Mouse Bone Marrow Mononuclear Cells Research Articles

Abstract 6535: Targeting estrogen receptor signaling in tumor associated myeloid cells to enhance anti-tumor immunity

Abstract Immune checkpoint inhibitors (ICB) have significantly improved outcomes across the spectrum of cancers, especially in patients with metastatic melanoma or non-small cell lung cancer (NSCLC). Despite encouraging efficacy in some patients, the development of primary and acquired resistance and severe toxicity associated with these treatment regimens, together with the overall low response rates, have limited their widespread use. Mining publicly available transcriptomic datasets, we have demonstrated that the response to ICB in patients with melanoma is influenced by the functionality of tumor associated macrophages (TAMs). Thus, it is of significance that we have demonstrated that 17-β estradiol (E2), working through myeloid cell intrinsic estrogen receptor alpha (ERα), promotes tumor growth in preclinical mouse models by polarizing tumor associated macrophages towards an immune-suppressive state in a manner that suppresses CD8+T cell functionality and limits ICB efficacy independent of gender. Transcriptomic analysis of tumor infiltrating myeloid cells isolated from E2-treated syngeneic tumors revealed that the type I interferon (IFN) signaling pathway was suppressed and that this activity could be reversed by the addition of fulvestrant a Selective Estrogen Receptor Downregulator. The importance of type I IFN signaling in E2-regulated tumor growth was confirmed in syngeneic mouse tumor models (B16F10-melanoma and LLC1-NSCLC cells) by showing that inhibiting type I interferon receptor (IFNAR) activity using blocking antibodies reversed the protective effects of E2 deprivation (ovariectomy) on tumor growth. Further, using in vitro cultures of macrophages from mouse bone marrow or peripheral blood mononuclear cells from deidentified human donors, we demonstrated that E2-dependent inhibition of type I IFN signaling could be attributed to increased efferocytosis by myeloid cells. These findings were recapitulated in vivo using an IFN reporter mouse (Mx1-egfp) implanted with labelled tumor cells (mCherry-Spectrin labelled LLC1), where we demonstrated that E2 mediated-suppression of myeloid cell intrinsic interferon signaling within the tumor microenvironment is contingent upon their engulfment of apoptotic cancer cells. Mechanistically, this enhanced efferocytosis was attributed to increased expression of the fractalkine receptor (CX3CR1) in E2-treated myeloid cells- as either genetic or pharmacological depletion of CX3CR1 in mouse reversed E2-mediated tumor growth (B16F10 and LLC1), myeloid cell polarization (from immunosuppressive to proinflammatory), myeloid cell intrinsic type I IFN signaling and reinvigorated CD8+T cell function. Taken together, our results highlight the potential clinical utility of targeting the E2/ER-CX3CR1 pathway as a means to enhance response to ICBs. Citation Format: Binita Chakraborty, Michael Brown, Prabuddha Chakraborty, Aditi Goyal, Ching Yi Chang, Donald McDonnell. Targeting estrogen receptor signaling in tumor associated myeloid cells to enhance anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6535.

F4/80+CD206+ M2-like macrophages contribute to bone erosion in collagen-induced arthritis by differentiating into osteoclasts

Objective Rheumatoid arthritis is an autoimmune disease characterized by synovial inflammation-driven cartilage and bone destruction, a process mainly mediated by osteoclasts. In recent years M2-like macrophages have been found to play an important role in the pathological process of RA by mediating pro-inflammatory effects, but their roles in the bone destruction of autoimmune arthritis have not been reported. Methods and Results In this study we identified that an abundant cell population of CD45+CD11b+Gr-1-F4/80+CD206+ cells, which were normally classified as M2-like macrophages, was present in synovium of collagen-induced arthritis (CIA) mice, and these cells had the potential to differentiate into osteoclasts. These M2-like macrophages sorted from CIA synovium highly expressed RANK and could be activated by RANKL and M-CSF to acquire osteoclast markers and bone resorption function both in vitro and in vivo. Furthermore, in vitro differentiated M2 macrophages from both CIA mouse bone marrow and RA patient peripheral blood mononuclear cells were also able to differentiate into osteoclasts, confirming the general osteoclastogenesis capability of M2 subtype macrophages. Conclusion All these results suggest that synovial F4/80+CD206+ M2-like macrophages in RA may be novel osteoclast precursors and contribute significantly to bone erosive changes seen in RA. Our studies provided new directions and targets for the diagnosis and treatment of rheumatoid arthritis.

Transcriptome analysis of hydrogen inhibits osteoclastogenesis of mouse bone marrow mononuclear cells.

