Università degli Studi di Pisa
PhD in Clinical and Translational Sciences
Director: Prof. Stefano Del Prato, MD
“The role of zinc in thymic regeneration after bone marrow
transplant”
PhD Candidate Lorenzo Iovino, MD
Supervisor Prof. Mario Petrini, MD, PhD Co-supervisor Prof. Jarrod Dudakov, PhD Prof. Sara Galimberti, MD, PhD
Summary
1. Introduction and background ... 4
1.1 Hematopoietic stem cell transplantation (HSCT). Historical perspective and modern challenges ... 4
1.2 Thymus and generation of T cell repertoire ... 5
1.3 Role of the thymus in immune reconstitution after HSCT ... 7
1.4 Zinc, cell functions and immunity ... 8
1.5 Rationale of the study ... 10
2. Materials and methods ... 11
2.1 Zinc deficient diet and zinc supplementation to experimental mice ... 11
2.2 Cell Isolation ... 12
2.3 Cell cultures ... 12
2.3.1 Ex-vivo propagated Endothelial cells (exEC) ... 12
2.3.2 C9 and TE-71 ... 13
2.3.3 Co-culture experiments ... 13
2.2 ELISA and Western Blot experiments ... 13
2.3 Flow cytometry and FACS sorting ... 14
2.4 Proliferation Assays ... 14 2.5 Microarray ... 15 2.6 PCR 15 2.7 ICP-MS ... 15 2.8 Statistics ... 16 3. Results ... 17
3.1 Zinc deficiency impairs thymic reconstitution after TBI ... 17
3.2 Zinc supplementation improves thymic regeneration after damage ... 17
3.3 The BMP4 pathway of thymic regeneration is targeted by zinc ... 18
3.4 Zinc is released in thymic extracellular space after stress-induced thymocyte death ... 19
3.5 Zinc released in the extracellular space stimulates endothelial cells to produce BMP4 ... 20
3.6 Clinical applications: zinc supplementation promotes the generation of recent thymic emigrants in a mouse model of allogeneic stem cell transplantation ... 21
4. Discussion ... 23 5. Conclusions... 32 6. Figures ... 33 6.1 Figure 1 ... 33 6.2 Figure 2 ... 34 6.3 Figure 3 ... 35 6.4 Figure 4 ... 36
6.4 Figure 5 ... 37
6.6 Figure 6 ... 38
6.7 Figure 7 ... 39
7 Legends ... 40
7.1 Figure 1. Short-term zinc deficient diet impairs thymic function and thymic reconstitution after TBI ... 40
7.2 Figure 2. Zinc supplementation improves thymic regeneration after TBI ... 41
7.3 Figure 3. Zinc is involved in the EC-TEC axis of regeneration mediated by BMP4 ... 41
7.4 Figure 4. Intracellular/extracellular zinc changes after damage and during reconstitution ... 42
7.5 Figure 5. Zinc released in extracellular space after TBI finds its receptor GPR39 on many stromal cells ... 43
7.6 Figure 6. Improved thymic reconstitution under zinc supplementation leads to higher levels of circulating recent thymic emigrants in a murine model of allogeneic bone marrow transplant ... 44
7.7 Figure 7. Graphical abstract ... 44
8 Aknowledgements ... 45
1. Introduction and background
1.1 Hematopoietic stem cell transplantation (HSCT). Historical perspective and modern challenges
Hematopoietic stem cell transplantation (HSCT) is a medical procedure based on the adoptive transfer of hematopoietic precursors derived from the bone marrow of healthy individuals, collected from bone marrow, peripheral blood or cord blood (1). HSCT has been developed in the early 1970s’ to offer a cure to hematological malignancies, such as leukemia, and both congenital and acquired bone marrow disorders(2). The introduction of HSCT as therapeutic tool for leukemia followed the demonstration that it was possible to induce bone marrow aplasia after radiation (3) and chemotherapy (4-6) and that death due to bone marrow failure could be prevented by the administration of hematopoietic progenitors derived from other individuals of the same species(7). HSCT not only “replaces” a sick bone marrow with a healthy one, but there is also an immunologic reaction of the donor-derived lymphocytes and NK cells against cancer cells. This latter effect is also called “graft versus leukemia” (GvL) effect(8). For this reason, HSCT is considered the beginning of immunotherapy.
The major limits to bone marrow transplant were, and still are, the toxicity related to the so called “conditioning regimen” (the combination of radio-chemotherapy needed to ablate the bone marrow of the recipient) and the presence of the human leukocyte antigen compatibility barrier (HLA)(9), called major histocompatibility complex (MHC) in other species. Toxicity manifestations of conditioning regimen vary from mild to severe mucositis of mouth and gut(10) and risk of severe sepsis and hemorrhage due to aplasia(11). HLA is a system of cell surface proteins expressed in a codominant way that are involved in recognition of self (class I molecules) and antigen presentation to T cells (class II)(12). The structural difference in HLA proteins, which are coded by highly variable alleles present in general population, among individuals of the same species leads to a phenomenon known as HLA-incompatibility or HLA-mismatch. This mismatch is a major contributor to solid organ graft rejection(13), but it also explaines large part of a HSCT-related immune-syndrome, described as “graft-versus host disease”
(GvHD)(14). Clinical manifestations of GvHD are divided into acute (within 100 days after HSCT) and chronic(15) and affect many organs and systems, mimicking clinical features of autoimmune diseases and increasing the risk of infections(16, 17). GvHD is dependent on T- and B-cell activation facilitated by HLA mismatch (18). More recently, minor HLA loci were discovered and their role on HSCT outcome was investigated(19). Even if GvHD is still considered at a certain extent protective against malignant recurrence(20), GvHD-related complications could be severe, and it would be very useful to separate GvHD from GvL effects, even if that is difficult(21). Strategies to prevent and treat GvHD include: selection of best donors based on the grade of HLA-mismatch(22-24), especially among the donors who are compatible at 50% for HLA loci (haplo-identical donors) (25); use of immune suppressant drugs, such as methotrexate, cyclosporine, tacrolimus, corticosteroids, post-HSCT cyclophosphamide, and many other molecules(26); depletion of T and B cells from the graft product(27) or infusion/expansion of T regulatory cells (28). To reduce the risk of conditioning-related side effects and extend the possibility of transplant to patients older and with comorbidities, reduced intensity conditioning regimens (RIC) was developed(29). So far, more than one million of patients have received allogeneic or autologous HSCT all around the world(30). Altogether, all the procedures used in HSCT lead to depression of immune function for a period lasting from months to years that increased the risk of infections, malignancy relapse, and GvHD (31). There is a crucial need of strategies to improve immune reconstitution after HSCT.
