Publications by authors named "Amanda K Huber"

34 Publications

Novel Lineage-Tracing System to Identify Site-Specific Ectopic Bone Precursor Cells.

Stem Cell Reports 2021 Mar 18;16(3):626-640. Epub 2021 Feb 18.

Center for Organogenesis and Trauma, Department of Surgery, University of Texas Southwestern, 6000 Harry Hines Boulevard, Dallas, TX 75235, USA. Electronic address:

Heterotopic ossification (HO) is a form of pathological cell-fate change of mesenchymal stem/precursor cells (MSCs) that occurs following traumatic injury, limiting range of motion in extremities and causing pain. MSCs have been shown to differentiate to form bone; however, their lineage and aberrant processes after trauma are not well understood. Utilizing a well-established mouse HO model and inducible lineage-tracing mouse (Hoxa11-CreER;ROSA26-LSL-TdTomato), we found that Hoxa11-lineage cells represent HO progenitors specifically in the zeugopod. Bioinformatic single-cell transcriptomic and epigenomic analyses showed Hoxa11-lineage cells are regionally restricted mesenchymal cells that, after injury, gain the potential to undergo differentiation toward chondrocytes, osteoblasts, and adipocytes. This study identifies Hoxa11-lineage cells as zeugopod-specific ectopic bone progenitors and elucidates the fate specification and multipotency that mesenchymal cells acquire after injury. Furthermore, this highlights homeobox patterning genes as useful tools to trace region-specific progenitors and enable location-specific gene deletion.
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http://dx.doi.org/10.1016/j.stemcr.2021.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7940250PMC
March 2021

Mesenchymal VEGFA induces aberrant differentiation in heterotopic ossification.

Bone Res 2019 Dec 10;7(1):36. Epub 2019 Dec 10.

Department of Surgery, University of Michigan, Ann Arbor, MI, 48109, USA.

Heterotopic ossification (HO) is a debilitating condition characterized by the pathologic formation of ectopic bone. HO occurs commonly following orthopedic surgeries, burns, and neurologic injuries. While surgical excision may provide palliation, the procedure is often burdened with significant intra-operative blood loss due to a more robust contribution of blood supply to the pathologic bone than to native bone. Based on these clinical observations, we set out to examine the role of vascular signaling in HO. Vascular endothelial growth factor A (VEGFA) has previously been shown to be a crucial pro-angiogenic and pro-osteogenic cue during normal bone development and homeostasis. Our findings, using a validated mouse model of HO, demonstrate that HO lesions are highly vascular, and that VEGFA is critical to ectopic bone formation, despite lacking a contribution of endothelial cells within the developing anlagen.
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http://dx.doi.org/10.1038/s41413-019-0075-6DOI Listing
December 2019

The role of neutrophil extracellular traps and TLR signaling in skeletal muscle ischemia reperfusion injury.

FASEB J 2020 12 22;34(12):15753-15770. Epub 2020 Oct 22.

Department of Surgery, University of Michigan, Ann Arbor, MI, USA.

Ischemia reperfusion (IR) injury results in devastating skeletal muscle fibrosis. Here, we recapitulate this injury with a mouse model of hindlimb IR injury which leads to skeletal muscle fibrosis. Injury resulted in extensive immune infiltration with robust neutrophil extracellular trap (NET) formation in the skeletal muscle, however, direct targeting of NETs via the peptidylarginine deiminase 4 (PAD4) mechanism was insufficient to reduce muscle fibrosis. Circulating levels of IL-10 and TNFα were significantly elevated post injury, indicating toll-like receptor (TLR) signaling may be involved in muscle injury. Administration of hydroxychloroquine (HCQ), a small molecule inhibitor of TLR7/8/9, following injury reduced NET formation, IL-10, and TNFα levels and ultimately mitigated muscle fibrosis and improved myofiber regeneration following IR injury. HCQ treatment decreased fibroadipogenic progenitor cell proliferation and partially inhibited ERK1/2 phosphorylation in the injured tissue, suggesting it may act through a combination of TLR7/8/9 and ERK signaling mechanisms. We demonstrate that treatment with FDA-approved HCQ leads to decreased muscle fibrosis and increased myofiber regeneration following IR injury, suggesting short-term HCQ treatment may be a viable treatment to prevent muscle fibrosis in ischemia reperfusion and traumatic extremity injury.
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http://dx.doi.org/10.1096/fj.202000994RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8054227PMC
December 2020

Immobilization after injury alters extracellular matrix and stem cell fate.

J Clin Invest 2020 10;130(10):5444-5460

Section of Plastic Surgery, Department of Surgery.

Cells sense the extracellular environment and mechanical stimuli and translate these signals into intracellular responses through mechanotransduction, which alters cell maintenance, proliferation, and differentiation. Here we use a mouse model of trauma-induced heterotopic ossification (HO) to examine how cell-extrinsic forces impact mesenchymal progenitor cell (MPC) fate. After injury, single-cell (sc) RNA sequencing of the injury site reveals an early increase in MPC genes associated with pathways of cell adhesion and ECM-receptor interactions, and MPC trajectories to cartilage and bone. Immunostaining uncovers active mechanotransduction after injury with increased focal adhesion kinase signaling and nuclear translocation of transcriptional coactivator TAZ, inhibition of which mitigates HO. Similarly, joint immobilization decreases mechanotransductive signaling, and completely inhibits HO. Joint immobilization decreases collagen alignment and increases adipogenesis. Further, scRNA sequencing of the HO site after injury with or without immobilization identifies gene signatures in mobile MPCs correlating with osteogenesis, and signatures from immobile MPCs with adipogenesis. scATAC-seq in these same MPCs confirm that in mobile MPCs, chromatin regions around osteogenic genes are open, whereas in immobile MPCs, regions around adipogenic genes are open. Together these data suggest that joint immobilization after injury results in decreased ECM alignment, altered MPC mechanotransduction, and changes in genomic architecture favoring adipogenesis over osteogenesis, resulting in decreased formation of HO.
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http://dx.doi.org/10.1172/JCI136142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524473PMC
October 2020

Small molecule inhibition of non-canonical (TAK1-mediated) BMP signaling results in reduced chondrogenic ossification and heterotopic ossification in a rat model of blast-associated combat-related lower limb trauma.

Bone 2020 10 2;139:115517. Epub 2020 Jul 2.

