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Macrophage-Mediated Phagocytosis and Dissolution of Amyloid-Like Fibrils in Mice, Monitored by Optical Imaging

2019; Elsevier BV; Volume: 189; Issue: 5 Linguagem: Inglês

10.1016/j.ajpath.2019.01.011

ISSN

1525-2191

Autores

Tina Richey, James S. Foster, Angela D. Williams, Anna B. Williams, Alexa Stroh, Sallie Macy, Craig Wooliver, R. Eric Heidel, Siva Karthik Varanasi, Elizabeth N. Ergen, Dianne J. Trent, Stephen A. Kania, Stephen J. Kennel, Emily B. Martin, Jonathan S. Wall,

Tópico(s)

Dermatological and Skeletal Disorders

Resumo

Light chain–associated amyloidosis is characterized by the extracellular deposition of amyloid fibrils in abdominothoracic organs, skin, soft tissue, and peripheral nerves. Phagocytic cells of the innate immune system appear to be ineffective at clearing the material; however, human light chain amyloid extract, injected subcutaneously into mice, is rapidly cleared in a process that requires neutrophil activity. To better elucidate the phagocytosis of light chain fibrils, a potential method of cell-mediated dissolution, amyloid-like fibrils were labeled with the pH-sensitive dye pHrodo red and a near infrared fluorophore. After injecting this material subcutaneously in mice, optical imaging was used to quantitatively monitor phagocytosis and dissolution of fibrils concurrently. Histologic evaluation of the residual fibril masses revealed the presence of CD68+, F4/80+, ionized calcium binding adaptor molecule 1− macrophages containing Congo red–stained fibrils as well as neutrophil-associated proteins with no evidence of intact neutrophils. These data suggest an early infiltration of neutrophils, followed by extensive phagocytosis of the light chain fibrils by macrophages, leading to dissolution of the mass. Optical imaging of this novel murine model, coupled with histologic evaluation, can be used to study the cellular mechanisms underlying dissolution of synthetic amyloid-like fibrils and human amyloid extracts. In addition, it may serve as a test bed to evaluate investigational opsonizing agents that might serve as therapeutic agents for light chain–associated amyloidosis. Light chain–associated amyloidosis is characterized by the extracellular deposition of amyloid fibrils in abdominothoracic organs, skin, soft tissue, and peripheral nerves. Phagocytic cells of the innate immune system appear to be ineffective at clearing the material; however, human light chain amyloid extract, injected subcutaneously into mice, is rapidly cleared in a process that requires neutrophil activity. To better elucidate the phagocytosis of light chain fibrils, a potential method of cell-mediated dissolution, amyloid-like fibrils were labeled with the pH-sensitive dye pHrodo red and a near infrared fluorophore. After injecting this material subcutaneously in mice, optical imaging was used to quantitatively monitor phagocytosis and dissolution of fibrils concurrently. Histologic evaluation of the residual fibril masses revealed the presence of CD68+, F4/80+, ionized calcium binding adaptor molecule 1− macrophages containing Congo red–stained fibrils as well as neutrophil-associated proteins with no evidence of intact neutrophils. These data suggest an early infiltration of neutrophils, followed by extensive phagocytosis of the light chain fibrils by macrophages, leading to dissolution of the mass. Optical imaging of this novel murine model, coupled with histologic evaluation, can be used to study the cellular mechanisms underlying dissolution of synthetic amyloid-like fibrils and human amyloid extracts. In addition, it may serve as a test bed to evaluate investigational opsonizing agents that might serve as therapeutic agents for light chain–associated amyloidosis. Immunoglobulin