SIRPα in macrophages with the aim to eliminate tumor cells by macrophages in co-culture experiments.MethodsTHP-1 cells were differentiated to macrophages with PMA. The mRNA levels of differentiation markers CD14 and Mcl-1 mRNA and pro-inflammatory cytokines (IL-1β, IL-18, IL-6, IL-8 and TNF-α) were measured by qRT-PCR. The ability of PPRHs to silence CD47 and SIRPα was evaluated at the mRNA level by qRT-PCR and at the protein level by Western Blot. Macrophages were co-cultured with tumor cells in the presence of PPRHs to silence CD47 and/or SIRPα. Cell viability was assessed by MTT assays.ResultsTHP-1 cells differentiated to macrophages with PMA showed an increase in macrophage surface markers (CD14, Mcl-1) and pro-inflammatory cytokines (IL-1β, IL-18, IL-6, IL-8 and TNF-α). PPRHs were able to decrease both CD47 expression in MCF-7 cell line and SIRPα expression in macrophages at the mRNA and protein levels. In the presence of PPRHs, MCF-7 cells were eliminated by macrophages in co-culture experiments, whereas they survived in the absence of PPRHs.ConclusionsOur data support the usage of PPRHs to diminish CD47/SIRPα interaction by decreasing the expression of both molecules thus resulting in an enhanced killing of MCF-7 cells by macrophages, which might translate into beneficial effects in cancer therapy. These results indicate that PPRHs could represent a new approach with immunotherapeutic applications.

Role of dendritic cells in maintenance of the skin structural homeostasis.

Investigation of mechanisms that determine structural and immunological homeostasis under physiological conditions, alteration of neurohumoral and metabolic control, allows to pose that dendritic cells are the most important modulators of skin structure. This view is focused on current data about classification, molecular hallmarks, mechanisms and effects of their participation in skin structural homeostasis maintaining. As the most powerful and professional antigen-presenting cells, dendritic cells play a crucial role in innate and adaptive immunity, as well as in switching between immunity and tolerance depending on their activation and maturation states. Cutaneous dendritic cells represent a wide spectrum of phenotypically and functionally different subtypes of cells. They are diverse in terms of origin, structure, expression of receptors and co-stimulatory molecules. According to current data, dendritic cells of the skin are classified into several types, including Langerhans cells that express Langerin, CD1a, E-cadherin, etc. In addition, there are several types of dermal dendritic cells. The most numerous are CD11b + cells that represent about 65% of dermal dendritic cells. In addition there are Langerin+ dermal dendritic cells that demonstrate high XCR1 and lack of EpCam and Sirp1α expression. A few number of dermal DC are cells that express neither Langerin, no XCR1. They have low expression of CD11b and Sirpα, but positive in CX3CR1. In addition to conventional subsets, there is a separate group of plasmacytoid dendritic cells that enter skin under inflammation. This type of cells expresses CD123, CD45RA, CD303 (BDCA2). Authors highlight the functional differences and plasticity of skin dendritic cells populations, and discuss the role of dendritic cells in immune reactions, determination of polarity of immune response, tolerance to antigen and neo-antigens, impact of photoaging and skin aging.

Abstract 3287: Expression of CD14 and SIRP-α defines distinct populations of intratumoral monocytes/macrophages in B-cell non-Hodgkin lymphomas

