IL-10 KO Research Articles

Lipocalin 2 prevents intestinal inflammation by enhancing phagocytic bacterial clearance in macrophages.

Lipocalin 2 (Lcn2), also called neutrophil gelatinase B-associated lipocalin (NGAL), is an anti-microbial peptide originally identified in neutrophil granules. Although Lcn2/NGAL expression is increased in the inflamed intestinal tissues of patients with inflammatory bowel disease, the role of Lcn2/NGAL in the development of intestinal inflammation remains unclear. Here we investigated the role of Lcn2/NGAL in intestinal inflammation using a spontaneous mouse colitis model, interleukin-10 knock out (IL-10 KO) mice. Lcn2 expression in the colonic tissues of IL-10 KO mice increased with the development of colitis. Lcn2/IL-10 double-KO mice showed a more rapid onset and development of colitis compared to IL-10 KO mice. Lcn2 enhanced phagocytic bacterial clearance in macrophages in vitro after infection with Escherichia coli. Transfer of Lcn2-repleted macrophages prevented the development of colitis in Lcn2/IL-10 double-KO mice in vivo. Our findings revealed that Lcn2 prevents the development of intestinal inflammation. One crucial factor seems to be the enhancement of phagocytic bacterial clearance in macrophages by Lcn2.

IL-10 plays an important role in the control of inflammation but not in the bacterial burden in S. epidermidis CNS catheter infection.

BackgroundShunt infection is a frequent and serious complication in the surgical treatment in hydrocephalus. Previous studies have shown an attenuated immune response to these biofilm-mediated infections. We proposed that IL-10 reduces the inflammatory response to Staphylococcus epidermidis (S. epidermidis) CNS catheter infection.MethodsIn this study, a murine model of catheter-associated S. epidermidis biofilm infection in the CNS was generated based on a well-established similar model for S. aureus. The catheters were pre-coated with a clinically derived biofilm-forming strain of S. epidermidis (strain 1457) which were then stereotactically implanted into the lateral left ventricle of 8-week-old C57BL/6 and IL-10 knockout (IL-10 knockout) mice. Bacterial titers as well as cytokine and chemokine levels were measured at days 3, 5, 7, and 10 in mice implanted with sterile and S. epidermidis-coated catheters.ResultsCultures demonstrated a catheter-associated and parenchymal infection that persisted through 10 days following infection. Cytokine analysis of the tissue surrounding the catheters revealed greater levels of IL-10, an anti-inflammatory cytokine, in the infected group compared to the sterile. In IL-10 KO mice, we noted no change in bacterial burdens, showing that IL-10 is not needed to control the infection in a CNS catheter infection model. However, IL-10 KO mice had increased levels of pro-inflammatory mediators in the tissues immediately adjacent to the infected catheter, as well as an increase in weight loss.ConclusionsTogether our results indicate that IL-10 plays a key role in regulating the inflammatory response to CNS catheter infection but not in control of bacterial burdens. Therefore, IL-10 may be a useful therapeutic target for immune modulation in CNS catheter infection but this should be used in conjunction with antibiotic therapy for bacterial eradication.

The role of recipient derived interleukin-17A in a murine orthotopic lung transplant model of restrictive chronic lung allograft dysfunction

The single most important cause of late mortality after lung transplantation is chronic lung allograft dysfunction (CLAD). However, the pathological development of CLAD was not as simple as previously presumed and subclassification phenotypes, bronchiolitis obliterans syndrome (BOS) and restrictive CLAD (rCLAD), have been introduced. We want to re-investigate how CLAD manifests in the murine orthotopic lung transplant model and investigate the role of interleukin 17A (IL-17A) within this model.Orthotopic LTx was performed in CB57BL/6, IL-17 WT and IL-17 KO mice. In a first experiment, CB57BL/6 mice receiving an isograft (CB57BL/6) or allograft (BALB/C) were compared. In a second experiment IL-17 WT and IL-17 KO mice (both CB57BL/6 background) received an allograft (BALB/C). Mice received daily immunosuppression with steroids and cyclosporine and were sacrificed 10weeks after transplantation for histopathological analysis by an experienced lung pathologist.After murine orthotopic lung transplantation, the allograft histopathologically presented features of human rCLAD (i.e. overt inflammation, pleural/parenchymal fibrosis and obliterative bronchiolitis). In the IL-17A KO group, less inflammation in the bronchovascular axis (p=0.03) was observed and a non-significant trend towards less bronchovascular fibrosis, pleural/septal inflammation and fibrosis, and parenchymal inflammation and fibrosis when compared to WT mice.The major mismatch orthotopic lung transplant model resembles features of human rCLAD. IL-17A mediated immunity is involved in the inflammatory component, but had little influence on the degree of fibrosis. Further mechanistic and therapeutic studies in this mouse model are needed to fully understand the mechanisms in rCLAD.