Hydrogen (H2) is a major biodegradation product of implanted magnesium (Mg) alloys that are commonly used in the healing of bone fractures. Our earlier study showed that H2 can inhibit mouse bone marrow mononuclear cell (BMMC) osteoclastogenesis during the differentiation of these cells into osteoclasts, thereby facilitating fracture healing. However, the way by which H2 inhibits osteoclastogenesis remains to be elucidated. The present study used RNA-sequencing to study the transcriptome of H2-exposed BMMCs in an osteoclast-induced environment and identified the target genes and signaling pathways through which H2 exerts its biological effects. Several upregulated genes were identified: Fos, Dusp1, Cxcl1, Reln, Itga2b, Plin2, Lif, Thbs1, Vegfa and Gadd45a. Several downregulated genes were also revealed: Hspa1b, Gm4951, F830016B08Rik, Fads2, Hspa1a, Slc27a6, Cacna1b, Scd2, Lama3 and Col4a5. These differentially expressed genes were mainly involved in osteoclast differentiation cascades, as well as PI3K-AKT, Forkhead box O (FoxO), MAPK, peroxisome proliferator-activated receptor (PPAR), TNF, TGF-β, JAK-STAT, RAS, VEGF, hypoxia-inducible factor (HIF-1) and AMPK signaling pathways. In summary, the present study revealed the key genes and signaling pathways involved in the H2-mediated inhibition of osteoclastogenesis, thereby providing a theoretical basis for the significance of H2 and an experimental basis for the application of Mg alloys in the treatment of osteoporosis.

Lycium barbarum polysaccharide inhibits the mitochondrial pathway of apoptosis in mouse bone marrow mononuclear cells after radiation injury

Lycium barbarum polysaccharide inhibits the mitochondrial pathway of apoptosis in mouse bone marrow mononuclear cells after radiation injury

The Novel Role of Checkpoint Inhibitor PD-1H/VISTA in Osteoclast Activation and Multiple Myeloma Bone Disease

IntroductionMultiple myeloma (MM)-induced bone disease remains one of its most devastating complications, caused by increased bone resorption by overactivated osteoclasts coupled with impaired bone formation. MM cells produce osteoclast-activating factors that induce osteoclast activation and extensive bone resorption. Our previous work demonstrated that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor that is highly secreted by MM cells (Fu J etc. JCI. 2016). We also identified that the checkpoint inhibitor, programmed death-1 homolog (PD-1H/VISTA), serves as the MMP-13 receptor in osteoclasts and mediates MMP-13-dependent osteoclastogenic function which is largely blocked in Pd-1h -/-osteoclasts (Fu J etc. ASH 2019, 2020). While the inhibitory role of PD-1H/VISTA in T-cells has recently been described (ElTanbouly MA etc. Science 2020), its cellular binding proteins remain unclear, and its role in osteoclast activation andMM bone disease have not been addressed.Methods and ResultsTo identify its interacting proteins, PD-1H-His 6 recombinant protein was expressed in mouse bone marrow mononuclear cells, the associated proteins pulled down by Ni-NTA agarose beads from cell lysates and identified by mass spectrometry. Functional annotation charting of the 75 proteins enriched in PD-1H pull-down samples (with signal ratio of PD-1H-His 6 pull-down vs control >2) indicated that almost 30% of the interacting targets were either cytoskeletal or cytoskeleton-associated proteins.Given that the F-actin cytoskeleton undergoes dynamic reorganization during osteoclast differentiation and plays critical roles in bone resorption, we further addressed the role of PD-1H in F-actin cytoskeleton regulation. Initially, osteoclasts form F-actin-rich adhesive structures, termed podosomes. At later stages, podosomes collectively rearrange into clusters and rings and finally into sealing belts to mediate osteoclast spreading, migration and bone resorption (Teitelbaum SL. Ann N Y Acad Sci. 2011). By confocal immunofluorescence microscopy, we found that PD-1H co-localized with F-actin podosome clusters, rings and sealing belts during osteoclast differentiation (Figure 1A). The functional role of PD-1H in F-actin cytoskeleton reorganization was addressed using Pd-1h -/- osteoclast wherein Pd-1h knockout lead to the disruption of podosome clusters at early stages relative to WT controls, while at later stages, Pd-1h -/-osteoclasts exhibited significantly fewer F-actin rings and belts (Figure 1B). Further, binding of MMP-13 to PD-1H increased the number of osteoclasts forming F-actin rings and belts, as well as the size of F-actin belts, which was blocked in Pd-1h -/- osteoclasts.To determine the role of PD-1H in the development of myeloma-induced lytic bone lesions, 5TGM1 myeloma cells were bilaterally intratibially injected into Pd-1h wtRag2 -/- or Pd-1h -/-Rag2 -/- mice (n=10) to induce lytic bone lesions. Three weeks following intratibial 5TGM1 injection, tibiae were harvested for micro-computed tomography. Subsequent quantitative histomorphology analyses of the trabecular and cortical bones confirmed that the knockout of Pd-1h reduced MM-induced bone destruction with significantly less decrease in trabecular bone volume (Tb. [BV/TV]), trabecular bone number (Tb. N.), trabecular bone thickness (Tb. Th.), as well as less increase in trabecular bone spacing (Tb. Sp.) and bone specific surface (Tb. [BS/BV]) compared to Pd-1h wtRag2 -/- mice. Similar effects were observed in cortical bone with less decrease in cortical bone thickness (CT. Th.), cortical bone area fraction (CT. [BA/TA]), and cortical tissue mineral density (CT. TMD) in 5TGM1 bearing Pd-1h -/-Rag2 -/- mice vs Pd-1h wtRag2 -/- mice (Table 1).ConclusionsTaken together, our study, for the first time, reveals the novel role of checkpoint inhibitor, PD-1H/VISTA, in osteoclasts and myeloma bone disease. PD-1H associates with cytoskeleton proteins and regulates the F-actin cytoskeleton reorganization which is critical for osteoclast bone resorption activity. Further, PD-1H mediates MMP-13-induced osteoclast fusion, F-actin belts formation, and osteoclast activation. Pd-1h -/-in recipient mice significantly impairs MM-induced bone loss, demonstrating that PD-1H/VISTA plays a critical role in MM bone disease. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