Regenerative Medicine Department, Naval Medical Research Center, Silver Spring, MD, United States of America; Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, United States of America. Electronic address:

Heterotopic ossification (HO) is defined as ectopic bone formation around joints and in soft tissues following trauma, particularly blast-related extremity injuries, thermal injuries, central nerve injuries, or orthopaedic surgeries, leading to increased pain and diminished quality of life. Current treatment options include pharmacotherapy with non-steroidal anti-inflammatory drugs, radiotherapy, and surgical excision, but these treatments have limited efficacy and have associated complication profiles. In contrast, small molecule inhibitors have been shown to have higher specificity and less systemic cytotoxicity. Previous studies have shown that bone morphogenetic protein (BMP) signaling and downstream non-canonical (SMAD-independent) BMP signaling mediated induction of TGF-β activated kinase-1 (TAK1) contributes to HO. In the current study, small molecule inhibition of TAK1, NG-25, was evaluated for its efficacy in limiting ectopic bone formation following a rat blast-associated lower limb trauma and a murine burn tenotomy injury model. A significant decrease in total HO volume in the rat blast injury model was observed by microCT imaging with no systemic complications following NG-25 therapy. Furthermore, tissue-resident mesenchymal progenitor cells (MPCs) harvested from rats treated with NG-25 demonstrated decreased proliferation, limited osteogenic differentiation capacity, and reduced gene expression of Tac1, Col10a1, Ibsp, Smad3, and Sox2 (P < 0.05). Single cell RNA-sequencing of murine cells harvested from the injury site in a burn tenotomy injury model showed increased expression of these genes in MPCs during stages of chondrogenic differentiation. Additional in vitro cell cultures of murine tissue-resident MPCs and osteochondrogenic progenitors (OCPs) treated with NG-25 demonstrated reduced chondrogenic differentiation by 10.2-fold (P < 0.001) and 133.3-fold (P < 0.001), respectively, as well as associated reduction in chondrogenic gene expression. Induction of HO in Tak1 knockout mice demonstrated a 7.1-fold (P < 0.001) and 2.7-fold reduction (P < 0.001) in chondrogenic differentiation of murine MPCs and OCPs, respectively, with reduced chondrogenic gene expression. Together, our in vivo models and in vitro cell culture studies demonstrate the importance of TAK1 signaling in chondrogenic differentiation and HO formation and suggest that small molecule inhibition of TAK1 is a promising therapy to limit the formation and progression of HO.
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http://dx.doi.org/10.1016/j.bone.2020.115517DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7945876PMC
October 2020

Activin A does not drive post-traumatic heterotopic ossification.

Bone 2020 09 15;138:115473. Epub 2020 Jun 15.

Department of Surgery, University of Michigan, Ann Arbor, MI, United States of America; Division of Plastic Surgery, Department of Surgery, University of Michigan Health System, 1500 E Medical Center Drive, SPC 5340, Ann Arbor, MI 48109-5340, United States of America. Electronic address:

Heterotopic ossification (HO), the formation of ectopic bone in soft tissues, has been extensively studied in its two primary forms: post-traumatic HO (tHO) typically found in patients who have experienced musculoskeletal or neurogenic injury and in fibrodysplasia ossificans progressiva (FOP), where it is genetically driven. Given that in both diseases HO arises via endochondral ossification, the molecular mechanisms behind both diseases have been postulated to be manifestations of similar pathways including those activated by BMP/TGFβ superfamily ligands. A significant step towards understanding the molecular mechanism by which HO arises in FOP was the discovery that FOP causing ACVR1 variants trigger HO in response to activin A, a ligand that does not activate signaling from wild type ACVR1, and that is not inherently osteogenic in wild type settings. The physiological significance of this finding was demonstrated by showing that activin A neutralizing antibodies stop HO in two different genetically accurate mouse models of FOP. In order to explore the role of activin A in tHO, we performed single cell RNA sequencing and compared the expression of activin A as well as other BMP pathway genes in tHO and FOP HO. We show that activin A is expressed in response to injury in both settings, but by different types of cells. Given that wild type ACVR1 does not transduce signal when engaged by activin A, we hypothesized that inhibition of activin A will not block tHO. Nonetheless, as activin A was expressed in tHO lesions, we tested its inhibition and compared it with inhibition of BMPs. We show here that anti-activin A does not block tHO, whereas agents such as antibodies that neutralize ACVR1 or ALK3-Fc (which blocks osteogenic BMPs) are beneficial, though not completely curative. These results demonstrate that inhibition of activin A should not be considered as a therapeutic strategy for ameliorating tHO.
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http://dx.doi.org/10.1016/j.bone.2020.115473DOI Listing
September 2020

Perivascular Fibro-Adipogenic Progenitor Tracing during Post-Traumatic Osteoarthritis.

Am J Pathol 2020 09 10;190(9):1909-1920. Epub 2020 Jun 10.

Department of Pathology, Johns Hopkins University, Baltimore, Maryland; Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, UCLA and Orthopaedic Hospital, Los Angeles, California. Electronic address:

Perivascular mural cells surround capillaries and microvessels and have diverse regenerative or fibrotic functions after tissue injury. Subsynovial fibrosis is a well-known pathologic feature of osteoarthritis, yet transgenic animals for use in visualizing perivascular cell contribution to fibrosis during arthritic changes have not been developed. Here, inducible Pdgfra-CreER reporter mice were subjected to joint-destabilization surgery to induce arthritic changes, and cell lineage was traced over an 8-week period with a focus on the joint-associated fat pad. Results showed that, at baseline, inducible Pdgfra reporter activity highlighted adventitial and, to a lesser extent, pericytic cells within the infrapatellar fat pad. Joint-destabilization surgery was associated with marked fibrosis of the infrapatellar fat pad, accompanied by an expansion of perivascular Pdgfra-expressing cellular descendants, many of which adopted α-smooth muscle actin expression. Gene expression analysis of microdissected infrapatellar fat pad confirmed enrichment in membrane-bound green fluorescent protein/Pdgfra-expressing cells, along with a gene signature that corresponded with injury-associated fibro-adipogenic progenitors. Our results highlight dynamic changes in joint-associated perivascular fibro-adipogenic progenitors during osteoarthritis.
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http://dx.doi.org/10.1016/j.ajpath.2020.05.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456743PMC
September 2020

Tuning Macrophage Phenotype to Mitigate Skeletal Muscle Fibrosis.