Endogenous IL-17 as a factor determining the severity of Clostridium difficile infection in mice.

Clostridium difficile infection (CDI) is a toxin-mediated intestinal disease. Toxin A, toxin B and binary toxin are believed to be responsible for the pathogenesis of CDI, which is characterized by massive infiltration of neutrophils at the infected intestinal mucosa. IL-17 is one of the cytokines that play critical roles in several inflammatory and immunological diseases through various actions, including promoting neutrophil recruitment. The aim of this study was to examine the role of this cytokine in CDI by employing IL-17 A and F double knockout (IL-17 KO) mice for the CDI model. We demonstrated that IL-17 KO mice were more resistant to CDI than WT mice using several factors, such as diarrhoea score, weight change and survival rate. Although the bacterial numbers of C. difficile in faeces were not different, the inflammatory mediator levels at the large intestine on day 3 post-infection were attenuated in IL-17 KO mice. Finally, we showed that infiltration of neutrophils, but not macrophages, in the large intestine was significantly decreased in IL-17 KO mice compared to WT mice. In conclusion, the data demonstrate that endogenous IL-17 may be a factor determining the severity of CDI in mice. Although the mechanism is totally unknown, IL-17-mediated inflammatory responses, such as cytokine/chemokine production and neutrophil accumulation, may be plausible targets for future investigations.

Abstract 99: IL10-inhibits Fibroblast Progenitor Cell-mediated Cardiac Fibrosis in Pressure-overloaded Myocardium

Background: Activated fibroblasts (myoFBs) play critical role in cardiac fibrosis, however, their origin in diseased heart remains uncertain. Recent studies suggest the contribution of bone marrow fibroblasts progenitor cells (BM-FPC) in pressure overload (PO)-induced cardiac fibrosis. Previously we have shown that interleukin-10 suppress PO-induced cardiac fibrosis, however, its role on inhibition of BM-FPC-mediated fibrosis is not known. Thus, we hypothesized that IL-10 inhibits PO-induced homing and transition of BM-FPC to myoFBs and therefore, attenuates cardiac fibrosis. Methods and Results: Cardiac fibrosis was induced in Wild-type (WT) and IL-10-knockout (KO) mice by transverse aortic constriction (TAC). TAC-induced left ventricular (LV) dysfunction and fibrosis were further exaggerated in KO mice. Systemic recombinant IL-10 administration markedly improved LV function and inhibited PO-induced cardiac fibrosis. PO-enhanced FPC (Prominin1 + cells) mobilization and homing in IL-10 KO mice compared to WT mice. Furthermore, bone marrow transplantation (BMT) experiment was performed wherein WT marrow from GFP mice was repopulated in IL-10 KO mice. FPC mobilization was significantly reduced in BMT-IL10 KO mice compared to IL-10 KO mice after TAC. Furthermore, immunofluorescence result in BMT mice showed that subsets of myoFBs are derived from BM after TAC. To identify the molecular mechanism, wild type BM-FPC were treated with TGFβ 2 with or without IL10. IL10 treatment significantly inhibits TGFβ 2 -induced FPC to myoFBs transition. As miRNAs are key players in cardiac fibrosis, next we performed fibrosis-associated miRNA profiling using miRNA array kit. TGFβ 2 -induced miR-208, 155, 21 and 145 expression was markedly inhibited by IL-10. Conclusion: Taken together, our findings suggest that both reduced homing to heart and transition of FPC to myofibroblasts mediate anti-fibrotic effect of IL10 during PO-induced heart failure. Ongoing investigations using molecular approaches will provide a better understanding on the mechanistic and therapeutic aspects of IL10 on PO-induced cardiac fibrosis and heart failure.

Abstract 4055: Interleukin-12 deficiency increases modulation of epigenetic regulators and silencing of tumor suppressor genes in UVB-exposed mouse skin and skin tumors

