153

Preview:

Citation preview

recombinant IFNg into STAT4 -/- mice increase the serum ALT, but it does not reachthe level observed in WT mice, suggesting a non-IFNg mediated protection in STAT4-/- mice. Surprisingly, there were no significant difference in FAS and FasL transcriptin ConA injected liver tissue in WT and STAT4 -/- mice. In addition, anti-FAS antibodyinjections lead to significant necrotic changes in WT, but not in STAT4 -/- mice. Ourdata strongly indicate absence of STAT4 signaling potentially in hepatocytes regulatescell survival, which is further confirmed by significantly more pSTAT3 staining in livertissue of STAT4 -/-. STAT3 phosphorylation can be regulated by IL-22 or IL-6. Interest-ingly, serum IL-22 but not IL-6 was significantly elevated in STAT4 -/- mice. Our datastrongly suggest targeting STAT4 in auto-immune hepatitis may have a therapeuticbenefit.

http://dx.doi.org/10.1016/j.cyto.2013.06.155

153HMGB1 and TNF-alpha in an experimental model of acute lung injury (ALI) underprotective conventional mechanical ventilation (CMV)

Cilmery Suemi Kurokawa a, José Roberto Fioretto a, João Pessoa de Araújo Júnior b,Rafaelle Batistella Pires a, Marcos A Moraes a, Susiane O Klefens a, Maria ReginaMoretto a, Cristina Maria Teixeira Fortes a, Rossano Cesar Bonatto a, Mario FerreiraCarpi a, a Department of Pediatrics, Botucatu Medical School, Brazil, b Department ofMicrobiology and Immunology, Botucatu Bioscience Institute, Sao Paulo State University,UNESP, Brazil

Recently, HMGB1, a macrophage cytokine and nonhistone chromatin-associatedprotein, has been associated with sustained inflammatory response and productionof cytokines in lung injury. Therefore, the aim of this study was to measure HMGB1and TNF-alpha levels in bronchoalveolar lavage from rabbits with severe acute lunginjury (ALI) induced by tracheal infusion of warm saline. Methods: Thirty rabbitswere instrumented and randomly assigned into two groups: (1) animals with noALI (Control Group-CG; n = 15); and (2) animals with severe ALI (Severe Group-SG;n = 15). The groups were ventilated during 4 h with CMV. After ventilation, broncho-alveolar lavage fluid (BAL) was collected so that HMGB1, TNF-alpha levels and per-centage of white cell were determined. Left lung was collected for histologicalanalysis and confirmation of lesion. Results: Lung injury decreased pulmonary com-pliance (SG before: 1.86 ± 0.576 > SG after: 0.67 ± 0.24) and oxygenation (PaO2 – SGbefore: 427.92 ± 89.90 > SG after: 68.18 ± 19.08 mmHg). Higher levels of HMGB1were found in SG group as compared to CG group [SG: median-62 (p25–p75 = 20–79) > CG: 15 (13–25) ng/mL; p = 0.035] and TNF-alpha level was similar to HMGB1level in SG and CG groups [SG: 1078 (381–1224) > CG: 45 (25–86) pg/mL;p 6 0.001]. HMGB1 and TNF-alpha showed inverse correlation with oxygenationand positive correlation between them. Neutrophil percentage and histological scorewere higher in SG than CG (p = 0.001 and p = 0.0032, respectively). Conclusions:HMGB1 and TNF-alpha are found in alveolar space and correlation is found betweenthem and with oxygenation in this experimental model. Fapesp grants: 2011/15144-2.

http://dx.doi.org/10.1016/j.cyto.2013.06.156

154The IRF8-KLF4 transcription factor cascade is essential for the development ofmonocytes

Daisuke Kurotaki a, Naoki Osato a, Akira Nishiyama a, Michio Yamamoto a, TatsumaBan a, Hideaki Sato a, Jun Nakabayashi a, Marina Umehara a, Masatoshi Nakazawa b,Noriko Miyake c, Naomichi Matsumoto c, Keiko Ozato d, Tomohiko Tamura a,a Department of Immunology, Yokohama City University Graduate School of Medicine,Yokohama, Japan, b Department of Experimental Animal Science, Yokohama CityUniversity Graduate School of Medicine, Yokohama, Japan, c Department of HumanGenetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan,d Program in Genomics of Differentiation, Eunice Kennedy Shriver, National Institute ofChild Health and Human Development, National Institutes of Health, Bethesda, MD, USA

