Elisabetta Mariani

Plasma cells require autophagy for sustainable immunoglobulin production

The role of autophagy in plasma cells is unknown. Here we found notable autophagic activity in both differentiating and long-lived plasma cells and investigated its function through the use of mice with conditional deficiency in the essential autophagic molecule Atg5 in B cells. Atg5−/− differentiating plasma cells had a larger endoplasmic reticulum (ER) and more ER stress signaling than did their wild-type counterparts, which led to higher expression of the transcriptional repressor Blimp-1 and immunoglobulins and more antibody secretion. The enhanced immunoglobulin synthesis was associated with less intracellular ATP and more death of mutant plasma cells, which identified an unsuspected autophagy-dependent cytoprotective trade-off between immunoglobulin synthesis and viability. In vivo, mice with conditional deficiency in Atg5 in B cells had defective antibody responses, complete selection in the bone marrow for plasma cells that escaped Atg5 deletion and fewer antigen-specific long-lived bone marrow plasma cells than did wild-type mice, despite having normal germinal center responses. Thus, autophagy is specifically required for plasma cell homeostasis and long-lived humoral immunity.

Muscle inflammation and MHC class I up-regulation in muscular dystrophy with lack of dysferlin: an immunopathological study

Muscle inflammation is characteristic of inflammatory myopathies but also occurs in muscular dystrophy with lack of the sarcolemmal protein dysferlin. We quantified inflammatory cells and major histocompatibility complex (MHC) expression in muscle from 10 patients with dysferlinopathy. Infiltrating cells were always present although numbers varied considerably; macrophages were more common than T cells, T cytotoxicity was absent, and MHC class I was overexpressed on muscle fibers. These findings differ from polymyositis (PM) but are closely similar to those in SJL/J mice (which lack dysferlin) and emphasize the relationship between absence of dysferlin and immune system abnormalities in muscle.

Dampening Ab responses using proteasome inhibitors following in vivo B cell activation

Upon encounter with Ag, B lymphocytes undergo terminal differentiation into plasma cells, highly specialized Ab secretors that mediate humoral immune responses. Profound changes adapt cellular morphology and proteome to the new secretory functions. Although a massive secretory activity is expected to require an efficient ubiquitin‐proteasome degradation system, recent in vitro studies have surprisingly revealed that the proteasome function sharply decreases during plasma cell development, thereby limiting the proteolytic capacity. We challenged this paradigm in mouse models of B cell activation, and observed that following polyclonal activation, proteasome activity decreases more than previously reported in vitro. This decrease is linked to enhanced apoptosis after treatment with the potent anti‐myeloma proteasome inhibitor PS‐341. Accordingly, in vivo treatment with PS‐341 decreases Ab titres in T‐dependent and ‐independent mouse immunization models. This study provides the rationale for limiting the activity of Ab‐secreting cells in vivo by impacting proteasome function.

Magnesium-Enriched Hydroxyapatite Versus Autologous Bone in Maxillary Sinus Grafting: Combining Histomorphometry With Osteoblast Gene Expression Profiles Ex Vivo

BACKGROUND Many biomaterials are proposed for sinus floor lifting and grafting in the posterior maxilla for insufficient bone volume (BV). The aim of this study was to compare the use of magnesium-enriched hydroxyapatite (mHA) versus autogenous bone graft (AB) for maxillary sinus lift procedures by histomorphometric and ex vivo gene expression profiling. METHODS Fifteen patients requiring bilateral maxillary sinus augmentation received autologous bone particles (group A) and mHA (group B) (split-mouth design). Five months later, implants were placed, and biopsies were obtained. Bone specimens were analyzed by histomorphometry, BV and vital bone (VB) percentages were calculated, and ex vivo osteoblast expansion followed by highly sensitive osteoblast specific gene expression profiling for cbfa1, osteocalcin, osteopontin, collagen type I, receptor activator of nuclear factor-kappa B ligand (RANKL), and osteoprotegerin (OPG) by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) were performed. Comparisons were made using the Student t test. RESULTS After healing with no complications, BV was comparable in the two groups (80.79% +/- 14.27% for autologous versus 76.72% +/- 11.47% for mHA; P = not statistically significant), but VB was lower in the mHA group (29.65% +/- 9.81% versus 78.40% +/- 16.72%; P <0.05). Real-time RT-PCR analyses showed significantly higher expression of the osteoblast differentiation factor Cbfa1 and the matrix formation marker osteocalcin in the mHA group compared to the AB group, whereas type I collagen was comparable, and osteopontin was decreased. Attesting to a lower osteoclastogenic potential, the RANKL/OPG ratio was diminished. CONCLUSIONS Autogenous bone samples provided higher vital over comparable total bone levels than mHA-grafted sites. Osteoblast gene expression profiles from mHA grafts revealed higher expression of certain specific markers of osteoblast differentiation and bone formation, associated with a lower osteoclastogenic potential.