J Immunol 2020 04 11;204(8):2203-2215. Epub 2020 Mar 11.

Department of Surgery, University of Michigan, Ann Arbor, MI 48109;

Myeloid cells are critical to the development of fibrosis following muscle injury; however, the mechanism of their role in fibrosis formation remains unclear. In this study, we demonstrate that myeloid cell-derived TGF-β1 signaling is increased in a profibrotic ischemia reperfusion and cardiotoxin muscle injury model. We found that myeloid-specific deletion of abrogates the fibrotic response in this injury model and reduces fibro/adipogenic progenitor cell proliferation while simultaneously enhancing muscle regeneration, which is abrogated by adaptive transfer of normal macrophages. Similarly, a murine TGFBRII-Fc ligand trap administered after injury significantly reduced muscle fibrosis and improved muscle regeneration. This study ultimately demonstrates that infiltrating myeloid cell TGF-β1 is responsible for the development of traumatic muscle fibrosis, and its blockade offers a promising therapeutic target for preventing muscle fibrosis after ischemic injury.
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http://dx.doi.org/10.4049/jimmunol.1900814DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8080967PMC
April 2020

Regulation of heterotopic ossification by monocytes in a mouse model of aberrant wound healing.

Nat Commun 2020 02 5;11(1):722. Epub 2020 Feb 5.

Section of Plastic Surgery, Department of Surgery, University of Michigan, Ann Arbor, MI, 48109, USA.

Heterotopic ossification (HO) is an aberrant regenerative process with ectopic bone induction in response to musculoskeletal trauma, in which mesenchymal stem cells (MSC) differentiate into osteochondrogenic cells instead of myocytes or tenocytes. Despite frequent cases of hospitalized musculoskeletal trauma, the inflammatory responses and cell population dynamics that regulate subsequent wound healing and tissue regeneration are still unclear. Here we examine, using a mouse model of trauma-induced HO, the local microenvironment of the initial post-injury inflammatory response. Single cell transcriptome analyses identify distinct monocyte/macrophage populations at the injury site, with their dynamic changes over time elucidated using trajectory analyses. Mechanistically, transforming growth factor beta-1 (TGFβ1)-producing monocytes/macrophages are associated with HO and aberrant chondrogenic progenitor cell differentiation, while CD47-activating peptides that reduce systemic macrophage TGFβ levels and help ameliorate HO. Our data thus implicate CD47 activation as a therapeutic approach for modulating monocyte/macrophage phenotypes, MSC differentiation and HO formation during wound healing.
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http://dx.doi.org/10.1038/s41467-019-14172-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7002453PMC
February 2020

Mesenchymal VEGFA induces aberrant differentiation in heterotopic ossification.

Bone Res 2019 10;7:36. Epub 2019 Dec 10.

1Department of Surgery, University of Michigan, Ann Arbor, MI 48109 USA.

Heterotopic ossification (HO) is a debilitating condition characterized by the pathologic formation of ectopic bone. HO occurs commonly following orthopedic surgeries, burns, and neurologic injuries. While surgical excision may provide palliation, the procedure is often burdened with significant intra-operative blood loss due to a more robust contribution of blood supply to the pathologic bone than to native bone. Based on these clinical observations, we set out to examine the role of vascular signaling in HO. Vascular endothelial growth factor A (VEGFA) has previously been shown to be a crucial pro-angiogenic and pro-osteogenic cue during normal bone development and homeostasis. Our findings, using a validated mouse model of HO, demonstrate that HO lesions are highly vascular, and that VEGFA is critical to ectopic bone formation, despite lacking a contribution of endothelial cells within the developing anlagen.
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http://dx.doi.org/10.1038/s41413-019-0075-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904752PMC
December 2019

An IFNγ/CXCL2 regulatory pathway determines lesion localization during EAE.

J Neuroinflammation 2018 Jul 16;15(1):208. Epub 2018 Jul 16.

Holtom-Garrett Program in Neuroimmunology and Multiple Sclerosis Center, Department of Neurology, University of Michigan School of Medicine, Ann Arbor, MI, 48109, USA.

Background: Myelin oligodendrocyte glycoprotein (MOG)-reactive T-helper (Th)1 cells induce conventional experimental autoimmune encephalomyelitis (cEAE), characterized by ascending paralysis and monocyte-predominant spinal cord infiltrates, in C57BL/6 wildtype (WT) hosts. The same T cells induce an atypical form of EAE (aEAE), characterized by ataxia and neutrophil-predominant brainstem infiltrates, in syngeneic IFNγ receptor (IFNγR)-deficient hosts. Production of ELR+ CXC chemokines within the CNS is required for the development of aEAE, but not cEAE. The cellular source(s) and localization of ELR+ CXC chemokines in the CNS and the IFNγ-dependent pathways that regulate their production remain to be elucidated.

Methods: The spatial distribution of inflammatory lesions and CNS expression of the ELR+ CXC chemokines, CXCL1 and CXCL2, were determined via immunohistochemistry and/or in situ hybridization. Levels of CXCL1 and CXCL2, and their cognate receptor CXCR2, were measured in/on leukocyte subsets by flow cytometric and quantitative PCR (qPCR) analysis. Bone marrow neutrophils and macrophages were cultured with inflammatory stimuli in vitro prior to measurement of CXCL2 and CXCR2 by qPCR or flow cytometry.

Results: CNS-infiltrating neutrophils and monocytes, and resident microglia, are a prominent source of CXCL2 in the brainstem of IFNγRKO adoptive transfer recipients during aEAE. In WT transfer recipients, IFNγ directly suppresses CXCL2 transcription in microglia and myeloid cells, and CXCR2 transcription in CNS-infiltrating neutrophils. Consequently, infiltration of the brainstem parenchyma from the adjacent meninges is blocked during cEAE. CXCL2 directly stimulates its own expression in cultured neutrophils, which is enhanced by IL-1 and suppressed by IFNγ.

