Proteome of Geobacter sulfurreducens in the presence of U(VI).

Geobacter species often play an important role in the in situ bioremediation of uranium-contaminated groundwater, but little is known about how these microbes avoid uranium toxicity. To evaluate this further, the proteome of Geobacter sulfurreducens exposed to 100 µM U(VI) acetate was compared to control cells not exposed to U(VI). Of the 1363 proteins detected from these cultures, 203 proteins had higher abundance during exposure to U(VI) compared with the control cells and 148 proteins had lower abundance. U(VI)-exposed cultures expressed lower levels of proteins involved in growth, protein and amino acid biosynthesis, as well as key central metabolism enzymes as a result of the deleterious effect of U(VI) on the growth of G. sulfurreducens. In contrast, proteins involved in detoxification, such as several efflux pumps belonging to the RND (resistance-nodulation-cell division) family, and membrane protection, and other proteins, such as chaperones and proteins involved in secretion systems, were found in higher abundance in cells exposed to U(VI). Exposing G. sulfurreducens to U(VI) resulted in a higher abundance of many proteins associated with the oxidative stress response, such as superoxide dismutase and superoxide reductase. A strain in which the gene for superoxide dismutase was deleted grew more slowly than the WT strain in the presence of U(VI), but not in its absence. The results suggested that there is no specific mechanism for uranium detoxification. Rather, multiple general stress responses are induced, which presumably enable Geobacter species to tolerate high uranium concentrations.

U(VI) reduction by diverse outer surface c-type cytochromes of Geobacter sulfurreducens.

Early studies with Geobacter sulfurreducens suggested that outer-surface c-type cytochromes might play a role in U(VI) reduction, but it has recently been suggested that there is substantial U(VI) reduction at the surface of the electrically conductive pili known as microbial nanowires. This phenomenon was further investigated. A strain of G. sulfurreducens, known as Aro-5, which produces pili with substantially reduced conductivity reduced U(VI) nearly as well as the wild type, as did a strain in which the gene for PilA, the structural pilin protein, was deleted. In order to reduce rates of U(VI) reduction to levels less than 20% of the wild-type rates, it was necessary to delete the genes for the five most abundant outer surface c-type cytochromes of G. sulfurreducens. X-ray absorption near-edge structure spectroscopy demonstrated that whereas 83% ± 10% of the uranium associated with wild-type cells correspond to U(IV) after 4 h of incubation, with the quintuple mutant, 89% ± 10% of uranium was U(VI). Transmission electron microscopy and X-ray energy dispersion spectroscopy revealed that wild-type cells did not precipitate uranium along pili as previously reported, but U(IV) was precipitated at the outer cell surface. These findings are consistent with those of previous studies, which have suggested that G. sulfurreducens requires outer-surface c-type cytochromes but not pili for the reduction of soluble extracellular electron acceptors.

Biochemical characterization of purified OmcS, a c-type cytochrome required for insoluble Fe(III) reduction in Geobacter sulfurreducens.

Previous studies with Geobacter sulfurreducens have demonstrated that OmcS, an abundant c-type cytochrome that is only loosely bound to the outer surface, plays an important role in electron transfer to Fe(III) oxides as well as other extracellular electron acceptors. In order to further investigate the function of OmcS, it was purified from a strain that overproduces the protein. Purified OmcS had a molecular mass of 47015 Da, and six low-spin bis-histidinyl hexacoordinated heme groups. Its midpoint redox potential was -212 mV. A thermal stability analysis showed that the cooperative melting of purified OmcS occurs in the range of 65-82 °C. Far UV circular dichroism spectroscopy indicated that the secondary structure of purified OmcS consists of about 10% α-helix and abundant disordered structures. Dithionite-reduced OmcS was able to transfer electrons to a variety of substrates of environmental importance including insoluble Fe(III) oxide, Mn(IV) oxide and humic substances. Stopped flow analysis revealed that the reaction rate of OmcS oxidation has a hyperbolic dependence on the concentration of the studied substrates. A ten-fold faster reaction rate with anthraquinone-2,6-disulfonate (AQDS) (25.2 s⁻¹) was observed as compared to that with Fe(III) citrate (2.9 s⁻¹). The results, coupled with previous localization and gene deletion studies, suggest that OmcS is well-suited to play an important role in extracellular electron transfer.

