Wild-type Hosts Research Articles

Embryogenesis of the First Circulating Endothelial Cells

Prior to this study, the earliest appearance of circulating endothelial cells in warm-blooded animals was unknown. Time-lapse imaging of germ-line transformed Tie1-YFP reporter quail embryos combined with the endothelial marker antibody QH1 provides definitive evidence for the existence of circulating endothelial cells – from the very beginning of blood flow. Blood-smear counts of circulating cells from Tie1-YFP embryos showed that up to 30% of blood-borne cells are Tie1 positive; though cells expressing low levels of YFP were also positive for benzidine, a hemoglobin stain, suggesting that these cells were differentiating into erythroblasts. Electroporation-based time-lapse experiments, exclusively targeting the intra-embryonic mesoderm were combined with QH1 immunostaining. The latter antibody marks quail endothelial cells. Together the optical data provide conclusive evidence that endothelial cells can enter blood flow from vessels of the embryo proper, as well as from extra-embryonic areas. When Tie1-YFP positive cells and tissues are transplanted to wild type host embryos, fluorescent cells emigrate from such transplants and join host vessels; subsequently a few YFP cells are shed into circulation. These data establish that entering circulation is a commonplace activity of embryonic vascular endothelial cells. We conclude that in the class of vertebrates most closely related to mammals a normal component of primary vasculogenesis is production of endothelial cells that enter circulation from all vessels, both intra- and extra-embryonic.

Abstract 1436: TNF-alpha-dependent tumor reprogramming and promotion by microenvironment: Potential involvement of endothelial cells.

Abstract Interactions of tumor clones with the host microenvironment have profound effects on tumor growth, response to radio/chemotherapy and formation of metastases. TNFα is a potent factor driving and orchestrating such interactions. We used mice with disrupted TNFα receptor I/II signaling to investigate phenotypic and genomic changes induced in tumors by TNFα-deficient or wild type (WT) host microenvironment. At the basal level, disruption of host stromal TNFα signaling resulted in the over-expression of tumor genes involved in extracellular matrix remodeling and angiogenesis. Phenotypically, these tumors exhibited a reduced vascular density, which induced the expression of oxidative stress signaling pathways and increased levels of tissue hypoxia and necrosis. These alterations were paralleled by the suppression of genes involved in chromosomal stability and key effectors of the ATM-dependent/ non-homologous end-joining pathways, which resulted in greater DNA double-strand breaks and caspase 3 activation following IR treatment. We further investigated tumor-associated endothelial cells (TAEC) obtained from tumors grown in WT and TNFα RI/II-/- animals. Differentially expressed genes (DEGs) primarily represented TNFα/Type I IFN-signaling. These genes significantly separated cohorts of patients with chronic inflammation (RA, IBD and liver cirrhosis) from normal samples. We trained these genes in human cancer datasets and defined a six-gene inflammatory signature that predicted significantly reduced overall survival in breast cancer, colon cancer, lung cancer, and glioma. Consistent with these findings, conditioned culture media from human endothelial cells stimulated by pro-inflammatory cytokines accelerated the growth of human colon and breast tumors in immunodeficient mice as compared with conditioned media from untreated endothelial cells. Based on these studies we came to two important conclusions. First, the host stroma may determine phenotypic and transcriptomic properties of tumor cells, and TNFα might be an important component of such microenvironment-dependent tumor reprogramming (MDTR). Second, endothelial cells may be an important cellular component of MDTR and tumor promotion. Citation Format: Nikolai N. Khodarev, Sean P. Pitroda, Ralph R. Weichselbaum. TNF-alpha-dependent tumor reprogramming and promotion by microenvironment: Potential involvement of endothelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1436. doi:10.1158/1538-7445.AM2013-1436

P190-B RhoGAP regulates the functional composition of the mesenchymal microenvironment

