Genetic architecture underlying IgG-RF production is distinct from that of IgM-RF.

OBJECTIVE: HLA-DRB1 alleles, particularly the shared epitope (SE) alleles, are strongly associated with RA. Different genetic structures underlie the production of the various autoantibodies in RA. While extensive genetic analyses have been conducted to generate a detailed profile of ACPA, a representative autoantibody in RA, the genetic architecture underlying subfractions of RF other than IgM-RF, namely IgG-RF, known to be associated with rheumatoid vasculitis, is not well understood. METHODS: We enrolled a total of 743 RA subjects whose detailed autoantibody (IgG-RF, IgM-RF, and ACPA) data were available. We evaluated co-presence and correlations of the levels of these autoantibodies. We analysed associations between the presence or levels of the autoantibodies and HLA-DRB1 alleles for the 743 RA patients and 2008 healthy controls. RESULTS: We found both IgG-RF(+) and IgG-RF(-) RA subjects showed comparable associations with SE alleles, which was not observed for the other autoantibodies. Furthermore, there was a clear difference in SE allele associations between IgG-RF(+) and (-) subsets: the association with the IgG-RF(+) subsets was solely driven by HLA-DRB1*04:05, the most frequent SE allele in the Japanese population, while not only HLA-DRB1*04:05 but also HLA-DRB1*04:01, less frequent in the Japanese population but the most frequent SE allele in Europeans, were main drivers of the association in the IgG-RF(-) subset. We confirmed that these associations were irrespective of ACPA presence. CONCLUSION: We found a unique genetic architecture for IgG-RF(-) RA, which showed a strong association with a SE allele not frequent in the Japanese population but the most frequent SE allele in Europeans. The findings could shed light on uncovered RA pathology.

A Novel CD135+ Subset of Mouse Monocytes with a Distinct Differentiation Pathway and Antigen-Presenting Properties.

The mononuclear phagocyte system (MPS), composed of monocytes/macrophages and dendritic cells (DCs), plays a critical role at the interface of the innate and adaptive immune systems. However, the simplicity of MPS has been challenged recently by discoveries of novel cellular components. In the current study, we identified the CD135+ subset of monocytes as a novel class of APCs in mice. CD135+ monocytes were readily found in the bone marrow, spleen, and peripheral blood at steady state, and they expressed markers specific to DCs, including MHC class II and CD209a, along with markers for monocytes/macrophages. In addition, this subset phagocytosed bacteria and activated naive T lymphocytes, fulfilling the criteria for APCs. CD135+ monocytes were derived directly from macrophage DC progenitors, not from common monocyte progenitors or other monocytes, suggesting that these are distinct from conventional monocytes. These findings facilitate our understanding of the MPS network that regulates immune responses for host defense.

Co-occurrence of relapsing polychondritis and autoimmune thyroid diseases.

BACKGROUND: Relapsing polychondritis (RP) is a rare inflammatory disease characterized by recurrent inflammation and destruction of cartilaginous tissues. RP has characteristics of autoimmune disease and some reports have noted co-occurrence with autoimmune thyroid disease (AITD), consisting of Graves' disease (GD) and Hashimoto thyroiditis (HT). However, there have been no detailed studies on the co-occurrence of RP and AITD. In this study, we aimed to determine whether patients with RP tend to be complicated with AITD. We also analyzed the clinical and genetic profiles of patients in whom these diseases co-occur. METHODS: We recruited 117 patients with RP and reviewed their medical records. Furthermore, we genotyped Human Leucocyte Antigen (HLA)-A, B Cw, DRB1, DQB1, and DPB1 alleles for 93 of the 117 patients. The prevalence of AITD among the patients with RP was compared with that among the general Japanese population. We also analyzed the clinical and genetic features of the patients with both RP and AITD. RESULTS: The prevalence of GD among the patients with RP was 4.3% (5 among 117 patients), significantly higher than that among Japanese (0.11%) (p = 2.44 × 10-7, binomial test). RP patients with GD tended to have nasal involvement (p = 0.023) (odds ratio (OR) 2.58) and HLA-DPB1*02:02 (p = 0.035, OR 10.41). We did not find significant enrichment of HT in patients with RP. CONCLUSIONS: Patients with RP appear to be at elevated risk of GD. Nasal involvement and HLA-DPB1*02:02 characterize the subset of RP patients with GD, which may guide attempts to characterize a distinct subtype of RP for precision medicine.