Abstract Ultraviolet (UV) radiation has variety of deleterious effects on mammalian skin, and triggers several causative factors such as DNA damage, inflammation, immune suppression and genetic and epigenetic alterations which leads to the development of skin cancer. Earlier, we have shown that the deficiency of interleukin (IL)-12, an immunoregulatory cytokine, enhances photocarcinogenesis in mice through inhibition of repair of damaged DNA. In further studies we have tried to explore and examine the effect of IL-12 on epigenetic regulators in UV-exposed skin and UV-induced skin tumors using IL-12 knock out (IL-12 KO) mice as an in vivo animal model. The exposure of IL-12 KO mice with UVB (200 mJ/cm2) radiation resulted in higher levels of DNA methylation, DNA methyltransferase (Dnmt) activity, Ten-eleven translocations (TETs), a family of methylcytosine dioxygenase, histone deacetylases (HDAC), and lower levels of histone acetylase (HAT) activity than their wild type counterparts. To verify that IL-12 deficiency is associated with DNA hypermethylation and higher HDAC activity in UVB exposed skin, IL-12-KO mice were treated with endotoxin-free murine rIL-12 (50 ng/mouse/100 μl PBS; subcutaneous injection) 3 h before UVB exposure. Our results indicate that significant reduction in the levels of global DNA methylation, Dnmt and HDAC activity were observed compared with non-rIL-12 treated mice. Conversely, UVB exposed IL-12 wild type mice were treated with anti-IL-12 antibody (500 ng/mouse/100 μl PBS; subcutaneous injection). This treatment resulted in increased levels of DNA methylation, Dnmt and HDAC activities. These observations were also noted and compared between skin tumors from IL-12 KO mice and their wild type counterparts. To further explore the role of IL-12 deficiency on epigenetic modification-mediated photocarcinogenesis, we determined its effect on methylated and unmethylated levels of tumor suppressor genes, such as p16INK4a and RASSF1A, in UV-exposed skin and UV-induced skin tumors. It was observed that the levels of methylated tumor suppressor genes were higher in IL-12 KO mice skin than their wild type counterparts. Together, these findings indicate that IL-12 deficiency promotes: (i) DNA hypermethylation, (ii) HDAC activity and (iii) silencing of tumor suppressor genes in UV exposed mice and that makes the mice more susceptible to UV radiation-induced skin cancer risk. Together, these observations suggest that augmentation of IL-12 should be considered as a strategy for the prevention and treatment of UV radiation-induced skin cancer risk. Citation Format: Ram Prasad, Tripti Singh, Santosh K. Katiyar. Interleukin-12 deficiency increases modulation of epigenetic regulators and silencing of tumor suppressor genes in UVB-exposed mouse skin and skin tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4055.

The Role of Neutrophils in the Induction of Specific Th1 and Th17 during Vaccination against Tuberculosis.

Mycobacterium tuberculosis causes tuberculosis (TB), a disease that killed more than 1.5 million people worldwide in 2014, and the Bacillus Calmette Guérin (BCG) vaccine is the only currently available vaccine against TB. However, it does not protect adults. Th1 and Th17 cells are crucial for TB control, as well as the neutrophils that are directly involved in DC trafficking to the draining lymph nodes and the activation of T lymphocytes during infection. Although several studies have shown the importance of neutrophils during M. tuberculosis infection, none have shown its role in the development of a specific response to a vaccine. The vaccine mc2-CMX was shown to protect mice against M. tuberculosis challenge, mainly due to specific Th1 and Th17 cells. This study evaluated the importance of neutrophils in the generation of the Th1- and Th17-specific responses elicited by this vaccine. The vaccine injection induced a neutrophil rich lesion with a necrotic central area. The IL-17 KO mice did not generate vaccine-specific Th1 cells. The vaccinated IL-22 KO mice exhibited Th1- and Th17-specific responses. Neutrophil depletion during vaccination abrogated the induction of Th1-specific responses and prohibited the bacterial load reduction observed in the vaccinated animals. The results show, for the first time, the role of neutrophils in the generation of specific Th1 and Th17 cells in response to a tuberculosis vaccine.

A novel PAK1/Notch-1 axis contributes to intestinal inflammation and cancer

Background: PAK1 belongs to a family of serine-threonine kinase and contributes to various oncogenic pathways like MAPK and Wnt-β catenin. Our lab recently linked activation of p21-activated kinase (PAK1) to IBD as well as colitis-associated cancer (Khare V, Inflamm Bowel Dis 2015). Here we tested the effects of PAK1 deletion on intestinal inflammation and carcinogenesis in IL-10 KO mice. Methods: IL-10 KO mice were crossed with Pak1 KO mice to generate double-knockout mice (DKO). Inflammation was triggered with piroxicam (200ppm mixed to chow for 14 d). After 12 wks mice were euthanized, intestines were prepared and length was measured. Paraffin embedded Swiss rolls were stained with HE MAPK pathway activation, as shown by increased pERK1/2 staining and an increase in γH2AX positive nuclei, indicating more DNA double-strand breaks. A reduced differentiation of goblet cells and Paneth cells was marked by PAS- and Lysozyme staining, together with an expansion of the stem cell compartment (shown by increased activated Notch1 and LGR5 staining) in the colonic crypts of DKOs. Interestingly NFκB was found to be reduced in DKO mice. Conclusion: Our data suggest a novel interaction of PAK-1 with the Notch pathway in regulating crypt cell stemness, proliferation and differentiation. Lack of mature goblet cells and mucus can exacerbate colitis in IL-10 KO mice. Hyperproliferation may promote dysplastic lesions independent of NFκB.

