Patent Application
20210260157
Throughout this application various publications are referenced by the last name of the first author and the year of publication. Full citations for these publications are set forth in a section entitled References immediately preceding the claims. The disclosures of all referenced publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the invention relates.
This application incorporates-by-reference nucleotide and/or amino acid sequences which are present in the file named “210510_90169-PCT-US_Sequence_Listing_AWG.txt,” which is 1.34 kilobytes in size, and which was created Apr. 5, 2021 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, which is contained in the text file filed May 10, 2021 as part of this application.
Adult, stem cells are an essential component of tissue homeostasis with indispensable roles in both physiological tissue renewal and tissue repair following injury (Weissman 2000). The regenerative potential of stem cells has been very successful for haematological disorders (Gratwohl 2015). In contrast, there has been comparatively little clinical impact on enhancing the regeneration of solid organs despite the continuing major scientific and public interest (Brooks 2017). Strategies that rely on ex vivo expansion of autologous stem cells on an individual patient basis are prohibitively expensive (Trainor 2014) and success in animal models has often failed to translate in late phase clinical trials. The use of allogeneic cells would overcome the problems of limited supply but commonly entails risky lifelong immunosuppressive therapy. Some safety concerns remain about induced pluripotent stem cells (Dimmeler 2014). Furthermore, successful engraftment of erogenous stem cells to sites of tissue injury requires a supportive inductive niche and the typical proinflammatory scarred bed in damaged recipient tissues is sub-optimal (Forbes 2014) and cells that do engraft appear to largely act by release of paracrine factors rather than functional replacement of damaged cells (Ilic 2012).
An attractive alternative strategy, which overcomes many of the limitations described above, is to promote repair by directly harnessing the regenerative potential of endogenous stem cells (Dimmeler 2014, Lane 2014). This requires identification of key soluble mediators that enhance the activity of stem cells and can be administered systemically (Zhang 2015, Smith 2017). An interesting observation was made in 1970 that a priming injury at a distant site at the time or before the second trauma resulted in accelerated healing (Joseph 1970, Davis 2005). This phenomenon was only explained recently, when it was shown that a soluble mediator is released following the priming tissue injury which transitions stem cells in the contralateral limb to a state the authors termed GAlert (Rodgers 2014), which is intermediate between G0 and G1. In the presence of activating factors the primed GAlert cells enter the cell cycle more rapidly than quiescent stem cells, leading to accelerated tissue repair (Rodgers 2014). However, the identity of the soluble mediator(s) that transition stem cell to GAlert remain to be clarified.
The present invention provides a method of preventing in a subject a consequence of an anticipated injury to the subject which comprises administering to the subject an amount of either (a) the fully reduced (all thiol) form of HMGB1, or (b) a truncated form of HMGB1 having the biological activity of the fully reduced. form of HMGB1, effective to prevent the consequence of the anticipated injury in the subject.
As used herein, and unless stated otherwise, each of the following terms shall have the definition set forth below.
As used herein, including the appended claims, the singular forms of words such as “a,” “an,” and “the,” include their corresponding plural references unless the context clearly dictates otherwise.
As used herein, “effective” as in an amount effective to achieve an end means the quantity of a component that is sufficient to yield an indicated therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this disclosure. For example, an amount effective to treat a patient undergoing chemotherapy. The specific effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives.
As used herein, an “amount” of a compound as measured in milligrams refers to the milligrams of compound present in a preparation, regardless of the form of the preparation. An “amount of compound which is 90 mg” means the amount of the compound in a preparation is 90 mg, regardless of the form of the preparation. Thus, when in the form with a carrier, the weight of the carrier necessary to provide a dose of 90 mg compound would be greater than 90 mg due to the presence of the carrier.
As used herein, “about” in the context of a numerical value or range means ±10% of the numerical value or range recited or claimed.
As used herein, to “treat” or “treating” encompasses, e.g., inducing inhibition, regression, or stasis of the disorder and/or disease or promotion of repair and regeneration or recovery. As used herein, “inhibition” of disease progression or disease complication in a subject means preventing or reducing or reversing the disease progression and/or disease complication in the subject.
As used herein, “a biologically active truncated form of HMGB1” shall be understood to include all biologically active truncated forms of HMGB1 described in the prior art as of the filing date of this application.
The present invention provides a method of preventing a consequence of an anticipated injury in a subject which comprises administering to the subject a therapeutically effective amount of the fully reduced form of HMGB1 or a biologically active truncated form of HMGB1, so as to prevent the consequence of the anticipated injury.
In one embodiment, the fully reduced form of HMGB1, or the biologically active truncated form of HMGB1 forms a heterocomplex with CXCL12 capable of binding to CXCR4.
In some embodiments, the heterocomplex binds to CXCR4.
In one embodiment the fully reduced form of HMGB1, or the biologically active truncated form of HMGB1 acts on a tissue of the subject which relies on repair by stem cells or other cells with reparative potential that express the surface receptor CXCR4.
In another embodiment the anticipated injury is an injury to a tissue of the subject which relies on repair by stem cells or other cells with reparative potential that express the surface receptor CXCR4.
In one embodiment the anticipated injury is surgery. In some embodiments the surgery is an elective surgery. In some embodiments the elective surgery involves tissues that rely on repair by CXCR4 positive cells.