Conclusions: We provide evidence for an IFNγ-regulated CXCR2/CXCL2 autocrine/paracrine feedback loop in innate immune cells that determines the location of CNS infiltrates during Th1-mediated EAE. When IFNγ signaling is impaired, myeloid cell production of CXCL2 increases, which promotes brainstem inflammation and results in clinical ataxia. IFNγ, produced within the CNS of WT recipients, suppresses myeloid cell CXCR2 and CXCL2 production, thereby skewing the location of neuroinflammatory infiltrates to the spinal cord and the clinical phenotype to an ascending paralysis. These data reveal a novel mechanism by which IFNγ and CXCL2 interact to direct regional recruitment of leukocytes in the CNS, resulting in distinct clinical presentations.
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http://dx.doi.org/10.1186/s12974-018-1237-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048869PMC
July 2018

A randomized, subject and rater-blinded, placebo-controlled trial of dimethyl fumarate for obstructive sleep apnea.

Sleep 2018 08;41(8)

Department of Neurology, Sleep Disorders Center, University of Michigan, Ann Arbor, MI.

Study Objectives: To investigate the therapeutic effect of dimethyl fumarate (DMF, an immunomodulatory agent) on obstructive sleep apnea (OSA), and potential influence of any such effect by selected proinflammatory molecules.

Methods: Patients with OSA who deferred positive airway pressure therapy were randomized (2:1) to receive DMF or placebo for 4 months. Participants underwent polysomnography before randomization and at 4 months. Blood was collected monthly. The primary outcome was the mean group change in respiratory disturbance index (δ-RDI). Secondary analyses focused on the association between treatment effect of DMF (on RDI) and expression of plasma cytokines and chemokines, or nuclear factor κ-B (NFκB) signaling molecules in peripheral blood mononuclear cells.

Results: N = 65 participants were randomized. N = 50 participants (DMF = 35, placebo = 15) had complete data for final analyses. The mean difference in δ-RDI between groups was 13.3 respiratory events/hour of sleep: -3.1+/-12.9 vs. 10.2+/-13.1 in DMF and placebo groups, respectively (mixed-effects model treatment effect: β = -0.14, SE = 0.062, p = 0.033). Plasma levels of TNF-α showed only nonsignificant decreases, and IL-10 and IL-13 only nonsignificant increases, in DMF-treated participants compared with placebo. No significant interaction or main effect on RDI for selected cytokines and chemokines was found. Participants with a therapeutic response to DMF did experience significant reductions in intracellular NFκB signaling molecules at 4 months. Overall, DMF was well-tolerated.

Conclusions: The immunomodulatory drug DMF partially ameliorates OSA severity. Suppression of systemic inflammation through reduction of NFκB signaling may mediate this effect.

Clinical Trials: ClinicalTrials.gov, NCT02438137, https://clinicaltrials.gov/ct2/show/NCT02438137?term=NCT02438137&rank=1.
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http://dx.doi.org/10.1093/sleep/zsy109DOI Listing
August 2018

GM-CSF Promotes Chronic Disability in Experimental Autoimmune Encephalomyelitis by Altering the Composition of Central Nervous System-Infiltrating Cells, but Is Dispensable for Disease Induction.

J Immunol 2018 02 29;200(3):966-973. Epub 2017 Dec 29.

Holtom-Garrett Program in Neuroimmunology, Department of Neurology, University of Michigan School of Medicine, Ann Arbor, MI 48109;

GM-CSF has been portrayed as a critical cytokine in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) and, ostensibly, in multiple sclerosis. C57BL/6 mice deficient in GM-CSF are resistant to EAE induced by immunization with myelin oligodendrocyte glycoprotein (MOG) The mechanism of action of GM-CSF in EAE is poorly understood. In this study, we show that GM-CSF augments the accumulation of MOG-specific T cells in the skin draining lymph nodes of primed mice, but it is not required for the development of encephalitogenic T cells. Abrogation of GM-CSF receptor signaling in adoptive transfer recipients of MOG-specific T cells did not alter the incidence of EAE or the trajectory of its initial clinical course, but it limited the extent of chronic CNS tissue damage and neurologic disability. The attenuated clinical course was associated with a relative dearth of MOG-specific T cells, myeloid dendritic cells, and neutrophils, as well as an abundance of B cells, within CNS infiltrates. Our data indicate that GM-CSF drives chronic tissue damage and disability in EAE via pleiotropic pathways, but it is dispensable during early lesion formation and the onset of neurologic deficits.
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http://dx.doi.org/10.4049/jimmunol.1701484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5780231PMC
February 2018

An emerging role for eotaxins in neurodegenerative disease.

Clin Immunol 2018 04 21;189:29-33. Epub 2016 Sep 21.

Holtom-Garrett Program in Neuroimmunology, Department of Neurology, University of Michigan Medical School, Ann Arbor, MI, USA. Electronic address:

Eotaxins are C-C motif chemokines first identified as potent eosinophil chemoattractants. They facilitate eosinophil recruitment to sites of inflammation in response to parasitic infections as well as allergic and autoimmune diseases such as asthma, atopic dermatitis, and inflammatory bowel disease. The eotaxin family currently includes three members: eotaxin-1 (CCL11), eotaxin-2 (CCL24), and eotaxin-3 (CCL26). Despite having only ~30% sequence homology to one another, each was identified based on its ability to bind the chemokine receptor, CCR3. Beyond their role in innate immunity, recent studies have shown that CCL11 and related molecules may directly contribute to degenerative processes in the central nervous system (CNS). CCL11 levels increase in the plasma and cerebrospinal fluid of both mice and humans as part of normal aging. In mice, these increases are associated with declining neurogenesis and impaired cognition and memory. In humans, elevated plasma levels of CCL11 have been observed in Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, and secondary progressive multiple sclerosis when compared to age-matched, healthy controls. Since CCL11 is capable of crossing the blood-brain barrier of normal mice, it is plausible that eotaxins generated in the periphery may exert physiological and pathological actions in the CNS. Here, we briefly review known functions of eotaxin family members during innate immunity, and then focus on whether and how these molecules might participate in the progression of neurodegenerative diseases.
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http://dx.doi.org/10.1016/j.clim.2016.09.010DOI Listing
April 2018

Type-1 angiotensin receptor signaling in central nervous system myeloid cells is pathogenic during fatal alphavirus encephalitis in mice.

J Neuroinflammation 2016 08 25;13(1):196. Epub 2016 Aug 25.

Holtom-Garrett Program in Neuroimmunology, Department of Neurology, University of Michigan Medical School, Room 4007, A. Alfred Taubman Biomedical Sciences Research Building, 109 Zina Pitcher Place, Ann Arbor, MI, 48109-2200, USA.

