METHODS OF BOOSTING THYMIC REGENERATION IN PATIENTS SUFFERING FROM A THYMIC INJURY BY USING RANKL
20220265768 · 2022-08-25
Inventors
Cpc classification
A61K38/16
HUMAN NECESSITIES
A61K31/663
HUMAN NECESSITIES
C07K2319/30
CHEMISTRY; METALLURGY
A61K38/1793
HUMAN NECESSITIES
International classification
A61K31/663
HUMAN NECESSITIES
Abstract
Cytoablative treatments lead to severe damages on thymic epithelial cells (TECs), which result in delayed de novo thymopoiesis and a prolonged period of T-cell immunodeficiency. Understanding the mechanisms that govern thymic regeneration is of paramount interest for the recovery of a functional immune system notably after bone marrow transplantation (BMT). Here, the inventors show that administration of RANK ligand (RANKL) after total body irradiation and BMT boosts thymic regeneration. Notably, this treatment is also beneficial upon BMT in aged individuals. The inventors show that RANKL can improve thymopoiesis in aged individuals affected by thymic involution. Finally, the inventors show that RANK receptor is conserved in the human thymus. Accordingly, one aspect of the present invention relates to a method of boosting thymic regeneration in a patient suffering from a thymic injury comprising administering to the subject a therapeutically effective amount of a RANKL polypeptide.
Claims
1. A method of boosting thymic regeneration in a patient suffering from a thymic injury comprising administering to the patient a therapeutically effective amount of a RANKL polypeptide.
2. The method of claim 1 wherein the thymic injury results from cytoablative therapy, complications related to HIV/AIDS, aging process, malnutrition, and radiation poisoning due to nuclear disaster.
3. The method of claim 1 wherein the patient is selected from the group consisting of children, young adults, middle aged adults, and elderly adults.
4. The method of claim 1 wherein the patient suffers from a cancer and has undergone a cytoablative therapy which caused thymic injury.
5. The method of claim 4 wherein the cytoablative therapy is radiotherapy or chemotherapy.
6. The method of claim 4 wherein the administration of the RANKL polypeptide is performed after bone marrow transplantation.
7. The method of claim 1 wherein the RANKL polypeptide is a polypeptide comprising an amino acid sequence having at least 80% of identity with SEQ ID NO:1 or SEQ ID NO:2.
8. The method of claim 1 wherein the RANKL polypeptide is fused to an immunoglobulin domain to form an immunoadhesin.
9. The method of claim 1 wherein the RANKL polypeptide is administered to the patient in combination with bisphosphonate to prevent bone resorption.
10. The method of claim 1, wherein the thymic injury is due to cytoablative conditioning and the step of administering prevents immunodeficiency caused by the cytoablative conditioning.
11. The method of claim 1 wherein the thymic injury is thymic involution due to aging.
12. A method for the prophylactic treatment of infectious diseases, autoimmunity or cancer relapse in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a RANKL polypeptide.
13. The method according to claim 1 wherein the patient is a human.
14. The method of claim 8, wherein the immunoglobulin domain is a Fc region
15. The method of claim 12, wherein the step of administering boosts or enhances long-term recovery of T-cell functions.
Description
FIGURES
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EXAMPLE
[0038] Material & Methods
[0039] Human Sample
[0040] Human thymic fragments were obtained from normal male and female patients (3 months to 15 years old) during cardiovascular surgery at the Necker Hospital (Paris, France). All tissue samples were fast-frozen in liquid nitrogen within 30 minutes of their excision from patients.
[0041] Mice
[0042] CD45.1 and CD45.2 WT (Janvier), CD45.2 LTα α.sup.−/− (34), Rag2.sup.−/− (Shinkai Y et al. Cell. 1992. PMID: 1547487), ZAP-70.sup.−/− (Kadlecek T A et al. J Immunol. 1998. PMID: 9794398) and Rorc.sup.GFP/GFP knock-in (58) mice were on B6 background and maintained under specific pathogen-free conditions at the CIML (France). Chimeras were generated at 6-8 weeks or 6-8 months of age. WT mice of 12 months of age were used to analyze the effects of RANKL treatment on thymic involution.
[0043] Thymic Damage and BM Chimeras
[0044] TBI was performed with a Cs-137 γ-radiation source. Sublethal-TBI (SL-TBI) was performed with 500 rads with no hematopoietic rescue and lethal-TBI (L-TBI) with 2 doses of 500 rads. For the generation of chimeras, 10.sup.7 BM cells of CD45.1 origin were injected i.v. into lethally irradiated (2×500 rads) CD45.2 WT or LTα.sup.−/− recipient mice.
