- Email: [email protected]
PS2-31 Host MicroRNA regulation during influenza virus infection
56
Abstracts / Cytokine 52 (2010) 50–67
by 3-methyl adenine. As a potential mechanistic explanation it could be demonstrated that the polymorphism affects ATG16L1 protein expression upon NOD2stimulation. The present study is the first to link the ATG16L1 polymorphism with an excessive production of IL-1b and IL-6 in humans, which may explain the effects of this polymorphism on the inflammatory process in Crohn’s disease.
PS2-29 IL-17 and its Jekyll-and-Hyde role in the regulation of fat and bone Mushtaq Ahmed, Jaya Goswami, Nydiaris Hernández-Santos, Sarah L. Gaffen, University of Pittsburgh, Department of Medicine, Division of Rheumatology, Pittsburgh, PA USA
doi:10.1016/j.cyto.2010.07.231
Post-menopausal osteoporosis is considered to be an inflammatory process, in which numerous pro-inflammatory and T-cell-derived cytokines play a bone-destructive role. IL-17A is the signature cytokine of the pro-inflammatory Th17 population and plays dichotomous roles in diseases that affect bone turnover. Although IL-17A promotes bone loss in rheumatoid arthritis, it is protective against pathogen-induced bone destruction in a periodontal disease model. We used a model of ovariectomyinduced osteoporosis (OVX) in IL-17 receptor (IL-17RA)/ mice to evaluate the role of the IL-17A in bone loss caused by estrogen deficiency. Unexpectedly, IL17RA/mice were consistently and markedly more susceptible to OVX-induced bone loss than controls. There were no changes in prototypical Th1, Th2 or Th17 cytokines in serum that could account for increased bone loss. However, IL-17RA/ mice exhibited constitutively elevated leptin, which further increased following OVX. Consistently, IL-17A and IL-17F treatment of 3T3-L1 and ST-2 preadipocytes inhibited adipogenesis, leading to reduced production of leptin. In addition to its role in regulating metabolism and satiety, leptin can regulate bone turnover. Accordingly, these data show that IL-17A negatively regulates adipogenesis and subsequent leptin expression, which correlates with increased bone destruction during OVX. Since the mechanisms by which IL-17 influences adipocyte differentiation are entirely unknown, we aim to further investigate the molecular events involved during inhibition of adipogenesis by IL-17A signaling.
PS2-27 Bioactive Oncostatin M is present in induced-sputum samples from house dust mite sensitive asthmatic subjects Carl D. Richards 1, Matt Scott 1, Christine Kerr 1, Dominik Fritz 1, Param Nair 2, G. Gauvreau 2, P. O’Byrne 2, 1 Centre for Gene Therapeutics, Department of Pathology and Molecular Medicine, 2 Department of Medicine, McMaster University, Hamilton, Ontario, Canada Oncostatin M (OSM) is a gp130 cytokine that can regulate chemokine and cytokine responses in lung fibroblasts, epithelial cells and airway smooth muscle cells. The role of OSM and related gp130 cytokines, such as IL-31, LIF and IL-6, in asthma are still to be fully elucidated. OSM interacts with a receptor complex that includes gp130 and the OSMRbeta chain, where as IL-31 interacts with a complex composed of OSMRbeta chain and the gp130-like protein. In this study, we have assessed the levels of OSM and IL-31 in induced-sputum samples from house dust mite (HDM)sensitive subjects with mild asthma. We then assessed the ability of sputum samples (obtained at time 0, 7 and 24 hours) to regulate cytokine production by human airway smooth muscle cells (HASMC) in vitro. For each individual (n=7 subjects), induced sputum samples upon aerosol challenge with HDM contained elevated levels of OSM (as detected by ELISA) at 7 hours and 24 hours after aerosol challenge, however IL-31 was not detectable (ELISA). Sputum samples from subjects without aerosol challenge showed no detectable OSM. In selected samples with significant OSM levels (as detected by ELISA), neutralizing antibody (R& D Systems) to OSM was found to significantly reduce the ability of induced-sputum to elevate HASMC responses. Using recombinant cytokines in vitro, OSM was able to synergistically regulate eotaxin-1 and MCP-1 levels detected in HASMC supernatants at 24 hours when added in combination with IL-4 or IL-13. LIF or IL-31 did not show this activity. In addition, exogenously added OSM caused a synergistic increase in the activity of induced sputum samples to regulate HASMC production of chemokines. These results suggest that asthmatic subjects, when challenged by aerosol HDM antigen, express elevated levels of bioactive OSM in sputum that can initiate HASMC responses. Furthermore, OSM (but not LIF or IL-31) can regulate responses in HASMC synergistically in context of other cytokines likely present in Th2-skewed immune responses. This work was supported by the Canadian Institutes for Health Research. These results support unique functions of OSM in inflammatory responses by HASMC in vitro. Since biologically significant levels of OSM are expressed in asthmatic subjects in vivo, collectively these results suggest an important role of OSM in exacerbation of allergic airway inflammatory disease. doi:10.1016/j.cyto.2010.07.232