RUNX1 Deficiency and SRSF2 Mutation Cooperate to Promote Myelodysplastic Syndrome Development

Myelodysplastic syndromes (MDS) are genetically complex diseases that require multiple mutations during disease development. Acquisition of somatic mutations results in clonal diversity and different responses to therapy. Genes encoding the spliceosomal proteins SRSF2, SF3B1, U2AF1, and ZRSR2 are frequently mutated in MDS, together affecting around 60% of patients. SRSF2 mutations are found in 10-13% of MDS patients and almost always present as heterozygous missense mutations. Several patient studies have shown that SRSF2 mutations are closely associated with RUNX1, ASXL1, IDH1, and IDH2 mutations. RUNX1 is an important transcription factor for hematopoiesis and mutations of RUNX1 are detected in 10-15% of MDS. Although RUNX1 is one of the most frequently co-mutated genes with SRSF2 in MDS patients, it remains unknown how this combination of genetic alterations affect hematopoiesis. Here we hypothesize that RUNX1 and SRSF2 mutations cooperate in MDS development and explored this biologically in vivo.To investigate whether the coexistence of SRSF2 mutation and RUNX1 deficiency could impair hematopoiesis in vivo, we utilized previously reported Mx1-Cre based conditional knock-in Srsf2-P95H mutation (P95H/+) mice (which confers an MDS phenotype), and Mx1-Cre based Runx1 conditional knockout mice (Runx1 f/f) (which demonstrates a mild myeloproliferative phenotype). We crossed these two strains to establish a new mouse model (Srsf2 P95H/+ Runx1 f/f Mx1-Cre). To determine whether the phenotypic effects from the mutations are hematopoietic cell-intrinsic manner, we performed non-competitive bone marrow transplantation experiments by transplanting mouse bone marrow mononuclear cells collected from Mx1-Cre, Runx1 f/f Mx1-Cre, Srsf2 P95H/+ Mx1-Cre, and Srsf2 P95H/+ Runx1 f/f Mx1-Cre mice into lethally irradiated recipients. Four weeks after transplantation, we induced Cre expression by pIpC injection. RUNX1 loss and SRSF2 P95H together altered multi-lineage hematopoiesis in mice. Double mutant mice showed MDS features including severe leukopenia in multiple lineages, mild anemia, mild thrombocytopenia, and dysplastic morphology, such as hyposegmented/hypersegmented neutrophils and red blood cells with Howell-Jolly bodies in peripheral blood. Double mutant mice also displayed more dramatic skewing to the myeloid lineage at expense of the B cell lineage when compared to single mutant mice. Flow cytometric analysis of the peripheral blood cell lineages revealed reduced B cell percentages (Mean of B220+ percentage Mx1-Cre: 51.6%, Runx1 f/f Mx1-Cre: 40.5%, Srsf2 P95H/+ Mx1-Cre: 38.6%, Srsf2 P95H/+ Runx1 f/f Mx1-Cre:20%, N = 7-12 each genotype, 28 weeks after bone marrow transplantation) and increased myeloid percentages (Mean of CD11b+ percentage: 20.1%, 24.3%, 18.9%, 35.5% respectively). At 20-weeks post transplantation, we analyzed the spleen and bone marrow compartments of the mice. In the spleen, we found the same enhanced skewing from B-cells to myeloid lineage as in the peripheral blood compartment. However, in the bone marrow, single and double deficiency mice both demonstrated similar decreases in B cell percentage. To evaluate stem and progenitor function in vivo, we performed competitive bone marrow transplantation. We mixed CD45.1 bone marrow and CD45.2 (Mx1-Cre, Runx1 f/f Mx1-Cre, Srsf2 P95H/+ Mx1-Cre, Srsf2 P95H/+ Runx1 f/f Mx1-Cre) bone marrow in a 1:1 ratio, transplanted to lethally irradiated recipients, and injected pIpC 4 weeks after transplantation. The repopulation assays showed that SRSF2 P95H with RUNX1 deficiency confer a competitive disadvantage. Our findings from these mouse models indicate that RUNX1 deficiency and SRSF2 P95H mutation together impair multi-lineage hematopoiesis and exacerbate disease phenotypes caused by SRSF2 P95H mutation. DisclosuresNo relevant conflicts of interest to declare.

