Pro-inflammatory cytokine blockade attenuates myeloid expansion in a murine model of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a debilitating autoimmune disease characterized by chronic inflammation and progressive destruction of joint tissue. It is also characterized by aberrant blood phenotypes including anemia and suppressed lymphopoiesis that contribute to morbidity in RA patients. However, the impact of RA on hematopoietic stem cells (HSC) has not been fully elucidated. Using a collagen-induced mouse model of human RA, we identified systemic inflammation and myeloid overproduction associated with activation of a myeloid differentiation gene program in HSC. Surprisingly, despite ongoing inflammation, HSC from arthritic mice remain in a quiescent state associated with activation of a proliferation arrest gene program. Strikingly, we found that inflammatory cytokine blockade using the interleukin-1 receptor antagonist anakinra led to an attenuation of inflammatory arthritis and myeloid expansion in the bone marrow of arthritic mice. In addition, anakinra reduced expression of inflammation-driven myeloid lineage and proliferation arrest gene programs in HSC of arthritic mice. Altogether, our findings show that inflammatory cytokine blockade can contribute to normalization of hematopoiesis in the context of chronic autoimmune arthritis.

CHD4 is essential for transcriptional repression and lineage progression in B lymphopoiesis.

Cell lineage specification is a tightly regulated process that is dependent on appropriate expression of lineage and developmental stage-specific transcriptional programs. Here, we show that Chromodomain Helicase DNA-binding protein 4 (CHD4), a major ATPase/helicase subunit of Nucleosome Remodeling and Deacetylase Complexes (NuRD) in lymphocytes, is essential for specification of the early B cell lineage transcriptional program. In the absence of CHD4 in B cell progenitors in vivo, development of these cells is arrested at an early pro-B-like stage that is unresponsive to IL-7 receptor signaling and unable to efficiently complete V(D)J rearrangements at Igh loci. Our studies confirm that chromatin accessibility and transcription of thousands of gene loci are controlled dynamically by CHD4 during early B cell development. Strikingly, CHD4-deficient pro-B cells express transcripts of many non-B cell lineage genes, including genes that are characteristic of other hematopoietic lineages, neuronal cells, and the CNS, lung, pancreas, and other cell types. We conclude that CHD4 inhibits inappropriate transcription in pro-B cells. Together, our data demonstrate the importance of CHD4 in establishing and maintaining an appropriate transcriptome in early B lymphopoiesis via chromatin accessibility.

Zinc Finger Protein 521 Regulates Early Hematopoiesis through Cell-Extrinsic Mechanisms in the Bone Marrow Microenvironment.

Zinc finger protein 521 (ZFP521), a DNA-binding protein containing 30 Krüppel-like zinc fingers, has been implicated in the differentiation of multiple cell types, including hematopoietic stem and progenitor cells (HSPC) and B lymphocytes. Here, we report a novel role for ZFP521 in regulating the earliest stages of hematopoiesis and lymphoid cell development via a cell-extrinsic mechanism. Mice with inactivated Zfp521 genes (Zfp521-/-) possess reduced frequencies and numbers of hematopoietic stem and progenitor cells, common lymphoid progenitors, and B and T cell precursors. Notably, ZFP521 deficiency changes bone marrow microenvironment cytokine levels and gene expression within resident HSPC, consistent with a skewing of hematopoiesis away from lymphopoiesis. These results advance our understanding of ZFP521's role in normal hematopoiesis, justifying further research to assess its potential as a target for cancer therapies.

Evolved Cellular Mechanisms to Respond to Genotoxic Insults: Implications for Radiation-Induced Hematologic Malignancies.