Hematopoiesis is regulated by components of the microenvironment, so-called niche. Here, we show that p190-B GTPase Activating Protein (p190-B) deletion in mice causes hematopoietic failure during ontogeny, in p190-B−/− fetal liver and bones, and in p190-B+/− adult bones and spleen. These defects are non-cell autonomous, since we previously showed that transplantation of p190-B−/− hematopoietic cells into wild-type hosts leads to normal hematopoiesis. Coculture of mesenchymal stem/progenitor cells (MSC) and wild-type bone marrow cells reveals that p190-B−/− MSCs are dysfunctional in supporting hematopoiesis due to impaired Wnt signaling. Furthermore, p190-B loss causes alteration in bone marrow niche composition, including abnormal CFU-fibroblast, CFU-adipocyte and CFU-osteoblast numbers. This is due to altered MSC lineage fate specification to osteoblast and adipocyte lineages. Thus, p190-B organizes a functional mesenchymal/microenvironment for normal hematopoiesis during development.

Aire Deficiency Promotes TRP-1–Specific Immune Rejection of Melanoma

The thymic transcription factor autoimmune regulator (Aire) prevents autoimmunity in part by promoting expression of tissue-specific self-antigens, which include many cancer antigens. For example, AIRE-deficient patients are predisposed to vitiligo, an autoimmune disease of melanocytes that is often triggered by efficacious immunotherapies against melanoma. Therefore, we hypothesized that Aire deficiency in mice may elevate immune responses to cancer and provide insights into how such responses might be triggered. In this study, we show that Aire deficiency decreases thymic expression of TRP-1 (TYRP1), which is a self-antigen in melanocytes and a cancer antigen in melanomas. Aire deficiency resulted in defective negative selection of TRP-1-specific T cells without affecting thymic numbers of regulatory T cells. Aire-deficient mice displayed elevated T-cell immune responses that were associated with suppression of melanoma outgrowth. Furthermore, transplantation of Aire-deficient thymic stroma was sufficient to confer more effective immune rejection of melanoma in an otherwise Aire wild-type host. Together, our work showed how Aire deficiency can enhance immune responses against melanoma and how manipulating TRP-1-specific T-cell negative selection may offer a logical strategy to enhance immune rejection of melanoma.

The Drosophila Toll Pathway Controls but Does Not Clear Candida glabrata Infections

The pathogenicity of Candida glabrata to patients remains poorly understood for lack of convenient animal models to screen large numbers of mutants for altered virulence. In this study, we explore the minihost model Drosophila melanogaster from the dual perspective of host and pathogen. As in vertebrates, wild-type flies contain C. glabrata systemic infections yet are unable to kill the injected yeasts. As for other fungal infections in Drosophila, the Toll pathway restrains C. glabrata proliferation. Persistent C. glabrata yeasts in wild-type flies do not appear to be able to take shelter in hemocytes from the action of the Toll pathway, the effectors of which remain to be identified. Toll pathway mutant flies succumb to injected C. glabrata. In this immunosuppressed background, cellular defenses provide a residual level of protection. Although both the Gram-negative binding protein 3 pattern recognition receptor and the Persephone protease-dependent detection pathway are required for Toll pathway activation by C. glabrata, only GNBP3, and not psh mutants, are susceptible to the infection. Both Candida albicans and C. glabrata are restrained by the Toll pathway, yet the comparative study of phenoloxidase activation reveals a differential activity of the Toll pathway against these two fungal pathogens. Finally, we establish that the high-osmolarity glycerol pathway and yapsins are required for virulence of C. glabrata in this model. Unexpectedly, yapsins do not appear to be required to counteract the cellular immune response but are needed for the colonization of the wild-type host.

Myeloid-Specific Expression of Ron Receptor Kinase Promotes Prostate Tumor Growth