light chain–associated amyloidosis (AL) is a plasma cell disorder wherein high concentrations of monoclonal light chain proteins are secreted into the circulation, resulting in the systemic deposition of extracellular amyloid.1Dispenzieri A. Gertz M.A. Buadi F. What do I need to know about immunoglobulin light chain (AL) amyloidosis?.Blood Rev. 2012; 26: 137-154Crossref PubMed Scopus (103) Google Scholar, 2Merlini G. Bellotti V. Molecular mechanisms of amyloidosis.N Engl J Med. 2003; 349: 583-596Crossref PubMed Scopus (1441) Google Scholar, 3Wechalekar A.D. Gillmore J.D. Hawkins P.N. Systemic amyloidosis.Lancet. 2016; 387: 2641-2654Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar Amyloid is a complex, compositionally heterogeneous, acellular material composed of light chain fibrils in association with extracellular matrix components and >20 serum proteins (notably, serum amyloid P component and apolipoproteins).4Dogan A. Amyloidosis: insights from proteomics.Annu Rev Pathol. 2017; 12: 277-304Crossref PubMed Scopus (61) Google Scholar The light chains comprising the fibrils adopt a nonnative structure enabling ordered self-association.5Raffen R. Dieckman L.J. Szpunar M. Wunschl C. Pokkuluri P.R. Dave P. Wilkins Stevens P. Cai X. Schiffer M. Stevens F.J. Physicochemical consequences of amino acid variations that contribute to fibril formation by immunoglobulin light chains.Protein Sci. 1999; 8: 509-517Crossref PubMed Scopus (150) Google Scholar, 6Khurana R. Gillespie J.R. Talapatra A. Minert L.J. Ionescu-Zanetti C. Millett I. Fink A.L. Partially folded intermediates as critical precursors of light chain amyloid fibrils and amorphous aggregates.Biochemistry. 2001; 40: 3525-3535Crossref PubMed Scopus (290) Google Scholar, 7Velazquez-Lopez I. Valdes-Garcia G. Romero Romero S. Maya Martinez R. Leal-Cervantes A.I. Costas M. Sanchez-Lopez R. Amero C. Pastor N. Fernandez Velasco D.A. Localized conformational changes trigger the pH-induced fibrillogenesis of an amyloidogenic lambda light chain protein.Biochim Biophys Acta Gen Subj. 2018; 1862: 1656-1666Crossref PubMed Scopus (6) Google Scholar The structural modifications of the fibrillar light chains result in the presentation of neoepitopes that can be targeted by monoclonal Igs.8O'Nuallain B. Allen A. Kennel S.J. Weiss D.T. Solomon A. Wall J.S. Localization of a conformational epitope common to non-native and fibrillar immunoglobulin light chains.Biochemistry. 2007; 46: 1240-1247Crossref PubMed Scopus (41) Google Scholar, 9O'Nuallain B. Murphy C.L. Wolfenbarger D.A. Kennel S.J. Solomon A. Wall J.S. Grateau G. Kyle R.A. Skinner M. The amyloid-reactive monoclonal antibody 11-1F4 binds a cryptic epitope on fibrils and partially denatured immunoglobulin light chains and inhibits fibrillogenesis. Amyloid and Amyloidosis. CRC Press, Tours, France2005: 482-484Google Scholar, 10Wall J.S. Kennel S.J. Williams A. Richey T. Stuckey A. Huang Y. Macy S. Donnell R. Barbour R. Seubert P. Schenk D. AL amyloid imaging and therapy with a monoclonal antibody to a cryptic epitope on amyloid fibrils.PLoS One. 2012; 7: e52686Crossref PubMed Scopus (56) Google Scholar Despite the nonnative features of AL fibrils in pathologic amyloid deposits, phagocytic cells of the innate immune system do not eliminate the material. Consequently, amyloid accumulation is generally progressive, leading to severe organ dysfunction and failure, notably of the heart, nerves, and kidneys. In most clinical cases, patients present late with renal or cardiac symptoms.11Martinez-Naharro A. Hawkins P.N. Fontana M. Cardiac amyloidosis.Clin Med. 2018; 18: s30-s35Crossref PubMed Scopus (71) Google Scholar Treatment for amyloidosis currently focuses on reducing the concentration of the amyloid precursor protein using antiplasma