Cells Of Hematopoietic Origin Research Articles

Reciprocal stabilization of coagulation factor XIII-A and -B subunits is a determinant of plasma FXIII concentration

AbstractTransglutaminase factor XIII (FXIII) is essential for hemostasis, wound healing, and pregnancy maintenance. Plasma FXIII is composed of A and B subunit dimers synthesized in cells of hematopoietic origin and hepatocytes, respectively. The subunits associate tightly in circulation as FXIII-A2B2. FXIII-B2 stabilizes the (pro)active site-containing FXIII-A subunits. Interestingly, people with genetic FXIII-A deficiency have decreased FXIII-B2, and therapeutic infusion of recombinant FXIII-A2 (rFXIII-A2) increases FXIII-B2, suggesting FXIII-A regulates FXIII-B secretion, production, and/or clearance. We analyzed humans and mice with genetic FXIII-A deficiency and developed a mouse model of rFXIII-A2 infusion to define mechanisms mediating plasma FXIII-B levels. Like humans with FXIII-A deficiency, mice with genetic FXIII-A deficiency had reduced circulating FXIII-B2, and infusion of FXIII-A2 increased FXIII-B2. FXIII-A-deficient mice had normal hepatic function and did not store FXIII-B in liver, indicating FXIII-A does not mediate FXIII-B secretion. Transcriptional analysis and polysome profiling indicated similar F13b levels and ribosome occupancy in FXIII-A-sufficient and -deficient mice and in FXIII-A-deficient mice infused with rFXIII-A2, indicating FXIII-A does not induce de novo FXIII-B synthesis. Unexpectedly, pharmacokinetic/pharmacodynamic modeling of FXIII-B antigen after rFXIII-A2 infusion in humans and mice suggested FXIII-A2 slows FXIII-B2 loss from plasma. Accordingly, comparison of free FXIII-B2 vs FXIII-A2-complexed FXIII-B2 (FXIII-A2B2) infused into mice revealed faster clearance of free FXIII-B2. These data show FXIII-A2 prevents FXIII-B2 loss from circulation and establish the mechanism underlying FXIII-B2 behavior in FXIII-A deficiency and during rFXIII-A2 therapy. Our findings reveal a unique, reciprocal relationship between independently synthesized subunits that mediate an essential hemostatic protein in circulation. This trial was registered at www.ClinicalTrials.com as #NCT00978380.

Erythroid Cells as Full Participants in the Tumor Microenvironment.

The tumor microenvironment is an important factor that can determine the success or failure of antitumor therapy. Cells of hematopoietic origin are one of the most important mediators of the tumor-host interaction and, depending on the cell type and functional state, exert pro- or antitumor effects in the tumor microenvironment or in adjacent tissues. Erythroid cells can be full members of the tumor microenvironment and exhibit immunoregulatory properties. Tumor growth is accompanied by the need to obtain growth factors and oxygen, which stimulates the appearance of the foci of extramedullary erythropoiesis. Tumor cells create conditions to maintain the long-term proliferation and viability of erythroid cells. In turn, tumor erythroid cells have a number of mechanisms to suppress the antitumor immune response. This review considers current data on the existence of erythroid cells in the tumor microenvironment, formation of angiogenic clusters, and creation of optimal conditions for tumor growth. Despite being the most important life-support function of the body, erythroid cells support tumor growth and do not work against it. The study of various signaling mechanisms linking tumor growth with the mobilization of erythroid cells and the phenotypic and functional differences between erythroid cells of different origin allows us to identify potential targets for immunotherapy.

The Sts Proteins: Modulators of Host Immunity.