Human exposure to ionizing radiation is highly associated with adverse health effects, including reduced hematopoietic cell function and increased risk of carcinogenesis. The hematopoietic deficits manifest across blood cell types and persist for years after radiation exposure, suggesting a long-lived and multi-potent cellular reservoir for radiation-induced effects. As such, research has focused on identifying both the immediate and latent hematopoietic stem cell responses to radiation exposure. Radiation-associated effects on hematopoietic function and malignancy development have generally been attributed to the direct induction of mutations resulting from radiation-induced DNA damage. Other studies have illuminated the role of cellular programs that both limit and enhance radiation-induced tissue phenotypes and carcinogenesis. In this review, distinct but collaborative cellular responses to genotoxic insults are highlighted, with an emphasis on how these programmed responses impact hematopoietic cellular fitness and competition. These radiation-induced cellular programs include apoptosis, senescence and impaired self-renewal within the hematopoietic stem cell (HSC) pool. In the context of sporadic DNA damage to a cell, these cellular responses act in concert to restore tissue function and prevent selection for adaptive oncogenic mutations. But in the contexts of whole-tissue exposure or whole-body exposure to genotoxins, such as radiotherapy or chemotherapy, we propose that these programs can contribute to long-lasting tissue impairment and increased carcinogenesis.

Contrasting roles for C/EBPα and Notch in irradiation-induced multipotent hematopoietic progenitor cell defects.

Ionizing radiation (IR) is associated with reduced hematopoietic function and increased risk of hematopoietic malignancies, although the mechanisms behind these relationships remain poorly understood. Both effects of IR have been commonly attributed to the direct induction of DNA mutations, but evidence supporting these hypotheses is largely lacking. Here we demonstrate that IR causes long-term, somatically heritable, cell-intrinsic reductions in hematopoietic stem cell (HSC) and multipotent hematopoietic progenitor cell (mHPC) self-renewal that are mediated by C/EBPα and reversed by Notch. mHPC from previously irradiated (>9 weeks prior), homeostatically restored mice exhibit gene expression profiles consistent with their precocious differentiation phenotype, including decreased expression of HSC-specific genes and increased expression of myeloid program genes (including C/EBPα). These gene expression changes are reversed by ligand-mediated activation of Notch. Loss of C/EBPα expression is selected for within previously irradiated HSC and mHPC pools and is associated with reversal of IR-dependent precocious differentiation and restoration of self-renewal. Remarkably, restoration of mHPC self-renewal by ligand-mediated activation of Notch prevents selection for C/EBPα loss of function in previously irradiated mHPC pools. We propose that environmental insults prompt HSC to initiate a program limiting their self-renewal, leading to loss of the damaged HSC from the pool while allowing this HSC to temporarily contribute to differentiated cell pools. This "programmed mediocrity" is advantageous for the sporadic genotoxic insults animals have evolved to deal with but becomes tumor promoting when the entire HSC compartment is damaged, such as during total body irradiation, by increasing selective pressure for adaptive oncogenic mutations.

Ionizing radiation and hematopoietic malignancies: altering the adaptive landscape.

Somatic evolution, which underlies tumor progression, is driven by two essential components: (1) diversification of phenotypes through heritable mutations and epigenetic changes and (2) selection for mutant clones which possess higher fitness. Exposure to ionizing radiation (IR ) is highly associated with increased risk of carcinogenesis. This link is traditionally attributed to causation of oncogenic mutations through the mutagenic effects of irradiation. On the other hand, potential effects of irradiation on altering fitness and increasing selection for mutant clones are frequently ignored. Recent studies bring the effects of irradiation on fitness and selection into focus, demonstrating that IR exposure results in stable reductions in the fitness of hematopoietic stem and progenitor cell populations. These reductions of fitness are associated with alteration of the adaptive landscape, increasing the selective advantages conferred by certain oncogenic mutations. Therefore, the link between irradiation and carcinogenesis might be more complex than traditionally appreciated: while mutagenic effects of irradiation should increase the probability of occurrence of oncogenic mutations, IR can also work as a tumor promoter, increasing the selective expansion of clones bearing mutations which become advantageous in the irradiation-altered environment, such as activated mutations in Notch1 or disrupting mutations in p53.

Exploiting cross-priming to generate protective CD8 T-cell immunity rapidly.