Ron receptor kinase (MST1R) is important in promoting epithelial tumorigenesis, but the potential contributions of its specific expression in stromal cells have not been examined. Herein, we show that the Ron receptor is expressed in mouse and human stromal cells of the prostate tumor microenvironment. To test the significance of stromal Ron expression, prostate cancer cells were orthotopically implanted into the prostates of either wild-type or Ron tyrosine kinase deficient (TK(-/-); Mst1r(-/-)) hosts. In TK(-/-) hosts, prostate cancer cell growth was significantly reduced as compared with tumor growth in TK(+/+) hosts. Prostate tumors in TK(-/-) hosts exhibited an increase in tumor cell apoptosis, macrophage infiltration and altered cytokine expression. Reciprocal bone marrow transplantation studies and myeloid cell-specific ablation of Ron showed that loss of Ron in myeloid cells is sufficient to inhibit prostate cancer cell growth. Interestingly, depletion of CD8(+) T cells, but not CD4(+) T cells, was able to restore prostate tumor growth in hosts devoid of myeloid-specific Ron expression. These studies show a critical role for the Ron receptor in the tumor microenvironment, whereby Ron loss in tumor-associated macrophages inhibits prostate cancer cell growth, at least in part, by derepressing the activity of CD8(+) T cells.

Neurokinin-1 Receptor Signalling Impacts Bone Marrow Repopulation Efficiency

Tachykinins are a large group of neuropeptides with both central and peripheral activity. Despite the increasing number of studies reporting a growth supportive effect of tachykinin peptides in various in vitro stem cell systems, it remains unclear whether these findings are applicable in vivo. To determine how neurokinin-1 receptor (NK-1R) deficient hematopoietic stem cells would behave in a normal in vivo environment, we tested their reconstitution efficiency using competitive bone marrow repopulation assays. We show here that bone marrow taken from NK-1R deficient mice (Tacr1−/−) showed lineage specific B and T cell engraftment deficits compared to wild-type competitor bone marrow cells, providing evidence for an involvement of NK-1R signalling in adult hematopoiesis. Tachykinin knockout mice lacking the peptides SP and/or HK-1 (Tac1 −/−, Tac4 −/− and Tac1 −/−/Tac4 −/− mice) repopulated a lethally irradiated wild-type host with similar efficiency as competing wild-type bone marrow. The difference between peptide and receptor deficient mice indicates a paracrine and/or endocrine mechanism of action rather than autocrine signalling, as tachykinin peptides are supplied by the host environment.

Persistent Antigen and Germinal Center B Cells Sustain T Follicular Helper Cell Responses and Phenotype

T follicular helper (Tfh) cells provide help to B cells and are crucial for establishment of germinal center (GC) reactions, including production of high-affinity antibodies and generation of memory B cells and long-lived plasma cells. Here we report that the magnitude of the Tfh cell response was dictated by the amount of antigen and directly correlated with the magnitude of the GC B cell response. In addition, maintenance of the Tfh cell phenotype required sustained antigenic stimulation by GC B cells. In lymphopenic conditions, a strong and prolonged Tfh cell response led to bystander B cell activation, hypergammaglobulinemia, and production of poly- and self-reactive antibodies. These data demonstrate that antigen dose determines the size and duration of the Tfh cell response and GC reaction, highlight the transient nature of the Tfh cell phenotype, and suggest a link between overstimulation of Tfh cells and the development of dysregulated humoral immune responses.

Local Autoantigen Expression as Essential Gatekeeper of Memory T-Cell Recruitment to Islet Grafts in Diabetic Hosts

It is generally believed that inflammatory cues can attract noncognate, “bystander” T-cell specificities to sites of inflammation. We have shown that recruitment of naive and in vitro activated autoreactive CD8+ T cells into endogenous islets requires local autoantigen expression. Here, we demonstrate that absence of an autoantigen in syngeneic extrapancreatic islet grafts in diabetic hosts renders the grafts “invisible” to cognate memory (and naive) T cells. We monitored the recruitment of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206–214-reactive CD8+ T cells into IGRP206–214-competent and IGRP206–214-deficient islet grafts in diabetic wild-type or IGRP206–214−/− nonobese diabetic hosts (harboring either naive and memory T cells or only naive IGRP206–214-specific T-cells, respectively). All four host–donor combinations had development of recurrent diabetes within 2 weeks. Wild-type hosts recruited IGRP206–214-specific T cells into IGRP206–214+/+ but not IGRP206–214−/− grafts. In IGRP206–214−/− hosts, there was no recruitment of IGRP206–214-specific T cells, regardless of donor type. Graft-derived IGRP206–214 activated naive IGRP206–214-specific T cells, but graft destruction invariably predated their recruitment. These results indicate that recurrent diabetes is exclusively driven by autoreactive T cells primed during the primary autoimmune response, and demonstrate that local antigen expression is a sine qua non requirement for accumulation of memory T cells into islet grafts. These findings underscore the importance of tackling autoreactive T-cell memory after β-cell replacement therapy.