The suppressor of TCR signaling (Sts) proteins, Sts-1 and Sts-2, are a pair of closely related signaling molecules that belong to the histidine phosphatase (HP) family of enzymes by virtue of an evolutionarily conserved C-terminal phosphatase domain. HPs derive their name from a conserved histidine that is important for catalytic activity and the current evidence indicates that the Sts HP domain plays a critical functional role. Sts-1HP has been shown to possess a readily measurable protein tyrosine phosphatase activity that regulates a number of important tyrosine-kinase-mediated signaling pathways. The in vitro catalytic activity of Sts-2HP is significantly lower than that of Sts-1HP, and its signaling role is less characterized. The highly conserved unique structure of the Sts proteins, in which additional domains, including one that exhibits a novel phosphodiesterase activity, are juxtaposed together with the phosphatase domain, suggesting that Sts-1 and -2 occupy a specialized intracellular signaling niche. To date, the analysis of Sts function has centered predominately around the role of Sts-1 and -2 in regulating host immunity and other responses associated with cells of hematopoietic origin. This includes their negative regulatory role in T cells, platelets, mast cells and other cell types, as well as their less defined roles in regulating host responses to microbial infection. Regarding the latter, the use of a mouse model lacking Sts expression has been used to demonstrate that Sts contributes non-redundantly to the regulation of host immunity toward a fungal pathogen (C. albicans) and a Gram-negative bacterial pathogen (F. tularensis). In particular, Sts-/- animals demonstrate significant resistance to lethal infections of both pathogens, a phenotype that is correlated with some heightened anti-microbial responses of phagocytes derived from mutant mice. Altogether, the past several years have seen steady progress in our understanding of Sts biology.

BTK Isoforms p80 and p65 Are Expressed in Head and Neck Squamous Cell Carcinoma (HNSCC) and Involved in Tumor Progression

Here, we describe the expression of Bruton's Tyrosine Kinase (BTK) in head and neck squamous cell carcinoma (HNSCC) cell lines as well as in primary HNSCC samples. BTK is a kinase initially thought to be expressed exclusively in cells of hematopoietic origin. Apart from the 77 kDa BTK isoform expressed in immune cells, particularly in B cells, we identified the 80 kDa and 65 kDa BTK isoforms in HNSCC, recently described as oncogenic. Importantly, we revealed that both isoforms are products of the same mRNA. By investigating the mechanism regulating oncogenic BTK-p80/p65 expression in HNSSC versus healthy or benign tissues, our data suggests that the epigenetic process of methylation might be responsible for the initiation of BTK-p80/p65 expression in HNSCC. Our findings demonstrate that chemical or genetic abrogation of BTK activity leads to inhibition of tumor progression in terms of proliferation and vascularization in vitro and in vivo. These observations were associated with cell cycle arrest and increased apoptosis and autophagy. Together, these data indicate BTK-p80 and BTK-p65 as novel HNSCC-associated oncogenes. Owing to the fact that abundant BTK expression is a characteristic feature of primary and metastatic HNSCC, targeting BTK activity appears as a promising therapeutic option for HNSCC patients.

Origin, Phenotype, and Function of Mouse Dendritic Cell Subsets.

Dendritic cells are cells of hematopoietic origin that are specialized in antigen presentation and instruction of innate and adaptive immune responses. They are a heterogenous group of cells populating lymphoid organs and most tissues. Dendritic cells are commonly separated in three main subsets that differ in their developmental paths, phenotype, and functions. Most studies on dendritic cells were done primarily in mice; therefore, in this chapter, we propose to summarize the current knowledge and recent progress on mouse dendritic cell subsets' development, phenotype, and functions.

Abstract OEC102: Unbiased Discovery Of Allograft Inflammatory Factor 1 As A New And Critical Immunometabolic Regulatory Node During Cardiac Injury