The Long Non-coding RNA NEAT1/miR-224-5p/IL-33 Axis Modulates Macrophage M2a Polarization and A1 Astrocyte Activation.

To identify potential regulators and investigate the molecular mechanism of macrophage polarization affecting astrocyte activation from the perspective of non-coding RNA regulation, we isolated mouse bone marrow mononuclear cells (BMMNCs)-induced macrophages toward M1 or M2a polarization. Long non-coding RNA NEAT1 and IL-33 expression levels were significantly upregulated in M2a macrophages; NEAT1 knockdown in M2a macrophages markedly reduced the protein levels of IL-33 and M2a markers, IL-4 and IL-13 concentrations, and the bacterial killing capacity of M2a macrophages. NEAT1 acted as a competing endogenous RNA (ceRNA) to regulate IL-33 expression by sponging miR-224-5p in M2a macrophages; NEAT1 knockdown upregulated miR-224-5p expression, while miR-224-5p inhibition increased the protein content and concentration of IL-33. miR-224-5p inhibition exerted the opposite effects on the protein levels of IL-33 and M2a markers, IL-4 and IL-13 concentrations, and the bacterial killing capacity of M2a macrophages compared to NEAT1 knockdown; the effects of NEAT1 knockdown were significantly reversed by miR-224-5p inhibition. M2a macrophage conditioned medium (CM) significantly suppressed the activation of A1 astrocytes. NEAT1 knockdown M2a macrophage CM led to enhanced A1 astrocyte activation while miR-224-5p-silenced M2a macrophage CM led to a blockade of A1 astrocyte activation; the effects of NEAT1 knockdown M2a macrophage CM on A1 astrocyte activation were significantly reversed by miR-224-5p inhibition in M2a macrophages. The NEAT1/miR-224-5p/IL-33 axis modulates macrophage M2a polarization, therefore affecting A1 astrocyte activation.

Mutant U2AF1-induced differential alternative splicing causes an oxidative stress in bone marrow stromal cells.

Alternative splicing (AS) is a critical regulatory process of gene expression. In bone marrow microenvironment, AS plays a critical role in mesenchymal stem cells fate determination by forming distinct isoforms of important regulators. As a spliceosome factor, U2AF1 is essential for the catalysis of pre-mRNA splicing, and its mutation can cause differential AS events. In the present study, by forced expression of mutant U2AF1 (U2AF1S34F) in the mouse bone marrow stroma OP9 cells, we determine AS changes in U2AF1S34F transduced OP9 cells and investigate their role in stroma cell biological functions. We find that abundant differential RNA splicing events are induced by U2AF1S34F in OP9 cells. U2AF1S34F causes increased generation of hydrogen peroxide, promotes production of cytokines and chemokines. U2AF1S34F transduced OP9 cells also exhibit dysfunction of mitochondria. RNA-seq data, gene ontology (GO), and gene set enrichment analysis reveal that differentially expressed genes downregulated in response to U2AF1S34F are enriched in peroxisome component and function. U2AF1S34F can also cause release of hydrogen peroxide from OP9 cells. Furthermore, we investigate the influence of U2AF1S34F-induced oxidative stress in stromal cells on hematopoietic cells. When co-culturing mouse bone marrow mononuclear cells with OP9 cells, the U2AF1S34F expressing OP9 cells induce phosphorylation of histone H2AX in hematopoietic cells. Collectively, our results reveal that mutant U2AF1-induced differential AS events cause oxidative stress in bone marrow stromal cells and can further lead to DNA damage and genomic instability in hematopoietic cells.