Impact of 2 Gy γ-irradiation on the hallmark characteristics of human bone marrow-derived MSCs.

Two gray γ-irradiation is a widely employed basic module for total body irradiation (TBI) in allogeneic hematopoietic cell transplantation (HCT). The effects of γ-irradiation on hematopoietic and immune cells have been well investigated, but its effects on the bone marrow microenvironment (BMM) are unknown. Given the crucial contribution of mesenchymal/stromal stem cells (MSCs) in the BMM to hematopoiesis and osteogenesis, we investigated whether γ-irradiation affects the hallmark characteristics of human bone marrow-derived MSCs (BM-MSCs). Expansion of 2 Gy γ-irradiated BM-MSCs was delayed but eventually recovered. Colony formation and osteogenic, adipogenic, and chondrogenic differentiation capabilities of these cells were extensively suppressed. Irradiation of BM-MSCs did not affect the expansion of CD34 + hematopoietic stem and progenitor cells or production of CD11b + mature myeloid cells in co-cultures. However, it reduced production of CD19 + B-cells, as well as expression of CXCL12 and interleukin-7, which are essential for B-cell lymphopoiesis, in 2 Gy γ-irradiated BM-MSCs. Collectively, colony formation, osteogenic differentiation, and B-cell lymphopoiesis-supportive capabilities of γ-irradiated BM-MSCs were reduced. These effects may predispose survivors receiving HCT with TBI to defective bone formation and a perturbed humoral immune response.

Long-term follow-up of patients with anti-cyclic citrullinated peptide antibody-positive connective tissue disease: a retrospective observational study including information on the HLA-DRB1 allele and citrullination dependency.

BACKGROUND: The anti-cyclic citrullinated peptide (CCP) antibody is a diagnostic biomarker of rheumatoid arthritis (RA). However, some non-RA connective tissue disease (CTD) patients also test positive for the anti-CCP antibody and, thus, may ultimately develop RA. We retrospectively investigated whether anti-CCP-positive non-RA CTD patients developed RA and attempted to identify factors that may differentiate RA-overlapping CTD from pure CTD. METHODS: In total, 842 CTD patients with a primary diagnosis that was not RA were selected from our CTD database as of December 2012. Anti-CCP antibody titers were obtained from a retrospective chart review or measured using stored sera. RA was diagnosed according to the 1987 revised American College of Rheumatology classification criteria. Thirty-three anti-CCP-positive non-RA CTD patients were retrospectively followed up for the development of RA. Bone erosions on the hands and feet were assessed by X-ray. Citrullination dependency was evaluated by an in-house ELISA, the HLA-DRB1 allele was typed, and the results obtained were then compared between RA-overlapping and non-RA anti-CCP-positive CTD patients. RESULTS: Two out of 33 anti-CCP-positive CTD patients (6.1%) developed RA during a mean follow-up period of 8.9 years. X-rays were examined in 27 out of the 33 patients, and only one (3.7%) showed bone erosions. The frequency of the HLA-DRB1 shared epitope (SE) and anti-CCP antibody titers were both significantly higher in anti-CCP-positive RA-overlapping CTD patients than in anti-CCP-positive non-RA CTD patients, while no significant differences were observed in citrullination dependency. CONCLUSIONS: Anti-CCP-positive non-RA CTD patients rarely developed RA. HLA-DRB1 SE and anti-CCP antibody titers may facilitate the differentiation of RA-overlapping CTD from anti-CCP-positive non-RA CTD.

Correction to: Menatetrenone facilitates hematopoietic cell generation in a manner that is dependent on human bone marrow mesenchymal stromal/stem cells.

In the original publication of the article, the Figs. 4 C, F and 5 B, C were published with unexpected appearance of dots.

C/EBPß isoforms sequentially regulate regenerating mouse hematopoietic stem/progenitor cells.