In one embodiment the CXCR4 positive cells are CXCR4+ stem cells. The surgery may be knee, hip, ankle, shoulder, elbow, wrist, digital, pelvic or spinal surgery. In one embodiment the surgery is joint replacement surgery or fusion surgery. In another embodiment the surgery is surgery to, or affecting, the skin, abdomen and viscera therein, thoracic viscera, abdominal viscera, pelvis viscera, head and neck, brain, eye, spinal cord and associated nerves or blood vessels.
In one embodiment the anticipated injury is an injury to a bone or a bone tissue. The anticipated injury may be a bone fracture. In some embodiments the method improves bone fracture healing in the subject after the anticipated injury.
In some embodiments the anticipated injury is an injury to the heart or other muscle. The other muscle may be skeletal muscle.
In some embodiments the anticipated injury is an injury to skin. In other embodiments the anticipated injury is an injury to cartilage. In another embodiment the anticipated injury is an injury to cartilage in joints. In additional embodiments the anticipated injury is an injury to ligaments or tendons. In further embodiments the anticipated injury is an injury to an eye. In another embodiment, the anticipated injury is an injury to the brain. In another embodiment, the anticipated injury is an injury to the abdomen and/or abdominal viscera. In a further embodiment, the anticipated injury is an injury to thorax and/or thoracic viscera. In an additional embodiment, the anticipated injury is an injury to the skin, abdomen and pelvis and/or viscera therein, thoracic viscera, pelvic viscrea, brain, eye, spinal cord and/or associated nerves, head and/or neck, peripheral nerves, or blood vessels.
In one embodiment the method improves blood cell regeneration in the subject after the anticipated injury. In another embodiment the method improves bone marrow regeneration in the subject after the anticipated injury. In a further embodiment the method improves tissue regeneration in the subject after the anticipated injury.
In one embodiment the tissue is bone tissue, haematopoietic tissue, or muscle tissue. In another embodiment, the tissue is epithelial, connective, or nervous tissue. In a further embodiment, the method improves regeneration of organ tissue in the subject after the anticipated injury.
In some embodiments the consequence is tissue damage or tissue loss, or blood damage or blood loss. In other embodiments the consequence is delayed fracture healing or abnormal regeneration.
In one embodiment the subject is anticipated to be involved in an activity known to cause an increased risk of sustaining the anticipated injury. In another embodiment the subject is anticipated to be involved in sports activities or in military combat.
In some embodiments the administration is systemic. In other embodiments the administration is local.
In one embodiment the fully reduced form of HMGB1 is administered to the subject. In another embodiment the biologically active truncated form of HMGB1 is administered to the subject. In another embodiment the fully reduced form of HMGB1, or the biologically active truncated form of HMGB1, is a fully reduced (FR) all-thiol HMGB1 (FR-HMGB1).
In one embodiment the fully reduced form of HMGB1, or the biologically active truncated form of HMGB1, is a recombinant non-oxidizable one-serine form (1S) of HMGB1 (1S-HMGB1) in which a cysteine at one of C23, C45, or C106 is replaced by a serine.
In another embodiment the fully reduced form of HMGB1, or the biologically active truncated form of HMGB1 is a recombinant non-oxidizable two-serine form (2S) of HMGB1 (2S-HMGB1) in which the cysteines at both C23 and C45 or both C45 and C106 are replaced by a serine.
In a further embodiment the fully reduced form of HMGB1, or the biologically active truncated form of HMGB1, is a recombinant non-oxidizable all-serine form (3S) of HMGB1 (3S-HMGB1) in which the cysteines at each of C23, C45, and C106 are replaced by a serine.
In a further embodiment the administration of the fully reduced form of HMGB1, or the biologically active truncated form of HMGB1, is one day to one month prior to the anticipated injury.
This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.
Alarmins are a group of evolutionarily unrelated endogenous molecules with diverse homeostatic intracellular roles, which when released from dying, injured or activated cells trigger an immune/inflammatory response (Harry 2008, Glass 2011, and Chan 2015). Much effort has been focused on their deleterious role in autoimmune and inflammatory conditions (Chan 2015, Scaffidi 2002, Terrando 2010, Harris 2012, and Horiuchi 2017). Of the few studies (Chan 2012, Tirone 2018) that have investigated the role of alarmins in tissue repair, none have used a combination of human tissues and multiple animal injury models to characterize their effects on precise flow cytometry-defined endogenous adult stem cells in vivo. In the following examples, it has been demonstrated that High Mobility Group Box 1 (HMGB1) is a key upstream mediator of tissue regeneration which acts by transitioning CXCR4+ skeletal, haematopoietic and muscle stem cells from Go to GAlert. The following examples also demonstrate that, in the presence of appropriate activating factors, exogenous administration before or at the time of injury leads to accelerated tissue repair.
The objective of this study was to understand the role of alarmins in tissue regeneration in vivo through their effects on adult stem cells, and the translational relevance of these findings. We used human samples and primary human cells and multiple murine models of injury and regeneration. For prospective multi-parameter flow cytometry assays, we used well-established skeletal, haematopoietic and muscle stem cell-surface markers, and published isolation protocols (Chan 2015, Wilson 2008, Liu 2015). Sample size (n values) are reported as biological replicates of human donors and mice. The magnitude of the effect and variability in the measurements were used to determine sample size and replication of data. The genotypes and experimental conditions of each mouse/sample were not readily known to the experimenters during sample processing and data collection. Animals were excluded from the study only if their health status was compromised.