Monocytes are circulating blood leukocytes that regulate host defenses, inflam-mation and tissue homeostasis. Monocytes originate from bone marrow myeloid pro-genitors such as monocyte-dendritic cell progenitors (MDPs). The transcription factorInterferon Regulatory Factor-8 (IRF8) stimulates monocyte/macrophage differentia-tion, yet a genome-wide understanding of the differentiation program initiated byIRF8 has not been achieved. By combining chromatin immunoprecipitation-sequenc-ing with gene expression profiling, we show here that during IRF8-dependent mono-cyte/macrophage differentiation, the binding of IRF8 occurs at both promoter-proximal and distal regions and is associated with gene induction. At the distal sites,binding of IRF8 leads to increases in histone H3 lysine 4 monomethylation and PU.1

binding, signatures for enhancers in monocytes/macrophages. However, about half ofIRF8-induced genes were not bound by IRF8, suggesting the involvement of down-stream transcription factors. DNA motif analyses of these ‘indirect’ IRF8 target genespredicted Krüppel-like factor-4 (KLF4), essential for the development of Ly6C+ mono-cyte subset, to be one such transcription factor. IRF8 bound promoter-distal locus ofKlf4 gene and directly induced mRNA expression. Indeed, Irf8�/� MDPs do not expressKlf4 mRNA, and Irf8�/� mice lack Ly6C+ monocytes and have much lower counts ofLy6C� monocytes. Ectopic expression of KLF4 into an Irf8�/� myeloid progenitor cellline induced a subset of IRF8 target genes and partially restored monocyte differenti-ation. Based on these results, we concluded that the IRF8-KLF4 transcription factorcascade is essential for monocyte development.

http://dx.doi.org/10.1016/j.cyto.2013.06.157

155IRF8 and IRF1: Genomic regulators of innate immunity

David Langlais, Philippe Gros, Biochemistry Dept, Complex Traits Group, McGillUniversity, Montreal, Canada

IRF8 transcription factor is essential for proper macrophage and dendritic cell(DC) differentiation, activation and function. We recently identified IRF8 mutationsthat are associated with a novel primary myeloid immunodeficiency, coupled withmycobacterial infections susceptibility (MSMD). Interestingly, mice bearing Irf8R294C

loss-of-function alleles exhibit a similar phenotype; i.e. development of a chronicmyelogenous leukemia-like syndrome characterized by a severe loss of dendritic cellsand expansion of immature myeloid precursors, concomitant with susceptibility tointracellular infections. Recent GWAS and eQTL mapping studies associated IRF8locus with various inflammatory diseases. Our objective is to define transcriptionalnetworks controlled by these important inflammatory checkpoints (IRF8 and its bind-ing partners IRF1 and PU.1), in order to identify novel genes implicated in MSMD andin susceptibility to Mycobacterium tuberculosis infection. To identify these networks,we used a combination high-throughput sequencing techniques. Primary macro-phages, prior and after IFNc activation, were subjected to (1) RNA-seq to identifyIFNc-regulated genes and (2) chromatin immunoprecipitation (ChIP-seq) to localizeIRF8, IRF1 and PU.1 genomic binding sites. The comparison of ChIP-seq results forthese well-known binding partners confirmed their co-recruitment to regulatory ele-ments. Interestingly however, they are not obligate partners as we uncovered manydifferent binding schemes. Furthermore, IFNc triggers de novo IRF1 DNA binding,but had limited influence on IRF8 and PU.1 that are already occupying their bindingsites. The overlap of ChIP-seq and RNA-seq data revealed that IFNc-upregulated genesare strongly associated with IRF binding sites, as opposed to repressed genes. ChIP-qPCR and RNA-seq in IRF8R294C and IRF1�/� primary macrophages confirmed theinterdependence of IRF8 and IRF1 co-recruitment and their respective importancefor IFNc-regulated genes subsets. Genes identified in these transcriptional pathwaysrepresent the ‘‘core myeloid transcriptome” and are likely to play critical roles in mac-rophage function and may represent valuable candidates for myeloid immunodefi-ciencies and susceptibility to mycobacterial infections.

http://dx.doi.org/10.1016/j.cyto.2013.06.158

156What does evolution have to do with inflammation?

Brenda Laster, Department of Nuclear Engineering, Ben Gurion University, Beer Sheva84105, Israel

Our evolutionary history suggests that hydrogen peroxide (H2O2), a derivative ofreactive oxygen species (ROS) generated in mitochondria after exposure to environ-mental threats, such as pathogens, chemical toxins, or radiation, might be used tocontrol inflammation. The transition from anaerobic to aerobic respiration must havebeen accompanied by oxidative damage because, some time later, an antioxidant sys-tem developed that remains, until today, as a positive natural selection. The con-served antioxidant enzymes (GSH, Trx, peroxiredoxins) convert the highly energeticand damaging ROS species, superoxide, to H2O2. The multiple and highly concentra-tion-dependent roles of H2O2in inflammation range from inducing the gene expres-sion of heme oxygenase and its antioxidant by-products CO and biliverdin at lowconcentrations, to the induction of a systemic inflammatory response syndrome athigh concentrations. Exposure to environmental threats causes lipid peroxidation thatincreases ROS production in mitochondria. The ROS species, superoxide, is convertedto H2O2by superoxide dismutase enzymes enabling H2O2to enter the cytoplasmwhere it strongly influences the outcome of the NF-jB and MAPK oxidative stress sig-naling pathways that are also evolutionarily-conserved. By reversibly oxidizing cyste-ines in the catalytic core of phosphatases, it prolongs or curtails the activation of

Abstract / Cytokine 63 (2013) 243–314 279