Abstract BACKGROUND Monocytes and macrophages (mo/mΦ) are part of composition of tumor microenvironment in B-cell non-Hodgkin lymphoma (B-NHL). CD14+HLA-DRlow monocytes are immunosuppressive and this phenotype is promoted by increased expression of IL-10 in lymphoma. Despite their association with poor outcome, monocytes retain phagocytic function particularly in the presence of monoclonal antibodies and can be associated with an improved outcome in patients treated with rituximab. The aim of this study was to determine the prevalence of CD14+HLA-DRlow mo/mΦ in B-NHL tissue compared to normal tissue, and to determine whether their phenotype suggests that they are capable of phagocytic function. METHODS Tissue mo/mΦ were isolated by negative selection from 13 diagnostic B-NHL biopsy specimens and 6 normal tissues using a monocyte enrichment kit. Morphological and immunophenotypic characteristics of isolated mo/mΦ were determined by flow cytometry, immunocytochemistry and cytometry by time-of-flight (CyTOF). The purity of isolated tissue mo/mΦ was more than 95%, being confirmed by Giemsa stain and flow cytometry. RESULTS Increased numbers of CD68+ cells in initial B-NHL biopsies when compared to normal tissue was confirmed by immunohistochemistry and flow cytometry. However, CD14 expression was substantially lower than CD68 expression in the tissues suggesting that many CD68+ mo/mΦ are CD14 negative. Using a monocyte enrichment kit to isolate all mo/mΦ, we found that CD14 negative mo/mΦ constituted half the tissue mo/mΦ. Furthermore, we found by flow cytometry, CyTOF and Giemsa stain that CD14- mo/mΦ constituted 2 populations: a more frequent population of larger cells and a less common population of smaller cells. While all cells expressed CD68, the larger cells had increased expression of CD32, CD64 and HLA-DR. The CD14+ cells typically also expressed CD163 and CD33 and were a subset of the larger monocyte population, while the population of small cells was positive only for CD68 and CD45. Using CD14 and SIRP-α, we could identify 3 populations of mo/mΦ: CD14+SIRP-αhigh, CD14-SIRP-αlow and CD14-SIRP-α- cells. CD14+SIRP-αhigh cells and CD14-SIRP-αlow cells typically constitute the population of larger cells, while CD14-SIRP-α- cells constituted the population of smaller cells. Interestingly, while the CD14-SIRP-α- cells lack the typical phenotypic markers of mo/mΦ, they morphologically have the appearance of monocytic cells and this population appears expanded in lymphoma tissue. CONCLUSIONS We have identified a unique population of small mo/mΦ that have an immature phenotype and lack expression of CD14, SIRP-α, CD163, and other FcγR markers. This subset of mo/mΦ is prevalent in lymphoma and may have limited phagocytic function. This CD68+CD14-SIRP-α- mo/mΦ subpopulation may account for prognostic differences in the outcome of lymphoma patients treated with or without monoclonal antibodies. Citation Format: Ya-Ping Chen, Zhi-Zhang Yang, Jose C. Villasboas, Tammy Price-Troska, Anne J. Novak, Stephen M. Ansell. Expression of CD14 and SIRP-α defines distinct populations of intratumoral monocytes/macrophages in B-cell non-Hodgkin lymphomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3287.

Cleavage of Signal Regulatory Protein α (SIRPα) Enhances Inflammatory Signaling

Signal regulatory protein α (SIRPα) is a membrane glycoprotein immunoreceptor abundant in cells of monocyte lineage. SIRPα ligation by a broadly expressed transmembrane protein, CD47, results in phosphorylation of the cytoplasmic immunoreceptor tyrosine-based inhibitory motifs, resulting in the inhibition of NF-κB signaling in macrophages. Here we observed that proteolysis of SIRPα during inflammation is regulated by a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), resulting in the generation of a membrane-associated cleavage fragment in both THP-1 monocytes and human lung epithelia. We mapped a charge-dependent putative cleavage site near the membrane-proximal domain necessary for ADAM10-mediated cleavage. In addition, a secondary proteolytic cleavage within the membrane-associated SIRPα fragment by γ-secretase was identified. Ectopic expression of a SIRPα mutant plasmid encoding a proteolytically resistant form in HeLa cells inhibited activation of the NF-κB pathway and suppressed STAT1 phosphorylation in response to TNFα to a greater extent than expression of wild-type SIRPα. Conversely, overexpression of plasmids encoding the proteolytically cleaved SIRPα fragments in cells resulted in enhanced STAT-1 and NF-κB pathway activation. Thus, the data suggest that combinatorial actions of ADAM10 and γ-secretase on SIRPα cleavage promote inflammatory signaling.

Hepatitis E virus infection activates signal regulator protein α to down-regulate type I interferon.

Hepatitis E virus (HEV) is a major cause of enterically transmitted acute hepatitis worldwide. However, the mechanism of HEV replication is unclear. Type I interferon is the first defense line of host against viral infection. Signal regulator protein α (SIRP-α) plays an important role in negative regulation of innate immunity. In the present study, HEV infection significantly activated the expression of SIRP-α and down-regulated phosphorylation of IRF3, consequently resulted in suppression of type I interferon (IFN-β). In conclusion, HEV exploited SIRP-α to negative regulated IFN-β of the host innate immune system to promote viral infection. It suggested that interfering with the functions of SIRP-α should be considered as a potential therapeutic approach to the prevention and treatment of HEV infection.