Human and murine plasma: Plasma samples from patients who had sustained femoral fractures and from healthy unfractured controls were obtained from the John Radcliffe Hospital (REC: 16/SW/0263, PID: 12229, IRAS: 213014). The human plasma samples were from the patient's first in-hospital blood sampling, typically within 4 hours post-fracture. Murine plasma was collected 3 hours post-femoral fracture via cardiac puncture from 12 week old female C57B16/J wild type, Hmgb1f1/f1, Hmgb1−/− mice, and from healthy unfractured controls. For the circulating levels of HMGB1, S100A8/A9 and HMGB1-CXCL12 heterocomplex over a 4 week period, murine plasma samples were collected from 12 week old female C57B16/J wild. type at 1 hour, 3 hours, 6 hours, 10 hours, 5 days, 7 days, and 28 days after fracture injury. To assess the induction of inflammation-related cytokines by HMGB1, plasma samples were collected via cardiac puncture from 12 week old female C57B16/J wild type mice at 0.5 hours, 1 hour, 3 hours, 18 hours, 48 hours, and 2 weeks post intravenous (i.v.) administration of 0.75 mg/kg of FR, or 3S-HMGB1. Samples were collected at 3 hours post i.v. administration of 0.75 mg/kg of DS-HMGB1, or 0.5 μg/kg of LPS. All human and murine samples were aliquoted, frozen, and stored at −80° C. before being thawed and assayed.
Mice: All animal procedures were approved by the institutional ethics committee and the United Kingdom Home Office (PLL 71/7161, and PLL 30/3330), and were performed on skeletally mature 12-14 week old female C57BL/6J (Charles River), and transgenic mice. Hmgb1−/− mice were generated crossing Hmgb1f1/f1 (Riken) with Rosa-CreERT2 mice (Jackson Laboratory), and at 10 weeks of age administering 3 intraperitoneal (i.p.) injections of 1.5 mg tamoxifen (Sigma) on alternate days over a 6 day period, in a mixture of sunflower seed oil (Sigma) and 10% ethanol (VWR). Mice were used 7 days after the last tamoxifen injection. Hmgb1−/− mice were obtained at the expected Mendelian ratio with no adverse phenotypic side effects, and Hmgb1f1/f1 mice (not crossed with Rosa Cre-ERT2+/+ mice) treated with tamoxifen were used as controls. Animals were genotyped by PCR of earclip DNA, with the primer sequences in Table 1 below, using the HotStart Mouse Genotyping Kit (Kapa Biosystems).
Animals were anesthetized by aerosolized 2% isoflurane, given analgesia and transferred to a warming pad. The right upper hind limb was shaved and skin prepared with povidone iodine solution. After incising the skin, the femur was exposed by blunt dissecting through the fascia lata between the biceps femoris and gluteus superficialis muscles. A commercial external fixator jig was fitted (RISystem) and a 0.5 mm osteotomy created in the femoral diaphysis with a Gigli wire. The wound was closed with interrupted non-absorbable 6/0 Prolene sutures (Ethicon). Immediately postoperatively all mice were given subcutaneous hydration, analgesia and allowed to mobilize freely. Postoperative analgesia continued for 2 days. Mice were treated locally at the time of injury with an injection into the fascial pocket surrounding the osteotomy of 0.75 mg/kg FR-HMGB1 (HMGBiotech), 0.075 mg/kg, 0.75 mg/kg, or 7.5 mg/kg 3S-HMGB1 (HMGBiotech), 0.075 mg/kg CXCL12 (R&D), or 50 μl PBS vehicle control; 50 ml/kg glycyrrhizin (Sigma), or 50 μl DMSO:PBS 1:1 vehicle control; 3 mg/kg AMD3100 (Abcam), or 50 μl PBS vehicle control; 4 mg/kg rapamycin (LC Laboratories), or 50 μl DMSO:PBS 1:1 vehicle control. Glycyrrhizin was used to disrupt the formation of the HMGB1-CXCL12 heterocomplex as it is the only known specific inhibitor for blocking the binding site of CXCL12 on HMGB1 (Schiraldi 2012, Mollica 2007). Antibodies to HMGB1 do not specifically block the interaction with CXCL12 and may have other off target effects. AMD3100 was used to disrupt the binding of CXCL12 to CXCR4 as it is a specific and clinically approved inhibitor of the CXCL12-CXCR4 interaction. It was used to determine the receptor through which the HMGB1-CXCL12 heterocomplex acted, using the rate of fracture healing as a measure of this interaction. AMD3100 or other inhibitors, such as anti-CXCL12, of the CXCL12-CXCR4 axis for cellular level characterizations of the GAlert state were not used as this would have resulted in activation and release of stem cells from their niche, CXCL12-CXCR4 signaling being well known for enforcing the quiescent G0 state (Peled 1999, Sugiyama 2006, Nie 2008, Tzeng 2011, Ding 2013, Greenbaum 2013). For priming experiments, mice were treated systemically 2 weeks prior to injury with an i.v. injection of 0.75 mg/kg FR-HMGB1, 0.75 mg/kg 3S-HMGB1, or 50 μl PBS vehicle control.