PS2-28 MSCs and TSG-6 abort the inflammatory cascade of zymosan-induced peritonitis by binding to CD44 on resident macrophages to inhibit NFjB signaling Ryang Hwa Lee *, Hosoon Choi *, Nikolay Bazhanov, Dong-Ki Kim, Darwin J. Prockop, Texas A& M Health Science Center, College of Medicine, Institute for Regenerative Medicine at Scott & White, Temple, TX 76502, USA Human multipotent stromal cells (hMSCs) repair tissues through multiple actions that include secretion of the anti-inflammatory protein TSG-6. We examined the effects of hMSCs in the zymosan-induced mouse model for peritonitis. Both hMSCs and rhTSG-6 decreased cellular infiltration into the peritoneum and serum levels of plasmin and IL-6. hMSCs transduced with an siRNA for TSG-6 were ineffective. hMSCs and TSG-6 but not hMSCs with a TSG-6 siRNA inhibited TNF-a secretion by a line of macrophages (RW) stimulated with zymosan by binding to CD44 and inhibiting NFjB signaling. Inhibition of the macrophages in turn decreased amplification of the inflammatory response of mesothelial cells. hMSCs and TSG-6 also inhibited zymosan activation of resident macrophages isolated from the peritoneum of wildtype mice but not from CD44-/- transgenic mice. hMSCs or TSG-6 may be particularly effective in inhibiting inflammation because they abort the inflammatory cascade as it is initiated by resident macrophages. doi:10.1016/j.cyto.2010.07.233
*
Authors with equal contributions.
doi:10.1016/j.cyto.2010.07.234
PS2-30 PTX3 is expressed in the skin and is regulated by members of TGF-b family Andrea Doni 1, Virginia Maina 1, Marina Sironi 1, Ivan Cuccovillo 1, Irene Cambieri 2, Tiziana Musso 3, Carlotta Castagnoli 2, Cecilia Garlanda 1, Barbara Bottazzi 1, Alberto Mantovani 1, 1 Istituto Clinico Humanitas, IRCCS, Milan, Italy, 2 Dept. Plastic Surgery and Burn Unit Skin Bank, CTO Hospital, Turin, Italy, 3 Dept. Public Health and Microbiology, University of Turin, Italy The long pentraxin 3 (PTX3) is member of a complex superfamily of multifunctional proteins characterized by a cyclic multimeric structure. PTX3 is highly conserved in evolution and is produced by innate-immunity cells in response to proinflammatory signals and Toll-like receptor engagement. PTX3 plays complex, nonredundant functions in vivo, acting as a predecessor of antibodies, recognizing microbes, activating complement, facilitating pathogen recognition by phagocytes, and hence, playing a nonredundant role in resistance against selected pathogens. In addition, PTX3 is essential in female fertility acting as a nodal point for the assembly of the cumulus oophorus hyaluronan-rich extracellular matrix. Here we have investigated PTX3 expression and localization in human skin, and its regulation by growth factors. PTX3 is constitutively expressed in normal skin (NS) where it localized at the basal layer and, weakly, in the dermis; PTX3 expression was further analyzed in inflamed/fibrotic conditions, such as active-phase hypertrophic scars (HS), remission-phase HS and keloids. A striking increase of PTX3 expression was found in the HS, in particular in the interstitial dermis, compared to NS. PTX3 expression was found increased and further localized to blood vessels, in the interstitial matrix of the dermis and associated to cells scattered in matrix. In active-phase HS we found several infiltrated cells double positive for PTX3 and MMP-9, while in remissionphase HS and keloids PTX3 localized predominantly in the interstitial matrix, where MMP-9+ cells were also found. A similar PTX3 expression was further observed in mouse NS. Members of TGF-b family play an important role in wound healing and their altered production affects the different phase of scar evolution in hypertrophic scarring, a skin disorder characterized by persistent inflammation and fibrosis. We evaluated the regulatory effect of members of the TGF-b family on PTX3 production by a normal human fibroblast cell line. PTX3 production was strongly inhibited by Activin, but not Follistatin, and by the two TGF-b isoforms, TGF-b1 and TGFb-3. All were also able to inhibit the TNF-a-induced production. PTX3 expression in inflamed/fibrotic HS, thus prompt us to evaluate a possible role of this pentraxin in a mouse model of tissue regeneration. doi:10.1016/j.cyto.2010.07.235
MicroRNAs and Post-Transcriptional Regulation PS2-31 Host microRNA regulation during influenza virus infection Karen E. Johnson, Curt M. Horvath, Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL USA
Abstracts / Cytokine 52 (2010) 50–67 MicroRNAs are short RNAs that negatively regulate protein expression by inhibiting mRNA translation. MicroRNAs are vital in the antiviral immune responses of plants and invertebrates, but their role in the antiviral responses of mammals has not been extensively studied. Recent studies have shown that some microRNAs play antiviral roles by directly targeting viral genomes or mRNAs and that some are actually required for efficient virus replication. In this study we address regulation of microRNA expression in response to infection of human cells with influenza virus. To closely approximate human lung infection with influenza virus, we have used human airway epithelial (HAE) cells. We show that these cells can be infected with the A/Udorn/72 strain of influenza virus and that infection causes the expression of known innate immune factors, including interferon (IFN) b and retinoic acid-inducible gene I protein (RIG-I), which were induced to over 400 times and over 20 times the expression in mock-infected cells, respectively. We have identified cellular microRNAs that are differentially expressed in HAE cells, lung cancer cells (A549), and transformed human kidney cells (293) in response to influenza virus infection. These microRNAs include some that have previously been implicated in viral infection and the innate immune response. Differential expression of a subset of the microRNAs we identified was verified using quantitative RT-PCR assays. Though some of these microRNAs have been implicated in influenza virus infection in prior studies in mice, we provide evidence of some differences in the microRNA regulation in infection of human cells, and have identified several additional microRNAs with changes in expression following infection with influenza virus. Understanding the effects of influenza virus infection on microRNA expression could lead to a better understanding of the role of microRNAs in virus infection of mammalian cells and of the regulation of microRNA expression, in general. doi:10.1016/j.cyto.2010.07.236
PS2-32 MicroRNA regulation in response to human influenza virus infection William A. Buggele, Curt M. Horvath, Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois USA The cellular response to virus infection is initiated by recognition of an invading pathogen and subsequent changes in gene expression. The changes in gene expression are known to involve both transcriptional and translational mechanisms, and result in the establishment of an antiviral state that is hostile to virus replication. In addition to the well-established paradigms that control gene expression in response to virus infections, it has become clearly established that small RNAs contribute to antiviral responses in lower organisms via RNA interference (RNAi). However, there is little compelling evidence to suggest that RNAi mediates specific antiviral responses in mammalian hosts. Nonetheless, the understanding that microRNAs, endogenously encoded small RNAs capable of repressing translation in a sequence specific manner, are a significant element of protein expression has led to the hypothesis that microRNAs may contribute to the regulation of antiviral gene expression. To test this hypothesis, human lung cell lines (A549 and BEAS-2B) were infected with influenza virus (A/Udorn/72 and A/WSN/33) and the abundance of cellular microRNAs was evaluated with microarrays. Several microRNAs were found to change in abundance in response to influenza virus infection, and were independently verified using quantitative RT- PCR assays. These findings clearly demonstrate microRNA regulation in response to influenza virus infection in human cells. Understanding the role of microRNAs in the regulation of the cellular response to influenza virus infection will lead to a more complete understanding of the host response and may yield important therapeutic targets to combat virus infections. doi:10.1016/j.cyto.2010.07.237
Interferons in the Treatment of Diseases PS2-33 Expression of the cytosolic pyrimidine 5’-nucleotidase PN-I (NT5C3) in nonhematopoietic cells: a novel Type-I interferon-inducible gene Latifa Al-Haj, Khalid S.A. Khabar, BioMolecular Research Program, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia Pyrimidine 50 -nucleotidase (PN-I or NT5C3) is a member of the 5’-nucleotidase family of the enzymes that catalyze the dephosphorylation of nucleoside monophosphates to their corresponding nucleosides and regulate the nucleoside pools inside the cell. The NT5C3 expression has been reported to be erythrocyte-specific that possess nucleotidase and phosphotransferase activities. We have identified NT5C3 as a highly inducible gene in cells sensitive to IFN action. The present study was designed to evaluate the alterations of NT5C3 expression after type-I interferon treatment in vitro and to characterize the signaling pathway that mediate its induction. We describe enhanced expression of NT5C3 in different non-hematopoietic cell lines. Addition of Type-I (IFN-a, IFN-b, and IFN-Omega) but not type-II interferon (IFN-c)
57