Quantification of azacitidine incorporation into human DNA/RNA by accelerator mass spectrometry as direct measure of target engagement

We report an accelerator mass spectrometry (AMS) assay to quantify azacitidine (Aza) incorporation into DNA and RNA from human acute myeloid leukemia (AML) cells, mouse bone marrow (BM) and peripheral blood mononuclear cells (PBMCs). Aza, a cytidine nucleoside analogue, is a disease modifying pharmacological agent used for treatment of myelodysplastic syndromes (MDS) and AML. Our assay was able to directly quantify the complex of Aza incorporated into DNA/RNA, via isolation of DNA/RNA from matrix (i.e., cancer cells, BM and PBMC) and subsequent measurement of total radioactivity (i.e., 14C-Aza) by using AMS. The sensitivity of the method was able to quantify as little as a single Aza molecule incorporated into DNA with approximately 2 × 107 nucleotides from PBMCs. An in vivo mouse model was used for establishing the lower limits of quantification (LLOQs) for Aza incorporated into DNA/RNA in mouse PBMCs (∼ 3.7 × 105) and BM (∼27.8 mg) collected 24 h post-dose after total exposure of 18 nCi/mouse (Aza 1 mg/kg). The LLOQs for PBMC analysis were 2.5 picogram equivalents per microgram (pgEq/μg) DNA and 0.22 pgEq/μg RNA, and for BM analysis were 1.7 pgEq/μg DNA and 0.22 pgEq/μg RNA. A linear relationship (i.e., ∼10-fold) was established of radioactive dose from 14C-Aza 17 nCi/mouse to 188 nCi/mouse and AMS response (i.e., 14C/12C ratio ranging from 2.45 × 10−11 to 2.50 × 10-10), as Aza was incorporated into DNA in mouse BM. The current method enables the direct measurement of Aza incorporation into DNA and RNA from patient PBMCs and BM to provide dosing optimization, and to assess target engagement with as little as ∼5 mL whole blood and ∼3 mL of BM from patients.

A20 inhibits osteoclastogenesis via TRAF6-dependent autophagy in human periodontal ligament cells under hypoxia.

ObjectivesA20 exerts an anti‐osteoclastogenic effect through the inhibition of NF‐κB signalling in periodontitis. It also regulates autophagy via ubiquitin modification. This study was aimed at exploring the relationship between A20 and autophagy in anti‐osteoclastogenesis in human periodontal ligament cells (hPDLCs) under hypoxia.Materials and MethodsReal‐time PCR and Western blot were used to detect relative mRNA and protein levels. The formation of autophagosomes was measured by TEM. Osteoclastic differentiation was assessed by TRAP staining and hydroxyapatite resorption assay. The interactions between different proteins were observed by co‐IP.ResultsCells cultured under 2% O₂ exhibited decreased A20 expression and increased RANKL/OPG (R/O) ratio. There was a negative correlation between A20 and TRAF6, and similar results were found with autophagic flux. A20 delayed the increase in R/O ratio under hypoxia. Autophagy in hPDLCs and osteoclast differentiation and hydroxyapatite resorption areas in mouse bone marrow mononuclear cells (BMMCs) were inhibited by A20. Moreover, inhibition of autophagy using 3‐MA resulted in increased expression of A20 and decreased number and function of osteoclasts. In addition, A20 inhibited polyubiquitination at K63 and enhanced that at K48 in TRAF6 to suppress autophagy under hypoxic conditions.ConclusionsA20 inhibits osteoclastogenesis via inhibition of TRAF6‐dependent autophagy in hPDLCs under hypoxia. These findings suggest that A20 may be a key gene target during bone loss in periodontitis via TRAF6‐mediated inhibition of autophagy.

Hydrogen inhibits the osteoclastogenesis of mouse bone marrow mononuclear cells.