The transcription factor CCAAT enhancer-binding protein ß (C/EBPß) is required for stress-induced granulopoiesis at the level of hematopoietic stem/progenitor cells (HSPCs); however, its role and mechanisms of action in HSPCs are unknown. In this study, we assessed the regulation and functions of C/EBPß in HSPCs, especially under stress conditions. After 5-fluorouracil treatment or bone marrow transplantation, Cebpb-/- HSPCs exhibited impaired cell-cycle activation and myeloid differentiation at the early and late phases of regeneration, respectively, whereas at steady state, Cebpb deficiency did not affect HSPCs. C/EBPß was upregulated in response to hematopoietic stress, especially in CD150high long term-hematopoietic stem cells (LT-HSCs). Intracellular flow cytometric analysis that detected distinct domains of C/EBPß revealed that, among the 3 isoforms of C/EBPß, liver-enriched inhibitory protein (LIP) was upregulated in LT-HSCs prior to liver-enriched activating protein (LAP)/LAP* during regeneration. Early upregulation of LIP promoted cell-cycle entry of LT-HSCs by positively regulating Myc and expanded the HSPCs pool. Subsequent myeloid differentiation of amplified HSPCs was mediated by LAP/LAP*, which were upregulated at a later phase of regeneration. Collectively, our findings show that stress-induced sequential upregulation of C/EBPß isoforms is critical for fine-tuning the proliferation and differentiation of regenerating HSPCs.

Menatetrenone facilitates hematopoietic cell generation in a manner that is dependent on human bone marrow mesenchymal stromal/stem cells.

Vitamin K2 in the form of menatetrenone has clinical benefits for osteoporosis and cytopenia. Given the dominant role of mesenchymal-osteolineage cells in the regulation of hematopoiesis, we investigated whether menatetrenone alters the hematopoiesis-supportive capability of human bone marrow mesenchymal stromal/stem cells (BM-MSCs). Menatetrenone up-regulated fibronectin protein expression in BM-MSCs without affecting their proliferation and differentiation capabilities. In addition, menatetrenone treatment of BM-MSCs enhanced generation of the CD34+ cell population in co-cultures through acceleration of the cell cycle. This effect was associated with cell-cell interactions mediated by VLA-4 and fibronectin. This proposal was supported by cytokine array and quantitative real-time PCR analyses, in which there were no significant differences between the expression levels of hematopoiesis-associated soluble factors in naïve and menatetrenone-treated BM-MSCs. Profiling of hematopoietic cells in co-cultures with menatetrenone-treated BM-MSCs demonstrated that they included significantly more CD34+CD38+ hematopoietic progenitor cells and cells skewed toward myeloid and megakaryocytic lineages than those in co-cultures with untreated BM-MSCs. Notably, myelodysplastic syndrome-derived cells were induced to undergo apoptosis when co-cultured with BM-MSCs, and this effect was enhanced by menatetrenone. Overall, our findings indicate that pharmacological treatment with menatetrenone bestows a unique hematopoiesis-supportive capability on BM-MSCs, which may contribute to the clinical improvement of cytopenia.

Successful granulocyte apheresis using medium molecular weight hydroxyethyl starch.

Granulocyte transfusion (GTX) is a therapeutic option for severe bacterial or fungal infection in patients with sustained neutropenia after chemotherapy or stem cell transplantation. However, high molecular weight hydroxyethyl starch (HES), which has been used for selective sedimentation of red blood cells during apheresis, is not easily available in many countries including Japan. In this study, we evaluated the efficiency of granulocyte collection using medium molecular weight HES (130 kDa) in combination with the Spectra Optia apheresis system. Apheresis was performed for 2 consecutive days from seven donors and the mean total neutrophil yield from the first and second apheresis was 5.27 ± 3.10 × 1010 and 2.91 ± 2.92 × 1010, respectively. Infusion of concentrates from the first apheresis resulted in a significant neutrophil count increase and concentrates from the second apheresis were enough for maintenance of the neutrophil counts in all the recipients. Although the number of cases is limited, our results clearly show that sufficient number of granulocytes can be harvested by using medium molecular weight HES and this strategy is a safe and effective clinical practice in countries where high molecular weight HES is not available.