cell chemotherapy or immunotherapy,12Comenzo R.L. Steingart R.M. Cohen A.D. High-dose melphalan versus melphalan plus dexamethasone for AL amyloidosis.N Engl J Med. 2008; 358: 92PubMed Google Scholar, 13Sanchorawala V. Wright D.G. Seldin D.C. Falk R.H. Finn K.T. Dember L.M. Berk J.L. Quillen K. Anderson J.J. Comenzo R.L. Skinner M. High-dose intravenous melphalan and autologous stem cell transplantation as initial therapy or following two cycles of oral chemotherapy for the treatment of AL amyloidosis: results of a prospective randomized trial.Bone Marrow Transplant. 2004; 33: 381-388Crossref PubMed Scopus (102) Google Scholar, 14Sidiqi M.H. Gertz M.A. Daratumumab for the treatment of AL amyloidosis.Leuk Lymphoma. 2018; ([Epub ahead of print] doi:10.1080/10428194.2018.1485914s)Google Scholar autologous stem cell transplantation,15Chaulagain C.P. Comenzo R.L. How we treat systemic light-chain amyloidosis.Clin Adv Hematol Oncol. 2015; 13: 315-324PubMed Google Scholar, 16D'Souza A. Dispenzieri A. Wirk B. Zhang M.J. Huang J. Gertz M.A. Kyle R.A. Kumar S. Comenzo R.L. Peter Gale R. Lazarus H.M. Savani B.N. Cornell R.F. Weiss B.M. Vogl D.T. Freytes C.O. Scott E.C. Landau H.J. Moreb J.S. Costa L.J. Ramanathan M. Callander N.S. Kamble R.T. Olsson R.F. Ganguly S. Nishihori T. Kindwall-Keller T.L. Wood W.A. Mark T.M. Hari P. Improved outcomes after autologous hematopoietic cell transplantation for light chain amyloidosis: a Center for International Blood and Marrow Transplant Research study.J Clin Oncol. 2015; 33: 3741-3749Crossref PubMed Scopus (142) Google Scholar and protease inhibitors.17Lamm W. Willenbacher W. Lang A. Zojer N. Muldur E. Ludwig H. Schauer-Stalzer B. Zielinski C.C. Drach J. Efficacy of the combination of bortezomib and dexamethasone in systemic AL amyloidosis.Ann Hematol. 2011; 90: 201-206Crossref PubMed Scopus (51) Google Scholar In addition, methods to remove tissue amyloid deposits are being developed in hopes that this may promote organ recovery, improve quality of life, and extend patient survival.18Edwards C.V. Gould J. Langer A.L. Mapara M. Radhakrishnan J. Maurer M.S. Raza S. Mears J.G. Wall J. Solomon A. Lentzsch S. Interim analysis of the phase 1a/b study of chimeric fibril-reactive monoclonal antibody 11-1F4 in patients with AL amyloidosis.Amyloid. 2017; 24: 58-59Crossref PubMed Scopus (42) Google Scholar, 19Foster J.S. Williams A.D. Macy S. Richey T. Stuckey A. Wooliver D.C. Koul-Tiwari R. Martin E.B. Kennel S.J. Wall J.S. A peptide-Fc opsonin with pan-amyloid reactivity.Front Immunol. 2017; 8: 1082Crossref PubMed Scopus (4) Google Scholar, 20Richards D.B. Cookson L.M. Barton S.V. Liefaard L. Lane T. Hutt D.F. Ritter J.M. Fontana M. Moon J.C. Gillmore J.D. Wechalekar A. Hawkins P.N. Pepys M.B. Repeat doses of antibody to serum amyloid P component clear amyloid deposits in patients with systemic amyloidosis.Sci Transl Med. 2018; 10 (pii: eaan3128)Crossref PubMed Scopus (79) Google Scholar, 21Wall J.S. Williams A.D. Foster J.S. Richey T. Stuckey A. Macy S. Wooliver C. Campagna S.R. Tague E.D. Farmer A.T. Lands R.H. Martin E.B. Heidel R.E. Kennel S.J. Bifunctional amyloid-reactive peptide promotes binding of antibody 11-1F4 to diverse amyloid types and enhances therapeutic efficacy.Proc Natl Acad Sci U S A. 2018; 115: E10839-E10848Crossref PubMed Scopus (7) Google Scholar At present, there are no experimental animal models capable of recapitulating the development, progression, and regression, in response to therapy, of systemic, tissue-infiltrating AL-associated amyloidosis. Injection of patient-derived light chain proteins into mice, often in large amounts, has resulted in demonstrable pathology22Solomon A. Weiss D.T. Pepys M.B. Induction in mice of human light-chain-associated amyloidosis.Am J Pathol. 