Introduction: Myocardial infarction (MI) mobilizes macrophages, which are the central protagonists of tissue repair in the infarcted heart. While macrophages are necessary for repair after MI, they can also contribute to adverse remodeling and progression to heart failure. We sought to identify novel mediators of maladaptive macrophage responses by performing single cell RNA sequencing of a patient with ischemic cardiomyopathy. This led to the unbiased identification of Allograft inflammatory factor 1 ( AIF1 ) as the most significant differentially expressed gene in macrophages compared to other cardiac cells during human heart failure. Hypothesis: Since AIF1 was specifically expressed in macrophages during ischemic heart failure, we next tested the hypothesis that AIF1 promoted maladaptive macrophage responses after MI. Methods and Results: Using autopsy samples from human acute MI patients or a murine model of permanent occlusion MI, we found that AIF1 was increased in macrophages, but not neutrophils or monocytes, after MI. To test the functional significance of AIF1 expression in macrophages after MI, we generated bone marrow chimeric mice where Aif1 -deficiency was restricted to only cells of hematopoietic origin. Despite comparable levels of surgically induced injury, loss of Aif1 was associated with increased proreparative macrophages, smaller infarct sizes, and preservation of cardiac function compared to wild-type controls. Therapeutic targeting of Aif1 with anti-sense oligonucleotides also conferred cardioprotective responses after MI. Mechanistically, AIF1 promoted actin polymerization in bone marrow-derived macrophages after TLR4 stimulation leading to increased glycolytic metabolism and inflammatory IL-6 and TNF-α production. AIF1 also suppressed calcium uptake in the mitochondria to limit mitochondrial metabolism and anti-inflammatory IL-10 production. Conclusion: These data newly reveal a proinflammatory role for AIF1 in the infarcted heart through inflammatory glycolytic reprogramming of macrophages, which required both calcium mobilization and actin polymerization. Targeted knockdown of AIF1 led to cardioprotection supporting further exploration of macrophage AIF1 as a therapeutic target after MI.

Inflammasomes-New Contributors to Blood Diseases.

Inflammasomes are intracellular multimeric complexes that cleave the precursors of the IL-1 family of cytokines and various proteins, found predominantly in cells of hematopoietic origin. They consist of pattern-recognition receptors, adaptor domains, and the enzymatic caspase-1 domain. Inflammasomes become activated upon stimulation by various exogenous and endogenous agents, subsequently promoting and enhancing inflammatory responses. To date, their function has been associated with numerous pathologies. Most recently, many studies have focused on inflammasomes’ contribution to hematological diseases. Due to aberrant expression levels, NLRP3, NLRP1, and NLRC4 inflammasomes were indicated as predominantly involved. The NLRP3 inflammasome correlated with the pathogenesis of non-Hodgkin lymphomas, multiple myeloma, acute myeloid leukemia, lymphoid leukemias, myelodysplastic neoplasms, graft-versus-host-disease, and sickle cell anemia. The NLRP1 inflammasome was associated with myeloma and chronic myeloid leukemia, whereas NLRC4 was associated with hemophagocytic lymphohistiocytosis. Moreover, specific gene variants of the inflammasomes were linked to disease susceptibility. Despite the incomplete understanding of these correlations and the lack of definite conclusions regarding the therapeutic utility of inflammasome inhibitors, the available results provide a valuable basis for clinical applications and precede upcoming breakthroughs in the field of innovative treatments. This review summarizes the latest knowledge on inflammasomes in hematological diseases, indicates the potential limitations of the current research approaches, and presents future perspectives.

Beta 2-Adrenergic Receptor in Circulating Cancer-Associated Cells Predicts for Increases in Stromal Macrophages in Circulation and Patient Survival in Metastatic Breast Cancer.

The usage of beta blockers in breast cancer (BC) patients is implicated in the reduction in distant metastases, cancer recurrence, and cancer mortality. Studies suggest that the adrenergic pathway is directly involved in sympathetic-driven hematopoietic activation of pro-tumor microenvironmental proliferation and tumor cell trafficking into the circulation. Cancer-associated macrophage-like cells (CAMLs) are pro-tumor polynucleated monocytic cells of hematopoietic origin emanating from tumors which may aid in circulating tumor cell (CTC) dissemination into the circulation. We examined the linkage between Beta-2 adrenergic receptor (B2AR) signaling in CAMLs and CTCs by establishing expression profiles in a model BC cell line (MDA-MB-231). We compared the model to CAMLs and CTCs found in patents. Although internalization events were observed in patients, differences were found in the expression of B2AR between the tumor cell lines and the CAMLs found in patients. High B2AR expression on patients’ CAMLs was correlated with significantly more CAMLs in the circulation (p = 0.0093), but CTCs had no numerical relationship (p = 0.1565). High B2AR CAML expression was also significantly associated with a larger size of CAMLs (p = 0.0073), as well as being significantly associated with shorter progression-free survival (p = 0.0097) and overall survival (p = 0.0265). These data suggest that B2AR expression on CAMLs is closely related to the activation, intravasation, and growth of CAMLs in the circulation.