Chemokine (C-C Motif) Receptor 2 Mediates Dendritic Cell Recruitment to the Human Colon but Is Not Responsible for Differences Observed in Dendritic Cell Subsets, Phenotype, and Function Between the Proximal and Distal Colon

Background & AimsMost knowledge about gastrointestinal (GI)-tract dendritic cells (DC) relies on murine studies where CD103+ DC specialize in generating immune tolerance with the functionality of CD11b+/− subsets being unclear. Information about human GI-DC is scarce, especially regarding regional specifications. Here, we characterized human DC properties throughout the human colon.MethodsPaired proximal (right/ascending) and distal (left/descending) human colonic biopsies from 95 healthy subjects were taken; DC were assessed by flow cytometry and microbiota composition assessed by 16S rRNA gene sequencing.ResultsColonic DC identified were myeloid (mDC, CD11c+CD123−) and further divided based on CD103 and SIRPα (human analog of murine CD11b) expression. CD103-SIRPα+ DC were the major population and with CD103+SIRPα+ DC were CD1c+ILT3+CCR2+ (although CCR2 was not expressed on all CD103+SIRPα+ DC). CD103+SIRPα- DC constituted a minor subset that were CD141+ILT3−CCR2−. Proximal colon samples had higher total DC counts and fewer CD103+SIRPα+ cells. Proximal colon DC were more mature than distal DC with higher stimulatory capacity for CD4+CD45RA+ T-cells. However, DC and DC-invoked T-cell expression of mucosal homing markers (β7, CCR9) was lower for proximal DC. CCR2 was expressed on circulating CD1c+, but not CD141+ mDC, and mediated DC recruitment by colonic culture supernatants in transwell assays. Proximal colon DC produced higher levels of cytokines. Mucosal microbiota profiling showed a lower microbiota load in the proximal colon, but with no differences in microbiota composition between compartments.ConclusionsProximal colonic DC subsets differ from those in distal colon and are more mature. Targeted immunotherapy using DC in T-cell mediated GI tract inflammation may therefore need to reflect this immune compartmentalization.

“Velcro” Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein α (SIRPα) Antagonists That Enhance Antibody-dependent Cellular Phagocytosis

CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the "don't-eat-me" signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined "Velcro" engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that "Velcro" engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy.

Dendritic cell SIRPα regulates homeostasis of dendritic cells in lymphoid organs

Signal regulatory protein α (SIRPα), an immunoglobulin superfamily protein that is expressed predominantly in myeloid lineage cells such as dendritic cells (DCs) or macrophages, mediates cell-cell signaling. In the immune system, SIRPα is thought to be important for homeostasis of DCs, but it remains unclear whether SIRPα intrinsic to DCs is indeed indispensable for such functional role. Thus, we here generated the mice, in which SIRPα was specifically ablated in CD11c(+) DCs (Sirpa(Δ) (DC) ). Sirpa(Δ) (DC) mice manifested a marked reduction of CD4(+) CD8α(-) conventional DCs (cDCs) in the secondary lymphoid organs, as well as of Langerhans cells in the epidermis. Such reduction of cDCs in Sirpa(Δ) (DC) mice was comparable to that apparent with the mice, in which SIRPα was systemically ablated. Expression of SIRPα in DCs was well correlated with that of either endothelial cell-selective adhesion molecule (ESAM) or Epstein-Barr virus-induced molecule 2 (EBI2), both of which were also implicated in the regulation of DC homeostasis. Indeed, ESAM(+) or EBI2(+) cDCs were markedly reduced in the spleen of Sirpa(Δ) (DC) mice. Thus, our results suggest that SIRPα intrinsic to CD11c(+) DCs is essential for homeostasis of cDCs in the secondary lymphoid organs and skin.