The number of memory CD8 T cells generated by infection or vaccination correlates strongly with the degree of protection observed in infection and tumor models. Therefore, rapid induction of protective numbers of effector and memory CD8 T cells may be crucial in the case of malignancy, pandemic infection, or bioterrorism. Many studies have shown that amplifying T-cell numbers by prime-boost vaccination is most effective with a substantial time interval between immunizations. In contrast, immunization with peptide-coated mature dendritic cells (DCs) results in a CD8 T-cell response exhibiting accelerated acquisition of memory characteristics, including the ability to respond to booster immunization within days of initial priming. However, personalized DC immunization is too costly, labor intensive, and time-consuming for large-scale vaccination. Here, we demonstrate that in vivo cross-priming with cell-associated antigens or antigen-coated, biodegradable microspheres in the absence of adjuvant quickly generates CD8 T cells that display the phenotype and function of long-term memory populations. Importantly, cross-primed CD8 T cells can respond to booster immunization within days of the initial immunization to generate rapidly large numbers of effector and memory T cells that can protect against bacterial, viral, and parasitic infections, including lethal influenza and malaria-causing Plasmodium infection. Thus, accelerated CD8 T-cell memory after in vivo cross-priming in the absence of adjuvant is generalizable and can be exploited to generate protective immunity rapidly.

Alum induces innate immune responses through macrophage and mast cell sensors, but these sensors are not required for alum to act as an adjuvant for specific immunity.

To understand more about how the body recognizes alum we characterized the early innate and adaptive responses in mice injected with the adjuvant. Within hours of exposure, alum induces a type 2 innate response characterized by an influx of eosinophils, monocytes, neutrophils, DCs, NK cells and NKT cells. In addition, at least 13 cytokines and chemokines are produced within 4 h of injection including IL-1beta and IL-5. Optimal production of some of these, including IL-1beta, depends upon both macrophages and mast cells, whereas production of others, such as IL-5, depends on mast cells only, suggesting that both of these cell types can detect alum. Alum induces eosinophil accumulation partly through the production of mast cell derived IL-5 and histamine. Alum greatly enhances priming of endogenous CD4 and CD8 T cells independently of mast cells, macrophages, and of eosinophils. In addition, Ab levels and Th2 bias was similar in the absence of these cells. We found that the inflammation induced by alum was unchanged in caspase-1-deficient mice, which cannot produce IL-1beta. Furthermore, endogenous CD4 and CD8 T cell responses, Ab responses and the Th2 bias were also not impacted by the absence of caspase-1 or NLRP3. These data suggest that activation of the inflammasome and the type 2 innate response orchestrated by macrophages and mast cells in vivo are not required for adjuvant effect of alum on endogenous T and B cell responses.

DNA-like class R inhibitory oligonucleotides (INH-ODNs) preferentially block autoantigen-induced B-cell and dendritic cell activation in vitro and autoantibody production in lupus-prone MRL-Fas(lpr/lpr) mice in vivo.

INTRODUCTION: B cells have many different roles in systemic lupus erythematosus (SLE), ranging from autoantigen recognition and processing to effector functions (for example, autoantibody and cytokine secretion). Recent studies have shown that intracellular nucleic acid-sensing receptors, Toll-like receptor (TLR) 7 and TLR9, play an important role in the pathogenesis of SLE. Dual engagement of rheumatoid factor-specific AM14 B cells through the B-cell receptor (BCR) and TLR7/9 results in marked proliferation of autoimmune B cells. Thus, strategies to preferentially block innate activation through TLRs in autoimmune B cells may be preferred over non-selective B-cell depletion. METHODS: We have developed a new generation of DNA-like compounds named class R inhibitory oligonucleotides (INH-ODNs). We tested their effectiveness in autoimmune B cells and interferon-alpha-producing dendritic cells in vitro and in lupus-prone MRL-Faslpr/lpr mice in vivo. RESULTS: Class R INH-ODNs have 10- to 30-fold higher inhibitory potency when autoreactive B cells are synergistically activated through the BCR and associated TLR7 or 9 than when stimulation occurs via non-BCR-engaged TLR7/9. Inhibition of TLR9 requires the presence of both CCT and GGG triplets in an INH-ODN, whereas the inhibition of the TLR7 pathway appears to be sequence-independent but dependent on the phosphorothioate backbone. This difference was also observed in the MRL-Faslpr/lpr mice in vivo, where the prototypic class R INH-ODN was more effective in curtailing abnormal autoantibody secretion and prolonging survival. CONCLUSIONS: The increased potency of class R INH-ODNs for autoreactive B cells and dendritic cells may be beneficial for lupus patients by providing pathway-specific inhibition yet allowing them to generate protective immune response when needed.