Supercapacitors based on c-type cytochromes using conductive nanostructured networks of living bacteria.

Supercapacitors have attracted interest in energy storage because they have the potential to complement or replace batteries. Here, we report that c-type cytochromes, naturally immersed in a living, electrically conductive microbial biofilm, greatly enhance the device capacitance by over two orders of magnitude. We employ genetic engineering, protein unfolding and Nernstian modeling for in vivo demonstration of charge storage capacity of c-type cytochromes and perform electrochemical impedance spectroscopy, cyclic voltammetry and charge-discharge cycling to confirm the pseudocapacitive, redox nature of biofilm capacitance. The biofilms also show low self-discharge and good charge/discharge reversibility. The superior electrochemical performance of the biofilm is related to its high abundance of cytochromes, providing large electron storage capacity, its nanostructured network with metallic-like conductivity, and its porous architecture with hydrous nature, offering prospects for future low cost and environmentally sustainable energy storage devices.

Improvement of asymmetric thyroid eye disease with teprotumumab.

PURPOSE: Teprotumumab, a specific blocking antibody to the insulin like growth factor 1 receptor, significantly reduced proptosis in patients with thyroid eye disease (TED) in recent clinical trials. Given its specificity, we expect it to demonstrate greater efficacy on the worse affected orbit, in patients with asymmetric TED. Herein, we investigate the differential impact of teprotumumab on the orbits of such patients. METHODS: In this pooled analysis of patients who were enrolled in the recent phase 2 (NCT01868997) and phase 3 (NCT03298867) trials, all patients with asymmetric TED (difference in exophthalmometry of ≥3 mm) were screened for eligibility. The primary outcomes of the trials, proptosis, diplopia and Clinical Activity Score (CAS) response, were evaluated in both orbits of patients who had received treatment or placebo, to examine the differential response from baseline to week 24. RESULTS: From a pooled group of 84 patients randomised to receive teprotumumab and 87 randomised to placebo, 10 (12%) and 12 (14%), respectively, met the inclusion criteria. The teprotumumab-treated patients demonstrated significant reductions in proptosis, CAS and diplopia in both orbits of each patient and this was not seen with placebo. The reduction in proptosis and CAS was significantly greater in the worse affected orbit, improving symmetry. In the placebo arm, while the mean CAS in the study eye reduced over time, proptosis and diplopia did not change in either orbit. CONCLUSION: The findings in this study suggest the differential impact of teprotumumab on orbits that are clinically more affected by TED, suggesting that teprotumumab reduces asymmetry.

Teprotumumab for thyroid eye disease: early response is not required for benefit.

PURPOSE: In recent trials, 50% of patients treated with teprotumumab for thyroid eye disease had significant improvements in proptosis at 6 weeks. However, a small subgroup of patients did not have a significant response by week 12. We examine the outcomes at week 24 in patients from both trials who had little or no proptosis response at week 12. DESIGN: In this post hoc analysis, data from teprotumumab-treated patients in the placebo-controlled randomized phases 2 and 3 trials were reviewed. METHODS: Patients treated with teprotumumab or placebo with a ≤2 mm reduction from baseline in proptosis at week 12 and completed assessments at both the weeks 12 and 24 visits were included. The main outcome measures were a change in proptosis, clinical activity score (CAS) and diplopia in response to teprotumumab therapy at baseline and weeks 6, 12, 18, and 24. RESULTS: From the phases 2 and 3 studies, 24 patients from the treated and placebo groups were included for analysis (48 total). In the teprotumumab group, of the 24 who had no improvement in proptosis (≥2 mm from baseline) at 12 weeks, 15 (63%) demonstrated a clinically significant improvement at week 24. No patients from the 24 placebo patients had a clinically significant improvement in proptosis at 12 weeks, and 24 weeks. At week 12, 22 patients (92%) in the teprotumumab group had a significant reduction in the CAS (≥2 points) and at 24 weeks all patients achieved this reduction. At week 12, 11 (46%) patients from the placebo group had a significant improvement, while 10 (42%) had a significant improvement at 24 weeks. 22 of the 24 patients (92%) in the teprotumumab group had a diplopia grade > 0 at baseline. At week 12, 12 of the 22 (55%) had improvement in diplopia ≥ 1 grade. By week 24, 16 patients (73%) had an improvement in diplopia ≥ 1 grade. In the placebo group, 15 (63%) had significant diplopia. At week 12, 3 (20%) from this group had improvement in diplopia ≥ 1 grade, while at 24 weeks this number rose to 4 (27%). CONCLUSIONS: There is variability in the time taken to manifest a clinically significant response to teprotumumab, some patients my need a longer time to respond.