Donor-But Not Recipient-Derived Cells Produce Collagen-1 in Chronically Rejected Cardiac Allografts.

Fibrosis is a prominent feature of chronic allograft rejection, caused by an excessive production of matrix proteins, including collagen-1. Several cell types produce collagen-1, including mesenchymal fibroblasts and cells of hematopoietic origin. Here, we sought to determine whether tissue-resident donor-derived cells or allograft-infiltrating recipient-derived cells are responsible for allograft fibrosis, and whether hematopoietic cells contribute to collagen production. A fully MHC-mismatched mouse heterotopic heart transplantation model was used, with transient depletion of CD4+ T cells to prevent acute rejection. Collagen-1 was selectively knocked out in recipients or donors. In addition, collagen-1 was specifically deleted in hematopoietic cells. Tissue-resident macrophages were depleted using anti-CSF1R antibody. Allograft fibrosis and inflammation were quantified 20 days post-transplantation. Selective collagen-1 knock-out in recipients or donors showed that tissue-resident cells from donor hearts, but not infiltrating recipient-derived cells, are responsible for production of collagen-1 in allografts. Cell-type-specific knock-out experiments showed that hematopoietic tissue-resident cells in donor hearts substantially contributed to graft fibrosis. Tissue resident macrophages, however, were not responsible for collagen-production, as their deletion worsened allograft fibrosis. Donor-derived cells including those of hematopoietic origin determine allograft fibrosis, making them attractive targets for organ preconditioning to improve long-term transplantation outcomes.

A bioinformatics approach to investigate serum and hematopoietic cell-specific therapeutic microRNAs targeting the 3' UTRs of all four Dengue virus serotypes.

Hyperendemic circulation of all four Dengue virus (DENV) serotypes is a severe global public health problem, so any vaccine or therapeutics should be able to target all four of them. Cells of hemopoietic origin are believed to be primary sites of DENV replication. This study aimed to identify potential host miRNAs that target 3' UTR of all four DENV serotypes, thereby directly regulating viral gene expression or indirectly modulating the host system at different virus infection steps. We used four prediction algorithms viz. miRanda, RNA22, RNAhybrid and StarMir for predicting miRNA, targeting 3'UTR of all four DENV serotypes. Statistically, the most significant miRNA targets were screened based on their Log10 P-value (> 0.0001) of Gene Ontology (GO) term and Kyoto Encyclopaedia of Gene and Genome (KEGG) pathway enrichment analysis. The intersection test of at least three prediction tools identified a total of 30 miRNAs, which could bind to 3'UTR of all four DENV serotypes. Of the 30, eight miRNAs were of hematopoietic cell origin. GO term enrichment and KEGG analysis showed four hemopoietic origin miRNAs target genes of the biological processes mainly involved in the innate immune response, mRNA 3'-end processing, antigen processing and presentation and nuclear-transcribed mRNA catabolic process.

194 Phospholipase Cγ2 is Essential in Autoantibody-induced Skin Blistering

Phospholipase Cγ2 (PLCγ2) is a signaling molecule of tyrosine kinase-coupled receptors in cells of hematopoietic origin. Gain-of-function mutations of PLCγ2 have been reported to cause a complex syndrome involving inflammation and bullous skin eruptions in both mice and humans. Previously we showed that PLCγ2 deficient mice remained protected in an autoimmune skin blistering model induced by the passive transfer of autoantibodies against collagen type VII (C7), which is a key anchoring fibril in the dermal-epidermal junction.

Fluorescence-Based Real-Time Analysis of Osteoclast Development.

