IL-1 Mediates Microbiome-Induced Inflamm-Ageing of Hematopoietic Stem Cells in Mice

Ageing is associated with impaired hematopoietic and immune function. This is caused in part by decreased hematopoietic stem cell (HSC) population fitness and an increased myeloid differentiation bias. The reasons for this aging-associated HSC impairment are incompletely understood. We here demonstrate that aged specific pathogen free (SPF) wild-type mice in contrast to young SPF mice produce more IL-1a/b in steady-state bone marrow (BM), with most of IL-1a/b being derived from myeloid BM cells. Further, blood of steady-state aged SPF wild-type mice contains higher levels of microbe associated molecular patterns (MAMPs), specifically TLR4 and TLR8 ligands. Also, BM myeloid cells from aged mice produce more IL-1b in vitro, and aged mice show higher and more durable IL-1a/b responses upon LPS stimulation in vivo. To test if HSC ageing is driven via IL-1a/b, we evaluated HSCs from IL-1 receptor 1 (IL-1R1) knock-out mice. Indeed, aged HSCs from IL-1R1 knock-out mice show significantly mitigated ageing-associated inflammatory signatures. Moreover, HSCs from aged IL-1R1KO and also from germ-free mice maintain unbiased lympho-myeloid hematopoietic differentiation upon transplantation, thus resembling this functionality of young HSCs. Importantly, in vivo antibiotic suppression of microbiota or pharmacologic blockade of IL-1 signaling in aged wild-type mice was similarly sufficient to reverse myeloid biased output of their HSC populations. Collectively, our data defines the microbiome-IL-1/IL-1R1 axis as a key, self-sustaining, but also therapeutically partially reversible driver of HSC inflamm-ageing.

Download Full-text

Disturbed Endothelial Blood-Bone Marrow-Barrier In Nox4 Deficient Mice

Blood ◽

10.1182/blood.v122.21.1169.1169 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1169-1169

Author(s):

Maren Weisser ◽

Kerstin B. Kaufmann ◽

Tomer Itkin ◽

Linping Chen-Wichmann ◽

Tsvee Lapidot ◽

...

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Stem Cell ◽

Steady State ◽

Wild Type ◽

Hematopoietic Stem ◽

Knock Out ◽

Deficient Mice ◽

Permeability State

Abstract Reactive oxygen species (ROS) have been implicated in the regulation of stemness of hematopoietic stem cells (HSC). HSC with long-term repopulating capabilities are characterized by low ROS levels, whereas increased ROS levels correlate with lineage specification and differentiation. Several tightly regulated sources of ROS production are well known among which are the NADPH oxidases (Nox). HSC are known to express Nox1, Nox2 and Nox4, however, their role in maintenance of stem cell potential or in the activation of differentiation programs are poorly understood. While Nox2 is activated in response to various extrinsic and intrinsic stimuli, mainly during infection and inflammation, Nox4 is constitutively active and is considered to be responsible for steady-state ROS production. Consequently, Nox4 deficiency might lower ROS levels at steady-state hematopoiesis and thereby could have an impact on HSC physiology. In this work we studied HSC homeostasis in Nox4 knock-out mice. Analysis of the hematopoietic stem and progenitor cell (HSPC) pool in the bone marrow (BM) revealed no significant differences in the levels of Lineage marker negative (Lin-) Sca-1+ ckit+ (LSK) and LSK-SLAM (LSK CD150+ CD48-) cells in Nox4 deficient mice compared to wild type (WT) C57BL/6J mice. HSPC frequency upon primary and secondary BM transplantation was comparable between Nox4 deficient and WT mice. In addition, the frequency of colony forming cells in the BM under steady-state conditions did not differ between both mouse groups. However, Nox4 deficient mice possess more functional HSCs as observed in in vivo competitive repopulating unit (CRU) assays. Lin- cells derived from Nox4 knock out (KO) mice showed an increased CRU frequency and superior multilineage engraftment upon secondary transplantation. Surprisingly, ROS levels in different HSPC subsets of NOX4 KO mice were comparable to WT cells, implying that the absence of Nox4 in HSCs does not have a major intrinsic impact on HSC physiology via ROS. Therefore, the increased levels of functional HSCs observed in our studies may suggest a contribution of the BM microenvironment to steady-state hematopoiesis in the BM of Nox4 KO animals. Recent observations suggest a regulation of the BM stem cell pool by BM endothelial cells, in particular by the permeability state of the blood-bone marrow-barrier (Itkin T et al., ASH Annual Meeting Abstracts, 2012). Endothelial cells interact with HSCs predominantly via paracrine effects and control stem cell retention, egress and homing as well as stem cell activation. As Nox4 is highly expressed in endothelial cells and is involved in angiogenesis, we reasoned that the absence of NOX4 could affect HSC homeostasis through altered BM endothelium properties and barrier permeability state. Indeed, in preliminary assays we found reduced short-term homing of BM mononuclear cells into the BM of Nox4 deficient mice as compared to wild type hosts. Furthermore, in vivo administration of Evans Blue dye revealed reduced dye penetration into Nox4-/- BM compared to wild type mice upon intravenous injection. Taken together, these data indicate a reduced endothelial permeability in Nox4 KO mice. Ongoing experiments aim at further characterization of the Nox4-/- phenotype in BM sinusoidal and arteriolar endothelial cells, the impact of Nox4 deletion on BM hematopoietic and mesenchymal stem cells, and in deciphering the role of Nox4 in the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Changes in Liver Gene Expression of Azin1 Knock-out Mice