Estradiol Enhances CD4+ T-Cell Anti-Viral Immunity by Priming Vaginal DCs to Induce Th17 Responses via an IL-1-Dependent Pathway.

Clinical and experimental studies have shown that estradiol (E2) confers protection against HIV and other sexually transmitted infections. Here, we investigated the underlying mechanism. Better protection in E2-treated mice, immunized against genital HSV-2, coincided with earlier recruitment and higher proportions of Th1 and Th17 effector cells in the vagina post-challenge, compared to placebo-treated controls. Vaginal APCs isolated from E2-treated mice induced 10-fold higher Th17 and Th1 responses, compared to APCs from progesterone-treated, placebo-treated, and estradiol-receptor knockout mice in APC-T cell co-cultures. CD11c+ DCs in the vagina were the predominant APC population responsible for priming these Th17 responses, and a potent source of IL-6 and IL-1β, important factors for Th17 differentiation. Th17 responses were abrogated in APC-T cell co-cultures containing IL-1β KO, but not IL-6 KO vaginal DCs, showing that IL-1β is a critical factor for Th17 induction in the genital tract. E2 treatment in vivo directly induced high expression of IL-1β in vaginal DCs, and addition of IL-1β restored Th17 induction by IL-1β KO APCs in co-cultures. Finally, we examined the role of IL-17 in anti-HSV-2 memory T cell responses. IL-17 KO mice were more susceptible to intravaginal HSV-2 challenge, compared to WT controls, and vaginal DCs from these mice were defective at priming efficient Th1 responses in vitro, indicating that IL-17 is important for the generation of efficient anti-viral memory responses. We conclude that the genital mucosa has a unique microenvironment whereby E2 enhances CD4+ T cell anti-viral immunity by priming vaginal DCs to induce Th17 responses through an IL-1-dependent pathway.

The contribution of the HSCs niche to bone marrow failure in the IL-2 deficient model

Abstract Bone marrow failure (BMF) is a devastating disease where regeneration of peripheral blood lineages is compromised, resulting in anemia and mortality. In IL-2 KO BALB/c mice, the balance between early myeloid and lymphoid progenitor cell populations is skewed resulting in insufficient myelopoiesis with accumulation of CD8 T cells in the BM. It is widely accepted that crosstalk between cells of the bone microenvironment and hematopoietic cells affects each other’s behavior. However, it remains unknown how the BM niche in this mouse model contribute to the BMF. Multipotent stromal cells (MSCs), osteoblasts (OBs) and endothelial cells (ECs) are the three types of BM stromal cells (BMSCs) that may serve as the distinct functional niche to maintain HSCs. Repots have shown the interplay between CD8 T cells and MSCs cause a change in myelopoiesis and that MSCs promote the formation of Tregs. Our data also indicates that IL-2 KO CD8 T cells are important for the expansion of CD4 T cells in the bone. Since IL-2 KO bones are extremely brittle and CD4 T cells produce RANKL, the ligand needed for osteoclast (OC) differentiation, we evaluated the frequency of BMSCs and OCs. Our preliminary data shows clear differences in BMSCs profiles of IL-2 KO mice, namely elevated stromal cell numbers and a shift in Sca-1 expression in ECs and CD51 expression in OBs. Additionally, OC precursor frequency is elevated in the BM, indicating an increase in bone resorption. The ability of the different subsets of the BMSCs to support the differentiation of HSCs in vitro and the role of CD8 T cells in promoting these changes in vivo is being currently evaluated. Overall, our preliminary data suggests that the change in the bone microenvironment may contribute to the BMF observed in these mice.

Microbiota-inducible Innate Immune, Siderophore Binding Protein Lipocalin 2 is Critical for Intestinal Homeostasis.

