DT-treated Mice Research Articles

Abstract 262: Gastrin-dependent suppression of CCK2R+ antrum stem cells inhibits gastric atrophy and cancer initiation in C57BL/6 mice

Abstract Background The cholecystokin-2/gastrin receptor (CCK2R) is expressed by rare epithelial cells in the gastric antrum that are bone fide +4 gastric stem cells. CCK2R+ antral stem cells are relatively quiescent, divide asymmetrically, and lineage trace entire antral glands. Their stemness is sustained over time through gastrin/CCK2R signaling (Cell Stem Cell 26, 739-754, May 7, 2020). However, the role of gastrin/CCK2R signaling in antral stem cells during mucosal injury and regeneration has not been explored. Methods We generated CCK2R-CreERT2; Gastrin-BAC-DTR-P2A-TdTomato; Rosa26-ZsGreen mice in a C57BL/6 background. Hypogastrinemia was induced through diphtheria toxin-dependent ablation of antral G cells, while hypergastrinemia was achieved via Alzet pump gastrin infusion. Lineage tracing from antrum CCK2R+ stem cells was assessed quantitatively during homeostasis and also during injury in chronic H. pylori infection and MNU injury models. Results Our findings revealed that G cell ablation increased lineage tracing of CCK2R+ antral stem cells at day 14 and increased proliferation of antral epithelial cells (Ki67+ cells) (P<0.05). Moreover, sorted antrum CCK2R+ cells (24 hours post-tamoxifen induction) from DT-treated mice had increased organoids forming capacity and in vitro cell growth, consistent with an increase in active progenitors, while hypergastrinemia reduced their growth (P<0.05). Numb staining showed that G cell ablation led to increased symmetric division of CCK2R+ stem cells, while gastrin infusion maintained asymmetric division (P<0.05). In the chronic H. pylori infection model, after 3 months of infection, the G cell ablation group exhibited more severe gastric antral atrophy compared to controls. In the MNU injury model, after 18 weeks of injury, the G cell ablation group had a higher dysplasia score, with dysplastic lesions largely derived from CCK2R+ cells (P<0.05). In our ongoing studies of MNU injury, the G cell ablation group at 36 weeks showed significantly greater tumor number and size than the control groups. Single-cell RNA-seq data showed that G cell ablation led to the proliferation and expansion of CCK2R+ antral stem/progenitor cells. Conclusions In conclusion, while gastrin maintains long-term antral stem cell quiescence and self-renewal, hypogastrinemia over the short-term leads to expansion and proliferation of CCK2R+ antral stem cells, thus predisposing over the long-term to antral atrophy and cancer initiation. In C57BL/6 mice, hypergastrinemia appears to be a protective factor against antral cancer development. Citation Format: Biyun Zheng, Guodong Lian, Ermanno Malagola, Hiroki Kobayashi, Ruhong Tu, Feijing Wu, Jin Qian, Quin T. Waterbury, Yosuke Ochiai, Leah B Zamechek, Timothy C Wang. Gastrin-dependent suppression of CCK2R+ antrum stem cells inhibits gastric atrophy and cancer initiation in C57BL/6 mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 262.

Understanding the cellular and molecular mechanism of anti-CTLA4 mediated tumor rejection

Abstract Although the clinical success of checkpoint inhibitors treatment is significant to prolong the patient survival, there is a substantial gap in our understanding of their mechanism in patients due to several technical and ethical limitations. Recent work suggests that the therapeutic effect of anti-CTLA-4 antibodies may not only block CTLA-4 interaction with its ligands, but also selectively deplete intratumoral Tregs (the highest expresser of CTLA-4) through ADCC. This releases the immunosuppressive state of the tumor microenvironment, thereby, promoting the expansion of effector CD4 and CD8, and increasing the secretion of IFN-g and TNF to reject the tumors. We observed a rapid Treg reduction characterized with fragile phenotype as early as 24 hours after anti-CTLA4 treatment. Using FOXP3-DTR mice, we confirmed that Treg depletion is the major contributor against tumor progression as anti-CTLA4 effect was abrogated in the DT-treated mice, and Treg function was maintained in the FcR KO mice. This suggested that FcR engagement is responsible for the Treg weakness. We hypothesized that there might be additional signal being transmitted when anti-CTLA4 is engaging with FcR which is capable of inducing Treg reduction. We made this hypothesis when we found the important receptor CD25 expressed on the Treg was significantly decreased after engagement. This receptor can also sense surrounding IL2 to maintain Treg cell survival, function, and proliferation. Interestingly, the CD25 expression was not inhibited when lacking FcR to induce CTLA4 engagement. Hence, there is still a need to study the underlying mechanism to see why Treg cells express lower CD25 after CTLA4 engagement.