Zeitschrift für Naturforschung C ◽

10.1515/znc-2010-7-816 ◽

2010 ◽

Vol 65 (7-8) ◽

pp. 519-527 ◽

Cited By ~ 3

Author(s):

Tao Wan ◽

Yuan Hu ◽

Ailong Huang ◽

Ken-ichi Yamamura ◽

Hua Tang

Keyword(s):

Ornithine Decarboxylase ◽

Regulatory Protein ◽

Fold Change ◽

Oligonucleotide Array ◽

Wild Type ◽

Polyamine Biosynthesis ◽

Knock Out ◽

Knock Out Mice

The ornithine decarboxylase antizyme inhibitor (AZI) was discovered as a protein that binds to the regulatory protein antizyme and inhibits the ability of antizyme to interact with the enzyme ornithine decarboxylase (ODC). Several studies showed that the AZI protein is important for cell growth in vitro. However, the function of this gene in vivo remained unclear. In our study, we analyzed the transcriptional profiles of livers on the 19th day of pregnancy of Azin1 knock-out mice and wild-type mice using the Agilent oligonucleotide array. Compared to the wild-type mice, in the liver of Azin1 knock-out mice 1812 upregulated genes (fold change ≥ 2) and 1466 downregulated genes (fold change ≤ 0.5) were showed in the microarray data. Altered genes were then assigned to functional categories and mapped to signaling pathways. These genes have functions such as regulation of the metabolism, transcription and translation, polyamine biosynthesis, embryonic morphogenesis, regulation of cell cycle and proliferation signal transduction cascades, immune response and apoptosis. Real-time PCR was used to confirm the differential expression of some selected genes. Overall, our study provides novel understanding of the biological functions of AZI in vivo.

