Introduction: Progranulin (PGRN) is the precursor of granulin (GRN), a soluble cofactor for toll-like receptor 9 (TLR9) signaling evoked by oligonucleotide (CpG)-DNA. Because TLR9 signaling plays an important role in systemic lupus erythematosus (SLE), we investigated whether PGRN is involved in the pathogenesis of SLE. Methods: We measured concentrations of serum PGRN and interleukin-6 (IL-6) with enzyme-linked immunosorbent assay (ELISA) in patients with SLE (n = 68) and in healthy controls (n = 60). We assessed the correlation between the serum PGRN levels and established disease-activity indexes. The sera from the patients with high PGRN titers (>80 ng/ml) at the initial evaluation were reevaluated after the disease was ameliorated by treatment. We also measured the IL-6 concentration secreted by peripheral blood mononuclear cells (PBMCs) incubated with (a) oligonucleotide (CpG-B) in the presence or absence of recombinant human PGRN (rhPGRN); and (b) lupus sera in the presence or absence of a neutralizing anti-PGRN antibody. Results: Serum PGRN levels were significantly higher in SLE patients than healthy controls. Their levels were significantly associated with activity of clinical symptoms. They also significantly correlated with values of clinical parameters, including the SLE Disease Activity Index and anti-double-stranded DNA antibody titers, and inversely with CH50, C3, and C4 levels. Moreover, serum PGRN levels significantly decreased after successful treatment of SLE. The rhPGRN significantly upregulated the production of IL-6 by PBMCs stimulated with CpG-B. Patients' sera stimulated production of IL-6 from PBMCs, which was significantly impaired by neutralization of PGRN. The serum PGRN levels significantly correlated with the serum IL-6 levels. Conclusions: Serum PGRN could be a useful biomarker for disease activity of SLE. PGRN may be involved in the pathogenesis of SLE partly by enhancing the TLR9 signaling.

Recent studies show that innate lymphoid cells (ILCs) contribute to the development of chronic inflammation and autoimmune disease. In this study, we assessed the ILC function in the development of rheumatoid arthritis (RA). In a mouse model of collagen-induced arthritis (CIA), we identified and purified the ILC subsets in peripheral blood (PB), local lymph nodes (LNs), and joints by fluorescence-activated cell sorting and used quantitative PCR to assess the expression levels of representative cytokines. We also correlated the frequencies of each ILC subset in synovial fluid (SF) with clinical parameters in RA patients. In the CIA model, the proportion of CCR6+ ILC3s to total ILCs in joints with active inflammation significantly increased relative to non-arthritic joints (median 29.6% vs 16.7%, p = 0.035). CCR6+ ILC3s from mice with arthritis expressed significantly higher levels of IL-17A and IL-22 mRNA than did comparable cells from control mice (p < 0.0001 and p = 0.015). In RA patients, the proportion of CCR6+ ILCs in SF was positively correlated with tender joint counts (TJC) and swollen joint counts (SJC) (ρ=0.689, p = 0.0032 and ρ=0.644, p = 0.0071, respectively). Levels of CC chemokine ligand 20 (CCL20) increased in SF of patients with RA and were significantly correlated with CCR6+ ILC number (ρ=0.697, p = 0.0001). CCR6+ ILC3s may play some roles in the development of RA through the production of IL-17 and IL-22.

The efficacy of B cell-depleting therapies for rheumatoid arthritis underscores antibody-independent functions of effector B cells such as cognate T–B interactions and production of pro-inflammatory cytokines. Receptor activator of nuclear factor κB ligand (RANKL) is a key cytokine involved in bone destruction and is highly expressed in synovial fluid B cells in patients with rheumatoid arthritis. In this study we sought to clarify the generation mechanism of RANKL+ effector B cells and their impacts on osteoclast differentiation. Peripheral blood and synovial fluid B cells from healthy controls and patients with rheumatoid arthritis were isolated using cell sorter. mRNA expression of RANKL, osteoprotegerin, tumor necrosis factor (TNF)-α, and Blimp-1 was analyzed by quantitative real-time polymerase chain reaction. Levels of RANKL, CD80, CD86, and CXCR3 were analyzed using flow cytometry. Functional analysis of osteoclastogenesis was carried out in the co-culture system using macrophage RAW264 reporter cells. RANKL expression was accentuated in CD80+CD86+ B cells, a highly activated B-cell subset more abundantly observed in patients with rheumatoid arthritis. Upon activation via B-cell receptor and CD40, switched-memory B cells predominantly expressed RANKL, which was further augmented by interferon-γ (IFN-γ) but suppressed by interleukin-21. Strikingly, IFN-γ also enhanced TNF-α expression, while it strongly suppressed osteoprotegerin expression in B cells. IFN-γ increased the generation of CXCR3+RANKL+ effector B cells, mimicking the synovial B cell phenotype in patients with rheumatoid arthritis. Finally, RANKL+ effector B cells in concert with TNF-α facilitated osteoclast differentiation in vitro. Our current findings have shed light on the generation mechanism of pathogenic RANKL+ effector B cells that would be an ideal therapeutic target for rheumatoid arthritis in the future.