1.2 Thymus and generation of T cell repertoire
The thymus is the primary organ of T cell maturation(32). It derives from the third and the fourth pharyngeal pouch during embryogenesis and it is composed by a variety of cells of different origin. The main resident population are the thymic epithelial cells (TEC) that are divided into two populations based on their localization: an external layer of cells, called cortical thymic epithelial cells (cTEC), and an internal population called medullary thymic epithelial cells (mTEC)(33). The origin of TEC is still largely discussed and remains unclear(34). TEC have many cell-cell interactions that allow them to support T-cell lymphopoiesis(35, 36). Cells committed to lymphoid lineage (c-kit+) come from the bone marrow in a low number per day and enter the thymus through the blood vessels at the cortico-medullary junction (CMJ) and enter a process of differentiation in
which they undergo transcriptional changes(37). Four stages of differentiation of thymocytes are classically described: double negative thymocytes (DN), which lack the expression of both the mature T-cell surface markers CD4 and CD8 (CD4-/CD8-), double positive (DP) thymocytes (CD4+/CD8+), and single positive (SP) CD4+ and CD8+ thymocytes(38). During the T cell maturation process, also known as “thymopoiesis”, T-cell precursors are induced to maturate from the stimuli coming from TEC. cTEC interact with the DN, leading to the progression from DN1 thymocytes (CD44+/CD25-) to DN4 (CD44-/CD25+/IL7R+) through four stages. Between DN4 and DP stage, thymocytes undergo a process of expansion and active replication. DP maturate in contact with mTEC, and their final fate will be the stage of CD4+ or CD8+ SP. The Notch signaling is crucial in this maturation process(39), where TEC express on their surface Notch ligands (40) such as Dll4(41). The transcription factor Foxn1 is necessary for thymic organogenesis and Notch ligands expression in TEC(42). Absence of Foxn1 causes complete thymic atrophy and subsequent severe combined immune deficiency (SCID) in humans and mice(43). During thymopoiesis, TEC orchestrate the two processes known as “positive” and “negative” selection of T-cell repertoire (44): based on the “affinity” model, the interaction between MHC expressed on TECs and pre-T cell receptor (pre-TCR), generated by random rearrangement of TCR genes, is able to select only the clones with functional TCR at medium affinity(45). This mechanism guarantees the “central tolerance”(46) operated by thymus towards autoimmunity: in thymic medulla, the expression of AIRE makes mTEC able to express a huge variety of antigens normally present in all the tissues(47). In the same way, thymus allows the generation of a huge variety of potential TCR against many antigens selecting only those that are functionally active(48). Also the production of T regulatory cells is thymus-dependent(49). For the above mentioned reasons, thymopoiesis is considered very expensive from an energetic point of view: in fact, only a small proportion of T cell progenitors generated during this process exit the thymus as single positive naïve CD4+ or CD8+ T cell, also called recent thymic emigrants (RTE), whereas the great majority of them undergo apoptosis(48). Generation of T-cell repertoire starts during fetal life and reaches its peak in the childhood. Then, starting from the puberty, the thymus undergoes atrophy in all species(50). In humans, thymic aging is considered a physiological phenomenon(51) whose causes still remain poorly understood, but there is huge variability within the general population with individuals that preserve functional
thymus also in elderly (52, 53). Other cell populations play a role in T cell development and central tolerance, such as dendritic cells (DC)(54).
1.3 Role of the thymus in immune reconstitution after HSCT
Delayed immune reconstitution after hematopoietic stem cell transplantation (HSCT) and chemotherapy is an important side effect of cancer treatment (18) and leads to prolonged T cell deficiency, precipitating high morbidity and mortality from opportunistic infections and may even facilitate cancer relapse (55). There are two pathways of T cell reconstitution after HSCT: a thymic-independent and a thymic-dependent way. The thymic independent pathway is sustained by the expansion of mature T lymphocytes infused together with the graft, or residual host-derived T cells that survived the conditioning regimen and immunesuppressive therapies. This process is also called homeostatic peripheral expansion (HPE). HPE is important to reach a sufficient number of circulating lymphocytes in the first weeks after transplant, when severe lymphopenia permits opportunistic infections, but the memory phenotype of the expanded T cells and the restriction of TCR repertoire are not sufficient to give persistent protection(56, 57). Thymic-dependent pathway instead recapitulates the generation of T-cell repertoire that occurs in the childhood. There are two major limitations to thymic-dependent T cell reconstitution: first, the thymus is highly sensitive to many insults, which can come in the form of stress (corticosteroids), cytoreductive chemotherapy, infection, sex hormones, surgery, and irradiation(58). However, thymus also has a remarkable capacity for repair. In fact, the general phenomenon of endogenous thymic regeneration has been known for longer even than its immunological function(59-61). However, the underlying mechanisms controlling this process remain largely unstudied. Furthermore, the continuous decline in thymic function with age drastically reduces the regenerative capacity of the thymus(62-64). Poor thymic function negatively impacts post-HSCT outcome (59, 65), leading to higher risk of GvHD (31, 66), whereas preserved thymic function is related to better outcome, especially in T-depleted grafts (59). Poor thymic function also leads to decreased production of T regulatory cells (T reg), which are involved in protection from GvHD(67), increasing the risk of GvHD(60). Moreover, thymus itself is a target of GvHD, this way leading to a vicious circle(68, 69). In fact, functional thymus allows lymphoid cells coming from the bone marrow to be educated and selected by thymic microenvironnement: central tolerance to a “new self” reduces
the risk of GvHD. There is therefore a clear clinical need for therapeutic strategies to mediate rapid regeneration of thymic function following acute immune damage. Thymic generation of T-cell repertoire after HSCT facilitates the production of T cell clones potentially active towards infection agents and tumor antigens(58). Strategies to target thymus may have an impact in reducing mortality after HSCT(70), but only a little is known about pathways of thymic regeneration.
1.4 Zinc, cell functions and immunity
Zinc is a trace element acting as a co-factor of more than 300 proteins involved in cell cycle, apoptosis, inflammation and immune responses (71, 72). There are many crucial functions that are dependent on zinc ions: for example, zinc finger proteins, a group of ubiquitous proteins that are involved in DNA recognition, RNA packaging, transcriptional activation, regulation of apoptosis, protein folding and assembly, and lipid binding, structurally depend on the presence of a zinc ion in the zinc-finger motifs, zinc availability in the organism being crucial for their activity(73). Zinc levels in intra- and extra-cellular space of multicellular organisms are tightly regulated by a complex machinery: in mammalians, there are 14 proteins of the SLC39a family, also called “ZIP” (74). The role of these proteins is to increase zinc intracellular levels by importing zinc from the extracellular space or releasing zinc stored in endoplasmic reticulum, Golgi, and lysosomes. Conversely, members of Slc30a, (ZnT) are responsible for extrusion of zinc from cytosol and internalization into organelles. Expression of ZIPs and ZnTs is under control of many transcription factors dependent on cell metabolic status and zinc availability(75). Furthermore, there is a family of thiol-proteins that act as zinc-buffer and zinc-storing proteins, called metallothioneins (MT). Every MT can bind up to seven zinc ions. The expression of two MT isoforms, MT1 and MT2, is inducible from zinc and cell stress(76). MT3 is instead constitutively expressed in many tissues. MTs are able to accumulate zinc in intracellular space and interact with many other proteins, included downstream signaling of growth factors, protein kinase and phosphatase(77). MT are also released in extracellular space under peculiar conditions and have different interactions with other proteins depending on the number of zinc ions that they bind (78). The expression of MTs and other zinc-dependent proteins is regulated by many transcription factors, including MTF-1 (79).
Zinc is important for many physiologic processes, acting as structural element, intra- and extra-cellular messenger, and cofactor of enzyme reactions(80).
B and T lymphocytes internalize zinc in cytosol after physiological activation and during proliferation (81, 82). In T cells, zinc is required for downstream signaling upon interleukin-2 receptor (IL-2R) is activated by its physiological ligand IL-2(83). It is well known that zinc deficiency (ZD), a severe clinical condition with multi-organ involvement, is a cause of immunedepression and thymic atrophy with a reduction in the number of circulating recent thymic emigrants (RTE)(84). The familial form of ZD, due to inherited loss-of-function mutation of the intestinal zinc transporter ZIP4, has the complete spectrum of Achrodermatitis entheropathica, if untreated(85, 86). Thymic function and immune response, as well as other clinical manifestations of ZD, can be successfully treated with zinc supplementation(87, 88). Furthermore, mild ZD is considered as one of the causes of the reduction in thymic function in the elderly(89, 90). The precise role of zinc on thymic function remains to be elucidated: a seminal paper showed that zinc concentration in maternal and cord blood correlates with thimic size at birth(91). ZD was shown to increase inflammation in mice, this way causing increase in cortisol levels and contributing to premature intrathymic death of thymocytes(92). Moreover, hematopoietic stem cells are reprogrammed to differentiate towards myeloid lineage instead of lymphoid progenitors under ZD (93). It is important to note that there are no validated tests to diagnose ZD in its early stages: serum levels are below the normal range only under severe ZD(94). Intra-erythrocyte zinc levels and measure of MT on circulating T cells are proposed as the most sensitive markers of zinc status(95, 96). Factors contributing to ZD are age itself(97), diet poor in meat and fish, malnutrition/malabsorption, diarrhea, and use of corticosteroids(86). The world health organization (WHO) reports that more than two billion people all around the world are affected by ZD(98). Conversely, zinc intoxication is a rare condition, even if potentially severe (99).