Cytokine analysis: Enzyme-linked immosorbent assays (ELISAs) were used to measure levels of TNF, S100A8/A9 (R&D), HMGB1 (IBL International), and HMGB1-CXCL12 heterocomplex (R&D; IBL International) in human monocyte supernatant, and human and murine plasma samples. These were ‘sandwich’ ELISAs where the antigen of interest was quantified between two layers of antibodies: the capture and the detection antibody. For S100A8/A9 and HMGB1, commercial kits were used according to manufacturer's instructions. For HMGB1-CXCL12, we used the heterocomplex hybrid ELISA (Venereau 2012, Schiraidi 2012). The reagents for the TNF and HMGB1-CXCL12 ELISA are listed in Table 2 below. Further immunoassays to quantify circulating levels of inflammation-related cytokines, INF, IL-6, and IL-10, in mouse plasma following i.v. administration of FR, 3S or DS-HMGB1, or LPS were performed using commercial kits based on electrochemiluminescense (MesoScale Discovery) as per manufacturer's instructions.
Human MSCs (Lonza) were maintained in DMEM (Gibco), supplemented with 10% PBS (Gibco), 1% L-Glutamine (GE), and 1% penicillin/streptomycin (GE), in standard tissue culture conditions (37° C.; 5% CO2), and used between passages 3-5. Human monocytes were isolated from human peripheral blood leucocyte cones (John Radcliffe Hospital, NHS Blood and Transplant) by positive selection with CD14 MACS microbeads (Miltenyi Biotech) and an autoMACS machine. To determine the direct effect of the alarmins, S100A8, S100A9 (supplied by T. Vogl, Münster), FR-HMGB1, DS-HMGB1, and 3S-HMGB1(HMGBiotech), or LPS (ALEXIS Biochemicals), on hMSC osteogenesis, 104 hMSCs were plated in triplicate into wells of a 96 well plate with various concentration of alarmins, or LPS, in 200 μl of osteogenic media. The latter consisted of maintenance media supplemented with 100 nM dexamethasone (Sigma), 50 μg/ml ascorbic acid 2-phosphate (Sigma), and 10 mM β-glycerophosphate (Sigma). Treatment with oncostatin 10 ng/ml (Peprotech) was used as a positive control. To determine the effects of alarmins on hMSC osteogenesis in the presence of monocytes or their products, monocytes were co-cultured with hMSCs in a ratio of 10:1 (10 monocytes: 104 hMSCs) in osteogenic media with various concentrations of alarmins or LPS; or monocytes were incubated with various concentrations of alarmins or LPS, for 16 hours and the resulting supernatant was subsequently applied onto hMSCs. To determine the effects of priming hMSC with alarmins, hMSCs were plated in maintenance media with various concentrations of alarmins; after 16 hours this was changed to osteogenic media alone. For all permutations, the respective media was replaced at day 3, and at day 7 the media was removed, cells lysed in 20 μl NP-40 lysis buffer, and alkaline phosphatase (ALP) activity, which is a marker of osteogenic differentiation, was quantified using a commercial kit (WAKO Chemicals) as per manufacturer's instructions.
In vivo micro computed tomography (CT) setup and analyses: In routine orthopaedic practice, and in clinical trials, longitudinal radiographic investigations are the most widely used tool for assessing the progression of fracture healing. Therefore, similar assessment of murine models of fracture healing would have increased translational relevance. Radiographic assessments of bone tissue are also well-known to correlate highly with histological findings (Gregor 2012, Particelli 2012), and have the added advantage of being non-destructive, thereby allowing longitudinal assessment of each animal. MicroCT imaging was performed using a high-speed rotating gantry based system (PerkinElmer, Quantum FX). Animals were anaesthetised briefly with aerosolised isofluorane 2% for each 3 minute scan. The X-ray source was set to a current of 200 μA, voltage of 90 kVp, and a field of view of 5 mm to encompass the two fixator pins closest to the osteotomy gap, for a voxel resolution of 10 μm. After the scans, mice were revived in a heated box and returned to their cages. Scans were analyzed using a commercially available microCT software package Analyze12 (AnalyzeDirect), which permitted co-registration of scans acquired over a time course. The region of interest was defined as the bridging callus, which included only the tissue that formed in the osteotomy gap (
Isolation of stem cells: BD LSRFortessa X-20 and BD FACSAria III were used for flow cytometry and fluorescence activated cell sorting (FACS) respectively. Subsequent data analyses were performed with the FlowJo V10 software (TreeStar), Murine skeletal, muscle, and haematopoietic stem cells were defined and freshly isolated according to previously reported protocols (Chan 2015, Wilson 2008, Liu 2015). Bone, bone marrow, and muscle cell suspensions were created by respectively crushing femurs and enzymatically digesting with collagenase 800 U/ml (Worthington-Biochem), or extracting bone marrow plugs by flushing femurs with FACS buffer (Miltenyi Biotec) using a 25 gauge needle, or mincing thigh muscles and enzymatically digesting with collagenase 800 U/ml and disease 1 U/ml (Gibco). Bone and bone marrow cell suspensions were also enriched by treatment for 5 minutes with red blood cell lysis buffer (Sigma). Thereafter all suspensions were strained through 70 μm and 40 μm filters (Greiner Bio-One) and stained with respective antibodies. Definitions were: mSSC, CD45−Ter119−Tie2−AlphaV−Thy−6C3−CD105−CD200−; mMuSC, CD31−CD45−Sca-1−VCAM1+; mHSC, Lineage−(CD2−CD3−CD4−CD5−CD8−CD11a−CD11b−TER119−B220−Gr-1−)c-Kit+Sca-1+CD34−CD48−CD150+. Antibodies were: mSSC, CD45 (30-F11, BD), TER-119 (TER-119, BD), Tie2 [CD202b] (TEK4, Biolegend), AlphaV [CD51] (RMV-7, Biolegend), Thy1.1 [CD90.1] (OX-7, Biolegend), Thy1.2 [CD90.2] (30-H12, Biolegend), 6C3 [Ly-51] (6C3, Biolegend), CD105 (MJ7/18, Biolegend), CD200 (OX-90, BD); mMuSC, CD31 (MEC13.3, Biolegend), CD45 (30-F11, Biolegend), Sca-1 (D7, Biolegend), VCAM [CD106] (429, Biolegend); HSC CD2 (RM2-5, Biolegend), CD3 (17A2, Biolegend), CD4 (RM4-5, Biolegend), CD5 (53-7.3, Biolegend), CD8 (53-6.7, Biolegend), CD11a (M17/4, Biolegend), CD11b (M1-70, Biolegend), B220 [CD45R] (RA3-6B2, Biolegend), Gr-1 (RB6-8C5, Biolegend), TER-119 (TER-119, Biolegend), c-Kit [CD117] (208, Biolegend), Sca-1 (D7, Biolegend), CD34 (HM34, Biolegend), CD48 (HM48-1, Biolegend), CD150 (TC15-12F12.2, Biolegend). Stem cells were also stained for the presence of surface CXCR4 (2B11, BD), and intracellular HMGB1 (3E8, Biolegend). Human CD34+ haematopoietic stem and progenitor cells were isolated from human peripheral blood leucocyte cones (John Radcliffe Hospital, NHS Blood and Transplant) by magnetically activated cell sorting (MACS) (Peytour 2010) using the CD34 MicroBead Hit (Miltenyi Biotech) and an autoMACS machine.