Teprotumumab for non-inflammatory thyroid eye disease (TED): evidence for increased IGF-1R expression.

PURPOSE: Teprotumumab, a blocking antibody to the insulin like growth factor 1 receptor (IGF-1R) has been shown to significantly reduce proptosis in recent phase 2 and 3 trials in patients with inflammatory thyroid eye disease (TED). Herein, we investigate the impact of teprotumumab on patients with non-inflammatory TED. We also investigate the expression of the IGF-1R on orbital tissues from patients with inflammatory and non-inflammatory TED compared to controls. METHODS: Consecutive patients with non-inflammatory TED (clinical activity score, CAS ≤ 1, for at least 4 months, were treated with teprotumumab. They received a complete course (total eight infusions) of teprotumumab (10 mg/kg for the first infusion and 20 mg/kg for subsequent infusions every 3 weeks). The primary outcome was a proptosis response at week 24. Further, IGF-1R α and ß expression was evaluated on orbital tissue from patients with inflammatory and non-inflammatory TED, as well as healthy controls. Non-biased histological analysis of IGF-1R expression was performed using ImageJ. RESULTS: Four patients met eligibility criteria for the clinical study, with a mean (SD) CAS of 0 (0). Following teprotumumab treatment, there was a mean (SD) reduction in proptosis of 2.6 mm (1.2). Five patients were included for each group of the histological study; inflammatory TED, non-inflammatory TED and controls. IGF-1Rα and IGF-1Rß expression was significantly greater in the orbital tissues of patients with inflammatory TED and non-inflammatory TED, when compared to controls. CONCLUSION: Our findings demonstrate for the first time, that teprotumumab, a blocking antibody to the IGF-1R reduces proptosis in a series of patients with non-inflammatory TED. Overexpression of the IGF-1R in orbital tissue from patients with non-inflammatory disease compared to controls may be an important consideration for effect.

Alignment of the c-type cytochrome OmcS along pili of Geobacter sulfurreducens.

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.

Purification and characterization of OmcZ, an outer-surface, octaheme c-type cytochrome essential for optimal current production by Geobacter sulfurreducens.

Previous studies have demonstrated that Geobacter sulfurreducens requires the c-type cytochrome OmcZ, which is present in large (OmcZ(L); 50-kDa) and small (OmcZ(S); 30-kDa) forms, for optimal current production in microbial fuel cells. This protein was further characterized to aid in understanding its role in current production. Subcellular-localization studies suggested that OmcZ(S) was the predominant extracellular form of OmcZ. N- and C-terminal amino acid sequence analysis of purified OmcZ(S) and molecular weight measurements indicated that OmcZ(S) is a cleaved product of OmcZ(L) retaining all 8 hemes, including 1 heme with the unusual c-type heme-binding motif CX(14)CH. The purified OmcZ(S) was remarkably thermally stable (thermal-denaturing temperature, 94.2 degrees C). Redox titration analysis revealed that the midpoint reduction potential of OmcZ(S) is approximately -220 mV (versus the standard hydrogen electrode [SHE]) with nonequivalent heme groups that cover a large reduction potential range (-420 to -60 mV). OmcZ(S) transferred electrons in vitro to a diversity of potential extracellular electron acceptors, such as Fe(III) citrate, U(VI), Cr(VI), Au(III), Mn(IV) oxide, and the humic substance analogue anthraquinone-2,6-disulfonate, but not Fe(III) oxide. The biochemical properties and extracellular localization of OmcZ suggest that it is well suited for promoting electron transfer in current-producing biofilms of G. sulfurreducens.