Download Full-text

Negative Regulation by Interleukin-3 (IL-3) of Mouse Early B-Cell Progenitors and Stem Cells in Culture: Transduction of the Negative Signals by βc and βIL-3 Proteins of IL-3 Receptor and Absence of Negative Regulation by Granulocyte-Macrophage Colony-Stimulating Factor

Blood ◽

10.1182/blood.v92.3.901.415a25_901_907 ◽

1998 ◽

Vol 92 (3) ◽

pp. 901-907 ◽

Cited By ~ 3

Author(s):

Takuya Matsunaga ◽

Fumiya Hirayama ◽

Yuji Yonemura ◽

Richard Murray ◽

Makio Ogawa

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cultured Cells ◽

Negative Regulation ◽

Wild Type ◽

Hematopoietic Stem ◽

Knock Out ◽

Knock Out Mice ◽

Marrow Cells

The receptors for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5 share a common signaling subunit βc. However, in the mouse, there is an additional IL-3 signaling protein, βIL-3, which is specific for IL-3. We have previously reported that IL-3 abrogates the lymphoid potentials of murine lymphohematopoietic progenitors and the reconstituting ability of hematopoietic stem cells. We used bone marrow cells from βc- and βIL-3–knock-out mice to examine the relative contributions of the receptor proteins to the negative regulation by IL-3. First, we tested the effects of IL-3 on lymphohematopoietic progenitors by using lineage-negative (Lin−) marrow cells of 5-fluorouracil (5-FU)-treated mice in the two-step methylcellulose culture we reported previously. Addition of IL-3 to the combination of steel factor (SF, c-kit ligand) and IL-11 abrogated the B-lymphoid potential of the marrow cells of both types of knock-out mice as well as wild-type mice. Next, we investigated the effects of IL-3 on in vitro expansion of the hematopoietic stem cells. We cultured Lin−Sca-1–positive, c-kit–positive marrow cells from 5-FU–treated mice in suspension in the presence of SF and IL-11 with or without IL-3 for 7 days and tested the reconstituting ability of the cultured cells by transplanting the cells into lethally irradiated Ly-5 congenic mice together with “compromised” marrow cells. Presence of IL-3 in culture abrogated the reconstituting ability of the cells from both types of knock-out mice and the wild-type mice. In contrast, addition of GM-CSF to the suspension culture abrogated neither B-cell potential nor reconstituting abilities of the cultured cells of wild-type mice. These observations may have implications in the choice of cytokines for use in in vitro expansion of human hematopoietic stem cells and progenitors. © 1998 by The American Society of Hematology.

Download Full-text

Negative Regulation by Interleukin-3 (IL-3) of Mouse Early B-Cell Progenitors and Stem Cells in Culture: Transduction of the Negative Signals by βc and βIL-3 Proteins of IL-3 Receptor and Absence of Negative Regulation by Granulocyte-Macrophage Colony-Stimulating Factor

Blood ◽

10.1182/blood.v92.3.901 ◽

1998 ◽

Vol 92 (3) ◽

pp. 901-907 ◽

Cited By ~ 29

Author(s):

Takuya Matsunaga ◽

Fumiya Hirayama ◽

Yuji Yonemura ◽

Richard Murray ◽

Makio Ogawa

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cultured Cells ◽

Negative Regulation ◽

Wild Type ◽

Hematopoietic Stem ◽

Knock Out ◽

Knock Out Mice ◽

Marrow Cells

Abstract The receptors for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5 share a common signaling subunit βc. However, in the mouse, there is an additional IL-3 signaling protein, βIL-3, which is specific for IL-3. We have previously reported that IL-3 abrogates the lymphoid potentials of murine lymphohematopoietic progenitors and the reconstituting ability of hematopoietic stem cells. We used bone marrow cells from βc- and βIL-3–knock-out mice to examine the relative contributions of the receptor proteins to the negative regulation by IL-3. First, we tested the effects of IL-3 on lymphohematopoietic progenitors by using lineage-negative (Lin−) marrow cells of 5-fluorouracil (5-FU)-treated mice in the two-step methylcellulose culture we reported previously. Addition of IL-3 to the combination of steel factor (SF, c-kit ligand) and IL-11 abrogated the B-lymphoid potential of the marrow cells of both types of knock-out mice as well as wild-type mice. Next, we investigated the effects of IL-3 on in vitro expansion of the hematopoietic stem cells. We cultured Lin−Sca-1–positive, c-kit–positive marrow cells from 5-FU–treated mice in suspension in the presence of SF and IL-11 with or without IL-3 for 7 days and tested the reconstituting ability of the cultured cells by transplanting the cells into lethally irradiated Ly-5 congenic mice together with “compromised” marrow cells. Presence of IL-3 in culture abrogated the reconstituting ability of the cells from both types of knock-out mice and the wild-type mice. In contrast, addition of GM-CSF to the suspension culture abrogated neither B-cell potential nor reconstituting abilities of the cultured cells of wild-type mice. These observations may have implications in the choice of cytokines for use in in vitro expansion of human hematopoietic stem cells and progenitors. © 1998 by The American Society of Hematology.

