All Publications

  • 4-Methylumbelliferyl glucuronide contributes to hyaluronan synthesis inhibition JOURNAL OF BIOLOGICAL CHEMISTRY Nagy, N., Gurevich, I., Kuipers, H. F., Ruppert, S. M., Marshall, P. L., Xie, B. J., Sun, W., Malkovskiy, A. V., Rajadas, J., Grandoch, M., Fischer, J. W., Frymoyer, A. R., Kaber, G., Bollyky, P. L. 2019; 294 (19): 7864–77

    View details for DOI 10.1074/jbc.RA118.006166

    View details for Web of Science ID 000470153300027

  • 4-Methylumbelliferyl glucuronide contributes to hyaluronan synthesis inhibition. The Journal of biological chemistry Nagy, N., Gurevich, I., Kuipers, H. F., Ruppert, S. M., Marshall, P. L., Xie, B. J., Sun, W., Malkovskiy, A. V., Rajadas, J., Grandoch, M., Fischer, J. W., Frymoyer, A. R., Kaber, G., Bollyky, P. L. 2019

    Abstract

    4-Methylumbelliferone (4-MU) inhibits hyaluronan (HA) synthesis and is an approved drug used for managing biliary spasm. However, rapid and efficient glucuronidation is thought to limit its utility for systemically inhibiting HA synthesis. In particular, 4-MU in mice has a short half-life causing most of the drug to be present as the metabolite 4-methylumbelliferyl glucuronide (4-MUG), which makes it remarkable that 4-MU is effective at all. We report here that 4-MUG contributes to HA synthesis inhibition. We observed that oral administration of 4-MUG to mice inhibits HA synthesis, promotes FoxP3+ regulatory T-cell expansion, and prevents autoimmune diabetes. Mice fed either 4-MUG or 4-MU had equivalent 4-MU:4-MUG ratios in serum, liver and pancreas, indicating that 4-MU and 4-MUG reach an equilibrium in these tissues. LC-tandem MS experiments revealed that 4-MUG is hydrolyzed to 4-MU in serum, thereby greatly increasing the effective bioavailability of 4-MU. Moreover, using intra-vital 2-photon microscopy, we found that 4-MUG (a non-fluorescent molecule) undergoes conversion into 4-MU (a fluorescent molecule) and that 4-MU is extensively tissue bound in the liver, fat, muscle, and pancreas of treated mice. 4-MUG also suppressed HA synthesis independently of its conversion into 4-MU and without depletion of the HA precursor UDP-glucuronic acid (GlcUA). Together, these results indicate that 4-MUG both directly and indirectly inhibits HA synthesis and that the effective bioavailability of 4-MU is higher than previously thought. These findings greatly alter the experimental and therapeutic possibilities for HA synthesis inhibition.

    View details for PubMedID 30914479

  • Natural Tr1-like cells do not confer long-term tolerogenic memory. eLife Yadava, K. n., Medina, C. O., Ishak, H. n., Gurevich, I. n., Kuipers, H. n., Shamskhou, E. A., Koliesnik, I. O., Moon, J. J., Weaver, C. n., Nadeau, K. C., Bollyky, P. L. 2019; 8

    Abstract

    IL-10-producing Tr1 cells promote tolerance but their contributions to tolerogenic memory are unclear. Using 10BiT mice that carry a Foxp3-eGFP reporter and stably express CD90.1 following IL-10 production, we characterized the spatiotemporal dynamics of Tr1 cells in a house dust mite model of allergic airway inflammation. CD90.1+Foxp3-IL-10+ Tr1 cells arise from memory cells and rejoin the tissue-resident memory T-cell pool after cessation of IL-10 production. Persistent antigenic stimulation is necessary to sustain IL-10 production and Irf1 and Batf expression distinguishes CD90.1+Foxp3-IL-10+ Tr1 cells from CD90.1+Foxp3-IL-10- 'former' Tr1. Depletion of Tr1-like cells after primary sensitization exacerbates allergic airway inflammation. However, neither transfer nor depletion of former Tr1 cells influences either Tr1 numbers or the inflammatory response during subsequent allergen memory re-challenge weeks later. Together these data suggest that naturally-arising Tr1 cells do not necessarily give rise to more Tr1 upon allergen re-challenge or contribute to tolerogenic memory. This phenotypic instability may limit efforts to re-establish tolerance by expanding Tr1 in vivo.

    View details for DOI 10.7554/eLife.44821

    View details for PubMedID 31603425

  • Active dissemination of cellular antigens by DCs facilitates CD8(+) T-cell priming in lymph nodes EUROPEAN JOURNAL OF IMMUNOLOGY Gurevich, I., Feferman, T., Milo, I., Tal, O., Golani, O., Drexler, I., Shakhar, G. 2017; 47 (10): 1802–18

    Abstract

    Antigen (Ag) specific activation of naïve T cells by migrating dendritic cells (DCs) is a highly efficient process, although the chances for their colocalization in lymph nodes (LNs) appear low. Ag presentation may be delegated from Ag-donor DCs to the abundant resident DCs, but the routes of Ag transfer and how it facilitates T-cell activation remain unclear. We visualized CD8+ T cell-DC interactions to study the sites, routes, and cells mediating Ag transfer in mice. In vitro, Ag transfer from isolated ovalbumin (OVA)+ bone marrow (BM) DCs triggered widespread arrest, Ca2+ flux, and CD69 upregulation in OT-I T cells contacting recipient DCs. Intravital two-photon imaging revealed that survival of Ag-donor DCs in LNs was required for Ag dissemination among resident CD11c+ DCs. Upon interaction with recipient DCs, CD8+ T cells clustered, upregulated CD69, proliferated and differentiated into effectors. Few DCs sufficed for activation, and for efficient Ag dissemination lymphocyte function associated antigen 1 (LFA-1) expression on recipient DCs was essential. Similar findings characterized DCs infected with a replication-deficient OVA-expressing Vaccinia virus known to downregulate MHC-I. Overall, active Ag dissemination from live incoming DCs helped activate CD8+ T cells by increasing the number of effective presenting cells and salvaged T-cell priming when Ag-donor DCs could not present Ag.

    View details for DOI 10.1002/eji.201747042

    View details for Web of Science ID 000415363800008

    View details for PubMedID 28872666

  • Hyaluronan synthesis is necessary for autoreactive T-cell trafficking, activation, and Th1 polarization. Proceedings of the National Academy of Sciences of the United States of America Kuipers, H. F., Rieck, M., Gurevich, I., Nagy, N., Butte, M. J., Negrin, R. S., Wight, T. N., Steinman, L., Bollyky, P. L. 2016; 113 (5): 1339-1344

    Abstract

    The extracellular matrix polysaccharide hyaluronan (HA) accumulates at sites of autoimmune inflammation, including white matter lesions in multiple sclerosis (MS), but its functional importance in pathogenesis is unclear. We have evaluated the impact of 4-methylumbelliferone (4-MU), an oral inhibitor of HA synthesis, on disease progression in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Treatment with 4-MU decreases the incidence of EAE, delays its onset, and reduces the severity of established disease. 4-MU inhibits the activation of autoreactive T cells and prevents their polarization toward a Th1 phenotype. Instead, 4-MU promotes polarization toward a Th2 phenotpye and induction of Foxp3(+) regulatory T cells. Further, 4-MU hastens trafficking of T cells through secondary lymphoid organs, impairs the infiltration of T cells into the CNS parenchyma, and limits astrogliosis. Together, these data suggest that HA synthesis is necessary for disease progression in EAE and that treatment with 4-MU may be a potential therapeutic strategy in CNS autoimmunity. Considering that 4-MU is already a therapeutic, called hymecromone, that is approved to treat biliary spasm in humans, we propose that it could be repurposed to treat MS.

    View details for DOI 10.1073/pnas.1525086113

    View details for PubMedID 26787861

  • Hyaluronan synthesis is necessary for autoreactive T-cell trafficking, activation, and Th1 polarization. Proceedings of the National Academy of Sciences of the United States of America Kuipers, H. F., Rieck, M., Gurevich, I., Nagy, N., Butte, M. J., Negrin, R. S., Wight, T. N., Steinman, L., Bollyky, P. L. 2016

    Abstract

    The extracellular matrix polysaccharide hyaluronan (HA) accumulates at sites of autoimmune inflammation, including white matter lesions in multiple sclerosis (MS), but its functional importance in pathogenesis is unclear. We have evaluated the impact of 4-methylumbelliferone (4-MU), an oral inhibitor of HA synthesis, on disease progression in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Treatment with 4-MU decreases the incidence of EAE, delays its onset, and reduces the severity of established disease. 4-MU inhibits the activation of autoreactive T cells and prevents their polarization toward a Th1 phenotype. Instead, 4-MU promotes polarization toward a Th2 phenotpye and induction of Foxp3(+) regulatory T cells. Further, 4-MU hastens trafficking of T cells through secondary lymphoid organs, impairs the infiltration of T cells into the CNS parenchyma, and limits astrogliosis. Together, these data suggest that HA synthesis is necessary for disease progression in EAE and that treatment with 4-MU may be a potential therapeutic strategy in CNS autoimmunity. Considering that 4-MU is already a therapeutic, called hymecromone, that is approved to treat biliary spasm in humans, we propose that it could be repurposed to treat MS.