367-OR: Elucidating Mechanisms of α-Cell Dysfunction in T1D

We previously showed that α cell function and gene expression in islets from donors with type 1 diabetes (T1D) was significantly compromised. The reason (s) for these intrinsic α cell changes are unknown but may include loss of α-to-β cell contact, chronic hyperglycemia, and/or repeated hypoglycemic events. To test these concurrent hypotheses, we modeled human T1D islets using α cell-enriched (“T1D-like") human pseudoislets. The synchronous acquisition of intracellular Ca2+ and hormone response showed impaired intracellular Ca2+ signaling and glucagon secretion in T1D-like pseudoislets compared to controls containing both α and β cells. For modeling of chronic hyperglycemia, we transplanted pseudoislets into Nod-SCID-IL2Rγnull; RIP-Diphtheria Toxin Receptor mice made diabetic by diphtheria toxin (DT) -induced β cell depletion. In DT-treated mice, α cells in T1D-like pseudoislets maintained expression of MAFB and ARX but began to express the β cell-enriched transcription factor NKX6.1 (control vs. T1D-like, 3.43±2.49 vs. 54.8±20.8% NKX6.1+ α cells; P=0.046, N=3-5 mice, 2 donors/group) . To mimic repeated hypoglycemic events, we intermittently exposed T1D-like pseudoislets to low glucose (1.7 mM) (3 events x 3hr each) in vitro, using those remaining in basal glucose (5 mM) as control. Following multiple low glucose (MLG) exposures, T1D-like pseudoislets had reduced glucagon secretion in the presence of both 1.7 mM (control vs. MLG, 1.51±0.18 vs. 0.79±0.13 % glucagon content; P=0.011, N=4 donors) and 16.7 mM glucose (control vs. MLG, 0.79±0.13 vs. 0.39±0.% glucagon content; P=0.023, N=4) . Native human islets exposed to MLG had similar changes in glucagon response (control vs. MLG, 1.7mM: 0.63±0.vs. 0.22±0.% glucagon content, P=0.007; 16.7mM: 0.18±0.vs. 0.05±0.% glucagon content, P=0.029; N=4 donors) . These data suggest that loss of α-to-β cell contact and repeated exposure to low glucose impair α cell function. Further, exposure to chronic hyperglycemia may lead to changes in α cell identity state after β cell loss. Disclosure Y.D.Pettway: None. C.Dai: None. T.M.Richardson: None. J.T.Walker: None. R.Aramandla: None. G.Poffenberger: None. A.Bradley: None. A.C.Powers: None. M.Brissova: None.

Transient Depletion of Foxp3+ Regulatory T Cells Selectively Promotes Aggressive β Cell Autoimmunity in Genetically Susceptible DEREG Mice.

Type 1 diabetes (T1D) represents a hallmark of the fatal multiorgan autoimmune syndrome affecting humans with abrogated Foxp3+ regulatory T (Treg) cell function due to Foxp3 gene mutations, but whether the loss of Foxp3+ Treg cell activity is indeed sufficient to promote β cell autoimmunity requires further scrutiny. As opposed to human Treg cell deficiency, β cell autoimmunity has not been observed in non-autoimmune-prone mice with constitutive Foxp3 deficiency or after diphtheria toxin receptor (DTR)-mediated ablation of Foxp3+ Treg cells. In the spontaneous nonobese diabetic (NOD) mouse model of T1D, constitutive Foxp3 deficiency did not result in invasive insulitis and hyperglycemia, and previous studies on Foxp3+ Treg cell ablation focused on Foxp3DTR NOD mice, in which expression of a transgenic BDC2.5 T cell receptor (TCR) restricted the CD4+ TCR repertoire to a single diabetogenic specificity. Here we revisited the effect of acute Foxp3+ Treg cell ablation on β cell autoimmunity in NOD mice in the context of a polyclonal TCR repertoire. For this, we took advantage of the well-established DTR/GFP transgene of DEREG mice, which allows for specific ablation of Foxp3+ Treg cells without promoting catastrophic autoimmune diseases. We show that the transient loss of Foxp3+ Treg cells in prediabetic NOD.DEREG mice is sufficient to precipitate severe insulitis and persistent hyperglycemia within 5 days after DT administration. Importantly, DT-treated NOD.DEREG mice preserved many clinical features of spontaneous diabetes progression in the NOD model, including a prominent role of diabetogenic CD8+ T cells in terminal β cell destruction. Despite the severity of destructive β cell autoimmunity, anti-CD3 mAb therapy of DT-treated mice interfered with the progression to overt diabetes, indicating that the novel NOD.DEREG model can be exploited for preclinical studies on T1D under experimental conditions of synchronized, advanced β cell autoimmunity. Overall, our studies highlight the continuous requirement of Foxp3+ Treg cell activity for the control of genetically pre-installed autoimmune diabetes.

