IL-15 Tg Research Articles

Eosinophilic inflammation promotes CCL6-dependent metastatic tumor growth.

Compelling evidence suggests that inflammatory components contribute to cancer development. However, eosinophils, involved in several inflammatory diseases, were not fully explored in cancer metastasis. We show that airway inflammatory eosinophilia and colonic inflammation with eosinophil infiltration are both associated with increased metastasis in mice. Eosinophilia is responsible for increased bone metastasis in eosinophil-enriched Cd3δ-Il-5 transgenic (Il-5 Tg) mice. We also observe increased eosinophils in the malignant pleural effusion of cancer patients with pleural metastasis. Mechanistically, eosinophils promote tumor cell migration and metastasis formation through secreting C-C motif chemokine ligand 6 (CCL6). Genetic knockout of Ccl6 in Il-5 Tg mice remarkably attenuates bone metastasis. Moreover, inhibition of C-C chemokine receptor 1 (CCR1, the receptor of CCL6) in tumor cells reduces tumor cell migration and metastasis. Thus, our study identifies a CCL6-dependent prometastatic activity of eosinophils, which can be inhibited by targeting CCR1 and represent an approach to preventing metastatic disease.

Dysregulated microRNA expression in IL-4 transgenic mice, an animal model of atopic dermatitis.

IL-4 plays an important role in the pathogenesis of atopic dermatitis (AD). Previously we showed that the expression of genes in chemotaxis, angiogenesis, inflammation and barrier functions is dysregulated in IL-4 transgenic (Tg) mice, a well-characterized AD mouse model. In this study, we aim to study differential expression of microRNAs in IL-4 Tg mice. As compared with wild-type mice, we found that 10 and79 microRNAs are dysregulated in the skin of IL-4 mice before and after the onset of skin lesions, respectively. Bioinformatic analysis and previous reports show that these dysregulated microRNAs may be involved in the NF-κB, TLRs, IL-4/IL-13, MAPK and other pathways. We also found that miR-139-5p and miR-196b-3p are significantly up-regulated in the peripheral blood of IL-4 Tg mice. Taken together, our data have identified many dysregulated microRNAs in IL-4 Tg mice, which may play important roles in AD pathogenesis and pathophysiology.

A Novel Class of Bifunctional Immunotherapeutic That Exploits a Universal Antibody Binding Terminus (uABT) to Recruit Endogenous Antibodies to Cell Expressing CD38 Demonstrate In Vivo efficacy in Three Distinct Animal Models

Background: Antibody recruiting molecules (ARM) represent a new modality in immunotherapy of cancer. These are bifunctional molecules composed of two active termini connected by a linker. One of the termini binds to a target molecule on a cancer cell. The other terminus can recruit endogenous IgG antibodies independent of their antigen binding specificity representing a breakthrough improvement to previous approaches (Murelli et al.J Am Chem Soc. 2009). We named this active moiety a universal antibody binding terminus (uABT). As a result of antibody recruitment to the cell surface, the target cell is "opsonized" by antibodies which then bring in the immune effector cells to eliminate the target through various antibody-dependent destruction mechanisms. Multiple myeloma is a neoplasm that arises from terminally differentiated immunoglobulin producing long-lived plasma cells with 32,000 new cases diagnosed each year. Kleo Pharmaceuticals has developed a series of compounds, CD38-ARM which target human CD38 highly expressed by multiple myeloma cells. CD38 -ARM compounds are therapeutically active in three distinct in vivo models without depleting CD38 expressing immune effector cells like existing therapeutic antibodies such as Daratumumab. Methods: CD38-ARM compounds were tested in three independent in vivo models. In the first model, intraperitoneal Daudi xenograft in SCID mice, 20x106 fluorescently labelled Burkitt lymphoma CD38-expressing Daudi cells were injected into SCID mice and treated with 3 mg/kg of compounds. Peritoneal exudates were examined 24 hours later for percentages and absolute numbers of Daudi cells recovered. Activity of compounds was further tested in a MOLP-8 multiple myeloma cell xenograft model in nude mice. Animals received 106 MOLP8 cells subcutaneously, and were treated with a daily dose of 10 mg/kg after tumor volumes of 150 mm3 were reached. Finally, CD38-ARM efficacy was examined in hu IL-15 transgenic NOG mice that have been preconditioned with busulfan and reconstituted i.p. with 2x106human NK cells. Three weeks after reconstitution, 5x106RAJI cells expressing CD38 were implanted s.c. and treatment commenced a week later with 10 mg/kg QDx14. Mice were monitored for NK cell levels and activation status in the blood during the whole study duration by flow cytometry. Results: CD38-uAbt compounds are able to induce clearance of Burkitt's lymphoma Daudi cells expressing high levels of CD38 in a SCID mouse intraperitoneal model. In addition, we show efficacy of one of these compounds in a multiple myeloma xenograft model in nude mice. Using the MOLP8 subcutaneously implanted tumor model we show that administration of our lead molecule is able to induce 50% tumor growth inhibition (TGI) after a daily dosing schedule against this tumor. This activity is comparable to a pharmacologically relevant dose of Daratumumab. Finally, we demonstrate that CD38-ARM treatment shows significant efficacy in humanized mouse model, where IL-15 Tg NOG mice have been reconstituted with human NK cells prior to tumor implantation. In this model daily dosing with the CD38-ARM resulted in up to 70% TGI when compared to untreated control groups. Using this dose/schedule, the molecule did not elicit NK cell depletion as noted in the Daratumumab group. Conclusions: We demonstrated that CD38-ARM compounds are therapeutically active in three distinct in vivo models. Depletion of Daudi cells in a peritoneal SCID model provides strong evidence for the CD38-ARM's capacity to engage macrophage effector functions. Profound inhibition of tumor growth in the NK cell centered humanized hIL-15 Tg NOG mouse model indicates that killing of target cells is executed by human NK cells and provides a foundation to advancing our compounds towards the clinic. Overall, our results demonstrate value of CD38-ARM molecules both as stand-alone therapeutic as well as a platform to develop compounds tailored to a specific indication, by varying target binding moiety of the molecule. Our results also indicate that CD38-ARM compounds engage a variety of effector mechanisms involved in tumor clearance and tumor growth delay, indicating therapeutic potential across a wide range of clinical settings. Disclosures Bunin: Kleo pharmaceuticals: Employment, Equity Ownership. McGrath:Kleo pharmaceuticals: Employment, Equity Ownership. Rossi:Kleo pharmaceuticals: Employment, Equity Ownership. Welsch:Kleo pharmaceuticals: Employment, Equity Ownership. Vidal:Kleo pharmaceuticals: Employment, Equity Ownership. Trinh:Kleo pharmaceuticals: Employment, Equity Ownership. Spiegel:Kleo pharmaceuticals: Equity Ownership. Rastelli:Kleo pharmaceuticals: Employment, Equity Ownership. Alvarez:Kleo pharmaceuticals: Employment, Equity Ownership.