CD70 Deficiency Impairs Effector CD8 T Cell Generation and Viral Clearance but Is Dispensable for the Recall Response to Lymphocytic Choriomeningitis Virus

CD27 interactions with its ligand, CD70, are thought to be necessary for optimal primary and memory adaptive immune responses to a variety of pathogens. Thus far, all studies addressing the function of the CD27-CD70 axis have been performed in mice lacking CD27, in those overexpressing CD70, or in those in which these molecules were blocked or mimicked by Abs or recombinant soluble CD70. Because these methods have in some cases led to divergent results, we generated CD70-deficient mice to directly assess its role in vivo. We find that lack of CD70-mediated stimulation during primary responses to lymphocytic choriomeningitis virus lowered the magnitude of CD8 Ag-specific T cell response, resulting in impaired viral clearance, without affecting CD4 T cell responses. Unexpectedly, CD70-CD27 costimulation was not needed for memory CD8 T cell generation or the ability to mount a recall response to lymphocytic choriomeningitis virus. Adoptive transfers of wild-type memory T cells into CD70(-/-) or wild-type hosts also showed no need for CD70-mediated stimulation during the course of the recall response. Moreover, CD70 expression by CD8 T cells could not rescue endogenous CD70(-/-) cells from defective expansion, arguing against a role for CD70-mediated T:T help in this model. Therefore, CD70 appears to be an important factor in the initiation of a robust and effective primary response but dispensable for CD8 T cell memory responses.

Bacteriophage 434 Hex Protein Prevents RecA-Mediated Repressor Autocleavage

In a λimm434 lysogen, two proteins are expressed from the integrated prophage. Both are encoded by the same mRNA whose transcription initiates at the PRM promoter. One protein is the 434 repressor, needed for the establishment and maintenance of lysogeny. The other is Hex which is translated from an open reading frame that apparently partially overlaps the 434 repressor coding region. In the wild type host, disruption of the gene encoding Hex destabilizes λimm434 lysogens. However, the hex mutation has no effect on lysogen stability in a recA− host. These observations suggest that Hex functions by modulating the ability of RecA to stimulate 434 repressor autocleavage. We tested this hypothesis by identifying and purifying Hex to determine if this protein inhibited RecA‑stimulated autocleavage of 434 repressor in vitro. Our results show that in vitro a fragment of Hex prevents RecA-stimulated autocleavage of 434 repressor, as well as the repressors of the closely related phage P22. Surprisingly, Hex does not prevent RecA‑stimulated autocleavage of phage lambda repressor, nor the E. coli LexA repressor.

Late IL-2 signals limit effector CD4 T cell contraction and serve as a checkpoint for transition to memory