Next Generation Humanized Mice Support Engraftment of Myelofibrosis CD34+ Cells

IntroductionWhile the key transforming genetic events occur in the developing cancerous cell, this cell is dependent on its environmental context and interaction for competitive outgrowth and subsequent tumor-development. Myelofibrosis (MF) represents a model cancer disease with stepwise development from a chronic state that depends on microenvironmental interactions to a more aggressive disease. Engraftment of primary MF patient cells in murine xenograft models is poor (Wang et al., JCI 2012) and is possibly explained by the lack of supportive microenvironmental factors. Thrombopoietin (TPO) has been implicated in the pathogenesis of MF (Schepers et al., Cell Stem Cell 2013, Dadfarnia et al., Blood 2014, Abdel-Wahab et al., Annu Rev Med 2009). Also, the interaction between human hematopoietic cells and SIRPα expressed on mouse macrophages is critical for human engraftment in xenografts (Takenaka et al., Nature Immunology 2007). We hypothesized that the constitutive expression of human TPO and human SIRPα may promote the development of the human MF clone in mouse xenografts.MethodsPurified peripheral blood CD34+ cells were collected from six patients with primary MF or post-PV/ET MF and low to intermediate 2 risk disease according to the dynamic international prognostic scoring system (DIPSS). Four patients carried a JAK2-V617F mutation and two patients carried a calreticulin (CALR) mutation. CD34+ cells were intrahepatically transplanted into sublethally irradiated newborn humanSIRPα-transgenic/humanTPO-knockin Rag2-/- gamma-/- (TPO-SIRPα) mice (Rongvaux et al., Ann Rev. Immunol 2013). NSG mice were used as controls and injected with the same number of CD34+ cells. Two to three mice were injected with ≥1 million CD34+ cells from the same patient sample each. Mice were sacrificed 12-16 weeks after transplantation and human engraftment and hematopoietic cell lineage distribution was assessed by flow cytometry using human specific antibodies. Tissues were collected for immunohistochemistry, assessment of fibrosis and spleen weight. DNA was extracted from whole bone marrow and a qualitative PCR was performed to determine the presence of the JAK2-V617F or CALR-mutations.ResultsThree out of six samples generated a human graft of ≥20% human CD45+ cells, while the three other samples generated engraftment of 0.1-3%. The human graft was mainly composed of myeloid cells and monocytic differentiation was observed. In 2/2 experiments analysed, a JAK2-V617F and a CALR type 2 mutation were detected in the bone marrow of engrafted mice transplanted with the respective patient sample. Development of fibrosis was not observed three months post-transplantation, presumably due to the short observation time. Spleen weight was significantly increased in mice engrafted with human MF and was the consequence of increased murine extramedullary hematopoiesis. We then aimed to identify factors that could predict human MF engraftment in TPO-SIRPα mice. While neither the DIPSS, nor the presence of myeloid precursors in the peripheral blood (blasts excluded) were predictive of human MF engraftment, the presence of blasts in the peripheral blood significantly correlated with engraftment potential. Importantly, none of the patients developed acute leukemia during follow-up. Finally, preliminary evidence suggests that TPO-SIRPα mice are more supportive of human MF engraftment than NSG mice.ConclusionsThis is the first xenograft model that supports robust engraftment of human peripheral blood MF cells and further supports a role for TPO in the pathogenesis of MF. In contrast to previous models TPO-SIRPα mice strongly promote myeloid rather than lymphoid engraftment. The tight correlation between the presence of peripheral blood blasts and the human MF engraftment potential suggests that human MF stem cells reside in the blast population. In summary, the xenograft model presented here constitutes a powerful tool to assess heterogeneity regarding MF biology, microenvironmental dependence of the MF clone and likely also therapeutic response of MF in vivo. DisclosuresNo relevant conflicts of interest to declare.

Abstract 19583: Autonomous and Non-Autonomous Defects Underlie Hypertrophic Cardiomyopathy in BRAF-Mutant hiPSC-Derived Cardiomyocytes

Hypertrophic cardiomyopathy (HCM) is a pathological disorder predominantly due to mutations in sarcomeric components. Germline mutations in BRAF cause a developmental syndrome called cardio-facio-cutaneous syndrome (CFCS), in which 40% of patients also develop HCM. Since the role of the RAS/MAPK pathway in HCM is still unclear, we generated a human induced pluripotent stem cell model for CFCS from three patients with activating BRAF T599R or Q257R mutations. In order to examine hiPSC-derived cell-type specific phenotypes and cellular interactions underpinning HCM, we generated a method to purify cardiomyocytes and non-cardiomyocytes simultaneously by cell sorting based on SIRPα and CD90 expression. Purified BRAF-mutant SIRPα+/CD90- cells were >95% cardiomyocytes, displayed cellular hypertrophy with pro-hypertrophic gene expression, and dysregulation of the RAS/MAPK pathway. BRAF-mutant cardiomyocytes also displayed intrinsic calcium handling defects, including increased calcium transient irregularity and increased stored calcium within the sarcoplasmic reticulum. In addition, purified BRAF-mutant SIRPα-/CD90+ cells, which were fibroblast-like, displayed activation of the RAS/MAPK pathway and exhibited a pro-fibrotic phenotype. Cross-culture studies revealed that BRAF-mutant fibroblast-like cells critically modulate cardiomyocyte hypertrophy through TGFβ paracrine signaling, as TGFβ inhibition prevented cardiomyocyte hypertrophy induced by BRAF-mutant fibroblast-like cells. Additionally, inhibition of RAS/MAPK signaling was capable of rescuing BRAF-mutant cardiomyocyte hypertrophy and cardiomyocyte-intrinsic calcium handling abnormalities. Thus, we show for the first time that cell autonomous and non-autonomous defects underlie RASopathy-associated HCM. As fibroblast activation has been documented previously in sarcomeric HCM, our findings suggest that cardiac fibroblasts may contribute to pathologic hypertrophy in addition to causing fibrosis in primary forms of HCM. TGFβ inhibition may be a useful therapeutic option for patients with HCM due to RASopathies or other etiologies.