Hydrogen (H2) is one of the major biodegradation products of magnesium (Mg) alloys implanted for bony fracture healing and reconstruction; H2 thus plays a significant role in the regulation of local microenvironment and the biology of resident cells. The interactions between the H2 and the local cells are of great interest, and a full understanding of the effect of H2 on bone marrow mononuclear cells (BMMCs) would accelerate the development of effective strategies for successful bony healing. This study investigates how H2, with different concentrations and durations, regulates the osteoclastogenesis of mouse BMMCs. First, using H2 with five concentrations (0%, 2%, 25%, 50% and 75%) and three durations (5, 7 and 10days), the osteoclastogenesis of mouse BMMCs in these H2 conditions were measured using TRAP staining, F-actin ring formation assay, pit formation assay and RT-qPCR analysis. Based on these findings, the proliferation assay, apoptosis assay, western blot analysis and ELISA assay of BMMCs after osteoclast induction were performed. The findings showed that H2 (especially the 50% and 75% H2) obviously inhibited the osteoclast formation, function and osteoclast-related genes expression of osteoclast-induced BMMCs; additionally, H2 (50%) was found to reduce the proliferation, promote the apoptosis and inhibit the expression of osteoclast-related proteins of BMMCs with the presence of osteoclast-induced medium. Therefore, H2 significantly inhibited the osteoclastogenesis of mouse BMMCs, which may become a new therapeutic agent for anti-bony resorption and open new avenues for the translational research of Mg alloys.

Short-term ex-vivo exposure to hydrogen sulfide enhances murine hematopoietic stem and progenitor cell migration, homing, and proliferation

ABSTRACT For successful transplantation of Hematopoietic Stem cells (HSCs), it is quite necessary that efficient homing, engraftment and retention of HSC self-renewal capacity takes place, which is often restricted due to inadequate number of adult HSCs. Here, we report that short-term ex-vivo treatment of mouse bone marrow mononuclear cells (BMMNCs) to Sodium Hydrogen Sulfide (NaHS, hydrogen sulfide-H2S donor) can be used as a possible strategy to overcome such hurdle. H2S increases the expression of CXCR4 on HSPCs, enhancing their migration toward SDF-1α in-vitro and thus homing to BM niche. . Additionally, in-vitro studies revealed that H2S has a role in activating mitochondria, thus, pushing quiescent HSCs into division. These results suggest a readily available and cost-effective method to facilitate efficient HSC transplantation.

Abstract 718: Reparative Macrophage Transplantation for Myocardial Repair: A Refinement of Bone Marrow Mononuclear Cell-Based Therapy

Background: Reparative (alternatively activated or M2-like) macrophages play an important role in post-myocardial infarction (MI) cardiac repair. Transplantation of bone marrow mononuclear cells (BM-MNCs) is an emerging therapy for MI while its therapeutic efficacy in previous clinical trials is not satisfactory. We hypothesized that induced differentiation/polarisation of BM-MNCs to reparative macrophages before transplantation may enhance the effect of BM-MNC transplantation. Purpose: This study aimed to develop a robust in vitro protocol to produce reparative macrophages from BM-MNCs and to establish the pre-clinical proof of concept data for reparative macrophage transplantation for the treatment of MI. Methods & Results: Mouse BM-MNCs were treated with M-CSF plus IL-4, IL-10, TGF-β1 or combinations of these in vitro . The concomitant M-CSF+IL-4 protocol produced the highest rate and number of CD11b + F4/80 + CD206 + macrophages. Expression and secretion of tissue repair-related factors of the produced cells, including IGF-1, TGF-β1, VEGF and IL1-ra, were more extensive compared to BM-MNCs. Then, reparative macrophages, BM-MNCs or PBS only were intramyocardially injected in a mouse MI model. At 4 weeks after treatment, echocardiography demonstrated that reparative macrophage transplantation markedly improved cardiac function (left ventricular ejection fraction; 57.2±1.6%, n=11) compared to both BM-MNC transplantation (48.4±1.3%, n=9) and control group (44.4±2.0%, n=9). Histological studies showed that infarct size was the smallest after reparative macrophage transplantation in association with the greatest tissue repair in the peri-infarct myocardium, including augmented microvascular formation, reduced cardiomyocyte hypertrophy and reduced pathological interstitial fibrosis. It was also found that reparative macrophage transplantation increased host-derived reparative macrophages through TGF-β1 secretion. Conclusion: M-CSF+IL-4 treatment was effective in producing reparative macrophages from BM-MNCs in vitro . Addition of this pre-treatment improved the therapeutic effect of BM-MNC transplantation. Further pre-clinical and clinical development of this advanced cell therapy is warranted.

