Data-driven modeling predicts gene regulatory network dynamics during the differentiation of multipotential hematopoietic progenitors

Cellular differentiation during hematopoiesis is guided by gene regulatory networks (GRNs) comprising transcription factors (TFs) and the effectors of cytokine signaling. Based largely on analyses conducted at steady state, these GRNs are thought to be organized as a hierarchy of bistable switches, with antagonism between Gata1 and PU.1 driving red- and white-blood cell differentiation. Here, we utilize transient gene expression patterns to infer the genetic architecture—the type and strength of regulatory interconnections—and dynamics of a twelve-gene GRN including key TFs and cytokine receptors. We trained gene circuits, dynamical models that learn genetic architecture, on high temporal-resolution gene-expression data from the differentiation of an inducible cell line into erythrocytes and neutrophils. The model is able to predict the consequences of gene knockout, knockdown, and overexpression experiments and the inferred interconnections are largely consistent with prior empirical evidence. The inferred genetic architecture is densely interconnected rather than hierarchical, featuring extensive cross-antagonism between genes from alternative lineages and positive feedback from cytokine receptors. The analysis of the dynamics of gene regulation in the model reveals that PU.1 is one of the last genes to be upregulated in neutrophil conditions and that the upregulation of PU.1 and other neutrophil genes is driven by Cebpa and Gfi1 instead. This model inference is confirmed in an independent single-cell RNA-Seq dataset from mouse bone marrow in which Cebpa and Gfi1 expression precedes the neutrophil-specific upregulation of PU.1 during differentiation. These results demonstrate that full PU.1 upregulation during neutrophil development involves regulatory influences extrinsic to the Gata1-PU.1 bistable switch. Furthermore, although there is extensive cross-antagonism between erythroid and neutrophil genes, it does not have a hierarchical structure. More generally, we show that the combination of high-resolution time series data and data-driven dynamical modeling can uncover the dynamics and causality of developmental events that might otherwise be obscured.

Bifidobacterium longum attenuates ovariectomy-induced bone loss via modulating the Immunoporotic Breg-Treg-Th17 cell axis

Discoveries in the last few years have emphasized the existence of an enormous breadth of communication between osteo-immune system. These discoveries fuel novel approaches for the treatment of several bone-pathologies including osteoporosis, an inflammatory bone anomaly affecting more than 500 million people globally. Bifidobacterium longum (BL) is preferred probiotic of choice due to its varied immunomodulatory potential in alleviating various inflammatory diseases. Here, we evaluate the effect of BL in ovariectomy (ovx)-induced post-menopausal osteoporotic mice model. Our in vitro findings reveal that BL suppresses the differentiation and functional activity of RANKL-induced osteoclastogenesis in both mouse bone marrow cells and human PBMCs. Our in vivo data clearly establish that BL exhibits osteoprotective potential via modulating the immunoporotic Breg-Treg-Th17 cell-axis. Furthermore, micro-CT and bone mechanical strength data support that BL supplementation significantly enhanced bone mass and strength, and improved microarchitecture in ovx mice. Remarkably, alteration in frequencies of CD19+CD1dhiCD5+ Bregs, CD4+Foxp3+IL-10+ Tregs, and CD4+Rorgt+IL-17+ Th17 immune cells in distinct lymphoid organs along with serum-cytokine data (enhanced anti-osteoclastogenic cytokines IFN-g; and IL-10 and reduced osteoclastogenic-cytokines IL-6, IL-17, and TNF-a) strongly support the immunomodulatory potential of BL. Altogether our findings establish a novel osteo-protective and immunoporotic potential of BL in augmenting bone health under osteoporotic conditions.

Wnt5A supports antigen cross-presentation and CD8 T cell activation.

