Edwin J. W. Geven

Central and peripheral integration of interrenal and thyroid axes signals in common carp (Cyprinus carpio L.).

In teleostean fishes the hypothalamic-pituitary-thyroid axis (HPT axis) and the hypothalamic-pituitary-interrenal axis (HPI axis) regulate the release of thyroid hormones (THs) and cortisol respectively. Since many actions of both hormones are involved in the regulation of metabolic processes, communication between both signal pathways can be anticipated. In this study, we describe central and peripheral sites for direct interaction between mediators of both neuroendocrine axes in the common carp (Cyprinus carpio). Despite suggestions in the literature that CRH is thyrotropic in some fish; we were not able to establish stimulatory effects of CRH on the expression of the pituitary TSHbeta subunit gene. In preoptic area tissue incubated with 10(-7) M thyroxine (T(4)) a 2 x 9-fold increase in the expression of CRH-binding protein (CRHBP) was observed. Thus, T(4) could reduce the bioavailable hypothalamic crh via the up regulation of crhbp expression and hence down regulate the HPI axis. At the peripheral level, cortisol (10(-6) M), ACTH (10(-7) M), and alpha-MSH (10(-7) M) stimulate the release of T(4) from kidney and head kidney fragments, which contain all functional thyroid follicles in carp, by two- to fourfold. The substantiation of three pituitary thyrotropic factors, viz. TSH, ACTH, and alpha-MSH, in common carp, allows for an integration of central thyrotropic signals. Clearly, two sites for interaction between the HPT axis, the HPI axis, and alpha-MSH are present in common carp. These interactions may be key to the proper regulation of general metabolism in this fish.

The teleost head kidney: Integrating thyroid and immune signalling

ABSTRACT The head kidney, analogous to the mammalian adrenal gland, is an organ unique for teleost fish. It comprises cytokine‐producing lymphoid cells from the immune system and endocrine cells secreting cortisol, catecholamines, and thyroid hormones. The intimate organization of the immune system and endocrine system in one single organ makes bidirectional signalling between these possible. In this review we explore putative interactions between the thyroid and immune system in the head kidney. We give a short overview of the thyroid system, and consider the evidence for the presence of thyroid follicles in the head kidney as a normal, healthy trait in fishes. From mammalian studies we gather data on the effects of three important pro‐inflammatory cytokines (TNF&agr;, IL‐1&bgr;, IL‐6) on the thyroid system. A general picture that emerges is that pro‐inflammatory cytokines inhibit the activity of the thyroid system at different targets. Extrapolating from these studies, we suggest that the interaction of the thyroid system by paracrine actions of cytokines in the head kidney is involved in fine‐tuning the availability and redistribution of energy substrates during acclimation processes such as an immune response or stress response. HighlightsThe intimate organization of the immune system and endocrine system in the teleost head kidney makes bidirectional signalling between these possible.The presence of thyroid follicles in the head kidney as a normal, healthy trait in fishes.In mammals, the pro‐inflammatory cytokines TNF&agr;, IL‐1&bgr;, and IL‐6 have, in general, an inhibitory effect on the thyroid system.The interaction of the thyroid system by paracrine actions of cytokines in the head kidney is likely to be involved in fine‐tuning the availability of energy substrates during an immune or stress response.

The Involvement of Thyroid Hormone Metabolism in Gilthead Sea Bream (Sparus auratus) Osmoregulation

Abstract: We have investigated the effect of adaptation to low salinity water on the thyroid status of the euryhaline teleost, Sparus auratus. We show that, following low salinity adaptation, the plasma T4 concentration increases and branchial deiodination activities of T4, T3, and rT3 decrease. Moreover, branchial and hepatic enzyme activities that are putatively involved in thyroid hormone metabolism respond differentially in low salinity conditions. Our results indicate the involvement of thyroid hormones in Sparus auratus osmoregulation. Moreover, the gills appear well equipped to play an important role in the modulation of plasma thyroid hormone titers.

Therapy response monitoring of the early effects of a new BRAF inhibitor on melanoma xenograft in mice: evaluation of (18) F-FDG-PET and (18) F-FLT-PET.