Download Full-text

FcγRIII (CD16)-Deficient Mice Show IgG Isotype-Dependent Protection to Experimental Autoimmune Hemolytic Anemia

Blood ◽

10.1182/blood.v92.11.3997 ◽

1998 ◽

Vol 92 (11) ◽

pp. 3997-4002 ◽

Cited By ~ 92

Author(s):

Dirk Meyer ◽

Carsten Schiller ◽

Jürgen Westermann ◽

Shozo Izui ◽

Wouter L. W. Hazenbos ◽

...

Keyword(s):

Hemolytic Anemia ◽

Autoimmune Hemolytic Anemia ◽

Knock Out ◽

Effector Cells ◽

Igg Isotypes ◽

Knock Out Mice ◽

Deficient Mice ◽

Experimental Autoimmune

Abstract In autoimmune hemolytic anemia (AIHA), there is accumulating evidence for an involvement of FcγR expressed by phagocytic effector cells, but demonstration of a causal relationship between individual FcγRs and IgG isotypes for disease development is lacking. Although the relevance of IgG isotypes to human AIHA is limited, we could show a clear IgG isotype dependency in murine AIHA using pathogenic IgG1 (105-2H) and IgG2a (34-3C) autoreactive anti–red blood cell antibodies in mice defective for FcγRIII, and comparing the clinical outcome to those in wild-type mice. FcγRIII-deficient mice were completely resistent to the pathogenic effects of 105-2H monoclonal antibody, as shown by a lack of IgG1-mediated erythrophagocytosis in vitro and in vivo. In addition, the IgG2a response by 34-3C induced a less severe but persistent AIHA in FcγRIII knock-out mice, as documented by a decrease in hematocrit. Blocking studies indicated that the residual anemic phenotype induced by 34-3C in the absence of FcγRIII reflects an activation of FcγRI that is normally coexpressed with FcγRIII on macrophages. Together these results show that the pathogenesis of AIHA through IgG1-dependent erythrophagocytosis is exclusively mediated by FcγRIII and further suggest that FcγRI, in addition to FcγRIII, contributes to this autoimmune disease when other IgG isotypes such as IgG2a are involved.

Download Full-text

Knock out of sHSP genes determines some modifications in the probiotic attitude of Lactiplantibacillus plantarum

Biotechnology Letters ◽

10.1007/s10529-020-03041-6 ◽

2020 ◽

Author(s):

Angela Longo ◽

Pasquale Russo ◽

Vittorio Capozzi ◽

Giuseppe Spano ◽

Daniela Fiocco

Keyword(s):