Differential expression of inflammation-related genes in IL-4 transgenic mice before and after the onset of atopic dermatitis skin lesions

IL-4 plays an important role in the pathogenesis of atopic dermatitis (AD), a common chronic inflammatory skin disease. We have generated IL-4 transgenic (Tg) mice by over-expressing IL-4 in the epidermis. These mice spontaneously develop chronic pruritic inflammatory skin lesions, which meet the clinical and histological diagnostic criteria for human AD. Systemic survey of immune-related genes in this mouse model, however, has not been performed. In this study, we utilize PCR array technique to examine hundreds of inflammation-related genes in the IL-4 Tg mice before and after the onset of skin lesions as well as in their wild type (WT) littermates. Only those genes with at least 2-fold up-regulation or down-regulation and with a P-value of less than 0.05 in comparison to WT controls were identified and analyzed. In the skin lesions, many chemokines, pro-inflammatory cytokines, and other AD-related factors are dysregulated compared to the wild type mice. Particularly, CXCL5, IL-1β, IL-24, IL-6, oncostatin M, PTGS2, FPR1 and REG3γ are up-regulated several hundred-fold. In the pre-lesional group that shows no obvious skin abnormality on clinical observation, 30 dysregulated genes are nevertheless identified though the fold changes are much less than that of the lesional group, including CCL6, CCL8, CCL11, CCL17, CXCL13, CXCL14, CXCR3 and IL-12Rβ2. Finally using ELISA, we demonstrate that 4 most dramatically up-regulated factors in the skin are also elevated in the peripheral blood of the IL-4 Tg mice. Taken together, our data have identified hundreds of dysregulated factors in the IL-4 Tg mice before and after the onset of skin lesions. Future detailed examination of these factors will shed light on our understanding of the development and progression of AD and help to discover important biomarkers for clinical AD diagnosis and treatment.

Interleukin-4 up-regulation of epidermal interleukin-19 expression in keratinocytes involves the binding of signal transducer and activator of transcription 6 (Stat6) to the imperfect Stat6 sites.

Interleukin-19 (IL-19) plays an important role in asthma by stimulating T helper type 2 (Th2) cytokine production. Interestingly, IL-4, a key Th2 cytokine, in turn up-regulates IL-19 expression in bronchial epithelial cells, so forming a positive feedback loop. In atopic dermatitis (AD), another Th2 disease closely related to asthma, IL-19 is up-regulated in the skin. We propose to use IL-4 transgenic (Tg) mice and human keratinocyte culture to delineate the molecular mechanisms involved in the up-regulation of IL-19 in AD. IL-19 is similarly up-regulated in the skin of IL-4 Tg mice as in human AD. Next we show that IL-4 up-regulates IL-19 expression in keratinocytes. Interestingly, the up-regulation was suppressed by a pan-Janus kinase (Jak) inhibitor, suggesting that the Jak-signal transducer and activator of transcription (Jak-STAT) pathway may be involved. Dominant negative studies further indicate that STAT6, but not other STATs, mediates the up-regulation. Serial 5' deletion of the IL-19 promoter and mutagenesis studies demonstrate that IL-4 up-regulation of IL-19 in keratinocytes involves two imperfect STAT6 response elements. Finally, chromatin immunoprecipitation assay studies indicate that IL-4 increases the binding of STAT6 to its response elements in the IL-19 promoter. Taken together, we delineate the detailed molecular pathway for IL-4 up-regulation of IL-19 in keratinocytes, which may play an important role in AD pathogenesis.