Reparative macrophage transplantation for myocardial repair: a refinement of bone marrow mononuclear cell-based therapy

Reparative macrophages play an important role in cardiac repair post-myocardial infarction (MI). Bone marrow mononuclear cells (BM-MNCs) have been investigated as a donor for cell therapy but with limited clinical success. These cells, however, may be utilized as a source for reparative macrophages. This translational study aimed to establish a robust in vitro protocol to produce functional reparative macrophages from BM-MNCs and to establish pre-clinical evidence of the efficacy of reparative macrophage transplantation for the treatment of MI. Mouse BM-MNCs were treated with M-CSF plus IL-4, IL-10, TGF-β1 or combinations of these in vitro. The concomitant administration of M-CSF and IL-4 produced the highest rate and largest number of CD11b+F4/80+CD206+ reparative macrophages. Expression and secretion of tissue repair-related factors including IGF-1, TGF-β1, VEGF and IL1-ra were remarkably enhanced in reparative macrophages compared to BM-MNCs. These cells were transplanted in a mouse MI model, resulting in evident improvement in cardiac function recovery, compared to BM-MNC transplantation. Histological studies showed that reparative macrophage transplantation enhanced myocardial tissue repair including augmented microvascular formation, reduced cardiomyocyte hypertrophy and attenuated interstitial fibrosis. Moreover, survival of reparative macrophages in the heart post-transplantation was increased compared to BM-MNCs. Reparative macrophage transplantation also increased host-derived reparative macrophages in part through TGF-β secretion. In conclusion, concomitant M-CSF + IL-4 treatment effectively produced reparative macrophages from BM-MNCs in vitro. Transplantation of produced reparative macrophage achieved a superior therapeutic efficacy, compared to BM-MNC transplantation, through the enhanced quantity and quality of donor cell engraftment. Further development of this advanced cell-based therapy is warranted.

Assessment of mouse BMMNC DNA damage with a two-tailed comet assay after X-ray and carbon ion total body irradiation

The two-tailed comet assay (2T-comet assay) is a method for simultaneously evaluating DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). In the present study, the endonuclease DNase I and hydrogen peroxide were used to induce DSBs and SSBs in bone marrow mononuclear cells (BMMNCs) from mice, and the damaged DNAs were assessed with a 2T-comet assay. The results demonstrated that this method can detect and discriminate between BMMNC DNA SSBs and DSBs simultaneously. Using this method, we studied DNA damage in BMMNCs from female BALB/c mice after total body irradiation with X-rays and carbon ions. The results indicated that these two types of radiation induced serious DNA damage in BMMNCs in a dose-dependent manner. The DNA damage induced by carbon ions was more severe than that induced by X-rays at the same dose, and a high dose of carbon ion radiation was more likely to cause death in mice. The DSBs and SSBs induced by X-rays were the highest on the 3rd day post-IR. For carbon ion radiation, DSBs were the most serious on the 3rd day, while SSBs were more obvious on the 3rd day and 13th day post-IR. The ratio of DSBs/SSBs was clearly related to the different types of radiation.

Structural and immunomodulatory differences among lactobacilli exopolysaccharides isolated from intestines of mice with experimentally induced inflammatory bowel disease.

Characteristic changes in the microbiota biostructure and a decreased tolerance to intestinal bacteria have been associated with inflammatory bowel disease (IBD). However, few studies have examined the constituents of the intestinal microbiota, including the surface molecules of the bacteria, in healthy and IBD subsets. Here, we compare the chemical structures and immunomodulatory properties of the exopolysaccharides (EPS) of lactobacilli isolated from mice with induced IBD (IBD “+”) versus those of healthy mice (IBD “−”). Classical structural analyses were performed using nuclear magnetic resonance spectroscopy and mass spectrometry. Immunomodulatory properties were assessed by stimulation of dendritic cells derived from mouse bone marrow or human peripheral mononuclear blood cells. Our results revealed that EPS produced by IBD “+” species are structurally different from those isolated from IBD “−”. Moreover, the structurally different EPS generate different immune responses by dendritic cells. We speculate that resident strains could, upon gut inflammation, switch to producing EPS with specific motifs that are absent from lactobacilli IBD “−”, and/or that bacteria with a particular EPS structure might inhabit the inflamed intestinal mucosa. This study may shed light on the role of EPS in IBD and help the development of a specific probiotic therapy for this disease.

Performance of Keyhole Limpet Hemocyanin (KLH) as an Antigen Carrier for Protein Antigens Depends on KLH Property and Conjugation Route