Small Heat Shock Protein ◽

Cellular Adhesion ◽

Probiotic Properties ◽

Wild Type ◽

Knock Out ◽

Human Enterocyte ◽

Transcriptional Induction ◽

Pro Inflammatory Cytokines

Abstract Objective We investigated whether the knock out of small heat shock protein (sHSP) genes (hsp1, hsp2 and hsp3) impact on probiotic features of Lactiplantibacillus plantarum WCFS1, aiming to find specific microbial effectors involved in microbe-host interplay. Results The probiotic properties of L. plantarum WCFS1 wild type, hsp1, hsp2 and hsp3 mutant clones were evaluated and compared through in vitro trials. Oro-gastro-intestinal assays pointed to significantly lower survival for hsp1 and hsp2 mutants under stomach-like conditions, and for hsp3 mutant under intestinal stress. Adhesion to human enterocyte-like cells was similar for all clones, though the hsp2 mutant exhibited higher adhesiveness. L. plantarum cells attenuated the transcriptional induction of pro-inflammatory cytokines on lipopolysaccharide-treated human macrophages, with some exception for the hsp1 mutant. Intriguingly, this clone also induced a higher IL10/IL12 ratio, which is assumed to indicate the anti-inflammatory potential of probiotics. Conclusions sHSP genes deletion determined some differences in gut stress resistance, cellular adhesion and immuno-modulation, also implying effects on in vivo interaction with the host. HSP1 might contribute to immunomodulatory mechanisms, though additional experiments are necessary to test this feature.

Download Full-text

The Rho Family GTPase Cdc42 Regulates Multiple Hematopoietic Stem/Progenitor Cell Functions and Erythropoiesis.

Blood ◽

10.1182/blood.v104.11.32.32 ◽

2004 ◽

Vol 104 (11) ◽

pp. 32-32

Author(s):

Lei Wang ◽

Linda Yang ◽

Marie–Dominique Filippi ◽

David A. Williams ◽

Yi Zheng

Keyword(s):

Bone Marrow ◽

Fetal Liver ◽

Brdu Incorporation ◽

Low Density ◽

Actin Assembly ◽

Wild Type ◽

Hematopoietic Stem ◽

Rho Family

Abstract The Rho family GTPase Cdc42 has emerged as a key signal transducer in cell regulation. To investigate its physiologic function in hematopoiesis, we have generated mice carrying a gene targeted null allele of cdc42gap, a major negative regulatory gene of Cdc42 and mice with conditional targeted cdc42 allele (cdc42flox/flox). Deletion of the respective gene products in mice was confirmed by PCR genotyping and Western blotting. Low-density fetal liver or bone marrow cells from Cdc42GAP−/− mice displayed ~3 fold elevated Cdc42 activity and normal RhoA, Rac1 or Rac2 activity, indicating that cdc42gap deletion has a specific effect on Cdc42 activity. The Cdc42GAP-deficient hematopoietic stem/progenitor cells (HSC/Ps, Lin−c-Kit+) generated from Cdc42GAP−/− E14.5 fetal liver and the Cdc42−/− HSC/Ps derived by in vitro expression of Cre via a retrovirus vector from Cdc42flox/flox low density bone marrow showed a growth defect in liquid culture that was associated with increased apoptosis but normal cell cycle progression. Cdc42GAP-deficient HSC/Ps displayed impaired cortical F-actin assembly with extended actin protrusions upon exposure to SDF–1 in vitro and a punctuated actin structure after SCF stimulation while Cdc42−/− but not wild type HSC/Ps responded to SDF-1 in inducing membrane protrusions. Both Cdc42−/− and Cdc42GAP−/− HSC/Ps were markedly decreased in adhesion to fibronectin. Moreover, both Cdc42−/− and Cdc42GAP−/− HSC/Ps showed impaired migration in response to SDF-1. These results demonstrate that Cdc42 regulation is essential for multiple HSC/P functions. To understand the in vivo hematopoietic function of Cdc42, we have characterized the Cdc42GAP−/− mice further. The embryos and newborns of homozygous showed a ~30% reduction in hematopoietic organ (i.e. liver, bone marrow, thymus and spleen) cellularity, consistent with the reduced sizes of the animals. This was attributed to the increased spontaneous apoptosis associated with elevated Cdc42/JNK/Bid activities but not to a proliferative defect as revealed by in vivo TUNEL and BrdU incorporation assays. ~80% of Cdc42GAP−/− mice died one week after birth, and the surviving pups attained adulthood but were anemic. Whereas Cdc42GAP−/− mice contained small reduction in the frequency of HSC markers and normal CFU-G, CFU-M, and CFU-GM activities, the frequency of BFU-E and CFU-E were significantly reduced. These results suggest an important role of Cdc42 in erythropoiesis in vivo. Taken together, we propose that Cdc42 is essential for multiple HSC/P functions including survival, actin cytoskeleton regulation, adhesion and migration, and that deregulation of its activity can have a significant impact on erythropoiesis. Cdc42 regulates HSC/P functions and erythropoiesis Genotype/phenotype Apoptosis increase Adhesion decrease Migration decrease F-actin assembly HSC frequency decrease BFU-E, CFU-E decrease The numbers were indicated as fold difference compared with wild type. ND:not determined yet. Cdc42GAP−/− 2.43, p<0.005 0.97, p<0.01 1.01, p<0.01 protrusion (SDF-1); punctruated (SCF) 0.34, p<0.05 0.92, p<0.01; 0.38, p<0 Cdc42−/− 3.68, p<0.005 0.98, p<0.001 3.85, p<0.005 protrusion (SDF-1) ND ND