to epithelial cell lines revealed increase expression of NT5C3 in a time and a dose dependant manner. In addition, IFN-a treatment induces the activation of GFP construct fused with interferon stimulated element (ISRE) found in the 50 un-translated region (50 UTR) of NT5C3. Moreover, reporter experiments with GFP construct fused with NT5C3 AU-rich element (ARE) and IFN treatment show ARE mediate destabilization with wild type but not with mutant ARE containing construct. Using Signal Transducers and Activators of Transcription STAT1-deficient (U3A) and tyrosine kinase 2-deficient (U1A) fibrosarcoma cells, we observed that IFN induced expression of NT5C3 is STAT1- and Tyk2- dependent. Moreover, functional assessment using gemcitabine (20 , 20 -difluorodeoxy-cytidine) and human fibrosarcoma cancer cells revealed increased resistance to gemcitabine in cells overexpressing NT5C3. Overall, these results suggest that type-I IFN is a regulator of NT5C3 in non-hematopoietic cells, and NT5C3 is involved in gemcitabine-resistance. Current work is focused on elucidating the functional consequences of NT5C3 during IFN response, and its role in controlling inflammation via nucleoside production. doi:10.1016/j.cyto.2010.07.238
PS2-34 Evaluation of IFN-k responsiveness on renal cell carcinoma Kevin Kotredes, Yolanda Lopez, Ana Gamero, Temple University, Department of Biochemistry, Philadelphia, PA USA Interferon lambda 1 (IFN-k) is a recently discovered type III interferon (IFN-k, also called IL-28/IL-29) that shares many of the biological activities of type I IFNs (IFN-a). Type I IFNs and type III IFNs alike activate the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway leading to expression of IFN-related genes. Presently, IFN-a is used to treat certain malignancies but its use in patients is constrained due to severe toxicity. Treatment with type III IFNs may prove to be less toxic as receptors are expressed only in certain cell types, whereas receptors for type I IFNs are ubiquitously expressed. To date, the antigrowth effects of IFN-k have been studied in melanoma, esophageal, hepatocellular carcinoma cell lines. Due to the presence of IFN-k receptor (IFN-kR1) in kidney cells, the aim of this study is to assess the responsiveness of IFN-k on a panel of renal cell carcinoma lines. Our data showed that IFN-k treatment induced tyrosine phosphorylation of STAT1 and STAT2 in 5 out of 11 cell lines tested. Interestingly, of the five demonstrating activation, only one showed IFN-k-induced upregulation of MHC class I. We then found that supplementing IFN-k treatment with a receptor tyrosine kinase inhibitor, sunitinib, showed a moderate antiproliferative effect on renal cell carcinoma. Our studies have shown that the correlation between IFN-k responsiveness and the presence of IFN-kR1 mRNA transcripts is not always consistent due to the expression of different truncated forms of IFN-kR1, which may possess inhibitory activity. We investigated the existence of these altered forms of the IFN-kR1 and our results revealed three separate isoforms are found within various classes of renal cell carcinoma. Therefore, our data analysis shows that there may be more factors determining the response of renal cell carcinomas to IFN-k. doi:10.1016/j.cyto.2010.07.239
PS2-35 Reduced interferon receptor levels resulting from either knockdown or prolonged Type I interferon treatment promote a shift in cellular responsiveness Daniel Harari 1,2, Doron Levin 1,2, Renana Abramovich 1, Gideon Schreiber 1, 1 Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel, 2 D.H. and D.L. contributed equally to this study Patients undergoing Type I IFN cancer therapy are treated over extended periods, whereas in antiviral and anti-proliferative bioassays, studies are completed within days. We thus sought to examine the behavior of cancer cells undergoing prolonged IFN treatment. IFN administered to the cell lines WISH and MDA-MB-231 resulted in a maximum IFN-induced cell death of 70% within the first three days of culturing. The surviving cells however remained dormant for another 2-3 weeks. This dormancy period could be abrogated upon IFN removal. Further maintenance of these cells with IFN however resulted in their eventual breaking out of quiescence and return to growth. Examination of IFNAR1 and IFNAR2 surface expression by FACS revealed that with prolonged exposure to IFN, receptor levels were significantly reduced. To quantitatively examine the importance of IFNAR levels in IFN responsiveness, we performed a dose-responsive depletion of IFNAR1 or IFNAR2 by siRNA knockdown. 50% IFNAR depletion resulted in a significant reduction of cells undergoing anti-proliferation, whereas anti-viral activity remained stable. Further depletion of IFNARs to below a critical threshold however, rendered the cells unresponsive to both anti-viral and anti-proliferative activity. Our data provides a likely explanation as to why long term IFN administration results in loss of antiproliferative responsiveness with time. Our EC50 data clearly shows that the relationship between receptor levels and activity is a binary, zero and one response that is not dependant on the affinity or type of
Abstracts / Cytokine 52 (2010) 50–67
by 3-methyl adenine. As a potential mechanistic explanation it could be demonstrated that the polymorphism affects ATG16L1 protein expression upon NOD2stimulation. The present study is the first to link the ATG16L1 polymorphism with an excessive production of IL-1b and IL-6 in humans, which may explain the effects of this polymorphism on the inflammatory process in Crohn’s disease.