Osteoclasts are multinucleated cells of hematopoietic origin which are critically involved in physiological and pathological bone resorption. They develop from myeloid progenitors through characteristic gene expression changes and intercellular fusion. This process is directed by M-CSF and RANKL which are also able to trigger osteoclast development from bone marrow cells in vitro. Osteoclasts are conventionally visualized by histochemical staining followed by manual counting, which hinders kinetic studies and automated quantification. Here we describe two fluorescence-based assays for the real-time analysis of myeloid cell to osteoclast development (FRAMCO) in primary mouse bone marrow cell cultures. Both assays rely on red-to-green fluorescence conversion of the membrane-targeted tdTomato/membrane-targeted eGFP (mTmG) transgene by Cre recombinase driven by the osteoclast-specific cathepsin K promoter (Ctsk-Cre). In the first assay (FRAMCO1.1), osteoclast-specific gene expression triggers red-to-green color conversion of cells carrying both the Ctsk-Cre and mTmG transgenes. In the second assay (FRAMCO1.2), red-to-green fluorescence conversion is triggered by fusion of neighboring co-cultured bone marrow cells separately carrying either the Ctsk-Cre or the mTmG transgenes. The two assays were tested using a high-content confocal fluorescence imaging system, followed by automated quantification. The FRAMCO1.1 assay showed robust red-to-green fluorescence conversion of more than 50% of the culture (including mononuclear cells) within 3 days under osteoclastogenic conditions. The FRAMCO1.2 assay showed a less robust but still readily measurable red-to-green color conversion in multinuclear cells within 5 days of differentiation. The assays required both the Ctsk-Cre and the mTmG transgenes and gave no signals in parallel macrophage cultures. The proper functioning of the two assays was also confirmed at the DNA, mRNA and bulk protein level. The assay systems were validated using lisophosphatidylcholine, a previously reported inhibitor of preosteoclast fusion. Taken together, our assays allow high-throughput automated real-time analysis of two critical aspects of osteoclast development, facilitating the screening for novel drug candidates for the pharmacological control of osteoclast-mediated bone resorption.

Abstract 1649: Mechanistic understanding of HPK1 inhibition on enhanced human T cell activation and tumor immunity in a syngeneic model

Abstract Background: HPK1, a member of the MAP4K family of protein serine/threonine kinases, is involved in negatively regulating signal transduction cascades in cells of hematopoietic origin. Recent data generated using potent and highly selective HPK1 inhibitors underscores the role of HPK1 in negatively regulating T, B, and dendritic cell activation and thus orchestrating a comprehensive anti-tumor immune response. In the present study, we seek to further understand the biological effects of HPK1 inhibition in primary human T cells and in syngeneic models. Methods: RNA sequencing and multiplex cytokine analyses were utilized to profile both primary human T cells and the MC38 syngeneic model. In vivo efficacy, target engagement and pharmacodynamic data were generated using murine syngeneic tumor models. Results: In vitro, HPK1 small molecule inhibition resulted in enhanced cytokine production in primary human T cells. In vivo, HPK1 inhibition abrogated T cell receptor-stimulated phospho-SLP-76, enhanced cytokine production, and mediated robust tumor growth inhibition in a murine syngeneic tumor model. Conclusion: Pharmacological blockade of HPK1 kinase activity represents a novel and potentially valuable immunomodulatory approach for anti-tumor immunity. Citation Format: David Ciccone, Vad Lazari, Ian Linney, Michael Briggs, Samantha Carreiro, Christine Loh, Peter Tummino, Joshua McElwee, Alan Collis, Neelu Kaila. Mechanistic understanding of HPK1 inhibition on enhanced human T cell activation and tumor immunity in a syngeneic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1649.

Abstract 1760: Role of Batf-dependant Th17 immune response in PTEN-null mouse model