Proteome of Geobacter sulfurreducens grown with Fe(III) oxide or Fe(III) citrate as the electron acceptor.

The mechanisms for Fe(III) oxide reduction in Geobacter species are of interest because Fe(III) oxides are the most abundant form of Fe(III) in many soils and sediments and Geobacter species are prevalent Fe(III)-reducing microorganisms in many of these environments. Protein abundance in G. sulfurreducens grown on poorly crystalline Fe(III) oxide or on soluble Fe(III) citrate was compared with a global accurate mass and time tag proteomic approach in order to identify proteins that might be specifically associated with Fe(III) oxide reduction. A total of 2991 proteins were detected in G. sulfurreducens grown with acetate as the electron donor and either Fe(III) oxide or soluble Fe(III) citrate as the electron acceptor, resulting in 86% recovery of the genes predicted to encode proteins. Of the total expressed proteins 76% were less abundant in Fe(III) oxide cultures than in Fe(III) citrate cultures, which is consistent with the overall slower rate of metabolism during growth with an insoluble electron acceptor. A total of 269 proteins were more abundant in Fe(III) oxide-grown cells than in cells grown on Fe(III) citrate. Most of these proteins were in the energy metabolism category: primarily electron transport proteins, including 13 c-type cytochromes and PilA, the structural protein for electrically conductive pili. Several of the cytochromes that were more abundant in Fe(III) oxide-grown cells were previously shown with genetic approaches to be essential for optimal Fe(III) oxide reduction. Other proteins that were more abundant during growth on Fe(III) oxide included transport and binding proteins, proteins involved in regulation and signal transduction, cell envelope proteins, and enzymes for amino acid and protein biosynthesis, among others. There were also a substantial number of proteins of unknown function that were more abundant during growth on Fe(III) oxide. These results indicate that electron transport to Fe(III) oxide requires additional and/or different proteins than electron transfer to soluble, chelated Fe(III) and suggest proteins whose functions should be further investigated in order to better understand the mechanisms of electron transfer to Fe(III) oxide in G. sulfurreducens.

CD34- Orbital Fibroblasts From Patients With Thyroid-Associated Ophthalmopathy Modulate TNF-α Expression in CD34+ Fibroblasts and Fibrocytes.

Purpose: Orbital fibroblasts from patients with Graves' disease (GD-OF) express many different cytokines when treated with bovine thyrotropin (bTSH). The present study aimed to determine why TNF-α cannot be induced by bTSH in GD-OF. Methods: Fibrocytes and GD-OFs were cultivated from donors who were patients in a busy academic medical center practice. Real-time PCR, Western blot analysis, reporter gene assays, cell transfections, mRNA stability assays, ELISA, and flow cytometry were performed. Results: We found that bTSH induces TNF-α dramatically in fibrocytes but is undetectable in GD-OF. The induction in fibrocytes is a consequence of increased TNF-α gene promoter activity and is independent of ongoing protein synthesis. It could be attenuated by dexamethasone and the IGF-1 receptor inhibiting antibody, teprotumumab. When separated into pure CD34+ OF and CD34- OF subsets, TNF-α mRNA became highly inducible by bTSH in CD34+ OF but remained undetectable in CD34- OF. Conditioned medium from CD34- OF inhibited induction of TNF-α in fibrocytes. Conclusions: Our data indicate that CD34- OF appear to release a soluble(s) factor that downregulates expression and induction by bTSH of TNF-α in fibrocytes and their derivative CD34+ OF. We proffer that CD34- OF produce an unidentified modulatory factor that attenuates TNF-α expression in GD-OF and may do so in the TAO orbit.

The proteome of dissimilatory metal-reducing microorganism Geobacter sulfurreducens under various growth conditions.