Polymorphism in the innate immune receptor SIRPα controls CD47 binding and autoimmunity in the nonobese diabetic mouse.

The signal regulatory protein (SIRP) locus encodes a family of paired receptors that mediate both activating and inhibitory signals and is associated with type 1 diabetes (T1D) risk. The NOD mouse model recapitulates multiple features of human T1D and enables mechanistic analysis of the impact of genetic variations on disease. In this study, we identify Sirpa encoding an inhibitory receptor on myeloid cells as a gene in the insulin-dependent diabetes locus 13.2 (Idd13.2) that drives islet inflammation and T1D. Compared to T1D-resistant strains, the NOD variant of SIRPα displayed greater binding to its ligand CD47, as well as enhanced T cell proliferation and diabetogenic potency. Myeloid cell-restricted expression of a Sirpa transgene accelerated disease in a dose-dependent manner and displayed genetic and functional interaction with the Idd5 locus to potentiate insulitis progression. Our study demonstrates that variations in both SIRPα sequence and expression level modulate T1D immunopathogenesis. Thus, we identify Sirpa as a T1D risk gene and provide insight into the complex mechanisms by which disease-associated variants act in concert to drive defined stages in disease progression.

Signal regulatory protein alpha (SIRPα) regulates the homeostasis of CD103+CD11b+DCs in the intestinal lamina propria

Signal regulatory protein alpha (SIRPα/CD172a) is a conserved transmembrane protein thought to play an inhibitory role in immune function by binding the ubiquitous ligand CD47. SIRPα expression has been used to identify dendritic cell subsets across species and here we examined its expression and function on intestinal DCs in mice. Normal mucosa contains four subsets of DCs based on their expression of CD103 and CD11b and three of these express SIRPα. However, loss of SIRPα signaling in mice leads to a selective reduction in the CD103+CD11b+ subset of DCs in the small intestine, colon, and among migratory DCs in the mesenteric lymph node. In parallel, these mice have reduced numbers of TH17 cells in steady-state intestinal mucosa, and a defective TH17 response to Citrobacter infection. Identical results were obtained in CD47KO mice. DC precursors from SIRPα mutant mice had an enhanced ability to generate CD103+CD11b+ DCs in vivo, but CD103+CD11b+ DCs from mutant mice were more prone to die by apoptosis. These data show a previously unappreciated and crucial role for SIRPα in the homeostasis of CD103+CD11b+ DCs in the intestine, as well as providing further evidence that this subset of DCs is critical for the development of mucosal TH17 responses.

Human hematopoietic reconstitution and HLA-restricted responses in nonpermissive alymphoid mice.

We generated a new humanized mouse model to study HLA-restricted immune responses. For this purpose, we created unique murine hosts by enforcing the expression of human SIRPα by murine phagocytes in murine MHC-deficient HLA-transgenic alymphoid hosts, an approach that allowed the immune reconstitution of nonpermissive mice following injection of human hematopoietic stem cells. We showed that these mouse/human chimeras were able to generate HLA-restricted responses to immunization. These new humanized mice may offer attractive models to study immune responses to human diseases, such as HIV and EBV infections, as well as to assay new vaccine strategies.

Ontogenic, Phenotypic, and Functional Characterization of XCR1(+) Dendritic Cells Leads to a Consistent Classification of Intestinal Dendritic Cells Based on the Expression of XCR1 and SIRPα.