Background: Alphaviruses can cause fatal encephalitis in humans. Natural infections occur via the bite of infected mosquitos, but aerosol transmissibility makes some of these viruses potential bioterrorism agents. Central nervous system (CNS) host responses contribute to alphavirus pathogenesis in experimental models and are logical therapeutic targets. We investigated whether reactive oxygen species (ROS) generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) activity within the CNS contributes to fatal alphavirus encephalitis in mice.

Methods: Infected animals were treated systemically with the angiotensin receptor-blocking drug, telmisartan, given its ability to cross the blood-brain barrier, selectively block type-1 angiotensin receptors (AT1R), and inhibit Nox-derived ROS production in vascular smooth muscle and other extraneural tissues. Clinical, virological, biochemical, and histopathological outcomes were followed over time.

Results: The importance of the angiotensin II (Ang II)/AT1R axis in disease pathogenesis was confirmed by demonstrating increased Ang II levels in the CNS following infection, enhanced disease survival when CNS Ang II production was suppressed, increased AT1R expression on microglia and tissue-infiltrating myeloid cells, and enhanced disease survival in AT1R-deficient mice compared to wild-type (WT) controls. Systemic administration of telmisartan protected WT mice from lethal encephalitis caused by two different alphaviruses in a dose-dependent manner without altering virus replication or exerting any anti-inflammatory effects in the CNS. Infection triggered up-regulation of multiple Nox subunits in the CNS, while drug treatment inhibited local Nox activity, ROS production, and oxidative neuronal damage. Telmisartan proved ineffective in Nox-deficient mice, demonstrating that this enzyme is its main target in this experimental setting.

Conclusions: Nox-derived ROS, likely arising from CNS myeloid cells triggered by AT1R signaling, are pathogenic during fatal alphavirus encephalitis in mice. Systemically administered telmisartan at non-hypotensive doses targets Nox activity in the CNS to exert a neuroprotective effect. Disruption of this pathway may have broader implications for the treatment of related infections as well as for other CNS diseases driven by oxidative injury.
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http://dx.doi.org/10.1186/s12974-016-0683-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5000512PMC
August 2016

Targeting CXCL13 During Neuroinflammation.

Adv Neuroimmune Biol 2015;6(1):1-8. Epub 2015 Nov 13.

Department of Neurology, University of Michigan Medical School, Ann Arbor, MI, USA.

The chemokine, C-X-C motif ligand 13 (CXCL13), is constitutively expressed in lymphoid organs and controls the recruitment and compartmentalization of lymphocytes and antigen presenting cells within these specialized structures. Recent data, however, also find induction of this molecule during central nervous system (CNS) inflammation under a variety of circumstances. While its role(s) in the pathogenesis of neoplastic, infectious and autoimmune disorders of the CNS remain incompletely understood, several lines of evidence suggest that CXCL13 could become a relevant therapeutic target in at least some of these diseases. This review focuses on how CXCL13 contributes to the pathogenesis of selected CNS disorders involving both experimental animals and humans, paying particular attention to the issue of whether (and if so, how) blockade of this ligand or its receptor might benefit the host. Current blocking strategies largely involve the use of monoclonal antibodies, but an improved understanding of downstream signaling pathways makes small molecule inhibition a future possibility.
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http://dx.doi.org/10.3233/NIB-150101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4743661PMC
November 2015

Loss of the Ubiquitin-conjugating Enzyme UBE2W Results in Susceptibility to Early Postnatal Lethality and Defects in Skin, Immune, and Male Reproductive Systems.

J Biol Chem 2016 Feb 24;291(6):3030-42. Epub 2015 Nov 24.

From the Departments of Neurology,

UBE2W ubiquitinates N termini of proteins rather than internal lysine residues, showing a preference for substrates with intrinsically disordered N termini. The in vivo functions of this intriguing E2, however, remain unknown. We generated Ube2w germ line KO mice that proved to be susceptible to early postnatal lethality without obvious developmental abnormalities. Although the basis of early death is uncertain, several organ systems manifest changes in Ube2w KO mice. Newborn Ube2w KO mice often show altered epidermal maturation with reduced expression of differentiation markers. Mirroring higher UBE2W expression levels in testis and thymus, Ube2w KO mice showed a disproportionate decrease in weight of these two organs (~50%), suggesting a functional role for UBE2W in the immune and male reproductive systems. Indeed, Ube2w KO mice displayed sustained neutrophilia accompanied by increased G-CSF signaling and testicular vacuolation associated with decreased fertility. Proteomic analysis of a vulnerable organ, presymptomatic testis, showed a preferential accumulation of disordered proteins in the absence of UBE2W, consistent with the view that UBE2W preferentially targets disordered polypeptides. These mice further allowed us to establish that UBE2W is ubiquitously expressed as a single isoform localized to the cytoplasm and that the absence of UBE2W does not alter cell viability in response to various stressors. Our results establish that UBE2W is an important, albeit not essential, protein for early postnatal survival and normal functioning of multiple organ systems.
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http://dx.doi.org/10.1074/jbc.M115.676601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4742764PMC
February 2016

Th Cell Diversity in Experimental Autoimmune Encephalomyelitis and Multiple Sclerosis.

J Immunol 2015 Sep 3;195(6):2552-9. Epub 2015 Aug 3.

Holtom-Garrett Program in Neuroimmunology, Department of Neurology, University of Michigan, Ann Arbor, MI 48109; Graduate Program in Immunology, University of Michigan, Ann Arbor, MI 48109; Neurology Service, VA Ann Arbor Healthcare System, Ann Arbor, MI 48105

Multiple sclerosis (MS) is believed to be initiated by myelin-reactive CD4(+) Th cells. IL-12-polarized Th1 cells, IL-23-polarized Th17 cells, and Th17 cells that acquire Th1 characteristics were each implicated in autoimmune pathogenesis. It is debated whether Th cells that can drive the development of demyelinating lesions are phenotypically diverse or arise from a single lineage. In the current study, we assessed the requirement of IL-12 or IL-23 stimulation, as well as Th plasticity, for the differentiation of T cells capable of inducing CNS axon damage. We found that stable murine Th1 and Th17 cells independently transfer experimental autoimmune encephalomyelitis (widely used as an animal model of MS) in the absence of IL-23 and IL-12, respectively. Plastic Th17 cells are particularly potent mediators of demyelination and axonopathy. In parallel studies, we identified MS patients who consistently mount either IFN-γ- or IL-17-skewed responses to myelin basic protein over the course of a year. Brain magnetic resonance imaging revealed that patients with mixed IFN-γ and IL-17 responses have relatively high T1 lesion burden, a measure of permanent axon damage. Our data challenge the dogma that IL-23 and Th17 plasticity are universally required for the development of experimental autoimmune encephalomyelitis. This study definitively demonstrates that autoimmune demyelinating disease can be driven by distinct Th-polarizing factors and effector subsets, underscoring the importance of a customized approach to the pharmaceutical management of MS.
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http://dx.doi.org/10.4049/jimmunol.1501097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4561206PMC
September 2015

Is the concept of central nervous system immune privilege irrelevant in the setting of acute infection?