PS2-29 IL-17 and its Jekyll-and-Hyde role in the regulation of fat and bone Mushtaq Ahmed, Jaya Goswami, Nydiaris Hernández-Santos, Sarah L. Gaffen, University of Pittsburgh, Department of Medicine, Division of Rheumatology, Pittsburgh, PA USA
doi:10.1016/j.cyto.2010.07.231
Post-menopausal osteoporosis is considered to be an inflammatory process, in which numerous pro-inflammatory and T-cell-derived cytokines play a bone-destructive role. IL-17A is the signature cytokine of the pro-inflammatory Th17 population and plays dichotomous roles in diseases that affect bone turnover. Although IL-17A promotes bone loss in rheumatoid arthritis, it is protective against pathogen-induced bone destruction in a periodontal disease model. We used a model of ovariectomyinduced osteoporosis (OVX) in IL-17 receptor (IL-17RA)/ mice to evaluate the role of the IL-17A in bone loss caused by estrogen deficiency. Unexpectedly, IL17RA/mice were consistently and markedly more susceptible to OVX-induced bone loss than controls. There were no changes in prototypical Th1, Th2 or Th17 cytokines in serum that could account for increased bone loss. However, IL-17RA/ mice exhibited constitutively elevated leptin, which further increased following OVX. Consistently, IL-17A and IL-17F treatment of 3T3-L1 and ST-2 preadipocytes inhibited adipogenesis, leading to reduced production of leptin. In addition to its role in regulating metabolism and satiety, leptin can regulate bone turnover. Accordingly, these data show that IL-17A negatively regulates adipogenesis and subsequent leptin expression, which correlates with increased bone destruction during OVX. Since the mechanisms by which IL-17 influences adipocyte differentiation are entirely unknown, we aim to further investigate the molecular events involved during inhibition of adipogenesis by IL-17A signaling.
PS2-27 Bioactive Oncostatin M is present in induced-sputum samples from house dust mite sensitive asthmatic subjects Carl D. Richards 1, Matt Scott 1, Christine Kerr 1, Dominik Fritz 1, Param Nair 2, G. Gauvreau 2, P. O’Byrne 2, 1 Centre for Gene Therapeutics, Department of Pathology and Molecular Medicine, 2 Department of Medicine, McMaster University, Hamilton, Ontario, Canada Oncostatin M (OSM) is a gp130 cytokine that can regulate chemokine and cytokine responses in lung fibroblasts, epithelial cells and airway smooth muscle cells. The role of OSM and related gp130 cytokines, such as IL-31, LIF and IL-6, in asthma are still to be fully elucidated. OSM interacts with a receptor complex that includes gp130 and the OSMRbeta chain, where as IL-31 interacts with a complex composed of OSMRbeta chain and the gp130-like protein. In this study, we have assessed the levels of OSM and IL-31 in induced-sputum samples from house dust mite (HDM)sensitive subjects with mild asthma. We then assessed the ability of sputum samples (obtained at time 0, 7 and 24 hours) to regulate cytokine production by human airway smooth muscle cells (HASMC) in vitro. For each individual (n=7 subjects), induced sputum samples upon aerosol challenge with HDM contained elevated levels of OSM (as detected by ELISA) at 7 hours and 24 hours after aerosol challenge, however IL-31 was not detectable (ELISA). Sputum samples from subjects without aerosol challenge showed no detectable OSM. In selected samples with significant OSM levels (as detected by ELISA), neutralizing antibody (R& D Systems) to OSM was found to significantly reduce the ability of induced-sputum to elevate HASMC responses. Using recombinant cytokines in vitro, OSM was able to synergistically regulate eotaxin-1 and MCP-1 levels detected in HASMC supernatants at 24 hours when added in combination with IL-4 or IL-13. LIF or IL-31 did not show this activity. In addition, exogenously added OSM caused a synergistic increase in the activity of induced sputum samples to regulate HASMC production of chemokines. These results suggest that asthmatic subjects, when challenged by aerosol HDM antigen, express elevated levels of bioactive OSM in sputum that can initiate HASMC responses. Furthermore, OSM (but not LIF or IL-31) can regulate responses in HASMC synergistically in context of other cytokines likely present in Th2-skewed immune responses. This work was supported by the Canadian Institutes for Health Research. These results support unique functions of OSM in inflammatory responses by HASMC in vitro. Since biologically significant levels of OSM are expressed in asthmatic subjects in vivo, collectively these results suggest an important role of OSM in exacerbation of allergic airway inflammatory disease. doi:10.1016/j.cyto.2010.07.232