Abstract Basic leucine zipper transcription factor ATF-like (BATF) belongs to the activator protein 1 (AP-1) family of transcription factors and has been shown to be predominantly expressed in cells of hematopoietic origin, especially in T cells. Mice deficient for BATF were first described to lack T helper cell type 17 (Th17). Th17 cells are a distinct lineage of CD4+ T helper cells that provide protective immunity to infections but are also involved in chronic inflammation and autoimmunity. However, the role of Th17 response in prostate cancer remains controversial. In this study, we took a genetic approach to explore the role of BATF-dependent Th17 cells in prostate cancers by interbreeding BATF-deficient (Batf-/-) mice with mice that are conditionally mutant for PTEN, an established preclinical model for prostate cancer. Batf+/+;Pten-/- and Batf+/-;Pten-/- mice presented similar prostate phenotypes thus were put into one group (named Batf+), which were compared to the Batf-/-;Pten-/- group (named Batf-). We found that Batf- prostates were clearly smaller than Batf+ prostates at 30 weeks. The genitourinary bloc weight was significantly heavier in Batf+ mice than in Batf- mice at 12 and 30 weeks. Batf+ mice developed significantly higher percent of invasive adenocarcinomas in different prostate lobes at 9, 12, and 30 weeks, respectively compared to Batf- mice. Tumors in Batf- mice exhibited lower rates of cellular proliferation and higher apoptosis, as well as reduced inflammatory cell infiltration and angiogenesis in the prostatic stroma. Moreover, Batf- mice exhibited significantly reduced mRNA and protein levels of Th17 cytokines, AP-1 family members and IL-23R, and reduced NF-kB pathway activity in both spleen and prostate tissues. CD4+ T cells from Batf- mice cultured under Th17 conditions for 4 days, expressed reduced AP-1 family-regulated genes compared to Batf+ mice. Prostate cancer cells (LNCaP) with overexpression of BATF in vitro exhibited increased IL-23R expression. Prostate tissues from Batf- mice cultured under IL-6 or Th17 conditions for 4 days had reduced IL-23R expression compared to that from Batf+ mice. Taken together, our results suggest that Batf promotes the initiation and progression of prostate adenocarcinoma via Th17-mediated inflammatory immune responses and that the Batf-IL-23-IL-17 axis is a critical pathway that is needed for prostate adenocarcinoma development and progression. This work was supported by the NIH-National Institute of General Medical Sciences COBRE on Aging and Regenerative Medicine (P20GM103629, PD: S. Michal Jazwinski; PI: Q.Z.) and the Carol Lavin Bernick Faculty Grant from Tulane University (PI: Q.Z.). Citation Format: Qiuyang Zhang, Sen Liu, Bing Zhang. Role of Batf-dependant Th17 immune response in PTEN-null mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1760.

Wave-Complex-Dependent Actin Dynamics Controls Hematopoietic Stem Cell Bone Marrow Retention and Orderly Differentiation into Mature Hematopoietic Lineages

BackgroundSpatiotemporally controlled actin dynamics are crucial for a vast number of cellular functions including migration, proliferation, attachment or immunological synapse formation. Environmental signals activate cognate surface receptors which in turn engage specific small GTPases. Signal integrators (e.g. WASP, WAVE complex or WIP) propagate the signal to Arp2/3 complex and lead to immediate actin polymerization. Here we show that disruption of one of the actin polymerization pathways through specific targeting of WAVE complex disturbs hematopoietic stem and progenitor cells (HSPC) bone marrow retention and their sequential differentiation towards mature blood cells.MethodsWe made use of the recently described mouse model of Hem1 ablation. Hem1 is a strictly hematopoietic specific component of WAVE complex; its loss disrupts the entire WAVE complex exclusively in cells of hematopoietic origin. An array of in vitro assays was employed to test cell intrinsic effects of Hem1 ablation on actin polymerization dependent cellular functions. In vivo transplantation experiments were performed to study the impact of Hem1 ablation on immature and in consequence on mature hematopoiesis.ResultsHem1-/- HSPC display a bone marrow (BM) retention defect evidenced by strongly elevated numbers of circulating HSPC and 50% reduced BM cellularity. In line with that, migration towards CXCL12, a key BM anchor for HSPC, was completely abrogated in Hem1-/- HSPC. Attachment to feeder cells was also decreased by 35% compared to WT HSPC, although stress fiber formation upon CXCL12 stimulation or general ability to polarize was not affected. Next we tested whether this cellular phenotype had any impact on HSPC performance in vivo. Homing, the initial interaction of transplanted HSPC with BM environment, was not affected by Hem1 ablation, while engraftment, the ability to repopulate all mature hematopoietic lineages, was strongly diminished. Platelet and RBC engraftment was delayed, whereas Hem1-/- neutrophils did not engraft at all (Figure 1). Hem1-/- mice presented with chronic inflammation, hyperleukocytosis and elevated pro-inflammatory cytokine plasma levels. To rule out inflammation as causal for numeric and functional Hem1-/- HSPC depletion we performed co-transplantation experiments. Hem1-/- or WT BM was transplanted in a four-fold excess over GFP+ competitor BM. In Hem1-/- transplanted recipients, already 4 weeks after transplantation 60 to 90% of both myeloid and lymphoid compartments were GFP+, whereas, as expected, in the control experiment WT BM out-competed GFP+ BM over time. A similar picture emerged in secondary recipients. Again Hem1-/- BM completely lost against, whereas WT BM prevailed over GFP+ competitor. We furthermore confirmed that the Hem1-/- HSPC cell intrinsic defect is already apparent during embryonic development: As in BM of adult Hem1-/- mice, HSPC content in E14.5 old fetal livers (FL) was reduced by half compared to WT littermates. Also the competitiveness of FL HSPC was strongly impaired, similar to Hem1-/- adult BM HSPC. Whereas short term engraftment was provided by more mature competitor GFP+ BM in both recipients groups, already 4 weeks after transplantation, as expected, WT FL derived HSPC took over blood cell production and completely outcompeted GFP+ BM. In Hem1-/- FL recipients, by contrast, the contribution of competitor GFP+ BM to hematopoiesis remained high for the whole period tested without significant contribution of Hem1-/- FL hematopoiesis (Figure 2). Taken together, Hem1 (and, by extension, WAVE complex) governs HSPC BM retention and proper engraftment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Immunoproteasome Function in Normal and Malignant Hematopoiesis.

