This work was supported in part by the Lupus Research Institute, NCI grant CA138617, NIDDK grant DK090978, Charlotte Schmidlapp Award (to E.M.J.), and the Albert J. Ryan Fellowship (to J.K.). We are grateful for the support and instrumentation provided by the Research Flow Cytometry Core in the Division of Rheumatology at Cincinnati Children&#8217;s Hospital Medical Center, supported in part by NIH AR-47363, NIH DK78392 and NIH DK90971.

<ol>
	<li>Helmick, C. G., Felson, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Part+I.+Arthritis+Rheum.">Part I. Arthritis Rheum.</a> Estimates of the prevalence of arthritis and other rheumatic conditions in the United States. 58, 15-25 (2008).</li><li>Somers, E. C., Marder, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Population-based+incidence+and+prevalence+of+systemic+lupus+erythematosus:+The+Michigan+lupus+epidemiology+and+surveillance+program.">Population-based incidence and prevalence of systemic lupus erythematosus: The Michigan lupus epidemiology and surveillance program.</a> Arthritis and Rheumatol. 66, 369-378 (2014).</li><li>Perry, D., Sang, A., Yin, Y., Zheng, Y. -. Y., Morel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+models+of+systemic+lupus+erythematosus.">Murine models of systemic lupus erythematosus.</a> J Biomed Biotechnol. 2011, 271694 (2011).</li><li>Lindholm, L., Rydberg, L., Stranneg&#229;rd, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+host+plasma+cells+during+graft-versus-host+reactions+in+mice.">Development of host plasma cells during graft-versus-host reactions in mice.</a> Eur J Immunol. 3 (8), 511-515 (1973).</li><li>Fialkow, P. J., Gilchrist, C., Allison, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmunity+in+chronic+graft-versus-host+disease.">Autoimmunity in chronic graft-versus-host disease.</a> Clin Exp Immunol. 13, 479-486 (1973).</li><li>Streilein, J. W., Stone, M. J., Duncan, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+Specificity+of+Autoantibodies+Produced+in+Systemic+Graft-vs-Host+Disease.">Studies on the Specificity of Autoantibodies Produced in Systemic Graft-vs-Host Disease.</a> J Immunol. 114 (1), 255-260 (1975).</li><li>Gleichmann, E., Gleichmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diseases+caused+by+reactions+of+T+lymphocytes+to+in+compatible+structures+of+the+major+histocompatibility+complex.+I.+Autoimmune+hemolytic+anemia.">Diseases caused by reactions of T lymphocytes to in compatible structures of the major histocompatibility complex. I. Autoimmune hemolytic anemia.</a> Eur J Immunol. 6 (12), 899 (1976).</li><li>Morris, S., Cohen, P. L., Eisenberg, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+induction+of+systemic+lupus+erythematosus+by+recognition+of+foreign+Ia.">Experimental induction of systemic lupus erythematosus by recognition of foreign Ia.</a> Clin Immunol Immunopathol. 57 (2), 263-273 (1990).</li><li>Chen, F., Maldonado, M., Madaio, M., Eisenberg, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Host+(Endogenous)+T+Cells+in+Chronic+Graft-Versus-Host+Autoimmune+Disease.">The Role of Host (Endogenous) T Cells in Chronic Graft-Versus-Host Autoimmune Disease.</a> J Immunol. 161 (11), 5880-5885 (1998).</li><li>Klarquist, J., Hennies, C. M., Lehn, M. A., Reboulet, R. A., Feau, S., Janssen, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STING-Mediated+DNA+Sensing+Promotes+Antitumor+and+Autoimmune+Responses+to+Dying+Cells.">STING-Mediated DNA Sensing Promotes Antitumor and Autoimmune Responses to Dying Cells.</a> J Immunol. 193, 6124-6134 (2014).</li><li>Petri, M., Orbai, A. -. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+and+validation+of+systemic+lupus+international+collaborating+clinics+classification+criteria+for+systemic+lupus+erythematosus.">Derivation and validation of systemic lupus international collaborating clinics classification criteria for systemic lupus erythematosus.</a> Arthritis Rheum. 64 (8), 2677-2686 (2012).</li><li>Zangala, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+genomic+DNA+from+mouse+tails.">Isolation of genomic DNA from mouse tails.</a> J Vis Exp. (6), e246 (2007).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+Chain+Reaction:+Basic+Protocol+Plus+Troubleshooting+and+Optimization+Strategies.">Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies.</a> J Vis Exp. (63), e3998 (2012).</li><li>. Product information: Thermo Scientific FastDigest PsuI Available from: <a target="_blank" href="https://tools.lifetechnologies.com/content/sfs/manuals/MAN0012567_FastDigest_PsuI_UG.pdf">https://tools.lifetechnologies.com/content/sfs/manuals/MAN0012567_FastDigest_PsuI_UG.pdf</a> (2012)</li><li>Matheu, M. P., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+and+2-photon+imaging+of+peripheral+lymph+nodes+in+mice.">Dissection and 2-photon imaging of peripheral lymph nodes in mice.