Wild-type B6 Research Articles

Involvement of microglial CD40 in murine retrovirus-induced peripheral neuropathy (P6173)

Abstract The underlying mechanism of HIV-associated peripheral neuropathy is unclear. C57BL/6 (B6) mice infected with LP-BM5 develop severe immunodeficiency (termed as MAIDS) and peripheral neuropathy. It is known that CD154-CD40 interactions between CD4+ T and B cells are required for the manifestation of MAIDS post-LP-BM5 infection (p.i.) and that HIV encephalitis is associated with increased expression of CD40 on microglia. We aimed to determine whether spinal cord microglial CD40 signaling is involved in LP-BM5-induced peripheral neuropathy. B6-CD40 KO mice were adoptively transferred with either total leukocytes or B cells and examined for mechanical sensitivity, tissue viral loads, and cytokine responses along with the development of MAIDS p.i.. CD40 KO mice transferred either cell population developed MAIDS, the severity of which was correlated with peripheral cytokine responses. CD40 KO mice displayed transient and slightly increased mechanical sensitivity p.i. regardless of cell transfer. In all peripheral tissues examined, increased viral RNA was detected in all infected mice without significant group differences. However, in the lumbar spinal cord, infected wild type B6 mice had the highest viral load and cytokine responses among all groups. Neither type of cell transfer enhanced the LP-BM5-induced increase in viral load and cytokine responses in CD40 KO mice. Together, these data support the involvement of microglial CD40 in LP-BM5-induced peripheral neuropathy.

A novel role for IL-15 in the regulation of redox state in stressed mouse skin (P5086)

Abstract The association of IL-15 with a range of inflammatory disorders has implicated its function in innate immune response. Several isoforms of IL-15 mRNA generated by alternative splicing have been found constitutively expressed in normal mouse tissues at low level. An ENU mutagenized mouse (pedigree 191) bears a mutation in the exon 7 of IL-15 mRNA. Although the transcriptional level of the canonical IL-15 mRNA in P191 was comparable with B6 wild type skin, a transcript generated from alternatively spliced exon 7 of IL-15 mRNA (IL-15_ASE7) was increased by 8 folds in the epidermis of mutant mice. Mechanical stimulation induced downregulation of IL-15 mRNA in B6 but less reduced in P191 abraded skin which also exhibited attenuated dermal cellular infiltration and reduced expression of IL-1β, TNF-α and IL-6 transcripts. Moreover, genome-wide transcriptional profiling of B6 and P191 abraded skin by DNA microarray analysis showed that genes involved in oxidative phosphorylation and glutathione metabolism were distinguishably expressed between B6 and P191 abraded skin. Particularly, transcriptional levels of Uqcrb gene encoding components of the mitochondrial respiratory chain complex III and anti-oxidant Sod-1 gene were significantly reduced in P191 skin at 48 hours after abrasion. Detailed mechanisms by which IL-15 controls the redox state in epidermal keratinocytes as well as the regulatory role of IL-15_ASE7 on IL-15 action will be elucidated.

Glycolysis and Mitochondrial Respiration in Mouse LDHC-Null Sperm1

We demonstrated previously that a knockout (KO) of the lactate dehydrogenase type C (Ldhc) gene disrupted male fertility and caused a considerable reduction in sperm glucose consumption, ATP production, and motility. While that study used mice with a mixed genetic background, the present study used C57BL/6 (B6) and 129S6 (129) Ldhc KO mice. We found that B6 KO males were subfertile and 129 KO males were infertile. Sperm from 129 wild-type (WT) mice have a lower glycolytic rate than sperm from B6 WT mice, resulting in a greater reduction in ATP production in 129 KO sperm than in B6 KO sperm. The lower glycolytic rate in 129 sperm offered a novel opportunity to examine the role of mitochondrial respiration in sperm ATP production and motility. We observed that in media containing a mitochondrial substrate (pyruvate or lactate) as the sole energy source, ATP levels and progressive motility in 129 KO sperm were similar to those in 129 WT sperm. However, when glucose was added, lactate was unable to maintain ATP levels or progressive motility in 129 KO sperm. The rate of respiration (ZO2) was high when 129 KO or WT sperm were incubated with lactate alone, but addition of glucose caused a reduction in ZO2. These results indicate that in the absence of glucose, 129 sperm can produce ATP via oxidative phosphorylation, but in the presence of glucose, oxidative phosphorylation is suppressed and the sperm utilize aerobic glycolysis, a phenomenon known as the Crabtree effect.

