We thank Elsevier for their authorization (license number 4334770630127) to reproduce here Figure 2A from Unterreiner et al. (2017).

Experiments were conducted according to the guidelines and standards of the Novartis Human Research Ethics Committee.
1. Preparation of Peripheral Blood Mononuclear Cells (PBMCs) from Human Buffy Coats
NOTE: We received buffy coats from healthy volunteers one day after collection, in 50 mL bags. They were provided under informed consent and collected through the Interregionale Blutspende Schweizeriches Rotes Kreuz. We handled them using the p...

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R., 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=Dectin-1+is+required+for+beta-glucan+recognition+and+control+of+fungal+infection.">Dectin-1 is required for beta-glucan recognition and control of fungal infection.</a> Nature Immunology. 8 (1), 31-38 (2007).</li><li>Lanternier, F., 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=Primary+immunodeficiencies+underlying+fungal+infections.">Primary immunodeficiencies underlying fungal infections.</a> Current opinion in pediatrics. 25 (6), 736-747 (2013).</li><li>Thome, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifunctional+roles+for+MALT1+in+T-cell+activation.">Multifunctional roles for MALT1 in T-cell activation.</a> Nature reviews. Immunology. 8 (7), 495-500 (2008).</li><li>Unterreiner, A., Stoehr, N., Huppertz, C., Calzascia, T., Farady, C. J., Bornancin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+MALT1+paracaspase+inhibition+does+not+block+TNF-&#945;+production+downstream+of+TLR-4+in+myeloid+cells.">Selective MALT1 paracaspase inhibition does not block TNF-&#945; production downstream of TLR-4 in myeloid cells.</a> Immunology Letters. , 48-51 (2017).</li><li>Strasser, D., 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=Syk+Kinase-Coupled+C-type+Lectin+Receptors+Engage+Protein+Kinase+C-&#x3B4;+to+Elicit+Card9+Adaptor-Mediated+Innate+Immunity.">Syk Kinase-Coupled C-type Lectin Receptors Engage Protein Kinase C-&#x3B4; to Elicit Card9 Adaptor-Mediated Innate Immunity.</a> Immunity. 36 (1), 32-42 (2012).</li><li>Jaworski, 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=Malt1+protease+inactivation+efficiently+dampens+immune+responses+but+causes+spontaneous+autoimmunity.">Malt1 protease inactivation efficiently dampens immune responses but causes spontaneous autoimmunity.</a> The EMBO journal. 33 (23), 2765-2781 (2014).</li><li>Yu, J. 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=MALT1+protease+activity+is+required+for+innate+and+adaptive+immune+responses.">MALT1 protease activity is required for innate and adaptive immune responses.</a> PLoS ONE. 10 (5), 1-20 (2015).</li><li>Bardet, 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=The+T-cell+fingerprint+of+MALT1+paracaspase+revealed+by+selective+inhibition.">The T-cell fingerprint of MALT1 paracaspase revealed by selective inhibition.</a> Immunology and Cell Biology. 96 (1), 81-99 (2018).</li><li>Thoma, G., 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=Discovery+and+profiling+of+a+selective+and+efficacious+syk+inhibitor.">Discovery and profiling of a selective and efficacious syk inhibitor.</a> Journal of Medicinal Chemistry. 58 (4), 1950-1963 (2015).</li><li>Bauer, B., Steinle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HemITAM:+A+single+tyrosine+motif+that+packs+a+punch.">HemITAM: A single tyrosine motif that packs a punch.</a> Science Signaling. 10 (508), 1-10 (2017).</li><li>Gringhuis, S. I., 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=Selective+c-Rel+activation+via+Malt1+controls+anti-fungal+TH-17+immunity+by+dectin-1+and+dectin-2.">Selective c-Rel activation via Malt1 controls anti-fungal TH-17 immunity by dectin-1 and dectin-2.</a> PLoS Pathogens. 7 (1), (2011).</li><li>Dufner, A., Schamel, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cell+antigen+receptor-induced+activation+of+an+IRAK4-dependent+signaling+pathway+revealed+by+a+MALT1-IRAK4+double+knockout+mouse+model.">B cell antigen receptor-induced activation of an IRAK4-dependent signaling pathway revealed by a MALT1-IRAK4 double knockout mouse model.</a> Cell Communication and Signaling. 9 (1), 6 (2011).</li><li>Phelan, J. D., 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=A+multiprotein+supercomplex+controlling+oncogenic+signalling+in+lymphoma.">A multiprotein supercomplex controlling oncogenic signalling in lymphoma.</a> Nature. , (2018).</li></ol>

In this work, we used simple experimental settings to study signaling pathways in human and mouse innate cells, and interrogate their dependency on MALT1 proteolytic function. Expanding on previous work11, our study showed that MALT1 paracaspase activity controls C-type lectin-like receptor induced cytokine production, including TNF-&#945;. In contrast, TLR-4-induced TNF-&#945; was independent of MALT1 in both species. Collectively, these data corroborated the key and selective contribution of the...