Front Oncol 2015 28;5:99. Epub 2015 Apr 28.

Department of Neurology, University of Michigan Medical School , Ann Arbor, MI , USA.

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http://dx.doi.org/10.3389/fonc.2015.00099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4412012PMC
May 2015

The conundrum of interferon-β non-responsiveness in relapsing-remitting multiple sclerosis.

Cytokine 2015 Aug 15;74(2):228-36. Epub 2015 Feb 15.

Department of Neurology, University of Michigan Medical School, Ann Arbor, MI, USA. Electronic address:

A series of controlled clinical trials have shown that exogenous interferon-beta (IFN-β) benefits patients with relapsing-remitting multiple sclerosis (RRMS) by reducing relapse rate, disability progression, and the formation of new brain and spinal cord lesions on magnetic resonance imaging (MRI) scans. Unfortunately, however, the effectiveness of IFN-β is limited in this setting by the occurrence of treatment non-responsiveness in nearly 25% of patients. Furthermore, clinicians who care for RRMS patients remain unable to accurately identify IFN-β non-responders prior to the initiation of therapy, causing delays in the use of alternative treatments and sometimes requiring that patients turn to medications with more significant side effects to control their disease. Progress has been made toward understanding how both endogenous and exogenous IFN-β act to slow RRMS as well as the related mouse model, experimental autoimmune encephalomyelitis (EAE). Most studies point to its inhibitory actions on circulating immune cells as being important for suppressing both disorders, but multiple potential target cells and inflammatory pathways have been implicated and those essential to confer its benefits remain undefined. This review focuses on the role of both endogenous and exogenous IFN-β in RRMS, paying particular attention to the issue of why certain individuals appear refractory to its disease-modifying effects. A continued goal in this field remains the identification of a convenient biomarker that accurately predicts IFN-β treatment non-responsiveness in individual RRMS patients. Development of such an assay will allow clinicians to customize therapy for patients with this complex disorder.
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http://dx.doi.org/10.1016/j.cyto.2015.01.004DOI Listing
August 2015

Neutrophil-related factors as biomarkers in EAE and MS.

J Exp Med 2015 Jan 5;212(1):23-35. Epub 2015 Jan 5.

Holtom-Garrett Program in Neuroimmunology, Department of Neurology,Department of Radiology, Department of Biostatistics, and Graduate Program in Immunology, University of Michigan, Ann Arbor, MI 48109 Holtom-Garrett Program in Neuroimmunology, Department of Neurology,Department of Radiology, Department of Biostatistics, and Graduate Program in Immunology, University of Michigan, Ann Arbor, MI 48109 Neurology Service, VA Ann Arbor Healthcare System, Ann Arbor, MI 48105

A major function of T helper (Th) 17 cells is to induce the production of factors that activate and mobilize neutrophils. Although Th17 cells have been implicated in the pathogenesis of multiple sclerosis (MS) and the animal model experimental autoimmune encephalomyelitis (EAE), little attention has been focused on the role of granulocytes in those disorders. We show that neutrophils, as well as monocytes, expand in the bone marrow and accumulate in the circulation before the clinical onset of EAE, in response to systemic up-regulation of granulocyte colony-stimulating factor (G-CSF) and the ELR(+) CXC chemokine CXCL1. Neutrophils comprised a relatively high percentage of leukocytes infiltrating the central nervous system (CNS) early in disease development. G-CSF receptor deficiency and CXCL1 blockade suppressed myeloid cell accumulation in the blood and ameliorated the clinical course of mice that were injected with myelin-reactive Th17 cells. In relapsing MS patients, plasma levels of CXCL5, another ELR(+) CXC chemokine, were elevated during acute lesion formation. Systemic expression of CXCL1, CXCL5, and neutrophil elastase correlated with measures of MS lesion burden and clinical disability. Based on these results, we advocate that neutrophil-related molecules be further investigated as novel biomarkers and therapeutic targets in MS.
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http://dx.doi.org/10.1084/jem.20141015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4291533PMC
January 2015

Immune responses to non-tumor antigens in the central nervous system.

Front Oncol 2014 13;4:328. Epub 2014 Nov 13.

Department of Neurology, University of Michigan Medical School , Ann Arbor, MI , USA.

The central nervous system (CNS), once viewed as an immune-privileged site protected by the blood-brain barrier (BBB), is now known to be a dynamic immunological environment through which immune cells migrate to prevent and respond to events such as localized infection. During these responses, endogenous glial cells, including astrocytes and microglia, become highly reactive and may secrete inflammatory mediators that regulate BBB permeability and recruit additional circulating immune cells. Here, we discuss the various roles played by astrocytes, microglia, and infiltrating immune cells during host immunity to non-tumor antigens in the CNS, focusing first on bacterial and viral infections, and then turning to responses directed against self-antigens in the setting of CNS autoimmunity.
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http://dx.doi.org/10.3389/fonc.2014.00328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4230036PMC
November 2014

Dysregulation of the IL-23/IL-17 axis and myeloid factors in secondary progressive MS.

Neurology 2014 Oct 24;83(17):1500-7. Epub 2014 Sep 24.

From the Holtom-Garrett Program in Neuroimmunology and Multiple Sclerosis Center, Departments of Neurology (A.K.H., D.N.I., B.M.S.), Biostatistics (L.W.), and Radiology (A.S.), University of Michigan, Ann Arbor; Neurology Service (B.M.S.), VA Ann Arbor Healthcare System, MI; Department of Statistics and Actuarial Science (P.H.), University of Waterloo, Canada; Department of Mathematics and Statistics (X.Z.), Bowling Green State University, OH; and Department of Imaging Sciences (S.E.), University of Rochester Medical Center, NY.

