Depletion of slow-cycling PDGFRα+ADAM12+ mesenchymal cells promotes antitumor immunity by restricting macrophage efferocytosis.

The capacity to survive and thrive in conditions of limited resources and high inflammation is a major driver of tumor malignancy. Here we identified slow-cycling ADAM12+PDGFRα+ mesenchymal stromal cells (MSCs) induced at the tumor margins in mouse models of melanoma, pancreatic cancer and prostate cancer. Using inducible lineage tracing and transcriptomics, we demonstrated that metabolically altered ADAM12+ MSCs induced pathological angiogenesis and immunosuppression by promoting macrophage efferocytosis and polarization through overexpression of genes such as Gas6, Lgals3 and Csf1. Genetic depletion of ADAM12+ cells restored a functional tumor vasculature, reduced hypoxia and acidosis and normalized CAFs, inducing infiltration of effector T cells and growth inhibition of melanomas and pancreatic neuroendocrine cancer, in a process dependent on TGF-ß. In human cancer, ADAM12 stratifies patients with high levels of hypoxia and innate resistance mechanisms, as well as factors associated with a poor prognosis and drug resistance such as AXL. Altogether, our data show that depletion of tumor-induced slow-cycling PDGFRα+ MSCs through ADAM12 restores antitumor immunity.

Exploring natural genetic variation in photosynthesis-related traits of barley in the field.

Optimizing photosynthesis is considered an important strategy for improving crop yields to ensure food security. To evaluate the potential of using photosynthesis-related parameters in crop breeding programs, we measured chlorophyll fluorescence along with growth-related and morphological traits of 23 barley inbred lines across different developmental stages in field conditions. The photosynthesis-related parameters were highly variable, changing with light intensity and developmental progression of plants. Yet, the variation in photosystem II quantum yield observed among the inbred lines in the field largely reflected the variation in CO2 assimilation properties in controlled climate chamber conditions, confirming that the chlorophyll fluorescence-based technique can provide proxy parameters of photosynthesis to explore genetic variation under field conditions. Heritability (H2) of the photosynthesis-related parameters in the field ranged from 0.16 for the quantum yield of non-photochemical quenching to 0.78 for the fraction of open photosystem II center. Two parameters, the maximum photosystem II efficiency in the light-adapted state (H2=0.58) and the total non-photochemical quenching (H2=0.53), showed significant positive and negative correlations, respectively, with yield-related traits (dry weight per plant and net straw weight) in the barley inbred lines. These results indicate the possibility of improving crop yield through optimizing photosynthetic light use efficiency by conventional breeding programs.

An SNX10-dependent mechanism downregulates fusion between mature osteoclasts.

Homozygosity for the R51Q mutation in sorting nexin 10 (SNX10) inactivates osteoclasts (OCLs) and induces autosomal recessive osteopetrosis in humans and in mice. We show here that the fusion of wild-type murine monocytes to form OCLs is highly regulated, and that its extent is limited by blocking fusion between mature OCLs. In contrast, monocytes from homozygous R51Q SNX10 mice fuse uncontrollably, forming giant dysfunctional OCLs that can become 10- to 100-fold larger than their wild-type counterparts. Furthermore, mutant OCLs display reduced endocytotic activity, suggesting that their deregulated fusion is due to alterations in membrane homeostasis caused by loss of SNX10 function. This is supported by the finding that the R51Q SNX10 protein is unstable and exhibits altered lipid-binding properties, and is consistent with a key role for SNX10 in vesicular trafficking. We propose that OCL size and functionality are regulated by a cell-autonomous SNX10-dependent mechanism that downregulates fusion between mature OCLs. The R51Q mutation abolishes this regulatory activity, leading to excessive fusion, loss of bone resorption capacity and, consequently, to an osteopetrotic phenotype in vivo. This article has an associated First Person interview with the joint first authors of the paper.

Regulation of Energy Metabolism during Early B Lymphocyte Development.