1992; 140: 629-637PubMed Google Scholar, 23Teng J. Turbat-Herrera E.A. Herrera G.A. An animal model of glomerular light-chain-associated amyloidogenesis depicts the crucial role of lysosomes.Kidney Int. 2014; 86: 738-746Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar; however, these models were technically challenging and relied on nonrenewable, patient-derived resources. The human λ6 light chain transgenic murine model, generated by Ward et al,24Ward J.E. SooHoo P. Toraldo G. Jasuja R. Connors L.H. O'Hara C. Seldin D.C. Metabolic phenotype in an AL amyloidosis transgenic mouse model.Amyloid. 2011; 18 Suppl 1: 40-41Crossref PubMed Scopus (1) Google Scholar resulted in sporadic amyloid formation limited to vacuoles within the stomach wall. As a model tractable for studying amyloidolysis (the dissolution of amyloid) and the efficacy of amyloid-targeting biological agents, human AL-associated amyloid extracts (generally 20 to 100 mg per mouse), isolated from the liver or spleen, have been injected subcutaneously in mice to generate an amyloidoma.25Hrncic R. Wall J. Wolfenbarger D.A. Murphy C.L. Schell M. Weiss D.T. Solomon A. Antibody-mediated resolution of light chain-associated amyloid deposits.Am J Pathol. 2000; 157: 1239-1246Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar When administered to immunocompetent BALB/c mice, these materials were rapidly cleared within approximately 14 days, indicating that, despite being considered inert, components of the mammalian immune system were capable of amyloid removal.25Hrncic R. Wall J. Wolfenbarger D.A. Murphy C.L. Schell M. Weiss D.T. Solomon A. Antibody-mediated resolution of light chain-associated amyloid deposits.Am J Pathol. 2000; 157: 1239-1246Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar Rapid amyloidosis in this mouse model was associated with a robust humoral and innate immune response associated with neutrophil infiltration and the generation of antibodies to components of the human amyloid material. Subsequent studies with amyloid extract injection into immunocompromised severe combined immunodeficient mice avoided classic T- or B-cell responses, but components of the innate immune response were nonetheless capable of removing the amyloid material, albeit over a longer time frame.25Hrncic R. Wall J. Wolfenbarger D.A. Murphy C.L. Schell M. Weiss D.T. Solomon A. Antibody-mediated resolution of light chain-associated amyloid deposits.Am J Pathol. 2000; 157: 1239-1246Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar The murine amyloidoma model has been used to visualize, by using small animal positron emission tomography/computed tomography and single-photon emission computed tomography/computed tomography imaging, the target engagement of radiolabeled monoclonal antibodies (mAbs).10Wall J.S. Kennel S.J. Williams A. Richey T. Stuckey A. Huang Y. Macy S. Donnell R. Barbour R. Seubert P. Schenk D. AL amyloid imaging and therapy with a monoclonal antibody to a cryptic epitope on amyloid fibrils.PLoS One. 2012; 7: e52686Crossref PubMed Scopus (56) Google Scholar, 26O'Nuallain B. Hrncic R. Wall J.S. Weiss D.T. Solomon A. Diagnostic and therapeutic potential of amyloid-reactive IgG antibodies contained in human sera.J Immunol. 2006; 176: 7071-7078Crossref PubMed Scopus (70) Google Scholar, 27Wall J.S. Kennel S.J. Paulus M. Gregor J. Richey T. Avenell J. Yap J. Townsend D. Weiss D.T. Solomon A. Radioimaging of light chain amyloid with a fibril-reactive monoclonal antibody.J Nucl Med. 2006; 47: 2016-2024PubMed Google Scholar Treatment of the mice with these mAbs expedited the dissolution of the amyloidoma in severe combined immunodeficient mice, evidenced by a decrease in the wet weight of the residual amyloidoma, excised at necropsy, after 14 or 21 days of treatment.10Wall J.S. Kennel S.J. Williams A. Richey T. Stuckey A. Huang Y. Macy S. Donnell R. Barbour R. Seubert P. Schenk D. AL amyloid imaging and therapy with a monoclonal antibody to a cryptic epitope on amyloid fibrils.PLoS One. 2012; 7: e52686Crossref PubMed Scopus (56) Google Scholar, 25Hrncic R. Wall J. Wolfenbarger D.A. Murphy C.L. Schell M. Weiss D.T. Solomon A. Antibody-mediated resolution of light chain-associated amyloid deposits.Am J Pathol. 