Blood Group A Transgenic Mice: A Model for ABO-Incompatible (ABOi) Heart Transplantation (HTx)

Purpose Our group demonstrated that infant ABOi HTx results in development of immune tolerance to donor ABO antigen(s). We recently developed mice transgenic for expression of A- and H-glycosyltransferases (AH-Tg, C57BL/6 (B6) background). By lectin staining, AH-Tg mice express blood group A-antigen on vascular endothelium of solid organs including heart. Human ‘A into O’ HTx can be approximated using AH-Tg mice as donors and B6 wild-type (WT) mice as recipients. Herein we sought to characterize ‘natural’ and induced anti-A antibodies (Abs) in WT mice and binding to A-expressing AH-Tg hearts, and to investigate AMR following transplantation of AH-Tg heart grafts into WT mice with high anti-A titre. We hypothesize that serum from WT mice with high anti-A titre will bind to AH-Tg cardiac tissue, and that AH-Tg heart grafts will undergo AMR following transplantation into WT recipients with high anti-A Abs. Methods and Materials Juvenile WT B6 mice were induced to produce anti-A Abs by i.p. injection of blood group A red blood cells. Sera from WT and AH-Tg mice of different ages were assessed for anti-A Ab by hemagglutination assay. Binding of serum anti-A Abs to AH-Tg hearts was determined by immunohistochemistry (IHC). WT mice with high anti-A titres received AH-Tg heart transplants; AMR was assessed by IHC. Results Increasing anti-A Abs were detected with age in WT mice (n=108). No AH-Tg mice had detectable anti-A Abs (n=19). Using a commercial anti-A Ab, we detected A-antigen on vascular endothelium of hearts, kidneys, livers, and lungs from AH-Tg mice. Serum from older WT mice with natural anti-A Abs or from younger A-antigen-sensitized WT mice bound to AH-Tg but not WT cardiac tissue. Conclusions This study provides insight into the development of anti-A Abs in WT mice and confirms A-antigen expression on AH-Tg cardiac endothelium by serum anti-A Abs. Studies are ongoing to examine AMR following transplantation of AH-Tg heart grafts into WT mice with high anti-A titres.

Role of IRAK-M in Alcohol Induced Liver Injury

Increasing evidence suggests that innate immunity plays an important role in alcohol-induced liver injury and most studies have focused on positive regulation of innate immunity. The main objective of this study was to investigate the negative regulator of innate immunity, IL-1/Toll-like receptor (TLR) signaling pathways and interleukin receptor-associated kinase-M (IRAK-M) in alcoholic liver injury. We established an alcohol-induced liver injury model using wild type and IRAK-M deficient B6 mice and investigated the possible mechanisms. We found that in the absence of IRAK-M, liver damage by alcohol was worse with higher alanine transaminase (ALT), more immune cell infiltration and increased numbers of IFNγ producing cells. We also found enhanced phagocytic activity in CD68+ cells. Moreover, our results revealed altered gut bacteria after alcohol consumption and this was more striking in the absence of IRAK-M. Our study provides evidence that IRAK-M plays an important role in alcohol-induced liver injury and IRAK-M negatively regulates the innate and possibly the adaptive immune response in the liver reacting to acute insult by alcohol. In the absence of IRAK-M, the hosts developed worse liver injury, enhanced gut permeability and altered gut microbiota.

Endogenous Expression of B7-H4 Improves Long-term Murine Islet Allograft Survival

Allograft rejection is one of the main obstacles for islet transplantation. B7-H4 plays a key role in maintaining T-cell homeostasis by reducing T-cell proliferation and cytokine production. In this study, we investigated whether the endogenous expression of B7-H4 in β cells from B7-H4 transgenic mice enhances islet allograft survival. B7-H4 transgenic C57BL/6 (B6) mice (RIP.B7-H4) were developed by inserting the entire B7-H4 open reading frame under the rat insulin promoter (RIP). B7-H4 protein expression was examined by flow cytometric analysis and immunohistochemical staining. Islet allograft survival was investigated in streptozotocin-induced diabetic recipient BALB/c (H-2d) mice transplanted with 400 islets from RIP.B7-H4 (H-2b) mice under the kidney capsule. The recipient control group received islets from wild-type B6 donors. B7-H4 protein was significantly up-regulated in isolated islets from RIP.B7-H4 compared with wild-type B6 mice (56%±23% vs. 3%±1.2%). B7-H4 was coexpressed with insulin, but not glucagon, suggesting that B7-H4 is expressed in a β-cell-specific manner. Recipient BALB/c mice transplanted with RIP.B7-H4 islets established euglycemia for 42.3±18.4 days (mean±SD; n=9) compared with controls at 23.1±7.8 days (mean±SD; n=12; P<0.004, log-rank test). The endogenous expression of B7-H4 in donor β cells from transgenic mice prolongs islet allograft survival, confirming the negative role of B7-H4 in regulating alloreactive T-cell responses.