The proteome of Geobacter sulfurreducens, a model for the Geobacter species that predominate in many Fe(III)-reducing subsurface environments, was characterized with ultra high-pressure liquid chromatography and mass spectrometry using accurate mass and time (AMT) tags as well as with more traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Cells were grown under six different growth conditions in order to enhance the potential that a wide range of genes would be expressed. The AMT tag approach was able to identify a much greater number of proteins than could be detected with the 2-D PAGE approach. With the AMT approach over 3,000 gene products were identified, representing about 90% of the total predicted gene products in the genome. A high proportion of predicted proteins in most protein role categories were detected; the highest number of proteins was identified in the hypothetical protein role category. Furthermore, 91 c-type cytochromes of 111 predicted genes in the G. sulfurreducens genome were identified. Differences in the abundance of cytochromes and other proteins under different growth conditions provided information for future functional analysis of these proteins. These results demonstrate that a high percentage of the predicted proteins in the G. sulfurreducens genome are produced and that the AMT tag approach provides a rapid method for comparing differential expression of proteins under different growth conditions in this organism.

Evidence that OmcB and OmpB of Geobacter sulfurreducens are outer membrane surface proteins.

The c-type cytochrome (OmcB) and the multicopper protein (OmpB) required for Fe(III) oxide reduction by Geobacter sulfurreducens were predicted previously to be outer membrane proteins, but it is not clear whether they are positioned in a manner that permits the interaction with Fe(III). Treatment of whole cells with proteinase K inhibited Fe(III) reduction, but had no impact on the inner membrane-associated fumarate reduction. OmcB was digested by protease, resulting in a smaller peptide. However, immunogold labeling coupled with transmission electron microscopy did not detect OmcB, suggesting that it is only partially exposed on the cell surface. In contrast, OmpB was completely digested with protease. OmpB was loosely associated with the cell surface as a substantial portion of it was recovered in the culture supernatant. Immunogold labeling demonstrated that OmpB associated with the cell was evenly distributed on the cell surface rather than localized to one side of the cell like the conductive pili. Although several proteins required for Fe(III) oxide reduction are shown to be exposed on the outer surface of G. sulfurreducens, the finding that OmcB is also surface exposed is the first report of a protein required for optimal Fe(III) citrate reduction at least partially accessible on the cell surface.

CD40 Expression in Fibrocytes Is Induced by TSH: Potential Synergistic Immune Activation.

CONTEXT: Fibrocytes appear to participate in inflammation and tissue remodeling in patients with thyroid-associated ophthalmopathy (TAO). These patients have increased frequencies of circulating TSH receptor (TSHR)- and CD40-positive fibrocytes, suggesting TSHR and CD40 may play roles in proinflammatory cytokine production, which ultimately leads to orbital inflammation and tissue remodeling. OBJECTIVE: To investigate the potential interactions between the TSHR and CD40 signaling pathways and their roles in IL-6 and TNF-α production. DESIGN AND OUTCOME MEASURES: CD40 expression on fibrocytes was assessed using flow cytometry; IL-6 and TNF-α protein release using Luminex technology; increased IL-6 and TNF-α mRNA abundance, using real-time PCR; TSH- and CD40 ligand (CD40L)-stimulated Akt phosphorylation in fibrocytes, by western blot analysis; TSHR-CD40 protein-protein interaction, using co-immunoprecipitation, and CD40-TSHR co-localization, using immunocytochemistry. RESULTS: TSH enhances CD40 expression at a pre-translational level in fibrocytes. Production of IL-6 and TNF-α after costimulation with TSH and CD40L was greater than that after TSH or CD40L stimulation alone. TSH and CD40L costimulation also resulted in greater Akt phosphorylation. Akt and nuclear factor (NF)-κB inhibitors significantly reduced cytokine production after TSH and CD40L costimulation. TSHR and CD40L are colocalized on the cell surface and form a complex. CONCLUSIONS: TSHR and CD40 in fibrocytes appear to be physically and functionally related. TSH stimulates CD40 production on the fibrocyte surface. Cytokine expression upon simultaneous stimulation of TSHR and CD40 is greater than levels achieved with TSH or CD40L alone. Increased expression of CD40 by TSH is a potential mechanism for this process.