In the past, lack of lineage markers confounded the classification of dendritic cells (DC) in the intestine and impeded a full understanding of their location and function. We have recently shown that the chemokine receptor XCR1 is a lineage marker for cross-presenting DC in the spleen. Now, we provide evidence that intestinal XCR1+ DC largely, but not fully, overlap with CD103+ CD11b− DC, the hypothesized correlate of “cross-presenting DC” in the intestine, and are selectively dependent in their development on the transcription factor Batf3. XCR1+ DC are located in the villi of the lamina propria of the small intestine, the T cell zones of Peyer’s patches, and in the T cell zones and sinuses of the draining mesenteric lymph node. Functionally, we could demonstrate for the first time that XCR1+/CD103+ CD11b− DC excel in the cross-presentation of orally applied antigen. Together, our data show that XCR1 is a lineage marker for cross-presenting DC also in the intestinal immune system. Further, extensive phenotypic analyses reveal that expression of the integrin SIRPα consistently demarcates the XCR1− DC population. We propose a simplified and consistent classification system for intestinal DC based on the expression of XCR1 and SIRPα.

Suppressed Expression of Homotypic Multinucleation, Extracellular Domains of CD172α (SIRP‐α) and CD47 (IAP) Receptors in TAMs UpRegulated by Hsp70–Peptide Complex in Dalton's Lymphoma

CD172α and CD47 are members of glycoprotein expressed on macrophages and various immune cells, promote immune recognition and T cell stimulation that priming phagocytosis of pathogens and apoptotic bodies and malignant cell. Tumour-releasing immunosuppressive factor promotes tumour growth and transforms the tumour resident M1 phenotype of macrophage to M2 phenotype (TAMs) that promotes tumour progression by downregulating the expression of different surface receptor including CD172α and CD47. Recent studies have reported that CD172α and CD47 are involved in the pathogenesis and promote malignancies such as lymphoma, leukaemia, melanoma, lung cancer and multiple myeloma, and their expression varies during infection and malignancies. Autologous Hsp70 is well recognized for its role in activating macrophages leading to enhance production of inflammatory cytokines. It has been observed that Hsp70 derived from normal tissues do not elicit tumour immunity, while Hsp70 preparation from tumour cell was able to elicit tumour immunity. However, the role of exogenous autologous hsp70 on the formation of giant cells is completely unknown. Therefore, in the present study, we sought to investigate the effect of Hsp70-peptide complex on the expression of CD172α and CD47 receptors in normal peritoneal macrophages (NMO) and TAMs. Finding shows that the expression of CD172α and CD47 enhances in TAMs and it reverts back the suppressed function of TAMs into M1 state of immunoregulatory phenotype that promotes tumour regression by enhanced multinucleation and phagocytosis of malignant cells and significantly enhances the homotypic fusion of macrophages and polykaryon formation in vitro by enhancing the expression of SIRPα and IAP.

PTU-077 Novel Techniques To Unravel The Immune Mechanisms Driving Inflammatory Bowel Disease

IntroductionThe inflammatory bowel diseases (IBD), ulcerative colitis (UC) and Crohn’s Disease (CD) occur when failures of immune regulation result in an accumulation of immune effector cells that damage the intestine....

Microglial CX3CR1 required for M2 polarization and recovery after intracerebral hemorrhage (INC5P.322)

Abstract Intracerebral hemorrhage (ICH) occurs when a blood vessel in the brain ruptures, resulting in the activation of microglia. Upon activation, microglia polarize into a pro-inflammatory, M1 phenotype or an M2 phenotype associated with repair. Microglia and monocyte subsets express the chemokine receptor CX3CR1. CX3CR1-null microglia have been shown to have dysregulated inflammation. We hypothesize that microglial CX3CR1-deficiency promotes dysregulated, M1 microglia that do not aid in repair. C57BL/6 (WT) and CX3CR1GFP/GFP (CX3CR1-null) mice were irradiated and reconstituted with bone marrow from WT mice (CD45.1) to generate bone marrow chimeras (CD45.1→WT or CD45.1→CX3CR1-null). ICH was modeled by injecting 20μl of blood into the right striatum. Behavioral outcomes were measured using gait analysis, cylinder test, and beam walking. Mice were sacrificed 14 days after ICH; perihematomal regions were harvested for intracellular staining. The CD45.1→CX3CR1-null mice display greater neurological deficit 14 days after ICH by cylinder test (p=0.002), beam walking (p=<0.001) and gait analysis (p=0.02). By intracellular staining, WT microglia have produce more for IL-10 (p=0.002) and IL-4 (p=0.0003) than CX3CR1-null microglia. CX3CR1-null microglia have less SIRPα (p=0.02) and higher CD36 expression (p=0.0004) compared to WT microglia. Our results suggest CX3CR1 signaling is necessary for microglia to transition from M1 to M2 and contributes to recovery after ICH.