Inhibition of the V600E mutated BRAF kinase gene (BRAF(V600E) ) is an important and effective approach to treating melanomas. A new specific small molecule inhibitor of BRAF(V600E) , PLX3603, showed potent melanoma growth-inhibiting characteristics in preclinical studies and is currently under clinical investigation. In this study we investigated the feasibility of (18) F-FDG and (18) F-FLT-PET to monitor the early effects of the BRAF(V600E) inhibitor in mice with melanoma xenografts. SCID/beige mice with subcutaneous (s.c.) A375 melanoma xenografts, expressing BRAF(V600E) , received the BRAF(V600E) inhibitor twice daily orally (0, 25, 50 and 75 mg/kg). At 1, 3 and 7 days after start of therapy, the uptake of (18) F-FDG and (18) F-FLT in the tumor and normal tissues was determined in ex vivo tissue samples. Serial (18) F-FDG and (18) F-FLT-PET scans were acquired of animals at 1 day before and 1, 3 and 7 days after start of treatment with 75 mg/kg BRAF(V600E) inhibitor. A dose-dependent decrease in (18) F-FDG uptake in the A375 tumors was observed by ex vivo biodistribution analysis. Administration of 75 mg/kg BRAF inhibitor for 1, 3 and 7 days resulted in a significantly decreased (18) F-FDG uptake in A375 tumors (41, 35 and 51%, respectively). (18) F-FLT uptake in the A375 tumors was low at baseline and no significant changes in (18) F-FLT uptake were observed at any of the doses administered. These effects were corroborated by serial in vivo (18) F-FDG and (18) F-FLT-PET imaging. These data demonstrate that (18) F-FDG-PET can be used as an imaging biomarker to noninvasively evaluate the early effects of PLX3603.

S100A8/A9, a potent serum and molecular imaging biomarker for synovial inflammation and joint destruction in seronegative experimental arthritis

BackgroundSeronegative joint diseases are characterized by a lack of well-defined biomarkers since autoantibodies are not elevated. Calprotectin (S100A8/A9) is a damage-associated molecular pattern (DAMP) which is released by activated phagocytes, and high levels are found in seronegative arthritides. In this study, we investigated the biomarker potential of systemic and local levels of these S100 proteins to assess joint inflammation and joint destruction in an experimental model for seronegative arthritis.MethodsSerum levels of S100A8/A9 and various cytokines were monitored during disease development in interleukin-1 receptor antagonist (IL-1Ra)–/– mice using ELISA and multiplex bead-based immunoassay, and were correlated to macroscopic and microscopic parameters for joint inflammation, bone erosion, and cartilage damage. Local expression of S100A8 and S100A9 and matrix metalloproteinase (MMP)-mediated cartilage damage in the ankle joints were investigated by immunohistochemistry. In addition, local S100A8 and activated MMPs were monitored in vivo by optical imaging using anti-S100A8-Cy7 and AF489-Cy5.5, a specific tracer for activated MMPs.ResultsSerum levels of S100A8/A9 were significantly increased in IL-1Ra–/– mice and correlated with macroscopic joint swelling and histological inflammation, while serum levels of pro-inflammatory cytokines did not correlate with joint swelling. In addition, early serum S100A8/A9 levels were prognostic for disease outcome at a later stage. The increased serum S100A8/A9 levels were reflected by an increased expression of S100A8 and S100A9 within the ankle joint, as visualized by molecular imaging. Next to inflammatory processes, serum S100A8/A9 also correlated with histological parameters for bone erosion and cartilage damage. In addition, arthritic IL-1Ra–/– mice with increased synovial S100A8 and S100A9 expression showed increased cartilage damage that coincided with MMP-mediated neoepitope expression and in vivo imaging of activated MMPs.ConclusionsExpression of S100A8 and S100A9 in IL-1Ra–/– mice strongly correlates with synovial inflammation, bone erosion, and cartilage damage, underlining the potential of S100A8/A9 as a systemic and local biomarker in seronegative arthritis not only for assessing inflammation but also for assessing severity of inflammatory joint destruction.

S100A8/A9 increases the mobilization of pro-inflammatory Ly6Chigh monocytes to the synovium during experimental osteoarthritis

BackgroundMonocytes are dominant cells present within the inflamed synovium during osteoarthritis (OA). In mice, two functionally distinct monocyte subsets are described: pro-inflammatory Ly6Chigh and patrolling Ly6Clow monocytes. Alarmins S100A8/A9 locally released by the synovium during inflammatory OA for prolonged periods may be dominant proteins involved in stimulating recruitment of Ly6Chigh monocytes from the circulation to the joint. Our objective was to investigate the role of S100A8/A9 in the mobilization of Ly6Chigh and Ly6Clow monocytic populations to the inflamed joint in collagenase-induced OA (CiOA).MethodS100A8 was injected intra-articularly to investigate monocyte influx. CiOA was induced by injection of collagenase into knee joints of wild-type C57BL/6 (WT), and S100a9-/- mice. Mice were sacrificed together with age-matched saline-injected control mice (n = 6/group), and expression of monocyte markers, pro-inflammatory cytokines, and chemokines was determined in the synovium using ELISA and RT-qPCR. Cells were isolated from the bone marrow (BM), spleen, blood, and synovium and monocytes were identified using FACS.ResultsS100A8/A9 was highly expressed during CiOA. Intra-articular injection of S100A8 leads to elevated expression of monocyte markers and the monocyte-attracting chemokines CCL2 and CX3CL1 in the synovium. At day 7 (d7) after CiOA induction in WT mice, numbers of Ly6Chigh, but not Ly6Clow monocytes, were strongly increased (7.6-fold) in the synovium compared to saline-injected controls. This coincided with strong upregulation of CCL2, which preferentially attracts Ly6Chigh monocytes. In contrast, S100a9-/- mice showed a significant increase in Ly6Clow monocytes (twofold) within the synovium at CiOA d7, whereas the number of Ly6Chigh monocytes remained unaffected. In agreement with this finding, the Ly6Clow mobilization marker CX3CL1 was significantly higher within the synovium of S100a9-/- mice. Next, we studied the effect of S100A8/A9 on release of Ly6Chigh monocytes from the BM into the circulation. A 14% decrease in myeloid cells was found in WT BM at CiOA d7. No decrease in myeloid cells in S100a9-/- BM was found, suggesting that S100A8/A9 promotes the release of myeloid populations from the BM.ConclusionInduction of OA locally leads to strongly elevated S100A8/A9 expression and an elevated influx of Ly6Chigh monocytes from the BM to the synovium.

Abstract 486: Tumor-targeted, engineered IL-2 variant (IL-2v)-based immunocytokines for the immunotherapy of cancer.

IL-2 therapy can lead to durable responses in cancer patients, but is associated with significant toxicity. None of the described IL-2-based immunocytokines has progressed beyond Phase II trials due to various constraints in their design: 1) pM affinity for IL-2Rαβγ on immune cells and pulmonary vascular endothelium compromising tumor targeting due to the fusion of two wildtype IL-2 moieties to the antibody, together with FcγR binding on the same cells; 2) Rapid systemic clearance and short half-life due to high affinity IL-2Rαβγ binding; 3) Preferential activation of Tregs over immune effectors by wt IL-2. Here, we describe a novel monomeric tumor-targeted immunocytokine where a single, engineered IL-2 variant (IL-2v) with abolished IL-2Rα (CD25) binding is fused to the C-terminus of an antibody with a heterodimeric Fc-part. FcγR and C1q binding is completely abolished by a novel Fc mutation. For targeting, human(-ized) high affinity antibodies against CEA (GA504, CEA-IL2v) or FAP (GA501, FAP-IL2v) were selected. CEA- and FAP-IL2v were recombinantly produced and induction of P-STAT5, proliferation, activation induced cell death (AICD), activation markers and cytokines were determined on effector cells. Safety, pharmacokinetics (PK), tumor targeting, pharmacodynamics and anti-tumor efficacy were analyzed in SCID and immunocompetent C57Bl/6 mice. FAP- and CEA-IL-2v completely lack binding to CD25, but retain IL-Rβγ binding. They do not bind to CD25 or preferentially activate Tregs, and induce lower degree of AICD. However, IL-2Rβγ bioactivity is retained and they activate NK, CD4+ and CD8+ T cells as shown by induction of activation markers and proliferation. In particular, CEA- and FAP-IL2v expand and activate NK cells and skew the CD4:CD8 ratio towards CD8+ T cells in vivo. In C57Bl/6 mice, CEA- and FAP-IL2v demonstrate improved safety despite of higher exposure and circulatory half-life than the corresponding wt IL-2 immunocytokine. MicroSPECT/CT imaging revealed FAP-mediated tumor targeting of FAP-IL2v with low normal tissue uptake with FAP-IL2v tumor targeting being similar to the parental FAP antibody with low accumulation in lymphoid tissues and clearly superior to an FAP-targeted wt IL-2 immunocytokine that shows preferential spleen targeting. Studies in tumor-bearing mice showed dose dependent anti-tumor efficacy of CEA- and FAP-IL2v in established xenograft and syngeneic mouse models. Thus, CEA- and FAP-IL2v demonstrate superior safety, PK and tumor targeting, while lacking preferential induction of Tregs due to abolished CD25 and FcγR binding, monovalency and high-affinity tumor-targeting as compared to classical immunocytokines. They retain capacity to activate NK and T‐effector cells through IL‐2Rβγ; in particular once targeted to the tumor microenvironment. These data support their investigation for the immunotherapy of CEA/FAP-positive tumors. Citation Format: Christian Klein, Inja Waldhauer, Valeria Nicolini, Claire Dunn, Anne Freimoser-Grundschober, Sylvia Herter, Edwin Geven, Otto Boerman, Tapan Nayak, Erwin van Puijenbroek, David Wittig, Samuel Moser, Oliver Ast, Peter Bruenker, Ralf Hosse, Sabine Lang, Sebastian Neumann, Hubert Kettenberger, Adelbert Grossmann, Ingo Gorr, Stefan Evers, Pavel Pisa, Jennifer Fretland, Victor Levitsky, Christian Gerdes, Marina Bacac, Ekkehard Moessner, Pablo Umana. Tumor-targeted, engineered IL-2 variant (IL-2v)-based immunocytokines for the immunotherapy of cancer. [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 486. doi:10.1158/1538-7445.AM2013-486

Abstract PR8: Novel tumor-targeted, engineered IL-2 variant (IL-2v)-based immunocytokines for immunotherapy of cancer.

Introduction: IL-2 therapy can lead to durable responses in a modest proportion of cancer patients, but the treatment is associated with significant toxicity. Over the last decades, various IL-2-based immunocytokines have been generated by fusing IL-2 to tumor-targeting antibodies. However, none of these molecules have progressed beyond Phase II trials and they are hampered by various liabilities: 1) High functional affinity (low pM) for IL-2Rabg on immune cells and on pulmonary vascular endothelium (Krieg et al., PNAS, 2010) compromising preferential tumor targeting due to fusion of two IL-2 moieties to the antibody. This is further compounded when the immunocytokine binds to FcgRs on the same cells. 2) Rapid systemic clearance and short half-life due to high affinity IL-2Rabg binding. 3) Preferential activation of Tregs over immune effectors due to use of wildtype IL-2. Here, we describe a novel monomeric tumor-targeted immunocytokine where a single, engineered IL-2 variant (IL-2v) with abolished IL-2Ra (CD25) binding is fused to the C-terminus of a tumor-specific hIgG1 antibody with a heterodimeric Fc-part. FcgR and C1q binding is completely abolished by a novel Fc mutation. For targeting, human(-ized) high affinity antibodies against CEA (GA504, CEA-IL2v) or FAP (GA501, FAP-IL2v) were chosen. Experimental procedures: CEA- and FAP-IL2v were recombinantly produced and characterized by surface plasmon resonance. Induction of P-STAT5, proliferation, activation induced cell death (AICD), various activation markers and cytokine release were determined on effector cells. Safety, pharmacokinetics (PK), tumor targeting by imaging, immune-pharmacodynamics and anti-tumor efficacy were analyzed in immunocompromised Scid and immunocompetent C57BL/6 mice. Results: IL-2v completely lacks binding to CD25, but retains IL-Rbg binding. In line with this, FAP- and CEA-IL2v do not bind to CD25 or preferentially activate Tregs, and do not cause AICD. However, IL-2Rbg bioactivity is retained and they are still able to activate NK, CD4 and CD8 T cells as shown by concentration dependent increase in activation markers and induction of proliferation. In particular, CEA- and FAP-IL2v expand and activate NK cells and skew the CD4:CD8 ratio towards activated CD8 T cells in vivo. In C57BL/6 mice CEA- and FAP-IL2v demonstrate improved safety despite of ca. 2-fold higher exposure and t1/2 than a wildtype IL-2-based IgG immunocytokine. SPECT/CT imaging revealed FAP-mediated tumor targeting and accumulation of FAP-IL2v with low normal tissue uptake. Notably, FAP-IL2v tumor targeting was similar to the parental FAP antibody with low accumulation in lymphoid tissues; clearly superior to an FAP-targeted wt IL-2 immunocytokine that showed preferential homing to the spleen. Studies in tumor bearing mice showed dose dependent efficacy of CEA- and FAP-IL2v in established xenograft and immunocompetent syngeneic mouse models in terms of survival. Conclusion: CEA- and FAP-IL2v demonstrate superior safety, PK and tumor targeting, while lacking preferential induction of Tregs due to abolished CD25 and FcgR binding, monovalency and high-affinity tumor-targeting as compared to conventional immunocytokines. They retain the capacity to activate NK and T effector cells through IL 2Rbg; in particular once targeted and immobilized in the tumor microenvironment. These preclinical properties support further investigation for the immunotherapy of CEA/FAP-positive tumors. This abstract is also presented as Poster A23. Citation Format: Christian Klein, Waldhauer Inja, Valeria Nicolini, Dunn Claire, Anne Freimoser, Anne Freimoser, Sylvia Herter, Edwin Geven, Otto Boerman, Erwin van Puijenbroek, David Wittig, Samuel Moser, Oliver Ast, Ralf Hosse, Sabine Lang, Sebastian Neumann, Adelbert Grossmann, Ingo Gorr, Stefan Evers, Pavel Pisa, Jennifer Fretland, Victor Levitsky, Christian Gerdes, Marina Bacac, Ekkehard Moessner, Ekkehard Moessner, Pablo Umana. Novel tumor-targeted, engineered IL-2 variant (IL-2v)-based immunocytokines for immunotherapy of cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR8.

02.26 Increased expression of s100a9 regulates pain response during experimentally induced acute synovitis

Background Synovitis-associated pain is an important aspect of arthritis pathology. Inflammatory mediators released by the synovium have been implicated in the regulation of pain, including S100A8 and S100A9, which may act via stimulation of TLR4 on the nerve endings in the synovium. In this study we investigated the role of S100A9 in the pain response after induction of an acute synovitis using streptococcal cell walls (SCW) as a trigger, comparing S100A9-/- mice and their WT controls. Materials and methods Acute synovitis was induced by a single i.a. SCW injection in the knee joint of C57Bl6 (WT) and S100A9-/- mice. Serum S100A8/A9 levels were investigated by ELISA and S100A8 and S100A9 expression in synovium by immunohistochemistry. Joint swelling and cell influx was assessed by 99mTc accumulation and histology, respectively. Pain response was investigated by Incapacitance Tester (weight bearing), Catwalk (gait analysis) and von Frey’s filaments (mechanical allodynia). Gene expression of neuron activation markers in dorsal root ganglia (DRG) were determined by q-PCR. Results A single i.a. SCW injection resulted in increased synovial expression of S100A8 and S100A9 as well as significant increased serum S100A8/A9 levels (2.6-fold, p<0.001) 1 day p.i.. These increased levels however, did not contribute to the development of inflammation since joint swelling and cell influx were similar in both mice strains. WT mice showed a significant decrease in percentage of weight bearing on the SCW hindpaw (28%, p<0.001) 1 day p.i., while S100A9-/- mice showed no reduction. In line with that, gait analysis showed that the stand-phase of the unaffected paws was significantly increased in WT mice 1 day p.i., but not in S100A9-/- mice. No difference in mechanical allodynia was observed, both mouse strains showed a similar reduction of paw withdrawal threshold. Gene expression of neuron activation markers NAV1.7, ATF3 and GAP43 in DRG were significantly increased in SCW injected WT mice 1 day p.i (p=0.022, 0.004 and 0.030, respectively) but not in S100A9-/- mice. Conclusions These findings show that S100A9, which is released from the synovium upon inflammation, is an important mediator of pain response in the knee during the acute phase of inflammation.

03.09 Local application of adipose-derived mesenchymal stromal cells in experimental inflammatory oa results in interleukin-1β-mediated attraction of pmns and reduced s100a8/a9 release

Background Recent studies have shown that mild synovitis in early phases of osteoarthritis (OA) is conducive to development of joint damage. OA synovitis is characterized by elevated levels of pro-inflammatory factors like S100A8, S100A9, interleukin-6 (IL-6), and interleukin-1 beta (IL-1β). S100A8/A9 was found to be crucial in mediating joint destruction in inflammatory experimental OA. Previously we found that adipose-derived mesenchymal stromal cells (ASCs) exhibit immunosuppressive characteristics and reduce joint pathology after local application into mouse knee joints with experimental inflammatory OA. This protective effect is only perceived after intra-articular injection in early but not late stage OA, suggesting that the effect may be mediated by an inflammatory milieu. Objectives To examine the working mechanism of ASCs after early injection in experimental OA. Methods Experimental OA was induced by injection of collagenase into murine knee joints (CiOA). Total knee joints were stained with haematoxylin/eosin and the PMN-specific antibody NIMP-R14. ASCs were isolated from murine adipose tissue and stimulated for 24h with IL-1β or S100A8/A9. Gene expression in stimulated cells was analyzed using qPCR. Protein levels of chemokines and cytokines were measured in culture supernatant using Luminex. Migration of MACS isolated bone marrow (BM-) PMNs towards ASC-conditioned medium (CM) was examined using Transwell inserts. ASCs were co-cultured with BM-PMNs and analyzed using histology and Luminex. Results ASC injection into day 7 CiOA knee joints (when synovitis and IL-1β and S100A8/A9 levels are highest) caused a strong attraction of mainly PMN-like cells and their clustering around ASCs in the synovium shortly after injection (6h), which was confirmed by immunohistochemistry. IL-1β stimulation of ASCs in vitro strongly increased gene expression of PMN-attracting chemokines KC, CXCL5, and CXCL7 as well as protein levels of KC, whereas S100A8/A9 did not. The migration of BM-PMNs through Transwell inserts towards CM of IL-1β-stimulated ASCs was significantly increased (from 5% to 10%) when compared to CM of non-stimulated ASCs. Next, ASCs were co-cultured with BM-PMNs in the presence or absence of IL-1β. After 6h, a clear clustering of neutrophils around ASCs was observed, with a significant increase in the number of ASCs clustering with PMNs, as well as a significantly elevated number of clustering PMNs per ASC after IL-1β stimulation. Interestingly, association of PMNs with ASCs lead to a significantly lowered release of KC protein by ASCs (69% and 76% lower after 24h and 48h respectively), as well as a significantly reduced release of S100A8/A9 protein by the PMNs. This coincided with lowering of S100A8/A9 levels in washouts of inflamed synovium 6h and 48h after injection of ASCs in day 7 CiOA knee joints. Conclusions Local application of ASCs in inflamed CiOA knee joints results in attraction and clustering of PMNs with ASCs in the synovium. This presumably runs via IL-1β-mediated up-regulation of chemokine release by ASCs, and ultimately results in significantly lowered S100A8/A9 levels. Acknowledgements This research was supported by the Dutch Arthritis Association. Disclosure of Interest None declared