</a> J Vis Exp. (7), e265 (2007).</li><li>Harrell, M. I., Iritani, B. M., Ruddell, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymph+node+mapping+in+the+mouse.">Lymph node mapping in the mouse.</a> J Immunol Methods. 332 (1-2), 170-174 (2008).</li><li>Covelli, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3,+Internal+examination.">Chapter 3, Internal examination.</a> Guide to the necroscopy of the mouse. , (2009).</li><li>Quah, B. J. C., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+carboxyfluorescein+diacetate+succinimidyl+ester+(CFSE)+to+monitor+lymphocyte+proliferation.">The use of carboxyfluorescein diacetate succinimidyl ester (CFSE) to monitor lymphocyte proliferation.</a> J Vis Exp. (44), e2259 (2010).</li><li>Matheu, M. P., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+CD4++T+cells+from+mouse+lymph+nodes+using+Miltenyi+MACS+purification.">Isolation of CD4+ T cells from mouse lymph nodes using Miltenyi MACS purification.</a> J Vis Exp. (9), e53319 (2007).</li><li>Machholz, E., Mulder, G., Ruiz, C., Corning, B. F., Pritchett-Corning, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+Restraint+and+Common+Compound+Administration+Routes+in+Mice+and+Rats.">Manual Restraint and Common Compound Administration Routes in Mice and Rats.</a> J Vis Exp. (67), e2771 (2012).</li><li>Hoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+Blood+Collection+in+the+Mouse.">Methods of Blood Collection in the Mouse.</a> Lab Animal. 29 (10), 47-53 (2000).</li><li>Cohen, M., Varki, N. M., Jankowski, M. D., Gagneux, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Unfixed,+Frozen+Tissues+to+Study+Natural+Mucin+Distribution.">Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution.</a> J Vis Exp. (67), e3928 (2012).</li><li>Cohen, P. L., Maldonado, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+SLE.">Animal models for SLE.</a> Curr Protoc Immunol.. Chapter 15, Unit 15.20 (2003).</li><li>Seavey, M. M., Lu, L. D., Stump, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+systemic+lupus+erythematosus+(SLE)+and+ex+vivo+assay+design+for+drug+discovery.">Animal models of systemic lupus erythematosus (SLE) and ex vivo assay design for drug discovery.</a> Curr Protoc Pharmacol. Chapter 5, Unit 5 (2011).</li><li>McKenna, K. C., Vicetti Miguel, R. D., Beatty, K. M., Bilonick, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+caveat+for+T+cell+transfer+studies:+generation+of+cytotoxic+anti-Thy1.2+antibodies+in+Thy1.1+congenic+mice+given+Thy1.2++tumors+or+T+cells.">A caveat for T cell transfer studies: generation of cytotoxic anti-Thy1.2 antibodies in Thy1.1 congenic mice given Thy1.2+ tumors or T cells.</a> J Leukoc Biol. 89 (2), 291-300 (2011).</li><li>Scott, D. M., Ehrmann, I. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+mouse+male-specific+transplantation+antigen+H-Y..">Identification of a mouse male-specific transplantation antigen H-Y..</a> Nature. 376, 695-698 (1995).</li><li>Joly, E., Hudrisier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+trogocytosis+and+what+is+its+purpose.">What is trogocytosis and what is its purpose.</a> Nat Immunol. 4 (9), 815 (2003).</li><li>Brown, D. R., Calpe, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+an+NK+cell-independent+role+for+Slamf4+in+controlling+humoral+autoimmunity.">Cutting edge: an NK cell-independent role for Slamf4 in controlling humoral autoimmunity.</a> J Immunol. 187 (1), 21-25 (2011).</li><li>Morris, S. C., Cheek, R. L., Cohen, P. L., Eisenberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allotype-specific+immunoregulation+of+autoantibody+production+by+host+B+cells+in+chronic+graft-versus+host+disease.">Allotype-specific immunoregulation of autoantibody production by host B cells in chronic graft-versus host disease.</a> J Immunol. 144 (3), 916-922 (1990).</li><li>Choudhury, A., Cohen, P. L., Eisenberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cells+require+&#8220;nurturing&#8221;+by+CD4+T+cells+during+development+in+order+to+respond+in+chronic+graft-versus-host+model+of+systemic+lupus+erythematosus.">B cells require &#8220;nurturing&#8221; by CD4 T cells during development in order to respond in chronic graft-versus-host model of systemic lupus erythematosus.</a> Clin Immunol. 136 (1), 105-115 (2010).</li><li>Slifka, M. K., Antia, R., Whitmire, J. K., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humoral+immunity+due+to+long-lived+plasma+cells.">Humoral immunity due to long-lived plasma cells.</a> Immunity. 8 (3), 363-372 (1998).</li></ol>

