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is a key property that is exclusive to human blood monocytes. This disparity raisedthe possibility that distinct myeloid-specific transcription factor(s) may be involvedin the rapid induction of IFN-b in monocytes compared with non-myeloid cell types.We found that IFN-b was produced rapidly in primary human monocytes as a result ofcooperation between the myeloid-specific transcription factor IRF8 and the ubiqui-tous transcription factor IRF3. We provide evidence that IRF8 constitutively bindsto the IFN-b promoter region, and knockdown of IRF8 in monocytes abrogated IFN-b transcription. We uncovered a requirement for IRF3, a master regulator of IFN-bproduction, as a previously unidentified interaction partner of IRF8. We produced arange of deletion constructs of IRF3 and IRF8 mutants, and, using co-transfectionand co-immunoprecipitation, mapped the protein–protein interacting regions ofIRF3 and IRF8, and found that their interaction was independent of the DNA-bindingdomain (DBD) and the IRF association domain (IAD) of IRF8 and IRF3, respectively.Through these and other experiments, we have been able to demonstrate that IRF8directly synergizes with IRF3 in monocytes to facilitate faster IFN-b transcription afterpathogenic stimulation.

http://dx.doi.org/10.1016/j.cyto.2014.07.175

169IL-19, a novel SASP factor, is upregulated during senescence and in response toDSBs

Sara H Small 1, Ryan L. Ragland 1, Yaroslava Ruzankina 1, David W. Schoppy 1, F.B.Johnson 2, Eric J. Brown 1, 1 Cancer Biology, University of Pennsylvania, Philadelphia, PA,USA, 2 Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA,USA

ATR is a serine/threonine kinase that activates cell cycle checkpoints in response tostalled replication forks and resected double strand breaks (DSBs). Deletion of ATRcauses DSBs to be generated during S phase due to replication fork collapse. Notably,when ATR is deleted in a mosaic manner in p53�/� mice, the ATR-deleted cells persistin the tissues despite high levels of DSBs, and result in widespread inflammation anddefects in tissue regeneration. These results suggest that highly-damaged ATR-deletedcells generate extrinsic factors. To characterize the factors involved in this response, weperformed a gene expression microarray on p53�/� and p53�/�ATR[D/�] skin epithe-lial cells and identified several cytokines and growth factors that were selectivelyupregulated upon ATR deletion. These factors included a few factors previously identi-fied as members of the senescence-associated secretory phenotype (SASP), but moreprominently included IL-19, which has not previously been identified as a SASP factor.IL-19 is a member of a distinct grouping of cytokines known as the IL-10 family. In cul-tured cells, IL-19 is upregulated in response to DSBs or after passage- or oncogene-induced senescence. These treatments also lead in parallel to the induction of classicSASP factors, such as IL-1 and IL-6. Importantly, the degree of induction of IL-19 isfar greater than that of IL-6 or IL-8, and in fact IL-19 is required for ionizing radia-tion-induced SASP factor upregulation. Thus, the mechanism governing the regulationof IL-19 expression and its effects on the regulation of other cytokines will be discussed.


170Mechanisms behind immunoregulatory effects of probiotic yeasts

Ida M. Smith 1,2, Adam Baker 1, Nils Arneborg 2, Lene Jespersen 2, 1 Health & NutritionDivision, Chr. Hansen A/S, Hørsholm, Denmark, 2 Food Science, University of Copenhagen,Frederiksberg, Denmark

The concept of individual microorganisms influencing the makeup of T cell sub-sets through interactions with intestinal dendritic cells (DCs) appears to constitutethe foundation for immunoregulatory effects of probiotics, and several studies havereported probiotic strains resulting in reduction of intestinal inflammation throughmodulation of DC function. Consequent to a focus onSaccharomyces boulardii as thefundamental probiotic yeast, very little is known about non-Saccharomyces yeastsin terms of their interaction with the human gastrointestinal immune system.

We evaluated the immune stimulating capabilities of a diverse selection of non-Saccharomyces yeasts by incubation with human monocyte-derived DCs followed byDC incubation with autologous naive T cells. Quantification of secreted cytokine levelsrevealed yeasts with highly reproducible and distinct DC and T cell cytokine inductionprofiles, as compared to the established probiotic S. boulardii. The observed differ-ences in induced cytokine profiles, supported by T cell subset stains, indicate that cer-tain yeasts are capable of inducing an immune response dominated by Treg cells,whereas others appear to induce a more complex adaptive immune response involv-ing TH1, TH17, and Treg cells.