DNase 1–like 3 (DNase1L3), which belongs to DNase1 family, was originally identified as one of apoptosis- and necrosis-related endonucleases that fragmentate intranucleosomal DNA. A loss-of-function mutation has been reported in murine models of systemic lupus erythematosus (SLE) and in familial SLE patients. These reports suggest DNase1L3 plays an important role in the prevention of developing SLE; however, expression and function of DNase1L3 in human immune systems have been largely unclarified. As previous reports showed DNase1L3 is expressed in hematopoietic organs, we first analyzed expression levels of DNase1L3 in each subset of human peripheral blood cells by quantitative real-time PCR. Plasmacytoid dendritic cells showed the highest expression levels of DNase1L3 mRNA among peripheral blood cells. IL-4 enhanced DNase1L3 expression in monocytes, monocyte-derived dendritic cells, and monocyte-derived macrophages (MDMs), but not in T cells, B cells, or plasmacytoid dendritic cells. Together with IL-4, all-trans retinoic acid and apoptotic cells efficiently upregulated expression of DNalse1L3 in MDMs. As a result of intracellular signaling analysis, Jak1-IRS2-ERK/PI3K pathway was essential for IL-4–induced DNase1L3 expression. IL-4–treated monocyte-derived dendritic cells and MDMs secreted active DNase1L3 protein that could degrade liposome–DNA complexes, which were resistant to DNase1. Our results indicate DNase1L3 is secreted by innate immune cells and may play a critical role in the tissue homeostasis and on prevention of developing autoimmunity by degrading self-DNA.

The effectiveness of autologous haematopoietic stem cell transplantation (auto-HSCT) in treating severe systemic sclerosis (SSc) is established; however, the necessity of purified CD34+ cell grafts and the appropriate conditioning regimen remain unclear. This study aimed to compare the efficacy and safety of CD34-selected auto-HSCT with unmanipulated auto-HSCT to treat severe SSc. This study was a post hoc analysis of a phase I/II clinical trial conducted in Japan. Nineteen patients with severe SSc were enrolled. Peripheral blood stem cells (PBSCs) were mobilised with cyclophosphamide (4 g/m2) and filgrastim (10 μg/kg/day). Following PBSC collection by apheresis, CD34+ cells were immunologically selected in 11 patients. All patients were treated with high-dose cyclophosphamide (200 mg/kg) monotherapy as a conditioning regimen and received CD34-selected (n = 11) or unmanipulated auto-HSCT (n = 8). Changes in skin sclerosis and pulmonary function were assessed over an 8-year follow-up period. Differences in the changes, toxicity, progression-free survival (PFS) and overall survival were compared between patients who had received CD34-selected auto-HSCT and those who had received unmanipulated auto-HSCT. Skin sclerosis progressively improved after transplantation over an 8-year follow-up period in both groups, and the improvement was significantly greater in the CD34-selected group than in the unmanipulated group. Forced vital capacity in the CD34-selected group continuously increased over 8 years, whereas in the unmanipulated group it returned to baseline 3 years after transplantation. Toxicity and viral infections, such as cytomegalovirus infection and herpes zoster, were more frequently found in the CD34-selected group than in the unmanipulated group. The frequency of severe adverse events, such as bacterial infections or organ toxicity, was similar between the two groups. No treatment-related deaths occurred in either treatment group. PFS of the CD34-selected group was greater than that of the unmanipulated group, and the 5-year PFS rates of the CD34-selected and unmanipulated group were 81.8% and 50% respectively. CD34-selected auto-HSCT may produce favourable effects on improvement of skin sclerosis and pulmonary function compared with unmanipulated auto-HSCT. Use of CD34-selected auto-HSCT with high-dose cyclophosphamide monotherapy as a conditioning regimen may offer an excellent benefit-to-risk balance.