2000; 157: 1239-1246Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar Despite the utility of this mouse model for studying AL amyloid-targeting agents and evaluating the regression of the lesion in response to immunotherapy, it is not amenable to noninvasive quantitative, longitudinal monitoring of amyloid load. Moreover, the model is limited by the availability of large amounts of nonrenewable human amyloid extracts. As an alternative to in vivo studies, phagocytic cells grown in culture have been used to demonstrate uptake of human Alzheimer disease–associated β amyloid and AL-associated amyloid in the presence of opsonizing antibodies.21Wall J.S. Williams A.D. Foster J.S. Richey T. Stuckey A. Macy S. Wooliver C. Campagna S.R. Tague E.D. Farmer A.T. Lands R.H. Martin E.B. Heidel R.E. Kennel S.J. Bifunctional amyloid-reactive peptide promotes binding of antibody 11-1F4 to diverse amyloid types and enhances therapeutic efficacy.Proc Natl Acad Sci U S A. 2018; 115: E10839-E10848Crossref PubMed Scopus (7) Google Scholar, 28Renz M. Torres R. Dolan P.J. Tam S.J. Tapia J.R. Li L. Salmans J.R. Barbour R.M. Shughrue P.J. Nijjar T. Schenk D. Kinney G.G. Zago W. 2A4 binds soluble and insoluble light chain aggregates from AL amyloidosis patients and promotes clearance of amyloid deposits by phagocytosis (dagger).Amyloid. 2016; 23: 168-177Crossref PubMed Scopus (20) Google Scholar, 29Bard F. Cannon C. Barbour R. Burke R.L. Games D. Grajeda H. Guido T. Hu K. Huang J. Johnson-Wood K. Khan K. Kholodenko D. Lee M. Lieberburg I. Motter R. Nguyen M. Soriano F. Vasquez N. Weiss K. Welch B. Seubert P. Schenk D. Yednock T. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease.Nat Med. 2000; 6: 916-919Crossref PubMed Scopus (1806) Google Scholar Herein, we have addressed these limitations by generating a mouse model that uses fluorophore-labeled, renewable, synthetic ALλ6 amyloid-like fibrils (approximately 2 mg per mouse) and is amenable to longitudinal, quantitative monitoring of the lesion by optical imaging. In addition, by using dual-fluorophore imaging of individual mice, the dissolution of the fibrilloma was studied concurrently with phagocytosis of the fibrils by cells of the innate immune system using Dylight80021Wall J.S. Williams A.D. Foster J.S. Richey T. Stuckey A. Macy S. Wooliver C. Campagna S.R. Tague E.D. Farmer A.T. Lands R.H. Martin E.B. Heidel R.E. Kennel S.J. Bifunctional amyloid-reactive peptide promotes binding of antibody 11-1F4 to diverse amyloid types and enhances therapeutic efficacy.Proc Natl Acad Sci U S A. 2018; 115: E10839-E10848Crossref PubMed Scopus (7) Google Scholar and pHrodo red30Miksa M. Komura H. Wu R. Shah K.G. Wang P. A novel method to determine the engulfment of apoptotic cells by macrophages using pHrodo succinimidyl ester.J Immunol Methods. 2009; 342: 71-77Crossref PubMed Scopus (227) Google Scholar fluorophores. This model can be used to study phagocytosis and cell-mediated dissolution of amyloid material alone or in the context of investigational opsonizing agents that might serve as novel therapeutics for AL amyloidosis. The recombinant λ6 variable domain (rVλ6Wil) was synthesized in Escherichia coli and purified, as previously described.31Wall J. Schell M. Murphy C. Hrncic R. Stevens F.J. Solomon A. Thermodynamic instability of human lambda 6 light chains: correlation with fibrillogenicity.Biochemistry. 