Due to the evidence about the role of zinc in immune reactions, many clinical trials demonstrated that zinc supplementation improves thymic output and reduce the risk of infections in patients with compromised immune function or at high risk for ZD(100, 101). There are no data regarding the prevalence of ZD among cancer patients and patients under chemotherapy. Moreover, to the best of our knowledge, the role of zinc in immune reconstitution after transplant was not cover by any specific study.
1.5 Rationale of the study
Zinc is important for T cells, and zinc status impact thymic function(102). Thymic regeneration after damage is dependent on cross-talk between thymic stromal cells and thymocytes (103). Acute loss of thymocytes causes reduction in thymic size and cellularity (ref). The changes that occur in thymic microenvironnement after acute damage lead damage-resistant cells to release pro-regenerative factors in thymic microenvironment. We have recently described two such pathways centered on the production of interleukin-22 (IL-22) (104) and Bone morphogenetic protein 4 (BMP4) (105). Both the cytokines are significantly upregulated after damage, and mice deficient for them had a defect in their thymic regenerative capacity. In particular, BMP4, which is produced by thymic endothelial cells (EC) after damage, is able to trigger thymic epithelial cell (TEC) proliferation and induce expression of FOXN1, a transcription factor crucial in promoting thymocyte maturation (42). TEC regenerate, and allow this way the maturation of T-cell precursors coming from the bone marrow(106). Depending on the severity of the damage, in young mice thymus regenerates completely itself at pre-damage levels within 4-8 weeks. In older animals, and in humans, the capacity of regeneration of the thymus is dependent on pre-damage residual thymic function(107). In a pilot clinical trial, we demonstrated that patients receiving oral zinc supplementation after autologous HSCT showed increased thymic regeneration in the absence of adverse clinical events (108). For all the above-mentioned reasons, zinc could be an interesting supplement in helping immune reconstitution after HSCT.
In order to elucidate the underlying mechanisms of this process, we used a murine model to evaluate the effect of zinc supplementation in thymic reconstitution after acute damage.
2. Materials and methods
2.1 Zinc deficient diet and zinc supplementation to experimental mice
Inbred mice C57BL/6 mice were obtained from the Jackson Laboratories (Bar Harbor, USA). Zinc supplementation and zinc deficiency were started at the age of 4-6 weeks. To induce thymic damage, mice were given either sublethal and lethal TBI: sublethal-TBI (SL-sublethal-TBI), 1 x 550 cGy with no hematopoietic rescue was given in female C57BL/6 mice for all the SL-TBI experiments; lethal TBI (L-TBI) for allogeneic-HSCT (C57Bl/6 CD45.1+ female donors to CD45.2+ male recipient) was 2 x 550 cGy + 5 x 106 T cell depleted B10.Br BM cells. T cell depletion was performed with CD3 biotinylated and streptavidin-coated beads (Miltenyi Biotech). In the RAG2-GFP transplant, the procedure was the same of L-TBI and allogeneic-HSCT, but the donors were female mice expressing the green fluorescent protein (GFP) when the gene RAG2 is expressed, using the mice generated by Monroe and colleagues(109). Retro-orbital blood draws were performed after transplant. 100μl of peripheral blood (PB) were collected in EDTA-coated tubes. All TBI experiments were performed with a Cs-137 γ-radiation source. The BMP type I receptor inhibitor dorsomorphin dihydrochloride was given to indicated mice at a dose of 12.5mg/kg one day before TBI and then twice daily from day 1. Mice were maintained at Fred Hutchinson Cancer Research Center (Seattle, WA). Animals were allowed to acclimatize for at least 2 days before experimentation, which was performed according to Institutional Animal Care and Use Committee guidelines. Custom-made diets were purchased from Labdiet (St. Louis, MO). Diet for control mice was regular and balanced, whereas the 5SPA diet had the same content of all the nutrient expect of zinc content, which was 1 ppm compared to 35 ppm of the control one. Feed was irradiated. Zinc supplementation was administered orally by dissolving ultrapure zinc sulfate monohydrate purchased from Alfa Aesar (Haverhill, MA) in the drinking water. The dose dissolved in the water bottle was 1.06 g/ml, which was the dose necessary to deliver 300 mg/Kg/mouse every day based on the estimated average consumption of the zinc-supplemented water. Zinc deficient diet was administered for three weeks before TBI, and mice kept receiving the diet until the day of euthanasia. The mice that received zinc supplementation started receiving zinc 21 days before TBI and kept receiving it until the end of the experiment. In the “switch” experiment, mice under zinc-deficient diet were supplemented with zinc from day 0 (the
day of TBI) through drinking water at the same dose used in the zinc supplementation experiment.
2.2 Cell Isolation
Individual or pooled single cell suspensions of freshly dissected thymuses were obtained and either mechanically suspended or enzymatically digested as previously described. Cell counts were performed on Z2 particle counter (Beckman Coulter, Pasadena, CA), on Spark 10M chip reader (Tecan, Switzerland) or on hemocytometer. CD45− cells were enriched by magnetic bead separation using LS columns and CD45 beads (Miltenyi Biotech). Cell suspensions from spleen were prepared by tissue disruption with glass slides and filtered through a 40 μm filter. Peripheral blood samples were obtained from mice anesthetized with isoflurane and a drop of 0.5% proparacaine hydrochloride ophthalmic solution (Bausch & Lomb, Tampa, FL) applied 5 minutes prior to sampling. Blood was collected into EDTA capillary pipettes (Drummound Scientific, Broomall, PA). Anesthetized mice were restrained in a fashion that created proptosis of the eye, a capillary tube was inserted at the lateral canthus and the sinus punctured with gentle pressure and twisting motion. An aliquot of 20μl from each sample was cryopreserved for mass spectrometry analysis (see below). Peripheral blood counts were performed on Element Ht5 automatic counter (Heska, Loveland, CO).
2.3 Cell cultures
2.3.1 Ex-vivo propagated Endothelial cells (exEC)
ExEC were generated as previously described(110). Briefly, CD45−VE-Cadherin+ cells were FACS purified and incubated with lentivirus containing the E4ORF1 construct for 48 hours. Cell culture medium containing murine recombinant VEGF (10ng/ml) and FGF-2 (20ng/ml), endothelial growth supplement (bovine hypothalamus) (Alfa Aesar), sb431542 (Tocris Biosciences), heparin (50μg/ml) (Sigma-Aldrich), 1% Glutamax (Life Technologies), 1% non-essential amino acids (Life Technologies), 1% HEPES Buffer (Life Technologies), 1% antibiotic-antimycotic (Life Technologies) was replaced every 48h. Cells were cultured in presence of ultrapure zinc sulfate monohydrate purchased from Alfa Aesar (Haverhill, MA) at the concentration of 5μM, 10μM, 25μM, 50μM, 100
μM for 24 hours. Cells were also treated with zinc sulfate at the concentration of 1 nM, 2 nM, 5 nM and 10 nM in presence of the cell permeabilizer sodium pyrythione (Sigma Aldrich) at the concentration of 50 μM. The GPR39 agonist TC-G1008 was purchased from Tocris Biosciences (Bristol, UK).
2.3.2 C9 and TE-71
Mouse C9 (cTEC) and TE-71 (mTEC) cells were kindly provided by A. Farr, University of Washington. Cells were treated with zinc sulfate monohydrate purchased from Alfa Aesar (Haverhill, MA) at the concentration of 5μM, 10μM, 25μM, 50μM, 100 μM for 24 hours.
2.3.3 Co-culture experiments
Thymic extracellular fraction, also referred as “supernatants” (SN) were obtained by mechanically dissociating thymic tissue in defined volumes of PBS buffer. Thymi were collected from control and zinc-supplemented mice, at day 0 and day 2 after SL-TBI. Aliquots of 25 μl were incubated with exEC for 24h.
2.2 ELISA and Western Blot experiments
For detection of BMP4, whole thymus lysates were prepared by homogenizing tissue in RIPA buffer (50 mM Tris pH 7.6, 150 mM NaCl, 1% NP-40, 1% SDS, 0.01% sodium deoxycholate, 0.5 mM EDTA, and protease inhibitors (Thermo, A32955). ExEC were harvested after 48 hours of exposure to zinc directly into RIPA buffer. The resulting samples were quantified using the BMP4 ELISA kit (LSBio, LS-F13543) and read on a Spark 10M plate reader (Tecan, Switzerland) after loading a normalized equal amount of proteins (BCA quantification, Thermo Scientific, Waltham, MA). For Western Blot, the samples were lysate in the same way that we used for ELISA. GPR39 antibody was purchased by Novus Biological (Centennial, CO). The proteins were resolved on 12% SDS-PAGE gels, transferred onto PVDF membranes (BioRad). Following blocking with PBS containing 3% skim milk, the membranes were incubated overnight at 4°C with GPR39 primary antibody (1:1,000). Following incubation with peroxidase AffiniPure secondary antibodies (goat anti-mouse IgG, cat. no. 115-035-003; goat anti-rabbit IgG, cat. no. 111-035-003; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA,
USA) for 1 h at room temperature, the blots were then analyzed using the ECL detection system or scanned with an Odyssey Infrared Imager (LI-COR Biosciences, Lincoln, NE, USA).
2.3 Flow cytometry and FACS sorting
For flow cytometry and cell sorting, surface antibodies against CD45 (30-F11), CD31 (390 or MEC13.3), TER-119 (TER-119), CD4 (RM4-5 or GK1.5), CD8 (53-6.7), TCRβ (H57-597), CD3 (145-2C11), CD44 (IM7), CD25 (PC 61.5), CD62L (MEL14), MHC-II IA/IE (M5/114.15.2), EpCAM (G8.8), Ly51 (6C3), CD11c (HL3), IL-7Rα (A7R34), CCR6 (140706), CD45.1 (A20), CD45.2 (104), ki-67 (16A8), and PDGFRα (APA5) were purchased from BD Biosciences, BioLegend or eBioscience. Ulex europaeus agglutinin 1 (UEA-1), conjugated to FITC or Biotin, was purchased from Vector Laboratories (Burlingame, CA). GPR39 conjugated to FITC was polyclonal and purchased from Signalway Antibody (College Park, MD). For spleen and peripheral blood samples, erythrocyte lysis with ACK buffer (Thermo Scientific, Waltham, MA) was performed before staining. Flow cytometric analysis was performed on a Fortessa X50 (BD Biosciences) and cells were sorted on an Aria II (BD Biosciences) using FACSDiva (BD Biosciences) For all assays requiring analysis of intracellular cytokines or phosphoproteins, cells were fixed and permeabilized by using Fix Buffer I and Phospho-Perm Buffer III purchased from BD Bioscience. For zinc intracellular detection by flow cytometry, Fluozin-3 AM was purchased from Thermo Fisher (Waltham, MA) and cells were treated per protocol. Analysis of flow cytometry experiments was performed on FlowJo (Treestar Software).
2.4 Proliferation Assays
In vitro cell proliferation of exEC was measured using the CellTiter Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI). For peripheral T cell stimulation, cells were cultured for 48 hours in RPMI 1640 with 10% FBS in 96 well- CD3 coated plates, purchased from BD Biosciences, in presence of CD28 antibody. Lymphocyte proliferation was measured using the Aqueous One Solution Cell Proliferation Assay
from Promega (Madison, WI). To determine the absorbance, plates were read on a Spark 10M plate reader (Tecan, Switzerland).
2.5 Microarray
Thymic non-hematopoietic stromal cells were isolated using CD45 MACS cell depletion. Microarray analysis was performed on an Affymetrix MOE 430 A 2.0 platform in triplicate for untreated mice as well as day 4 and 7 after TBI. To obtain sufficient RNA for every timepoint, thymic ECs of several mice were pooled. All samples underwent a quality control on a bioanalyzer to exclude degradation of RNA. RNA extraction, control of RNA integrity with a bioanalyzer, and cRNA labeling and hybridization, was performed by the Integrated Genomics Core Facility of Memorial Sloan Kettering Cancer Center. GSEA analysis was performed using the GSEA tool v2.0 of the Broad Institute (http://software.broadinstitute.org/gsea). Comparisons were made to known signaling pathways from the Gene Ontogeny database (GO numbers: 0045446, 0010594, 0001938, 0001763, 0002040, 2000351, 0030510) The microarray data used in this study have been deposited in the GEO under accession number GSE106982.
2.6 PCR
Reverse transcription-PCR was performed with iScript clear gDNA cDNA synthesis kit (Bio Rad). PCR was done on CFX96 (Bio Rad) with iTaq Universal SYBR Green (Bio Rad). Relative amounts of mRNA were calculated by the comparative ∆C(t) method. SYBR Green gene expression assays for qPCR, including Bmp4 (Mm00432087_m1), Foxn1 (Mm00433948_m1), beta-actin, Top1 were all purchased from Life Technologies (Carlsbad, CA) and Bio Rad (Hercules, CA).
2.7 ICP-MS
Thymi were harvested and frozen immediately after. Before freezing them, all samples were weighted. Supernatants were generated by mechanical dissociation of the thymi and subsequent separation of the cellular fraction from the non-cellular fraction. Thymocytes were collected by mechanical dissociation of the thymi and separation of the stromal component. Cells were counted before freezing them. Aliquots of 20 μl from peripheral blood were collected from EDTA tubes. All samples were digested in a similar fashion. Initial and final weights were recorded and a 500uL aliquot added by the user was taken into account to determine concentrations in μg Zn/ L of solute. All samples
were weighed for a “tare” weight. 0.5mL of an aliquot containing 50% v/v ccTrace metal grade HNO3 and 10% ccH2O2 was added to every sample. (ccHNO3 68%, ccH2O2 30%). The samples were openly digested in a fume hood for 3 hours, vortexed then reheated for an additional 1 hour at 87-91C until visual solubilization was achieved. Samples were brought up to a ~1.5mL final volume and weighed so a mg Zn/g thymic tissue or supernatant could be calculated. All samples were analyzed at a 10x dilution on an ICAP RQ ICP-MS (University of Washington, Oceanography Lab) in 2% optima grade nitric acid. Three isotopes of zinc were analyzed and cross referenced for isobaric interferences Zn66 was chosen as the preferred isotope having the most signal with the least amount of interferences. All final results are internal standard (ISTD) corrected to rhodium for physical interferences inside the plasma.
2.8 Statistics
Statistical analysis between two groups was performed with the nonparametric, unpaired Mann-Whitney U test. Statistical comparison between 3 or more groups was performed with the nonparametric, unpaired Kruskall-Wallis test. In Fig. 4, statistics were generated using a two-way ANOVA with Tukey’s multiple comparison test.All statistics were calculated using Graphpad Prism and display graphs were generated in Graphpad Prism or R.
3. Results
3.1 Zinc deficiency impairs thymic reconstitution after TBI
Mice under zinc-free diet for three weeks showed lower thymic cellularity when compared to mice that received complete diet (Fig. 1 A-B). After sublethal TBI, zinc deficient mice showed impaired thymic reconstitution in terms of total cellularity and organ size (Fig. 1C). Thymic capacity of reconstitution can be restored in zinc deficient mice when zinc was supplemented with drinking water starting from day 0 (Fig. 1D). As expected, the most abundant cells were the thymocytes: the classic CD4/CD8 plot, gated on CD45+ events, showed a decrease in DP and SP thymocytes in mice under ZD (Fig. 1E). Interestingly, DN are reduced after three weeks of zinc restriction, but not significantly (Fig. 1F), whereas the total number of DN was sharply reduced in ZD mice after TBI. At steady state, a sub analysis of CD90+ DN (Thy1+) showed no significant different in these cells and through the four sub-stages of differentiation (from DN1 to DN4) between control and ZD (Fig. 1H-I). Conversely, DP, CD4+, and CD8+ were severely impacted from ZD before and after TBI (Fig 1 J-K). Also the principal stromal populations, the cTEC and mTEC, were negatively impacted by ZD (Fig 1L). These findings clearly demonstrated that pre-damage levels of zinc in the body cause reduction in thymic function, having a negative impact on post-damage thymic reconstitution. About the thymocytes, the early damage cause by ZD seems to affect the late maturation stage between DN and DP. Zinc administration can rescue thymic capacity of regeneration.
3.2 Zinc supplementation improves thymic regeneration after damage
Since ZD impairs thymic function and immune recovery after TBI, we wanted to test the effect of zinc supplementation on thymic reconstitution. Mice were put on zinc supplementation (300 mg/Kg/day of zinc sulfate monohydrate in the drinking water) (111). Mice were then treated with SL-TBI and kept under zinc supplementation until the day of harvesting (day 3,7,2, and 42) (Fig. 2A). No difference in thymic cellularity was observed after three weeks of treatment between control and zinc-supplemented mice, but mice that received zinc supplementation showed improved thymic reconstitution at all the timepoints after SL-TBI (Fig. 2 B). We did not observe any adverse event during
the whole course of treatment. In this model, DN thymocytes showed significantly increased numbers only four weeks after TBI, whereas DP and CD4+ tend to increase earlier (Fig. 2 C-F); stromal cells, especially TEC (Fig. 2 G-H), showed good response to zinc treatment. It is well documented that, after acute damage, TEC are the target of many regenerative factors and respond to these stimuli by replicating. Both the absolute number (Fig. 2I), and the proportion (Fig. 2H) of proliferating TEC, measured by the expression of Ki-67, were higher in the thymi from zinc treated mice. These data suggest that zinc improves regeneration of thymocytes after damage and triggers the production of regenerative factors.
3.3 The BMP4 pathway of thymic regeneration is targeted by zinc
Since we found increased proliferation of TEC after zinc supplementation, we tested the direct effect of zinc sulfate (10, 25, 50, and 100μ) on cell lines of mouse cortical (C9) and medullary (TE-71) TEC, but we did not observe increased proliferation after 24 and 48 hours (data not shown). We recently demonstrated that thymic endothelial cells (EC) represent a radio-resistant niche that is able to respond to acute damage with the production of bone morphogenetic protein 4 (BMP4), a cytokine that exerts angiocrine functions by stimulating the proliferation of TECs. We know from many papers that zinc plays an important role in vascular integrity and response to stress of ECs in many tissues(112). We thus aimed to measure BMP4 after SL-TBI in thymi of mice that received zinc supplementation: BMP4 protein was more expressed in zinc-treated mice after TBI, especially at day 10 (Fig. 3A-B). Moreover, Bmp4 expression at the mRNA level was significantly higher in EC sorted from zinc-treated mice at day 7 (Fig. 3C); there was a latency between the peak in mRNA levels of Bmp4 and the peak of BMP4 production at the protein level observed at day 10. As a confirmation of this, there was no difference in the expression of Foxn1, whose expression is stimulated by BMP4, in cTEC sorted from control and zinc-treated animals at day 7 (Fig. 3C). Altogether, these findings suggest that zinc may improve thymic regeneration by stimulating the production of BMP4 from EC. Using a technique to constitutively activate the Akt pathway in ECs using the prosurvival adenoviral gene E4ORF1 (29), EC can be propagated and expanded ex vivo (exEC) while maintaining their phenotype and vascular tube formation capacity. These exEC showed increased production of Bmp4 when treated for 24 hours with zinc sulfate at concentrations of 50 μM and 100 μM (Fig.
3E). The same finding was confirmed at the protein level, when exEC were harvested after 48 hours of incubation with zinc sulfate at 100 μM (Fig. 3F). Consistent with our hypothesis that zinc is involved in the in vivo pathway of BMP4 production, treatment with the pan-BMP-receptor inhibitor dorsomorphin dihydrochloride (12.5mg/kg) abrogated the effect of zinc supplementation on thymic cellularity (Fig. 3G-H). Furthermore, mice under zinc-deficient diet showed significantly lower levels of BMP4 at day 10 after TBI when compared to controls and mice that received zinc supplementation from day 0, after three weeks of ZD (“switch”) (Fig. 3I). These findings suggest that zinc plays an important role on the BMP4 pathway.
3.4 Zinc is released in thymic extracellular space after stress-induced thymocyte death
In order to clarify how zinc plays a role in thymic regeneration, we aimed to measure endogenous changes in zinc levels in control mice after TBI. Inductively coupled plasma mass spectrometry (ICP-MS) analysis of the samples showed that the total amount of zinc in the thymus followed the same trend of thymic cellularity (Fig. 4A); conversely, the concentration of zinc in the isolated thymic extracellular space, also referred as supernatant (SN), increased early after TBI and seven days after damage (Fig. 4B). The relative contribution of extracellular zinc to total thymic zinc increased after damage and went back to baseline at day 14, when approximately 75% of thymus is reconstituted (Fig. 4C). This trend suggests a mobilization of the pool of zinc from intra- to extracellular space after damage: this hypothesis was confirmed by the measure of the concentration of zinc/mg of thymic tissue, that showed a reduction between day 0 and day 4 after SL-TBI followed by return to baseline, and of the bioavailability of extracellular zinc normalized by the number of thymic cells that showed the opposite trend (Fig 4D). Based on all the phenotypic data that we collected, thymocyte are the population candidate to be responsible for the extracellular mobilization of the zinc pool after damage and its uptake during regeneration. In fact, thymocytes die after acute injury and start repopulating the thymus about day 7 after SL-TBI. If we consider the ratio between zinc in the SN/zinc in isolated thymocytes, there was a clear displacement of the zinc pool leading to sharp extracellular/intracellular increase after TBI (Fig 4E).
Observing what happened under zinc supplementation, zinc/mg of tissue is higher than control after three weeks of treatment; then, zinc concentrations followed the same trend
as the control, but at higher values (Fig 4F). This same dynamic was observed when we measured total zinc content in isolated thymocytes (Fig. 4G). Zinc in SN from zinc-treated mice mirrored the same trend as the controls, with slightly higher measurable levels of zinc (Fig. 4H). Since the acute depletion of double positive (DP) thymocytes after damage is known to be a potent signal for the production of regenerative factors such as interleukin-23(104), it is conceivable that dying thymocytes can trigger other cells to produce regenerative factors by releasing substances in the extracellular space. This mechanism was described for other substances, and zinc itself was demonstrated to be involved in such a pathway in other tissues (113). Of interest, when we measured zinc levels on peripheral blood (PB) before and after TBI, zinc did not show difference between mice that received zinc treatment and control mice (Fig. 4J). Of interest, zinc levels in PB went significantly down starting from day 7, remaining lower than baseline until later timepoints. In summary, the biological meaning of these findings could be: i) under normal conditions and during thymic regeneration, thymocytes internalize zinc, accounting for the majority of thymic zinc content; ii) after TBI, death of thymocytes leads to displacement of zinc from intra- to extracellular space, extracellular zinc potentially acting as a messenger for radio-resistant cells to produce regenerative factors such BMP4; iii) zinc supplementation may act by exacerbating this phenomenon; iv) PB is not reliable to assess variations in zinc status in mice.
3.5 Zinc released in the extracellular space stimulates endothelial cells to produce BMP4
Focusing on stromal cells, which are CD45-, we wanted to look at the changes in the expression of zinc-related genes with a RNA-macroarray. Among the members of Slc39a family, responsible for encoding the ZIP proteins, many of them were upregulated at day 4 and 7; the most upregulated was ZIP8 (Fig 5A). It is of interest that the expression of the zinc efflux transporter SLC30A1 (ZnT1) (Fig 1B) and of the inducible zinc-binding proteins MT1 and MT2 (Fig. 1C) increased at day 4 and day 7 after TBI. Recent reports showed that many functions of EC are regulated by zinc via the expression of a surface receptor called GPR39. This receptor is G-protein coupled and acts by translating extracellular zinc signals into release of intracellular second messengers such as IP3 and calcium (113). No data are available about the role of this receptor in thymus. According to our RNA-macroarray on CD45- cells, GPR39
expression increased significantly at day 4 and day 7 after TBI (Fig. 5D). In order to identify the potential site of the target of zinc administration (intra-, extracellular, or both) on EC, we selectively increased intracellular zinc concentration of exEC by treating them with the zinc ionophore sodium pyrythione, but we did not observe any increase of Bmp4 expression (Fig. 5E). The latter finding suggested that zinc cause BMP4 production from ECs by binding to a surface receptor more than through zinc internalization. To test the hypothesis that zinc released in extracellular space after damage induces production of BMP4 from EC, we co-cultured exECs in presence of supernatants collected from control and zinc-treated mice at day 0 and 48h after TBI: we observed no differences in Bmp4 production at day 0, whereas we observed increased production of Bmp4 in exEC cultured with supernatant harvested 48h after TBI from zinc-treated mice (Fig 5F). This latter finding suggests that zinc itself released in the supernatant mediates the increase of Bmp4 expression. These findings suggest a role for extracellular stimulation of GPR39. We tested GPR39 expression on different thymic subpopulations, and we found that, at baseline, only the stromal cells have significant expression of the receptor (Fig. 5G), whereas it was almost undetectable on the surface of the thymocytes. EC showed to have high expression of GPR39, increased expression at day 4 and stable higher expression at day 7 (Fig. 5H). Also, exEC were positive for GPR39 when tested with a western blot antibody (Fig. 5L). To clarify the specific role of this receptor in the response to zinc, we treated exECs with the selective GPR39 agonist TC-G1008: stimulation of exEC with this molecule for 24 hours produced increase in Bmp4 expression that was higher than with zinc treatment (Figure 5M). Bmp4 did not further increase by combining zinc and TC-G1008.
3.6 Clinical applications: zinc supplementation promotes the generation of recent thymic emigrants in a mouse model of allogeneic stem cell transplantation
The importance of thymic regeneration in the clinical setting is clear after myeloablative HSCT: severe damage of the thymus due to the combination of drugs and radiotherapy leads to prolonged lymphopenia and reduction in TCR diversity that have a bad impact on survival(55). We used a model of minor-mismatched murine allogeneic T-depleted HSCT (Fig. 6A): zinc supplementation promoted thymic reconstitution also in this setting
of severe damage. As in the SL-TBI model, thymic cellularity did not change after three weeks of zinc administration, but thymic cellularity was more rapidly reconstituted after HSCT in mice that received zinc treatment (Fig. 6B). The four populations of thymocytes were all stimulated to regenerate (Fig. 6C-F), and the same trend was observed for TEC (Fig. 6G-H). Thymic engraftment of donor-derived CD45.1+ thymocytes was more rapid in zinc-treated mice (data not shown). Of interest, in this model of myeloablative HSCT cTEC and mTEC reconstitute faster than the control under zinc treatment and in an earlier phase than thymocytes; in fact, increases of DP, CD4+, and CD8+ occurred later. In order to track the actual production of higher numbers of recent thymic emigrants (RTE) in periphery, we performed mouse HSCT by using donors expressing the green fluorescent protein (GFP) in thymus-derived naïve T cells (Gig. 6I). On peripheral blood, mice that received zinc supplementation showed higher levels of GFP+ CD4+ and CD8+ lymphocytes five weeks after HSCT, when the lymphocyte count starts coming back (Fig. 6J). The improved production of naïve T cells persisted in spleen harvested eight weeks after HSCT (Fig. 6K). We also performed a lymphocyte proliferation assay for CD4+ and CD8+ lymphocytes isolated from spleen at day 42 and day 63, but there were no differences between control and zinc-treated mice (data not shown). Taken together, these data suggest that improved thymic regeneration caused by zinc can help immune reconstitution after HSCT by increasing the production of thymic-derived naïve T cells.
4. Discussion
At the cellular level, zinc is essential for proliferation and differentiation of many cells, but zinc homeostasis is also involved in signal transduction and apoptosis. Cells depend on a regular supply of zinc and make use of a complex homeostatic regulation by many proteins. The plasma pool, which is required for the distribution of zinc, represents less than one percent of the total body content (114, 115). Despite its important function, the body has only limited zinc stores that are easily depleted and cannot compensate longer periods of zinc deficiency. Additionally, during inflammation there are changes in zinc homeostasis due to the action of cytokynes, leading to sequestration of zinc into liver cells and subsequently to hypozincemia (116). Alterations in zinc uptake, retention, sequestration, or secretion can quickly lead to zinc deficiency and affect zinc-dependent functions in virtually all tissues, and in particular in the immune system. In humans, the most prominent example for the effects of zinc deficiency is Acrodermatitis Enteropathica, a rare autosomal recessive inheritable disease that causes thymic atrophy and a high susceptibility to infections(117). All symptoms can be reversed by nutritional supplementation of excess zinc. One of the firsts well-known phenomena associated to zinc deficiency was immune depression: zinc deficiency does not affect just a single component of the immune system; the effects are complex, occur on many levels, and involve the expression of several hundred genes(118). T cells are the most sensitive to zinc levels(119).
One major mechanism by which zinc affects immunity is its role as a signaling ion. The first example was protein kinase C (PKC), which has been identified as a molecular interaction partner for zinc in T cells(120). Its N-terminal regulatory domain contains four Cys3His zinc binding motifs. Zinc treatment stimulates PKC kinase activity, its affinity to phorbol esters, and binding to the plasma membrane and cytoskeleton. Furthermore, zinc chelators inhibit the induction of these events by physiological activators of PKC(121). Zinc also plays a very important role as intracellular messenger upon IL-2 binds its receptor on T cell surface(83).
Interestingly, a comparison between alterations of the immune system during zinc deprivation and aging shows many similarities, indicating a possible relation between immunosenescence and zinc deficiency(122). In both cases, clinical manifestations are anergy, thymic atrophy, reduced cell-mediated cytotoxicity and T-helper activity. As expected, the decline in immune function in aged patients leads to augmented incidence
and mortality from infectious diseases, such as pneumonia and tuberculosis (123, 124), and re-infections with herpes zoster(125). Restriction of T cell repertoire, decline of thymic function, and T cell exhaustion during aging lead also to increased frequency of autoimmune diseases(126).
The precise mechanism through which zinc deficiency causes thymic atrophy is largely unknown. A reduction of zinc availability increases the rate of intrathymic death of T cell precursors, either by elevating glucocorticoid production, or via the loss of zinc-dependent regulation of apoptosis in immune cells(92, 127). The role of zinc on thymic autocrine hormones, as thymulin, remains largely unknown, but also these small peptides are zinc-dependent(128).
In our model of zinc deprivation, we show that even short-term deprivation of zinc in young animals has a strong impact on thymic function: the production of double positive (DP) and single positive (SP) thymocytes was strikingly reduced in absence of evident signs of disease in mice. Interestingly, double negatives (DN) were less affected than the other thymocyte subsets by zinc deficiency: the great intrathymic expansion of thymocytes occurs between the DN4 and the DP stage. This phase, and the later stages of differentiation, may be more sensistive to zinc deficiency than the earlier ones(129). The proportion of proliferating DN was in fact reduced in our data. In the work of Wong et coll., zinc supplementation led to increased proliferation of DN towards the DN4 stage in aged mice(111), which is consistent with our findings.
We also demonstrate, to the best of our knowledge for the first time in literature, that pre-TBI levels of zinc have a deep negative impact in thymic reconstitution.
Since zinc deficiency is difficult to diagnose due to the lack of specific tests, and given the potential underestimated prevalence of this condition among cancer patients, our study stress the need of accurate pre-transplant screening for nutritional status in HSCT candidates and the urgency of new diagnostic tool to diagnose poor zinc status.
After TBI, all thymocyte subsets showed sharp reduction and worse reconstitution under zinc deficiency when compared to mice under regular diet, but total DN thymocytes were significantly reduced only at day 7. For what concerns epithelial cells, mTEC were already reduced at steady state after three weeks of zinc deprivation, whereas cTEC showed only non-significant reduction. Nevertheless, after TBI both mTEC and cTEC regenerated worse than the control. The difference between the two epithelial
populations was observed also under different conditions: for example, cTEC decline prominently during age-related thymic involution(130). We recently demonstrated that cTEC are more resistant to acute thymic damage than mTEC(105), even if other reports showed that mTEC are more resistant than cTEC to DNA damage(131). cTEC support early stages of T-cell development and hence determine the overall lymphopoietic capacity of the thymus(132). Zinc is important for epithelial integrity, especially in intestine and skin(133, 134). A potential explanation for the higher sensitivity of mTEC to zinc deprivation could be their interdependence with the DP thymocytes, for the same mechanism that cause death of TEC after radiation-induced DP depletion (105). Epithelial-lymphoid interactions in thymic microenvironment are bidirectional, the lack of one population having negative effects on the other(40).
As a proof of the importance of zinc in post-TBI thymic reconstitution, zinc supplementation starting at day 0 after three weeks of zinc deprivation was able to partially restore thymic capacity of regeneration.
Given these results, and on the basis of many papers showing that zinc supplementation has a protective effect against acute cell stress(71, 135), we administered zinc sulfate starting from day -21 before TBI. At steady state, total thymic cellularity and all the subsets were not different between mice that received zinc supplement and controls. After SL-TBI, we observed improved regeneration both of the lymphoid and the epithelial component of the thymus in mice that received zinc. The regeneration of CD4+ and CD8+ thymocytes usually occurs after the repopulation of DP; in our data, we observed a better expansion of DP followed by improved production of SP CD4+ and CD8+ in zinc treated animals, this latter finding suggesting a physiological stimulation of thymopoiesis by zinc. Also in this setting, DN seemed to be less affected by zinc treatment. After damage, the blood-thymus barrier allows lymphoid-oriented precursors coming from bone marrow to enter the thymic cortico-medullary junction in higher numbers than under normal conditions(58). The increase in thymic cellularity that we observed in zinc treated mice depended mainly on higher number of thymocytes, and this could be a result of direct effect of zinc on the maturation of thymocytes, as shown in several papers(97, 111), or could be due to increased production of lymphoid-oriented progenitors in the bone marrow led by zinc(93). Further studies on the effect of zinc on bone marrow reconstitution are needed to clarify this point and will be object of future investigations.
The better reconstitution of TEC (both cTEC and mTEC), and the higher proportion of proliferating TEC after TBI in the zinc-treated mice suggest that zinc might promote reconstitution via the stimulation also of the thymic parenchyma. We did not find increased proliferation in cell lines of TEC when we treated them in vitro with different concentrations of zinc. So far, the only known mechanisms of thymic epithelial regeneration after damage depend on the production of cytokines from other cells, such as IL-22 from innate lymphoid cells (ILC), IL-23 from dendritic cells (DC)(104), and BMP4 from endothelial cells (EC)(105), that stimulate TEC and precursors of TEC to proliferate.
BMP4 is a member of bone morphogenic proteins, a family of peptides involved in embryogenesis and homeostasis of many tissues(136). BMP4 is necessary for thymic organogenesis and maintenance of Foxn1 expression in TEC of adult animals (137-139). EC are an important source of BMP4 under stress conditions(140), they being more resistant than other cells to acute thymic stress. We demonstrated that EC respond to acute depletion of thymocytes with increased release of BMP4 in thymic microenvironment, this way targeting regeneration of TEC(105). Zinc supplementation improves BMP4 production from thymic EC both in vivo and in vitro. We have two in vivo evidences for this zinc-BMP4 axis: injection of the BMP-receptor inhibitor dorsomorphin(141) is able to abrogate the effect of zinc supplementation on thymic cellularity at day 10 after TBI, and zinc deficiency negatively affects the capacity of producing BMP4 in response to damage. What remains to be elucidated is the relative contribution of intra- and extracellular zinc under treatment to this mechanism.
Zinc concentration in organs is kept under strict homeostasis in normal conditions (142). After acute damage, and during organ regeneration, zinc concentration changes in other tissues, such as liver(143). We demonstrate herein that total thymic zinc follows the same trend of thymic size, weight, and cellularity after acute stress, showing a rapid decrease and a following increase that brings zinc amount back to baseline. There is a net loss of tissue and a consequent loss of zinc. Interestingly, after TBI we observed concomitant increase of zinc levels in the extracellular fraction, the supernatant (SN). The rise of zinc in SN was even higher if we consider the relative contribution of extracellular zinc on total thymic zinc and zinc bioavailability normalized for the number of cells.
These data suggest that thymocytes, which represent about 90% of cellularity and are the population that is most sensitive to damage, are responsible for the decrease and the regrowth of total thymic zinc.
This is also consistent with our phenotype data showing strict dependency of thymocytes from zinc status. Of interest, we showed that also zinc concentration/mg of tissue decreased after TBI: this finding means that not only zinc-rich cells are lost after TBI, but also that zinc is releases from cells after TBI in an active or passive way. The increase of zinc in the extracellular space that we observe was probably less pronounced than in reality because of the wash-out of small molecules operated by blood stream. Taken together, these findings suggest a displacement of zinc from the intra- to extracellular space that normally occurs during thymocyte death. When we isolated the thymocytes, we confirmed that they effectively were the population that contained the highest part of thymic zinc. Zinc in thymocytes isolated from zinc-treated mice was higher than the controls after three weeks of treatment. After TBI, thymocytes from zinc-treated mice still showed zinc levels that were higher than the controls, but also zinc in SN was higher. The non-significant difference between zinc-treated and control samples can be explained by technical reasons and the above-mentioned phenomena of the wash out.
The exact mechanism of cell death of the thymocytes after acute damage is unknown: the large majority of thymocytes naturally undergo apoptosis during the intrathymic selection(48). This pathway of cell death usually does not trigger the production of regenerative factors from stromal cells involved in tissue rebuilding and is instead supposed to be essential for the survival of the TEC. Apoptotic death of T-cell precursors during selection does not cause release of intracellular content in the extracellular space(144). Conversely, other pathways of cell death, such as pyroptosis, necrosis, and necroptosis, are known to be sensitizing towards cells responsible for tissue repair (145). This mechanism, well described especially in skin and intestine, is based on the loss in the extracellular space of small substances that are normally sequestered intracellularly, such as, among others, ATP, HGMB1, Ca2+, uric acid, and formyl peptides(146). These substances, which are also called “alarmins”, have their receptors expressed on the surface of cells that trigger tissue regeneration(147), toll-like receptors (TLR) being the ones that are best characterized for this role(148). The presence of zinc in extracellular space was demonstrated to be crucial for epidermal integrity(149), where
zinc formulations are clinically used to promote wounds repair(150), and release of zinc also through exocytotic vesicles was described in many tissues under normal and pathologic conditions(151). In summary, mass spectrometry data from our experiments highlight a double role for zinc in thymus: thymocytes tend to use zinc for their intracellular machinery during maturation, whereas release of zinc in extracellular space can participate to mechanisms of tissue repair.