Download Full-text

Rhogtpase RAC2 Is Required for In Vivo Transformation Activity by P210 Bcr-Abl Fusion Oncogene.

Blood ◽

10.1182/blood.v104.11.717.717 ◽

2004 ◽

Vol 104 (11) ◽

pp. 717-717

Author(s):

Nithya Krishnan ◽

Jeff R. Bailey ◽

Victoria Summey-Harner ◽

Claudio Brunstein ◽

Catherine M. Verfaillie ◽

...

Keyword(s):

Mammalian Cells ◽

Philadelphia Chromosome ◽

Protein Domain ◽

Myelogenous Leukemia ◽

Wild Type ◽

Hematopoietic Stem ◽

Cell Functions ◽

Empty Vector

Abstract Bcr-Abl, the translocation product of the Philadelphia chromosome implicated in human chronic myelogenous leukemia (CML), is a kinase affecting hematopoietic stem cell (HSC) behavior with respect to proliferation, apoptosis, adhesion and migration. Rho GTPases, particularly the Rac subfamily, have been shown to regulate these same cell functions in normal HSC and also regulate gene expression in many mammalian cells. BCR contains a “GTPase-activating protein” domain and a guanine nucleotide exchange domain, the latter or which is preserved in p210 Bcr-Abl. Since HSC functions regulated by Bcr-Abl and Rac are similar, we studied the potential involvement of Rac activation in Bcr-Abl signaling cascade. Human CML samples demonstrate baseline activation of Rac proteins that is reversed by in vitro treatment with STI571. To study the specific involvement of Rac2, we used a gene targeted mouse model with Rac2 null bone marrow. Using retovirus-mediated gene transfer, we introduced p210 Bcr-Abl in the MSCV vector into wild-type or Rac2−/− HSC/P and studied the behavior of these cells in vitro and in vivo. Irradiated recipient mice injected with LDBM cells transduced with p210 developed a uniformly fatal myeloproliferative syndrome (Median survival: 45 days, N=12), while mice injected with p210 transduced Rac2−/− LDBM cells (N=12, 2 independent exp.) had 100% survival and no development of leukocytosis, splenomegaly or organ infiltration of hematopoietic cells. These data suggest that Rac GTPases are critical for the transformation of HSC by Bcr-Abl and provide an additional therapeutic target for intervention in CML. WILD TYPE Rac 2 −/− Empty Vector MSCV-p210 Empty vector MSCV-p210 *p < 0.01 vs WT-MIEG3, **p< 0.01 vs WT-p210 bcr-abl. Proliferation (CPM) Medium 562 ± 278 16,207± 1605* 819.7 ± 363 3,135.5 ± 498** SCF (100ng/ml) 856 ± 187 23,226 ± 2203* 853.7 ± 524 3,756.8 ± 207** Cytokines (SCF, GCSF, MGDF) 8011± 1412 42,711± 13393* 4833 ±1019 3,614.5 ± 1982** Migration (%) Fibronectin 7 ± 0.4 38 ± 1.9* 0.4 ± 0.0 0.8 ± 0.1** SDF-1α 30 ±2.8 13 ±1.1* 0.5 ± 0.0 0.6 ± 0.0** Adhesion (% ) Fibronectin 76± 2.9 40 ±3* 4 ±0.4 10 ±0.1 **

Download Full-text

Rac1 and Rac2 GTPases Play Distinct Roles and Are Essential for Full Osteoclast Differentiation.