The ubiquitin–proteasome system (UPS) is a central part of protein homeostasis, degrading not only misfolded or oxidized proteins but also proteins with essential functions. The fact that a healthy hematopoietic system relies on the regulation of protein homeostasis and that alterations in the UPS can lead to malignant transformation makes the UPS an attractive therapeutic target for the treatment of hematologic malignancies. Herein, inhibitors of the proteasome, the last and most important component of the UPS enzymatic cascade, have been approved for the treatment of these malignancies. However, their use has been associated with side effects, drug resistance, and relapse. Inhibitors of the immunoproteasome, a proteasomal variant constitutively expressed in the cells of hematopoietic origin, could potentially overcome the encountered problems of non-selective proteasome inhibition. Immunoproteasome inhibitors have demonstrated their efficacy and safety against inflammatory and autoimmune diseases, even though their development for the treatment of hematologic malignancies is still in the early phases. Various immunoproteasome inhibitors have shown promising preliminary results in pre-clinical studies, and one inhibitor is currently being investigated in clinical trials for the treatment of multiple myeloma. Here, we will review data on immunoproteasome function and inhibition in hematopoietic cells and hematologic cancers.

Balancing risk in a patient with primary bone histiocytic sarcoma during pregnancy: Case report and review of the literature.

Introduction and importanceHistiocytic sarcoma (HS) is a rare, aggressive malignant neoplasm of hematopoietic cell origin. Primary HS of the proximal humerus, without involvement of lymph nodes or bone marrow, or systemic features, is very rare.Case presentationWe report a rare case of primary bony HS of the proximal humerus without bone marrow involvement in a healthy 33-year-old pregnant woman. She was successfully treated with surgical resection during pregnancy and radiotherapy post-delivery.Clinical discussionThis is the first report of a patient with primary bony HS during pregnancy. This highlights the fact that although HS is a neoplasm of hemolymphoid cell lineage, it frequently arises in non-lymphoid organs.ConclusionThis case emphasizes the importance of a multidisciplinary approach and the need for balancing treatment risk in a patient with primary bone HS during pregnancy.

Hematopoietic Wnts Modulate Endochondral Ossification During Fracture Healing.