PS2-28 MSCs and TSG-6 abort the inflammatory cascade of zymosan-induced peritonitis by binding to CD44 on resident macrophages to inhibit NFjB signaling Ryang Hwa Lee *, Hosoon Choi *, Nikolay Bazhanov, Dong-Ki Kim, Darwin J. Prockop, Texas A& M Health Science Center, College of Medicine, Institute for Regenerative Medicine at Scott & White, Temple, TX 76502, USA Human multipotent stromal cells (hMSCs) repair tissues through multiple actions that include secretion of the anti-inflammatory protein TSG-6. We examined the effects of hMSCs in the zymosan-induced mouse model for peritonitis. Both hMSCs and rhTSG-6 decreased cellular infiltration into the peritoneum and serum levels of plasmin and IL-6. hMSCs transduced with an siRNA for TSG-6 were ineffective. hMSCs and TSG-6 but not hMSCs with a TSG-6 siRNA inhibited TNF-a secretion by a line of macrophages (RW) stimulated with zymosan by binding to CD44 and inhibiting NFjB signaling. Inhibition of the macrophages in turn decreased amplification of the inflammatory response of mesothelial cells. hMSCs and TSG-6 also inhibited zymosan activation of resident macrophages isolated from the peritoneum of wildtype mice but not from CD44-/- transgenic mice. hMSCs or TSG-6 may be particularly effective in inhibiting inflammation because they abort the inflammatory cascade as it is initiated by resident macrophages. doi:10.1016/j.cyto.2010.07.233
*
Authors with equal contributions.
doi:10.1016/j.cyto.2010.07.234
PS2-30 PTX3 is expressed in the skin and is regulated by members of TGF-b family Andrea Doni 1, Virginia Maina 1, Marina Sironi 1, Ivan Cuccovillo 1, Irene Cambieri 2, Tiziana Musso 3, Carlotta Castagnoli 2, Cecilia Garlanda 1, Barbara Bottazzi 1, Alberto Mantovani 1, 1 Istituto Clinico Humanitas, IRCCS, Milan, Italy, 2 Dept. Plastic Surgery and Burn Unit Skin Bank, CTO Hospital, Turin, Italy, 3 Dept. Public Health and Microbiology, University of Turin, Italy The long pentraxin 3 (PTX3) is member of a complex superfamily of multifunctional proteins characterized by a cyclic multimeric structure. PTX3 is highly conserved in evolution and is produced by innate-immunity cells in response to proinflammatory signals and Toll-like receptor engagement. PTX3 plays complex, nonredundant functions in vivo, acting as a predecessor of antibodies, recognizing microbes, activating complement, facilitating pathogen recognition by phagocytes, and hence, playing a nonredundant role in resistance against selected pathogens. In addition, PTX3 is essential in female fertility acting as a nodal point for the assembly of the cumulus oophorus hyaluronan-rich extracellular matrix. Here we have investigated PTX3 expression and localization in human skin, and its regulation by growth factors. PTX3 is constitutively expressed in normal skin (NS) where it localized at the basal layer and, weakly, in the dermis; PTX3 expression was further analyzed in inflamed/fibrotic conditions, such as active-phase hypertrophic scars (HS), remission-phase HS and keloids. A striking increase of PTX3 expression was found in the HS, in particular in the interstitial dermis, compared to NS. PTX3 expression was found increased and further localized to blood vessels, in the interstitial matrix of the dermis and associated to cells scattered in matrix. In active-phase HS we found several infiltrated cells double positive for PTX3 and MMP-9, while in remissionphase HS and keloids PTX3 localized predominantly in the interstitial matrix, where MMP-9+ cells were also found. A similar PTX3 expression was further observed in mouse NS. Members of TGF-b family play an important role in wound healing and their altered production affects the different phase of scar evolution in hypertrophic scarring, a skin disorder characterized by persistent inflammation and fibrosis. We evaluated the regulatory effect of members of the TGF-b family on PTX3 production by a normal human fibroblast cell line. PTX3 production was strongly inhibited by Activin, but not Follistatin, and by the two TGF-b isoforms, TGF-b1 and TGFb-3. All were also able to inhibit the TNF-a-induced production. PTX3 expression in inflamed/fibrotic HS, thus prompt us to evaluate a possible role of this pentraxin in a mouse model of tissue regeneration. doi:10.1016/j.cyto.2010.07.235
MicroRNAs and Post-Transcriptional Regulation PS2-31 Host microRNA regulation during influenza virus infection Karen E. Johnson, Curt M. Horvath, Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL USA