Melatonin and Selenium Suppress Docetaxel-Induced TRPV1 Activation, Neuropathic Pain andOxidativeNeurotoxicity in Mice.

Docetaxel (DT) has been reported to positive therapeutic actions in the treatment of glioblastoma, breast tumors, and prostate cancers. However, it can also induce peripheral neuropathic pain and neurotoxicity as adverse effects. Expression level of TRPV1 cation channel is high in dorsal root ganglion (DRG), and its activation via capsaicin and reactive oxygen species (ROS) mediates peripheral neuropathic pain in mice. As cancer is known to increase the levels of ROS, the protective roles of melatonin (MT) and selenium (Se) were evaluated on the TRPV1-mediated neurotoxicity and pain in the DT-treated mice. Mice and TRPV1 expressing SH-SY5Y cells were equally divided into control, MT, Se, DT, DT+MT, and DT+Se groups. In the results of pain tests in the mice, we observed a decrease in DT-mediated mechanical and heat neuropathic pain by MT and Se. The results of plate reader assay and laser confocal microscopy image analyses indicated a protective role of MT and Se on the DT-induced increase of mitochondrial ROS, cytosolic ROS, apoptosis, lipid peroxidation, intracellular free Zn2+, Ca2+, and caspase-3 and -9 levels in the DRG and SH-SY5Y cells. MT and Se modulated DT-induced decreases of total antioxidant status, reducedglutathione and glutathione peroxidase in the DRG. However, the effects of DT were not observed in the non-TRPV1 expressing SH-SY5Y cells. Hence, MT and Se mediated protective effects against DT-induced adverse peripheral oxidative neurotoxicity and peripheral pain. These effects may be attributed to potent antioxidant properties of MT and Se.

Emergency myelopoiesis contributes to immune cell exhaustion and pulmonary vascular remodelling.

Background and PurposePulmonary hypertension (PH) secondary to chronic lung disease (World Health Organization Group 3 PH) is deadly, with lung transplant being the only available long‐term treatment option. Myeloid‐derived cells are known to affect progression of both pulmonary fibrosis and PH, although the mechanism of action is unknown. Therefore, we investigated the effect of myeloid cell proliferation induced by emergency myelopoiesis on development of PH and therapy directed against programmed death‐ligand 1 (PD‐L1), expressed by myeloid cells in prevention of pulmonary vascular remodelling.Experimental ApproachLysM.Cre‐DTR (“mDTR”) mice were injected with bleomycin (0.018 U·g−1, i.p.) while receiving either vehicle or diphtheria toxin (DT; 100 ng, i.p.) to induce severe PH. Approximately 4 weeks after initiation of bleomycin protocol, right ventricular pressure measurements were performed and tissue samples collected for histologic assessment. In a separate experiment, DT‐treated mice were given anti‐PD‐L1 antibody (αPD‐L1; 500 μg, i.p.) preventive treatment before bleomycin administration.Key ResultsMice undergoing induction of emergency myelopoiesis displayed more severe PH, right ventricular remodelling and pulmonary vascular muscularization compared to controls, without a change in lung fibrosis. This worsening of PH was associated with increased pulmonary myeloid‐derived suppressor cell (MDSC), particularly polymorphonuclear MDSC (PMN‐MDSC). Treatment with αPD‐L1 normalized pulmonary pressures. PD‐L1 expression was likewise found to be elevated on circulating PMN‐MDSC from patients with interstitial lung disease and PH.Conclusions and ImplicationsPD‐L1 is a viable therapeutic target in PH, acting through a signalling axis involving MDSC.LINKED ARTICLESThis article is part of a themed issue on Risk factors, comorbidities, and comedications in cardioprotection. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.1/issuetoc