[0045] RANKL and IL-22 Stimulations
[0046] The recombinant mouse RANKL-GST protein was produced as previously described (24). RANKL-GST (5 mg/kg) or GST (5 mg/kg) proteins were administrated i.v. daily during three days after SL-TBI or at day 2, 4 and 6 after BMT. Unmanipulated WT mice of 9-12 months of age were administrated i.v. with GST (5 mg/kg) or RANKL-GST (5 mg/kg) proteins during three consecutive days. Recombinant mouse IL-22 protein (200 μg/kg; R&D Systems) was administrated i.v. at days 2, 4, and 6 after BMT in combination or not with RANKL-GST protein.
[0047] RANKL Neutralization Experiments
[0048] Endotoxin anf azide-Free (LEAF) neutralizing anti-RANKL antibody (7.5 mg/kg; IK22/5; BioLegend) or purified Rat IgG2a, κ isotype control (7.5 mg/kg; RTK2758; BioLegend) were administrated i.v. during three days after SL-TBI.
[0049] Risedronate Treatment
[0050] WT mice were lethally irradiated, BM transplanted at day 0 and treated i.p. with risedronate (30 μg/kg; Sigma Aldrich) at day −2, −1, 0, 2, 4 and 6 before and after BMT. These mice were co-treated i.v. with GST or RANKL-GST proteins (5 mg/kg) at day 2, 4 and 6 after BMT.
[0051] TRAP Staining
[0052] Mouse femurs fixed in 4% paraformaldehyde during 48 h were decalcified with 10% EDTA, pH 7.5 during two weeks. 5 μm sections were deparaffinized and stained for TRAP activity (Sigma Aldrich) according to the manufacturer's instructions. Sections were counterstained with hematoxylin and images were quantified with ImageJ software.
[0053] TEC Isolation
[0054] TECs were isolated as previously described (29) by enzymatic digestion with collagenase D and DNase I (Roche) and depletion of hematopoietic cells using anti-CD45 magnetic beads and AutoMACS (Miltenyi Biotec). Flow cytometry
[0055] CD4 (RM4.5), CD8α (53-6.7), CD45.1 (A20), LTα (AF.B3), IL-7Rα (SB/199), CD80 (16-10A1), Ly51 (BP-1), I-Ab (AF6-120.1), CD45 (30-F11), CD44 (IM7) and Sca-1 (D7) antibodies were from BD. CD25 (PC61), RANK (R12-31), RANKL (IK22/5), CD3ε (145-2C11), lineage cocktail (145-2C11, RB6-8C5, M1/70, RA3-6B2, Ter-119), CD11c (N418), α6-integrin (GoH3) and CD31 (390) were from BioLegend. Foxp3 (FJK-16s), Ki-67 (SolA15), EpCAM (G8.8), LTβR (ebio3C8), Aire (5H12), RORγt (B2D), CD117 (2B8) and PDGFRα (APA5) were from eBioscience. FITC-conjugated UEA-1 was from Vector Laboratories. For RANKL and LTα detection, cells were incubated for 3 h with Brefeldin A (Biosciences). Foxp3 and Ki-67 intracellular stainings were performed with the Foxp3 staining kit (eBioscience). Aire, LTα, RANKL and RORγt intracellular stainings were performed with BD Cytofix/Cytoperm and Perm/Wash buffers. For staining with LTβR-Fc, cells were incubated with LTβR-Fc (RnD systems) at 1 μg/10.sup.6 cells for 45 min on ice. LTβR-Fc staining was visualized using a Alexa Fluor 488-conjugated donkey anti-human IgG F(ab′).sub.2 fragment (Jackson ImmunoResearch). Flow cytometry analysis was performed with a FACSCanto II (BD) and data were analyzed with FlowJo software. Quantitative RT-PCR Total RNA was prepared with TRIzol (Invitrogen). cDNAs was synthesized with oligo(dT) using Superscript TT reverse transcriptase (Invitrogen). qPCR was performed with the ABI 7500 fast real-time PCR system (Applied Biosystem) and SYBR Premix Ex Taq master mix (Takara).
[0056] Immunofluorescence Staining Frozen thymic sections were stained with Alexa Fluor 488-conjugated anti-Aire (5H12, ebioscience) and anti-keratin 14 (AF64, Covance Research) revealed with Cy3-conjugated anti-rabbit (Invitrogen). Frozen mouse and human thymic sections were stained with anti-RANK antibodies (R12-31, BioLegend for mouse detection; 80707, RnD systems for human detection) revealed with Cy3-conjugated goat anti-rat IgG (BioLegend) and with Alexa Fluor 488-conjugated donkey anti-mouse (Thermofischer) for mouse and human staining, respectively. Sections were counterstained with 1 μg/ml DAPI as previously described (59). Images were acquired with a LSM 780 Leica SP5X confocal microscope and quantified with ImageJ software.