Antigen processing, cross-presentation, and antigen-specific CD8 T cell response form part and parcel of T cell-mediated immunity. Yet, lacunae remain in our understanding of antigen processing/presentation and CD8 T cell response. Given the association of Wnt5A signaling with immune homeostasis, we evaluated the utility of Wnt5A in antigen processing, cross-presentation, and CD8 T cell activation. Using mouse bone marrow-derived dendritic cells as antigen-presenting cells and ovalbumin as a model antigen we found that Wnt5A mediated regulation of actin and proteasome dynamics is inherently associated with antigen processing. A Wnt5A-Actin-Protasome axis also contributes to antigen cross-presentation and antigen responsive CD8 T cell expansion. In concurrence with these observations, we demonstrated impaired activation of ovalbumin-specific CD8 T cells in ovalbumin immunized Wnt5A heterozygous mice as illustrated by their poor CD8 T cell recall response to ovalbumin when compared to similarly immunized wild type cohorts. Our results suggest that Wnt5A signaling-directed antigen processing/presentation could be vital for generating CD8 T cell recall response to antigen, thus shedding light on a critical parameter of immunity.

GM-CSF impairs erythropoiesis by disrupting erythroblastic island formation via macrophages

Anemia is a significant complication of chronic inflammation and may be related to dysregulated activities among erythroblastic island (EBI) macrophages. GM-CSF was reported to be upregulated and attracted as a therapeutic target in many inflammatory diseases. Among EBIs, we found that the GM-CSF receptor is preferentially and highly expressed among EBI macrophages but not among erythroblasts. GM-CSF treatment significantly decreases human EBI formation in vitro by decreasing the adhesion molecule expression of CD163. RNA-sequence analysis suggests that GM-CSF treatment impairs the supporting function of human EBI macrophages during erythropoiesis. GM-CSF treatment also polarizes human EBI macrophages from M2-like type to M1-like type. In addition, GM-CSF decreases mouse bone marrow (BM) erythroblasts as well as EBI macrophages, leading to a reduction in EBI numbers. In defining the molecular mechanism at work, we found that GM-CSF treatment significantly decreases the adhesion molecule expression of CD163 and Vcam1 in vivo. Importantly, GM-CSF treatment also decreases the phagocytosis rate of EBI macrophages in mouse BM as well as decreases the expression of the engulfment-related molecules Mertk, Axl, and Timd4. In addition, GM-CSF treatment polarizes mouse BM EBI macrophages from M2-like type to M1-like type. Thus, we document that GM-CSF impairs EBI formation in mice and humans. Our findings support that targeting GM-CSF or reprogramming EBI macrophages might be a novel strategy to treat anemia resulting from inflammatory diseases.

Isolation of High Purity Mouse Mesenchymal Stem Cells through Depleting Macrophages Using Liposomal Clodronate

BACKGROUND: The use of mouse bone marrow mesenchymal stem cells (mBMSCs) represents a promising strategy for performing preclinical studies in the field of cell-based regenerative medicine; however, mBMSCs obtained via conventional isolation methods have two drawbacks, i.e., (i) they are heterogeneous due to frequent macrophage contamination, and (ii) they require long-term culturing for expansion. METHODS: In the present study, we report a novel strategy to generate highly pure mBMSCs using liposomal clodronate. This approach is based on the properties of the two cell populations, i.e., BMSCs (to adhere to the plasticware in culture dishes) and macrophages (to phagocytose liposomes). RESULTS: Liposomal clodronate added during the first passage of whole bone marrow culture was selectively engulfed by macrophages in the heterogeneous cell population, resulting in their effective elimination without affecting the MSCs. This method allowed the generation of numerous high-purity Sca-1+CD44+F4/80− mBMSCs (> 95%) with just one passaging. Comparative studies with mBMSCs obtained using conventional methods revealed that the mBMSCs obtained in the present study had remarkably improved experimental utilities, as demonstrated by in vitro multilineage differentiation and in vivo ectopic bone formation assays. CONCLUSION: Our newly developed method, which enables the isolation of mBMSCs using simple and convenient protocol, will aid preclinical studies based on the use of MSCs.