The Biology of IL-15: Implications for Cancer Therapy and the Treatment of Autoimmune Disorders

IL-15 has a pivotal role in life and death of natural killer (NK) and CD8 memory T cells. IL-15 signals through a heterotrimeric receptor involving the common gamma chain (γc) shared with IL-2, IL-4, IL-7, IL-9, and IL-21, IL-2/IL-15 receptor β (IL-15Rβ) shared with IL-2 and a private IL-15Rα subunit. IFN- or CD40 ligand-stimulated dendritic cells coordinately express IL-15 and IL-15Rα. Cell surface IL-15Rα presents IL-15 in trans to cells that express IL-2/IL-15Rβ and γc. IL-15 is being used to treat patients with metastatic malignancy. However, IL-15 is an inflammatory cytokine involved in immunological memory including that to self, thereby playing a role in autoimmune diseases. These insights provide the scientific basis for clinical strategies directed toward diminishing IL-15 action. Dysregulated IL-15 expression was demonstrated in patients with rheumatoid arthritis, inflammatory bowel disease, psoriasis, celiac disease, and alopecia areata. The monoclonal antibody Hu-Mik-β-1 targets the cytokine receptor subunit IL-2/IL-15Rβ (CD122), blocks IL-15 transpresentation, and is being used in clinical trials in patients with autoimmune diseases. In parallel, clinical trials have been initiated involving the Jak2/3 (Janus kinase-2/3) inhibitor tofacitinib and Jak1/2 inhibitor ruxolitinib to block IL-15 signaling.

Increased Expression Of IL-15 Promotes Cutaneous T-Cell Lymphomagenesis Via The Upregulation Of Histone Deacetylases: Evidence For Successful Preclinical Targeting

Cutaneous lymphoma is a heterogeneous group of neoplasms of skin-homing malignant lymphocytes. Cutaneous T-cell Lymphoma (CTCL) represents 70-80% of all cutaneous lymphoma and its pathogenesis is largely unknown. Previous studies have shown that interleukin (IL)-15 is a potent stimulant and growth factor for CTCL cells in vitro. In order to investigate the intrinsic levels of IL-15 in CTCL patients, malignant CD4+ T-cells were analyzed for expression of IL-15. Relative quantitation of IL-15 transcript in patient vs normal donor (ND) CD4+ cells showed overexpression of IL-15 in patients (fold increase mean ± SD = 5.36 ± 4.39, N=13, P<0.001). Increase in IL-15 transcript was directly proportional to disease severity in patients i.e. fold increase mean ± SD in IL-15 in Stage I =3.28 ± 1.42, N=3 each, P=0.0047 vs. Stage III patients = 7.42 ± 1.30, N=3 each, P=0.0073. Further, cutaneous lesions in patients stained positive for IL-15 protein in atypical lymphoid cells and Pautrier's microabscess. We next investigated the role of IL-15 in CTCL development using IL-15 transgenic (tg) mice. Within 4-6 weeks of birth, IL-15 tg mice developed extensive patch/plaque skin lesions, progressive alopecia, and severe pruritus. Adult IL-15 tg mice developed extensive involvement with cutaneous lymphoma that was fatal in 100% of the mice (P=0.0003). Antibodies staining revealed that CD4+ skin resident T-cells in IL-15 tg mice were CD3+CD62L-CD44hiCCR4+CLA+. Flow cytometric analysis of single cell suspension of skin showed ∼25-fold increase in CD3+ T-cells in IL-15 tg compared to WT controls (Mean ± SD of absolute number of cells= 3.80 ± 6.97, N=14 vs. 0.15 ± 0.26, N=8 respectively, P<0.001). Lymphoma cells from IL-15 tg mouse skin engrafted and mimicked the primary disease in immune deficient SCID mice upon adoptive transfer. CD4+ T-cells from CTCL patients showed increased histone deacetylases (HDAC) 1, HDAC2 and HDAC6 transcripts over ND CD4+ T-cells and immunoblot analysis of ND CD4+ T-cells exposed to 100ng/ml IL-15 showed upregulation of HDAC1, HDAC2 and HDAC6 ex vivo. IL-15 stimulation of ND CD4+ T-cells resulted in loss of expression of the downstream HDAC1/2 target tumor suppressor, p21 in vitro, and knock down of HDAC6 in IL-15 stimulated ND CD4+ T-cells inhibited their migration in vitro; suggesting that IL-15 mediated upregulation of HDAC6 is critical for T-cell migration. Considering these observations, we used specific novel HDAC inhibitors (HDACi) to target HDAC1/2 (JQ12) and/or HDAC6 (WT161) in IL-15 tg mice to determine if we could prevent CTCL in vivo. IL-15 tg mice were treated with 50mg/kg of either or both the inhibitors, 5 days/week for 4 weeks (n=4 each). While placebo treated IL-15 tg mice progressively developed lesions during the course of treatment, IL-15 tg mice treated with JQ12 and/or WT161 showed no clinical signs of disease. This was further corroborated by histopathology analysis of skin sections from treated mice (Figure 1). Thus, our data suggest that inhibiting HDAC1, HDAC2 and/or HDAC6 pathways inhibits the development of CTCL in IL-15tg mice. In addition to the prevention study, we assessed the ability of a novel pan-HDACi, AR42, to treat active and progressive disease in our model. IL-15 tg mice with established CTCL were randomized to receive either AR42 or placebo feed (n=6 each) for 12 days. The IL-15 tg mice treated with AR42 showed remarkable improvement compared to the placebo mice whose disease progressed. Histopathology analysis of the AR42-treated IL-15 tg mice showed an impressive clearance of the CD3+ and CD4+ atypical lymphocytic infiltrate compared to placebo-treated mice (Figure 2). In summary we provide evidence that IL-15 has a causal role in the pathogenesis of CTCL; that IL-15 tg mice provide a novel model for studying disease pathogenesis and for evaluating potential therapies; that HDACi targeting specific HDACs may be effective in preventing CTCL and a novel pan-HDACi can reverse severe dermatologic disease in this CTCL model. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Abstract 3857: Targeting HDAC1 in a novel model of cutaneous T-cell lymphoma.