Wnt signaling plays a critical role in bone formation, homeostasis, and injury repair. Multiple cell types in bone have been proposed to produce the Wnts required for these processes. The specific role of Wnts produced from cells of hematopoietic origin has not been previously characterized. Here, we examined if hematopoietic Wnts play a role in physiological musculoskeletal development and in fracture healing. Wnt secretion from hematopoietic cells was blocked by genetic knockout of the essential Wnt modifying enzyme PORCN, achieved by crossing Vav-Cre transgenic mice with Porcnflox mice. Knockout mice were compared with their wild-type littermates for musculoskeletal development including bone quantity and quality at maturation. Fracture healing including callus quality and quantity was assessed in a diaphyseal fracture model using quantitative micro computer-assisted tomographic scans, histological analysis, as well as biomechanical torsional and 4-point bending stress tests. The hematopoietic Porcn knockout mice had normal musculoskeletal development, with normal bone quantity and quality on micro-CT scans of the vertebrae. They also had normal gross skeletal dimensions and normal bone strength. Hematopoietic Wnt depletion in the healing fracture resulted in fewer osteoclasts in the fracture callus, with a resultant delay in callus remodeling. All calluses eventually progressed to full maturation. Hematopoietic Wnts, while not essential, modulate osteoclast numbers during fracture healing. These osteoclasts participate in callus maturation and remodeling. This demonstrates the importance of diverse Wnt sources in bone repair.

Synthesis and Biochemical Evaluation of Warhead-Decorated Psoralens as (Immuno)Proteasome Inhibitors.

The immunoproteasome is a multicatalytic protease that is predominantly expressed in cells of hematopoietic origin. Its elevated expression has been associated with autoimmune diseases, various types of cancer, and inflammatory diseases. Selective inhibition of its catalytic activities is therefore a viable approach for the treatment of these diseases. However, the development of immunoproteasome-selective inhibitors with non-peptidic scaffolds remains a challenging task. We previously reported 7H-furo[3,2-g]chromen-7-one (psoralen)-based compounds with an oxathiazolone warhead as selective inhibitors of the chymotrypsin-like (β5i) subunit of immunoproteasome. Here, we describe the influence of the electrophilic warhead variations at position 3 of the psoralen core on the inhibitory potencies. Despite mapping the chemical space with different warheads, all compounds showed decreased inhibition of the β5i subunit of immunoproteasome in comparison to the parent oxathiazolone-based compound. Although suboptimal, these results provide crucial information about structure–activity relationships that will serve as guidance for the further design of (immuno)proteasome inhibitors.

TAT-Beclin-1 induces severe synovial hyperplasia and does not protect from injury-induced osteoarthritis in mice

Autophagy maintains cartilage homeostasis and is compromised during osteoarthritis (OA), contributing to cartilage degeneration. We sought to determine if D-isomer TAT-Beclin-1, a potent inducer of autophagy, could attenuate post-traumatic OA in mice. 10-week-old mice underwent destabilization of the medial meniscus (DMM) surgery to induce post-traumatic OA, or sham surgery (control), and injected intra-articularly with D-isomer TAT-Beclin-1 (0.5-2mg/kg) or PBS 1 week post-surgery for up to 9 weeks. Mice were sacrificed at 2 or 10 weeks post-surgery. Knee joint sections were evaluated by histopathology for cartilage degeneration and synovitis, and immunostaining for key markers of autophagy (LC3B), cell proliferation (nuclear Ki67), activated fibroblasts (αSMA), and cells of hematopoietic origin (CD45). All D-isomer TAT-Beclin-1-treated DMM mice had no difference in the degree of cartilage degeneration compared to PBS-injected DMM mice. Surprisingly, all D-isomer TAT-Beclin-1-treated mice exhibited substantial synovial hyperplasia, with increased cellularity and ECM deposition (fibrosis-like phenotype), as compared to PBS-injected mice. Synovial effects of D-isomer TAT-Beclin-1 were dose- and injection frequency-dependent. An increased percentage of cells positive for LC3B and nuclear Ki67 were found in the synovial intima early after injection, which persisted after frequent injections. D-isomer TAT-Beclin-1 did not attenuate cartilage degeneration, but rather induced synovial hyperplasia associated with increased expression of key markers of autophagy and cell proliferation and a fibrosis-like phenotype, independent of markers of fibroblast activation or persistent hematopoietic-origin cell infiltration. These data suggest that, if not tissue-targeted, caution should be taken using autophagy activators due to diverse cellular responses in the joint.