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The risk of colon cancer is increased in patients with Crohn's disease and ulcerative colitis. Inflammation-induced DNA damage could be an important link between inflammation and cancer, although the pathways that link inflammation and DNA damage are incompletely defined. RAG2-deficient mice infected with Helicobacter hepaticus (Hh) develop colitis that progresses to lower bowel cancer. This process depends on nitric oxide (NO), a molecule with known mutagenic potential. We have previously hypothesized that production of NO by macrophages could be essential for Hh-driven carcinogenesis, however, whether Hh infection induces DNA damage in this model and whether this depends on NO has not been determined. Here we demonstrate that Hh infection of RAG2-deficient mice rapidly induces expression of iNOS and the development of DNA double-stranded breaks (DSBs) specifically in proliferating crypt epithelial cells. Generation of DSBs depended on iNOS activity, and further, induction of iNOS, the generation of DSBs, and the subsequent development of dysplasia were inhibited by depletion of the Hh-induced cytokine IL-22. These results demonstrate a strong association between Hh-induced DNA damage and the development of dysplasia, and further suggest that IL-22-dependent induction of iNOS within crypt epithelial cells rather than macrophages is a driving force in this process.
AIMS - To investigate the hypothesis that alteration in histone acetylation/deacetylation triggers aberrant STAT1/MyD88 expression in macrophages from diabetics. Increased STAT1/MyD88 expression is correlated with sterile inflammation in type 1 diabetic (T1D) mice.
METHODS - To induce diabetes, we injected low-doses of streptozotocin in C57BL/6 mice. Peritoneal or bone marrow-differentiated macrophages were cultured in 5mM (low) or 25mM (high glucose). ChIP analysis of macrophages from nondiabetic or diabetic mice was performed to determine acetylation of lysine 9 in histone H3 in MyD88 and STAT1 promoter regions. Macrophages from diabetic mice were treated with the histone acetyltransferase inhibitor anacardic acid (AA), followed by determination of mRNA expression by qPCR.
RESULTS - Increased STAT1 and MyD88 expression in macrophages from diabetic but not naive mice cultured in low glucose persisted for up to 6days. Macrophages from diabetic mice exhibited increased activity of histone acetyltransferases (HAT) and decreased histone deacetylases (HDAC) activity. We detected increased H3K9Ac binding to Stat1/Myd88 promoters in macrophages from T1D mice and AA in vitro treatment reduced STAT1 and MyD88 mRNA expression.
CONCLUSIONS/INTERPRETATION - These results indicate that histone acetylation drives elevated Stat1/Myd88 expression in macrophages from diabetic mice, and this mechanism may be involved in sterile inflammation and diabetes comorbidities.
Copyright © 2016 Elsevier Inc. All rights reserved.
EGF receptor (EGFR) in tumor cells serves as a tumor promoter. However, information about EGFR activation in macrophages in regulating M2 polarization and tumor development is limited. This study aimed to investigate the effects of EGFR activation in macrophages on M2 polarization and development of gastrointestinal tumors. IL-4, a cytokine to elicit M2 polarization, stimulated release of an EGFR ligand, HB-EGF, and transactivation and down-regulation of EGFR in Raw 264.7 cells and peritoneal macrophages from WT mice. Knockdown of HB-EGF in macrophages inhibited EGFR transactivation by IL-4. IL-4-stimulated STAT6 activation, Arg1 and YM1 gene expression, and HB-EGF production were further enhanced by inhibition of EGFR activity in Raw 264.7 cells using an EGFR kinase inhibitor and in peritoneal macrophages from Egfr(wa5) mice with kinase inactive EGFR and by knockdown of EGFR in peritoneal macrophages from Egfr(fl/fl) LysM-Cre mice with myeloid cell-specific EGFR deletion. Chitin induced a higher level of M2 polarization in peritoneal macrophages in Egfr(fl/fl) LysM-Cre mice than that in Egfr(fl/fl) mice. Accordingly, IL-4-conditioned medium stimulated growth and epithelial-to-mesenchymal transition in gastric epithelial and colonic tumor cells, which were suppressed by that from Raw 264.7 cells with HB-EGF knockdown but promoted by that from Egfr(wa5) and Egfr(fl/fl) LysM-Cre peritoneal macrophages. Clinical assessment revealed that the number of macrophages with EGFR expression became less, indicating decreased inhibitory effects on M2 polarization, in late stage of human gastric cancers. Thus, IL-4-stimulated HB-EGF-dependent transactivation of EGFR in macrophages may mediate inhibitory feedback for M2 polarization and HB-EGF production, thereby inhibiting gastrointestinal tumor growth.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Tityus serrulatus sting causes thousands of deaths annually worldwide. T. serrulatus-envenomed victims exhibit local or systemic reaction that culminates in pulmonary oedema, potentially leading to death. However, the molecular mechanisms underlying T. serrulatus venom (TsV) activity remain unknown. Here we show that TsV triggers NLRP3 inflammasome activation via K(+) efflux. Mechanistically, TsV triggers lung-resident cells to release PGE2, which induces IL-1β production via E prostanoid receptor 2/4-cAMP-PKA-NFκB-dependent mechanisms. IL-1β/IL-1R actions account for oedema and neutrophil recruitment to the lungs, leading to TsV-induced mortality. Inflammasome activation triggers LTB4 production and further PGE2 via IL-1β/IL-1R signalling. Activation of LTB4-BLT1/2 pathway decreases cAMP generation, controlling TsV-induced inflammation. Exogenous administration confirms LTB4 anti-inflammatory activity and abrogates TsV-induced mortality. These results suggest that the balance between LTB4 and PGE2 determines the amount of IL-1β inflammasome-dependent release and the outcome of envenomation. We suggest COX1/2 inhibition as an effective therapeutic intervention for scorpion envenomation.