C/EBPß is a critical mediator of IFN-α-induced exhaustion of chronic myeloid leukemia stem cells.

Even in the era of ABL tyrosine kinase inhibitors, eradication of chronic myeloid leukemia (CML) stem cells is necessary for complete cure of the disease. Interferon-α (IFN-α) has long been used for the treatment of chronic-phase CML, but its mechanisms of action against CML stem cells remain unclear. We found that IFN-α upregulated CCAAT/enhancer binding protein ß (C/EBPß) in BCR-ABL-expressing mouse cells by activating STAT1 and STAT5, which were recruited to a newly identified 3' distal enhancer of Cebpb that contains tandemly aligned IFN-γ-activated site elements. Suppression or deletion of the IFN-γ-activated site elements abrogated IFN-α-dependent upregulation of C/EBPß. IFN-α induced differentiation and exhaustion of CML stem cells, both in vitro and in vivo, in a C/EBPß-dependent manner. In addition, IFN-α upregulated C/EBPß and induced exhaustion of lineage- CD34+ cells from CML patients. Collectively, these results clearly indicate that C/EBPß is a critical mediator of IFN-α-induced differentiation and exhaustion of CML stem cells.

HLA-DRB1 Analysis Identified a Genetically Unique Subset within Rheumatoid Arthritis and Distinct Genetic Background of Rheumatoid Factor Levels from Anticyclic Citrullinated Peptide Antibodies.

OBJECTIVE: HLA-DRB1 is the most important locus associated with rheumatoid arthritis (RA) and anticitrullinated protein antibodies (ACPA). However, fluctuations of rheumatoid factor (RF) over the disease course have made it difficult to define fine subgroups according to consistent RF positivity for the analyses of genetic background and the levels of RF. METHODS: A total of 2873 patients with RA and 2008 healthy controls were recruited. We genotyped HLA-DRB1 alleles for the participants and collected consecutive data of RF in the case subjects. In addition to RF+ and RF- subsets, we classified the RF+ subjects into group 1 (constant RF+) and group 2 (seroconversion). We compared HLA-DRB1 alleles between the RA subsets and controls and performed linear regression analysis to identify HLA-DRB1 alleles associated with maximal RF levels. Omnibus tests were conducted to assess important amino acid positions. RESULTS: RF positivity was 88%, and 1372 and 970 RF+ subjects were classified into groups 1 and 2, respectively. RF+ and RF- showed similar genetic associations to ACPA+ and ACPA- RA, respectively. We found that shared epitope (SE) was more enriched in group 2 than 1, p = 2.0 × 10-5, and that amino acid position 11 showed a significant association between 1 and 2, p = 2.7 × 10-5. These associations were independent of ACPA positivity. SE showed a tendency to be negatively correlated with RF titer (p = 0.012). HLA-DRB1*09:01, which reduces ACPA titer, was not associated with RF levels (p = 0.70). CONCLUSION: The seroconversion group was shown to have distinct genetic characteristics. The genetic architecture of RF levels is different from that of ACPA.

Multiple myeloma cells adapted to long-exposure of hypoxia exhibit stem cell characters with TGF-ß/Smad pathway activation.

The emergence of new molecular targeting agents has improved the prognosis of patients with multiple myeloma (MM). However, MM remains incurable because MM stem cells are likely resistant to these agents. Thus, it is important to further investigate the biology of MM stem cells, which reside in the hypoxic bone marrow niche. In this study, we established and investigated the characteristics of hypoxia-adapted MM (HA-MM) cells, which could proliferate for more than six months under hypoxic conditions (1% O2). The G0 fraction of HA-MM cells was larger than that of parental MM cells under normoxic conditions (20% O2). HA-MM cells possess enhanced tumorigenicity in primary and secondary transplantation studies. HA-MM cells also exhibited increased mRNA levels of stem cell markers and an enhanced self-renewal ability, and thus demonstrated characteristics of MM stem cells. These cells overexpressed phosphorylated Smad2, and treatment with a transforming growth factor (TGF)-ß/Smad signaling inhibitor decreased their clonogenicity in a replating assay. In conclusion, MM cells adapted to long-exposure of hypoxia exhibit stem cell characters with TGF-ß/Smad pathway activation.