Thyrotropin and CD40L Stimulate Interleukin-12 Expression in Fibrocytes: Implications for Pathogenesis of Thyroid-Associated Ophthalmopathy.

BACKGROUND: Increased numbers of bone marrow-derived progenitor cells, known as fibrocytes, populate the peripheral circulation, orbit, and thyroid of patients with Graves' disease (GD). These cells have been implicated in the development of thyroid-associated ophthalmopathy. They can differentiate into myofibroblasts or adipocytes, produce inflammatory cytokines, and remodel tissue. This study sought to determine whether thyrotropin (TSH) and CD40 ligand (CD40L), implicated in the pathogenesis of GD, induce interleukin-12 (IL-12) in human fibrocytes. MATERIALS AND METHODS: IL-12 protein concentrations and mRNA levels were measured by Luminex and real-time polymerase chain reaction, respectively. Flow cytometry assessed intracellular IL-12 concentrations. Vector containing IL-12p40 promoter was transfected into cultured fibrocytes, and promoter activity was monitored using luciferase assay. RESULTS: TSH and CD40L stimulated intracellular IL-12 protein accumulation in peripheral blood fibrocytes. Inhibiting Akt and nuclear factor-κB (NF-κB) activity diminished IL-12 expression in fibrocytes, while TSH did not induce promoter activity. TSH-mediated IL-12 production required de novo synthesized proteins and augmented IL-12 mRNA stability. IL-12 production mediated by CD40L required tumor necrosis factor receptor-associated factor 6. CONCLUSION: TSH and CD40L induce IL-12 expression in fibrocytes, and Akt and NF-κB mediate this activity. Given the importance of IL-12 in immune function, its production by fibrocytes may promote an inflammatory immune response and tissue remodeling in thyroid-associated ophthalmopathy.

TSH-Mediated TNFα Production in Human Fibrocytes Is Inhibited by Teprotumumab, an IGF-1R Antagonist.

PURPOSE: Fibrocytes (FC) are bone marrow-derived progenitor cells that are more abundant and infiltrate the thyroid and orbit in Graves orbitopathy (GO). FCs express high levels of thyrotropin receptor (TSHR) and insulin-like growth factor-1 receptor (IGF-1R). These receptors are physically and functionally associated, but their role in GO pathogenesis is not fully delineated. Treatment of FCs with thyroid stimulating hormone (TSH) or M22 (activating antibody to TSHR) induces the production of numerous cytokines, including tumor necrosis factor α (TNFα). Teprotumumab (TMB) is a human monoclonal IGF-1R blocking antibody currently in clinical trial for GO and inhibits TSHR-mediated actions in FCs. AIM: To characterize the molecular mechanisms underlying TSH-induced TNFα production by FCs, and the role of IGF-1R blockade by TMB. DESIGN: FCs from healthy and GD patients were treated with combinations of TSH, M22, MG132 and AKTi (inhibitors of NF-κB and Akt, respectively), and TMB. TNFα protein production was measured by Luminex and flow cytometry. Messenger RNA expression was quantified by real time PCR. RESULTS: Treatment with TSH/M22 induced TNFα protein and mRNA production by FCs, both of which were reduced when FCs were pretreated with MG132 and AKTi (p<0.0001). TMB decreased TSH-induced TNFα protein production in circulating FCs from mean fluorescent index (MFI) value of 2.92 to 1.91, and mRNA expression in cultured FCs from 141- to 52-fold expression (p<0.0001). TMB also decreased M22-induced TNFα protein production from MFI of 1.67 to 1.12, and mRNA expression from 6- to 3-fold expression (p<0.0001). CONCLUSION: TSH/M22 stimulates FC production of TNFα mRNA and protein. This process involves the transcription factor NF-κB and its regulator Akt. Blocking IGF-1R attenuates TSH/M22-induced TNFα production. This further delineates the interaction of TSHR and IGF1-R signaling pathways. By modulating the proinflammatory properties of FCs such as TNFα production, TMB may be a promising therapeutic agent for GO.