Depletion of regulatory T cells in ongoing paracoccidioidomycosis rescues protective Th1/Th17 immunity and prevents fatal disease outcome

In human paracoccidioidomycosis (PCM), a primary fungal infection typically diagnosed when the disease is already established, regulatory T cells (Treg) cells are associated with disease severity. Experimental studies in pulmonary PCM confirmed the detrimental role of these cells, but in most studies, Tregs were depleted prior to or early during infection. These facts led us to study the effects of Treg cell depletion using a model of ongoing PCM. Therefore, Treg cell depletion was achieved by treatment of transgenic C57BL/6DTR/eGFP (DEREG) mice with diphtheria toxin (DT) after 3 weeks of intratracheal infection with 1 × 106Paracoccidioides brasiliensis yeasts. At weeks 6 and 10 post-infection, DT-treated DEREG mice showed a reduced number of Treg cells associated with decreased fungal burdens in the lungs, liver and spleen, reduced tissue pathology and mortality. Additionally, an increased influx of activated CD4+ and CD8+ T cells into the lungs and elevated production of Th1/Th17 cytokines was observed in DT-treated mice. Altogether, our data demonstrate for the first time that Treg cell depletion in ongoing PCM rescues infected hosts from progressive and potentially fatal PCM; furthermore, our data indicate that controlling Treg cells could be explored as a novel immunotherapeutic procedure.

Targeted Interneuron Ablation in the Mouse Hippocampus Can Cause Spontaneous Recurrent Seizures.

The death of GABAergic interneurons has long been hypothesized to contribute to acquired epilepsy. These experiments tested the hypothesis that focal interneuron lesions cause acute seizures [i.e., status epilepticus (SE)] and/or chronic epilepsy [i.e., persistent spontaneous recurrent seizures (SRSs)]. To selectively ablate interneurons, Gad2-ires-Cre mice were injected unilaterally in the CA1 area of the dorsal hippocampus with an adeno-associated virus containing the diphtheria toxin receptor (DTR). Simultaneously, an electrode, connected to a miniature telemetry device, was positioned at the injection site for chronic recordings of local field potentials (LFPs). Two weeks after virus transfection, intraperitoneal injection of DT consistently caused focal, specific, and extensive ablation of interneurons. Long-term, continuous monitoring revealed that all mice with DT-induced interneuron lesions had SRSs. Seizures lasted tens of seconds and interseizure intervals were several hours (or days); therefore, these interneuron lesions did not induce SE. The SRSs occurred 3-5 d after DT treatment, which is the estimated time required for DT-induced cell death; therefore, induction of SRSs occurred without the latent period typical of acquired epilepsy. In five of six DT-treated mice, SRSs stopped within days, suggesting that the DT-induced interneuron lesions did not usually cause epilepsy. In one mouse, however, SRSs occurred for ≥34 d after interneuron ablation, similar to epilepsy after experimental SE. Sham control mice had no detectable seizures, confirming that the SRSs were due to ablation of interneurons. These data show that selective interneuron ablation consistently caused SRSs but not SE; and, at least under the conditions used here, interneuron lesions rarely led to persistent SRSs (i.e., epilepsy).