Event Abstract Back to Event Late IL-2 signals limit effector CD4 T cell contraction and serve as a checkpoint for transition to memory Karl K. Mckinstry1*, Tara M. Strutt1, Andrea Cooper2 and Susan Swain1 1 University of Massachusetts Medical School, Pathology, United States 2 Trudeau Institute, United States Pathogen challenge causes the dramatic expansion of responding T cells resulting in large populations of activated effector cells at both secondary lymphoid sites where they originate, and at sites of infection and inflammation to which they migrate. Following the resolution of infection, most effector cells undergo apoptosis in the process known as ‘contraction’, while a cohort escapes death, transitions to a resting memory state, and persists for extended periods to provide long-term immunity. The factors and mechanisms determining the extent of T cell contraction and the efficiency of memory generation are unclear. We find that late, autocrine IL-2 during a narrow timeframe is required to limit the contraction of highly-differentiated CD4 T cell effectors generated by influenza A virus (IAV). We validate key findings by restoring IL-2 signals to IL-2-deficient CD4 T cells responding in wild type (WT) hosts, and by short-term neutralization of IL-2 in recipients of WT IAV-specific donor cells. Interestingly, late IL-2 is required to rescue effectors generated from both naïve and memory precursors from enhanced contraction. One of the few differences that distinguish WT and IL-2-deficient effectors at the peak of their response is higher expression of IL-7 receptor by WT cells. We find that late IL-2 is required for effectors to permanently increase IL-7 receptor expression, allowing ambient IL-7 to limit excessive contraction and to sustain their long-term persistence in the lung and in secondary lymphoid organs. This establishes a late IL-2-dependent checkpoint for memory CD4 T cell generation, with important implications for vaccine design. Acknowledgements This work was supported by NIH grants AI-46530 and NS-061014. Keywords: T cell memory, CD4 T cell, Influenza A virus, Cytokines, Interleukin-2 Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Adaptive Immunity Citation: Mckinstry KK, Strutt TM, Cooper A and Swain S (2013). Late IL-2 signals limit effector CD4 T cell contraction and serve as a checkpoint for transition to memory. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01203 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 12 Sep 2013; Published Online: 19 Sep 2013. * Correspondence: Dr. Karl K Mckinstry, University of Massachusetts Medical School, Pathology, Worcester, Massachusetts, United States, kai.mckinstry@ucf.edu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Karl K Mckinstry Tara M Strutt Andrea Cooper Susan Swain Google Karl K Mckinstry Tara M Strutt Andrea Cooper Susan Swain Google Scholar Karl K Mckinstry Tara M Strutt Andrea Cooper Susan Swain PubMed Karl K Mckinstry Tara M Strutt Andrea Cooper Susan Swain Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

A preclinical mouse model of invasive lobular breast cancer metastasis.

Metastatic disease accounts for more than 90% of cancer-related deaths, but the development of effective antimetastatic agents has been hampered by the paucity of clinically relevant preclinical models of human metastatic disease. Here, we report the development of a mouse model of spontaneous breast cancer metastasis, which recapitulates key events in its formation and clinical course. Specifically, using the conditional K14cre;Cdh1(F/F);Trp53(F/F) model of de novo mammary tumor formation, we orthotopically transplanted invasive lobular carcinoma (mILC) fragments into mammary glands of wild-type syngeneic hosts. Once primary tumors were established in recipient mice, we mimicked the clinical course of treatment by conducting a mastectomy. After surgery, recipient mice succumbed to widespread overt metastatic disease in lymph nodes, lungs, and gastrointestinal tract. Genomic profiling of paired mammary tumors and distant metastases showed that our model provides a unique tool to further explore the biology of metastatic disease. Neoadjuvant and adjuvant intervention studies using standard-of-care chemotherapeutics showed the value of this model in determining therapeutic agents that can target early- and late-stage metastatic disease. In obtaining a more accurate preclinical model of metastatic lobular breast cancer, our work offers advances supporting the development of more effective treatment strategies for metastatic disease.

CD166 (ALCAM): A Functional Marker of Primitive Murine and Human Hematopoietic Stem Cells and Cellular Elements of Their Niche