1999; 38: 14101-14108Crossref PubMed Scopus (157) Google Scholar Amyloid-like fibrils were prepared in phosphate-buffered saline with 0.05% sodium azide, as previously described.32Martin E.B. Williams A. Heidel E. Macy S. Kennel S.J. Wall J.S. Peptide p5 binds both heparinase-sensitive glycosaminoglycans and fibrils in patient-derived AL amyloid extracts.Biochem Biophys Res Commun. 2013; 436: 85-89Crossref PubMed Scopus (14) Google Scholar The buffer was exchanged by centrifugation (2000 × g, 15 minutes), followed by the addition of sterile, endotoxin-free phosphate-buffered saline. The presence of fibrils was confirmed using a thioflavin T assay.32Martin E.B. Williams A. Heidel E. Macy S. Kennel S.J. Wall J.S. Peptide p5 binds both heparinase-sensitive glycosaminoglycans and fibrils in patient-derived AL amyloid extracts.Biochem Biophys Res Commun. 2013; 436: 85-89Crossref PubMed Scopus (14) Google Scholar The fibrils were stored at 4°C until used. Aliquots of rVλ6Wil fibrils were labeled, according to manufacturer's instructions, with either the pH-sensitive dye, pHrodo red–STP ester (Life Technologies, Waltham, MA), or the NHS-DyLight800 NHS-ester near-infrared dye (Thermo-Pierce, Waltham, MA). Labeling was performed in 100 mmol/L sodium bicarbonate buffer, pH 8.3. For both fluorophores, the unbound dye was removed by buffer exchange after centrifugation at 2000 × g for 15 minutes using sterile phosphate-buffered saline, pH 7.2. Amyloid-like rVλ6Wil fibrils suspended in tris-buffered saline were dried on Formvar-coated copper grids and negatively stained using 4% uranyl acetate. Images were acquired using a Libra 200 MC transmission electron microscope (Zeiss, Thornwood, NY) with a current of 230 μA, a voltage of 200 kV, and a 1-second exposure. Raw 264.7 cells (1 × 106) were incubated with 20 μg of rVλ6Wil fibrils (20% w/w pHrodo red–labeled fibrils) for 2 hours at 37°C and analyzed using an Attune NxT acoustic focusing cytometer (Applied Biosystems, Carlsbad, CA) by gating first for intact cells using forward and side scatter parameters. Fluorescence data were obtained using an excitation at 488 nm, and the emission detected with a 574/26 cutoff filter. The cell population falling within the pHrodo fluorescence-positive gate was determined and is reported as a percentage of the intact cell population. A suspension of rVλ6Wil fibrils (2 mg rVλ6Wil in 0.2 mL), containing 20% (w/w) pHrodo red–labeled fibrils, was injected subcutaneously, on the dorsal flank, of female NU/NU mice (n = 4). Alternatively, 2 mg of rVλ6Wil fibrils [with 15% (w/w) pHrodo red– and 15% (w/w) DL800-labeled fibrils] was injected into female NU/NU mice (n = 3). Mice were imaged under isoflurane anesthesia, in an iBox Scientia small animal optical imaging system (Analytik Jena, Upland, CA), using a Cy5 excitation and emission filter set for pHrodo red and an 800-nm bandpass filter set for DL800 (2-second exposures with 1 × 1 binning) over 18 days after injection. Animals were euthanized by isoflurane overdose at day 18 after injection, and the residual fibril masses were harvested and fixed in 10% buffered formalin (24 hours) for histologic examination. The mean raw density of the fibril-associated fluorescence in the pHrodo red and DL800 images was performed initially using a standard elliptical region of interest (ROI) encompassing the fibril mass and a second ROI covering a fibril-free region of the mouse, which served as the mouse-specific background value. The final fluorescence signal intensity was calculated by subtraction of the background mean raw density. The data were also analyzed using a freeform ROI drawn around the borders of the rVλ6Wil fibril lesion. The data from the two mouse studies were analyzed by two or three