Graft-Versus-Host Disease Amelioration by Human Bone Marrow Mesenchymal Stromal/Stem Cell-Derived Extracellular Vesicles Is Associated with Peripheral Preservation of Naive T Cell Populations.

A substantial proportion of patients with acute graft-versus-host disease (aGVHD) respond to cell therapy with culture-expanded human bone marrow mesenchymal stromal/stem cells (BM-MSCs). However, the mechanisms by which these cells can ameliorate aGVHD-associated complications remain to be clarified. We show here that BM-MSC-derived extracellular vesicles (EVs) recapitulated the therapeutic effects of BM-MSCs against aGVHD. Systemic infusion of human BM-MSC-derived EVs prolonged the survival of mice with aGVHD and reduced the pathologic damage in multiple GVHD-targeted organs. In EV-treated GVHD mice, CD4+ and CD8+ T cells were suppressed. Importantly, the ratio of CD62L-CD44+ to CD62L + CD44- T cells was decreased, suggesting that BM-MSC-derived EVs suppressed the functional differentiation of T cells from a naive to an effector phenotype. BM-MSC-derived EVs also preserved CD4 + CD25 + Foxp3+ regulatory T cell populations. In a culture of CD3/CD28-stimulated human peripheral blood mononuclear cells with BM-MSC-derived EVs, CD3+ T cell activation was suppressed. However, these cells were not suppressed in cultures with EVs derived from normal human dermal fibroblasts (NHDFs). NHDF-derived EVs did not ameliorate the clinical or pathological characteristics of aGVHD in mice, suggesting an immunoregulatory function unique to BM-MSC-derived EVs. Microarray analysis of microRNAs in BM-MSC-derived EVs versus NHDF-derived EVs showed upregulation of miR-125a-3p and downregulation of cell proliferative processes, as identified by Gene Ontology enrichment analysis. Collectively, our findings provide the first evidence that amelioration of aGVHD by therapeutic infusion of BM-MSC-derived EVs is associated with the preservation of circulating naive T cells, possibly due to the unique microRNA profiles of BM-MSC-derived EVs. Stem Cells 2018;36:434-445.

Pharmacological targeting of bone marrow mesenchymal stromal/stem cells for the treatment of hematological disorders.

The therapeutic effects of mesenchymal stromal/stem cells (MSCs) are mainly based on three characteristics: immunomodulation, tissue regeneration, and hematopoietic support. Cell therapy using culture-expanded MSCs is effective in some intractable bone and hemato-immune disorders; however, its efficacy is limited. In this article, we review the previous efforts to improve the clinical outcomes of cell therapy using MSCs for such disorders. We describe pharmacological targeting of endogenous bone marrow-derived MSCs as a crucial quality-based intervention to establish more effective MSC-based therapies.

Effects of acute exposure to low-dose radiation on the characteristics of human bone marrow mesenchymal stromal/stem cells.

BACKGROUND: In recent years, increasing attention has been paid to the effects of low-dose irradiation on human health. We examined whether low-dose irradiation affected the functions of mesenchymal stromal/stem cells (MSCs), which are tissue/organ-supportive stem cells, derived from bone marrow (BM). METHODS: Normal human BM-MSCs from five healthy individuals were used in this study. Culture-expanded BM-MSCs were exposed to 0.1 gray (Gy) of γ-radiation (Cesium-137) at a rate of 0.8 Gy/min (Ir-MSCs), and their expansion, multi-differentiation, and hematopoiesis-supportive capabilities were investigated. RESULTS: The expansion of BM-MSCs was transiently delayed after low-dose γ-irradiation compared with that of non-irradiated BM-MSCs (non-Ir-MSCs) in two out of five lots. Adipogenic and osteogenic differentiation capabilities were not significantly affected by low-dose irradiation, although one lot of BM-MSCs tended to have transiently reduced differentiation. When human BM hematopoietic stem/progenitor cells (HPCs) were co-cultured with Ir-MSCs, the generation of CD34+CD38+ cells from HPCs was enhanced compared with that in co-cultures with non-Ir-MSCs in two out of five lots. The mRNA expression level of interleukin (IL)-6 was increased and those of stem cell factor (SCF) and fms-related tyrosine kinase 3 ligand (Flt3L) were decreased in the affected lots of Ir-MSCs. In the other three lots of BM-MSCs, a cell growth delay, enhanced generation of CD34+CD38+ cells from HPCs in co-culture, and a combination of increased expression of IL-6 and decreased expression of SCF and Flt3L were not observed. Of note, the characteristics of these affected Ir-MSCs recovered to a similar level as those of non-Ir-MSCs following culture for 3 weeks. CONCLUSIONS: Our results suggest that acute exposure to low-dose (0.1 Gy) radiation can transiently affect the functional characteristics of human BM-MSCs.