[0057] Statistical Analysis
[0058] Statistical significance was assessed with GraphPad Prism 6 software using Student's t test or Anova on multiple variable analyses *, P<0.05; **, P<0.01; ***, P<0.001, ****, P<0.0001. Correlations were calculated using the nonparametric Spearman correlation test. Error bars represent mean±SEM.
[0059] Study Approval
[0060] Experiments were performed in accordance with the animal care guidelines of the European Union and French laws. All animal procedures were approved by and performed with in accordance with guidelines of the Centre d'Immunologie de Marseille-Luminy (CIML).
[0061] Results:
[0062] RANKL is Upregulated During the Early Phase of Thymic Regeneration
[0063] Because at steady state RANKL has been reported as a potent regulator of mTEC differentiation (25, 27, 31), we investigated whether this cytokine plays a role in thymic regeneration. To this, we first analyzed RANKL expression in the thymus at day 3 after SL-TBI (d3 SL-TBI) and found that RANKL was strongly upregulated in CD45.sup.+ hematopoietic cells compared to untreated (UT) WT mice. Among hematopoietic cells, LTi cells, identified as CD4.sup.+CD3.sup.−IL-7Rα.sup.+RORγt*, and CD4.sup.+ SP cells were clearly the main producers of RANKL, which was upregulated in a radiation dose-dependent manner. To definitively confirm the cellular source of RANKL, we analyzed the thymus from Rorc.sup.−/− mice, defective in LTi cells (35). Under physiological conditions, the thymus from Rorc.sup.−/− mice showed similar levels of RANKL mRNA compared to WT mice. In contrast, at d3 SL-TBI, while the expression of RANKL was strongly upregulated in the WT thymus, thymus from Rorc.sup.−/− mice failed to increase RANKL, suggesting that LTi cells are required for RANKL upregulation after thymic damage. However, since Rorc.sup.−/− mice show reduced numbers of DP and CD4+ SP cells, we also analyzed RANKL expression in the thymus of UT in ZAP-70.sup.−/− mice, lacking SP cells, to determine the contribution of CD4+ SP cells in RANKL expression. Strikingly, thymus from ZAP-70.sup.−/− mice failed to increase RANKL after SL-TBI, indicating that CD4+ SP cells constitute the major source of RANKL. Furthermore, RANKL expression in the thymus of Rag2−/− mice, lacking both DP and CD4.sup.+ SP but having normal numbers of LTi cells, is upregulated after SL-TBI but to a lesser extent than in WT thymus at d3 SL-TBI. These data thus indicate that RANKL is mainly produced by CD4.sup.+ SP and LTi cells after irradiation. RANKL was upregulated in these two cell-types until day 10 after SL-TBI with no hematopoietic rescue, implying that RANKL upregulation occurs in the early phase of thymic regeneration.
[0064] The Administration of RANKL Boosts TEC Regeneration Upon Thymic Damage
[0065] The aforementioned data strongly suggest that RANKL could play a role in thymic regeneration after irradiation. To confirm this assumption, WT mice were treated with a neutralizing anti-RANKL antibody (IK22/5) during three days after SL-TBI. PBS- and isotype antibody-treated mice were used as controls. The administration of neutralizing anti-RANKL antibody led to an impaired TEC regeneration illustrating by a 2.5-fold decrease in numbers of total TECs (EpCAM.sup.+), cTECs (EpCAM.sup.+UEA-1.sup.−Ly51.sup.+) and mTECs (EpCAM.sup.+UEA-1.sup.+Ly51.sup.−) compared to controls. In a therapeutic perspective, we next investigated whether conversely the ex vivo administration of RANKL protein shows beneficial effects on TEC regeneration. WT mice were treated with RANKL-GST protein during three days after SL-TBI. PBS- and GST-treated mice were used as controls. Remarkably, RANKL-treated mice showed a two-fold increase in numbers of total TECs, cTECs and mTECs compared to controls (
[0066] To gain mechanistic insights into the mode of action of RANKL, we analyzed the proliferation status of cTECs, mTECs and TEPCs. Numbers of Ki-67.sup.+ cells in these three populations were increased in RANKL-treated mice. Purified mTECs showed reduced expression of Bax, Bid and Bak pro-apoptotic genes and cTECs reduced expression of Bax and increased expression of the Bcl-xl anti-apoptotic gene. Furthermore, the density of medullary Aire.sup.+ cells and the expression of Aire and Aire-dependent TRAs were enhanced. Interestingly, we also found that RANKL stimulated in cTECs the expression of Selp, ICAM-1 and CCL21, implicated in thymus homing of lymphoid progenitors. Altogether, these data show that RANKL administration boosts TEC proliferation, survival and differentiation and thus enhances TEC regeneration after thymic damage.
[0067] RANKL Administration Induces LTα Upregulation in LTi Cells after TBI
[0068] We next investigated the underlying mechanism(s) of RANKL treatment. Interestingly, we found that irradiation led to the upregulation of RANKL cognate receptor, RANK on LTi cells. Thus, a possible mechanism is that RANKL acts on this cell-type. Furthermore, during embryogenesis, in vitro experiments have shown that RANKL induces LTα in peripheral LTi cells (37). To investigate whether RANKL regulates in vivo LTα in thymic LTi cells after irradiation, WT mice were treated with RANKL-GST or GST proteins for three days after SL-TBI. Thymic LTi cells from RANKL-treated mice upregulated LTα compared to those from GST-treated mice. Conversely, the administration of a neutralizing anti-RANKL antibody inhibited LTα upregulation in LTi cells that was expressed at similar level than that observed in non-irradiated WT mice (UT). Moreover, in vitro stimulation with RANKL-GST also significantly stimulated LTα expression specifically in LTi cells while the addition of RANKL antagonist, RANK-Fc, fully blocked LTα induction, demonstrating the specificity of the treatment used. Consistently, LTα upregulation tightly correlated with that of RANKL during the course of BMT. Interestingly, both LTα and LTβ mRNAs were increased in the total thymus at d3 SL-TBI compared to UT WT mice. LTα was specifically induced in hematopoietic cells and LTi were the main producers of LTα and LTβ. We hypothesized that LTα could be expressed as a membrane anchored LTα1β2 heterocomplex, which only binds to LTβR (38). We used a soluble LTβR-Fc fusion protein, which detects the two LTβR ligands, LTα1β2 and LIGHT. In contrast to LTα and LTβ, LIGHT was slightly expressed and not upregulated after thymic injury, indicating that LTβR-Fc staining detects only LTα1β2 in LTi cells, which is upregulated in a radiation dose-dependent manner. Finally after BMT, LTα protein was selectively upregulated in LTi cells from recipient and not from donor origin until day 6 after thymic injury, showing the importance of the host LTi cells in LTα production. These data thus show that RANKL treatment induces LTα expression in LTi cells early after thymic injury.
[0069] LTα is Critical for TEC Regeneration and De Novo Thymopoiesis During the Course of BMT
[0070] We next addressed whether LTα upregulation in response to RANKL treatment after TBI is involved in thymic regeneration. In line with this assumption, total TECs, cTECs and mTECs but also TEPC-enriched cells upregulated LTβR at d3 SL-TBI. While at steady state, LTα.sup.−/− mice, in which the expression of LTα1β2 is fully lost, did not show any defect in thymocytes and TEC subsets (32, 34), numbers of cTECs, mTECs and mTEC subsets (CD80.sup.loAire.sup.−, CD80.sup.hiAire.sup.− and CD80.sup.hiAire.sup.+) as well as the density of Aire.sup.+ cells were dramatically reduced at d3 SL-TBI. Of note, no significant defect neither in CD45-PDGFRα.sup.+ fibroblasts nor in thymic LTi cells was observed in LTα.sup.−/− mice at d3 SL-TBI. To definitively address the role of LTα during thymic recovery after BMT, lethally irradiated CD45.2 WT or LTα.sup.−/− recipients were reconstituted with CD45.1 congenic BM cells (WT CD45.1:WT or WT CD45.1:LTα.sup.−/− mice) and TEC numbers were analyzed at day 10, 21 and 65 after BMT. We observed reduced numbers of total TECs, cTECs and mTECs as well as mTEC subsets in WT CD45.1:LTα.sup.−/− mice compared to WT CD45.1:WT controls at all time points analyzed. Moreover, cTEC.sup.hi, mTEC.sup.hi, TEC.sup.lo, mTEC.sub.lo and TEPC-enriched cells were also reduced. Importantly, total TECs, cTECs, mTECs and TEPCs were less proliferative. A reduced density of medullary Aire.sup.+ cells was still detectable at d65 after BMT and consequently, the expression of Aire-dependent TRAs (SP1 and SP2) was strongly affected. The expression of an Aire-independent TRA (casein β) and Fezf2 as well as its target genes (Apoc3, Fabp9 and Resp18) (5) were also reduced. These data thus reveal that LTα is critical for TEC regeneration including TEPCs during the course of BMT.
[0071] In line with these thymic environmental defects, thymocytes were reduced from double-negative (DN) to SP stage after BMT in WT CD45.1:LTα.sup.−/− mice. Consequently, numbers of peripheral CD4.sup.+ and CD8.sup.+ T cells as well as CD4.sup.+Foxp3.sup.+ regulatory T cells (Tregs) from CD45.1 donor origin were reduced in the blood and spleen of WT CD45.1:LTα.sup.−/− mice from d21 to d100 after BMT.
[0072] Since de novo thymopoiesis was impaired from DN1 stage in WT CD45.1:LTα.sup.−/− mice, we analyzed early T-lineage progenitors (ETPs; CD4.sup.−CD8.sup.−CD44.sup.+CD25.sup.−Lin.sup.−CD117.sup.+). Whereas numbers of ETPs were normal in LTα.sup.−/− thymus at steady state, ETPs from CD45.1 donor origin were reduced in WT CD45.1:LTα.sup.−/− chimeras until two months after BMT. This defect was not attributable to impaired hematopoietic progenitors because normal numbers of prethymic progenitors were observed in the BM of these mice. We hypothesized that reduced ETPs could be due to a reduced homing capacity of circulating T-cell progenitors. Thymus homing is controlled by a multistep adhesion cascade initiated by P-selectin slowing down T-cell progenitors and allowing them to respond to CCL25, CCL21/19 gradients and to engage with ICAM-1 and VCAM-1 expressed by the thymic stroma, leading to a firm arrest (39, 42). Strikingly, we found that purified CD31+ endothelial cells from WT CD45.1:LTα.sup.−/− mice showed a reduced expression of adhesion molecules such as ICAM-1, VCAM-1 and P-selectin at d21 after BMT. Furthermore, purified EpCAM+ TECs in these mice also showed a reduced expression of CCL19 and CCL21. Endothelial cells and TECs were thus defective in key molecules involved in thymus homing in absence of LTα during BMT. To firmly demonstrate that thymus homing of T-cell progenitors was altered in LTα.sup.−/− mice, short-term homing assays were performed by injecting CD45.1 congenic BM cells into irradiated WT and LTα.sup.−/− recipients. LTα.sup.−/− thymi imported three-fold less ETPs than WT thymi after thymic injury.
[0073] It is noteworthy that the effects of LTα on TEC and T-cell recovery were independent of those mediated by IL-23-regulated IL-22 described to be involved in thymic regeneration (43) since LTα.sup.−/− mice exhibited similar production of these two cytokines after TBI. Of note, RANKL was expressed at normal level at d3 SL-TBI in LTα.sup.−/− mice, suggesting that LTα did not regulate RANKL. Altogether, these results reveal that RANKL-regulated LTα constitutes an indispensable pathway for thymic regeneration.
[0074] We next investigated the respective efficiency of IL-22 and RANKL in thymic recovery during BMT. BM-transplanted mice were treated with IL-22 or RANKL at d2, d4, and d6 after BMT and thymic regeneration was analyzed at d21 (data not shown). We found that IL-22 and RANKL administrated alone increased similarly numbers of developing T cells including ETPs and ameliorated peripheral T-cell reconstitution. Importantly, IL-22 alone increased only numbers of CD80hi mTECs, whereas RANKL alone enhanced numbers of all TEC subsets including cTECs and mTECs from the immature CD80.sup.lo to the mature CD80hi stage. The numbers of cTEC.sup.hi, mTEC.sup.hi, TEC.sup.lo, mTEC.sup.lo, and TEPC-enriched cells were also increased only in RANKL-treated mice. Thus, RANKL and IL-22 do not exhibit the same effects on TEC regeneration with a preferential effect for IL-22 on only mTEC.sup.hi and a more large effect for RANKL on all TEC subsets. These data thus indicate that RANKL shows a superior ability than IL-22 to recover TECs after BMT.
[0075] RANKL Administration Enhances Thymic Regeneration Upon BMT in an LTα-Dependent Manner
[0076] Since RANKL treatment improves TEC regeneration upon SL-TBI (
[0077] Interestingly, RANKL in WT CD45.1:WT chimeras substantially increased numbers of total donor cells and thymocytes of CD45.1 origin from ETP to SP stages at d21 and d65 upon BMT. In contrast, RANKL administration in WT CD45.1:LTα.sup.−/− mice had a poor effect on de novo thymopoiesis. To decipher the mode of action of RANKL on T-cell reconstitution, we performed short-term homing assays in irradiated WT and LTα.sup.−/− mice treated with GST or RANKL. Strikingly, the receptivity capacity of circulating progenitors was substantially enhanced in RANKL-treated WT CD45.1:WT mice compared to GST-treated controls. Importantly, this was not due to increased numbers of prethymic progenitors in the BM upon RANKL treatment. In contrast, RANKL had no effect on ETP homing in WT CD45.1:LTα.sup.−/− mice. Consequently, RANKL treatment increased peripheral T-cell reconstitution only in WT CD45.1:WT mice after BMT. Altogether, these data demonstrate that LTα is critical for optimal effects of RANKL administration on TEC regeneration, thymus homing of lymphoid progenitors and T-cell reconstitution upon BMT.
[0078] Bisphosphonate Treatment Protects from Bone Resorption without Affecting RANKL-Improved Thymic Regeneration
[0079] Given that RANKL administration upon BMT increased primitive progenitors in the BM, which correlated with the development of active osteoclasts (Kollet O, Dar A, Shivtiel S, Kalinkovich A, Lapid K, Sztainberg Y, Tesio M, Samstein R M, Goichberg P, Spiegel A, Elson A, Lapidot T. (2006) Osteoclasts degrade endosteal components and promote mobilization of hematopoietic progenitor cells. Nat Med. 12(6):657-64), we have investigated whether combining RANKL with bisphosphonates, which is described to prevent bone resorption (Tomimori Y, Mori K, Koide M, Nakamichi Y, Ninomiya T, Udagawa N, Yasuda H. (2009) Evaluation of pharmaceuticals with a novel 50-hour animal model of bone loss. J Bone Miner Res. 2009 July; 24(7):1194-205), protects from the development of osteoclasts without interfering with RANKL beneficial effects on thymic regeneration during BMT. The development of osteoclasts and thymic regeneration were analyzed in WT mice treated with GST and RANKL as well as in mice co-treated with risedronate and RANKL. Active osteoclasts were identified by expression of the phosphatase TRAP 21 days after BMT. The areas stained for TRAP.sup.+ osteoclasts were increased in RANKL-treated mice compared to GST-treated mice (
[0080] RANKL Treatment has Also Beneficial Effects on Thymic Recovery Upon BMT in Aged Individuals
[0081] Because the recovery of T-cell functions upon BMT is known to be delayed and less efficient in elderly patients compared to young individuals (44), we finally investigated whether RANKL beneficial effects are persistent with age. To this, WT mice of 6-8 months, in which early thymic involution is characterized by a decline in TEC cellularity (45, 46), were subjected to the same treatment described in
[0082] The Administration of RANKL Reverses the Effects of Thymic Involution
[0083] Age-associated thymic involution results in decreased TEC cellularity and T-cell development, which leads to increased morbidity and mortality in a numerous clinical settings. Importantly, the thymus shows compelling plastic properties and thus the effects of thymic involution can be therapeutically reversed. Since we found that RANKL improve thymic regeneration upon BMT in young and aged individuals, we hypothesized that this treatment could increase thymopoietic capacity at steady state in aged mice to ameliorate the effects of age-associated thymic involution. 12 months of age WT mice were treated with GST or RANKL-GST during three consecutive days and thymus composition was analyzed 21 days later. Strikingly, the thymus size was increased in RANKL-treated mice compared to GST-treated mice (
[0084] The Expression of RANKL Receptor, RANK, is Conserved in the Thymic Medulla in Human
[0085] To determine whether the administration of RANKL protein could be conceivable in human, we first compared the expression of its receptor, RANK, on mouse and human thymic sections. Similarly to the mouse thymus, RANK was mainly expressed in the thymic medulla in human (
[0086] Discussion:
[0087] Pre-BMT conditioning induces severe damages on the thymic microenvironment, which results in delayed lymphocyte production. It is therefore of paramount clinical interest to discover new molecules that enhance thymic regeneration for an efficient recovery of the immune system (8, 11).
[0088] Our study demonstrates that the administration of RANKL substantially improves thymic recovery during BMT. We found that thymic LTi cells and CD4.sup.+ SP cells, described to be radio-resistant (43) (Tomoo Ueno et al. J Exp Med, 2004), constitute the major source of RANKL. Administration of RANKL induces LTα expression specifically in LTi cells after injury. Furthermore, thymic LTi cells from recipient origin upregulated both RANKL and LTα during the early phase of BMT. Thus, these cells change their phenotype upon stress-induced thymic damage. Thymic LTi cells are thus likely in a “quiescent stage” at steady state and are activated after irradiation to repair the injured tissue. When considering that LTi cells expressing RANKL and LTα are involved in the organogenesis of lymph nodes (47), our data suggest that thymic LTi cells likely reactivate an embryonic program to repair the thymus after irradiation.