Abstract Cutaneous T-cell lymphoma (CTCL) is a type of non-Hodgkin's lymphoma composed of a spectrum of diseases of malignant clonal helper CD4+ T lymphocytes. Previous studies have shown that IL-15 acts as viability factor for CTCL cells, inhibiting apoptosis, and may work in association with other factors to maintain the malignant infiltrate in the epidermis. IL-15 is highly expressed both at mRNA and protein levels in the skin of CTCL patients. Using an IL-15 transgenic (tg) mouse model for spontaneous CTCL that clinically and pathologically mimics the human malignancy, we described a system for studying lymphomagenesis in CTCL (97th Annual Meeting of the AACR; 2010. Abstract nr. 2858). Skin lesions in these mice revealed the presence of characteristic Pautrier's microabscesses and these mice had poor survival. Cutaneous CD4+ T-cells from these mice were capable of engrafting into the skin of secondary SCID recipients, producing histological lesions of similar pathology. Consistent with human CTCL, we found that these tg mice are predominantly CD62L- (P=0.0009, N=14) with overexpression of CCR4, CLA, and CD44 (P=0.0001, N=14). Interestingly, we also observed elevated histone deacetylase-1 (Hdac1) expression in IL-15 tg mice compared to the wild type (WT) cohort (P=0.0013, N=3). We hypothesized that IL-15 regulates epigenetic changes in normal CD4+ lymphocytes by modulating Hdac1 and Hdac6. We analyzed normal human- and WT mouse- CD4+ T-cells and found that IL-15 induces upregulation of Hdac1 protein and cytoplasmic translocalization of Hdac6. To elucidate the mechanism controlling Hdac1 induction, we tested whether IL-15 stimulation leads to autoregulation of Hdac1 in vitro. Chromatin immunoprecipitation (ChIP) PCR analysis of IL-15 stimulated CD4+ T-cells revealed that Hdac1 binds to its own promoter as early as 12 hours post incubation increasing its own transcription. Similar observations were made in CD4+ T-cells from CTCL patients when compared to normal donor CD4+ T-cells suggesting that Hdac1 could be upregulated in CD4+ T-cells as a result of autoregulation from endogenous IL-15. To test Hdac-inhibitor therapy in IL-15 tg CTCL mice, we performed a randomized, placebo-controlled trial of an oral HDAC inhibitor invented at The Ohio State University, AR-42 (Arno Therapeutics Inc., Flemington, NJ). Interestingly, 100% of mice treated with AR-42 showed clinical improvement and reduced dermal lymphocytic infiltration after the treatment when compared to the placebo arm. In summary, our study provides a murine model of spontaneous CTCL that mimics the human disease, and also, demonstrate for the first time IL-15 mediated autoregulation of a histone-modifying enzyme Hdac1 in CD4+ T-cells that may contribute towards pathogenesis of CTCL. Finally, we show that AR-42 has significant efficacy in this preclinical CTCL model, which has in part paved the way for its current evaluation in a Phase I human clinical trial. Citation Format: Anjali Mishra, Laura Sullivan, Gregory Sams, Jessica Johns, Douglas Curphey, Lauren Falkenberg, Heather Gibson, Shujun Liu, Laura Jaroncyk, Kathleen McConnell, Henry Wong, Krista La Perle, Ramiro Garzon, Guido Marcucci, Pierluigi Porcu, Michael Caligiuri. Targeting HDAC1 in a novel model of cutaneous T-cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3857. doi:10.1158/1538-7445.AM2013-3857