The most important feature of humoral immunity is the adaptation of the diversity of newly generated B cell receptors, that is, the antigen receptor repertoire, to the body's own and foreign structures. This includes the transient propagation of B progenitor cells and B cells, which possess receptors that are positively selected via anabolic signalling pathways under highly competitive conditions. The metabolic regulation of early B-cell development thus has important consequences for the expansion of normal or malignant pre-B cell clones. In addition, cellular senescence programs based on the expression of B cell identity factors, such as Pax5, act to prevent excessive proliferation and cellular deviation. Here, we review the basic mechanisms underlying the regulation of glycolysis and oxidative phosphorylation during early B cell development in bone marrow. We focus on the regulation of glycolysis and mitochondrial oxidative phosphorylation at the transition from non-transformed pro- to pre-B cells and discuss some ongoing issues. We introduce Swiprosin-2/EFhd1 as a potential regulator of glycolysis in pro-B cells that has also been linked to Ca2+-mediated mitoflashes. Mitoflashes are bioenergetic mitochondrial events that control mitochondrial metabolism and signalling in both healthy and disease states. We discuss how Ca2+ fluctuations in pro- and pre-B cells may translate into mitoflashes in early B cells and speculate about the consequences of these changes.

Reduced Fluorescence versus Forward Scatter Time-of-Flight and Increased Peak versus Integral Fluorescence Ratios Indicate Receptor Clustering in Flow Cytometry.

Clustering of surface receptors is often required to initiate signal transduction, receptor internalization, and cellular activation. To study the kinetics of clustering, we developed an economic high-throughput method using flow cytometry. The quantification of receptor clustering by flow cytometry is based on the following two observations: first, the fluorescence signal length (FL time-of-flight [ToF]) decreases relative to the forward scatter signal length (FSc-ToF), and second, the peak FL (FL-peak) increases relative to the integral FL (FL-integral) upon clustering of FL-labeled surface receptors. Receptor macroclustering can therefore be quantified using the ratios FL-ToF/FSc-ToF (method ToF) or FL-peak/FL-integral (method Peak). We have used these methods to analyze clustering of two immune receptors known to undergo different conformational and oligomeric states: the BCR and the complement receptor 3 (CR3), on murine splenocytes, purified B cells, and human neutrophils. Engagement of both the BCR and CR3, on immortalized as well as primary murine B cells and human neutrophil, respectively, resulted in decreased FL-ToF/FSc-ToF and increased FL-peak/FL-integral ratios. Manipulation of the actin-myosin cytoskeleton altered BCR clustering which could be measured using the established parameters. To confirm clustering of CR3 on neutrophils, we applied imaging flow cytometry. Because receptor engagement is as a biological process dependent on cell viability, energy metabolism, and temperature, receptor clustering can only be quantified by gating on viable cells under physiological conditions. In summary, with this novel method, receptor clustering on nonadherent cells can easily be monitored by high-throughput conventional flow cytometry.

Swiprosin-1/EFhd2 limits germinal center responses and humoral type 2 immunity.

Activated B cells are selected for in germinal centers by regulation of their apoptosis. The Ca2+ -binding cytoskeletal adaptor protein Swiprosin-1/EFhd2 (EFhd2) can promote apoptosis in activated B cells. We therefore hypothesized that EFhd2 might limit humoral immunity by repressing both the germinal center reaction and the expected enhancement of immune responses in the absence of EFhd2. Here, we established EFhd2(-/-) mice on a C57BL/6 background, which revealed normal B- and T-cell development, basal Ab levels, and T-cell independent type 1, and T-cell independent type 2 responses. However, T cell-dependent immunization with sheep red blood cells and infection with the helminth Nippostrongylus brasiliensis (N.b) increased production of antibodies of multiple isotypes, as well as germinal center formation in EFhd2(-/-) mice. In addition, serum IgE levels and numbers of IgE+ plasma cells were strongly increased in EFhd2(-/-) mice, both after primary as well as after secondary N.b infection. Finally, mixed bone marrow chimeras unraveled an EFhd2-dependent B cell-intrinsic contribution to increased IgE plasma cell numbers in N.b-infected mice. Hence, we established a role for EFhd2 as a negative regulator of germinal center-dependent humoral type 2 immunity, with implications for the generation of IgE.