A single non-synonymous polymorphism of TLR2 is sufficient to recapitulate the variability of Experimental Multiple Sclerosis

A single non-synonymous polymorphism of TLR2 is sufficient to recapitulate the variability of Experimental Multiple Sclerosis

The Early Rejection of Allogeneic BMC by Macrophages or NK Cells Is TRIF Signaling Dependent

Abstract 1883The barrier for rejection of allogeneic bone marrow cells (BMC) has been attributed primarily to adaptive immunity, especially T cell immune responses. Significant progress has been made in developing immune-based nonmyeloablative conditioning strategies to achieve mixed chimerism in bone marrow transplantation (BMT) with targeting of T cells. The role of the innate immune system in BMC allorejection has not been adequately addressed. The fact that when T cells are targeted, alloengraftment requires additional conditioning from nonspecific reagents, such as irradiation and immunosuppressive drugs, suggesting the existence of another barrier. As humoral immunity is unlikely a barrier for BMC in unprimed naïve recipients, the innate immune system is most likely another barrier in BMT. The present study focused on a role for components of the innate immune response in allogeneic BMT. Using T cell-deficient (TCR-β/δ−/−) mice, we found that rejection of transplanted allogeneic BMC occurred very early, well before the time required for T cell activation and was T cell independent, suggesting an effector role for innate immune cells in BMC rejection.How the innate immune system recognizes or responds to allogeneic BMC remains unknown. Toll-like receptors (TLR) are a class of proteins that play a key role in the innate immune system. The signaling function of TLR depends on intracellular adaptors of MyD88 and/or TRIF. We have demonstrated that TRIF signaling is the innate immune signaling in BMC allorejection by showing superior engraftment in mice deficient in TRIF but not MyD88. To further determine the cell populations of innate immunity in allogeneic BMC rejection mediated through TRIF signaling, TRIF deficient (TRIFLps2/Lps2) mice were used as recipients for in vivo cytotoxicity assays after adoptive transfer of wildtype innate immune cells: macrophages or NK cells. Wildtype F4/80+ macrophages were sorted from B6 spleens and peritoneal cavities and NK1.1+ NK cells from B6 spleens. The doses of transferred NK1.1+ and F4/80+ cells were 370,000 and 140,000 per recipient, respectively. One day after transfer, 20 × 106 CFSE-labeled BALB/c target (high intensity) and internal control B6 (low intensity) BMC were injected. TRIFLps2/Lps2 mice that did not receive transferred cells and wildtype B6 mice treated with saline served as controls.As expected, donor cells were rapidly eliminated in control wildtype B6 mice and rejection was complete by day 3. The rejection of donor cells was significantly less in TRIFLps2/Lps2 mice without receiving adoptively transferred cells compared with wildtype B6 controls, from marginal significance (P = 0.04) at 3 hr to the highest significance (P = 0.0001) at day 3 after cell infusion. At day 3, the killing rates were 91.4 ± 1.4% in TRIFLps2/Lps2 mice without transferred cells and 97.6 ± 1.5% in wildtype B6 controls. The eliminating rates of donor cells were increased in TRIFLps2/Lps2 recipients that received either F4/80+ or NK1.1+ cells, and the kinetics of elimination of donor cells was shifted to resemble B6 controls with no significant difference between them at these 3 time points (P values: 0.11 to 0.87). The cytotoxicity of donor cells was significantly increased in TRIFLps2/Lps2 recipients adoptively transferred with F4/80+ or NK1.1+ cells when compared with TRIFLps2/Lps2 controls at all time points (P values: 0.038 to 0.002), except the one at 3hr when compared between TRIFLps2/Lps2 recipients received NK1.1+ cells and TRIFLps2/Lps2 controls (P = 0.058). At day 1, the killing percentages of CFSE labeled BALB/c cells were 71.0 ± 5.1%, 69.1 ± 3.9%, or 61.1 ± 5.8% in TRIFLps2/Lps2 recipients that received either F4/80+ cells, NK1.1+ cells, or none, respectively. Taken together, the restored cytotoxicity in TRIF-deficient recipients transferred with wildtype F4/80+ or NK1.1+ cells suggests that TRIF signaling is essential for macrophage- and NK cell-mediated early rejection of allogeneic BMC, and that both cell types function as non-redundant effector cells in BMC rejection. Disclosures:Ildstad:Regenerex, LLC, a biotech start-up company: Equity Ownership.

CD24-Siglec-G Interaction Plays an Important in Reducing Experimental Graft-Versus-Host Disease (GVHD)