independent reviewers (J.S.W., E.B.M., and A.B.W.) to assess interrater reliability and bias associated with placement of the fibril and control ROIs. The freeform ROI data are presented herein. Sections (6 μm thick) of residual rVλ6Wil fibril masses, harvested from mice at 18 days after injection, were stained with hematoxylin and eosin (Fisher Scientific, Waltham, MA) or alkaline Congo red solution (0.8% w/v Congo red, 0.2% w/v KOH, and 80% ethanol) for 1 hour at room temperature, followed by counterstain with Mayer's hematoxylin for 2 minutes. Consecutive tissue sections were immunostained for the following: human free λ light chain (catalog number A10101; Dako, Carpinteria, CA; 1:20,000); CD68 (catalog number ab12512; Abcam, Cambridge, MA; 1:10,000); ionized calcium binding adaptor molecule 1 (Iba-1; catalog number 019-19741; Wako Pure Chemical Industries, Richmond, VA; 1:9000); F4/80 (catalog number MCA497R; Santa Cruz Biotechnology, Dallas, TX; 1:1000); lysosomal associated membrane protein 1 (LAMP1; catalog number sc-19992; Santa Cruz Biotechnology; 1:4000); Ly6G (catalog number sc53515; RB8-8C5; Santa Cruz Biotechnology; 1:5000); myeloperoxidase (catalog number PU496-UP; BioGenex, Fremont, CA; 1:25); and neutrophil elastase (catalog number ab68672; Abcam; 1:3000). Before staining, all tissue sections were subjected to antigen retrieval by boiling in citrate buffer (Dako) for 30 minutes. Slides were developed by addition of peroxidase-conjugated anti-rabbit IgG or anti-rat IgG (ImmPRESS; Vector, Burlingame, CA), as appropriate, for 50 minutes at room temperature, and diaminobenzidine solution (ImmPACT DAB; Vector) for 3 minutes at room temperature. Photomicrographs of Congo red birefringence were acquired using a Leica DM500 light microscope (Leica Microsystems, Wetzlar, Germany) with a cooled charge-coupled device camera (SPOT RT-Slider; Diagnostic Instruments, Sterling Heights, MI), fitted with cross-polarizing filters. All other images were acquired with a BZ-X700E microscope (Keyence, Atlanta, GA) using bright-field optics or a Texas red filter (for Congo red fluorescence). Typically, a 40× objective with a 2× digital zoom and an exposure time of 1/40 seconds was used for bright-field images, unless otherwise noted. For quantification of optical imaging data, interrater reliability was assessed by determining the intraclass correlation coefficient, using the two-way random model. Comparison of mean fluorescence intensity data between initial, peak, and final time points was performed by using a paired, two-tailed t-test. All statistical analyses were performed using Prism software version 6.07 (GraphPad Software Inc., San Diego, CA) or SPSS software version 25 (IBM Corp., Armonk, NY). Statistical significance was assumed at an α value of 0.05. Synthetic amyloid-like fibrils were generated using the recombinant λ6 variable domain from patient Wil by vigorously shaking the protein at 37°C. This resulted in a heterogeneous population of fibril structures (Figure 1A). Electron microscopic analysis revealed monodisperse fibril aggregates approximately 15 nm in diameter and approximately 200 nm in length observed together with macroscopic aggregates of fibrils that measured 1.2 μm along certain axes (Figure 1A). In these studies, synthetic rVλ6Wil amyloid-like fibrils were mixed with fibrils labeled with an appropriate fluorophore. The in vitro uptake of 20 μg of rVλ6Wil fibrils, with 10% w/w pHrodo red–labeled rVλ6Wil fibrils, by RAW 264.7 murine macrophages was monitored by flow cytometry (Figure 1, B and C). The pHrodo red fluorophore fluorescence emission (maximum, 585 nm) is enhanced in the acidic microenvironment of the phagolysosome. Cultured RAW 264.7 cells were used to gate side scatter and forward scatter parameters to