Blood ◽

10.1182/blood.v106.11.67.67 ◽

2005 ◽

Vol 106 (11) ◽

pp. 67-67

Author(s):

Matti Korhonen ◽

Haibo Zhao ◽

Roberta Faccio ◽

F. Patrick Ross ◽

Tracy M. Hopkins ◽

...

Keyword(s):

Bone Mass ◽

Signaling Pathways ◽

Osteoclast Differentiation ◽

Integrin Subunit ◽

Hematopoietic Stem ◽

Knock Out ◽

Osteoclast Activity ◽

Rac Gtpases

Abstract Bone-resorbing osteoclasts play a central role in bone remodeling, which occurs throughout life. Many skeletal diseases such as osteoporosis, Paget’s disease and the lytic lesions of multiple myeloma, display excess osteoclast activity. Thus, in addition to basic biological questions, there is considerable clinical interest in the control of osteoclast differentiation and function. Previously we have demonstrated that the small GTPases Rac1 and Rac2 have specific roles in the control of hematopoietic stem cell and neutrophil functions (Gu and Filippi et al., Science 2003; Filippi et al., Nat Immunol 2004; Cancelas et al., Nat Med 2005). During these studies, we noted differences in the bone structure of Rac-deficient mice, suggesting alterations in osteoclast activity. Furthermore we found that in hematopoietic stem cells Rac proteins regulate signaling pathways that are also known to control osteoclastogenesis. In this study, we have employed a genetic approach to analyze the roles of Rac proteins in osteoclast differentiation. We utilized constitutively Rac2-null mice in combination with cre-induced deletion of floxed Rac1 sequences to effect the loss of both Rac GTPases in hematopoietic cells. Macrophages from Rac2−/− mice generated normal numbers of osteoclasts in vitro. However, the full differentiation of these cells, as assayed by emergence of differentiation markers, was perturbed. Expression the TRAP (tartrate-resistant acid phosphatase) enzyme was delayed (12 +/−3% vs. 88 +/−8%, Rac2−/− vs. wt, n= 5, p<0.001) and the expression of the β3 integrin subunit was decreased (16% vs. 76%, Rac2−/− vs. wt, n=5). The number of cells having podosomes was reduced (8 +/−3 vs. 206 +/−48 cells with podosomes/well Rac2−/− vs. wt, p<0.001). Cell fusion, which accompanies osteoclastogenesis, was also reduced. In contrast Rac1−/− macrophages produced severely reduced numbers of osteoclasts in vitro (13 +/−8/well vs. 272 +/−52 Rac1−/− vs. wt, n=2, p<0.001). Rac1−/−Rac2−/− double knock-out cells essentially developed no osteoclasts in vitro. The p44/42, JNK (jun N-terminal kinase), Akt and p38 intracellular kinase signaling pathways have all been shown to be important for osteoclastogenesis. Activation of the p44/42 and JNK (jun N-terminal kinase) pathways in response to stimulation with M-CSF (macrophage colony stimulating factor) and RANKL (receptor activator of NF-κB ligand), cytokines critically involved in osteoclast differentiation, was reduced in the Rac2−/− macrophages. When Rac1−/− cells were stimulated with M-CSF, decreased activation of the Akt and JNK pathways was observed. To study the effect of Rac deficiency on bone mass in vivo, we generated Rac1−/−Rac2−/− double knock-out mice. These mice had significantly increased bone mass (bone volume/tissue volume 0.33 +/−0.03 vs. 0.13 +/−0.02 Rac1−/−Rac2−/− vs. wild-type; p<0.001). These results indicate that 1) Rac GTPases are critical to the differentiation of macrophages into osteoclasts, 2) in the absence of Rac2 osteoclastogenesis is perturbed while inhibition of Rac1 function leads to nearly complete inhibition osteoclastogenesis, 3) specific alterations in intracellular signaling pathways are seen in Rac-deficient osteoclast precursors, and 4) inhibition of Rac function in vivo leads to an increase in bone mass.