IL-4 up-regulates epidermal chemotactic, angiogenic, and pro-inflammatory genes and down-regulates antimicrobial genes in vivo and in vitro: Relevant in the pathogenesis of atopic dermatitis

Atopic dermatitis (AD) is a common chronic inflammatory skin disease. Although the pathogenesis of AD is not fully understood, we and others have shown that IL-4 plays a key role. In this study we aimed to identify keratinocyte genes regulated by IL-4 that may play important roles in the pathophysiology of AD. HaCat cells were treated with IL-4 at various concentrations for 24h, and PCR gene array on inflammation/autoimmunity was performed three times for analysis of differential gene expression. Of all the 370 genes examined, 32 and 53 genes are up- and down-regulated, respectively. Specifically related to AD, chemokines CCL3L1, CCL8, CCL24, CCL25, CCL26, CXCL6 and CXCL16 are up-regulated by IL-4. Pro-inflammatory factors, such as IL-19, IL-20, IL-1α, IL-12Rβ2, IL-25, IL-31RA, OSMR and nitric oxide synthase 2, are also up-regulated. In addition, IL-4 up-regulates VEGFA, a pro-angiogenic factor. In contrast, antimicrobial peptides (AMPs) or factors involved in APM production, such as IFN-κ, S100s, Toll-like receptors, and several chemokines are down-regulated. Similarly IL-4 also down-regulates TNF-α, lymphotoxin-β, an IgE suppressor, TNFSF18, a T-cells function regulator, and the glucocorticoid receptor. On the in vivo level, real-time RT-PCR on the selected genes confirmed that IL-4 up-regulates chemokines, proinflammatory cytokines while it suppresses AMP production related genes in the skin obtained from IL-4 Tg mice. Detailed examination of these genes will delineate their specific roles in chemotaxis, inflammation, angiogenesis and AMP production, all of which may contribute to the development and progression of AD.

Abstract 2858: A novel mouse model for cutaneous T-cell lymphoma reveals a role for IL-15 and activity of a new oral HDAC inhibitor

Abstract Cutaneous T-cell lymphoma (CTCL) is a type of non-Hodgkin's lymphoma representing a spectrum of diseases composed of malignant clonal CD4 (+) helper T lymphocytes. The etiology of CTCL remains to be elucidated and no animal models for CTCL are known. We quantified expression of IL-15 in patients with CTCL and demonstrated aberrant overexpression of IL-15 compared to normal CD4+ T cells (Fold increase = 7.476 ± 1.788 N=3, P=0.0073), that showed direct correlation with the stage of the disease (Fold increase in IL-15 in Stage I patients vs. Stage III patients = 3.283 ± 0.8246 vs. 7.476 ± 1.788, N=3 each, P=0.02). We engineered a transgenic (tg) mouse to constitutively and globally overexpress murine IL-15. These mice develop alopecia and cutaneous lesions in all mice within 6 weeks of birth, characterized by erythematous plaques/patches, erythroderma, pruritis and early death compared to wild type (WT) controls (49 weeks vs. 100 weeks, P=0.0003). Skin lesions showed an infiltration of atypical lymphocyte in the dermis and throughout the epidermis including the formation of classic Pautrier's microabscesses as seen in human CTCL. Immunophenotypic analysis of the skin T-cells from these mice revealed ∼25 fold increase in CD3+ T-cells compared to WT littermates. Intradermal CD4+ cells from CTCL mice were predominantly CD62L- and significantly higher than WT mice (P=0.007). Immunohistochemistry showed high expression of cutaneous lymphocyte antigen (CLA) and CCR4 on skin sections of CTCL mice, consistent with a skin-specific homing pattern as seen in human CTCL. Cells obtained from the skin of these IL-15tg CTCL mice could be successfully engrafted into skin of SCID recipient mice with reproducible histological lesions. Consistent with human studies, CTCL mice exhibited significantly increased levels of Hdac-1 in CD4+ T cells when compared to their WT counterparts (P = 0.0013). To investigate the importance of IL-15 in this process, we cultured sorted mouse WT (CD3+CD4+) T-cells in medium supplemented with 100ng/ml IL-15 and observed a significant increase in Hdac-1 expression at the mRNA and protein level within 10 days of culture, compared to freshly isolated WT CD4+ T cells (N=3 each, p =0.02). In a randomized, placebo-controlled trial of an orally administered novel HDAC inhibitor, AR-42 (Arno Therapeutics, Inc, Parsippany, NJ; Sargeant AM et al, Cancer Research, 2008) to IL-15 tg mice with CTCL, we observed an improvement in skin lesions and a marked reduction in T-cell dermal infiltrate as early as 12 days post-treatment. In conclusion, we provide evidence for a reproducible in vivo mouse model of spontaneous CTCL that appears to mimic the human condition. Further, we provide evidence for a role of aberrant IL-15 expression in the genesis of both human and experimental CTCL, and alterations in Hdac-1 expression that may to serve as an appropriate target for a novel oral HDAC inhibitor now in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2858. doi:10.1158/1538-7445.AM2011-2858