SNX10 regulates osteoclastogenic cell fusion and osteoclast size in mice.

Bone-resorbing osteoclasts (OCLs) are formed by differentiation and fusion of monocyte precursor cells, generating large multinucleated cells. Tightly regulated cell fusion during osteoclastogenesis leads to formation of resorption-competent OCLs, whose sizes fall within a predictable physiological range. The molecular mechanisms that regulate the onset of OCL fusion and its subsequent arrest are, however, largely unknown. We have previously shown that OCLs cultured from mice homozygous for the R51Q mutation in the vesicle trafficking-associated protein sorting nexin 10, a mutation that induces autosomal recessive osteopetrosis in humans and in mice, display deregulated and continuous fusion that generates gigantic, inactive OCLs. Fusion of mature OCLs is therefore arrested by an active, genetically encoded, cell-autonomous, and SNX10-dependent mechanism. To directly examine whether SNX10 performs a similar role in vivo, we generated SNX10-deficient (SKO) mice and demonstrated that they display massive osteopetrosis and that their OCLs fuse uncontrollably in culture, as do homozygous R51Q SNX10 (RQ/RQ) mice. OCLs that lack SNX10 exhibit persistent presence of DC-STAMP protein at their periphery, which may contribute to their uncontrolled fusion. To visualize endogenous SNX10-mutant OCLs in their native bone environment, we genetically labeled the OCLs of WT, SKO, and RQ/RQ mice with enhanced Green Fluorescent Protein (EGFP), and then visualized the 3D organization of resident OCLs and the pericellular bone matrix by 2-photon, confocal, and second harmonics generation microscopy. We show that the volumes, surface areas and, in particular, the numbers of nuclei in the OCLs of both mutant strains were on average 2-6-fold larger than those of OCLs from WT mice, indicating that deregulated, excessive fusion occurs in the mutant mice. We conclude that the fusion of OCLs, and consequently their size, is regulated in vivo by SNX10-dependent arrest of fusion of mature OCLs.

Osteoclasts (OCLs) are cells that degrade bone. These cells are generated by fusion of monocyte precursor cells, but the mechanisms that regulate this process and eventually arrest it are unknown. We had previously shown that OCLs cultured from mice carrying the R51Q mutation in the protein sorting nexin 10 (SNX10) lose their resorptive capacity and become gigantic due to uncontrolled fusion. To examine whether SNX10 is required for OCL fusion arrest also in vivo, we inactivated the Snx10 gene in mice and fluorescently labeled their OCLs and OCLs of R51Q SNX10 mice, isolated their femurs, and used advanced 3D microscopy methods to visualize OCLs within the bone matrix. As expected, mice lacking SNX10 exhibited excessive bone mass, indicating that their OCLs are inactive. OCLs within bones of both mutant mouse strains were on average 2­6-fold larger than in control mice and contained proportionally more nuclei. We conclude that OCL fusion is arrested in control, but not SNX10 mutant, mice, indicating that the sizes of mature OCLs are limited in vivo by an active, SNX10-dependent mechanism that suppresses cell fusion.

Targeting CDC42 reduces skeletal degeneration after hematopoietic stem cell transplantation.