The way T cells can accumulate zinc was described in many papers: T lymphocytes actively internalize zinc during replication via the expression of the transporter ZIP6: the knock-out of this transporter blocks zinc accumulation that is observed upon TCR stimulation and leads to impairment of T cell functions(82). This evidence fits with our observations that mice under zinc deficient diet showed slower maturation of thymocytes and mice under zinc supplementation accumulated zinc in their thymocytes. Cells buffer increases of zinc availability by promoting the expression of metallothioneins: these proteins are able to sequester zinc in order to keep the concentration of intracellular free-zinc within certain range(152).
Transcriptome data from RNA-macroarray of sorted CD45- cells from control animals showed increased expression of ZnT1 and MT1 and MT2 after TBI: this is a clear sign of exposure of stromal cells to higher concentrations of zinc(76). MT1 and MT2 were demonstrated to be important for tissue regeneration in other epithelial organs, such as liver(153), and these findings may suggest a similar mechanism also in thymic endogenous regeneration.
We cannot exclude that zinc is also internalized in stromal cells after damage, as the increase in some ZIP expression may suggest, but the fact that zinc stimulation with high extracellular, but not with increased intracellular levels, promotes BMP4 production in EC suggest that its prevalent action is through the interaction with some extracellular receptor.
There are no data in literature linking the production of BMP4 from EC and zinc stimulation, but mice that are germline-null for Slc39a13 (ZIP13) showed lack of response in tissues whose development is dependent on BMP/TGFβ pathway(74). Very little is known about the events that are upstream to the transcription of BMP4.
GPR39 was recently discovered as a “zinc sensor” or “zinc receptor”(154). Upon binding zinc in its extracellular domain, the conformational change of GPR39 intracellular
domain caused the release of the Gα subunit, which acts as a GTPase: this leads to the activation of phospholipase ϒ (PLCγ) and a cascade of intracellular second messengers as IP3, DAG and Ca2+. The final effect of GPR39 activation is the phosphorylation of many transcription factors such as MEKK, ERK1/2, and AKT (155). GPR39 is recognized as a regulator of mood via the interaction with extracellular zinc in neurons(156). The role of GPR39 in in tissue repair was demonstrated in gut, with a direct effect of zinc on epithelial stem cells in the crypts(157), and in skin, where zinc released from mast cells after damage was shown to trigger IL-6 production from GPR39+ fibroblasts and consequent wound healing (158). Of interest, there is only one paper focusing on the role of GPR39 in the immune system, and it reports reduced total thymic cellularity in mice with germline knock-out of GPR39(159). GPR39 is expressed on EC, where it regulates cell integrity, formation of vessels, expression of cell adhesion molecules and production of cytokines through the interaction with zinc ions(160, 161). In our data, we demonstrate for the first time that GPR39 is expressed in thymus, with expression in stromal cells being remarkably higher than in thymocytes. RNA macroarray on CD45- cells showed upregulation of Gpr39 after damage, and this finding was consistent with the observation of increased proportion of GPR39+ events in many populations that we found in flow cytometry. Moreover, also exEC resulted positive for GPR39 expression. Increased expression of this receptor could be induced by radiation damage itself and might represent an endogenous mechanism of protection against radiation injury.
Cocultures of exEC with SN harvested from different conditions (radiated and non-radiated, control and zinc-supplemented mice) showed that zinc released in the extracellular space is actually able to trigger BMP4 production in EC also ex vivo. The fact that exEC exposed to the selective GPR39 agonist TC-G 1008(161) showed higher production of Bmp4 with no further increase when treated together with zinc, suggests that GPR39 pathway is actually involved in this zinc-dependent axis of regeneration. These preliminary data are promising also from the point of view of their potential clinical applications, but they need to be confirmed in a specific project. It is very difficult to identify specific targets responsible for the effect of zinc due to its interaction with more than 3000 proteins and the variable expression of ZIP, ZnT, and MT in different cells and under different conditions. The hypothesis of one receptor responsible for the cross-talk between zinc-rich dying thymocytes and regeneration-initiating cells is very intriguing, but it could also be too simplistic. GPR39 is also expressed on the surface of
other cells involved in thymic regeneration, such as ILC and DC, but also on the surface of both cTEC and mTEC. Furthermore, zinc is able to stimulate BMP4 production about day 10, but we observed increased thymic cellularity also before then. There could be some other unknown pathway of regeneration which is involved in zinc response and still needs to be elucidated.
A detailed study of GPR39 downstream signaling in EC both in vivo and in vitro, the production of conditional GPR39 knock-out mouse models (in order to silence the expression only in EC after damage, for example), and the in vivo administration of different GPR39 agonists, alone or in combination with zinc supplementation or ZD diet, will be object of further studies.
Finally, thymic regeneration is clinically important especially after myeloablative bone marrow transplant. Zinc was able to promote thymic reconstitution also after minor-mismatched allogeneic HSCT. We decided to use a model of T-depleted graft in order to isolate the specific contribution of the thymic component to T cell regeneration. Furthermore, we chose an immunological low-risk model also because the primary purpose of this first project was not to investigate the role of zinc in protection from GvHD. Thymic reconstitution after HSCT was expectedly slower than in the SL-TBI model, but it showed the same dynamics. Zinc promoted better thymic regeneration after HSCT by improving thymocyte maturation and stromal regeneration. The confirmation of improved and accelerated production of recent thymic emigrants (RTE) came from the RAG2-GFP transplant: RAG1 and 2 are enzymes that are expressed during the recombination process regarding the B cell receptor (BCR) and the TCR genes. We used a minor MHC-mismatched C57B6→C57B6 HSCT model to take advantage of RAG2-GFP reporter bone marrow. Detection of GFP+ peripheral T cells in this transplant model indicates newly developed T cells that have undergone recent T-cell receptor gene rearrangement, a marker of RTE. This is the equivalent of T cell receptor excision circles (TRECs) that are used in clinical practice in order to assess immune reconstitution after HSCT(162). On peripheral blood five weeks after HSCT, and in spleen eight weeks after HSCT, we found more CD4+ and CD8+ RTE identified through the persistence of GFP. These findings demonstrate that zinc administration can improve thymopoiesis after allo-HSCT and promotes the development of new mature peripheral T cells, basically confirming in an animal model the preliminary finding of a pilot trial that we previously performed on a small number of aged patients that
received zinc supplement after auto-HSCT (108). Improved production of thymic-derived naïve T cells originated from the donor was demonstrated to be protective towards leukemia relapse after HSCT and GvHD(163) (164), whereas naïve T cells infused with the graft are potentially responsible for higher risk of GvHD(165, 166). Zinc was demonstrated to be effective in the control of allogenic reaction only in one study performed on mixed lymphocyte cultures (MLR)(167). The efficacy of zinc supplementation in preventing malignant relapse after HSCT and controlling GvHD needs to be demonstrated in pre-clinical models.
5. Conclusions
Our findings highlight the importance of zinc in thymopoiesis. Zinc deficiency has been known for a long time for its negative impact on immune cells. This work clarifies for the first time the importance of zinc status on immune reconstitution after bone marrow transplant. Zinc deficiency causes immune suppression by negatively affecting thymopoiesis and thymic rebuilding after acute damage. Zinc supplementation promotes the maturation of thymocytes, and, after acute stress, reinforces an endogenous mechanism of regeneration in which zinc released from damaged cells activates cells responsible for the production of regenerating factors. Endothelial cells in thymus play an eminent role in the regeneration of the organ via the production of BMP4, and zinc is part of this loop. The zinc receptor GPR39 could be an interesting therapeutic target to boost thymic regeneration.
Prospective clinical trials are needed in order to confirm the safety and the efficacy of zinc supplementation in improving thymic reconstitution after allo-HSCT in patients.
