Role of Clever-1 in Leukocyte Trafficking and Inflammation

Secondary lymphoid organs, including lymph nodes (LNs) and the spleen, play an important role in protecting us against invading pathogens. While LNs are connected to lymphatic vessels and receive antigens from the peripheral tissues, the spleen is connected to blood vessels and filters blood-borne antigens. Migration of antigen presenting cells, such as dendritic cells (DCs), and lymphocytes into secondary lymphoid organs is crucial for initiating a proper adaptive immune response. Common lymphatic endothelial and vascular endothelial receptor-1 (Clever-1) is a multifunctional scavenger and adhesion receptor expressed on a subset of macrophages as well as endothelial cells and has been shown to selectively regulate leukocyte migration into different tissues. The role of Clever-1 in lymphocyte and dendritic cell migration into secondary lymphoid organs has not been thoroughly investigated. My doctoral studies describe new roles for Clever-1 in leukocyte trafficking and immune response. In this thesis work, we showed that lymphocyte entry into the spleen is not a passive process forced by blood flow, but rather an active process regulated by Clever-1. We also discovered that lymphocytes preferentially enter the spleen via the vessels in the red pulp (RP) rather than marginal zone (MZ) sinuses or the white pulp (WP) vessels. These findings change the long-lasting dogma since 1973, which stated that lymphocytes enter the spleen via the MZ sinuses. We also identified Clever-1 as the first molecule that regulates lymphocyte migration into the spleen via the vasculature in the red pulp. In addition, we discovered that Clever-1 is expressed on peripheral lymphatic vessels and its absence reduces DC transmigration. Moreover, Clever-1, expressed on lymphatic endothelial cells (LECs), down-modulates DC activation and subsequently regulates the magnitude of antigen-specific T-cell responses in lymph nodes. In summary, this thesis work identified Clever-1 as a unique molecule that controls the migration of lymphocytes and DCs into secondary lymphoid organs, along with its crucial role in regulating antigen-specific immune responses.Toissijaiset imukudokset mukaan lukien imusolmukkeet ja perna suojelevat meitä taudinaiheuttajia vastaan. Imusolmukkeet ovat suoraan kytköksissä imusuoniin ja vastaanottavat antigeenejä ympäröivistä kudoksista. Perna puolestaan on yhteydessä verisuoniin ja suodattaa veren kuljettamia antigeenejä. Antigeenejä esittelevien solujen kuten dendriittisolujen (DC) ja lymfosyyttien migraatio toissijaisiin imukudoksiin on elintärkeää toimivan hankitun puolustusvasteen käynnistämiseksi. Clever-1 on monitoiminnallinen reseptori, joka sitoo ja poistaa elimistön eitoivottuja molekyylejä ja jota ilmennetään endoteelisoluissa. Clever-1:n on osoitettu valikoidusti säätelevän leukosyyttien migraatiota eri kudoksiin. Kuitenkin Clever1:n toimintaa lymfosyyttien ja dendriittisolujen migraatiossa ei ole riittävästi tutkittu. Väitöskirjatyössäni kuvaan Clever-1:n ennen tuntematonta toimintaa leukosyyttien liikehdinnässä ja puolustusvasteessa. Tässä väitöstyössä osoitin, että lymfosyytit eivät päädy pernaan vapaasti veren virtauksen voimalla vaan Clever-1 säätelee lymfosyyttien pääsyä pernaan. Havaitsin myös, että lymfosyytit kulkevat ensisijaisesti pernaan punaisen ytimen verisuonten kautta eivätkä valkoisen ytimen verisuonten tai marginaalialueen onteloiden kautta. Nämä löydökset kumoavat vuodelta 1973 lähtöisin olevan vallitsevan teorian, jonka mukaan lymfosyytit kulkevat pernaan marginaalialueen onteloiden kautta. Tutkimustulosteni perusteella Clever-1 on ensimmäinen tunnistettu molekyyli, joka säätelee lymfosyyttien siirtymistä pernaan. Tämän lisäksi havaitsin, että Clever-1:tä ilmennetään perifeerisissä imusuonissa, joissa se ohjaa dendriittisolujen transmigraatiota kudoksista endoteelisolujen läpi imusuonien onteloon. Ilmentyneenä endoteelisoluissa Clever-1 vähentää dendriittisolujen aktivaatiota ja sitä kautta säätelee T-soluriippuvaisten antigeenivasteiden suuruutta. Kokonaisuutena väitöskirjatyöni osoittaa Clever-1:n ainutlaatuisuuden lymfosyyttien ja dendriittisolujen muuttoliikkeen/migraation sekä antigeenispefisisten puolustusvasteiden säätelijänä

Iliac vein stenting in a patient with lower extremity swelling resulting from diffuse pelvic mass: A case report

We report a 66-year-old male patient with severe right lower extremity swelling resulting from diffuse pelvic mass with compression on right external iliac vein. The patient had papillary urothelial carcinoma of bladder seven years ago and radical cystectomy and ureterostomy was performed. Recurrence of malignancy had occurred five years after the operation. The patient had also bilateral diffuse lung metastasis. The external iliac vein had severe stenosis and invasion of pelvic mass into the vein was evident on venography. Venoplasty of external iliac vein was performed throughout the stenosis. A venous stent of 80 mm length and 12 mm diameter was introduced over the guidewire and deployed in the external iliac vein. Dramatic clinical response was evident since postoperative day two. Swelling of right lower extremity was resolved dramatically on three-month and six-month follow-up visits. We believe that endovascular venous recanalization of iliac veins is feasible and safe in patients with unresectable and diffuse pelvic masses

Melanocortin 1 Receptor Deficiency in Hematopoietic Cells Promotes the Expansion of Inflammatory Leukocytes in Atherosclerotic Mice

Melanocortin receptor 1 (MC1-R) is expressed in leukocytes, where it mediates anti-inflammatory actions. We have previously observed that global deficiency of MC1-R signaling perturbs cholesterol homeostasis, increases arterial leukocyte accumulation and accelerates atherosclerosis in apolipoprotein E knockout (Apoe-/-) mice. Since various cell types besides leukocytes express MC1-R, we aimed at investigating the specific contribution of leukocyte MC1-R to the development of atherosclerosis. For this purpose, male Apoe-/- mice were irradiated, received bone marrow from either female Apoe-/- mice or MC1-R deficient Apoe-/- mice (Apoe-/- Mc1re/e) and were analyzed for tissue leukocyte profiles and atherosclerotic plaque phenotype. Hematopoietic MC1-R deficiency significantly elevated total leukocyte counts in the blood, bone marrow and spleen, an effect that was amplified by feeding mice a cholesterol-rich diet. The increased leukocyte counts were largely attributable to expanded lymphocyte populations, particularly CD4+ T cells. Furthermore, the number of monocytes was elevated in Apoe-/- Mc1re/e chimeric mice and it paralleled an increase in hematopoietic stem cell count in the bone marrow. Despite robust leukocytosis, atherosclerotic plaque size and composition as well as arterial leukocyte counts were unaffected by MC1-R deficiency. To address this discrepancy, we performed an in vivo homing assay and found that MC1-R deficient CD4+ T cells and monocytes were preferentially entering the spleen rather than homing in peri-aortic lymph nodes. This was mechanistically associated with compromised chemokine receptor 5 (CCR5)-dependent migration of CD4+ T cells and a defect in the recycling capacity of CCR5. Finally, our data demonstrate for the first time that CD4+ T cells also express MC1-R. In conclusion, MC1-R regulates hematopoietic stem cell proliferation and tissue leukocyte counts but its deficiency in leukocytes impairs cell migration via a CCR5-dependent mechanism.</p

CD73 regulates zoledronate-induced lymphocyte infiltration in triple-negative breast cancer tumors and lung metastases

IntroductionBisphosphonates (BPs) are bone-protecting osteoclast inhibitors, typically used in the treatment of osteoporosis and skeletal complications of malignancies. When given in the adjuvant setting, these drugs may also prevent relapses and prolong overall survival in early breast cancer (EBC), specifically among postmenopausal patients. Because of these findings, adjuvant nitrogen-containing BPs (N-BPs), such as zoledronate (ZOL), are now the standard of care for high-risk EBC patients, but there are no benefit-associated biomarkers, and the efficacy remains low. BPs have been demonstrated to possess anti-tumor activities, but the mechanisms by which they provide the beneficial effects in EBC are not known. MethodsWe used stably transfected 4T1 breast cancer cells together with suppression of CD73 (sh-CD73) or control cells (sh-NT). We compared ZOL effects on tumor growth and infiltrating lymphocytes (TILs) into tumors and lung metastases using two mouse models. B cell depletion was performed using anti-CD20 antibody.ResultsSh-CD73 4T1 cells were significantly more sensitive to the growth inhibitory effects of n-BPs in vitro. However, while ZOL-induced growth inhibition was similar between the tumor groups in vivo, ZOL enhanced B and T lymphocyte infiltration into the orthotopic tumors with down-regulated CD73. A similar trend was detected in lung metastases. ZOL-induced tumor growth inhibition was found to be augmented with B cell depletion in sh-NT tumors, but not in sh-CD73 tumors. As an internal control, ZOL effects on bone were similar in mice bearing both tumor groups.DiscussionTaken together, these results indicate that ZOL modifies TILs in breast cancer, both in primary tumors and metastases. Our results further demonstrate that B cells may counteract the growth inhibitory effects of ZOL. However, all ZOL-induced TIL effects may be influenced by immunomodulatory characteristics of the tumor

Lymphatic Endothelial Cell Activation and Dendritic Cell Transmigration Is Modified by Genetic Deletion of Clever-1

Clever-1 also known as Stabilin-1 and FEEL-1 is a scavenger molecule expressed on a subpopulation of anti-inflammatory macrophages and lymphatic endothelial cells (LECs). However, its role in regulating dendritic cell (DC) trafficking and subsequent effects on immunity have remained unexplored. In this study, we demonstrate that DC trafficking from the skin into the draining lymph nodes is compromised in the absence of Clever-1. By adoptive transfer approaches we further show that the poor trafficking is due to the impaired entrance of DCs into afferent lymphatics. Despite this, injections of ovalbumin-loaded DCs into the footpads induced a stronger proliferative response of OT II T cells in the draining lymph nodes. This could be explained by the increased MHC II expression on DCs and a less tolerogenic phenotype of LECs in lymph nodes of Clever-1 knockout mice. Thus, although fewer DCs reach the nodes, they are more active in creating antigen-specific immune responses. This suggests that the DCs migrating to the draining lymph node within Clever-1 positive lymphatics experience immunosuppressive interactions with LECs. In conclusion, besides being a trafficking molecule on lymphatic vasculature Clever-1 is immunosuppressive towards migrating DCs and thus, regulates the magnitude of immune responses created by incoming DCs in the draining lymph nodes

CD73 regulates zoledronate-induced lymphocyte infiltration in triple-negative breast cancer tumors and lung metastases

Introduction: Bisphosphonates (BPs) are bone-protecting osteoclast inhibitors, typically used in the treatment of osteoporosis and skeletal complications of malignancies. When given in the adjuvant setting, these drugs may also prevent relapses and prolong overall survival in early breast cancer (EBC), specifically among postmenopausal patients. Because of these findings, adjuvant nitrogen-containing BPs (N-BPs), such as zoledronate (ZOL), are now the standard of care for high-risk EBC patients, but there are no benefit-associated biomarkers, and the efficacy remains low. BPs have been demonstrated to possess anti-tumor activities, but the mechanisms by which they provide the beneficial effects in EBC are not known. Methods: We used stably transfected 4T1 breast cancer cells together with suppression of CD73 (sh-CD73) or control cells (sh-NT). We compared ZOL effects on tumor growth and infiltrating lymphocytes (TILs) into tumors and lung metastases using two mouse models. B cell depletion was performed using anti-CD20 antibody. Results: Sh-CD73 4T1 cells were significantly more sensitive to the growth inhibitory effects of n-BPs in vitro. However, while ZOL-induced growth inhibition was similar between the tumor groups in vivo, ZOL enhanced B and T lymphocyte infiltration into the orthotopic tumors with down-regulated CD73. A similar trend was detected in lung metastases. ZOL-induced tumor growth inhibition was found to be augmented with B cell depletion in sh-NT tumors, but not in sh-CD73 tumors. As an internal control, ZOL effects on bone were similar in mice bearing both tumor groups. Discussion: Taken together, these results indicate that ZOL modifies TILs in breast cancer, both in primary tumors and metastases. Our results further demonstrate that B cells may counteract the growth inhibitory effects of ZOL. However, all ZOL-induced TIL effects may be influenced by immunomodulatory characteristics of the tumor.Peer reviewe

Vascular adhesion protein-1 defines a unique subpopulation of human hematopoietic stem cells and regulates their proliferation

Although the development of hematopoietic stem cells (HSC) has been studied in great detail, their heterogeneity and relationships to different cell lineages remain incompletely understood. Moreover, the role of Vascular Adhesion Protein-1 in bone marrow hematopoiesis has remained unknown. Here we show that VAP-1, an adhesin and a primary amine oxidase producing hydrogen peroxide, is expressed on a subset of human HSC and bone marrow vasculature forming a hematogenic niche. Bulk and single-cell RNAseq analyses reveal that VAP-1+ HSC represent a transcriptionally unique small subset of differentiated and proliferating HSC, while VAP-1− HSC are the most primitive HSC. VAP-1 generated hydrogen peroxide acts via the p53 signaling pathway to regulate HSC proliferation. HSC expansion and differentiation into colony-forming units are enhanced by inhibition of VAP-1. Contribution of VAP-1 to HSC proliferation was confirmed with mice deficient of VAP-1, mice expressing mutated VAP-1 and using an enzyme inhibitor. In conclusion, VAP-1 expression allows the characterization and prospective isolation of a new subset of human HSC. Since VAP-1 serves as a check point-like inhibitor in HSC differentiation, the use of VAP-1 inhibitors enables the expansion of HSC.Peer reviewe

DataSheet_1_CD73 regulates zoledronate-induced lymphocyte infiltration in triple-negative breast cancer tumors and lung metastases.docx

IntroductionBisphosphonates (BPs) are bone-protecting osteoclast inhibitors, typically used in the treatment of osteoporosis and skeletal complications of malignancies. When given in the adjuvant setting, these drugs may also prevent relapses and prolong overall survival in early breast cancer (EBC), specifically among postmenopausal patients. Because of these findings, adjuvant nitrogen-containing BPs (N-BPs), such as zoledronate (ZOL), are now the standard of care for high-risk EBC patients, but there are no benefit-associated biomarkers, and the efficacy remains low. BPs have been demonstrated to possess anti-tumor activities, but the mechanisms by which they provide the beneficial effects in EBC are not known. MethodsWe used stably transfected 4T1 breast cancer cells together with suppression of CD73 (sh-CD73) or control cells (sh-NT). We compared ZOL effects on tumor growth and infiltrating lymphocytes (TILs) into tumors and lung metastases using two mouse models. B cell depletion was performed using anti-CD20 antibody.ResultsSh-CD73 4T1 cells were significantly more sensitive to the growth inhibitory effects of n-BPs in vitro. However, while ZOL-induced growth inhibition was similar between the tumor groups in vivo, ZOL enhanced B and T lymphocyte infiltration into the orthotopic tumors with down-regulated CD73. A similar trend was detected in lung metastases. ZOL-induced tumor growth inhibition was found to be augmented with B cell depletion in sh-NT tumors, but not in sh-CD73 tumors. As an internal control, ZOL effects on bone were similar in mice bearing both tumor groups.DiscussionTaken together, these results indicate that ZOL modifies TILs in breast cancer, both in primary tumors and metastases. Our results further demonstrate that B cells may counteract the growth inhibitory effects of ZOL. However, all ZOL-induced TIL effects may be influenced by immunomodulatory characteristics of the tumor. </p