The Adjuvant Effect Of PLGA Is Mediated Via Dendritic Cells In Vivo

Event Abstract Back to Event The Adjuvant Effect Of PLGA Is Mediated Via Dendritic Cells In Vivo Sangeetha Srinivasan1 and Julia E. Babensee1 1 Georgia Institute of Technology, Wallace H. Coulter Department of Biomedical Engineering, United States Introduction: The number of applications using biomaterials is rapidly growing due to the diversity of physical and chemical properties they can offer; specifically, devices comprised of materials such as poly lactic-co-glycolic acid (PLGA) have been approved by the Food and Drug Administration (FDA) for clinical use. We have previously shown that PLGA is distinct in its interaction with the adaptive immune system, in that it acts as an adjuvant to a model antigen such as ovalbumin (OVA) to boost antigen-specific immunity[1]. To enable researchers to design improved devices using PLGA, we are interested in understanding its mechanism by which it acted as an adjuvant in vivo. We hypothesized that PLGA’s adjuvant effect in vivo is primarily mediated by its effects on dendritic cells – the most potent antigen-presenting cells of the immune system – in an antigen-specific manner. Accordingly, we used a CD11c-DTR mouse model[2] in which CD11c+ cells (of which DCs are the largest population) were conditionally ablated by delivering diphtheria toxin. These mice received OVA-laden PLGA scaffolds as dorsal subcutaneous implants while also having received an intravenous injection with fluorescently label OVA-specific CD4+ T-cells. After allowing sufficient time for the interaction each of these components in vivo, we examined mice spleens for clonal expansion of the exogenously delivered T-cells. Our results clearly indicate that the OVA-specific T-cells in the DT-treated CD11c-DTR mice had failed to proliferate in comparison with non-DT treated controls due to the lack of sufficient DCs in vivo to drive antigen presentation, thereby supporting our original hypothesis that PLGA’s adjuvant effect is mediated via DCs. Materials and Methods: CD11c-DTR, OT-II and wild-type C57BL/6 mice of 6-8 weeks were purchased from Jackson labs and quarantined for one week prior to any treatment. CD4+ OVA-specific T-cells were isolated from OT-II mice mice and fluorescently labeled with carboxy fluorescein succinimidyl ester (CFSE) (Invitrogen) and injected in mice intravenously. Diphtheria toxin (Sigma) was supplied as 4ng/g[2]of mouse body weight via intravenous tail vein injections once every two days in order to sustain CD11c+ depletion over a 6-day period. PLGA scaffolds were prepared as described before[3]. Briefly, 75:25 pellets were dissolved in dichloromethane and cast into Teflon dishes mixed with dehydrated OVA (Sigma) and 125 – 250um NaCl salt crystals acting as porogens. The scaffolds thus made were washed, dried and cut into 8mm disks and implanted in the dorsal subcutaneous pockets in mice – one scaffold per mouse. Mice were subsequently sacrificed and their splenocytes were isolated and analyzed using flow cytometry. Results: The DC depletion experiment was conducted over a 7-day period with the appropriate treatments and days depicted on figure 1. Splenocytes from 3 mice groups – DTR mice with DT treatment, (control) DTR mice without DT treatment and (control) C57BL/6 wild-type with DT treatment (all with a PLGA/OVA implant– were stained with fluorescent antibodies against specific T-cell markers. CFSE incorporates in the nucleus of cells and is propagated in their daughter cells on cell division, thereby reducing in the extent of fluorescence emitted by the subsequent progeny[2]. Figure 2 shows the fluorescence intensity of the originally injected cells (shown in black) while that of any of its progeny (shown in blue). The lack of attenuation of CFSE fluorescence signal in DT-treated DTR mice (fig. 2 - bottom right panel) compared to the control groups (fig. 2 – top and bottom left panels) indicate that OVA-specific T-cells were unable to proliferate in DT-treated mice and thereby due to the lack of CD11c+ cells. National Institute of Health Grant

Activation of DR3 Signaling Expands Recipient Derived Regulatory T Cells and Enhances Donor Immune Reconstitution Derived from Allogeneic Hematopoietic Stem Cells