CD34 Is Required for Infiltration of Eosinophils into the Colon and Pathology Associated with DSS-Induced Ulcerative Colitis

Eosinophil migration into the gut and the release of granular mediators plays a critical role in the pathogenesis of inflammatory bowel diseases, including ulcerative colitis. We recently demonstrated that eosinophil migration into the lung requires cell surface expression of the sialomucin CD34 on mast cells and eosinophils in an asthma model. Based on these findings, we investigated a similar role for CD34 in the migration of eosinophils and other inflammatory cells into the colon as well as explored the effects of CD34 ablation on disease development in a dextran sulfate sodium-induced model of ulcerative colitis. Our findings demonstrate decreased disease severity in dextran sulfate sodium-treated Cd34(-/-) mice, as assessed by weight loss, diarrhea, bleeding, colon shortening and tissue pathology, compared with wild-type controls. CD34 was predominantly expressed on eosinophils within inflamed colon tissues, and Cd34(-/-) animals exhibited drastically reduced colon eosinophil infiltration. Using chimeric animals, we demonstrated that decreased disease pathology resulted from loss of CD34 from bone marrow-derived cells and that eosinophilia in Cd34(-/-)IL5(Tg) animals was sufficient to overcome protection from disease. In addition, we demonstrated a decrease in peripheral blood eosinophil numbers following dextran sulfate sodium treatment. These findings demonstrate that CD34 was expressed on colon-infiltrating eosinophils and played a role in eosinophil migration. Further, our findings suggest CD34 is required for efficient eosinophil migration, but not proliferation or expansion, in the development of ulcerative colitis.

The roles of eotaxin and the STAT6 signalling pathway in eosinophil recruitment and host resistance to the nematodes Nippostrongylus brasiliensis and Heligmosomoides bakeri

Expulsion of adult Nippostrongylus brasiliensis worms from the small intestine is profoundly impaired in signal transducer and activator of transcription (STAT)6-deficient mice. IL-5 transgenic (Tg) mice with constitutive eosinophilia show profound early resistance in the skin and/or later pre-lung phase of primary infections with N. brasiliensis. This study was designed to assess the importance of the eosinophil chemokine eotaxin and the STAT6/interleukin (IL)-4/IL-13 signalling pathway in early resistance to N. brasiliensis. Eosinophil recruitment into the skin following injection of N. brasiliensis larvae was reduced in STAT6- or eotaxin-deficient/IL-5 Tg double mutant mice. While ablation of eotaxin did not impair resistance in the pre-lung phase of N. brasiliensis infections in IL-5 Tg mice, elimination of STAT6 caused a modest reduction in resistance in both primary and secondary infections on this genetic background. STAT6 −/−-, IL-13 −/−- and IL-4Rα −/−-deficient single mutant and IL-13 −/−/IL-4Rα −/− double mutant mice were more susceptible than WT mice during the pre-lung phase of secondary N. brasiliensis infections. In contrast, primary or secondary resistance were unaffected at either the pre-lung or gut stages of infection in eotaxin −/− single mutant mice. STAT6 −/− and eotaxin −/− mice with or without the IL-5 transgene, were no more susceptible than WT or IL-5 Tg mice to protracted primary infections with Heligmosomoides bakeri, a parasitic nematode that is restricted to the gut. Our data suggest that parasitic nematodes that transit through the skin and lungs en route to the gut may be susceptible to early (pre-lung) innate and adaptive immune mechanisms that are dependent on the STAT6/IL-4/IL-13 signalling pathway, and this may be important for the development of effective therapies and vaccines.

Characterization of An NKp46+ NKT Subset Which Is Susceptible to Malignant Transformation in Vivo.