Abstract KLH naturally exists either as high molecular weight (HMW) cylinders composed of 20 copies of subunits, or as separate subunits (Subunit). The molecular weight of KLH Subunit is about 400kDa. Owing mainly to its large size, and the abundant and complex carbohydrate structures on its peptide chains, KLH has been widely used for decades as an immune stimulant, antigen carrier, and vaccine component. But, how to characterize the properties of KLH as an antigen carrier through functional assays remains to be determined. At Stellar, we conjugated KLH of different formats and sources with ovalbumin (OVA) and fluorescence tag-conjugated OVA through different conjugation routes. Dendritic cells (DCs) derived from mouse bone marrow mononuclear cells and mouse DC line JAWSII were pulsed with these conjugates. By utilizing DC uptake assays and measuring the fluorescence intensity of DC lysate supernatant, the amount of KLH- OVA-fluorescence conjugates uptaken by DCs was quantitatively determined. With flow cytometry, both the DC maturation markers, and the amount of ovalbumin epitopes that are presented by MHC II molecules of DCs, were measured. To further compare the property of KLH-protein conjugates as potential vaccines, we vaccinated mice with OVA. OVA-specific memory T cells extracted from the vaccinated mice were stimulated with DCs that had been pulsed with KLH-OVA conjugates. T cell proliferation and cytokine release from these stimulated T cells were quantitatively measured and compared. To summarize, with the functional assays we designed, we found that the performance of KLH as a carrier for protein antigens depends on KLH property and conjugation route.

Sorting nexin 10 acting as a novel regulator of macrophage polarization mediates inflammatory response in experimental mouse colitis.

In response to changes in microenvironment, macrophages polarize into functionally distinct phenotypes, playing a crucial role in the pathogenesis of inflammatory bowel disease (IBD). Here, we investigated the effects of sorting nexin 10 (SNX10), a protein involved in endosomal trafficking and osteoclast maturation, on regulation of macrophage polarization and progression of mouse colitis. Our results revealed that SNX10 deficiency increased the population of M2-type monocytes/macrophages, and protected against colonic inflammation and pathological damage induced by dextran sulfate sodium (DSS). By in vitro study, we showed that deficiency of SNX10 polarized macrophages derived from mouse bone marrow or human peripheral blood mononuclear cells (PBMCs) towards an anti-inflammatory M2 phenotype, which partially reversed by SNX10 plasmid transfection. Adoptive transfer of SNX10−/− macrophages ameliorated colitis in WT mice. However, transfer of WT macrophages exacerbated colitis in SNX10−/− mice. Our data disclose a crucial role and novel function for SNX10 in macrophage polarization. Loss of SNX10 function may be a potential promising therapeutic strategy for IBD.

Measuring the properties of different forms of keyhole limpet hemocyan reveals important differences (VAC5P.1116)

Abstract KLH can exist as cylinders composed of 20 copies (didecamer) of subunits, or as separate subunits. It is a well-established immune stimulant and vaccine component. However, literature reports cite dramatically different immune responses to different preparations of KLH. We are developing functional assays to investigate the immunogenicity of different forms and preparations of KLH with the goal of improving the predictive outcome of its use as vaccine carrier or diagnostic agent for both research and clinical studies. Dendritic cells (DCs) derived from mouse bone marrow mononuclear cells, human PBMC, mouse DC line JAWSII, as well as human leukemia lines THP-1 and KG-1, were treated with different forms of KLH. To compare the efficiency of DCs to stimulate KLH-specific adaptive immune responses, KLH treated DCs were used to activate T cells extracted from spleens of mice that were immunized with KLH. DC antigen uptake efficiency, DC maturation, T cell proliferation and cytokine secretion were compared. Ovalbumin was conjugated to different forms of KLH. DCs derived from mouse bone marrow mononuclear cells were treated with the KLH-Ovalbumin conjugates for antigen uptake efficiency and ovalbumin-specific T cell activation studies. We found that didecameric KLH and subunit KLH have significantly different properties. We report here the results of these studies which suggest the need for methods to identify appropriate forms of KLH when used as hapten carrier or immunogen.

The combined use of cytokine-induced killer cells and cyclosporine a for the treatment of aplastic anemia in a mouse model.

In this study, we investigated the combined use of cytokine-induced killer (CIK) cells and cyclosporine A (CsA) to treat a mouse model of aplastic anemia (AA). CIK cells were cultured and injected alone or in combination with CsA into mice that had previously been induced into AA by busulfan and mouse interferon-γ (IFN-γ). The CIK cell-treated group had a survival rate of 55%, which was similar to the 60% survival rate observed in the CsA-treated group. The combination group showed a survival rate as high as 90%, while none of the mice in the no-treatment group survived to the end of the experiment. The CIK cells produced multiple cytokines, including several hematopoietic growth factors, which could promote the expansion of mouse bone marrow mononuclear cells in vitro. CsA reduced the proportion of CD4(+) T cells and the level of IFN-γ. The combined CIK cell and CsA treatment exhibited the best curative effect, a finding that might be due to the influence of these factors on both hematopoiesis and immunity. These data suggest that the combination of CIK cells and immunosuppressive therapy might be a candidate therapy for AA in the future.