Objective: In the current exploratory study, we longitudinally measured immune parameters in the blood of individuals with relapsing-remitting multiple sclerosis (RRMS) and secondary progressive multiple sclerosis (SPMS), and investigated their relationship to disease duration and clinical and radiologic measures of CNS injury.

Methods: Peripheral blood mononuclear cells (PBMCs) and plasma were obtained from subjects with RRMS, SPMS, and from healthy controls on a monthly basis over the course of 1 year. MRI and Expanded Disability Status Scale evaluations were performed serially. PBMCs were analyzed by enzyme-linked immunosorbent spot assay to enumerate myelin basic protein-specific interleukin (IL)-17- and interferon (IFN)-γ-producing cells. Plasma concentrations of proinflammatory factors were measured using customized Luminex panels.

Results: Frequencies of myelin basic protein-specific IL-17- and IFN-γ-producing PBMCs were higher in individuals with RRMS and SPMS compared to healthy controls. Patients with SPMS expressed elevated levels of IL-17-inducible chemokines that activate and recruit myeloid cells. In the cohort of patients with SPMS without inflammatory activity, upregulation of myeloid-related factors correlated directly with MRI T2 lesion burden and inversely with brain parenchymal tissue volume.

Conclusions: The results of this exploratory study raise the possibility that Th17 responses and IL-17-inducible myeloid factors are elevated during SPMS compared with RRMS, and correlate with lesion burden. Our data endorse further investigation of Th17- and myeloid-related factors as candidate therapeutic targets in SPMS.
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http://dx.doi.org/10.1212/WNL.0000000000000908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4222856PMC
October 2014

Site-specific chemokine expression regulates central nervous system inflammation and determines clinical phenotype in autoimmune encephalomyelitis.

J Immunol 2014 Jul 13;193(2):564-70. Epub 2014 Jun 13.

Department of Neurology, Holtom-Garrett Program in Neuroimmunology and Multiple Sclerosis Center, University of Michigan School of Medicine, Ann Arbor, MI 48109; Graduate Program in Immunology, University of Michigan School of Medicine, Ann Arbor, MI 48109; and Neuroscience Program, University of Michigan School of Medicine, Ann Arbor, MI 48109

The adoptive transfer of myelin-reactive T cells into wild-type hosts results in spinal cord inflammation and ascending paralysis, referred to as conventional experimental autoimmune encephalomyelitis (EAE), as opposed to brainstem inflammation and ataxia, which characterize disease in IFN-γRKO hosts (atypical EAE). In this article, we show that atypical EAE correlates with preferential upregulation of CXCL2 in the brainstem, and is driven by CXCR2-dependent recruitment of neutrophils. In contrast, conventional EAE is associated with upregulation of CCL2 in the spinal cord, and is driven by recruitment of monocytes via a partially CCR2-dependent pathway. This study illustrates how regional differences in chemokine expression within a target organ shape the spatial pattern and composition of autoimmune infiltrates, leading to disparate clinical outcomes.
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http://dx.doi.org/10.4049/jimmunol.1400825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4091641PMC
July 2014

Type-I interferons suppress microglial production of the lymphoid chemokine, CXCL13.

Glia 2014 Sep 14;62(9):1452-62. Epub 2014 May 14.

Department of Neurology, University of Michigan Medical School, Ann Arbor, Michigan.

Lymphoid chemokines are crucial for the development and maintenance of lymphoid organs, but their ectopic expression in non-lymphoid tissues is implicated in both local response to infection and chronic organ-specific autoimmunity. Production of one such chemokine, C-X-C motif ligand 13 (CXCL13), within the central nervous system (CNS) has been linked to the pathogenesis of multiple sclerosis (MS), although little is known about factors controlling its expression in different neural cell types and across a range of disease states. We provoked acute neuroinflammation in experimental animals without causing any associated demyelination using neuroadapted Sindbis virus (NSV) to better understand the sources and regulators of this chemokine in the CNS. We found that mice genetically deficient in the transcription factor, interferon (IFN) regulatory factor-7 (IRF7), made significantly higher CXCL13 protein levels in the CNS compared with wild-type (WT) controls. Microglia proved to be the main producer of CXCL13 in the brain during infection of both WT and IRF7(-/-) mice, and primary microglia cultured in vitro generated CXCL13 following stimulation with either virus particles or synthetic Toll-like receptor (TLR) ligands. Microglia cultured from IRF7(-/-) mice selectively overproduced CXCL13, and manipulation of extracellular type-I IFN levels demonstrated the existence of a negative feedback loop whereby type-I IFN receptor signaling specifically suppressed microglial CXCL13 release. Since IFN-β is used to treat patients with relapsing-remitting MS and yet acts through unknown mechanisms, we speculate that suppressed lymphoid chemokine production by microglia could contribute to its therapeutic effects.
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http://dx.doi.org/10.1002/glia.22692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4143141PMC
September 2014

Virus-induced CD8+ T cells accelerate the onset of experimental autoimmune encephalomyelitis: implications for how viral infections might trigger multiple sclerosis exacerbations.

J Neuroimmunol 2013 Jun 18;259(1-2):47-54. Epub 2013 Apr 18.

Department of Neurology, University of Michigan Medical School, 4007 Biomedical Sciences Research Building, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200, United States.

Viral infections can exacerbate multiple sclerosis (MS) through poorly defined mechanisms. We developed an experimental system whereby infection with an asymptomatic neurotropic alphavirus caused a transient acceleration of experimental autoimmune encephalomyelitis (EAE) without altering the expansion or differentiation of autoreactive CD4+ T cells. Instead, this effect on the clinical course of EAE depended on CD8+ T cells that neither participate in viral clearance nor induce neuropathology in infected mice without EAE. Our system should be useful to further unravel how certain viral infections trigger MS exacerbations and to understand how CD8+ T cells can exert pathogenic effects within active demyelinating lesions.
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http://dx.doi.org/10.1016/j.jneuroim.2013.03.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3654028PMC
June 2013

Hepatitis C virus infection of a thyroid cell line: implications for pathogenesis of hepatitis C virus and thyroiditis.

Thyroid 2013 Jul 21;23(7):863-70. Epub 2013 Jun 21.

Division of Digestive Diseases, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA.