NKp46 is likely the most specific marker for natural killer (NK) cells across species. NKp46 is also expressed on a minute fraction of NKT-like, but not CD1d-restricted, NKT cells. In our interleukin (IL)-15 transgenic (tg) leukemia C57BL/6 and FvB/NJ mouse models, we found that virtually all (90~98%, n = 5) NKT acute lymphoblastic leukemia (ALL) cells express NKp46. We therefore hypothesized that the NKp46(+) NKT subset, accounting for far less than 1% of lymphocytes in both mice and humans, is more susceptible to malignant transformation in an IL-15, proinflammatory environment when compared to the much more abundant NKp46(−) NKT subset. Consistent with this hypothesis, we observed that NKT leukemic incidence varies directly with percentage of NKp46(+) NKT cells in two murine strains: mice on the FvB/NJ background have 37.3 ± 8.9% (n = 3) NKp46(+) NKT cells and 30% NKT ALL, while C57/B6 mice have <2% NKp46(+) NKT cells and <1% NKT ALL. Functional and phenotypic characterization of murine NKp46(+) NKT cells indicated that:NKp46(+) NKT cells have higher in vivo proliferative capacity than NKp46(−) NKT cells in wild-type mice [19.7 ± 2.6% vs. 13.2 ± 1.8% BrdU(+) cells, p < 0.05, n = 5, by BrdU incorporation assay]; accordingly, when compared to total NKT from WT mice, the proportion of NKp46(+) NKT in polyclonal IL-15 tg mice increased 68%, while NKp46(−) NKT decreased 21% (p < 0.05, n = 3 for both subsets).Compared to NKp46(−) NKT cells, NKp46(+) NKT cells show higher expression of markers associated with maturation (e.g. Ly49D/G2) and activation (e.g. CD122, CD69) andproduce more IFN-γ after co-stimulation with IL-12 and 18 in vitro [37.2 ± 1.8 % vs. 21.0 ± 5.3 IFN-γ(+) cells, n = 3, p < 0.01, by intracellular flow analysis], and are more cytotoxic [39.1 ± 0.9 % vs. 23.5 ± 1.5 %, CD107a(+) cells, n = 3, p < 0.01, by degranulation assay].Similar studies of NKT in human peripheral blood demonstrated the scarcity of NKp46(+) NKT cells (1.3 ± 0.7% of NKT, n = 3), which produced more IFN-γ than NKp46(−) NKT cells after co-stimulation with IL-12 and IL-18 [21.1 ± 4.8% vs. 10.7 ± 3.8% IFN-γ(+) cells, n = 3, p < 0.05, by intracellular flow analysis], suggesting similar characteristics of human and murine NKp46(+) NKT cells. Finally, microarray data analysis indicated that blasts from 38 CD3(+) large granular lymphocyte (LGL) leukemia patients showed significantly higher NKp46 expression than that seen in either normal peripheral blood mononuclear cells or CD8 T cells, irrespective of whether the cells were resting or activated (p < 0.01 for all comparisons). In summary, we believe that NKp46(+) NKT cells, a minor NKT subset in mice and humans, are more mature, more activated, more functionally potent, more proliferative, and more responsive to IL-15 in vivo. At least some of these attributes may contribute to an increased susceptibility of the NKp46(+) subset to malignant transformation when IL-15 is over-expressed in vivo.

Toxocara canis larval excretory/secretory proteins impair eosinophil‐dependent resistance of mice to Nippostrongylus brasiliensis

Survival of parasitic helminths within a host requires immune evasion and excretory/secretory (ES) proteins may contribute to this process. Eosinophils are important effector cells in immunity of mice to the nematode Nippostrongylus brasiliensis and eosinophilic interleukin-5 transgenic (IL-5 Tg) mice are highly resistant to the earliest stages of primary infections. In contrast, Toxocara canis is largely resistant to eosinophils, with viable larvae encysted in tissues often surrounded by these and other leucocytes. The aim of this study was to investigate whether T. canis ES (TES) proteins inhibit eosinophil-dependent resistance to N. brasiliensis. Mouse serum pre-treated with TES had reduced capacity to mediate the adherence of leucocytes to N. brasiliensis infective-stage larvae (L3) and this correlated with reduced complement C3 deposition on the parasite. TES did not inhibit eosinophil survival or eotaxin-dependent eosinophil migration in vitro. Cellular inflammation and eosinophil degranulation in the skin in response to injection of L3 was also not impaired by TES. However, when TES was included with L3 in an inoculum given to IL-5 Tg mice, a greatly increased number of parasites migrated to the lung. This suggests that the early eosinophil-dependent resistance in these mice was suppressed, by mechanisms yet to be determined.

A regulatory role of interleukin 15 in wound healing and mucosal infection in mice

IL-15 plays a critical role in the development and maturation of gammadelta intraepithelial T lymphocytes (IEL), which are known to play important roles in wound healing and resolving inflammation in mice. In this study, we found that IL-15 transgenic (Tg) mice, under the control of a MHC Class I promoter, exhibited accelerated wound healing but were highly susceptible to genital infection with HSV-2. The IEL in the skin and reproductive organs of IL-15 Tg mice produced an aberrantly higher level of TGF-beta1 upon TCR triggering than in control mice. In vivo neutralization of TGF-beta ameliorated the susceptibility of IL-15 Tg mice to genital HSV-2 infection. Taken together, overexpression of IL-15 may stimulate IEL to produce TGF-beta1, promoting wound healing but impeding protection against genital HSV-2 infection.