Background & AimsLipocalin 2 (Lcn2) is a multifunctional innate immune protein whose expression closely correlates with the extent of intestinal inflammation. However, whether Lcn2 plays a role in the pathogenesis of gut inflammation is unknown. Herein, we investigated the extent to which Lcn2 regulates inflammation and gut bacterial dysbiosis in mouse models of IBD.MethodsLcn2 expression was monitored in murine colitis models and upon microbiota ablation/restoration. Wild-type (WT) and Lcn2 knockout (Lcn2KO) mice were analyzed for gut bacterial load, composition by 16S ribosomal RNA gene pyrosequencing, and their colitogenic potential by co-housing with interleukin (Il)10KO mice. Acute (dextran sodium sulfate) and chronic (IL10R neutralization and T-cell adoptive transfer) colitis were induced in WT and Lcn2KO mice with or without antibiotics.ResultsLcn2 expression was dramatically induced on inflammation and was dependent on the presence of a gut microbiota and MyD88 signaling. Use of bone marrow–chimeric mice showed that nonimmune cells are the major contributors of circulating Lcn2. Lcn2KO mice showed increased levels of entA-expressing gut bacteria burden, and, moreover, a broadly distinct bacterial community relative to WT littermates. Lcn2KO mice developed highly colitogenic T cells and showed exacerbated colitis on exposure to DSS or neutralization of IL10. Such exacerbated colitis could be prevented by antibiotic treatment. Moreover, exposure to the microbiota of Lcn2KO mice, via cohousing, resulted in severe colitis in Il10KO mice.ConclusionsLcn2 is a bacterially induced, MyD88-dependent protein that plays an important role in gut homeostasis and a pivotal role on challenge. Hence, therapeutic manipulation of Lcn2 levels may provide a strategy to help manage diseases driven by alteration of the gut microbiota.

Deletion of interleukin-6 alleviated interstitial fibrosis in streptozotocin-induced diabetic cardiomyopathy of mice through affecting TGFβ1 and miR-29 pathways.

Interleukin 6 (IL-6) has been shown to be an important regulator of cardiac interstitial fibrosis. In this study, we explored the role of interleukin-6 in the development of diabetic cardiomyopathy and the underlying mechanisms. Cardiac function of IL-6 knockout mice was significantly improved and interstitial fibrosis was apparently alleviated in comparison with wildtype (WT) diabetic mice induced by streptozotocin (STZ). Treatment with IL-6 significantly promoted the proliferation and collagen production of cultured cardiac fibroblasts (CFs). High glucose treatment increased collagen production, which were mitigated in CFs from IL-6 KO mice. Moreover, IL-6 knockout alleviated the up-regulation of TGFβ1 in diabetic hearts of mice and cultured CFs treated with high glucose or IL-6. Furthermore, the expression of miR-29 reduced upon IL-6 treatment, while increased in IL-6 KO hearts. Overexpression of miR-29 blocked the pro-fibrotic effects of IL-6 on cultured CFs. In summary, deletion of IL-6 is able to mitigate myocardial fibrosis and improve cardiac function of diabetic mice. The mechanism involves the regulation of IL-6 on TGFβ1 and miR-29 pathway. This study indicates the therapeutic potential of IL-6 suppression on diabetic cardiomyopathy disease associated with fibrosis.

Elevated levels of Interleukin (IL)-33 induce bone pathology but absence of IL-33 does not negatively impact normal bone homeostasis

Interleukin (IL)-33 is a member of the IL-1 family. IL-33 effects are mediated through its receptor, ST2 and IL-1RAcP, and its signaling induces the production of a number of pro-inflammatory mediators, including TNFα, IL-1β, IL-6, and IFN-γ. There are conflicting reports on the role of IL-33 in bone homeostasis, with some demonstrating a bone protective role for IL-33 whilst others show that IL-33 induces inflammatory arthritis with concurrent bone destruction. To better clarify the role IL-33 plays in bone biology in vivo, we studied IL-33 KO mice as well as mice in which the cytokine form of IL-33 was overexpressed. Mid-femur cortical bone mineral density (BMD) and bone strength were similar in the IL-33 KO mice compared to WT animals during the first 8months of life. However, in the absence of IL-33, we observed higher BMD in lumbar vertebrae and distal femur in female mice. In contrast, overexpression of IL-33 resulted in a marked and rapid reduction of bone volume, mineral density and strength. Moreover, this was associated with a robust increase in inflammatory cytokines (including IL-6 and IFN-γ), suggesting the bone pathology could be a direct effect of IL-33 or an indirect effect due to the induction of other mediators. Furthermore, the detrimental bone effects were accompanied by increases in osteoclast number and the bone resorption marker of C-terminal telopeptide collagen-I (CTX-I). Together, these results demonstrate that absence of IL-33 has no negative consequences in normal bone homeostasis while high levels of circulating IL-33 contributes to pathological bone loss.

Interleukin-15 Constrains Mucosal T Helper 17 Cell Generation: Influence of Mononuclear Phagocytes.