CD4+ Foxp3+ regulatory T cells (Treg) are a subpopulation of T cells which regulate the immune system, maintain the tolerance of self-antigens and enhance immune tolerance after transplantation. It was also reported that recipient derived Treg could provide immune privilege to allogeneic stem cells (HSC) after transplantation. However, the precise interaction with Treg and HSC has not been fully elucidated. In this study, we investigated the role of recipient derived Treg in the engraftment and immune reconstitution following transplantation of purified allogeneic HSC and the effectiveness of Treg expansion following activation of DR3 (Death receptor 3, also called as TNFRSF25) signaling in this model.We first tested the effect of Treg depletion using Foxp3-DTR mice in allogeneic HSC transplantation. In this system, FACS-sorted purified HSC (Lin-cKit+Sca1+ population) derived from WT-FVB mice (CD45.1+/H2kq+) were injected into lethally irradiated B6-Foxp3-DTR mice (CD45.2+/H2kb+) with or without pre-treatment of diphtheria toxin (DT). On day 0 and day 28 after transplantation decreased frequencies of Foxp3+ cells in residual recipient derived CD4+ T cells were observed in peripheral blood from the DT treated mice (P<0.001 on day 0, P<0.002 on day 28). Although total myeloid chimerism was comparable between control and DT-treated mice, the frequency of donor derived immune cells including CD4+ T cells (P<0.01 on day 56), CD8+ T cells (P<0.01 on day 56), and B220+ B cells (P<0.001 on day 56) was significantly decreased in DT-treated mice. These data suggested that recipient derived Treg play an important role in allogeneic immune reconstitution after transplantation.DR3 is a member of the TNF receptor superfamily and we previously reported the expansion of Treg by the activation of this signaling pathway (Kim et al, ASH abstract 2013). We next tested whether activation of DR3 signaling by its agonistic antibody would affect the donor immune reconstitution after allogeneic HSC transplantation. The frequencies of Foxp3+ cells in CD4+ T cells were significantly increased in thymus, spleen, peripheral blood, and bone marrow 4 days after antibody injection (P<0.01). Isolated Treg derived from antibody treated mice showed stronger suppressive function in the mixed lymphoid reaction compared with those from isotype antibody treated mice.The mice treated with antibody on day -4 were transplanted with purified allogeneic HSC on day 0. Antibody treated mice showed a higher frequency of donor derived CD4+ T cells (P<0.001 on day 28), CD8+ T cells (P<0.05 on day 28), and B220+ B cells (P<0.05 on day 28) in this allogeneic HSC transplantation model.In summary, our data suggest that recipient derived CD4+Foxp3+ Treg play an important role in donor immune reconstitution and the activation of DR3 signaling in recipient mice enhances donor immune reconstitution by expansion of recipient derived Treg.H.N and BS-K contributed equally to this work. DisclosuresNo relevant conflicts of interest to declare.

3,6′‐dithiothalidomide improves experimental stroke outcome by suppressing neuroinflammation

Tumor necrosis factor-α (TNF) plays a prominent role in the brain damage and functional deficits that result from ischemic stroke. It was recently reported that the thalidomide analog 3,6'-dithiothalidomide (3,6'-DT) can selectively inhibit the synthesis of TNF in cultured cells. We therefore tested the therapeutic potential of 3,6'-DT in a mouse model of focal ischemic stroke. Administration of 3,6'-DT immediately prior to a stroke or within 3 hr after the stroke reduced infarct volume, neuronal death, and neurological deficits, whereas thalidomide was effective only when administered prior to stroke. Neuroprotection was accompanied by decreased inflammation; 3,6'-DT-treated mice exhibited reduced expression of TNF, interleukin-1β, and inducible nitric oxide synthase; reduced numbers of activated microglia/macrophages, astrocytes, and neutrophils; and reduced expression of intercellular adhesion molecule-1 in the ischemic brain tissue. 3,6'-DT treatment attenuated stroke-induced disruption of the blood-brain barrier by a mechanism that appears to involve suppression of matrix metalloproteinase-9 and preservation of occludin. Treatment with 3,6'-DT did not reduce ischemic brain damage in mice lacking TNF receptors, consistent with a critical role for suppression of TNF production and TNF signaling in the therapeutic action of 3,6'-DT. These findings suggest that anti-inflammatory mechanisms underlie the therapeutic actions of 3,6-DT in an animal model of stroke.

Macrophages Are Essential for Maintenance of Corpus Luteum Function in Early Pregnancy.

Macrophages are abundant within the ovary, and have been identified in the corpus luteum (CL) of most species studied, including in rodents and human. Through their secretory products, macrophages are thought to be involved in ovarian tissue remodelling, including luteinization, and in regulating steroidogenesis. Macrophages co-cultured with granulosa or luteal cells act to stimulate progesterone secretion in vitro. To determine the impact of macrophage ablation during early pregnancy, we utilised CD11b-DTR transgenic mice to elicit transient systemic ablation of macrophages by administration of diphtheria toxin (DT). The effects of macrophage ablation during the pre-implantation phase of pregnancy were evaluated. Ablation of macrophages on day 1 pc or day 4 pc caused complete pregnancy loss in all DT-treated CD11b-DTR mice, while DT-treated wild-type mice or PBS-treated CD11b-DTR mice maintained viable pregnancies. Macrophage ablation on day 3 pc significantly reduced serum progesterone (P4) levels to 40% of control levels when measured 24 h later. Administration of exogenous P4 on each of day 4-7 pc prevented fetal loss in DT-treated CD11b-DTR mice, and pregnancy progressed with viable pups at late gestation, while no pregnancies remained viable in DT-treated mice administered vehicle only. Immunohistochemical evaluation with anti-CD31 mAb showed that macrophage ablation in the CD11b-DTR mice resulted in disappearance of endothelial cells from within the CL. This suggests that macrophages are essential for support of endothelial cells within the CL, either by provision of trophic support and/or by macrophage transdifferentiation into endothelial cells. In conclusion, these data indicate a critical role for macrophages in corpus luteum development and steroidogenic function in early pregnancy. Given that various nutritional and immune stressors can profoundly affect macrophage numbers and behaviour, it seems reasonable to speculate that macrophage-regulated CL development is a vulnerable event and may contribute to some forms of unexplained infertility. (platform)