include only intact cells; in the absence of pHrodo red rVλ6Wil fibrils, they exhibited no fluorescence greater than untreated cells in the red channel (Figure 1B). After a 2-hour incubation with fibrils, 27% of the cell population exhibited higher than background fluorescence in the Texas red filter channel (emission maximum, approximately 615 nm), indicating uptake and acidification of the pHrodo red–labeled fibrils (Figure 1C). These data demonstrate the pH-induced enhancement of pHrodo red fluorescence of the fibrils once engulfed by phagocytic cells. To further study the phagocytosis of rVλ6Wil fibrils in vivo, the uptake, into acidified phagolysosomes, of pHrodo red–labeled rVλ6Wil fibrils was monitored by using optical imaging of hairless, immunocompromised nude (NU/NU) mice (Figure 2). Mice (n = 4) were administered, subcutaneously, a 2-mg mass of rVλ6Wil fibrils (20% w/w pHrodo red fibrils) on the left dorsal flank. On day 1 after injection, no fluorescence was visible on the mouse flank (Figure 2A). In a representative mouse, by day 3 after injection, focal areas of enhanced fluorescence emission were readily visible using optical imaging, within a well-defined fibrilloma (Figure 2A). Serial imaging of the subject demonstrated an increase in the fluorescence emission, reaching a maximum at approximately day 7 after injection, followed by a decrease over the following 11 days, at which point a single focal area of fluorescence intensity remained visible (Figure 2A). At day 18 after injection, the residual fibrilloma was excised post-mortem and appeared as a well-defined lesion associated with the skin, which retained its fluorescence (Figure 2B). The fluorescence intensity associated with the amyloid lesion in each mouse was quantified from optical imaging data using a freeform ROI analysis, and the background-corrected mean raw density was calculated (Figure 2C). Analysis of the optical images is potentially open to subjective bias associated with placement of the ROIs. Therefore, the background-corrected mean raw density data calculated from the two mouse studies were generated by two or three independent reviewers (with only one reviewer in each case familiar with the study design; J.S.W., E.B.M., and A.B.W.), and an intraclass correlation coefficient was calculated to assess bias and consistency. Images analyzed by three reviewers yielded a significant intraclass correlation coefficient of 0.82 (P < 0.001), with a 95% CI of 0.62 to 0.92, and when two reviewers were used, the coefficient increased to 0.94 (P < 0.001), with a 95% CI of 0.86 to 0.98. This analysis indicated that the placement of the ROIs over the fibrilloma and background region of the mouse by the primary reviewer (J.S.W.) of the data was unbiased and consistent with reviewers (E.B.M. and A.B.W.) blinded to the study design. A significant increase in the fluorescence emission occurred between days 1 and 4 after injection, peaking at approximately day 7 after injection, which represented a rapid onset of phagocytosis of the rVλ6Wil fibrils. The fluorescence emission then decreased between days 7 and 18 after injection (Figure 2C), suggesting that after uptake, dissolution of the fibrils occurred and the fluorophore-labeled breakdown products were no longer present in the acidified compartment. Variability in the mean fluorescence intensity for the group was due to intersubject variation in the time to reach peak fluorescence intensity and the maximum fluorescence emission at the peak, as evidenced when the data for each mouse were plotted (Figure 2D). Despite individual variations, all of the mice exhibited a biphasic change in fluorescence emission (Figure 2D). At 18 days after injection, the residual fibri

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