Thyrotropin receptor and CD40 mediate interleukin-8 expression in fibrocytes: implications for thyroid-associated ophthalmopathy (an American Ophthalmological Society thesis).

PURPOSE: To better understand the pathogenesis of thyroid-associated orbitopathy (TAO) through elucidating the role of thyrotropin receptor (TSHR) and CD40 in the expression of interleukin-8 (IL-8) in peripheral blood fibrocytes. Fibrocytes infiltrate the orbit of patients with TAO, where they differentiate into fibroblasts. Fibrocyte precursors occur with increased frequency in the peripheral blood expressing TSHR and CD40 in TAO patients. We hypothesize that in vitro derived fibrocytes and peripheral blood fibrocyte precursors express proinflammatory chemoattractant molecules including IL-8 initiated by TSHR and CD40 signaling. Since nearly all TAO patients express activating antibodies to TSHR, this is particularly relevant for activation of peripheral blood fibrocytes. METHODS: TSHR and CD40 expression on peripheral blood fibrocytes was determined by flow cytometry. IL-8 RNA was quantitated by real-time polymerase chain reaction. IL-8 protein production was measured by Luminex and flow cytometry. Thyroid-stimulating hormone and CD40 ligand-stimulated phosphorylation of Akt in peripheral blood fibrocytes was studied by flow cytometry. RESULTS: Both TSHR- and CD40-mediated signaling lead to IL-8 expression in mature fibrocytes. Fibrocyte precursors assayed directly from circulating peripheral blood demonstrate intracellular IL-8 expression with addition of thyroid-stimulating hormone or CD40 ligand. TSHR- and CD40-induced IL-8 production is mediated by Akt phosphorylation. CONCLUSIONS: Peripheral blood TSHR(+) and CD40(+) fibrocytes express IL-8 and may promote the recruitment of inflammatory cells, mitogenesis, and tissue remodeling in TAO. TSHR- and CD40-mediated IL-8 signaling is mediated by Akt. Delineating the molecular mechanisms of fibrocyte immune function may provide potential therapeutic targets for TAO.

Expression of thyrotropin receptor, thyroglobulin, sodium-iodide symporter, and thyroperoxidase by fibrocytes depends on AIRE.

CONTEXT: CD34(+) fibrocytes, bone marrow-derived progenitor cells, infiltrate orbital connective tissue in thyroid-associated ophthalmopathy, a manifestation of Graves' disease. In the orbit, they become CD34(+) fibroblasts and coexist with native CD34(-) fibroblasts. Fibrocytes have been shown to express TSH receptor and thyroglobulin. OBJECTIVE: The objective of the study was to determine whether a broader repertoire of thyroid protein expression can be detected in fibrocytes and whether a common factor is responsible. DESIGN/SETTING/PARTICIPANTS: Fibrocytes and fibroblasts were collected and analyzed from healthy individuals and those with Graves' disease in an academic clinical practice. MAIN OUTCOME MEASURES: Real-time PCR, Western blot analysis, gene promoter analysis, cell transfections, and flow cytometric cell sorting were performed. RESULTS: We detect two additional thyroid proteins expressed by fibrocytes, namely sodium-iodide symporter and thyroperoxidase. The autoimmune regulator (AIRE) protein appears necessary for this expression. AIRE expression in fibrocytes results from an active AIRE gene promoter and stable AIRE mRNA. Knocking down AIRE with a targeting small interfering RNA reduces the expression of these thyroid proteins in fibrocytes as well as the transcription factors paired box-8 and thyroid transcription factor-1. When compared with an unaffected first-degree relative, levels of these proteins are substantially reduced in fibrocytes from an individual with an inactivating AIRE mutation. Levels of AIRE and the thyroid proteins are lower in orbital fibroblasts from patients with thyroid-associated ophthalmopathy than in fibrocytes. However, when mixed fibroblast populations are sorted into pure CD34(+) and CD34(-) subsets, the levels of these proteins are dramatically increased selectively in CD34(+) fibroblasts. CONCLUSIONS: Fibrocytes express four proteins, the aggregate expression of which was previously thought to be restricted to thyroid epithelium. These proteins represent the necessary molecular biosynthetic machinery necessary for thyroid hormone production. Our findings implicate AIRE in the promiscuous expression of thyroid proteins in fibrocytes.