Abstracts / Cytokine 52 (2010) 50–67 MicroRNAs are short RNAs that negatively regulate protein expression by inhibiting mRNA translation. MicroRNAs are vital in the antiviral immune responses of plants and invertebrates, but their role in the antiviral responses of mammals has not been extensively studied. Recent studies have shown that some microRNAs play antiviral roles by directly targeting viral genomes or mRNAs and that some are actually required for efficient virus replication. In this study we address regulation of microRNA expression in response to infection of human cells with influenza virus. To closely approximate human lung infection with influenza virus, we have used human airway epithelial (HAE) cells. We show that these cells can be infected with the A/Udorn/72 strain of influenza virus and that infection causes the expression of known innate immune factors, including interferon (IFN) b and retinoic acid-inducible gene I protein (RIG-I), which were induced to over 400 times and over 20 times the expression in mock-infected cells, respectively. We have identified cellular microRNAs that are differentially expressed in HAE cells, lung cancer cells (A549), and transformed human kidney cells (293) in response to influenza virus infection. These microRNAs include some that have previously been implicated in viral infection and the innate immune response. Differential expression of a subset of the microRNAs we identified was verified using quantitative RT-PCR assays. Though some of these microRNAs have been implicated in influenza virus infection in prior studies in mice, we provide evidence of some differences in the microRNA regulation in infection of human cells, and have identified several additional microRNAs with changes in expression following infection with influenza virus. Understanding the effects of influenza virus infection on microRNA expression could lead to a better understanding of the role of microRNAs in virus infection of mammalian cells and of the regulation of microRNA expression, in general. doi:10.1016/j.cyto.2010.07.236
PS2-32 MicroRNA regulation in response to human influenza virus infection William A. Buggele, Curt M. Horvath, Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois USA The cellular response to virus infection is initiated by recognition of an invading pathogen and subsequent changes in gene expression. The changes in gene expression are known to involve both transcriptional and translational mechanisms, and result in the establishment of an antiviral state that is hostile to virus replication. In addition to the well-established paradigms that control gene expression in response to virus infections, it has become clearly established that small RNAs contribute to antiviral responses in lower organisms via RNA interference (RNAi). However, there is little compelling evidence to suggest that RNAi mediates specific antiviral responses in mammalian hosts. Nonetheless, the understanding that microRNAs, endogenously encoded small RNAs capable of repressing translation in a sequence specific manner, are a significant element of protein expression has led to the hypothesis that microRNAs may contribute to the regulation of antiviral gene expression. To test this hypothesis, human lung cell lines (A549 and BEAS-2B) were infected with influenza virus (A/Udorn/72 and A/WSN/33) and the abundance of cellular microRNAs was evaluated with microarrays. Several microRNAs were found to change in abundance in response to influenza virus infection, and were independently verified using quantitative RT- PCR assays. These findings clearly demonstrate microRNA regulation in response to influenza virus infection in human cells. Understanding the role of microRNAs in the regulation of the cellular response to influenza virus infection will lead to a more complete understanding of the host response and may yield important therapeutic targets to combat virus infections. doi:10.1016/j.cyto.2010.07.237
Interferons in the Treatment of Diseases PS2-33 Expression of the cytosolic pyrimidine 5’-nucleotidase PN-I (NT5C3) in nonhematopoietic cells: a novel Type-I interferon-inducible gene Latifa Al-Haj, Khalid S.A. Khabar, BioMolecular Research Program, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia Pyrimidine 50 -nucleotidase (PN-I or NT5C3) is a member of the 5’-nucleotidase family of the enzymes that catalyze the dephosphorylation of nucleoside monophosphates to their corresponding nucleosides and regulate the nucleoside pools inside the cell. The NT5C3 expression has been reported to be erythrocyte-specific that possess nucleotidase and phosphotransferase activities. We have identified NT5C3 as a highly inducible gene in cells sensitive to IFN action. The present study was designed to evaluate the alterations of NT5C3 expression after type-I interferon treatment in vitro and to characterize the signaling pathway that mediate its induction. We describe enhanced expression of NT5C3 in different non-hematopoietic cell lines. Addition of Type-I (IFN-a, IFN-b, and IFN-Omega) but not type-II interferon (IFN-c)