The paracaspase activity of MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) was revealed in 20081,2. Since then, a number of studies have reported its critical contribution to antigen receptor responses in lymphocytes. Genetic models in the mouse as well as pharmacology data support a key role in T cells, in T-cell dependent autoimmunity and in B-cell lymphoma settings3,4. In lymphocytes, MALT1 paracaspase activation occurs upon assembly of a CARD11-BCL10-MALT1 complex5, which is triggered by antigen-receptor proximal signaling downstream of the T- or B- cell receptor. There is also ample evidence that a similar CARD9-BCL10-MALT1 complex is important for propagating signals downstream of C-type lectin-like receptors (CLLR), e.g., Dectin-1, Dectin-2 and MINCLE in myeloid cells6,7. Dectin-1 has been particularly well studied because this pathway is critical for host defense against fungal infections8,9. Implication of MALT1 in Toll-like receptor (TLR) pathways, however, has remained controversial10. Recent evidence in human myeloid cells ruled out a direct role for MALT1 paracaspase activity in the regulation of TNF- production downstream of TLR-411.
In the present work, we used various experimental settings and stimulatory conditions in human and mouse myeloid cells to probe innate signaling pathways, relying on specific pharmacological tool inhibitors and measurement of cytokine production.

<table>
	<tbody>
		<tr>
			<td>100 &#181;m nylon cell strainer</td>
			<td>Sigma</td>
			<td>CLS431752</td>
			<td></td>
		</tr>
		<tr>
			<td>14 ml Falcon tube</td>
			<td>BD Falcon</td>
			<td>352057</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Falcon tube</td>
			<td>Falcon</td>
			<td>352090</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Falcon tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>Costar</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well flat-bottom plate, with low evaporation lid</td>
			<td>Costar</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well V-bottom plate</td>
			<td>Costar</td>
			<td>734-1798</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Chloride - NH4Cl</td>
			<td>Sigma</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Assay diluent RD1-W ELISA</td>
			<td>R&#38;D</td>
			<td>895038</td>
			<td>Assay diluent</td>
		</tr>
		<tr>
			<td>Cell culture microplate, 384 well, black</td>
			<td>Greiner</td>
			<td>781986</td>
			<td></td>
		</tr>
		<tr>
			<td>Depleted Zymosan</td>
			<td>Invivogen</td>
			<td>tlrl-dzn</td>
			<td>now: tlrl-zyd</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td>DMSO</td>
		</tr>
		<tr>
			<td>EDTA-Na2</td>
			<td>Sigma</td>
			<td>E5134</td>
			<td>Ethylenediaminetetraacetic acid disodium salt dihydrate</td>
		</tr>
		<tr>
			<td>ELISA muTNF-&#945;</td>
			<td>R&#38;D</td>
			<td>SMTA00</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td>17-1440-03</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS C tubes</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-096-334</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS dissociator</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td></td>
		</tr>
		<tr>
			<td>GM-CSF</td>
			<td>Novartis</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat-inactivated Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10082</td>
			<td>FBS</td>
		</tr>
		<tr>
			<td>HTRF hu IL-23</td>
			<td>CisBio</td>
			<td>62HIL23PEG</td>
			<td></td>
		</tr>
		<tr>
			<td>HTRF hu TNF-&#945;</td>
			<td>CisBio</td>
			<td>62TNFPEC</td>
			<td></td>
		</tr>
		<tr>
			<td>HTRF reconstitution buffer</td>
			<td>CisBio</td>
			<td>62RB3RDE</td>
			<td>50mM Phosphate buffer, pH 7.0, 0.8M KF, 0.2% BSA</td>
		</tr>
		<tr>
			<td>IFN-&#947;</td>
			<td>R&#38;D</td>
			<td>L4516</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-4</td>
			<td>Novartis</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Abbott</td>
			<td>Forene</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharides (LPS)</td>
			<td>Sigma</td>
			<td>L4391</td>
			<td>LPS used in human samples</td>
		</tr>
		<tr>
			<td>Lipopolysaccharides</td>
			<td>Sigma</td>
			<td>L4516</td>
			<td>LPS used in murine samples</td>
		</tr>
		<tr>
			<td>Lysis buffer</td>
			<td>Self-made</td>
			<td>-</td>
			<td>155 mM NH4Cl, 10 mM KHCO3, 1 mM EDTA, pH 7.4</td>
		</tr>
		<tr>
			<td>Magnet</td>
			<td>Stemcell</td>
			<td>18001</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate, 384 well white</td>
			<td>Greiner</td>
			<td>784075</td>
			<td></td>
		</tr>
		<tr>
			<td>Monocytes enrichment kit</td>
			<td>Stemcell</td>
			<td>19059</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Mr. