Granulocytes are pivotal regulators of tissue injury. However, the transcriptional mechanisms that regulate granulopoiesis under inflammatory conditions are poorly understood. Here we show that the transcriptional coregulator B cell leukemia/lymphoma 3 (Bcl3) limits granulopoiesis under emergency (i.e., inflammatory) conditions, but not homeostatic conditions. Treatment of mouse myeloid progenitors with G-CSF — serum concentrations of which rise under inflammatory conditions — rapidly increased Bcl3 transcript accumulation in a STAT3-dependent manner. Bcl3-deficient myeloid progenitors demonstrated an enhanced capacity to proliferate and differentiate into granulocytes following G-CSF stimulation, whereas the accumulation of Bcl3 protein attenuated granulopoiesis in an NF-κB p50–dependent manner. In a clinically relevant model of transplant-mediated lung ischemia reperfusion injury, expression of Bcl3 in recipients inhibited emergency granulopoiesis and limited acute graft damage. These data demonstrate a critical role for Bcl3 in regulating emergency granulopoiesis and suggest that targeting the differentiation of myeloid progenitors may be a therapeutic strategy for preventing inflammatory lung injury.
Daniel Kreisel, Seiichiro Sugimoto, Jeremy Tietjens, Jihong Zhu, Sumiharu Yamamoto, Alexander S. Krupnick, Ruaidhri J. Carmody, Andrew E. Gelman
Chong Chen, Yu Liu, Yang Liu, Pan Zheng
The mammalian target of rapamycin (mTOR) is a signaling molecule that senses environmental cues, such as nutrient status and oxygen supply, to regulate cell growth, proliferation, and other functions. Unchecked, sustained mTOR activity results in defects in HSC function. Inflammatory conditions, such as autoimmune disease, are often associated with defective hematopoiesis. Here, we investigated whether hyperactivation of mTOR in HSCs contributes to hematopoietic defects in autoimmunity and inflammation. We found that in mice deficient in Foxp3 (scurfy mice), a model of autoimmunity, the development of autoimmune disease correlated with progressive bone marrow loss and impaired regenerative capacity of HSCs in competitive bone marrow transplantation. Similarly, LPS-mediated inflammation in C57BL/6 mice led to massive bone marrow cell death and impaired HSC function. Importantly, treatment with rapamycin in both models corrected bone marrow hypocellularity and partially restored hematopoietic activity. In cultured mouse bone marrow cells, treatment with either of the inflammatory cytokines IL-6 or TNF-α was sufficient to activate mTOR, while preventing mTOR activation in vivo required simultaneous inhibition of CCL2, IL-6, and TNF-α. These data strongly suggest that mTOR activation in HSCs by inflammatory cytokines underlies defective hematopoiesis in autoimmune disease and inflammation.
Chong Chen, Yu Liu, Yang Liu, Pan Zheng
The alternative pathway (AP) of complement activation is constitutively active and must be regulated by host proteins to prevent autologous tissue injury. Dysfunction of AP regulatory proteins has been linked to several human inflammatory disorders. Properdin is a positive regulator of AP complement activation that has been shown to extend the half-life of cell surface–bound C3 convertase C3bBb; it may also initiate AP complement activation. Here, we demonstrate a critical role for properdin in autologous tissue injury mediated by AP complement activation. We identified myeloid lineage cells as the principal source of plasma properdin by generating mice with global and tissue-specific knockout of Cfp (which encodes properdin) and by generating BM chimeric mice. Properdin deficiency rescued mice from AP complement–mediated embryonic lethality caused by deficiency of the membrane complement regulator Crry and markedly reduced disease severity in the K/BxN model of arthritis. Ab neutralization of properdin in WT mice similarly ameliorated arthritis development, whereas reconstitution of properdin-null mice with exogenous properdin restored arthritis sensitivity. These data implicate systemic properdin as a key contributor to AP complement–mediated injury and support its therapeutic targeting in complement-dependent human diseases.