The authors have nothing to disclose.

Il modello inducibile BM12 è un modo relativamente semplice ed efficace per studiare i processi cellulari e molecolari del LES. Attivazione cronica di adoptively trasferiti cellule T CD4 diretti contro antigeni auto porta all&#39;accumulo di Tfh, cellule GC B, e le plasmacellule che può essere misurato mediante citometria di flusso, come descritto qui. Futuri studi che utilizzano questo modello può interrogare rapidamente e facilmente il ruolo dei geni candidati e nuove terapie nei processi centro germinale autoimmun...

Il lupus eritematoso sistemico (LES) è una malattia autoimmune complessa caratterizzata prototipicamente da anticorpi anti-nucleo (ANA) la produzione e la glomerulonefrite. Numerose altre sequele, tra cui dermico, cardio-polmonare e lesioni epatiche sono associati con la malattia in alcuni individui. Le stime sulla prevalenza negli Stati Uniti variano ampiamente, da 150,000-1,500,000 1,2, con incidenza particolarmente elevata nelle donne e le minoranze 3. Sebbene l&#39;eziologia del LES è stato difficile da discernere, si pensa a sorgere dall&#39;interazione di diversi fattori genetici e ambientali, che culminano in autoimmunità sistemica. Numerosi modelli animali sono stati impiegati per studiare i fattori di insorgenza della malattia e la progressione. Modelli classici murini di SLE includono ceppi di topi geneticamente predisposti, tra cui il modello di X NZB NZW F1 e suoi derivati ​​di potenza NZM, il ceppo / lpr MLR, e il ceppo BXSB / Yaa, e sistemi inducibili, come il pristane e malattia cronica del trapianto contro l&#39;ospite (GVHD cronica) modelli 4. I primi rapporti di produzione di autoanticorpi nei modelli GVHD usati diversi ceppi di topi o ceppi criceto per genitore in trasferimenti F1 5 - 8; metodi più comuni utilizzati per studiare le malattie simil-lupus attualmente includono il genitore DBA / 2 → (C57BL / 6 x DBA / 2) F1, ed il modello di trasferimento BM12 descritto qui. Ogni modello ha i suoi avvertimenti, ma in genere condividono un set comune di funzioni che sono correlate alle caratteristiche cliniche della malattia umana. I parametri più spesso segnalati in modelli murini includono splenomegalia, linfoadenopatia, nefrite, la produzione ANA, e a livello cellulare, l&#39;espansione delle cellule T helper follicolari (TFH), le cellule B germinali centro (GC), e le cellule del plasma. Il modello BM12 inducibile si ottiene il trasferimento adottivo di linfociti da IA BM12 B6 (C) - H2 - Ab1 BM12 / KhEgJ (BM12) topo, un ceppo identical a C57BL / 6 ad eccezione di 3 aminoacidi sostituzioni su MHC di classe II, in IA b C57BL / 6 (B6) topi. Alloactivation di donatori cellule T CD4 di destinatario APC porta a cGVHD con sintomi molto simili LES. In particolare, questi includono l&#39;espansione di Tfh donatore-derivati, espansione delle cellule B GC destinatario di derivazione e plasmacellule, e la produzione di ANA tra cui anti-dsDNA, anti-ssDNA, anti-cromatina, e gli anticorpi anti-RBC 9. Nel corso del tempo, topi riceventi sviluppano glomerulonefrite associata a depositi di IgG nel glomerulare, interstiziale e regioni vascolari dei reni 10. Abbiamo recentemente dimostrato che, simile alla malattia umana, c&#39;è anche un ruolo critico per tipo IFN in questo modello 11. In particolare, i criteri di definizione per il LES umano includono lo sviluppo di nefrite compatibile con SLE ​​in presenza di anticorpi anti-dsDNA anticorpi 12, entrambi i quali sono caratteristiche importanti di questo modello di topo. Ci sono sevantaggi Veral del modello BM12 rispetto ai modelli spontanee. Modelli classici che sviluppano segni SLE-come spontaneamente si basano su entrambi i ceppi di topi ibridi, ceppi inbred di