AbstractAbstract 640Phenotypic definition of murine and human hematopoietic stem cells (HSC) relies on a large number of markers. Few surface antigens with functional importance have been identified as key common markers on adult murine and human HSC. Intimate interactions between HSC and elements of the hematopoietic niche (HN) depend on ligand-counter ligand molecules co-expressed independently on niche components or putative HSC. To date, not a single surface marker has been identified as a common marker expressed on murine and human HSC and on cells of the HN. We previously demonstrated that CD166 is expressed on osteoblasts (OB) that mediate a hematopoiesis enhancing activity (HEA). Given that CD166 is a member of the immunoglobulin superfamily that can mediate homophilic cell-cell interactions, we investigated the role of CD166 in identifying HSC and the impact of CD166 on hematopoiesis, stem cell engraftment, and the HN. Interestingly, CD166+, but not CD166- fractions of murine and human repopulating HSC identified by a rigorous hierarchical classification for each species mediated robust long-term engraftment. In the murine system, 25 sorted Lineage- Sca1+ ckit+ (LSK) CD48-CD150+CD166+ cells mediated 69.5 ± 7.3% chimerism 4 months post-transplantation (PT) while donor-derived chimerism supported by 25 sorted LSKCD48-CD150+CD166- cells was only 13.6 ± 11.6% (p<0.01) suggesting that CD166 identifies long-term repopulating cells beyond what is possible with SLAM markers. In the human system, 1000 cord blood-derived Lin-CD34+CD38-CD49f-CD166+ cells and Lin-CD34+CD38-CD49f+CD166+ cells engrafted at 44.5 ± 9.7% and 38.4 ± 8.9%, respectively 16 weeks PT in conditioned NSG mice. More importantly, chimerism derived from Lin-CD34+CD38-CD49f+CD166- cells was 1.6 ± 0.1% (p<0.01 vs both fractions) demonstrating that the CD166+ but not the CD166- fraction of CD34+Lin-CD38- cells (regardless of the status of CD49f expression) contains long-term engrafting human HSC. In CD166 knockout (KO) mice, numbers of LSKCD48-CD150+ cells in the bone marrow and Lin-CD48- cells in the peripheral blood were significantly reduced relative to wild-type (WT) controls although other hematopoietic parameters in KO mice were within normal ranges. Phenotypically defined HSC from CD166−/− mice failed to engraft in lethally irradiated WT recipients. Levels of engraftment 4mo PT of 1,000 LSK cells from WT donors into WT recipients was 71.8% ± 8.3% while that obtained from a similar number of KO cells was 5.8% ± 2.8% (p<0.01). To permit direct comparison of KO and WT mice as recipients, both genotypes were transplanted with purified LSK cells from GFP C57BL/6 mice. While short-term repopulating GFP cells engrafted efficiently in KO mice 1mo PT (66.0% ± 6.5%), reconstitution declined substantially 2mo PT and was 10.3% ± 2.7% at 3mo PT (compared to 52.6% ± 10.4% in WT hosts, p<0.01) and less than 5% at 4mo PT, demonstrating that the CD166−/− hematopoietic niche can not support long-term repopulating cells. We used our previously described co-culture system to assess the impact of homophilic CD166 interactions on the HEA of OB. The highest HEA was reached when both OB and LSK cells expressed CD166. However, when either or both cell types lacked CD166 expression, the degree of HEA was significantly lower demonstrating that homophilic CD166 interactions are critical to maintaining HSC function. Since Stat3 has 3 binding sites on the CD166 promoter, we examined the relationship between expression of Stat3 and CD166. HSC from Stat3−/− mice which do not engraft efficiently in WT recipients expressed very low levels of CD166. In addition, pharmacologic inhibition of Stat3 expression led to a simultaneous inhibition of CD166 expression. Reconstitution kinetics data and survival of KO mice under hematopoietic stress conditions suggested that CD166−/− HSC have an intrinsic self-renewal capacity precluding them from both rapid proliferation and expansion and maintenance of the stem cell pool in the HN. Our data illustrate for the first time, that CD166 is a universal marker of both murine and human HSC and OB within the HN and suggest that CD166 may modulate HSC-niche interactions and impact stem cell fate. The conserved homology between murine and human CD166 provides an excellent bridge between human and murine studies for efficient translational investigations and interventions for enhancing stem cell engraftment and clinical utility. Disclosures:Broxmeyer:CordUse: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy.

Regulatory Role of Host IL-17 Via Control of Host Macrophage Activation Contributes to Less Acute Gvhd