    View details for DOI 10.1073/pnas.1525086113

    View details for PubMedID 26787861

  • Heparanase of murine effector lymphocytes and neutrophils is not required for their diapedesis into sites of inflammation FASEB JOURNAL Stoler-Barak, L., Petrovich, E., Aychek, T., Gurevich, I., Tal, O., Hatzav, M., Ilan, N., Feigelson, S. W., Shakhar, G., Vlodavsky, I., Alon, R. 2015; 29 (5): 2010-2021

    Abstract

    Heparanase, the exclusive mammalian heparan sulfate-degrading enzyme, has been suggested to be utilized by leukocytes to penetrate through the dense basement membranes surrounding blood venules. Despite its established role in tumor cell invasion, heparanase function in leukocyte extravasation has never been demonstrated. We found that TH1/TC1-type effector T cells are highly enriched for this enzyme, with a 3.6-fold higher heparanase mRNA expression compared with naive lymphocytes. Using adoptive transfer of wild-type and heparanase-deficient effector T cells into inflamed mice, we show that T-cell heparanase was not required for extravasation inside inflamed lymph nodes or skin. Leukocyte extravasation through acute inflamed skin vessels was also heparanase independent. Furthermore, neutrophils emigrated to the inflamed peritoneal cavity independently of heparanase expression on either the leukocytes or on the endothelial and mesothelial barriers, and overexpression of the enzyme on neutrophils did not facilitate their emigration. However, heparanase absence significantly reduced monocyte emigration into the inflamed peritoneal cavity. These results collectively suggest that neither leukocyte nor endothelial heparanase is required for T-cell and neutrophil extravasation through inflamed vascular barriers, whereas this enzyme is required for optimal monocyte recruitment to inflamed peritoneum.

    View details for DOI 10.1096/fj.14-265447

    View details for Web of Science ID 000354114600034

    View details for PubMedID 25634957

  • The integrin coactivator Kindlin-3 is not required for lymphocyte diapedesis BLOOD Cohen, S. J., Gurevich, I., Feigelson, S. W., Petrovich, E., Moser, M., Shakhar, G., Fassler, R., Alon, R. 2013; 122 (15): 2609-2617

    Abstract

    Kindlin-3 is an integrin-binding focal adhesion adaptor absent in patients with leukocyte and platelet adhesion deficiency syndrome and is critical for firm integrin-dependent leukocyte adhesion. The role of this adaptor in leukocyte diapedesis has never been investigated. In the present study, the functions of Kindlin-3 in this process were investigated in effector T lymphocytes trafficking to various lymphoid and nonlymphoid tissues. In vitro, Kindlin-3-deficient T cells displayed severely impaired lymphocyte function antigen-1-dependent lymphocyte adhesion but partially conserved very late antigen-4 adhesiveness. In vivo, the number of adoptively transferred Kindlin-3-deficient T effectors was dramatically elevated in the circulating pool compared with normal effectors, and the Kindlin-3 mutant effectors failed to enter inflamed skin lesions. The frequency of Kindlin-3-deficient T effectors arrested on vessel walls within inflamed skin-draining lymph nodes was also reduced. Strikingly, however, Kindlin-3-deficient effector T cells accumulated inside these vessels at significantly higher numbers than their wild-type lymphocyte counterparts and successfully extravasated into inflamed lymph nodes. Nevertheless, on entering these organs, the interstitial motility of these lymphocytes was impaired. This is the first in vivo demonstration that Kindlin-3-stabilized integrin adhesions, although essential for lymphocyte arrest on blood vessels and interstitial motility, are not obligatory for leukocyte diapedesis.

    View details for DOI 10.1182/blood-2013-04-495036

    View details for Web of Science ID 000326079400019

    View details for PubMedID 23980064

  • Luminal Bacteria Recruit CD103(+) Dendritic Cells into the Intestinal Epithelium to Sample Bacterial Antigens for Presentation IMMUNITY Farache, J., Koren, I., Milo, I., Gurevich, I., Kim, K., Zigmond, E., Furtado, G. C., Lira, S. A., Shakhar, G. 2013; 38 (3): 581-595

    Abstract

    CD103+ dendritic cells (DCs) carry bacteria from the small intestine and can present antigens to T cells. Yet they have not been recorded sampling luminal bacteria or presenting bacterial antigens in mesentery lymph nodes. We used 2-photon microscopy in live Cx3cr1(+/gfp) ×Cd11c-YFP mice to study these processes. At steady state, sparse CD103+ DCs occupied the epithelium. They patrolled among enterocytes while extending dendrites toward the lumen, likely using tight-junction proteins to penetrate the epithelium. Challenge with Salmonella triggered chemokine- and toll-like receptor (TLR)-dependent recruitment of additional DCs from the lamina propria (LP). The DCs efficiently phagocytosed the bacteria using intraepithelial dendrites. Noninvasive bacteria were similarly sampled. In contrast, CD103+ DCs sampled soluble luminal antigen inefficiently. In mice harboring CD103+ DCs, antigen-specific CD8 T cells were subsequently activated in MLNs. Intestinal CD103+ DCs are therefore equipped with unique mechanisms to independently complete the processes of uptake, transportation, and presentation of bacterial antigens.