topo non sullo sfondo B6, o grande loci genetici sullo sfondo B6, che rendono attraversando a knockout o topi geneticamente modificati altrimenti difficile e richiede tempo. Con il modello inducibile BM12, topi geneticamente modificati possono servire sia come donatore o ricevente, consentendo più rapida identificazione del compartimento cellulare in cui alcuni geni possono essere importanti per la malattia. Inoltre, lo sviluppo della malattia nel modello BM12 è molto più veloce, che richiede solo 2 settimane fino alla comparsa di ANA, rispetto a diversi mesi per i modelli più spontanee. Inoltre, a differenza dei modelli spontanei che sviluppano la malattia in diversi momenti, l&#39;insorgenza e la progressione della malattia nel modello B6 BM12 → è altamente sincronizzati. Questo permette la generazione di coorti di dimensioni appropriate che possono Be utilizzato per strategie di intervento o terapeutici in qualsiasi fase della sviluppo della malattia. Quello che segue è un protocollo dettagliato per l&#39;avvio SLE-come autoimmunità dal trasferimento adottivo di linfociti BM12 in topi C57BL / 6, o varianti genetiche sullo sfondo B6. Inoltre, si descrive un protocollo di colorazione citometria a flusso per l&#39;enumerazione Tfh, cellule GC B, e tipi di cellule del plasma-cella associata con la malattia umana. È importante sottolineare che questi protocolli possono anche essere utilizzati per caratterizzare la malattia nella maggior parte dei modelli murini di LES e identificare Tfh, cellule GC B le plasmacellule in altri modelli di malattia.

<table><tbody><tr><td>B6.SJL-Ptprca Pepcb/BoyJ</td><td>The Jackson Laboratory</td><td>001162</td><td>CD45.1+ BoyJ mouse strain</td></tr><tr><td>B6(C)-H2-Ab1bm12/KhEgJ</td><td>The Jackson Laboratory</td><td>001162</td><td>Bm12 mouse strain</td></tr><tr><td>FastDigest PsuI</td><td>Life Technologies</td><td>FD1554</td><td>Restriction digest enzyme for genotyping</td></tr><tr><td>1X RBC Lysis Buffer</td><td>eBioscience</td><td>00-4333-57</td><td></td></tr><tr><td>IMDM</td><td>GE Healthcare</td><td>SH30228.01</td><td></td></tr><tr><td>Plasma Separation Tube (PST)</td><td>BD</td><td>365974</td><td>Blood collection tube with Dipotassium EDTA</td></tr><tr><td>Serum Separation Tube (SST)</td><td>BD</td><td>365967</td><td>Blood collection tube with Clot activator / SST Gel</td></tr><tr><td>Ficoll</td><td>GE Healthcare</td><td>17-1440-02&amp;nbsp;</td><td>High density cell separation solution</td></tr><tr><td>Lympholyte-M</td><td>Cedarlane</td><td>CL5030</td><td>High density cell separation solution</td></tr><tr><td>GL-7-biotin</td><td>eBioscience</td><td>13-5902-82&amp;nbsp;</td><td></td></tr><tr><td>Streptavidin-BUV395</td><td>BD</td><td>564176</td><td></td></tr><tr><td>CD138-BV421</td><td>BioLegend</td><td>142508</td><td></td></tr><tr><td>CD4-BV510</td><td>BioLegend</td><td>100559</td><td></td></tr><tr><td>TCR&amp;beta;-BV605</td><td>BD</td><td>562840</td><td></td></tr><tr><td>CD45.1-BV711</td><td>BioLegend</td><td>110739</td><td></td></tr><tr><td>CD45.2-FITC</td><td>BioLegend</td><td>109806</td><td></td></tr><tr><td>PD-1-PE</td><td>BioLegend</td><td>135206</td><td></td></tr><tr><td>CD19-PerCP</td><td>BioLegend</td><td>115532</td><td></td></tr><tr><td>Fas-PE-Cy7</td><td>BD</td><td>557653</td><td></td></tr><tr><td>CXCR5-APC</td><td>BioLegend</td><td>145506</td><td></td></tr><tr><td>Fixable Viability Dye ef780</td><td>eBioscience</td><td>65-0865-18</td><td></td></tr><tr><td>CD4-BV421</td><td>BioLegend</td><td>100443</td><td></td></tr><tr><td>1.2 ml FACS tube inserts, racked</td><td>USA Scientific</td><td>1412-1400</td><td></td></tr><tr><td>BD Falcon&amp;trade; Round-Bottom Tubes</td><td>BD</td><td>352017</td><td></td></tr></tbody></table>