Abstract 453The role of host antigen presenting cells (APCs) on negatively regulating GVHD is not well understood. Members of the sialic acid binding Ig–like lectin-G (Siglec-G) is an immunoreceptor tyrosine-based inhibitory motifs (ITIM) or ITIM-like regions in its intracellular domain that negatively regulates immune activation induced by non-infectious damage associated molecules (DAMPs). Following conditioning for allogeneic BMT, several DAMPs are released which stimulate host APCs and enhance GVHD. But the role of negative regulators of DAMP associated immune activation, such as Siglecs, in regulating allo-reactivity is not known. We therefore utilized well defined clinically relevant murine models of allogeneic BMT to test the hypothesis that deficiency of a negative regulator of responses to DAMPs in the hosts, namely Siglec-G, will increase GVHD. B6 wild type (WT) and Siglec-G−/− animals were lethally irradiated (13Gy) and transplanted on day 0 with 5×106 bone marrow and 3×106 splenic CD90+T cells from either syngeneic WT-B6 or MHC mismatched BALB/c donors. The Siglec-G−/− animals showed significantly worse survival than the allo-WT animals (p=0.0045). The increased mortality was associated with an increase in GVHD specific clinical severity (p<0.05), donor T cell expansion (p<0.03), and serum levels of pro-inflammatory cytokines (TNFα, IFN-γ, p<0.05) on day +7 after BMT. We next evaluated whether this was because of Siglec-G deficiency only on the radiosensitive host APCs. To this end we generated [B6àB6Ly5.2] and [Siglec-G−/−àB6Ly5.2] BM chimeras and utilized them as recipients following lethal radiation. They were injected with 5×106 BM and 3×106 CD90+ T cells from either syngeneic WT B6 or allogeneic BALB/c donors. The allogeneic [Siglec-G−/−àB6Ly5.2] animals demonstrated significantly worse survival than the [B6àB6Ly5.2] animals (p<0.0001). We determined the converse, i.e. analyzed the role of Siglec-G on radio-resistant host APCs by generating [B6Ly5.2àB6] and [B6Ly5.2àSiglec-G−/−] BM chimeras and utilized them as recipients. [B6Ly5.2àSiglec-G−/−] chimeras demonstrated similar survival as [B6Ly5.2àB6] chimeras. These data collectively demonstrate that Siglec-G expression only on the host radiosensitive APCs is critical for protection from GVHD. To confirm the role of increased DAMPs in causing greater mortality we tested whether the intensity of conditioning affects the serum level of DAMPs (HMGB1, proinflammatory cytokines) and found that significantly greater levels of DAMPs were observed in the mice that received 13Gy than 8 Gy. Furthermore, consistent with the increased levels of DAMPs, Siglec-G−/− animals showed higher GVHD only after 13Gy radiation but not after 8Gy conditioning. Because responses to non-infectious DAMPs are regulated by Siglec-G through its interaction with CD24 we next hypothesized that enhanced CD24-Siglec-G interaction would mitigate GVHD following myeloablative conditioning. We first characterized stimulation of allogeneic T cell responses by Siglec-G−/− APCs. We utilized CD24−/− and WT BALB/c T cells as responders in an MLR with B6 and Siglec-G−/−stimulators. We found that Siglec-G−/− APCs expanded the CD24−/− T cells more than WT-B6 APCs. We next tested in vivo whether enhanced CD24-Siglec-G interaction would mitigate GVHD. We utilized a novel CD24 fusion protein (day-1, 100mg/mouse) and found that it decreased GVHD mortality only in the WT but not in the Siglec-G−/− animals. Together our data demonstrate a critical role for CD24-Siglec-G interactions in regulating GVHD and suggest that administration of the novel CD24 fusion protein may be an innovative strategy to mitigate GVHD. Disclosures:No relevant conflicts of interest to declare.

Inhibitory Ligands CD200, CD270, CD274 and CD276 Are Expressed On Eμ-TCL1 Transgenic Mouse Splenocytes and Are of Potential Relevance to Impaired T-Cell Function in Vivo

AbstractAbstract 313 Background:We have previously demonstrated that CD4 and CD8 T-cells from CLL patients show profound dysfunctions in multiple gene pathways, including the actin cytoskeleton, which impairs the formation of functional immunologic synapses between T cells and APCs. Functional screening assays on Mec-1 cells have identified CD200, CD270, CD274, and CD276 as inhibitory ligands which induce impaired actin synapse formation in both allogeneic and autologous T cells. We also demonstrated that the Eμ-TCL1 transgenic mouse model of CLL closely resembles the T-cell defects observed in humans, validating it as a valuable preclinical tool to examine changes in the microenvironment alongside the development of leukaemia. The aim of the current study is to investigate the role of CD200, CD270, CD274, and CD276 in the Eμ-TCL1 model. Methods:We used multiparameter flow cytometry to establish the expression of inhibitory ligands on CD19+/CD5+ unpurified splenocytes from Eμ-TCL1 mice on both the C57Bl/6 (B6) and the C3HB6-F1 background and compared this to unpurified splenocytes from age matched wild-type (WT) controls of the respective coisogenic strain. Results:A total of 19 leukemic Eμ-TCL1 (n=10 C57Bl/6 and n=9 C3HB6-F1 background) and 11 WT mice (n=6 C57Bl/6 and n=5 C3HB6-F1 background) were examined. CD19+/CD5+ CLL cells constituted 92% (range 62–97%) of the DAPI-negative lymphocyte population. On CD19+/CD5+ CLL cells, CD274 (mean 98% ± SEM 0.4) and CD200 (mean 84% ± SEM 2.9 were uniformly strongly expressed, while CD270 (mean 74% ± SEM 4.7) and CD276 (mean 50% ± SEM 6.6) showed a weaker and more diverse expression, with no significant differences between the two backgrounds (all p>.05). Similar expression patterns were observed in Eμ-TCL1 mice with spontaneously occurring CLL and transplanted transgenic mice, with no differences between spontaneous and induced CLL (all p>.05). We then compared transgenic CD19+/CD5+ CLL cells to the WT CD19+ and the WT CD19+/CD5+ B1a-like cell population. Eμ-TCL1 CLL splenocytes showed a significant higher expression of CD274 and CD276 compared to expression on WT CD19+ (p<.0001, p=.00349) splenocytes. When compared to WT B1a-like splenocytes, only CD274 was significantly higher expressed (p<.0001). To clarify the impact of genetic strain, B6 and C3HB6-F1 were investigated separately: transgenic mice on the B6 background showed significantly higher expression of CD274 compared to WT B6 CD19+ (p=.0015) and WT B6 B1a-like (p<.0001) splenocytes. In contrast, transgenic mice on the C3HB6-F1 background showed a significant higher expression of CD274 and CD276 compared to WT CD19+ (p=.0002, p=.00354) and WT B1a-like (p=.0005, p=.00384) splenocytes. These patterns substantiate differences of the expression of inhibitory ligands between the WT strains, but of note, these were not mirrored in TCL1 mice.In previous experiments, we used the Eμ-TCL1 model to investigate the polarization of F-actin and phosphotyrosine at the immune synapse between splenic autologous T-cells and APCs and subsequent effector function. Age-matched WT mice had a significantly higher accumulation than transgenic mice. To assess the functional role of inhibitory ligands, knock-down experiments using lentiviral shRNA and blocking antibodies are currently under way to assess if this restores immune synapse formation and T cell effector function in vivo. Conclusions:The inhibitory ligands CD200, CD270, CD274 and CD276 are expressed in vivo and appear to be of functional relevance for the anti-cancer immune response. They therefore represent attractive targets to restore T-cell effector function, which might be achieved by gene therapy approaches and blocking antibodies. Disclosures:Gribben:Celgene: Honoraria.