    View details for DOI 10.1016/j.immuni.2013.01.009

    View details for Web of Science ID 000330941500021

    View details for PubMedID 23395676

  • Murine anti-third-party central-memory CD8(+) T cells promote hematopoietic chimerism under mild conditioning: lymph-node sequestration and deletion of anti-donor T cells BLOOD Ophir, E., Or-Geva, N., Gurevich, I., Tal, O., Eidelstein, Y., Shezen, E., Margalit, R., Lask, A., Shakhar, G., Hagin, D., Bachar-Lustig, E., Reich-Zeliger, S., Beilhack, A., Negrin, R., Reisner, Y. 2013; 121 (7): 1220-1228

    Abstract

    Transplantation of T cell-depleted BM (TDBM) under mild conditioning, associated with minimal toxicity and reduced risk of GVHD, offers an attractive therapeutic option for patients with nonmalignant hematologic disorders and can mediate immune tolerance to subsequent organ transplantation. However, overcoming TDBM rejection after reduced conditioning remains a challenge. Here, we address this barrier using donorderived central memory CD8(+) T cells (Tcms), directed against third-party antigens. Our results show that fully allogeneic or (hostXdonor)F1-Tcm, support donor chimerism (> 6 months) in sublethally irradiated (5.5Gy) mice, without GVHD symptoms. Chimerism under yet lower irradiation (4.5Gy) was achieved by combining Tcm with short-term administration of low-dose Rapamycin. Importantly, this chimerism resulted in successful donor skin acceptance, whereas third-party skin was rejected. Tracking of host anti-donor T cells (HADTCs), that mediate TDBMT rejection, in a novel bioluminescence-imaging model revealed that Tcms both induce accumulation and eradicate HADTCs in the LNs,concomitant with their elimination from other organs, including the BM. Further analysis with 2-photon microcopy revealed that Tcms form conjugates with HADTCs, resulting in decelerated and confined movement of HADTCs within the LNs in an antigen-specific manner. Thus, anti-third-party Tcms support TDBMT engraftment under reduced-conditioning through lymph-node sequestration and deletion of HADTCs, offering a novel and potentially safe approach for attaining stable hematopoietic chimerism.

    View details for DOI 10.1182/blood-2012-07-441493

    View details for Web of Science ID 000314870700026

    View details for PubMedID 23223359

  • DC mobilization from the skin requires docking to immobilized CCL21 on lymphatic endothelium and intralymphatic crawling JOURNAL OF EXPERIMENTAL MEDICINE Tal, O., Lim, H. Y., Gurevich, I., Milo, I., Shipony, Z., Ng, L. G., Angeli, V., Shakhar, G. 2011; 208 (10): 2141-2153

    Abstract

    Dendritic cells (DCs) must travel through lymphatics to carry skin antigens into lymph nodes. The processes controlling their mobilization and migration have not been completely delineated. We studied how DCs in live mice respond to skin inflammation, transmigrate through lymphatic endothelium, and propagate in initial lymphatics. At steady state, dermal DCs remain sessile along blood vessels. Inflammation mobilizes them, accelerating their interstitial motility 2.5-fold. CCR7-deficient BMDCs crawl as fast as wild-type DCs but less persistently. We observed discrete depositions of CCL21 complexed with collagen-IV on the basement membrane of initial lymphatics. Activated DCs move directionally toward lymphatics, contact CCL21 puncta, and migrate through portals into the lumen. CCR7-deficient DCs arrive at lymphatics through random migration but fail to dock and transmigrate. Once inside vessels, wild-type DCs use lamellipodia to crawl along lymphatic endothelium and, sensing lymph flow, proceed downstream. DCs start drifting freely only in collecting lymphatics. These results demonstrate in vivo that the CCL21-CCR7 axis plays a dual role in DC mobilization: promoting both chemotaxis and arrest of DCs on lymphatic endothelium. Intralymphatic crawling, in which DCs combine active adhesion-based migration and directional cues from lymph flow, represents a new step in DC mobilization which may be amenable to regulation.

    View details for DOI 10.1084/jem.20102392

    View details for Web of Science ID 000295318900018

    View details for PubMedID 21930767