the bm12 inducible model of systemic lupus erythematosus (sle) in c57bl/6 mice

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible animal models mirror common components of human disease, including the bm12 transfer model. Upon transfer of bm12 splenocytes or purified CD4 T cells, C57BL/6 mice rapidly develop large frequencies of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells followed by high levels of circulating anti-nuclear antibodies. Since this model utilizes mice on a pure C57BL/6 background, researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time. Here we describe protocols for the induction of the model and the quantitation Tfh, GC B cells, and plasma cells by multi-color flow cytometry. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells, and plasma cells in other disease models.

Topi malati sviluppano splenomegalia in appena 14 giorni, esibendo milza 2-3 volte le dimensioni di topi sani in termini di massa e cellularità (Figura 2). Splenociti sono sequenzialmente gate sulla dispersione della luce (FSC-A da SSC-A), l&#39;eliminazione di doppiette (FSC-W o -H da FSC-A), cellule vitali (basso colorazione della vitalità tintura) e CD4 + TCRβ + (Figura 3A). Cellule del donatore si distinguono dalle cellule riceve...

Watch this Scientific Journal Video about The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice at JoVE.com

The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

Il BM12 inducibile modello di Lupus Eritematoso Sistemico (LES) in C57BL / 6 Mice

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible ...

the-bm12-inducible-model-systemic-lupus-erythematosus-sle-c57bl6

Research

JoVE Journal

Medicine

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

17.3K Views.  Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine. The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

Video: The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

Induzione di ipofisite autoimmune sperimentale nei topi SJL

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

Ricerca

Immunologia e infezioni

The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease 3-5. However, in various models the induction of disease was rather complex and often hepatitis was only transient3-5. Therefore, we have developed a straightforward mouse model that uses the major human autoantigen in type 2 autoimmune hepatitis (AIH-2), namely hCYP2D6, as a trigger6. Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-27,8. Delivery of hCYP2D6 into wildtype FVB or C57BL/6 mice was by an Adenovirus construct (Ad-2D6) that ensures a direct delivery of the triggering antigen to the liver. Thus, the ensuing local inflammation generates a fertile field9 for the subsequent development of autoimmunity. A combination of intravenous and intraperitoneal injection of Ad-2D6 is the most effective route to induce a long-lasting autoimmune damage to the liver (section 1). Here we provide a detailed protocol on how autoimmune liver disease is induced in the CYP2D6 model and how the different aspects of liver damage can be assessed. First, the serum levels of markers indicating hepatocyte destruction, such as aminotransferases, as well as the titers of hCYP2D6 antibodies are determined by sampling blood retroorbitaly (section 2). Second, the hCYP2D6-specific T cell response is characterized by collecting lymphocytes from the spleen and the liver. In order to obtain pure liver lymphocytes, the livers are perfused by PBS via the portal vein (section 3), digested in collagen and purified over a Percoll gradient (section 4). The frequency of hCYP2D6-specific T cells is analyzed by stimulation with hCYP2D6 peptides and identification of IFN&gamma;-producing cells by flow cytometry (section 5). Third, cellular infiltration and fibrosis is determined by immunohistochemistry of liver sections (section 6). Such analysis regimen has to be conducted at several times after initiation of the disease in order to prove the chronic nature of the model. The magnitude of the immune response characterized by the frequency and activity of hCYP2D6-specific T and/or B cells and the degree of the liver damage and fibrosis have to be assessed for a subsequent evaluation of possible treatments to prevent, delay or abrogate the autodestructive process of the liver.

