MYC-mediated early glycolysis negatively regulates proinflammatory responses by controlling IRF4 in inflammatory macrophages.

MYC activates different metabolic programs in a cell-type- and cell-status-dependent manner. However, the role of MYC in inflammatory macrophages has not yet been determined. Metabolic and molecular analyses reveal that MYC, but not hypoxia inducible factor 1 (HIF1), is involved in enhancing early glycolytic flux during inflammatory macrophage polarization. Ablation of MYC decreases lactate production by regulating lactate dehydrogenase (LDH) activity and causes increased inflammatory cytokines by regulating interferon regulatory factor 4 (IRF4) in response to lipopolysaccharide. Moreover, myeloid-specific deletion of MYC and pharmacological inhibition of the MYC/LDH axis enhance inflammation and the bacterial clearance in vivo. These results elucidate the potential role of the MYC/LDH/IRF4 axis in inflammatory macrophages by connecting early glycolysis with inflammatory responses and suggest that modulating early glycolytic flux mediated by the MYC/LDH axis can be used to open avenues for the therapeutic modulation of macrophage polarization to fight against bacterial infection.

The Cytokine TNF Promotes Transcription Factor SREBP Activity and Binding to Inflammatory Genes to Activate Macrophages and Limit Tissue Repair.

Cytokine tumor necrosis factor (TNF)-mediated macrophage polarization is important for inflammatory disease pathogenesis, but the mechanisms regulating polarization are not clear. We performed transcriptomic and epigenomic analysis of the TNF response in primary human macrophages and revealed late-phase activation of SREBP2, the master regulator of cholesterol biosynthesis genes. TNF stimulation extended the genomic profile of SREBP2 occupancy to include binding to and activation of inflammatory and interferon response genes independently of its functions in sterol metabolism. Genetic ablation of SREBP function shifted the balance of macrophage polarization from an inflammatory to a reparative phenotype in peritonitis and skin wound healing models. Genetic ablation of SREBP activity in myeloid cells or topical pharmacological inhibition of SREBP improved skin wound healing under homeostatic and chronic inflammatory conditions. Our results identify a function and mechanism of action for SREBPs in augmenting TNF-induced macrophage activation and inflammation and open therapeutic avenues for promoting wound repair.

Type I interferons and the cytokine TNF cooperatively reprogram the macrophage epigenome to promote inflammatory activation.

Cross-regulation of Toll-like receptor (TLR) responses by cytokines is essential for effective host defense, avoidance of toxicity and homeostasis, but the underlying mechanisms are not well understood. Our comprehensive epigenomics approach to the analysis of human macrophages showed that the proinflammatory cytokines TNF and type I interferons induced transcriptional cascades that altered chromatin states to broadly reprogram responses induced by TLR4. TNF tolerized genes encoding inflammatory molecules to prevent toxicity while preserving the induction of genes encoding antiviral and metabolic molecules. Type I interferons potentiated the inflammatory function of TNF by priming chromatin to prevent the silencing of target genes of the transcription factor NF-κB that encode inflammatory molecules. The priming of chromatin enabled robust transcriptional responses to weak upstream signals. Similar chromatin regulation occurred in human diseases. Our findings reveal that signaling crosstalk between interferons and TNF is integrated at the level of chromatin to reprogram inflammatory responses, and identify previously unknown functions and mechanisms of action of these cytokines.

Interferon-γ Represses M2 Gene Expression in Human Macrophages by Disassembling Enhancers Bound by the Transcription Factor MAF.

Mechanisms by which interferon (IFN)-γ activates genes to promote macrophage activation are well studied, but little is known about mechanisms and functions of IFN-γ-mediated gene repression. We used an integrated transcriptomic and epigenomic approach to analyze chromatin accessibility, histone modifications, transcription-factor binding, and gene expression in IFN-γ-primed human macrophages. IFN-γ suppressed basal expression of genes corresponding to an "M2"-like homeostatic and reparative phenotype. IFN-γ repressed genes by suppressing the function of enhancers enriched for binding by transcription factor MAF. Mechanistically, IFN-γ disassembled a subset of enhancers by inducing coordinate suppression of binding by MAF, lineage-determining transcription factors, and chromatin accessibility. Genes associated with MAF-binding enhancers were suppressed in macrophages isolated from rheumatoid-arthritis patients, revealing a disease-associated signature of IFN-γ-mediated repression. These results identify enhancer inactivation and disassembly as a mechanism of IFN-γ-mediated gene repression and reveal that MAF regulates the macrophage enhancer landscape and is suppressed by IFN-γ to augment macrophage activation.

Tumor Necrosis Factor dynamically regulates the mRNA stabilome in rheumatoid arthritis fibroblast-like synoviocytes.

During rheumatoid arthritis (RA), Tumor Necrosis Factor (TNF) activates fibroblast-like synoviocytes (FLS) inducing in a temporal order a constellation of genes, which perpetuate synovial inflammation. Although the molecular mechanisms regulating TNF-induced transcription are well characterized, little is known about the impact of mRNA stability on gene expression and the impact of TNF on decay rates of mRNA transcripts in FLS. To address these issues we performed RNA sequencing and genome-wide analysis of the mRNA stabilome in RA FLS. We found that TNF induces a biphasic gene expression program: initially, the inducible transcriptome consists primarily of unstable transcripts but progressively switches and becomes dominated by very stable transcripts. This temporal switch is due to: a) TNF-induced prolonged stabilization of previously unstable transcripts that enables progressive transcript accumulation over days and b) sustained expression and late induction of very stable transcripts. TNF-induced mRNA stabilization in RA FLS occurs during the late phase of TNF response, is MAPK-dependent, and involves several genes with pathogenic potential such as IL6, CXCL1, CXCL3, CXCL8/IL8, CCL2, and PTGS2. These results provide the first insights into genome-wide regulation of mRNA stability in RA FLS and highlight the potential contribution of dynamic regulation of the mRNA stabilome by TNF to chronic synovitis.

Hypoxia-Sensitive COMMD1 Integrates Signaling and Cellular Metabolism in Human Macrophages and Suppresses Osteoclastogenesis.

Hypoxia augments inflammatory responses and osteoclastogenesis by incompletely understood mechanisms. We identified COMMD1 as a cell-intrinsic negative regulator of osteoclastogenesis that is suppressed by hypoxia. In human macrophages, COMMD1 restrained induction of NF-κB signaling and a transcription factor E2F1-dependent metabolic pathway by the cytokine RANKL. Downregulation of COMMD1 protein expression by hypoxia augmented RANKL-induced expression of inflammatory and E2F1 target genes and downstream osteoclastogenesis. E2F1 targets included glycolysis and metabolic genes including CKB that enabled cells to meet metabolic demands in challenging environments, as well as inflammatory cytokine-driven target genes. Expression quantitative trait locus analysis linked increased COMMD1 expression with decreased bone erosion in rheumatoid arthritis. Myeloid deletion of Commd1 resulted in increased osteoclastogenesis in arthritis and inflammatory osteolysis models. These results identify COMMD1 and an E2F-metabolic pathway as key regulators of osteoclastogenic responses under pathological inflammatory conditions and provide a mechanism by which hypoxia augments inflammation and bone destruction.

MYC-dependent oxidative metabolism regulates osteoclastogenesis via nuclear receptor ERRα.

Osteoporosis is a metabolic bone disorder associated with compromised bone strength and an increased risk of fracture. Inhibition of the differentiation of bone-resorbing osteoclasts is an effective strategy for the treatment of osteoporosis. Prior work by our laboratory and others has shown that MYC promotes osteoclastogenesis in vitro, but the underlying mechanisms are not well understood. In addition, the in vivo importance of osteoclast-expressed MYC in physiological and pathological bone loss is not known. Here, we have demonstrated that deletion of Myc in osteoclasts increases bone mass and protects mice from ovariectomy-induced (OVX-induced) osteoporosis. Transcriptomic analysis revealed that MYC drives metabolic reprogramming during osteoclast differentiation and functions as a metabolic switch to an oxidative state. We identified a role for MYC action in the transcriptional induction of estrogen receptor-related receptor α (ERRα), a nuclear receptor that cooperates with the transcription factor nuclear factor of activated T cells, c1 (NFATc1) to drive osteoclastogenesis. Accordingly, pharmacological inhibition of ERRα attenuated OVX-induced bone loss in mice. Our findings highlight a MYC/ERRα pathway that contributes to physiological and pathological bone loss by integrating the MYC/ERRα axis to drive metabolic reprogramming during osteoclast differentiation.

IFN-γ Induces Histone 3 Lysine 27 Trimethylation in a Small Subset of Promoters to Stably Silence Gene Expression in Human Macrophages.

The mechanisms by which IFN-γ activates expression of interferon-stimulated genes that have inflammatory and host defense functions are well understood. In contrast, little is known about how IFN-γ represses gene expression. By using transcriptomic and epigenomic analysis, we found that stable repression of a small group of genes by IFN-γ is associated with recruitment of the histone methyltransferase EZH2 and deposition of the negative mark histone 3 lysine 27 trimethylation (H3K27me3) at their promoters. Repressed genes included MERTK, PPARG, and RANK, which have anti-inflammatory functions and promote osteoclast differentiation. Gene repression and H3K27me3 persisted after IFN-γ signaling was terminated, and these silenced genes were no longer responsive to glucocorticoids, IL-4, and M-CSF. These results identify cytokine-induced H3K27 trimethylation as a mechanism that stabilizes gene silencing in macrophages. IFN-γ-induced macrophage activation is thus reinforced by a chromatin-based mechanism that blocks anti-inflammatory and opposing pathways.

Divergent clonal evolution of castration-resistant neuroendocrine prostate cancer.

An increasingly recognized resistance mechanism to androgen receptor (AR)-directed therapy in prostate cancer involves epithelial plasticity, in which tumor cells demonstrate low to absent AR expression and often have neuroendocrine features. The etiology and molecular basis for this 'alternative' treatment-resistant cell state remain incompletely understood. Here, by analyzing whole-exome sequencing data of metastatic biopsies from patients, we observed substantial genomic overlap between castration-resistant tumors that were histologically characterized as prostate adenocarcinomas (CRPC-Adeno) and neuroendocrine prostate cancer (CRPC-NE); analysis of biopsy samples from the same individuals over time points to a model most consistent with divergent clonal evolution. Genome-wide DNA methylation analysis revealed marked epigenetic differences between CRPC-NE tumors and CRPC-Adeno, and also designated samples of CRPC-Adeno with clinical features of AR independence as CRPC-NE, suggesting that epigenetic modifiers may play a role in the induction and/or maintenance of this treatment-resistant state. This study supports the emergence of an alternative, 'AR-indifferent' cell state through divergent clonal evolution as a mechanism of treatment resistance in advanced prostate cancer.

Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH.

More than half of human colorectal cancers (CRCs) carry either KRAS or BRAF mutations and are often refractory to approved targeted therapies. We found that cultured human CRC cells harboring KRAS or BRAF mutations are selectively killed when exposed to high levels of vitamin C. This effect is due to increased uptake of the oxidized form of vitamin C, dehydroascorbate (DHA), via the GLUT1 glucose transporter. Increased DHA uptake causes oxidative stress as intracellular DHA is reduced to vitamin C, depleting glutathione. Thus, reactive oxygen species accumulate and inactivate glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Inhibition of GAPDH in highly glycolytic KRAS or BRAF mutant cells leads to an energetic crisis and cell death not seen in KRAS and BRAF wild-type cells. High-dose vitamin C impairs tumor growth in Apc/Kras(G12D) mutant mice. These results provide a mechanistic rationale for exploring the therapeutic use of vitamin C for CRCs with KRAS or BRAF mutations.

Interferon-γ regulates cellular metabolism and mRNA translation to potentiate macrophage activation.

Interferon-γ (IFN-γ) primes macrophages for enhanced microbial killing and inflammatory activation by Toll-like receptors (TLRs), but little is known about the regulation of cell metabolism or mRNA translation during this priming. We found that IFN-γ regulated the metabolism and mRNA translation of human macrophages by targeting the kinases mTORC1 and MNK, both of which converge on the selective regulator of translation initiation eIF4E. Physiological downregulation of mTORC1 by IFN-γ was associated with autophagy and translational suppression of repressors of inflammation such as HES1. Genome-wide ribosome profiling in TLR2-stimulated macrophages showed that IFN-γ selectively modulated the macrophage translatome to promote inflammation, further reprogram metabolic pathways and modulate protein synthesis. These results show that IFN-γ-mediated metabolic reprogramming and translational regulation are key components of classical inflammatory macrophage activation.