Download Full-text

In Vitro and In Vivo Characterization of Full-Length rFVIII Modified with PEG Via Coupling to Primary Amino Groups.

Blood ◽

10.1182/blood.v110.11.3147.3147 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3147-3147 ◽

Cited By ~ 7

Author(s):

Peter L. Turecek ◽

Jürgen Siekmann ◽

Herbert Gritsch ◽

Katalin Váradi ◽

Rafi-Uddin Ahmad ◽

...

Keyword(s):

Half Life ◽

Hemophilia A ◽

Specific Activity ◽

Exchange Chromatography ◽

Full Length ◽

Thrombin Inhibitor ◽

Knock Out ◽

Knock Out Mice

Abstract Chemical modification of recombinant therapeutic proteins with PEG has been shown to enhance the biological half-life. Here we assess the effect of PEGylation on FVIII. Full-length rFVIII bulk drug substance from protein-free fermentation (Advate process, Baxter) was conditioned into a buffer suitable for coupling to polyethylene glycol succinimidyl succinate (linear PEG, 5 kDa PEG chain length). PEG was covalently bound by amine coupling preferentially to lysine residues of FVIII at neutral pH. PEG was removed by ion-exchange chromatography and the PEG-FVIII derivative was concentrated by ultra-diafiltration. The conjugates thus obtained retained about 30–40% of the activity of non-modified rFVIII. The specific activity decreased with the amount of PEG linked to the FVIII molecule. In SDS-PAGE and immunoblot studies PEGylated rFVIII showed a band pattern similar to unmodified FVIII with full-length, heavy chain fragments of 180 kDa and 120 kDa and the light chain fragment of 80 kDa. PEGylation also occurred to a high extent in the B domain of FVIII. All bands appeared broadened due to the attachment of polymeric PEG. The maintenance of functionality of FVIII was demonstrated by its potential to be activated and inactivated by thrombin. In the assay PEGylated and unmodified FVIII were incubated with 1 nM thrombin. Sub-samples were drawn at intervals up to 40 minutes and added to a mixture of FIXa, FX, phospholipid vesicles and Ca2+ containing a thrombin inhibitor. After 3 minutes incubation at 37°C the amount of activated FX (FXa) was measured using a FXa-specific chromogenic substrate. Unmodified rFVIII showed a typical picture of an immediate increase in FXa activity and a subsequent decline with no further FXa generation after 15 minutes. PEGylated rFVIII was activated to the same extent as unmodified FVIII but the decay in FXa generation was slower and did not reach the zero level, even 40 minutes after incubation. The formation of the typical thrombin cleavage fragments, with unmodified as well as PEGylated rFVIII, was demonstrated in a Western blot analysis. The slower inactivation by thrombin was also seen there. The pharmacokinetic properties of PEGylated rFVIII compared with rFVIII were investigated in hemophilia A knock-out mice. Both preparations were applied at a dose of 200 IU rFVIII/kg and groups of mice (n=5) were exsanguinated at several time points up to 24 hours. Terminal half-life for PEGylated rFVIII was calculated at 4.9 hours compared with 1.9 hours for unmodified rFVIII in hemophilia A knock-out mice. AUC was approximately doubled. These results indicate that rFVIII can be biochemically modified with PEG whilst at least partly retaining its major functions, but at the same time prolonging its survival in the circulation of hemophilic mice.

Download Full-text