Deregulation of CCND1 or BCL2 in the Immature B-Cell Stage Determines the Resulting Lymphoma Histology

AbstractAbstract 416In B-cell lymphomas, several chromosomal translocations are associated with specific histological subtypes. CCND1/IGH is detected in more than 90 % of the cases with mantle cell lymphoma (MCL), and BCL2/IGH is characteristically observed in follicular lymphoma and diffuse large B-cell lymphoma (DLBCL) of germinal center (GC) B-cell origin. Although these strong correlations are clinically recognized, their biological mechanism is not clearly explained so far. According to the results of translocation breakpoint mapping, both CCND1 and BCL2 translocations are considered to be generated by an error during physiological VDJ rearrangement of the IGH gene in the precursor-B cell stage. We hypothesized that the occurrence of these translocations in the immature B-cell stage, probably as an initial genetic event, should have a special impact on the determination of resulting lymphoma histology.We generated a mouse model mimicking human lymphoma with CCND1 or BCL2 translocation by lentivirally introducing these genes into Tp53+/− B6 mouse bone marrow cells and transplanting them to lethally irradiated wild-type B6 mice. In this model, CCND1 or BCL2 is expressed from immature to mature B cell stage under the control of the CD19 promoter, and subsequently mutations accumulate in the background of Tp53 haploinsufficiency. Both mice developed B-cell lymphomas several months after the transplantation, but their tumors showed some different features. CCND1-Tp53+/− mice developed B220lowCD5+CD23− tumors that expand into the B-cell area with sparing the GC, whereas BCL2-Tp53+/− mice preferentially developed B220highCD5−CD23+ tumors that tend to localize in the GC. Additionally, somatic hypermutation (SHM) analysis of the IGH gene of the tumor cells revealed obviously higher mutation frequency in the BCL2-Tp53+/− mice than in the CCND1-Tp53+/- mice (p<0.004). These results indicate that the primary gene deregulation of CCND1 and BCL2 determines the upcoming lymphoma of MCL-type and GC B-cell lymphoma-type, respectively. MCL has been postulated to be derived from naïve pre-GC B cells, with few SHM introduced in the immunoglobulin variable region. But the MCL-like tumor cells generated in CCND1-Tp53+/−mice seem to be originated from B-1a B cells, a distinct B cell population that does not enter GC and has few SHM in nature.There has been a debate whether B-1 cell population exists in humans, but it is recently proposed that the phenotype of human B-1 cells is CD20+CD27+CD43+CD70− by testing sort-purified B cell fractions for fundamental B-1 cell functions based on mouse studies (J Exp Med 2011;208:67–80). Interestingly, flow cytometric analysis of the human MCL tumor cells has shown that they mostly express this provisional B-1 cell phenotype, supporting the idea that human MCL is also derived from B-1 cells.Our mouse model precisely reproduces the link between CCND1 and BCL2 translocations and the resulting lymphoma subtypes in humans. It is assumed that these translocations trigger the cell expansion of different B-cell subgroups, which consequently leads to the development of lymphoma of distinct histology. Our findings provide new insights into the mechanism of lymphoma subtype determination. Disclosures:No relevant conflicts of interest to declare.