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to epithelial cell lines revealed increase expression of NT5C3 in a time and a dose dependant manner. In addition, IFN-a treatment induces the activation of GFP construct fused with interferon stimulated element (ISRE) found in the 50 un-translated region (50 UTR) of NT5C3. Moreover, reporter experiments with GFP construct fused with NT5C3 AU-rich element (ARE) and IFN treatment show ARE mediate destabilization with wild type but not with mutant ARE containing construct. Using Signal Transducers and Activators of Transcription STAT1-deficient (U3A) and tyrosine kinase 2-deficient (U1A) fibrosarcoma cells, we observed that IFN induced expression of NT5C3 is STAT1- and Tyk2- dependent. Moreover, functional assessment using gemcitabine (20 , 20 -difluorodeoxy-cytidine) and human fibrosarcoma cancer cells revealed increased resistance to gemcitabine in cells overexpressing NT5C3. Overall, these results suggest that type-I IFN is a regulator of NT5C3 in non-hematopoietic cells, and NT5C3 is involved in gemcitabine-resistance. Current work is focused on elucidating the functional consequences of NT5C3 during IFN response, and its role in controlling inflammation via nucleoside production. doi:10.1016/j.cyto.2010.07.238
PS2-34 Evaluation of IFN-k responsiveness on renal cell carcinoma Kevin Kotredes, Yolanda Lopez, Ana Gamero, Temple University, Department of Biochemistry, Philadelphia, PA USA Interferon lambda 1 (IFN-k) is a recently discovered type III interferon (IFN-k, also called IL-28/IL-29) that shares many of the biological activities of type I IFNs (IFN-a). Type I IFNs and type III IFNs alike activate the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway leading to expression of IFN-related genes. Presently, IFN-a is used to treat certain malignancies but its use in patients is constrained due to severe toxicity. Treatment with type III IFNs may prove to be less toxic as receptors are expressed only in certain cell types, whereas receptors for type I IFNs are ubiquitously expressed. To date, the antigrowth effects of IFN-k have been studied in melanoma, esophageal, hepatocellular carcinoma cell lines. Due to the presence of IFN-k receptor (IFN-kR1) in kidney cells, the aim of this study is to assess the responsiveness of IFN-k on a panel of renal cell carcinoma lines. Our data showed that IFN-k treatment induced tyrosine phosphorylation of STAT1 and STAT2 in 5 out of 11 cell lines tested. Interestingly, of the five demonstrating activation, only one showed IFN-k-induced upregulation of MHC class I. We then found that supplementing IFN-k treatment with a receptor tyrosine kinase inhibitor, sunitinib, showed a moderate antiproliferative effect on renal cell carcinoma. Our studies have shown that the correlation between IFN-k responsiveness and the presence of IFN-kR1 mRNA transcripts is not always consistent due to the expression of different truncated forms of IFN-kR1, which may possess inhibitory activity. We investigated the existence of these altered forms of the IFN-kR1 and our results revealed three separate isoforms are found within various classes of renal cell carcinoma. Therefore, our data analysis shows that there may be more factors determining the response of renal cell carcinomas to IFN-k. doi:10.1016/j.cyto.2010.07.239
PS2-35 Reduced interferon receptor levels resulting from either knockdown or prolonged Type I interferon treatment promote a shift in cellular responsiveness Daniel Harari 1,2, Doron Levin 1,2, Renana Abramovich 1, Gideon Schreiber 1, 1 Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel, 2 D.H. and D.L. contributed equally to this study Patients undergoing Type I IFN cancer therapy are treated over extended periods, whereas in antiviral and anti-proliferative bioassays, studies are completed within days. We thus sought to examine the behavior of cancer cells undergoing prolonged IFN treatment. IFN administered to the cell lines WISH and MDA-MB-231 resulted in a maximum IFN-induced cell death of 70% within the first three days of culturing. The surviving cells however remained dormant for another 2-3 weeks. This dormancy period could be abrogated upon IFN removal. Further maintenance of these cells with IFN however resulted in their eventual breaking out of quiescence and return to growth. Examination of IFNAR1 and IFNAR2 surface expression by FACS revealed that with prolonged exposure to IFN, receptor levels were significantly reduced. To quantitatively examine the importance of IFNAR levels in IFN responsiveness, we performed a dose-responsive depletion of IFNAR1 or IFNAR2 by siRNA knockdown. 50% IFNAR depletion resulted in a significant reduction of cells undergoing anti-proliferation, whereas anti-viral activity remained stable. Further depletion of IFNARs to below a critical threshold however, rendered the cells unresponsive to both anti-viral and anti-proliferative activity. Our data provides a likely explanation as to why long term IFN administration results in loss of antiproliferative responsiveness with time. Our EC50 data clearly shows that the relationship between receptor levels and activity is a binary, zero and one response that is not dependant on the affinity or type of