AbstractAbstract 4669IL-17 has emerged as a key pro-inflammatory cytokine, however, recent studies demonstrated that IL-17 has also anti-inflammatory effects. In addition, a wide variety of IL-17 producing cells are identified not only in T-cells but also in subpopulation of monocytes and macrophages, mast cells, Paneth cell in the gut so on. Therefore, IL-17 mediated immune responses are becoming much more complicated. In hematopoietic stem cell transplantation, we have reported that host-derived IL-17 has a protective effect against acute graft-versus-host disease (GVHD) using rodent models, in contrast, donor-BM-derived IL-17 exacerbates chronic GVHD. In acute GVHD model, lethally irradiated IL-17 knockout (KO) recipient mice receiving allogeneic BM with low dose splenocytes developed more severe acute gut GVHD compared to wild type (WT) host mice and finally half of them died (p<0.05). Much higher number of infiltrated monocyte/macrophage cluster was observed in spleen and gut in IL-17 KO host mice. Furthermore, residual host-typed peritoneal macrophages in IL-17 KO host mice were highly activated with the expression of TNF-α, while activation of donor-typed macrophages was much less with the production of anti-inflammatory cytokine IL-10. It suggested that these activated and increased macrophages might deeply involve in the pathogenesis of lethal GVHD. To test this, IL-17 KO recipient mice were given lipoCL2MDP intraperitoneally on day -7 before BMT to deplete host macrophages. As shown in the figure below, the macrophages depleted IL-17 KO host mice given splenocytes to induce lethal GVHD showed a significant survival benefit compared with IL-17 KO mice without depletion of macrophages. In conclusion, host IL-17 has a protective effect against acute GVHD with suppression of the activated macrophages. This is the first report that IL-17 has the immuno-regulatory role via control of activated host macrophages in acute GVHD. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

TLR7 Contributes to the Rapid Progression but Not to the Overall Fatal Outcome of Secondary Pneumococcal Disease following Influenza A Virus Infection

Increased risk for bacterial superinfections substantially contributes to the mortality caused by influenza A virus (IAV) epidemics. While the mechanistic basis for this lethal synergism is still insufficiently understood, immune modulation through the viral infection has been shown to be involved. Since the pattern-recognition receptor (PRR) toll-like receptor 7 (TLR7) is a major sensor for the viral genome, we studied how IAV recognition by TLR7 influences the development of secondary pneumococcal infection. In a mouse model of IAV, TLR7-deficient hosts induced a potent antiviral response and showed unchanged survival. In secondary pneumococcal infection during acute influenza, TLR7ko mice showed a fatal outcome similar to wild-type (WT) hosts, despite significantly delayed disease progression. Also, when bacterial superinfection occurred after virus clearance, WT and TLR7-deficient hosts showed similar mortality, even though we found the phagocytic activity of alveolar macrophages isolated from IAV-pre-infected hosts to be enhanced in TLR7ko over WT mice. Thus, we show that a virus-sensing PRR modulates the progression of secondary pneumococcal infection following IAV. However, the fatal overall outcome in WT as well as TLR7ko hosts suggests that processes distinct from TLR7-triggering override the contribution of this single PRR.

MMP9 regulates the cellular response to inflammation after skeletal injury

Like other tissue injuries, bone fracture triggers an inflammatory response, which plays an important role in skeletal repair. Inflammation is believed to have both positive and negative effects on bone repair, but the underlying cellular mechanisms are not well understood. To assess the role of inflammation on skeletal cell differentiation, we used mouse models of fracture repair that stimulate either intramembranous or endochondral ossification. In the first model, fractures are rigidly stabilized leading to direct bone formation, while in the second model, fracture instability causes cartilage and bone formation. We compared the inflammatory response in these two mechanical environments and found changes in the expression patterns of inflammatory genes and in the recruitment of inflammatory cells and osteoclasts. These results suggested that the inflammatory response could influence skeletal cell differentiation after fracture. We then exploited matrix metalloproteinase 9 (MMP9) that is expressed in inflammatory cells and osteoclasts, and which we previously showed is a potential regulator of cell fate decisions during fracture repair. Mmp9−/− mice heal stabilized fractures via endochondral ossification, while wild type mice heal via intramembranous ossification. In parallel, we observed increases in macrophages and T cells in the callus of Mmp9−/− compared to wild type mice. To assess the link between the profile of inflammatory cells and skeletal cell fate functionally, we transplanted Mmp9−/− mice with wild type bone marrow, to reconstitute a wild type hematopoietic lineage in interaction with the Mmp9−/− stroma and periosteum. Following transplantation, Mmp9−/− mice healed stabilized fractures via intramembranous ossification and exhibited a normal profile of inflammatory cells. Moreover, Mmp9−/− periosteal grafts healed via intramembranous ossification in wild type hosts, but healed via endochondral ossification in Mmp9−/− hosts. We observed that macrophages accumulated at the periosteal surface in Mmp9−/− mice, suggesting that cell differentiation in the periosteum is influenced by factors such as BMP2 that are produced locally by inflammatory cells. Taken together, these results show that MMP9 mediates indirect effects on skeletal cell differentiation by regulating the inflammatory response and the distribution of inflammatory cells, leading to the local regulation of periosteal cell differentiation.