146. MACROPHAGES ARE ESSENTIAL FOR MAINTENANCE OF EARLY PREGNANCY THROUGH REGULATION OF CORPUS LUTEUM FUNCTION

Macrophages are abundant within the ovary, and have been identified in the corpus luteum (CL) of most species studied, including in rodents and human. Through their secretory products, macrophages are thought to be involved in ovarian tissue remodelling, including luteinization, and in regulating steroidogenesis [1, 2]. Macrophages co-cultured with granulosa or luteal cells act to stimulate progesterone secretion [3]. To determine the impact of macrophage ablation during early pregnancy, we utilised the macrophage specific CD11b-DTR diphtheria toxin receptor (DTR) transgenic mouse to cause transient systemic ablation of macrophages via the administration of DT (25 ng/g). The effects of macrophage ablation during the pre-implantation phase of pregnancy were evaluated and implantation sites were counted. Ablation of macrophages on day 1 pc (n=13 wild-type; n=9 CD11b-DTR) or day 4 pc (n=6 CD11b-DTR; n=9 wild-type) caused complete pregnancy loss in all DT-treated CD11b-DTR mice, while wild-type mice maintained viable pregnancies [mean ± SEM implantation sites = 5.0 ± 1.4 (day 1 treated), and 6.0 ± 1.9 (day 4 treated)]. Serum progesterone was analysed 24 h following macrophage ablation. A single DT injection on day 3 pc significantly reduced serum progesterone (P4) levels [n=7 wild-type P4 (ng/ml)= 29.6 ± 3.3; n=8 CD11b-DTR = 11.1 ± 2.1]. The administration of exogenous P4 (2 mg) on each of day 4-7 pc prevented fetal loss in DT-treated CD11b-DTR mice (n=6; implantation sites = 7.8 ± 1.6), while no pregnancies remained viable in DT-treated mice administered vehicle only (n=9). In conclusion, luteal insufficiency appears to be the cause of pregnancy failure following macrophage ablation. These data indicate a critical role for macrophages in corpus luteum function in early pregnancy.

Abstract 3956: CD11b Positive Monocytes Are Required for Endothelial Regeneration

Bone marrow (BM)-derived circulating endothelial progenitor cells (EPC) contribute to endothelial cell (EC) regeneration. Systemic transfusion of EPC fosters the restoration of the endothelium after EC damage in mice. Transfusion of mononuclear cells (MNC) leads to an even more pronounced endothelial regeneration compared to EPCs. Here, we determine the role of monocytic cells and their interaction with circulating EPC on EC regeneration. To evaluate the effect of monocytes on re-endothelialization, neointima formation, and endothelial function, wild-type (WT) mice received repetitive intravenous injections of spleen-derived MNC, CD11b-depleted MNC, CD11b+ monocytes, or cell-free vehicle after EC denudation. Endothelial restoration was enhanced and neointima formation reduced after transfusion of MNC and CD11b+ cells. CD11b-depleted cells had no effect. Transfusion of CD11b+ cells in ApoE−/− mice improved endothelial-dependent vasorelaxation while CD11b-depleted cells had no effect. Next, WT mice were treated with clodronate liposomes (10μl/g bw) which induces the ablation of circulating monocytes. Re-endothelialization was diminished in clodronate- compared to vehicle-treated mice. Next, we selectively depleted CD11b+ monocytes using CD11b-DTR mice which express the human diphtheria toxin receptor (DTR) under the control of the CD11b promoter. Treatment with diphtheria toxin (DT) resulted in the complete ablation of circulating monocytes. Re-endothelialization was delayed in DT-treated compared to vehicle-treated mice. Systemic treatment of WT mice with Sca1+ EPC improves endothelial function. Repetitive transfusion of Sca1+ cells enhanced re-endothelialization in vehicle-treated CD11b-DTR mice compared to DT-treated mice. Rescue experiments showed that transfusion of CD11b+ cells in CD11b-DTR mice treated with either vehicle or DT resulted in a comparable enhancement of re-endothelialization. Our results indicate that circulating CD11b+ monocytes contribute to EC regeneration. Ablation of monocytes in vivo leads to a delay in re-endothelialization after focal EC damage which can not be rescued by transfusion of Sca1+ cells.