Inhibition of osteoclastogenesis and inflammatory bone resorption by targeting BET proteins and epigenetic regulation.

Emerging evidence suggests that RANKL-induced changes in chromatin state are important for osteoclastogenesis, but these epigenetic mechanisms are not well understood and have not been therapeutically targeted. In this study, we find that the small molecule I-BET151 that targets bromo and extra-terminal (BET) proteins that 'read' chromatin states by binding to acetylated histones strongly suppresses osteoclastogenesis. I-BET151 suppresses pathologic bone loss in TNF-induced inflammatory osteolysis, inflammatory arthritis and post-ovariectomy models. Transcriptome analysis identifies a MYC-NFAT axis important for osteoclastogenesis. Mechanistically, I-BET151 inhibits expression of the master osteoclast regulator NFATC1 by suppressing expression and recruitment of its newly identified upstream regulator MYC. MYC is elevated in rheumatoid arthritis macrophages and its induction by RANKL is important for osteoclastogenesis and TNF-induced bone resorption. These findings highlight the importance of an I-BET151-inhibited MYC-NFAT axis in osteoclastogenesis, and suggest targeting epigenetic chromatin regulators holds promise for treatment of inflammatory and oestrogen deficiency-mediated pathologic bone resorption.

The oestrogen receptor alpha-regulated lncRNA NEAT1 is a critical modulator of prostate cancer.

The androgen receptor (AR) plays a central role in establishing an oncogenic cascade that drives prostate cancer progression. Some prostate cancers escape androgen dependence and are often associated with an aggressive phenotype. The oestrogen receptor alpha (ERα) is expressed in prostate cancers, independent of AR status. However, the role of ERα remains elusive. Using a combination of chromatin immunoprecipitation (ChIP) and RNA-sequencing data, we identified an ERα-specific non-coding transcriptome signature. Among putatively ERα-regulated intergenic long non-coding RNAs (lncRNAs), we identified nuclear enriched abundant transcript 1 (NEAT1) as the most significantly overexpressed lncRNA in prostate cancer. Analysis of two large clinical cohorts also revealed that NEAT1 expression is associated with prostate cancer progression. Prostate cancer cells expressing high levels of NEAT1 were recalcitrant to androgen or AR antagonists. Finally, we provide evidence that NEAT1 drives oncogenic growth by altering the epigenetic landscape of target gene promoters to favour transcription.

RBP-J imposes a requirement for ITAM-mediated costimulation of osteoclastogenesis.

Osteoclastogenesis requires activation of RANK signaling as well as costimulatory signals from immunoreceptor tyrosine-based activation motif-containing (ITAM-containing) receptors/adaptors, predominantly tyrosine kinase-binding proteins DAP12 and FcRγ, in osteoclast precursors. It is not well understood how costimulatory signals are regulated and integrated with RANK signaling. Here, we found that osteopetrotic bone phenotypes in mice lacking DAP12 or DAP12 and FcRγ are mediated by the transcription factor RBP-J, as deletion of Rbpj in these mice substantially rescued the defects of bone remodeling. Using a TNF-α-induced model of inflammatory bone resorption, we determined that RBP-J deficiency enables TNF-α to induce osteoclast formation and bone resorption in DAP12-deficient animals. Thus, RBP-J imposes a requirement for ITAM-mediated costimulation of RANKL or TNF-α-induced osteoclastogenesis. Mechanistically, RBP-J suppressed induction of key osteoclastogenic factors NFATc1, BLIMP1, and c-FOS by inhibiting ITAM-mediated expression and function of PLCγ2 and activation of downstream calcium-CaMKK/PYK2 signaling. Moreover, RBP-J suppressed Plcg2 expression and downstream calcium oscillations indirectly by a TGF-ß/PLCγ2/calcium axis. Together, our findings indicate that RBP-J suppresses ITAM-mediated costimulation, thereby limiting crosstalk between ITAM and RANK/TNFR signaling and allowing fine tuning of osteoclastogenesis during bone homeostasis and under inflammatory conditions. Furthermore, these data suggest that environmental cues that regulate RBP-J expression/function potentially modulate the requirement for costimulatory signaling for osteoclast differentiation and bone remodeling.

Histone methyltransferase MMSET/NSD2 alters EZH2 binding and reprograms the myeloma epigenome through global and focal changes in H3K36 and H3K27 methylation.