The Role of VIP in Cornea

Exogenous vasoactive intestinal peptide (VIP) down-regulates pro-inflammatory but up-regulates anti-inflammatory cytokines, growth factors (GFs) and Toll-like receptors promoting healing in experimental Pseudomonas aeruginosa (P. aeruginosa) keratitis. Whether VIP is required for GF or GF receptor (R) expression in normal and infected corneas is unknown and is the purpose of this study. VIP knockout ((-/-)) and wild-type (WT) C57BL/6 (B6) mice were infected and tested using PCR array, real-time RT-PCR, ELISA, and immunostaining. VIP antagonist treatment studies also were done using B6 and BALB/c mice. Infected corneas of VIP(-/-) versus WT B6 mice perforated earlier (2 vs. 5 days postinfection [p.i.]), and array data showed that GFs were differentially changed between groups. RT-PCR revealed that the infected cornea of VIP(-/-) versus WT mice expressed higher mRNA levels of epidermal growth factor (EGF) and hepatocyte growth factor (HGF), reduced FGF, EGFR, and HGFR, with no difference in FGFR; differences between groups were not seen in normal cornea. Immunostaining for GF and GFR in the normal cornea of VIP(-/-) versus WT mice was similar. However, at 1 day p.i., VIP(-/-) versus WT mice had more intense EGF and HGF, similar FGFR, and reduced FGF, EGFR, and HGFR staining. VIP antagonist treatment decreased protein levels for GFR at 5 days p.i. in both B6 and BALB/c mice, with no significant changes in normal cornea. The data showed that endogenous VIP is not requisite for GF or GFR expression in the normal cornea but, after infection, its absence or reduction is critical for their regulation.

Assessment of Longevity of the Direct and Indirect CD4 T Cell Allorecognition Response

Introduction: Recipient CD4 T cells can recognise alloantigen ‘directly’, intact on donor antigen presenting cells (APCs), and ‘indirectly’, as self-restricted processed allopeptide. The relative duration of each pathway remains unclear, and further complicated by our recent description that the longevity of indirect responses differed according to target alloantigen. This work however examined different CD4 T cell responses in different murine models, and to control for differences in the immunogenicity of each, we developed a new model of chronic allograft vasculopathy that would allow definitive examination of all CD4 T cell allorecognition pathways operating in each recipient. Methods: Several mouse strains were intercrossed to generate a novel strain, bm12.Kd.IE, which differs from wild-type B6 by possessing additional MHC class I (H-2Kd) and MHC class II (IAbm12 and IE) molecules, but lacks the IAb MHC class II antigen. Heterotopic cardiac allografts were performed using bm12.Kd.IE donors into B6 recipients, and the model characterised by measuring rejection kinetics, degree of allograft vasculopathy, and production of alloantibody and autoantibody. The relative duration of direct and indirect allorecognition of different alloantigens was assessed by comparing division of monoclonal populations of alloreactive, CFSE-labelled TCR-transgenic CD4 T cells that were adoptively transferred either on day 0 or 28 post transplantation. Indirect allorecognition of MHC I was assessed using TCR75 CD4 T cells (I-Ab-restricted, H2-Kd peptide-specific); class II using TEa CD4 T cells (I-Ab-restricted, I-E peptide-specific) and minor antigen using Mar CD4 T cells (I-Ab-restricted, H-Y peptide-specific). Direct allorecognition of I-Abm12 MHC II was assessed using ABM CD4 T cells. Results: Bm12.Kd.IE hearts survived >50 days in B6 recipients, with histological evidence of chronic allograft vasculopathy and production of anti-Kd and anti-I-E alloantibody. Autoantibody, assessed by indirect immunfluosrescent binding to nuclear-antigen-expressing HEp-2 cells, also developed. Flow cytometric analysis of CFSE labelled transgenic CD4 T cells confirmed that direct pathway responses were robust immediately after transplant, with extensive division of the transferred ABM CD4 T cells, but were minimal four weeks later. Similarly, strong indirect pathway responses against MHC II alloantigen occurred immediately after transplant, but were surprisingly undetectable at later time points. In contrast, indirect alloresponses against MHC I alloantigen were equally strong at early and late times after transplant. Comparable results were obtained when indirect alloresponses against minor H-Y antigen were studied, although division at five weeks was less marked than immediately after transplant. Conclusions: Our results provide experimental evidence to support the paradigm that direct pathway CD4 T cells responses are short-lived, but also highlight that the duration of indirect pathway responses varies according to target alloantigen. The transient nature of anti-class II responses presumably reflects rapid loss of donor APC after transplantation.

Immunization with a peptide of Semliki Forest virus promotes remyelination in experimental autoimmune encephalomyelitis

Remyelination is one of the elusive topics in treatment of multiple sclerosis (MS). Our previous studies have shown that Semliki Forest virus (SFV)-infected δ-knock-out (KO) mice did not exhibit the extensive remyelination, seen in wild type (WT) B6 mice, after viral clearance and demyelination. The Remyelination in SFV-infected WT mice started on day 15 and was completed by day 35 post-infection (pi), whereas the KO mice remained partially demyelinated through day 42 pi. Treatment with E2 peptide2 in incomplete Freund's adjuvant (IFA), resulted in higher antibody production and earlier remyelination in SFV-infected KO (day 28 pi), than WT mice. This finding suggested that anti-E2 peptide2 antibody could play a part in remyelination. In the current study, the effect of E2 peptide2 treatment was evaluated in the experimental autoimmune encephalomyelitis (EAE) model. Mice with established EAE were treated with E2 peptide2 in IFA to develop antibody. Treated EAE mice made significantly higher anti-E2 peptide2 antibody than untreated EAE group. Average clinical disease scores were significantly lower in peptide treated compared to untreated EAE mice. Furthermore, histopathological and immunohistochemical studies demonstrated increased remyelinating areas and higher number of activated oligodendrocytes and astrocytes, in treated compared to untreated EAE groups. Moreover, the anti-E2 peptide2 antibody showed higher binding to the myelinated areas of treated than untreated EAE mice. We conclude that treatment with, or antibody to, SFV E2 peptide2 triggers some mechanism that promotes remyelination.