Yuko Kimura, Lin Zhou, Takashi Miwa, Wen-Chao Song
Activated protein C (aPC) therapy reduces mortality in adult patients with severe sepsis. In mouse endotoxemia and sepsis models, mortality reduction requires the cell signaling function of aPC, mediated through protease-activated receptor–1 (PAR1) and endothelial protein C receptor (EPCR; also known as Procr). Candidate cellular targets of aPC include vascular endothelial cells and leukocytes. Here, we show that expression of EPCR and PAR1 on hematopoietic cells is required in mice for an aPC variant that mediates full cell signaling activity but only minimal anticoagulant function (5A-aPC) to reduce the mortality of endotoxemia. Expression of EPCR in mature murine immune cells was limited to a subset of CD8+ conventional dendritic cells. Adoptive transfer of splenic CD11chiPDCA-1– dendritic cells from wild-type mice into animals with hematopoietic EPCR deficiency restored the therapeutic efficacy of aPC, whereas transfer of EPCR-deficient CD11chi dendritic cells or wild-type CD11chi dendritic cells depleted of EPCR+ cells did not. In addition, 5A-aPC inhibited the inflammatory response of conventional dendritic cells independent of EPCR and suppressed IFN-γ production by natural killer–like dendritic cells. These data reveal an essential role for EPCR and PAR1 on hematopoietic cells, identify EPCR-expressing dendritic immune cells as a critical target of aPC therapy, and document EPCR-independent antiinflammatory effects of aPC on innate immune cells.
Edward Kerschen, Irene Hernandez, Mark Zogg, Shuang Jia, Martin J. Hessner, Jose Fernandez, John H. Griffin, Claudia S. Huettner, Francis J. Castellino, Hartmut Weiler
Hepcidin is a peptide hormone that regulates iron homeostasis and acts as an antimicrobial peptide. It is expressed and secreted by a variety of cell types in response to iron loading and inflammation. Hepcidin mediates iron homeostasis by binding to the iron exporter ferroportin, inducing its internalization and degradation via activation of the protein kinase Jak2 and the subsequent phosphorylation of ferroportin. Here we have shown that hepcidin-activated Jak2 also phosphorylates the transcription factor Stat3, resulting in a transcriptional response. Hepcidin treatment of ferroportin-expressing mouse macrophages showed changes in mRNA expression levels of a wide variety of genes. The changes in transcript levels for half of these genes were a direct effect of hepcidin, as shown by cycloheximide insensitivity, and dependent on the presence of Stat3. Hepcidin-mediated transcriptional changes modulated LPS-induced transcription in both cultured macrophages and in vivo mouse models, as demonstrated by suppression of IL-6 and TNF-α transcript and secreted protein. Hepcidin-mediated transcription in mice also suppressed toxicity and morbidity due to single doses of LPS, poly(I:C), and turpentine, which is used to model chronic inflammatory disease. Most notably, we demonstrated that hepcidin pretreatment protected mice from a lethal dose of LPS and that hepcidin-knockout mice could be rescued from LPS toxicity by injection of hepcidin. The results of our study suggest a new function for hepcidin in modulating acute inflammatory responses.
Ivana De Domenico, Tian Y. Zhang, Curry L. Koening, Ryan W. Branch, Nyall London, Eric Lo, Raymond A. Daynes, James P. Kushner, Dean Li, Diane M. Ward, Jerry Kaplan
Necrosis stimulates inflammation, and this response is medically relevant because it contributes to the pathogenesis of a number of diseases. It is thought that necrosis stimulates inflammation because dying cells release proinflammatory molecules that are recognized by the immune system. However, relatively little is known about the molecular identity of these molecules and their contribution to responses in vivo. Here, we investigated the role of uric acid in the inflammatory response to necrotic cells in mice. We found that dead cells not only released intracellular stores of uric acid but also produced it in large amounts postmortem as nucleic acids were degraded. Using newly developed Tg mice that have reduced levels of uric acid either intracellularly and/or extracellularly, we found that uric acid depletion substantially reduces the cell death–induced inflammatory response. Similar results were obtained with pharmacological treatments that reduced uric acid levels either by blocking its synthesis or hydrolyzing it in the extracellular fluids. Importantly, uric acid depletion selectively inhibited the inflammatory response to dying cells but not to microbial molecules or sterile irritant particles. Collectively, our data identify uric acid as a proinflammatory molecule released from dying cells that contributes significantly to the cell death–induced inflammatory responses in vivo.