[0089] Interestingly, we found that RANKL administration after TBI boosts the regeneration of TEC subsets including TEPC-enriched cells. Conversely, the administration of a neutralizing anti-RANKL antibody leads to an impaired TEC regeneration. Furthermore, flow cytometry, histology and qPCR experiments indicated that RANKL administration also boosts the regeneration of Aire.sup.+ mTECs. Importantly, RANKL treatment during the early phase of BMT enhances numbers of TECs, ETPs and thymocytes. We demonstrated that thymus homing of T-cell progenitors and T-cell output are improved. Notably, although mice were treated during the early phase of BMT, RANKL treatment had long-term beneficial effects detectable until two months after BMT on both TEC and T-cell compartments. Improved T-cell reconstitution can be explained by increased stromal niches linked to increased TEC cellularity but also to enhanced thymus homing of lymphoid progenitors, which is a critical step for ameliorating T-cell recovery (19, 21). The latter effect is likely mediated by increased expression of adhesion molecules and chemokines involved in this process. Nevertheless, we cannot exclude that the enhanced thymus homing by RANKL administration is also favored by increased vasculature, which is important for thymus homing. Thus, our data reveal that RANKL plays distinct roles in the thymus at steady state and during BMT.
[0090] We further found by in vitro and in vivo stimulations that RANKL induces LTα in LTi cells, which express its cognate receptor, RANK. Importantly, the administration of a neutralizing RANKL-antibody inhibits LTα upregulation in LTi cells, indicating that RANKL specifically controls LTα upregulation after SL-TBI. In contrast, LTα did not regulate RANKL, indicating that LTα acts downstream of RANKL. Whereas LTα is dispensable at steady state for TEC and T-cell cellularity, we found that LTα is critical for the recovery of thymic function. TEC subsets including TEPCs were severely reduced in LTα.sup.−/−-transplanted recipients from up to two months after BMT. Moreover, all thymocyte subsets as well as ETPs were also reduced in these mice likely due to defective thymus homing capacity. In accordance with our data, it has been recently reported that LTβR regulates VCAM-1 and ICAM-1 on endothelial cells, known to promote T-cell progenitor entry in the thymus (40, 49, 50). Consistently, we found that LTα expression is important for the expression of adhesion molecules and chemokines in stromal cells during BMT. Given that in the steady state thymus, LTα expressed by SP thymocytes, is involved in the regular thymic architecture (12, 33, 51, 52), our data show that LTα expressed by LTi cells after thymic injury plays distinct roles.
[0091] Furthermore, it has been described that IL-22 participates to thymus recovery (43). Our data reveal that RANKL-regulated LTα in LTi cells represents a distinct mechanism of that mediated by IL-22 in thymic regeneration and thus highlighting that this cell-type uses different mechanisms for thymic repair. Importantly, although IL-22 treatment during BMT enhances only mature CD80hi mTECs, our data demonstrate that RANKL treatment ameliorates the regeneration of not only CD80hi mTECs but also CD80.sup.lo mTECs and cTECs. The administration of RANKL boosts also the recovery of TEPCs, which are essential for the renewal of stromal niches after BMT. These results thus show that in contrast to IL-22, RANKL has large spectrum effects on TECs with a superior ability to regenerate these cell populations. Importantly, whereas IL-22 treatment had no more effect on thymopoiesis at d28 after BMT, RANKL administration boosts thymic regeneration until d65 after BMT. Thus, RANKL constitutes an innovative therapy to enhance thymic regeneration after BMT by acting on both TEC and T-cell reconstitution. One would expect that thymic regeneration during the course of BMT is also defective in RANKL.sup.−/− mice. Unfortunately, we were unable to test this hypothesis since RANKL.sup.−/− mice show severe growth retardations (53) and exhibit a drastic reduction in mTECs (27).
[0092] In addition, the administration of fibroblast growth factor 7 (Fgf-7) before BMT has been shown to protect TECs and ameliorates the thymopoietic capacity (Min D, Taylor P A, Panoskaltsis-Mortari A, Chung B, Danilenko D M, Farrell C, Lacey D L, Blazar B R, Weinberg K I. (2002) Protection from thymic epithelial cell injury by keratinocyte growth factor: a new approach to improve thymic and peripheral T-cell reconstitution after bone marrow transplantation. Blood. 15; 99(12):4592-600). However, in contrast to RANKL, Fgf-7 shows a protective but not a regenerative effect during BMT.
[0093] Nevertheless, we demonstrate that RANKL administration has beneficial effects on thymic recovery. Moreover, RANKL treatment in BM-transplanted LTα.sup.−/− mice had only minor effects on TEC regeneration, numbers of ETPs and strikingly de novo thymopoiesis was not ameliorated. Interestingly, whereas LTα is dispensable for Aire.sup.+ mTEC differentiation at steady state (32), the regeneration of these cells induced by RANKL treatment critically depends on LTα. These data indicate that the mechanisms involved in Aire.sup.+ mTEC regeneration are distinguishable from those implicated in their emergence/differentiation at steady state. Importantly, beneficial effects of RANKL treatment observed on TEC regeneration are likely not due to a direct action on TECs because the effects of RANKL on these cells were modest in absence of LTα. Thus, these data argue in favor of model in which RANKL acts indirectly on TECs through LTi cells that express LTα. This notion is supported by the fact that the turnover rate of mTECs is of around 2 weeks (54, 55) and thus it is unlikely that RANKL injected early after BMT still acts on mTECs two months later.