Teprotumumab, an IGF-1R blocking monoclonal antibody inhibits TSH and IGF-1 action in fibrocytes.

CONTEXT: Thyroid-associated ophthalmopathy (TAO) is the component of Graves' disease characterized by orbital inflammation and connective tissue remodeling. The IGF-1 receptor (IGF-1R) and TSH receptor (TSHR) form a physical and functional complex in orbital fibroblasts. A subset of these fibroblasts is derived from infiltrating CD34(+) fibrocytes. Teprotumumab (RV 001, R1507) is a human monoclonal anti-IGF-1R blocking antibody currently undergoing a phase 2 clinical trial in patients with active TAO. OBJECTIVE: To determine whether teprotumumab inhibits the induction by TSH of IL-6 and IL-8 in fibrocytes. DESIGN: Fibrocytes were treated without or with teprotumumab in combination with IGF-1 or TSH. MAIN OUTCOME MEASURES: IL-6 and IL-8 mRNA expression and protein production were analyzed by real-time PCR and Luminex, respectively. Phosphorylated Akt (S473) levels were analyzed by Western blot. TSHR and IGF-1R display was assessed by flow cytometry. RESULTS: Fibrocyte display of IGF-1R and TSHR was reduced with teprotumumab, as were IGF-1- and TSH-dependent phosphorylated Akt levels. TSH induction of IL-6 and IL-8 mRNA and protein was also reduced by the monoclonal antibody. CONCLUSIONS: Teprotumumab attenuates the actions of both IGF-1 and TSH in fibrocytes. Specifically, it blocks the induction of proinflammatory cytokines by TSH. These results provide, at least in part, the molecular rationale for interrogating the therapeutic efficacy of this antibody in TAO.

Interleukin-6 production in CD40-engaged fibrocytes in thyroid-associated ophthalmopathy: involvement of Akt and NF-κB.

PURPOSE: CD40-CD40 ligand (CD40L) interactions appear to play pathogenic roles in autoimmune disease. Here we quantify CD40 expression on fibrocytes, circulating, and bone marrow-derived progenitor cells. The functional consequences of CD40 ligation are determined since these may promote tissue remodeling linked with thyroid-associated ophthalmopathy (TAO). METHODS: CD40 levels on cultivated fibrocytes and orbital fibroblasts (GOFB) from patients with Graves' disease (GD), as well as fibrocyte abundance, were determined by flow cytometry. CD40 mRNA expression was evaluated by real-time PCR, whereas response to CD40 ligation was measured by Luminex and RT-PCR. Protein kinase B (Akt) and nuclear factor (NF)-kappa B (NF-κB) signaling were determined by Western blot and immunofluorescence. RESULTS: Basal CD40 expression on fibrocytes is greater than that on GOFB. IFN-γ upregulates CD40 in both cell types and its actions are mediated at the pretranslational level. Fibrocytes produce high levels of cytokines, including interleukin-6 (IL-6), TNF-α, IL-8, MCP-1, and RANTES (Regulated on Activation, Normal T Cell Expressed and Secreted) in response to CD40L. IL-6 induction results from an increase in steady state IL-6 mRNA, and is mediated through Akt and NF-κB activation. Circulating CD40(+)CD45(+)Col1(+) fibrocytes are far more frequent in vivo in donors with TAO compared with healthy subjects. CONCLUSIONS: Particularly high levels of functional CD40 are displayed by fibrocytes. CD40L-provoked signaling results in the production of several cytokines. Among these, IL-6 expression is mediated through Akt and NF-κB pathways. The frequency of circulating CD40(+) fibrocytes is markedly increased in patients with TAO, suggesting that this receptor might represent a therapeutic target for TAO.