ABSTRACT: Osteopenia and osteoporosis are common long-term complications of the cytotoxic conditioning regimen for hematopoietic stem cell transplantation (HSCT). We examined mesenchymal stem and progenitor cells (MSPCs), which include skeletal progenitors, from mice undergoing HSCT. Such MSPCs showed reduced fibroblastic colony-forming units frequency, increased DNA damage, and enhanced occurrence of cellular senescence, whereas there was a reduced bone volume in animals that underwent HSCT. This reduced MSPC function correlated with elevated activation of the small Rho guanosine triphosphate hydrolase CDC42, disorganized F-actin distribution, mitochondrial abnormalities, and impaired mitophagy in MSPCs. Changes and defects similar to those in mice were also observed in MSPCs from humans undergoing HSCT. A pharmacological treatment that attenuated the elevated activation of CDC42 restored F-actin fiber alignment, mitochondrial function, and mitophagy in MSPCs in vitro. Finally, targeting CDC42 activity in vivo in animals undergoing transplants improved MSPC quality to increase both bone volume and trabecular bone thickness. Our study shows that attenuation of CDC42 activity is sufficient to attenuate reduced function of MSPCs in a BM transplant setting.

PREDIG: Web application to model and predict the enzymatic saccharification of plant cell wall.

Enzymatic digestion of lignocellulosic plant biomass is a key step in bio-refinery approaches for the production of biofuels and other valuable chemicals. However, the recalcitrance of this material in conjunction with its variability and heterogeneity strongly hampers the economic viability and profitability of biofuel production. To complement both academic and industrial experimental research in the field, we designed an advanced web application that encapsulates our in-house developed complex biophysical model of enzymatic plant cell wall degradation. PREDIG (https://predig.cs.hhu.de/) is a user-friendly, free, and fully open-source web application that allows the user to perform in silico experiments. Specifically, it uses a Gillespie algorithm to run stochastic simulations of the enzymatic saccharification of a lignocellulose microfibril, at the mesoscale, in three dimensions. Such simulations can for instance be used to test the action of distinct enzyme cocktails on the substrate. Additionally, PREDIG can fit the model parameters to uploaded experimental time-course data, thereby returning values that are intrinsically difficult to measure experimentally. This gives the user the possibility to learn which factors quantitatively explain the recalcitrance to saccharification of their specific biomass material.

Why Animal Experiments Are Still Indispensable in Bone Research: A Statement by the European Calcified Tissue Society.

Major achievements in bone research have always relied on animal models and in vitro systems derived from patient and animal material. However, the use of animals in research has drawn intense ethical debate and the complete abolition of animal experimentation is demanded by fractions of the population. This phenomenon is enhanced by the reproducibility crisis in science and the advance of in vitro and in silico techniques. 3D culture, organ-on-a-chip, and computer models have improved enormously over the last few years. Nevertheless, the overall complexity of bone tissue cross-talk and the systemic and local regulation of bone physiology can often only be addressed in entire vertebrates. Powerful genetic methods such as conditional mutagenesis, lineage tracing, and modeling of the diseases enhanced the understanding of the entire skeletal system. In this review endorsed by the European Calcified Tissue Society (ECTS), a working group of investigators from Europe and the US provides an overview of the strengths and limitations of experimental animal models, including rodents, fish, and large animals, as well the potential and shortcomings of in vitro and in silico technologies in skeletal research. We propose that the proper combination of the right animal model for a specific hypothesis and state-of-the-art in vitro and/or in silico technology is essential to solving remaining important questions in bone research. This is crucial for executing most efficiently the 3R principles to reduce, refine, and replace animal experimentation, for enhancing our knowledge of skeletal biology, and for the treatment of bone diseases that affect a large part of society. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

PTPRJ promotes osteoclast maturation and activity by inhibiting Cbl-mediated ubiquitination of NFATc1 in late osteoclastogenesis.