Infection of mice with an Adenovirus expressing the major human autoantigen cytochrome P450 2D6 (hCYP2D6) recognized by sera of patients suffering from type 2 autoimmune hepatitis results in a persistent form of autoimmune-mediated liver disease characterized by extensive hepatitis, fibrosis and generation of a CYP2D6-specific immune response.

Il CYP2D6 modello animale: come indurre epatite autoimmune in topi

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to ...

Medicina

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG35-55 is suspended in complete Freund's adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis. C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG35-55), resulting in an ascending flaccid paralysis caused by autoreactive immune cells in the central nervous system. Protocols for disease induction and monitoring will be discussed.

Mielina Oligodendrocyte glicoproteina (MOG<sub&gt; 35-55</sub&gt;) Indotta encefalomielite autoimmune sperimentale (EAE) in C57BL / 6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

Induzione di encefalomielite autoimmune sperimentale nei topi e valutazione della distribuzione delle malattie-dipendente delle cellule immunitarie in vari tessuti

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.

This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration.

La determinazione di soppressione del sistema immunitario contro il sistema nervoso centrale di protezione per farmacologici Interventi a demielinizzazione autoimmune

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in ...

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)106-125 or P0180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin. The ability to induce EAN in the C57BL/6 strain allows for the use of the numerous genetic tools that exist on this mouse background, and thus allows the sophisticated study of disease pathogenesis and interrogation of the mechanistic action of novel therapeutics in combination with transgenic approaches. In this study, we demonstrate a simple approach to successfully induce EAN using the P0180-199 peptide in C57BL/6 mice. We also outline a protocol for the assessment of functional deficits that occur in this model, accompanied by an array of neuropathological features. Thus, this model is a powerful experimental model to study the pathogenesis of human peripheral demyelinating neuropathies, and to determine the efficacy of potential therapies that aim to promote myelin repair and protect against nerve damage in autoimmune neuritis.

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund&#39;s complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

Un approccio semplice per indurre la neurite Autoimmune Sperimentale in topi C57BL/6 per le valutazioni funzionali e Neuropathological

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

Neuroscienze

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

Induzione dell'epatite autoimmune indotta da farmaci in topi BALB / c per lo studio dei suoi meccanismi patogenetici

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

Murino colostomia distale, un nuovo modello di colite di diversione in topi C57BL/6

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

Analyses of Proteinuria, Renal Infiltration of Leukocytes, and Renal Deposition of Proteins in Lupus-prone MRL/lpr Mice

Systemic lupus erythematosus (SLE) is an autoimmune disorder with no known cure and is characterized by persistent inflammation in many organs, including the kidneys. Under such circumstances, the kidney loses its ability to clean waste from the blood and regulate salt and fluid concentrations, eventually leading to renal failure. Women, particularly those of childbearing age, are diagnosed nine times more often than men. Kidney disease is the leading cause of mortality in SLE patients. The present protocol describes a quick and simple method to measure excreted protein levels in collected urine, tracking lupus progression over time. In addition, an approach to isolate kidney mononuclear cells is provided based on size and density selection to investigate renal infiltration of leukocytes. Furthermore, an immunohistochemical method has been developed to characterize protein deposition in the glomeruli and leukocyte infiltration in the tubulointerstitial space. Together, these methods can help investigate the progression of chronic inflammation associated with the kidneys of lupus-prone MRL/lpr mice.

The present protocol describes a method to track lupus progression in mice. Two additional procedures are presented to characterize lupus nephritis based on cell infiltration and protein deposition in the kidneys.

Analisi della proteinuria, infiltrazione renale dei leucociti e deposizione renale di proteine in topi MRL/lpr inclini al lupus

Systemic lupus erythematosus (SLE) is an autoimmune disorder with no known cure and is characterized by persistent inflammation in many organs, including ...