Interleukin (IL)-15 has multiple roles in innate and adaptive immunity, especially regarding CD8+ T cells and natural killer cells. However, the role of IL-15 in regulating differentiation of T helper cell subsets and mononuclear phagocytes (MPs) in different tissues in vivo is unknown. Here we report that IL-15 indirectly regulates Th17 but not other Th subsets in the intestinal lamina propria (LP), apparently through effects on MPs. Th17 cells in the LP were more prevalent in IL-15 KO mice than their wild-type counterparts, and less prevalent in IL-15 transgenic mice than their wild-type littermates, even co-caged. MPs from the LP of these mice were sufficient to mimic the in vivo finding in vitro by skewing of cocultured wild type OVA-specific CD4+ T cells. However, production of IL-15 or lack thereof by these MPs was not sufficient to explain the skewing, as addition or blockade of IL-15 in the cultures had no effect. Rather, a skewing of the relative proportion of CD11b+, CD103+ and double positive LP MP subsets in transgenic and KO could explain the differences in Th17 cells. Thus, IL-15 may influence MP subsets in the gut in a novel way that alters the frequency of LP Th17 cells.

Abstract 14287: Ang II-induced Pathological Autophagy is Inhibited by IL-10 via Akt Dependent Inhibition of Beclin 1 in Mice Heart

Although, autophagy is an essential cellular salvage process to maintain cellular homeostasis, pathological autophagy can lead to cardiac abnormalities and ultimately heart failure. Therefore, a tight regulation on autophagic process would be important to treat chronic heart failure. Previously, we have shown that IL-10 strongly improved cardiac function in chronic heart failure models, but the role of IL-10 in regulation of pathological autophagy is not yet investigated. We tested the hypothesis that IL-10 inhibits angiotensin II-induced pathological autophagy and thus improved cardiac function. Pathological autophagy was induced in wild type (WT) and IL10-knockout mice by angiotensin II infusion. Ang II-induced left ventricular dysfunction and hypertrophic remodeling were accentuated in IL-10 KO mice compared to WT mice. IL-10 KO mice showed exaggerated autophagy with reduced AKT phosphorylation. In neonatal rat ventricular cardiomyocytes, Ang II activated beclin1 and LC3 levels and inhibited AKT/mTORC1 and AKT-Bcl2 signaling. IL-10 inhibited Ang II-induced autophagic marker proteins. Additionally, IL-10 restored Ang II effects on AKT/mTORC1 and AKT-Bcl2 signaling. Both pharmacological/molecular inhibition of AKT via PI3K inhibitor (LY290002) or Akt siRNA, attenuated IL-10 effects on the Ang II-induced pathological autophagy, confirming that IL-10 mediated regulation of pathological autophagy is AKT dependent. Similar results were observed with mTORC1 inhibitor rapamycin. Chloroquine (a lysosome inhibitor) strongly inhibits Ang II-induced autophagic flux. However, chloroquine did not affect IL-10 effects on autophagic flux, suggesting that IL-10 inhibits stress-induced pathological autophagy. Finally, as physical interaction of Bcl2 with beclin 1 is important to inhibit autophagy and IL-10 is strong activator of Bcl2, we performed immunoprecipitation experiment. Immunoprecipitation data suggested that Ang II disrupt the physical interaction of beclin 1 with Bcl2 and IL-10 reestablished this physical interaction to reduce autophagy. Our data give a novel role of IL-10 in regulation of pathological autophagy and thus can act as a potential therapeutic molecule in treatment of chronic heart disease.

Abstract B39: Neutrophils G-CSF stimulated promotes specific protection against graft vs. host disease and keeps the graft vs. leukemia effect