[0094] To avoid any potential side effects of the systemic administration of RANKL, such as osteoporosis, a possible strategy would be to deliver directly this molecule intrathymically in patients after cytoablative conditioning or combined RANKL with bisphosphonate to prevent bone resorption (57). The administration of bisphosphonate leads to a decrease in osteoclast differentiation and in apoptosis of mature osteoclasts. Importantly, this drug that inhibits bone resorption does not affect RANKL signaling (Kim Y H, Kim G S, Jeong-Hwa B (2002) Inhibitory action of bisphosphonates on bone resorption does not involve the regulation of RANKL and OPG expression. Exp Mol Med 34: 145-151; Verde M E, Bermejo D, Gruppi A, Grenon M (2015) Effect of Bisphosphonates on the Levels of Rank1 and Opg in Gingival Crevicular Fluid of Patients With Periodontal Disease and Post-menopausal Osteoporosis. Acta Odontol Latinoam 28: 215-221). Furthermore, bisphosphonate combined with RANKL have been shown to suppress mouse and human osteoclast differentiation mediated by RANKL treatment (Tomimori Y, Mori K, Koide M, Nakamichi Y, Ninomiya T, Udagawa N, Yasuda H (2009) Evaluation of pharmaceuticals with a novel 50-hour animal model of bone loss. J Bone Miner Res 24: 1194-1205). Our data demonstrate that a co-treatment of risedronate and RANKL during BMT protects efficiently from bone resorption without interfering with RANKL beneficial effects on thymic regeneration. Thus, RANKL treatment in clinic is expected to be promising for enhancing the regeneration of immune functions in patients whose thymus has been severely damaged.
[0095] Interestingly, RANKL administration is also efficient for TEC and T-cell regeneration during BMT in older individuals in which thymic involution results in diminished TEC cellularity, disrupted thymic architecture and decreased T-cell output (45, 46). These results are of special interest for elderly patients in which the recovery of T-cell functions upon BMT is less efficient (44).
Age-induced thymic involution results in a reduced efficacy of the immune system to fight against opportunistic infections and also favors the development of autoimmunity and increases the incidence of cancer (Lynch H E, Goldberg G L, Chidgey A, Van den Brink M R, Boyd R, Sempowski G D (2009) Thymic involution and immune reconstitution. Trends Immunol. July; 30(7):366-73). Thymic function declines in the second year of life and is marked by a progressive diminution mainly in cTECs but also in mTECs (Steinmann G G, Klaus B, Müller-Hermelink H K (1985) The involution of the ageing human thymic epithelium is independent of puberty. A morphometric study. Scand J Immunol.; 22(5):563-75). The administration of insulin growth factor (IGF) or Ghrelin peptide hormone partly improves thymic compartments in aged mice (Montecino-Rodriguez El, Clark R, Dorshkind K. (1998) Effects of insulin-like growth factor administration and bone marrow transplantation on thymopoiesis in aged mice. Endocrinology. 139(10):4120-6; Dixit V D, Yang H, Sun Y, Weeraratna A T, Youm Y H, Smith R G, Taub D D. (2007) Ghrelin promotes thymopoiesis during aging. J Clin Invest. 117(10):2778-90). Fgf-7 plays also significant roles in the correction of thymus senescence in mice (Min D1, Panoskaltsis-Mortari A, Kuro-O M, Hollander G A, Blazar B R, Weinberg K I. (2007) Sustained thymopoiesis and improvement in functional immunity induced by exogenous KGF administration in murine models of aging. Blood. 5; 109(6):2529-37). However, the efficacy of Fgf-7 alone in clinical trials remains to be established. Our data demonstrate that a single three days course of RANKL in aged mice partially recovered the numbers of thymocytes until three weeks after treatment. Importantly, this treatment totally reversed the diminution of cTECs and mTECs but also ETPs observed upon thymic involution. Interestingly, we show that the expression of RANK is conserved in the thymic medulla in human, indicating that RANKL treatment could be also beneficial to restore thymic function in different clinical settings.
This study thus reveals that administration of RANKL offers an innovative therapeutic strategy to boost thymic recovery at several levels: TEC regeneration, thymus homing of T-cell progenitors and de novo thymopoiesis.
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