Induzione e valutazione dell'encefalomielite autoimmune sperimentale nel topo

Modeling Multiple Sclerosis in the Two Sexes: MOG35-55-Induced Experimental Autoimmune Encephalomyelitis

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different prevalence in the sexes, affecting more women than men, and different outcomes, showing more aggressive forms in men than in women. Furthermore, MS is highly heterogeneous in terms of clinical aspects, radiological, and pathological features. Thus, it is necessary to take advantage of experimental animal models that allow the investigation of as many aspects of the pathology as possible. Experimental autoimmune encephalomyelitis (EAE) represents one of the most used models of MS in mice, modeling different disease features, from the activation of the immune system to CNS damage. Here we describe a protocol for the induction of EAE in both male and female C57BL/6J mice using myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) immunization, which leads to the development of a chronic form of the disease. We also report the evaluation of the daily clinical score and motor performance of these mice for 28 days post immunization (28 dpi). Lastly, we illustrate some basic histological analysis at the CNS level, focusing on the spinal cord as the primary site of disease-induced damage.

Autore in primo piano: Creazione di un modello sperimentale versatile di encefalomielite autoimmune rilevante sia per i topi maschi che per le femmine 

Experimental autoimmune encephalomyelitis is one of the most widely used murine models of multiple sclerosis. In the current protocol, C57BL/6J mice of both sexes are immunized with myelin oligodendrocyte glycoprotein peptide, resulting mainly in ascending paresis of the tail and limbs. Here we discuss the protocol of EAE induction and evaluation.

Il BM12 inducibile modello di Lupus Eritematoso Sistemico (LES) in C57BL / 6 Mice

Riepilogo

Esplora Altri Video

Capitoli in questo video

Induzione di ipofisite autoimmune sperimentale nei topi SJL

Il CYP2D6 modello animale: come indurre epatite autoimmune in topi

Mielina Oligodendrocyte glicoproteina (MOG

Induzione di encefalomielite autoimmune sperimentale nei topi e valutazione della distribuzione delle malattie-dipendente delle cellule immunitarie in vari tessuti

La determinazione di soppressione del sistema immunitario contro il sistema nervoso centrale di protezione per farmacologici Interventi a demielinizzazione autoimmune

Un approccio semplice per indurre la neurite Autoimmune Sperimentale in topi C57BL/6 per le valutazioni funzionali e Neuropathological

Induzione dell'epatite autoimmune indotta da farmaci in topi BALB / c per lo studio dei suoi meccanismi patogenetici

Murino colostomia distale, un nuovo modello di colite di diversione in topi C57BL/6

Analisi della proteinuria, infiltrazione renale dei leucociti e deposizione renale di proteine in topi MRL/lpr inclini al lupus

Modellazione della sclerosi multipla nei due sessi: encefalomielite autoimmune sperimentale indotta da MOG_35-55

Il BM12 inducibile modello di Lupus Eritematoso Sistemico (LES) in C57BL / 6 Mice

Riepilogo

Esplora Altri Video

Capitoli in questo video

Induzione di ipofisite autoimmune sperimentale nei topi SJL

Il CYP2D6 modello animale: come indurre epatite autoimmune in topi

Mielina Oligodendrocyte glicoproteina (MOG

Induzione di encefalomielite autoimmune sperimentale nei topi e valutazione della distribuzione delle malattie-dipendente delle cellule immunitarie in vari tessuti

La determinazione di soppressione del sistema immunitario contro il sistema nervoso centrale di protezione per farmacologici Interventi a demielinizzazione autoimmune

Un approccio semplice per indurre la neurite Autoimmune Sperimentale in topi C57BL/6 per le valutazioni funzionali e Neuropathological

Induzione dell'epatite autoimmune indotta da farmaci in topi BALB / c per lo studio dei suoi meccanismi patogenetici

Murino colostomia distale, un nuovo modello di colite di diversione in topi C57BL/6

Analisi della proteinuria, infiltrazione renale dei leucociti e deposizione renale di proteine in topi MRL/lpr inclini al lupus

Modellazione della sclerosi multipla nei due sessi: encefalomielite autoimmune sperimentale indotta da MOG35-55

Modellazione della sclerosi multipla nei due sessi: encefalomielite autoimmune sperimentale indotta da MOG_35-55