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) is used to treat a series of hematological diseases. While the presence of T cells mediates the graft-versus-leukemia (GVL) effect and improves engraftment, it is also responsible for the graft-versus-host disease (GVHD), which is the main barrier of HSCT. Patients transplanted with cells from G-CSF treated donors show an unexpected low rate of acute GVHD given the high numbers of T cells present. We have previously shown that blood from G-CSF treated donors have high numbers of inhibitory granulocytes. Experimentally these granulocytes are able to prevent aGVHD in a semi-allogeneic mouse model. Here, our goal is to characterize the granulocytes and its mechanism of action. When we compare this granulocytes from the spleens of 5 day G-CSF treated donors and its untreated control, we observed on flow cytometry that the percentage of this granulocytes/neutrophils Ly6G+ increases from about 2% to about 20%. These cells have some differences among themselves after treatment as increased burst oxidative (measured by DHR) and phagocytosis of E.coli, as well as differences in the pattern of expression of some important surface molecules, such as MHC-II. Levels of myeloperoxidase are lower after treatment with G-CSF, and we confirmed by transmission electron microscopy that these neutrophils have a degranulated profile, with 83% fewer cytoplasmic granules than controls. Indeed, the spleens from 5 day G-CSF treated donors together with control bone marrow cells (G-B6) injected into irradiated F1(B6XBALB/c) mice shows survival rate of 100%, against 10% in non-G-B6 transplants. This protection depends on Gr1+ cells and presents milder pathological and clinical manifestations in gastrointestinal mucosa, skin and liver. However, when these G-B6 neutrophils are from IL-10 KO mice, protection is abolished both in survival curve and in clinical score. Surprisingly, 25 days after transplantation the percentage of the subtype of T regulatory Foxp3+ cells in protected chimeras is increased in all lymphoid organs analyzed and this can be associated with the long term specific tolerance observed. Finally, the anti-tumoral effect in G-B6 chimeras is as potent as in the B6 control chimera and this effect was maintained for more than 20 weeks after HSCT. So we can conclude that treatment with G-CSF generates degranulated neutrophils that reduce GVHD in an IL-10 dependent manner while keeping GVL effect opening a promising road in the prevention of human aGVHD. Financial Support: FAF, INCA, Swiss Bridge, INCT Cancer, FAPERJ, CNPQ, CAPES, Fiocruz. Citation Format: Suelen Martins Perobelli, Ana Carolina Terra Mercadante, Triciana Gonçalves-Silva, Rômulo Galvani, Antônio Pereira-Neves, Marlene Benchimol, Alberto Nobrega, Adriana Bonomo. Neutrophils G-CSF stimulated promotes specific protection against graft vs. host disease and keeps the graft vs. leukemia effect. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B39.

Osteopontin Deficiency Accelerates Spontaneous Colitis in Mice with Disrupted Gut Microbiota and Macrophage Phagocytic Activity.

BackgroundOsteopontin (OPN) is a multifunctional protein expressed in a variety of tissues and cells. Recent studies revealed increased OPN expression in the inflamed intestinal tissues of patients with inflammatory bowel disease (IBD). The role of OPN in the pathophysiology of IBD, however, remains unclear.AimsTo investigate the role of OPN in the development of intestinal inflammation using a murine model of IBD, interleukin-10 knock out (IL-10 KO) mice.MethodsWe compared the development of colitis between IL-10 KO and OPN/IL-10 double KO (DKO) mice. OPN expression in the colonic tissues of IL-10 KO mice was examined by fluorescence in situ hybridization (FISH) analysis. Enteric microbiota were compared between IL-10 KO and OPN/IL-10 DKO mice by terminal restriction fragment length polymorphism analysis. The effect of OPN on macrophage phagocytic function was evaluated by phagocytosis assay.ResultsOPN/IL-10 DKO mice had an accelerated onset of colitis compared to IL-10 KO mice. FISH analysis revealed enhanced OPN synthesis in the colonic epithelial cells of IL-10 KO mice. OPN/IL-10 DKO mice had a distinctly different enteric bacterial profile with a significantly lower abundance of Clostridium subcluster XIVa and a greater abundance of Clostridium cluster XVIII compared to IL-10 KO mice. Intracellular OPN deletion in macrophages impaired phagocytosis of fluorescence particle-conjugated Escherichia coli in vitro. Exogenous OPN enhanced phagocytosis by OPN-deleted macrophages when administered at doses of 1 to 100 ng/ml, but not 1000 ng/ml.ConclusionsOPN deficiency accelerated the spontaneous development of colitis in mice with disrupted gut microbiota and macrophage phagocytic activity.

Abstract 3170: IL17 favours carcinogenesis and limits the anti-tumor responses in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDA) is a fatal medical condition with few advances in therapy and patient survival. The role of IL17 and Th17 in cancer progression and anti-tumor immunity is still controversial. In this study we have investigated the role of IL17 and Th17 in PDA carcinogenesis and anti-tumor responses. IL-17 production and Th17 percentage in blood from PDA patients and control subjects as well from genetically engineered mice (GEM) that spontaneously develop PDA, were analysed by ELISA and FACS respectively. Survival of GEM mice crossed with IL-17 KO mice (GEM/IL17KO) was analysed by Kaplan-Meier analysis. WT or IL17KO mice that were prophylactically vaccinated with a plasmid coding for the PDA-associated antigen α-enolase (ENO1) (1-3), were subsequently injected with PDA cell line derived from GEM. Tumor growth and the induction of an antigen-specific immune response were monitored. We have found a statistically significant increase of peripheral Th17 cells and a decrease of Th1 in PDA patients compared to healthy subjects. GEM mice showed 3-fold more RORγT+ cells at the onset of duct transformation, which dropped to the level of control mice at 20 weeks and lasted until 35 weeks. Notably, GEM/IL17 KO mice survived significantly more than GEM mice, even if there were no changes in the tumor onset. In addition, in GEM/IL17 KO mice, we observed an increased fibrotic reaction compared to GEM mice starting from early stage of disease. Lastly, ENO-1 DNA vaccination in IL17KO mice significantly decreased PDA cells growth and enhanced the CD8 T cell response compared to that observed in WT mice. Our data suggest that the absence of IL17 does not affect the tumor onset but the progression, likely affecting the immune response, which seems to be able to better counteract tumor growth in the absence of this cytokine. Tomaino et al., J Proteome Res. 2007;6:4025-31; Cappello et al, Int. J. Cancer. 2009;125:639-48; Cappello P et al, Gastroenterology. 2013;144:1098-106. Citation Format: Paola Cappello, Roberta Curto, Gianluca Mucciolo, Simona Rolla, Elisabetta Tonoli, Francesco Novelli. IL17 favours carcinogenesis and limits the anti-tumor responses in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3170. doi:10.1158/1538-7445.AM2015-3170