OBJECTIVE - The IκB kinase (IKK) is an enzyme complex that initiates the nuclear factor κB transcription factor cascade, which is important in regulating multiple cellular responses. IKKα is directly associated with 2 major prosurvival pathways, PI3K/Akt and nuclear factor κB, but its role in cell survival is not clear. Macrophages play critical roles in the pathogenesis of atherosclerosis, yet the impact of IKKα signaling on macrophage survival and atherogenesis remains unclear.
APPROACH AND RESULTS - Here, we demonstrate that genetic IKKα deficiency, as well as pharmacological inhibition of IKK, in mouse macrophages significantly reduces Akt S(473) phosphorylation, which is accompanied by suppression of mTOR complex 2 signaling. Moreover, IKKα null macrophages treated with lipotoxic palmitic acid exhibited early exhaustion of Akt signaling compared with wild-type cells. This was accompanied by a dramatic decrease in the resistance of IKKα(-/-) monocytes and macrophages to different proapoptotic stimuli compared with wild-type cells. In vivo, IKKα deficiency increased macrophage apoptosis in atherosclerotic lesions and decreased early atherosclerosis in both female and male low-density lipoprotein receptor (LDLR)(-/-) mice reconstituted with IKKα(-/-) hematopoietic cells and fed with the Western diet for 8 weeks compared with control LDLR(-/-) mice transplanted with wild-type cells.
CONCLUSIONS - Hematopoietic IKKα deficiency in mouse suppresses Akt signaling, compromising monocyte/macrophage survival and this decreases early atherosclerosis.
© 2016 American Heart Association, Inc.
Infection with Helicobacter pylori (H. pylori) leads to inflammatory events that can promote gastric cancer development. Immune cells transition from the circulation into the infected mucosa through the interaction of their receptors and ligands in the endothelial compartment. CD44 expression is increased in advanced gastric lesions. However, the association of this molecule with the progression of these lesions over time has not been investigated. In addition, there is a lack of understanding of the CD44-dependent cellular processes that lead to gastritis, and possibly to gastric cancer. Here we studied H. pylori-positive subjects with gastric lesions that ranged from multifocal atrophic gastritis to dysplasia to determine gene expression changes associated with disease progression over a period of 6 years. We report that CD44 expression is significantly increased in individuals whose gastric lesions progressed along the gastric precancerous cascade. We also show that CD44-/- mice develop less severe and less extensive H. pylori-induced metaplasia, and show fewer infiltrating Gr1+ cells compared to wild type mice. We present data suggesting that CD44 is associated with disease progression. Mechanisms associated with these effects include induction of interferon gamma responses.
Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
Outer surface protein C (OspC) is one of the major lipoproteins expressed on the surface of Borrelia burgdorferi during tick feeding and the early phase of mammalian infection. OspC is required for B. burgdorferi to establish infection in both immunocompetent and SCID mice and has been proposed to facilitate evasion of innate immune defenses. However, the exact biological function of OspC remains elusive. In this study, we showed that the ospC-deficient spirochete could not establish infection in NOD-scid IL2rγ(null) mice that lack B cells, T cells, NK cells, and lytic complement. The ospC mutant also could not establish infection in anti-Ly6G-treated SCID and C3H/HeN mice (depletion of neutrophils). However, depletion of mononuclear phagocytes at the skin site of inoculation in SCID and C3H/HeN mice allowed the ospC mutant to establish infection in vivo. In phagocyte-depleted mice, the ospC mutant was able to colonize the joints and triggered neutrophilia during dissemination. Furthermore, we found that phagocytosis of green fluorescent protein (GFP)-expressing ospC mutant spirochetes by murine peritoneal macrophages and human THP-1 macrophage-like cells, but not in PMN-HL60, was significantly higher than parental wild-type B. burgdorferi strains, suggesting that OspC has an antiphagocytic property. In addition, overproduction of OspC in spirochetes also decreased the uptake of spirochetes by murine peritoneal macrophages. Together, our findings provide evidence that mononuclear phagocytes play a key role in clearance of the ospC mutant and that OspC promotes spirochetes' evasion of macrophages during early Lyme borreliosis.