Intracellular NAD+ Depletion Confers a Priming Signal for NLRP3 Inflammasome Activation

Nicotinamide adenine dinucleotide (NAD+) is an important cofactor in many redox and non-redox NAD+-consuming enzyme reactions. Intracellular NAD+ level steadily declines with age, but its role in the innate immune potential of myeloid cells remains elusive. In this study, we explored whether NAD+ depletion by FK866, a highly specific inhibitor of the NAD salvage pathway, can affect pattern recognition receptor-mediated responses in macrophages. NAD+-depleted mouse bone marrow-derived macrophages (BMDMs) exhibited similar levels of proinflammatory cytokine production in response to LPS or poly (I:C) stimulation compared with untreated cells. Instead, FK866 facilitated robust caspase-1 activation in BMDMs in the presence of NLRP3-activating signals such as ATP and nigericin, a potassium ionophore. However, this FK866-mediated caspase-1 activation was completely abolished in Nlrp3-deficient macrophages. FK866 plus nigericin stimulation caused an NLRP3-dependent assembly of inflammasome complex. In contrast, restoration of NAD+ level by supplementation with nicotinamide mononucleotide abrogated the FK866-mediated caspase-1 cleavage. FK866 did not induce or increase the expression levels of NLRP3 and interleukin (IL)-1β but drove mitochondrial retrograde transport into the perinuclear region. FK866-nigericin-induced mitochondrial transport is critical for caspase-1 cleavage in macrophages. Consistent with the in vitro experiments, intradermal coinjection of FK866 and ATP resulted in robust IL-1β expression and caspase-1 activation in the skin of wild-type, but not Nlrp3-deficient mice. Collectively, our data suggest that NAD+ depletion provides a non-transcriptional priming signal for NLRP3 activation via mitochondrial perinuclear clustering, and aging-associated NAD+ decline can trigger NLRP3 inflammasome activation in ATP-rich environments.

Antimutagenic Activity of Cassia Auriculata Linn Fractions along with Anticancer Activity in Male Albino Mice

Background: In recent years, there has been a surge in interest in studying plant-derived materials and their impact on DNA. Herbal products include a number of natural substances that may help protect cells against mutagen-induced cell damage. Aim: The purpose of this research was to assess the genotoxic effects of Cassia Auriculata Linn flavonoids (CAF) and Cassia Auriculata Linn saponin (CAS) rich fractions on mouse bone marrow cells utilizing chromosomal aberration test and micronucleus assay. Methodology: The suppressive impact of CAF and CAS on 7, 12-dimethylbenz (α) anthracene (DMBA) and Croton oil induced skin tumor promotion in mice with topical administration twice weekly for 18 weeks is also investigated in this work. Three dosages of 100 and 200 mg/kg body weight were used. Single oral dosages of CAF and CAS Fraction at the three levels did not enhance the number of micronucleate polychromatic erythrocytes in the micronucleus experiment. Result: In mice bone marrow cells, a single oral treatment of CAF and CAS fraction revealed no significant alterations in mitotic indices or chromosomal aberration induction. The clastogenicity of CYP was considerably decreased by pretreatment with CAF and CAS fraction. As a result, it can be stated that CAF and CAS fraction had no genotoxic impact on mouse bone marrow cells. Conclusions: The portions of Cassia Auriculata have been shown to be non-genotoxic and non-clastogenic at the quantities utilized in this investigation. CAF and CAS Fraction might possibly be a promising skin tumor promotion reducing agent, according to this research.

Hydroxyapatite composited PEEK with 3D porous surface enhances osteoblast differentiation through mediating NO by macrophage