Hajime Kono, Chun-Jen Chen, Fernando Ontiveros, Kenneth L. Rock
Activated protein C (APC), the only FDA-approved biotherapeutic drug for sepsis, possesses anticoagulant, antiinflammatory, and barrier-protective activities. However, the mechanisms underlying its antiinflammatory functions are not well defined. Here, we report that the antiinflammatory activity of APC on macrophages is dependent on integrin CD11b/CD18, but not on endothelial protein C receptor (EPCR). We showed that CD11b/CD18 bound APC within specialized membrane microdomains/lipid rafts and facilitated APC cleavage and activation of protease-activated receptor–1 (PAR1), leading to enhanced production of sphingosine-1-phosphate (S1P) and suppression of the proinflammatory response of activated macrophages. Deletion of the γ-carboxyglutamic acid domain of APC, a region critical for its anticoagulant activity and EPCR-dependent barrier protection, had no effect on its antiinflammatory function. Genetic inactivation of CD11b, PAR1, or sphingosine kinase–1, but not EPCR, abolished the ability of APC to suppress the macrophage inflammatory response in vitro. Using an LPS-induced mouse model of lethal endotoxemia, we showed that APC administration reduced the mortality of wild-type mice, but not CD11b-deficient mice. These data establish what we believe to be a novel mechanism underlying the antiinflammatory activity of APC in the setting of endotoxemia and provide clear evidence that the antiinflammatory function of APC is distinct from its barrier-protective function and anticoagulant activities.
Chunzhang Cao, Yamei Gao, Yang Li, Toni M. Antalis, Francis J. Castellino, Li Zhang
HSCs are BM-derived, self-renewing multipotent cells that develop into circulating blood cells. They have been implicated in the repair of inflamed parenchymal tissue, but the signals that regulate their trafficking to sites of inflammation are unknown. As monocytes are recruited to sites of inflammation via chemoattractants that activate CCR2 on their surface, we investigated whether HSCs are also recruited to sites of inflammation through CCR2. Initial analysis indicated that in mice, CCR2 was expressed on subsets of HSCs and hematopoietic progenitor cells (HPCs) and that freshly isolated primitive hematopoietic cells (Lin–c-Kit+ cells) responded to CCR2 ligands in vitro. In vivo analysis indicated that after instillation of thioglycollate to cause aseptic inflammation and after administration of acetaminophen to induce liver damage, endogenous HSCs/HPCs were actively recruited to the peritoneum and liver, respectively, in WT but not Ccr2–/– mice. HSCs/HPCs recovered from the peritoneum successfully engrafted into the BM of irradiated primary and secondary recipients, confirming their self renewal and multipotency. Importantly, administration of exogenous WT, but not Ccr2–/–, HSCs/HPCs accelerated resolution of acetaminophen-induced liver damage and triggered the expression of genes characteristic of the macrophage M2 or repair phenotype. These findings reveal what we believe to be a novel role for CCR2 in the homing of HSCs/HPCs to sites of inflammation and suggest new functions for chemokines in promoting tissue repair and regeneration.
Yue Si, Chia-Lin Tsou, Kelsey Croft, Israel F. Charo
Down syndrome critical region gene 1 (DSCR-1) short variant (DSCR-1s) is an inhibitor of calcineurin/NFAT signaling encoded by exons 4–7 of DSCR1. We previously reported that VEGF induces DSCR-1s expression in endothelial cells, which in turn negatively feeds back to attenuate endothelial cell activation. Here, in order to characterize the role of the promoter that drives DSCR-1s expression in mediating inducible expression in vivo and to determine the functional relevance of DSCR-1s in inflammation, we targeted a DNA construct containing 1.7 kb of the human DSCR1s promoter coupled to the lacZ reporter to the hypoxanthine guanine phosphoribosyl transferase (Hprt) locus of mice. We determined that lacZ was uniformly expressed in the endothelium of transgenic embryos but was markedly downregulated postnatally. Systemic administration of VEGF or LPS in adult mice resulted in cyclosporine A–sensitive reactivation of the DSCR1s promoter and endogenous gene expression in a subset of organs, including the heart and brain. The DSCR1s promoter was similarly induced in the endothelium of tumor xenografts. In a mouse model of endotoxemia, DSCR-1s–deficient mice demonstrated increased sepsis mortality, whereas adenovirus-mediated DSCR-1s overexpression protected against LPS-induced lethality. Collectively, these data suggest that the DSCR1s promoter directs vascular bed–specific expression in activated endothelium and that DSCR-1s serves to dampen the host response to infection.
Takashi Minami, Kiichiro Yano, Mai Miura, Mika Kobayashi, Jun-ichi Suehiro, Patrick C. Reid, Takao Hamakubo, Sandra Ryeom, William C. Aird, Tatsuhiko Kodama
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