To explore the mechanisms behind the observed cytokine induction, we blockedrelevant DC pattern recognition receptors and investigated the cytokine inducing prop-erties of yeast cell wall extracts. Our data identify the b-glucan receptor Dectin-1 as keyfor DC recognition of S. boulardii as well as non-Saccharomyces yeasts, initiating down-stream signaling pathways leading to the observed DC cytokine profiles. In contrast,TLR2 and DC-SIGN do not appear involved in the recognition. As expected based onthe identification of Dectin-1 as involved in yeast recognition, b-glucan containingyeast cell wall extracts induced robust DC cytokine secretion, an observation that par-allels recent in vivo findings and appears to support a hypothesis that yeast cell wallcomponents are responsible for the observed modulation of immune cell function.


171General strategy for modulating cytokine function: Development of potent, effi-cacious and selective antagonists of IL-6

Mark Smythe, Protagonist Therapeutics, St. Lucia, QLD, Australia

Rheumatoid arthritis (RA) is a chronic inflammatory disorder that affects synovialjoints. Anti-TNF-alpha and anti-IL-6 receptor monoclonal antibodies are among thetherapeutic approaches that have been successfully used in treatment of this disease.However, there is a need for additional and differentiated therapies, which may haveadvantages over the monoclonal antibody approach, including improved tissue pen-etration into the joints and a lower anti-drug antibody response. Here we present ageneric strategy for modulating cytokine function and focus on the preclinical devel-opment of an injectable anti-IL-6 peptide for the treatment of RA. Using a combina-tion of proprietary software, phage-display selection, and medicinal peptide-basedchemistry, we designed the disulfide-rich peptide, PN-1, a potent inhibitor of IL-6.We found that PN-1, and its 40 K-PEGylated analog, PN-2, potently inhibited IL-6induced pStat3 activation in the human monocyte cell line U937. A pharmacokineticanalysis of PN-2 demonstrated plasma half-life of >24 h, consistent with the knowneffect of PEGylation. Finally, we evaluated the in vivo efficacy of PN-2 in cynomolo-gous monkey and found that PN-2 decreased the levels of the IL-6 induced biomark-ers SAA and CRP in the monkey by 97% at 24 h after IL-6 challenge.


172Impact of microbiota on expression of mRNA of NF-kappa B family molecules inthe intestine of germ-free and conventional piglets

Igor Splichal, Alla Splichalova, Sava Klabackova, Institute of Microbiology, ASCR, Prague4 – Krc, Czech Republic

Aims: The gastrointestinal tract of a newborn is colonized by vaginal and fecal mic-robiota of the mother immediately during delivery. Nuclear factor kappa B (NF-kB)family molecules play a central role in inflammatory response. How a presence/absence of the microbiota stimulation of the immune system influence the expressionof NF-kB related molecules in the intestine?

Methods: Conventional naturally-born 1 and 7 days-old suckled piglets (CV) werebred in a pigpen. Their germ-free colostrum-deprived formula-fed counterparts(GF) were derived by hysterectomy and were reared in gnotobiological isolators.mRNA of NF-kB family molecules (RelA, cRel, RelB, NF-kB1 and NF-kB2) were mea-sured by RT-qPCR in the terminal ileum to study the influence of the microbiota onstimulation of inflammatory response. IL-8, TNF-alpha and HMGB1 in age-corre-sponding counterpart piglets served as control inflammation-induced genes.

Results: mRNA for IL-8 in the ileum were significantly higher in CV piglets then intheir GF counterparts. The situation in TNF-alpha was similar but less significant.NF-kB1, NF-kB2, and RelB in the ileum showed the same trend. In contrast, RelAshowed opposite ratio and cRel different relations in each age group.

Conclusions: Nuclear factor-kappa B is a central regulator of the transcriptionalresponses that is regulated by complex signal transduction pathways after stimuliof different origin. The hysterectomy-derived colostrum-deprived gnotobiotic pigletsmay be suitable experimental models for in vivo study of these signal transductionpathways.

Acknowledgements: This work was supported by Grants 13-08803S of the CzechScience Foundation, FR-TI4/504 of the Ministry of Industry and Trade of the CzechRep. and the Institutional Research Concept RVO: 61388971 of the Institute ofMicrobiology.


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