Overexpression of the histone methyltransferase MMSET in t(4;14)+ multiple myeloma patients is believed to be the driving factor in the pathogenesis of this subtype of myeloma. MMSET catalyzes dimethylation of lysine 36 on histone H3 (H3K36me2), and its overexpression causes a global increase in H3K36me2, redistributing this mark in a broad, elevated level across the genome. Here, we demonstrate that an increased level of MMSET also induces a global reduction of lysine 27 trimethylation on histone H3 (H3K27me3). Despite the net decrease in H3K27 methylation, specific genomic loci exhibit enhanced recruitment of the EZH2 histone methyltransferase and become hypermethylated on this residue. These effects likely contribute to the myeloma phenotype since MMSET-overexpressing cells displayed increased sensitivity to EZH2 inhibition. Furthermore, we demonstrate that such MMSET-mediated epigenetic changes require a number of functional domains within the protein, including PHD domains that mediate MMSET recruitment to chromatin. In vivo, targeting of MMSET by an inducible shRNA reversed histone methylation changes and led to regression of established tumors in athymic mice. Together, our work elucidates previously unrecognized interplay between MMSET and EZH2 in myeloma oncogenesis and identifies domains to be considered when designing inhibitors of MMSET function.

Modulation of TNF-induced macrophage polarization by synovial fibroblasts.

Mesenchymal stromal cells have emerged as powerful modulators of the immune system. In this study, we explored how the human macrophage response to TNF is regulated by human synovial fibroblasts, the representative stromal cell type in the synovial lining of joints that become activated during inflammatory arthritis. We found that synovial fibroblasts strongly suppressed TNF-mediated induction of an IFN-ß autocrine loop and downstream expression of IFN-stimulated genes (ISGs), including chemokines CXCL9 and CXCL10 that are characteristic of classical macrophage activation. TNF induced the production of soluble synovial fibroblast factors that suppressed the macrophage production of IFN-ß, and cooperated with TNF to limit the responsiveness of macrophages to IFN-ß by suppressing activation of Jak-STAT signaling. Genome-wide transcriptome analysis showed that cocultured synovial fibroblasts modulate the expression of approximately one third of TNF-regulated genes in macrophages, including genes in pathways important for macrophage survival and polarization toward an alternatively activated phenotype. Pathway analysis revealed that gene expression programs regulated by synovial fibroblasts in our coculture system were also regulated in rheumatoid arthritis synovial macrophages, suggesting that these fibroblast-mediated changes may contribute to rheumatoid arthritis pathogenesis. This work furthers our understanding of the interplay between innate immune and stromal cells during an inflammatory response, one that is particularly relevant to inflammatory arthritis. Our findings also identify modulation of macrophage phenotype as a new function for synovial fibroblasts that may prove to be a contributing factor in arthritis pathogenesis.

Synergistic activation of inflammatory cytokine genes by interferon-γ-induced chromatin remodeling and toll-like receptor signaling.

Synergistic activation of inflammatory cytokine genes by interferon-γ (IFN-γ) and Toll-like receptor (TLR) signaling is important for innate immunity and inflammatory disease pathogenesis. Enhancement of TLR signaling, a previously proposed mechanism, is insufficient to explain strong synergistic activation of cytokine production in human macrophages. Rather, we found that IFN-γ induced sustained occupancy of transcription factors STAT1, IRF-1, and associated histone acetylation at promoters and enhancers at the TNF, IL6, and IL12B loci. This priming of chromatin did not activate transcription but greatly increased and prolonged recruitment of TLR4-induced transcription factors and RNA polymerase II to gene promoters and enhancers. Priming sensitized cytokine transcription to suppression by Jak inhibitors. Genome-wide analysis revealed pervasive priming of regulatory elements by IFN-γ and linked coordinate priming of promoters and enhancers with synergistic induction of transcription. Our results provide a synergy mechanism whereby IFN-γ creates a primed chromatin environment to augment TLR-induced gene transcription.

A hybrid mechanism of action for BCL6 in B cells defined by formation of functionally distinct complexes at enhancers and promoters.