The adverse immune response mediated by macrophages is one of the main factors that are prone to lead poor osseointegration of polyetheretherketone (PEEK) implants in clinic. Hence, endowing PEEK with immunomodulatory ability to avoid the adverse immune response becomes a promise strategy to promote bone repair. In this work, sulfonation and hydrothermal treatment were used to fabricate a three-dimensional porous surface on PEEK and hydroxyapatite composited PEEK. The hydroxyapatite composited PEEK with three-dimensional porous surface inhibited macrophages polarizing to M1 phenotype and down-regulated iNOS protein expression, which led to a nitric oxide concentration reduction in culture medium of mouse bone marrow mesenchymal stem cells (mBMSCs) under co-culture condition. The decrease of nitric oxide concentration could help to increase bone formation related OSX and ALP genes expressions and decrease bone resorption related MMP-9 and MMP-13 genes expressions via cAMP-PKA-RUNX2 pathway in mBMSCs. In summary, the hydroxyapatite composited PEEK with three-dimensional porous surface has the potential to promote osteogenesis of PEEK through immunomodulation, which provides a promising strategy to improve the bone repair ability of PEEK.

Dicalcium silicate-induced mitochondrial dysfunction and autophagy-mediated macrophagic inflammation promotes osteogenic differentiation of BMSCs

Dicalcium silicate (Ca2SiO4, C2S) has osteogenic potential but induces macrophagic inflammation. Mitochondrial function plays a vital role in macrophage polarization and macrophagic inflammation. The mitochondrial function of C2S-treated macrophages is still unclear. This study hypothesized: (1) the C2S modulates mitochondrial function and autophagy in macrophages to regulate macrophagic inflammation, and (2) C2S-induced macrophagic inflammation regulates osteogenesis. We used RAW264.7 cells as a model of macrophage. The C2S (75-150 μg/mL) extract was used to analyze the macrophagic mitochondrial function and macrophage-mediated effect on osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (BMSCs). The results showed that C2S extract (150 μg/mL) induced TNF-α, IL-1β, and IL-6 production in macrophages. C2S extract (150 μg/mL) enhanced reactive oxygen species (ROS) level and intracellular calcium level but reduced mitochondrial membrane potential (MtMP) and ATP production. TEM images showed reduced mitochondrial abundance and altered the mitochondrial morphology in C2S (150 μg/mL)-treated macrophages. Protein level expression of PINK1, Parkin, Beclin1, and LC3 was upregulated but TOMM20 was downregulated. mRNA sequencing and KEGG analysis showed that C2S-induced differentially expressed mRNAs in macrophages were mainly distributed in the essential signaling pathways involved in mitochondrial function and autophagy. The conditioned medium from C2S-treated macrophage (C2S-CM) robustly promoted osteogenic differentiation in BMSCs. In conclusion, our results indicate mitochondrial dysfunction and autophagy as the possible mechanism of C2S-induced macrophagic inflammation. The promotion of osteogenic differentiation of BMSCs by the C2S-induced macrophagic inflammation suggests the potential application of C2S in developing immunomodulatory bone grafts.

DnaJ, A Promising Vaccine Candidate Against Ureaplasma Urealyticum Infection

Background: Ureaplasma urealyticum (Uu) is an important sexually transmitted pathogen that is responsible for diseases such as non-gonococcal urethritis, chorioamnionitis and neonatal respiratory diseases. The rapid emergence of multidrug-resistant bacteria threatens the effective treatment of U. urealyticum infections. Considering this, vaccination could be an efficacious medical intervention to prevent U. urealyticum infection and disease. As a highly conserved molecular chaperone, DnaJ is expressed and upregulated by pathogens soon after infection. Here, we assessed the potential of recombinant DnaJ vaccine in a mouse model and dendritic cells (DCs). Results: The results showed that intramuscular administration of recombinant DnaJ induced robust humoral- and T helper (Th) 1 cell-mediated immune responses and protected against cervical infection, inflammation, and the pathologic sequelae after U. urealyticum infection. Importantly, DnaJ also induced the maturation of mouse bone marrow-derived DCs (BMDCs), ultimately promoting naïve T-cell differentiation towards the Th1 phenotype. In addition, adoptive immunisation of DnaJ-pulsed BMDCs elicited antigen-specific immunoglobulin G2 antibodies as well as a Th1-biased cellular response in mice. Conclusion: We concluded that DnaJ can be a promising vaccine candidate to control U. urealyticum infections.