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes atherosclerosis by increasing low-density lipoprotein (LDL) cholesterol levels through degradation of hepatic LDL receptor (LDLR). Studies have described the systemic effects of PCSK9 on atherosclerosis, but whether PCSK9 has local and direct effects on the plaque is unknown. To study the local effect of human PCSK9 (hPCSK9) on atherosclerotic lesion composition, independently of changes in serum cholesterol levels, we generated chimeric mice expressing hPCSK9 exclusively from macrophages, using marrow from hPCSK9 transgenic (hPCSK9tg) mice transplanted into apoE(-/-) and LDLR(-/-) mice, which were then placed on a high-fat diet (HFD) for 8 weeks. We further characterized the effect of hPCSK9 expression on the inflammatory responses in the spleen and by mouse peritoneal macrophages (MPM) in vitro. We found that MPMs from transgenic mice express both murine (m) Pcsk9 and hPCSK9 and that the latter reduces macrophage LDLR and LRP1 surface levels. We detected hPCSK9 in the serum of mice transplanted with hPCSK9tg marrow, but did not influence lipid levels or atherosclerotic lesion size. However, marrow-derived PCSK9 progressively accumulated in lesions of apoE(-/-) recipient mice, while increasing the infiltration of Ly6C(hi) inflammatory monocytes by 32% compared with controls. Expression of hPCSK9 also increased CD11b- and Ly6C(hi) -positive cell numbers in spleens of apoE(-/-) mice. In vitro, expression of hPCSK9 in LPS-stimulated macrophages increased mRNA levels of the pro-inflammatory markers Tnf and Il1b (40% and 45%, respectively) and suppressed those of the anti-inflammatory markers Il10 and Arg1 (30% and 44%, respectively). All PCSK9 effects were LDLR-dependent, as PCSK9 protein was not detected in lesions of LDLR(-/-) recipient mice and did not affect macrophage or splenocyte inflammation. In conclusion, PCSK9 directly increases atherosclerotic lesion inflammation in an LDLR-dependent but cholesterol-independent mechanism, suggesting that therapeutic PCSK9 inhibition may have vascular benefits secondary to LDL reduction.
Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
OBJECTIVES - Toll-like Receptor 4 (TLR4) is implicated in modulating inflammatory cytokines though its role in atherosclerosis remains uncertain. We have recently described a non-foam cell macrophage phenotype driven by ingestion of hemoglobin:haptoglobin complexes (HH), via the scavenger receptor CD163, characterized by reduced inflammatory cytokine production. In this study, we examined the role of iron metabolism in modulating TLR4 signaling in these cells.
METHODS AND RESULTS - Areas in human atherosclerotic plaque with non-foam cell, CD163 positive macrophages demonstrated reduced expression of tumor necrosis factor alpha (TNF-α) and interferon-beta (INF-β) compared to foam cells. Human macrophages differentiated in hemoglobin:haptoglobin (HH) complexes expressed the CD163 positive non-foam cell phenotype and demonstrated significantly less TNF-α and INF-β compared to control macrophages when exposed to oxidized LDL (oxLDL) or lipopolysaccharide (LPS). LPS stimulated expression of TNF-α and INF-β could be restored in HH macrophages by pretreatment with hepcidin, an endogenous suppressor of ferroportin1 (FPN), or by genetic suppression of FPN in macrophages derived from myeloid specific FPN knockout mice. LPS stimulated control macrophages demonstrated increase in TLR4 trafficking to lipid rafts; this response was suppressed in HH macrophages but was restored upon pretreatment with hepcidin. Using a pharmacologic hepcidin suppressor, we observed a decrease in cytokine expression and TLR4-lipid raft trafficking in LPS-stimulated in a murine macrophage model.
CONCLUSION - TLR4 dependent macrophage signaling is controlled via hepcidin-ferroportin1 axis by influencing TLR4-lipid raft interactions. Pharmacologic manipulation of iron metabolism may represent a promising approach to limiting TLR4-mediated inflammatory responses.
Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
In preterm infants, exposure to inflammation increases the risk of bronchopulmonary dysplasia, a chronic, developmental lung disease. Although macrophages are the key cells that initiate lung inflammation, less is known about lung macrophage phenotype and maturation. We hypothesized that fetal lung macrophages mature into distinct subpopulations during mouse development, and that activation could influence macrophage maturation. Expression of the fetal macrophage markers CD68, CD86, CD206, Ym1, fibrinogen-like protein 2, and indolamine-2, 3-dioxygenase was developmentally regulated, with each marker having different temporal patterns. Flow cytometry analysis showed macrophages within the fetal lung were less diverse than the distinctly separate subpopulations in newborn and adult lungs. Similar to adult alveolar macrophages, fetal lung macrophages responded to the TLR4 agonist LPS and the alternative activation cytokines IL-4 and IL-13. Using a macrophage-specific constitutively active IκB Kinase transgenic model (IKFM), we demonstrated that macrophage activation increased proinflammatory gene expression and reduced the response of fetal lung macrophages to IL-4 and IL-13. Activation also increased fetal lung macrophage proliferation. Fetal IKFM lungs contained increased percentages of more mature, CD11b(low)F4/80(high) cells that also expressed higher levels of the alternative activation markers CD204 and CD206. Development of fetal lung macrophages into mature alveolar macrophages may therefore include features of both proinflammatory and alternative activation paradigms.
Copyright © 2014 by The American Association of Immunologists, Inc.