Bone-resorbing osteoclasts (OCLs) are multinucleated phagocytes, whose central roles in regulating bone formation and homeostasis are critical for normal health and development. OCLs are produced from precursor monocytes in a multistage process that includes initial differentiation, cell-cell fusion, and subsequent functional and morphological maturation; the molecular regulation of osteoclastogenesis is not fully understood. Here, we identify the receptor-type protein tyrosine phosphatase PTPRJ as an essential regulator specifically of OCL maturation. Monocytes from PTPRJ-deficient (JKO) mice differentiate and fuse normally, but their maturation into functional OCLs and their ability to degrade bone are severely inhibited. In agreement, mice lacking PTPRJ throughout their bodies or only in OCLs exhibit increased bone mass due to reduced OCL-mediated bone resorption. We further show that PTPRJ promotes OCL maturation by dephosphorylating the M-CSF receptor (M-CSFR) and Cbl, thus reducing the ubiquitination and degradation of the key osteoclastogenic transcription factor NFATc1. Loss of PTPRJ increases ubiquitination of NFATc1 and reduces its amounts at later stages of osteoclastogenesis, thereby inhibiting OCL maturation. PTPRJ thus fulfills an essential and cell-autonomous role in promoting OCL maturation by balancing between the pro- and anti-osteoclastogenic activities of the M-CSFR and maintaining NFATc1 expression during late osteoclastogenesis.

Sorting Nexin 10 as a Key Regulator of Membrane Trafficking in Bone-Resorbing Osteoclasts: Lessons Learned From Osteopetrosis.

Bone homeostasis is a complex, multi-step process, which is based primarily on a tightly orchestrated interplay between bone formation and bone resorption that is executed by osteoblasts and osteoclasts (OCLs), respectively. The essential physiological balance between these cells is maintained and controlled at multiple levels, ranging from regulated gene expression to endocrine signals, yet the underlying cellular and molecular mechanisms are still poorly understood. One approach for deciphering the mechanisms that regulate bone homeostasis is the characterization of relevant pathological states in which this balance is disturbed. In this article we describe one such "error of nature," namely the development of acute recessive osteopetrosis (ARO) in humans that is caused by mutations in sorting nexin 10 (SNX10) that affect OCL functioning. We hypothesize here that, by virtue of its specific roles in vesicular trafficking, SNX10 serves as a key selective regulator of the composition of diverse membrane compartments in OCLs, thereby affecting critical processes in the sequence of events that link the plasma membrane with formation of the ruffled border and with extracellular acidification. As a result, SNX10 determines multiple features of these cells either directly or, as in regulation of cell-cell fusion, indirectly. This hypothesis is further supported by the similarities between the cellular defects observed in OCLs form various models of ARO, induced by mutations in SNX10 and in other genes, which suggest that mutations in the known ARO-associated genes act by disrupting the same plasma membrane-to-ruffled border axis, albeit to different degrees. In this article, we describe the population genetics and spread of the original arginine-to-glutamine mutation at position 51 (R51Q) in SNX10 in the Palestinian community. We further review recent studies, conducted in animal and cellular model systems, that highlight the essential roles of SNX10 in critical membrane functions in OCLs, and discuss possible future research directions that are needed for challenging or substantiating our hypothesis.

Massive osteopetrosis caused by non-functional osteoclasts in R51Q SNX10 mutant mice.

The R51Q mutation in sorting nexin 10 (SNX10) was shown to cause a lethal genetic disease in humans, namely autosomal recessive osteopetrosis (ARO). We describe here the first R51Q SNX10 knock-in mouse model and show that mice homozygous for this mutation exhibit massive, early-onset, and widespread osteopetrosis. The mutant mice exhibit multiple additional characteristics of the corresponding human disease, including stunted growth, failure to thrive, missing or impacted teeth, occasional osteomyelitis, and a significantly-reduced lifespan. Osteopetrosis in this model is the result of osteoclast inactivity that, in turn, is caused by absence of ruffled borders in the mutant osteoclasts and by their inability to secrete protons. These results confirm that the R51Q mutation in SNX10 is a causative factor in ARO and provide a model system for studying this rare disease.

Phosphorylation of the phosphatase PTPROt at Tyr399 is a molecular switch that controls osteoclast activity and bone mass in vivo.