IL-15 receptor α signaling constrains the development of IL-17–producing γδ T cells

The development and homeostasis of γδ T cells is highly dependent on distinct cytokine networks. Here we examine the role of IL-15 and its unique receptor, IL-15Rα, in the development of IL-17-producing γδ (γδ-17) T cells. Phenotypic analysis has shown that CD44(high) γδ-17 cells express IL-15Rα and the common gamma chain (CD132), yet lack the IL-2/15Rβ chain (CD122). Surprisingly, we found an enlarged population of γδ-17 cells in the peripheral and mesenteric lymph nodes of adult IL-15Rα KO mice, but not of IL-15 KO mice. The generation of mixed chimeras from neonatal thymocytes indicated that cell-intrinsic IL-15Rα expression was required to limit IL-17 production by γδ T cells. γδ-17 cells also were increased in the peripheral lymph nodes of transgenic knock-in mice, where the IL-15Rα intracellular signaling domain was replaced with the intracellular portion of the IL-2Rα chain (that lacks signaling capacity). Finally, an analysis of neonatal thymi revealed that the CD44(lo/int) precursors of γδ-17 cells, which also expressed IL-15Rα, were increased in newborn mice deficient in IL-15Rα signaling, but not in IL-15 itself. Thus, these findings demonstrate that signaling through IL-15Rα regulates the development of γδ-17 cells early in ontogeny, with long-term effects on their peripheral homeostasis in the adult.

Abstract 172: Interleukin-10-mediated Activation of AKT and Bcl2 Inhibits Chronic Angiotensin II-induced Pathological Autophagy

Rationale: Although, autophagy is an essential cellular salvage process to maintain cellular homeostasis, pathological (stress-induced exaggerated/defective) autophagy can lead to cardiac abnormalities and ultimately heart failure. Therefore, a tight regulation of autophagic process would be important to treat chronic heart failure. Previously, we have shown that IL-10 strongly inhibited pressure overload-induced hypertrophy and heart failure, but role of IL-10 in regulation of pathological autophagy is not known. Hypothesis: We tested the hypothesis that IL-10 inhibits angiotensin II-induced pathological autophagy and this process, in part, led to improved cardiac function. Methods and Results: Pathological autophagy was induced in wild type (WT) and IL10-knockout (IL-10 KO) mice by angiotensin II (Ang II for 28 days) infusion. Ang II-induced left ventricular (LV) dysfunction and hypertrophic remodeling were accentuated in IL-10 KO mice compared to WT mice. IL-10 KO mice showed exaggerated autophagy as observed by Electron Microscopy and Western blotting (beclin 1, LC3 II/I and CHOP) with reduced AKT phosphorylation at serine-473. In neonatal rat ventricular cardiomyocytes (NRCM), Ang II treatment enhanced beclin1, LC3 and CHOP protein levels and inhibited AKT and 4EBP1 phosphorylation and Bcl2 levels. Interestingly, IL-10 inhibited Ang II-induced autophagic marker proteins. Additionally, IL-10 restored Ang II-induced suppression of AKT and 4EBP1 phosphrylation and restoration of Bcl2 protein level. Pharmacological inhibition of AKT via PI3K inhibitor (LY290002), reversed IL-10 responses on the Ang II-induced pathological autophagy, confirming that IL-10 mediated inhibition of autophagy is AKT dependent. Finally, as physical interaction of Bcl2 with beclin 1 is important to inhibit autophagy, we performed immunoprecipitation pull-down experiments, which showed Ang II disrupts the physical interaction of beclin 1 with Bcl2 and IL-10 reestablished this physical interaction to reduce autophagy. Conclusion: Our data provides a novel role of IL-10 in regulation of pathological autophagy and thus can act as a potential therapeutic molecule in treatment of chronic heart disease.