Quantitative real-time PCR (qRT-PCR): Total RNA was isolated using TR1 reagent (Zymo Research) from cells from whole bone, bone marrow, and muscle cell suspensions using Direct-zol™ RNA MiniPrep (Zymo Research) as per manufacturer's instructions. HMGB1 gene expression was determined by qRT-PCR and normalised to Gapdh. The amplifying primers were as follows, Gapdh (TaqMan, Mouse: Mm99999915_gl Gapdh) and Hmgb1 (TaqMan, Mouse: Mm00849805_gH Hmgb1). All reactions were performed in an ViiA7 Real Time PCR System (Applied Biosystems) using TaqMan Fast Advanced MasterMix (Applied Biosystems) according to the manufacturer's instructions.
Cell cycle kinetics: To evaluate cell cycle propensity, pulse labelling with BrdU (Abeam) was performed with animals injected with 10 mg of BrdU i.p. 10 hours before cell isolation from whole femurs. Mice were treated locally at the time of fracture with 15 mg/kg FR-HMGB1, 15 mg/kg 3S-HMGB1, 15 mg/kg BMP2 (Peprotech), or 50 μl of PBS vehicle control. To evaluate speed of entry to cell cycle, continuous labelling with BrdU was performed by administering 6.5 mg/ml in their drinking water with 5% sucrose for the indicated period. BrdU incorporation was quantified with the commercially available BrdU FlowKit (BD) as per manufacturer's instructions. Following cell isolation and staining, cells were fixed and permeabilized with Cytofix/Cytoperm (BD) for 15 minutes at room temperature, buffered with Permeablization Buffer Plus (BD) for 10 minutes at 4° C., re-fixed with Cytofix/Cytoperm for 5 minutes at room temperature, then treated with 30 μg/ml DNase (BD) for 1 hour at 37° C. to expose incorporated BrdU, and lastly stained with anti-BrdU (BD). Mice were treated systemically at the initiation of continuous BrdU administration with an i.v. injection of 15 mg/kg FR-HMGB1, 15 mg/kg 3S-HMGB1, or 100 μl of PBS vehicle control. The cells from these mice were compared to cells from the fractured side of injured mice who had also been administered continuous BrdU.
Cell migration: In vivo cell migration to the fracture site was determined by quantifying the number of BrdU− cells in fractured femurs 12 hours post-fracture using flow cytometry and Precision Count Beads (Biolegend). Mice were administered 10 mg of BrdU i.p. at the time of fracture and treated locally with 0.075 mg/kg CXCL12 or 50 μl PBS vehicle. Subsequently, BrdU incorporation in the bone and bone marrow cell suspensions from the fractured femurs was determined using the commercially available BrdU FlowKit (BD) as per manufacturer's instructions.
In vitro cell migration of mSSCs was determined by placing 1000 freshly FACS isolated mSSCs in 6 μl of DMEM in the middle observation channel of collagen coated μ-Slide Chemotaxis slides (Ibidi). A chemotactic gradient was established across the observation channel by pipetting 70 μl DMEM 0% FBS into the left reservoir, and into the right reservoir either 0.15 μg/ml or 1.5 μg/ml CXCL12, or 0% or 20% BBS controls. The channels and reservoirs were plugged to prevent evaporation and cell migration was followed by time-lapse microscopy using an automated xyz motorized stage (Prior Scientific, Prior Proscan II), a climate chamber at 37° C., 5% CO2 with humidity (Solent Scientific), a spinning disk Nikon Eclipse TE2000-U microscope with a 10× objective, and Volocity 6.3 (PerkinElmer) recording software. Cells were monitored over a period of 22 hours by capture of brightfield images every 5 minutes. Migration of 50 cells was analyzed using the automatic tracking function within the Imaris 6.7 (Bitplane) software, and represented using the Chemotaxis and Migration Tool 2.0 (Ibidi). Cells were excluded if track length was less than 50 μm.
Mitochondrial DNA: DNA was extracted from 1000 freshly FACS isolated mSSCs, mMuSCs, and mHSCs, and from 10000 trypsinised hMSCs, and 10000 MACS isolated hHSPCs, using the QIAamp DNA Micro Kit (Qiagen) as per manufacturer's instructions. mtDNA was quantified by qRT-PCR using primers amplifying the Cytochrome B region on mtDNA (Taqman, Mouse: Mm04225271_g1 CYTB; Human: Hs 02596367_s1 MT_CYB) relative to the β-globin region on gDNA (Taqman, Mouse: Mm 01611268_g1 Hbb-b1; Human: 00758889_s1 HBB). Mice were treated systemically with an i.v. injection of 0.75 mg/kg FR-HMGB1, 0.75 mg/kg 3S-HMGB1, or 100 μl of PBS vehicle control, The cells from these mice were compared to cells from the uninjured contralateral side of fractured animals. hMSCs were treated for 16 hours with 10 μg/ml FR-HMGB1 in DMEM, 10 μg/ml 3S-HMGB1 in DMEM, DMEM vehicle control, or osteogenic media supplemented with 10 μg/ml BMP2. Whole human peripheral blood leucocyte cones were treated for 2 hours with 1.5 μg/ml FR-HMGB1, 1.5 μg/ml 3S-HMGB1, 10 ng/ml IFN-γ (Miltenvi Biotec), or RPMI (Lonza) vehicle control.
Cellular ATP: Cellular ATP levels of 1000 freshly FACS isolated. mESCs, mMuSCs, and mESCs, and from, 10000 trypsinised hMSCs, and 10000 MACS isolated hHSPCs, were quantified using the commercially available ATP Bioluminescence Assay Kit CLS II (Roche), and used as per manufacturer's instructions. Cells were pelleted, boiled in 100 mM Tris, 4 mM EDTA, pH 7.75 for 2 minutes, pelleted again, and luciferase reagent was added to the supernatant. This was then read on a FLUOstar Omega (BMG Labtech) spectrophotometer, with the luminescence optic. Mice were treated systemically with an i.v. injection of 0.75 mg/kg FR-HMGB1, 0.75 mg/kg 3S-HMGB1, 0.075 mg/kg CXCL12 or 100 μl of PBS vehicle control. The cells from these mice were compared to cells from the uninjured contralateral side of fractured animals. hMSCs were treated for 16 hours with 10 μg/ml FR-HMGB1 in DMEM, 10 μg/ml 3S-HMGB1 in DMEM, DMEM vehicle control, or osteogenic media supplemented with 10 μg/ml BMP2. Whole human peripheral blood leucocyte cones were treated for 2 hours with 1.5 μg/ml FR-HMGB1, 1.5 μg/ml 3S-HMGB1, 10 ng/ml IFN-γ (Miltenyi Biotec), or RPMI (Lonza) vehicle control.
Cell size: Freshly FACS isolated mSSCs, mMuSCs, and mHSCs, trypsinised hMSCs, and MACS isolated hESPCs, were placed onto a haemocytometer and stained with 0.4% trypan blue solution (Sigma). Bright field images of the haemocytometer were acquired with an Olympus CKX41 microscope using a 40× objective lens. The analysis of cell diameter was manually performed using the Fiji distribution of ImageJ2 software (NIH) (Schindelin 2012). Mice were treated systemically with an i.v. injection of 0.75 mg/kg FR-HMGB1, 0.75 mg/kg 3S-HMGB1, or 100 μl of PBS vehicle control. The cells from these mice were compared to cells from the uninjured contralateral side of fractured animals.
cMet inhibition: Mice were treated i.p. twice a day for 5 consecutive days with 7.5 mg/kg of the c-Met inhibitor PHA 665752 (Selleckchem), or 7.5 μl DMSO in 400 μl of PBS vehicle control, or they were treated i.p. once a day for 2 consecutive days with 0.5 mg/kg anti-cMet (R&D), or 0.5 mg/kg goat IgG isotype control (R&D) in 400 μl of PBS. Following the treatment period mice were sacrificed, mMuSCs isolated, and stained for CXCR4 surface expression.
Haematological injury model: Animals were warmed up in a heating box, transferred to a restraining device, and a single i.v. injection of 150 mg/kg 5-fluorouracil (Sigma) was administered via the tail vein. 40 μl of peripheral blood was collected at the times indicated from the tail vein with EDTA-containing Microvettes (Sarstedt). 10 μl of this sample was smeared onto slides, air-dried, stained with Giemsa (Sigma) and May Grunwald solutions (RA Lamb), and neutrophils and leucocytes were counted with light microscopy using an Olympus BX51 microscope and a 40× objective lens to determine the differential neutrophil count. The remainder of the sample was treated for 5 minutes with red blood cell lysis buffer (Sigma), stained with 0.4% trypan blue solution (Sigma), and leucocytes were counted with a haemocytometer to quantify total peripheral leucocytes. Together with the differential neutrophil count as above, the total neutrophil count was also determined. Mice were treated systemically at the time of injury or systemically 2 weeks prior to injury with an i.v. injection of 0.75 mg/kg FR-HMGB1, 0.75 mg/kg 3S-HMGB1, or 100 μl of PBS vehicle control.
Muscle injury model: Animals were anesthetized by aerosolised 2% isoflurane, given analgegia, transferred to a warming pad and the right lower hindlimb was shaved and skin was prepared with povidine iodine. 80 μl of 1.2% BaCl2 (Sigma) was injected into and along the length of the tibialis anterior (TA) muscle (Rodgers 2014). Immediately postoperatively all mice were given analgesia and allowed to mobilize freely, and given postoperative analgesia for 2 days. Mice were euthanized and TA muscles extracted at the times indicated, fixed in 4% paraformaldehyde (Santa Cruz Biotechnology) for 24 hours, embedded in paraffin, sectioned, stained with haematoxylin and eosin to identify centrally nucleated fibres, and imaged with an Olympus BM51 using a 40× objective lens. The cross-sectional area (CSA) of the fibres that were approximately midway along the proximal-distal axis of the TA muscle belly was manually measured using the Fiji distribution of ImageJ2 software (NIH) (Schindelin 2012). Mice were injected intramuscularly at the time of injury or intravenously 2 weeks prior to injury, with 0.75 mg/kg FR-HMGB1, 0.75 mg/kg 3S-HMGB1, or 50 μl or 100 μl of PBS vehicle control respectively.
Statistical analysis: Statistical analyses were performed using GraphPad Prism 7 (GraphPad Software). Unless stated otherwise, significance was calculated using two-tailed unpaired Student's t-tests. For microCT callus volumes, bone mineral density, and in vivo cycling to continuous BrdU administration, significance was calculated using non-linear curve fitting and the F-test (
are shown as mean±95% CI. Results were considered statistically significant when p<0.05. Significant results were expressed using asterisks, where *p<0.05, **p<0.01, ***p<0.001, ****p<0,0001. This convention was used throughout.
Fracture healing is a good model of tissue regeneration (Einhorn 2015) and based on studies of the early events in fracture healing (Glass 2011), including the key role of neutrophils (Chan 2015), we postulated that the alarmins HMGB1 and S100A3/A9 may play key roles in tissue regeneration. HMGB1 is a highly conserved ubiquitous and abundant non-histone nuclear architectural protein that forms part of the transcription machinery (Harris 2012). S100A8/A9 proteins are calcium binding proteins that make up 40% of neutrophil cytoplasmic content (Edgeworth 1991). Both these alarmins have been associated with regulating skeletal cells (Chan 2012, Zreigat 2007). Elevated levels of HMGB1 and S100A8/A9 were found in the circulation following fracture both in human patients and mice (
The regenerative potential of these alarmins were screened in humans by assessing the osteogenic differentiation of primary human mesenchymal stromal/stem cells (hMSCs) (
A murine fracture model (Zwingenberger 2013) was optimized to permit longitudinal in vivo analysis over time (
Subsequently, the signaling pathways through which HMGB1 promoted regeneration were delineated. FR-HMGB1 is known to form a heterocomplex with CXCL12 (Venereau 2012, Schiraldi 2012), a chemokine, which in turn binds to the receptor, CXCR4 (Venereau 2012, Schiraldi 2012). Elevated plasma levels of the HMGB1-CXCL12 heterocomplex were found in both human patients and mice following fracture injury (
Exogenous HMGB1 led to a sustained increase in mSSC cell cycling in vivo.
Apart from regulating chemotaxis, the CXCL12-CXCR4 axis also influences the cycling of haematopoietic stem cells by enforcing quiescence (Peled 1999, Sugiyama 2006, Nie 2008, Tzeng 2011, Ding 2013, Greenbaum 2013). Therefore, whether the HMGB1-CXCL12-CXCR4 axis additionally affects the cell cycle of stem cells to promote tissue regeneration was investigated. The propensity to cycle of mSSCs from the fractured bones of mice that had been pulse-labelled with BrdU (
An elegant series of experiments recently demonstrated that systemic mediator(s) can transition stem cells distant to the site of initial injury to a dynamic state of the cell cycle, intermediate between G0 and G1, termed GAlert (Rodgers 2014). In contrast to deeply quiescent G0 stem cells, GAlert cells are more metabolically active as evidenced by increased cellular levels of ATP and are poised to enter the cell cycle when exposed to activating signals. As HMGB1 enhanced the in vivo cycling of mSSCs exposed to secondary activating signals, together with the elevated systemic levels of HMGB1 and HMGB1-CXCL12 post-injury in humans and mice, and observations of accelerated fracture healing with exogenous HMGB1 treatment, it is hypothesized that HMGB1 may in part accelerate fracture healing by transitioning mSSCs to the recently defined GAlert state. It is also postulated that these effects may pertain to other previously well-identified and characterized stem cells known to express CXCR4, including murine haematopoietic (mHSCs) (Peled 1999, Sugiyama 2006, Nie 2003, Tzeng 2011, Ding 2013, Greenbaum 2013) and muscle stem cells (mMuSCs) (Maesner 2016) (
The essential criteria describing the state are increased ATP levels, mitochondrial DNA, cell size, faster entry to cell cycle, and mTORC1 dependency (Rodgers 2014). We found that the clinically approved mTORC1 inhibitor, rapamycin, abolished the accelerated healing effects of exogenous HMGB1 (
HMGB1 Accelerates Healing of Multiple Tissues, Even if Administered 2 Weeks Before Injury
It was hypothesized that HMGB1 would also lead to accelerated tissue regeneration in other tissues where stem cells could transition to GAlert, for example blood and muscle. In mice myeloablated with a common chemotherapeutic agent, 5-fluouracil (5-FU) (
HMGB1 has been identified as a therapeutic target that acts on multiple endogenous adult stem cells to accelerate the physiological regenerative response to current or future injuries. These findings have broad relevance to the fields of stem cell biology and regenerative medicine and suggest a novel therapeutic approach to promote tissue repair. The existence of the GAlert phase, which is intermediate between G0 and G1, was described previously (Rodgers 2014). It was noted that stem cells in GAlert enter the cell cycle faster compared to those in G0 and initiators of this transition would have wide-ranging implications for the field of regenerative medicine by accelerating repair.