MHC class II transactivator is an in vivo regulator of osteoclast differentiation and bone homeostasis co-opted from adaptive immunity.

The molecular networks controlling bone homeostasis are not fully understood. The common evolution of bone and adaptive immunity encourages the investigation of shared regulatory circuits. MHC Class II Transactivator (CIITA) is a master transcriptional co‐activator believed to be exclusively dedicated for antigen presentation. CIITA is expressed in osteoclast precursors, and its expression is accentuated in osteoporotic mice. We thus asked whether CIITA plays a role in bone biology. To this aim, we fully characterized the bone phenotype of two mouse models of CIITA overexpression, respectively systemic and restricted to the monocyte‐osteoclast lineage. Both CIITA‐overexpressing mouse models revealed severe spontaneous osteoporosis, as assessed by micro‐computed tomography and histomorphometry, associated with increased osteoclast numbers and enhanced in vivo bone resorption, whereas osteoblast numbers and in vivo bone‐forming activity were unaffected. To understand the underlying cellular and molecular bases, we investigated ex vivo the differentiation of mutant bone marrow monocytes into osteoclasts and immune effectors, as well as osteoclastogenic signaling pathways. CIITA‐overexpressing monocytes differentiated normally into effector macrophages or dendritic cells but showed enhanced osteoclastogenesis, whereas CIITA ablation suppressed osteoclast differentiation. Increased c‐fms and receptor activator of NF‐κB (RANK) signaling underlay enhanced osteoclast differentiation from CIITA‐overexpressing precursors. Moreover, by extending selected phenotypic and cellular analyses to additional genetic mouse models, namely MHC Class II deficient mice and a transgenic mouse line lacking a specific CIITA promoter and re‐expressing CIITA in the thymus, we excluded MHC Class II expression and T cells from contributing to the observed skeletal phenotype. Altogether, our study provides compelling genetic evidence that CIITA, the molecular switch of antigen presentation, plays a novel, unexpected function in skeletal homeostasis, independent of MHC Class II expression and T cells, by exerting a selective and intrinsic control of osteoclast differentiation and bone resorption in vivo.

Assessing heterogeneity of osteolytic lesions in multiple myeloma by1H HR-MAS NMR metabolomics

Multiple myeloma (MM) is a malignancy of plasma cells characterized by multifocal osteolytic bone lesions. Macroscopic and genetic heterogeneity has been documented within MM lesions. Understanding the bases of such heterogeneity may unveil relevant features of MM pathobiology. To this aim, we deployed unbiased 1H high-resolution magic-angle spinning (HR-MAS) nuclear magnetic resonance (NMR) metabolomics to analyze multiple biopsy specimens of osteolytic lesions from one case of pathological fracture caused by MM. Multivariate analyses on normalized metabolite peak integrals allowed clusterization of samples in accordance with a posteriori histological findings. We investigated the relationship between morphological and NMR features by merging morphological data and metabolite profiling into a single correlation matrix. Data-merging addressed tissue heterogeneity, and greatly facilitated the mapping of lesions and nearby healthy tissues. Our proof-of-principle study reveals integrated metabolomics and histomorphology as a promising approach for the targeted study of osteolytic lesions.