Background: Autoimmune and non-autoimmune thyroiditis frequently occur in persons with hepatitis C virus (HCV) infection. Treatment with interferon alpha (IFNα) is also associated with significant risk for the development of thyroiditis. To explore HCV-thyroid interactions at a cellular level, we evaluated whether a human thyroid cell line (ML1) could be infected productively with HCV in vitro.

Methods And Results: ML1 cells showed robust surface expression of the major HCV receptor CD81. Using a highly sensitive, strand-specific reverse transcription polymerase chain reaction assay, positive-sense and negative-sense HCV RNA were detected in ML1 cell lysates at days 3, 7, and 14 postinfection with HCV. HCV core protein was expressed at high levels in ML1 supernatants at days 1, 3, 5, 7, and 14 postinfection. The nonstructural protein NS5A was also detected in ML1 cell lysates by Western blotting. HCV entry into ML1 cells was shown to be dependent on the HCV entry factors CD81 and SR-B1/CLA1, while IFNα inhibited HCV replication in ML1 cells in a dose-dependent manner. Supernatants from HCV-infected ML1 cells were able to infect fresh ML1 cells productively, suggesting that infectious virions could be transferred from infected to naïve thyroid cells in vivo. Additionally, HCV infection of ML1 cells led to increased expression of the pro-inflammatory cytokine IL-8.

Conclusions: For the first time, we have demonstrated that HCV can infect human thyroid cells in vitro. These findings strongly suggest that HCV infection of thyrocytes may play a role in the association between chronic HCV infection and thyroid autoimmunity. Furthermore, the thyroid may serve as an extrahepatic reservoir for HCV viral replication, thus contributing to the persistence of viral infection and to the development of thyroid autoimmunity.
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http://dx.doi.org/10.1089/thy.2012.0507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3704108PMC
July 2013

Hyperthyroid-associated osteoporosis is exacerbated by the loss of TSH signaling.

J Clin Invest 2012 Oct 17;122(10):3737-41. Epub 2012 Sep 17.

Thyroid Research Unit and The Mount Sinai Bone Program, Department of Medicine, Mount Sinai School of Medicine, and James J. Peters VA Medical Center, New York, New York 10029, USA.

The osteoporosis associated with human hyperthyroidism has traditionally been attributed to elevated thyroid hormone levels. There is evidence, however, that thyroid-stimulating hormone (TSH), which is low in most hyperthyroid states, directly affects the skeleton. Importantly, Tshr-knockout mice are osteopenic. In order to determine whether low TSH levels contribute to bone loss in hyperthyroidism, we compared the skeletal phenotypes of wild-type and Tshr-knockout mice that were rendered hyperthyroid. We found that hyperthyroid mice lacking TSHR had greater bone loss and resorption than hyperthyroid wild-type mice, thereby demonstrating that the absence of TSH signaling contributes to bone loss. Further, we identified a TSH-like factor that may confer osteoprotection. These studies suggest that therapeutic suppression of TSH to very low levels may contribute to bone loss in people.
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http://dx.doi.org/10.1172/JCI63948DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3461920PMC
October 2012

Genetically driven target tissue overexpression of CD40: a novel mechanism in autoimmune disease.

J Immunol 2012 Sep 10;189(6):3043-53. Epub 2012 Aug 10.

Division of Endocrinology, Mount Sinai School of Medicine, New York, NY 10029, USA.

The CD40 gene, an important immune regulatory gene, is also expressed and functional on nonmyeloid-derived cells, many of which are targets for tissue-specific autoimmune diseases, including β cells in type 1 diabetes, intestinal epithelial cells in Crohn's disease, and thyroid follicular cells in Graves' disease (GD). Whether target tissue CD40 expression plays a role in autoimmune disease etiology has yet to be determined. In this study, we show that target tissue overexpression of CD40 plays a key role in the etiology of autoimmunity. Using a murine model of GD, we demonstrated that thyroidal CD40 overexpression augmented the production of thyroid-specific Abs, resulting in more severe experimental autoimmune GD (EAGD), whereas deletion of thyroidal CD40 suppressed disease. Using transcriptome and immune-pathway analyses, we showed that in both EAGD mouse thyroids and human primary thyrocytes, CD40 mediates this effect by activating downstream cytokines and chemokines, most notably IL-6. To translate these findings into therapy, we blocked IL-6 during EAGD induction in the setting of thyroidal CD40 overexpression and showed decreased levels of thyroid stimulating hormone receptor-stimulating Abs and frequency of disease. We conclude that target tissue overexpression of CD40 plays a key role in the etiology of organ-specific autoimmune disease.
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http://dx.doi.org/10.4049/jimmunol.1200311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3436983PMC
September 2012

Novel variant of thyroglobulin promoter triggers thyroid autoimmunity through an epigenetic interferon alpha-modulated mechanism.

J Biol Chem 2011 Sep 12;286(36):31168-79. Epub 2011 Jul 12.

Division of Endocrinology, Department of Medicine, Mount Sinai Medical Center, New York, New York 10029, USA.

Autoimmune thyroid diseases (AITD) arise from complex interactions between genetic, epigenetic, and environmental factors. Whole genome linkage scans and association studies have established thyroglobulin (TG) as a major AITD susceptibility gene. However, the causative TG variants and the pathogenic mechanisms are unknown. Here, we describe a genetic/epigenetic mechanism by which a newly identified TG promoter single-nucleotide polymorphism (SNP) variant predisposes to AITD. Sequencing analyses followed by case control and family-based association studies identified an SNP (-1623A→G) that was associated with AITD in the Caucasian population (p = 0.006). We show that the nucleotide substitution introduced by SNP (-1623A/G) modified a binding site for interferon regulatory factor-1 (IRF-1), a major interferon-induced transcription factor. Using chromatin immunoprecipitation, we demonstrated that IRF-1 binds to the 5' TG promoter motif, and the transcription factor binding correlates with active chromatin structure and is marked by enrichment of mono-methylated Lys-4 residue of histone H3, a signature of active transcriptional enhancers. Using reporter mutations and siRNA approaches, we demonstrate that the disease-associated allele (G) conferred increased TG promoter activity through IRF-1 binding. Finally, treatment of thyroid cells with interferon α, a known trigger of AITD, increased TG promoter activity only when it interacted with the disease-associated variant through IRF-1 binding. These results reveal a new mechanism of interaction between environmental (IFNα) and genetic (TG) factors to trigger AITD.
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http://dx.doi.org/10.1074/jbc.M111.247510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3173071PMC
September 2011