C/EBPß is required for survival of Ly6C- monocytes.

The transcription factor CCAAT/enhancer-binding protein ß (C/EBPß) is highly expressed in monocytes/macrophages. However, its roles in monopoiesis are largely unknown. Here, we investigated the roles of C/EBPß in monopoiesis. Further subdivision of monocytes revealed that Cebpb messenger RNA was highly upregulated in Ly6C- monocytes in bone marrow. Accordingly, the number of Ly6C- monocytes was significantly reduced in Cebpb-/- mice. Bone marrow chimera experiments and Mx1-Cre-mediated deletion of Cebpb revealed a cell-intrinsic and monocyte-specific requirement for C/EBPß in monopoiesis. In Cebpb-/- mice, turnover of Ly6C- monocytes was highly accelerated and apoptosis of Ly6C- monocytes was increased. Expression of Csf1r, which encodes a receptor for macrophage colony-stimulating factor, was significantly reduced in Ly6C- monocytes of Cebpb-/- mice. C/EBPß bound to positive regulatory elements of Csf1r and promoted its transcription. Collectively, these results indicate that C/EBPß is a critical factor for Ly6C- monocyte survival, at least in part through upregulation of Csf1r.

A Rare Case of Adult Autoimmune Neutropenia Successfully Treated with Prednisolone.

Autoimmune neutropenia (AIN) is a rare disorder that may cause life-threatening infections. In adults, most cases are secondary to other pathological conditions, and primary AIN is extremely rare. We herein report a case involving a 57-year-old woman diagnosed with AIN. A granulocyte immunofluorescence test detected autoantibodies against human neutrophil antigens in her serum, while various examinations revealed no other causes of neutropenia, suggesting her AIN was primary. She was refractory to granulocyte-colony-stimulating factor but responded to prednisolone. Her neutrophil count remained normal after gradual discontinuation of prednisolone. Diagnostic procedures and optimal treatments for this disorder need to be established.

Isolation of mesenchymal stromal/stem cells from cryopreserved umbilical cord blood cells.

Umbilical cord blood (UCB) has advantages over other tissues because it can be obtained without an invasive procedure and complex processing. We explored the availability of cryopreserved UCB cells as a source of mesenchymal stromal/stem cells (MSCs). MSCs were successfully isolated from six of 30 UCB units (median volume, 34.0 mL; median nucleated cell number, 4.4×108) that were processed and cryopreserved using CP-1/human serum albumin. This isolation rate was lower than that (57%) from non-cryopreserved UCB cells. The number of nucleated cells before and after hydroxyethyl starch separation, UCB unit volume, and cell viability after thawing did not significantly differ between UCB units from which MSCs were successfully isolated and those from which they were not. When CryoSure-DEX40 was used as a cryoprotectant, MSCs were isolated from two of ten UCB units. Logistic regression analysis demonstrated that the cryopreservation method was not significantly associated with the success of MSC isolation. The isolated MSCs had a similar morphology and surface marker expression profile as bone marrow-derived MSCs and were able to differentiate into osteogenic, adipogenic, and chondrogenic cells. In summary, MSCs can be isolated from cryopreserved UCB cells. However, the cryopreservation process reduces the isolation rate; therefore, freshly donated UCB cells are preferable for the isolation of MSCs.