IL‐15 exacerbates collagen‐induced arthritis with an enhanced CD4+ T cell response to produce IL‐17

IL-15 is thought to be involved in the pathogenesis of rheumatoid arthritis (RA). We found that IL-15 plays an important role in the development of murine collagen-induced arthritis (CIA). The incidence and severity of CIA were slightly decreased in IL-15 KO mice but were increased in IL-15 Tg mice compared with wild-type (WT) mice. The levels of type II collagen (CII)-specific IL-17 production were significantly increased in IL-15 Tg mice compared with WT mice with CIA. Expression of IL-23R was up-regulated in CD4(+) T cells in IL-15 Tg mice but down-regulated in IL-15 KO mice compared with WT mice. In correlation with the expression levels of IL-23R, IL-17 production by CD4(+) T cells in response to exogenous IL-23 was increased in IL-15 Tg mice compared with WT mice. Furthermore, exogenous IL-15 synergized with IL-23 to induce CII-specific IL-17 production by CD4(+) T cells in vitro. Taken together, these results indicate that IL-15 plays an important role in the progression of CIA through increasing antigen-specific IL-17 production by CD4(+) T cells.

Strain-dependent resistance to allergen-induced lung pathophysiology in mice correlates with rate of apoptosis of lung-derived eosinophils

Although exposed to similar allergic and environmental stimuli, not all humans develop asthma. Similarly, mouse strains vary in the degree of pathophysiology seen following induction of experimental asthma. Three mouse strains (CBA/Ca, BALB/c, and C57BL/6) were used to determine if the extent and duration of inflammation influenced the degree of lung tissue damage in an OVA-induced allergic asthma model. Airways obstruction, leukocyte infiltration, edema, eosinophil accumulation, and degranulation were less severe in wild-type (wt) CBA/Ca mice than wt BALB/c and C57BL/6 mice. F1 hybrids of CBA/Ca mice crossed with BALB/c or C57BL/6 mice had bronchoalveolar lavage leukocyte (BAL) and cell-free protein profiles similar to those of the respective disease-susceptible parental strain. IL-5 transgene expression on each of the three genetic backgrounds accentuated the difference between CBA/Ca and the other two strains. Importantly, even when overexpressing IL-5, CBA/Ca mice did not develop substantial airways obstruction. Eosinophils recovered from the airways of allergic wt and IL-5 transgenic (Tg) CBA/Ca mice entered apoptosis at a faster rate than eosinophils from the other parental strains and F1 hybrids. In contrast, eosinophils harvested from the peritoneal cavities of untreated CBA/Ca IL-5 Tg mice had a relatively low rate of apoptosis in vitro. The CBA/Ca mouse strain is therefore relatively resistant to experimental asthma, and this may be a consequence of a propensity for apoptosis of eosinophils recruited into the allergic lung. Restricting survival of a key effector cell may thus limit pathogenesis in this experimental model and in humans.

IL-4 Suppresses UVB-induced Apoptosis in Skin

In this study, cutaneous role of IL-4 in UVB-induced apoptosis was investigated using transgenic mice with skin-specific expression of IL-4 (IL-4 Tg mice). The transgenic mice did not show any gross clinical abnormalities. However, epidermis was thickened and increased MHC class II positive cells were detected as well as enhanced expression of inflammatory cytokines such as IL-1 and TNF-alpha in skin. In addition, histological analysis revealed increased infiltration of lymphocytes, acanthosis, hyperkeratosis, and parakeratosis in skin of IL-4 Tg mice. The physiological effect of IL-4 overexpression in skin against environmental stimulus such as UVB was investigated by irradiating wild-type and IL-4 Tg mice with UVB followed by evaluation of apoptosis. The result demonstrated suppressed apoptosis in epidermis of IL-4 Tg mice compared with wild-type mice. To further assess anti-apoptotic function of IL-4 in keratinocytes, stable cell clones were made where IL-4 was constitutively overexpressed and examined for UVB-induced apoptosis. The results showed that apoptosis was remarkably decreased in IL-4 over-expressing cell clones compared with that in mock transfected cells. Collectively, data presented here shows that IL-4 has an inhibitory effect against UVB-induced apoptosis in keratinocytes, suggesting that IL-4 may be an important regulator in cutaneous immunity against UVB.

Commentary 1

Commentary 1

CCL27 is a critical factor for the development of atopic dermatitis in the keratin-14 IL-4 transgenic mouse model

The keratin-14 IL-4 transgenic (Tg) mouse model of atopic dermatitis (AD) is characterized by skin infiltration of T cells, early up-regulation of T(h)2 cytokines and late surge of T(h)1 cytokines. In the present study, we investigated the role of CCL27, a T cell skin-homing chemokine known to be elevated in sera of human AD patients, in disease development in our animal model of AD. The results showed that the mRNA and protein levels of CCL27 in the skin and serum were significantly increased in IL-4 Tg mice. The percentage of T cells expressing CCR10 in skin draining lymph nodes of IL-4 Tg mice was increased, consistent with the findings of >80% of skin-infiltrating T cells in Tg mice expressing CCR10. Chemotaxis transmigration assay demonstrated that CCL27 promotes a greater degree of migration of T cells in diseased Tg mice. Subcutaneous injection of neutralizing anti-CCL27 to IL-4 Tg mice with early skin lesions resulted in reduced clinical progression of inflammation, accompanied with decreased T cell and mast cell infiltration in the skin, and down-regulation of inflammatory cytokines. In conclusion, CCL27 and CCR10 interaction is important for the development of skin inflammation in our AD model.