749 COMPLEMENT 3 ACTIVATES THE RENAL RENIN-ANGIOTENSIN SYSTEM BY THE EPITHELIAL-TO-MESENCHYMAL TRANSITION

Objective: Recent studies have suggested that complement 3 (C3) excerts the pleiotrophic effects to maintain the stemness and the synthetic phenotype of mesenchymal cells. We have demonstrated that mesenchymal cells such as VSMCs and mesangial cells from SHR genetically produce C3. Mature tubular epithelial cells in adult kidney canundergo epithelial-to-mesenchymal transition (EMT), a phenotypicconversion that is fundamentally linked to the pathogenesisof renal interstitial fibrosis. We investigated role of C3 in EMT phenomena and the renal renin-angiotensin (RA) systems. Methods: Mice TCMK-1 epithelial cells were incubated with C3a in vitro. We induced EMT by UUO in wild type B6 or C3 KO mice. EMT was evaluated by decreases in E-cadherin and increases in α-SMA of h-caldesmon.The expression for C3 and renin was examined by immunocytochemistry, Western Blot and real time PCR. Expressions for C3, renin, LXRα and rorenin receptor were evaluated by immunohistochemistry and real time PCR. Results: C3a induced EMT and in TCMK-1 cells in which renin was expressed with nuclear localization of LXRα. In wild type mice, C3 and renin were strongly stained in the degenerated nephrotubulus at same localization with increases in expression of LXRα and rorenin receptor. In C3 KO mice, the EMT phenomen was suppressed with no expression of renin and C3. Conclusion: C3 induce the EMT to dedifferentiate the epithelial cells that produce renin through the induction of LXRα in kidney. C3 may be a primary factor to activate the renal RA systems to induce hypertension.

CX(3)CR1(+) macrophages support IL-22 production by innate lymphoid cells during infection with Citrobacter rodentium.

Innate immune cells, such as intestinal epithelial cells, dendritic cells (DCs), macrophages, granulocytes, and innate lymphoid cells provide a first line of defence to enteric pathogens. To study the role of CX3CR1+ DCs and macrophages in host defence, we infected CX3CR1-GFP animals with Citrobacter rodentium. When transgenic CX3CR1-GFP animals are infected with the natural mouse pathogen C. rodentium, CX3CR1−/− animals showed a delayed clearance of C. rodentium as compared with (age- and sex-matched) wild-type B6 animals. The delayed clearance of C. rodentium is associated with reduced interleukin (IL)-22 expression. In C. rodentium-infected CX3CR1-GFP animals, IL-22 producing lymphoid-tissue inducer cells (LTi cells) were selectively reduced in the absence of CX3CR1. The reduced IL-22 expression correlates with decreased expression of the antimicrobial peptides RegIIIβ and RegIIIγ. The depletion of CX3CR1+ cells by diphtheria toxin injection in CX3CR1-GFP × CD11c.DOG animals confirmed the role of CX3CR1+ phagocytes in establishing IL-22 production, supporting the clearance of a C. rodentium infection.

Salivary gland hypofunction induced by activation of innate immunity is dependent on type I interferon signaling

Activation of innate immunity through polyinosinic:polycytidylic acid [poly(I:C)] causes acute salivary gland hypofunction. As a major consequence of poly(I:C) treatment is type I interferon (IFN) production, this study was undertaken to investigate their role in salivary gland dysfunction. Different strains of mice deficient in either interferon alpha receptor (IFNAR1(-/-)) or IL-6(-/-), or IL-10(-/-), or EBI3(-/-) were treated with poly(I:C). Salivary gland function was determined by measuring pilocarpine-induced saliva volume. Gene expression levels were measured by real-time PCR. Ca(2+) mobilization studies were performed using ex-vivo acinar cells. A single injection of poly(I:C) rapidly induced salivary gland hypofunction in wild-type B6 mice (41% drop in saliva volumes compared to PBS-treated mice). In contrast, the loss of function in poly(I:C)-treated IFNAR(-/-) mice was only 9.6%. Gene expression analysis showed reduced levels of Il-6, Il-10, and Il-27 in submandibular glands of poly(I:C)-treated IFNAR(-/-) mice. While salivary gland dysfunction in poly(I:C)-treated IL-10(-/-) and EBI3(-/-) mice was comparable to wild-type mice, the IL-6(-/-) mice were more resistant, with only a 21% drop in function. Pilocarpine-induced Ca(2+) flux was significantly suppressed in acinar cells obtained from poly(I:C)-treated wild-type mice. Our data demonstrate that a combined action of type I IFNs and IL-6 contributes toward salivary gland hypofunction. This happens through interference with Ca(2+) mobilization within acinar cells. Thus, in acute viral infections and diseases like Sjögren's syndrome, elevated levels of type I IFNs and IL-6 can directly affect glandular function.