The BCL6 transcriptional repressor is required for the development of germinal center (GC) B cells and diffuse large B cell lymphomas (DLBCLs). Although BCL6 can recruit multiple corepressors, its transcriptional repression mechanism of action in normal and malignant B cells is unknown. We find that in B cells, BCL6 mostly functions through two independent mechanisms that are collectively essential to GC formation and DLBCL, both mediated through its N-terminal BTB domain. These are (1) the formation of a unique ternary BCOR-SMRT complex at promoters, with each corepressor binding to symmetrical sites on BCL6 homodimers linked to specific epigenetic chromatin features, and (2) the "toggling" of active enhancers to a poised but not erased conformation through SMRT-dependent H3K27 deacetylation, which is mediated by HDAC3 and opposed by p300 histone acetyltransferase. Dynamic toggling of enhancers provides a basis for B cells to undergo rapid transcriptional and phenotypic changes in response to signaling or environmental cues.

Epigenomic alterations in localized and advanced prostate cancer.

Although prostate cancer (PCa) is the second leading cause of cancer death among men worldwide, not all men diagnosed with PCa will die from the disease. A critical challenge, therefore, is to distinguish indolent PCa from more advanced forms to guide appropriate treatment decisions. We used Enhanced Reduced Representation Bisulfite Sequencing, a genome-wide high-coverage single-base resolution DNA methylation method to profile seven localized PCa samples, seven matched benign prostate tissues, and six aggressive castration-resistant prostate cancer (CRPC) samples. We integrated these data with RNA-seq and whole-genome DNA-seq data to comprehensively characterize the PCa methylome, detect changes associated with disease progression, and identify novel candidate prognostic biomarkers. Our analyses revealed the correlation of cytosine guanine dinucleotide island (CGI)-specific hypermethylation with disease severity and association of certain breakpoints (deletion, tandem duplications, and interchromosomal translocations) with DNA methylation. Furthermore, integrative analysis of methylation and single-nucleotide polymorphisms (SNPs) uncovered widespread allele-specific methylation (ASM) for the first time in PCa. We found that most DNA methylation changes occurred in the context of ASM, suggesting that variations in tumor epigenetic landscape of individuals are partly mediated by genetic differences, which may affect PCa disease progression. We further selected a panel of 13 CGIs demonstrating increased DNA methylation with disease progression and validated this panel in an independent cohort of 20 benign prostate tissues, 16 PCa, and 8 aggressive CRPCs. These results warrant clinical evaluation in larger cohorts to help distinguish indolent PCa from advanced disease.

NFIB is a governor of epithelial-melanocyte stem cell behaviour in a shared niche.

Adult stem cells reside in specialized niches where they receive environmental cues to maintain tissue homeostasis. In mammals, the stem cell niche within hair follicles is home to epithelial hair follicle stem cells and melanocyte stem cells, which sustain cyclical bouts of hair regeneration and pigmentation. To generate pigmented hairs, synchrony is achieved such that upon initiation of a new hair cycle, stem cells of each type activate lineage commitment. Dissecting the inter-stem-cell crosstalk governing this intricate coordination has been difficult, because mutations affecting one lineage often affect the other. Here we identify transcription factor NFIB as an unanticipated coordinator of stem cell behaviour. Hair follicle stem-cell-specific conditional targeting of Nfib in mice uncouples stem cell synchrony. Remarkably, this happens not by perturbing hair cycle and follicle architecture, but rather by promoting melanocyte stem cell proliferation and differentiation. The early production of melanin is restricted to melanocyte stem cells at the niche base. Melanocyte stem cells more distant from the dermal papilla are unscathed, thereby preventing hair greying typical of melanocyte stem cell differentiation mutants. Furthermore, we pinpoint KIT-ligand as a dermal papilla signal promoting melanocyte stem cell differentiation. Additionally, through chromatin-immunoprecipitation with high-throughput-sequencing and transcriptional profiling, we identify endothelin 2 (Edn2) as an NFIB target aberrantly activated in NFIB-deficient hair follicle stem cells. Ectopically induced Edn2 recapitulates NFIB-deficient phenotypes in wild-type mice. Conversely, endothelin receptor antagonists and/or KIT blocking antibodies prevent precocious melanocyte stem cell differentiation in the NFIB-deficient niche. Our findings reveal how melanocyte and hair follicle stem cell behaviours maintain reliance upon cooperative factors within the niche, and how this can be uncoupled in injury, stress and disease states.