Galectin-3 Expression and Secretion Links Macrophages to the Promotion of Renal Fibrosis

Macrophages have been proposed as a key cell type in the pathogenesis of renal fibrosis; however, the mechanism by which macrophages drive fibrosis is still unclear. We show that expression of galectin-3, a beta-galactoside-binding lectin, is up-regulated in a mouse model of progressive renal fibrosis (unilateral ureteric obstruction, UUO), and absence of galectin-3 protects against renal myofibroblast accumulation/activation and fibrosis. Furthermore, specific depletion of macrophages using CD11b-DTR mice reduces fibrosis severity after UUO demonstrating that macrophages are key cells in the pathogenesis of renal fibrosis. Disruption of the galectin-3 gene does not affect macrophage recruitment after UUO, or macrophage proinflammatory cytokine profiles in response to interferon-gamma/lipopolysaccharide. In addition, absence of galectin-3 does not affect transforming growth factor-beta expression or Smad 2/3 phosphorylation in obstructed kidneys. Adoptive transfer of wild-type but not galectin-3(-/-) macrophages did, however, restore the fibrotic phenotype in galectin-3(-/-) mice. Cross-over experiments using wild-type and galectin-3(-/-) macrophage supernatants and renal fibroblasts confirmed that secretion of galectin-3 by macrophages is critical in the activation of renal fibroblasts to a profibrotic phenotype. Therefore, we demonstrate for the first time that galectin-3 expression and secretion by macrophages is a major mechanism linking macrophages to the promotion of renal fibrosis.

Critical role of macrophages in the marginal zone in the suppression of immune responses to apoptotic cell–associated antigens

Injection of apoptotic cells can induce suppression of immune responses to cell-associated antigens. Here, we show that intravenous injection of apoptotic cells expressing a fragment of myelin oligodendrocyte glycoprotein (MOG) reduced MOG-specific T cell response and prevented the development of EAE. Since injected apoptotic cells accumulated initially in the splenic marginal zone (MZ), the role of macrophages in the MZ in immune suppression was examined using transgenic mice in which these cells could be transiently deleted by diphtheria toxin (DT) injection. DT-treated mice became susceptible to EAE even though MOG-expressing apoptotic cells were preinjected. Deletion of the macrophages caused delayed clearance of injected dying cells in the MZ. In wild-type mice, injected apoptotic cells were selectively engulfed by CD8 alpha(+) DCs, which are responsible for suppression of immune responses to cell-associated antigens. In contrast, deletion of macrophages in the MZ caused aberrant phagocytosis of injected dying cells by CD8 alpha(-)CD11b(+) DCs. These results indicate that macrophages in the MZ regulate not only efficient clearance of apoptotic cells but also selective engulfment of dying cells by CD8 alpha(+) DCs and that functional failure of these unique macrophages impairs suppression of immune responses to cell-associated antigens.

Correlation of cytotoxicity with elimination of iodine-125 from nude mice inoculated with prelabeled human melanoma cells.

BRO human melanoma cells were prelabeled in vitro with [125I]5-iodo-2'-deoxyuridine ([125I]IdUrd) and inoculated into NIH-II nude mice ip, im, sc, or iv. Saline or diphtheria toxin (DT), which is selectively toxic to human cells compared to those of mice, was injected, and the loss of 125I from the animals was monitored daily with a whole-body gamma scintillation detector. For most of the inoculation sites DT accelerated the rate of 125I excretion and in all cases was cytotoxic for the inoculated cells as determined by host survival or measurement of visible tumor growth. Differences between the rates of 125I loss for DT-treated mice compared to untreated mice were most evident for cells inoculated ip or im. These results indicate that [125I]IdUrd prelabeling of human tumor cells inoculated in nude mice offers a rapid method for determination of cytotoxicity in vivo.