Mouse background and IVIG dosage are critical in establishing the role of inhibitory Fcγ receptor for the amelioration of experimental ITP

A recognized paradigm for the therapeutic action of intravenous immunoglobulin (IVIG) in immune thrombocytopenia (ITP) involves up-regulation of the inhibitory Fcγ receptor (FcγRIIB) in splenic macrophages. However, published data have indicated that opposing results are obtained when using FcγRIIB-deficient mice on different strain backgrounds. Herein we show BALB/c FcγRIIB(-/-) and wild-type, with or without spleens, all recover ITP with similar dynamics after IVIG (1 g/kg) treatment; however, this was not the case for C57BL/6 (B6) FcγRIIB(-/-). In investigating this conundrum, we found that wild-type B6 mice are much less sensitive than BALB/c to IVIG-mediated amelioration of ITP, requiring approximately 2- to 2.5-fold more IVIG than BALB/c. When using 2.5 g/kg IVIG in FcγRIIB(-/-) B6 mice, amelioration of ITP was as in wild-type in all animals. Our findings led us to the conclusion that different strains of mice respond differently to IVIG and that FcγRIIB plays no role in the mechanism of effect of IVIG in experimental ITP.

TNF but not IL-12 or interferon-alpha (IFN-a) plays a critical signal 3 role in in vivo CD8 cytotoxic T lymphocyte (CTL) maturation in the parent-into-F1 (P→F1) model. (126.7)

Abstract In addition to TcR signaling and costimulation, CD8 T cells require a third signal to mature into CTL. In pathogen models, IFN-a and IL-12 serve as signal 3 however in the P→F1 model, B6→B6D2F1 mice exhibit robust donor anti-host CD8 CTL in the absence of exogenous pathogens resulting in &amp;gt;90% elimination of host splenocytes i.e. acute graft-vs.host disease (GVHD). Selective inhibition of donor CD8 (but not donor CD4) activation converts acute GVHD to chronic GVHD with splenocyte expansion. To determine whether signal 3 molecules IL-12 or IFN-a are required to directly act on donor T cells in signal 3 manner, we transferred IL-12 or IFN-a receptor defective B6 donor cells into normal B6D2F1 mice. In neither transfer was acute GVHD phenotype significantly impaired compared to wild type (WT) B6→F1 mice. Moreover, peak CD8 CTL effectors were not impaired quantitatively or qualitatively. Similarly, blocking of WT B6→F1 with anti-IL-12 mAb did not alter phenotype. Significant IL-12 or IFN-a related gene expression was not detected by RT-PCR in WT B6→F1 mice prior to day 7. By contrast, TNF receptor 2 KO (TNFR2KO)→F1 but not TNFR1KO→F1 mice convert phenotype from acute to chronic GVHD. Thus, in the absence of pathogens, IFN-a and IL-12 play no significant signal 3 role alone. Instead, endogenous TNF signaling through TNFR2 plays a critical signal 3 role. These results have implications for CTL formation in the non-pathogen setting i.e. tumors, autoantigens and transplantation.

Neutrophil Gelatinase Associated Lipocalin (NGAL) deficiency accelerates the onset of serum autoantibodies in pristane induced lupus (60.10)

Abstract NGAL (lipocalin-2), a member of the lipocalin superfamily, is expressed by neutrophils, hepatocytes, and renal resident cells following ischemia or inflammatory conditions &amp; is involved in innate immune responses during infection. However, its role in adaptive immunity has not been described. Previously, we had found that NGAL is instrumental in the pathogenesis of antibody mediated nephritis. To investigate a possible role for NGAL in the generation of autoreactive antibody responses, we examined its effect in an experimental lupus model. We injected pristane (0.5 ml per mouse i.p.) into wild type B6 (n=10) and NGAL-deficient (NGAL-/-) (n=10) mice and followed the course of disease. Analyzing the levels of serum autoantibodies at 3 months post injection, NGAL-/- mice had significant increases in IgG2a (p=0.007) and IgG2b (p=0.019) anti-double stranded DNA antibodies, and IgG2a (p=0.002) &amp; IgG2b (p=0.010) anti-single stranded DNA antibodies, and IgG anti-histone antibodies (p=0.025) as compared to pristane challenged, NGAL sufficient mice. Immunoprecipitation studies showed elevated serum anti-Sm/RNP antibodies in NGAL-/- mice compared to the WT mice at 3 months post injection, with an increase in titer and epitope diversity of this response at 6 months post injection. In conclusion, NGAL delays the onset of anti-nuclear antibodies in pristane induced lupus. The mechanisms by which NGAL plays a protective role in humoral autoimmunity are under investigation.