Bone resorption by osteoclasts is essential for bone homeostasis. The kinase Src promotes osteoclast activity and is activated in osteoclasts by the receptor-type tyrosine phosphatase PTPROt. In other contexts, however, PTPROt can inhibit Src activity. Through in vivo and in vitro experiments, we show that PTPROt is bifunctional and can dephosphorylate Src both at its inhibitory residue Tyr527 and its activating residue Tyr416 Whereas wild-type and PTPROt knockout mice exhibited similar bone masses, mice in which a putative C-terminal phosphorylation site, Tyr399, in endogenous PTPROt was replaced with phenylalanine had increased bone mass and reduced osteoclast activity. Osteoclasts from the knock-in mice also showed reduced Src activity. Experiments in cultured cells and in osteoclasts derived from both mouse strains demonstrated that the absence of phosphorylation at Tyr399 caused PTPROt to dephosphorylate Src at the activating site pTyr416 In contrast, phosphorylation of PTPROt at Tyr399 enabled PTPROt to recruit Src through Grb2 and to dephosphorylate Src at the inhibitory site Tyr527, thus stimulating Src activity. We conclude that reversible phosphorylation of PTPROt at Tyr399 is a molecular switch that selects between its opposing activities toward Src and maintains a coherent signaling output, and that blocking this phosphorylation event can induce physiological effects in vivo. Because most receptor-type tyrosine phosphatases contain potential phosphorylation sites at their C termini, we propose that preventing phosphorylation at these sites or its consequences may offer an alternative to inhibiting their catalytic activity to achieve therapeutic benefit.

Fra-2 regulates B cell development by enhancing IRF4 and Foxo1 transcription.

The role of AP-1 transcription factors in early B cell development and function is still incompletely characterized. Here we address the role of Fra-2 in B cell differentiation. Deletion of Fra-2 leads to impaired B cell proliferation in the bone marrow. In addition, IL-7-stimulated pro-B cell cultures revealed a reduced differentiation from large pre-B cells to small B cells and immature B cells. Gene profiling and chromatin immunoprecipitation sequencing analyses unraveled a transcriptional reduction of the transcription factors Foxo1, Irf4, Ikaros, and Aiolos in Fra-2-deficient B cells. Moreover, expression of IL7Rα and Rag 1/2, downstream targets of Irf4 and Foxo1, were also reduced in the absence of Fra-2. Pro-B cell proliferation and small pre-B cell differentiation were fully rescued by expression of Foxo1 and Irf4 in Fra-2-deficient pro-B cells. Hence, Fra-2 is a key upstream regulator of Foxo1 and Irf4 expression and influences proliferation and differentiation of B cells at multiple stages.

A defined metabolic state in pre B cells governs B-cell development and is counterbalanced by Swiprosin-2/EFhd1.

B-cell development in the bone marrow comprises proliferative and resting phases in different niches. We asked whether B-cell metabolism relates to these changes. Compared to pro B and small pre B cells, large pre B cells revealed the highest glucose uptake and ROS but not mitochondrial mass, whereas small pre B cells exhibited the lowest mitochondrial membrane potential. Small pre B cells from Rag1-/-;33.C9 µ heavy chain knock-in mice revealed decreased glycolysis (ECAR) and mitochondrial spare capacity compared to pro B cells from Rag1-/- mice. We were interested in the step regulating this metabolic switch from pro to pre B cells and uncovered that Swiprosin-2/EFhd1, a Ca2+-binding protein of the inner mitochondrial membrane involved in Ca2+-induced mitoflashes, is expressed in pro B cells, but downregulated by surface pre B-cell receptor expression. Knockdown and knockout of EFhd1 in 38B9 pro B cells decreased the oxidative phosphorylation/glycolysis (OCR/ECAR) ratio by increasing glycolysis, glycolytic capacity and reserve. Prolonged expression of EFhd1 in EFhd1 transgenic mice beyond the pro B cell stage increased expression of the mitochondrial co-activator PGC-1α in primary pre B cells, but reduced mitochondrial ATP production at the pro to pre B cell transition in IL-7 cultures. Transgenic EFhd1 expression caused a B-cell intrinsic developmental disadvantage for pro and pre B cells. Hence, coordinated expression of EFhd1 in pro B cells and by the pre BCR regulates metabolic changes and pro/pre B-cell development.