HMGB1 has been demonstrated to accelerate healing of multiple tissue types by forming a heterocomplex with CXCL12, which then binds to CXCR4, to transition quiescent stem cells in three different tissues to GAlert. A recent publication (Tirone 2018) showed that HMGB1 promotes repair in a murine model of muscle injury in part by modulating the immune response. We utilized prospective multi-parameter flow cytometry isolation methodologies to study the cycling of well-defined endogenous adult stem cell populations in vivo to reduce potential in vitro artefacts and identified a novel mechanism of action of FR-HMGB1 during tissue repair via the initiation of the GAlert state. Furthermore, we demonstrated that this also pertains to human stem and progenitor cells,
Whilst this work has focused on endogenous adult stem cells, it is possible that the transition to GAlert by HMGB1 may also pertain to other cell types that are usually quiescent in the steady state, can express CXCR4 and are capable of re-entering the cell cycle to effect tissue repair, such as mature hepatocytes. Indeed, it was recently observed that HMGB1 treatment results in enhanced proliferation of hepatocytes following injury (Tirone 2018). Using clinically relevant injury models of fracture repair, the response to chemotherapy and muscle regeneration, in conjunction with human tissues and cells, applicants have demonstrated that FR-HMGB1 leads to accelerated regeneration of multiple tissues by transitioning the respective stem cells to GAlert.
HMGB1 has critical intracellular and extracellular functions as demonstrated by the lethality of the constitutive global knockout (Kang 2014). In the nucleus HMGB1 interacts with nucleosomes, transcription factors and histones and thus regulates gene transcription. It has recently been shown that muscle regeneration. is compromised in partial Hmgb1−/− mice (Tirone 2018). Fracture healing has been shown to be dramatically impaired in conditional Hmgb1−/− with robust intracellular and extracellular protein knockdown, and that stem cells fail to transition to GAlert. At the cellular level, exogenous HMGB1 can rescue the GAlert phenotype but did not evaluate the rescue at tissue healing level as exogenous HMGB1 addition would not compensate for the critical intra-nuclear roles of HMGB1 (Kang 2014).
Whilst extracellular FR-HMGB1 enhances cell migration by forming a heterocomplex with the relatively abundant CXCL12 that is produced following injury, our data shows that the enhanced regenerative effects of the heterocomplex extend beyond those explained by increased chemotaxis. Indeed, the novel finding that systemic pre-treatment with HMGB1 two weeks prior to injury also accelerates tissue regeneration, with stem cells remaining in GAlert at this time point. (
In summary, a major discovery of recent decades has been the existence of stem cells and their potential to repair many, if not most, tissues. With the aging population, many attempts have been made to use exogenous stem cells to promote tissue repair, so far with limited success. An alternative approach, which may be more effective and far less costly, is to promote tissue regeneration by targeting endogenous stem cells. However, ways of enhancing endogenous stem cell function remain poorly defined. Injury leads to the release of danger signals which are known to modulate the immune response, but their role in stem cell-mediated repair in vivo remains to be clarified. In this application it has demonstrated that high mobility Q:9 group box 1 (HMGB1) is released following fracture in both humans and mice, forms a heterocomplex with CXCL12, and acts via CXCR4 to accelerate skeletal, hematopoietic, and muscle regeneration in vivo. Pretreatment with HMGB1 2 weeks before injury also accelerated tissue regeneration, indicating an acquired proregenerative signature. HMGB1 led to sustained increase in cell cycling in vivo, and using Hmgb1−/− mice we identified the underlying mechanism as the transition of multiple quiescent stem cells from G0 to GAlert. HMGB1 also transitions human stem and progenitor cells to GAlert. Therefore, exogenous HMGB1 benefits patients in many scenarios, including trauma, chemotherapy, and elective surgery.
This invention is significant because while stem cell therapy has become the standard of care for hematological disorders, challenges remain for the treatment of solid organ injuries. Targeting endogenous cells would overcome many hurdles associated with exogenous stem cell therapy. Alarmins are released upon tissue damage, and here it is described Glow upregulation of a physiological pathway by exogenous administration a single dose of HMGB1, either locally or systemically, promotes tissue repair by targeting endogenous stem cells. It is shown that HMGB1 complexed with CXCL12 transitions stem cells that express CXCR4 from G0 to GAlert. These primed cells rapidly respond to appropriate activating factors released upon injury. HMGB1 promotes healing even if administered 2 weeks before injury, thereby expanding its translational benefit for diverse clinical scenarios.
A model is developed in which a highly-conserved injury signal, HMGB1, acts via a well-established maintenance signaling pathway, CXCL12-CXCR4, to promote tissue regeneration as depicted in
This application is a § 371 national stage of PCT International Application No. PCT/IB2019/000364, filed Apr. 9, 2019, claiming the benefit of U.S. Provisional Application No. 62/655,736, filed Apr. 10, 2018, the contents of each of which are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2019/000384 | 4/9/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62655736 | Apr 2018 | US |