Transgenic Analysis of Signaling Pathways Required for Xenopus Tadpole Spinal Cord and Muscle Regeneration

The Xenopus tadpole has the capacity fully to regenerate its tail after amputation. Previously, we have established that this regeneration process requires the operation of several signaling pathways including the bone morphogenic protein, Wnt, and Fgf pathways. Here, we have addressed the signaling requirements for spinal cord and muscle regeneration in a tissue-specific manner. Two methods were used namely grafts of transgenic spinal cord to a wild type host, and the use of the Tet-on conditional transgenic system to express inhibitors in the individual tissues. For the grafting experiments, the tail was amputated through the graft, which contained a temperature inducible inhibitor of the Wnt-β-catenin pathway. For the Tet-on experiments, treatment with doxycycline was used to induce cell autonomous inhibitors of the Wnt-β-catenin or the Fgf pathway in either spinal cord or muscle. The results show that both spinal cord and muscle regeneration depend on both the Wnt-β-catenin and the Fgf pathways. This experimental design also enables us to observe the effect of inhibition of regeneration of one tissue on the regeneration of the others. Regardless of the method of inhibition, we find that reduction of spinal cord regeneration reduces regeneration of other parts of the tail, including the myotomal muscles. In contrast, reduction of muscle regeneration has no effect on the regeneration of the spinal cord. In common with other regeneration systems, this indicates that soluble factors from the spinal cord are needed to promote the regeneration of the other tissues in the tail.

Ectopic formation of primordial germ cells by transplantation of the germ plasm: Direct evidence for germ cell determinant in Xenopus

In many animals, the germ line is specified by a distinct cytoplasmic structure called germ plasm (GP). GP is necessary for primordial germ cell (PGC) formation in anuran amphibians including Xenopus. However, it is unclear whether GP is a direct germ cell determinant in vertebrates. Here we demonstrate that GP acts autonomously for germ cell formation in Xenopus.EGFP-labeled GP from the vegetal pole was transplanted into animal hemisphere of recipient embryos. Cells carrying transplanted GP (T-GP) at the ectopic position showed characteristics similar to the endogenous normal PGCs in subcellular distribution of GP and presence of germ plasm specific molecules. However, T-GP-carrying-cells in the ectopic tissue did not migrate towards the genital ridge. T-GP-carrying cells from gastrula or tailbud embryos were transferred into the endoderm of wild-type hosts. From there, they migrated into the developing gonad. To clarify whether ectopic T-GP-carrying cells can produce functional germ cells, they were identified by changing the recipients, from the wild-type Xenopus to transgenic Xenopus expressing DsRed2. After transferring T-GP carrying cells labeled genetically with DsRed2 into wild-type hosts, we could find chimeric gonads in mature hosts. Furthermore, the spermatozoa and eggs derived from T-GP-carrying cells were fertile. Thus, we have demonstrated that Xenopus germ plasm is sufficient for germ cell determination.

Suppressing autoimmunity by TGF-β: not just through Treg cells

Transforming growth factor-β (TGF-β) is a pleiotropic factor. It regulates many aspects of T-cell biology including thymocyte development, T cell differentiation and effector T cell function.1 Previous work has shown that the loss of TGF-β signaling in the T-cell lineage leads to severe lymphoproliferative autoimmunity.2,3 Until now, it has been unclear to what extent the autoimmunity observed in those mice is driven by TGF–unresponsive conventional T cells, as TGF- is also crucial for the maintenance and function of regulatory T cells (Treg). A recent study published in Nature Immunology revealed a novel role of TGF-β signaling in non-regulatory naive T cells. It was found that TGF-β controls lymphopenia-driven T-cell proliferation in neonatal mice, thereby preventing the development of autoimmunity.4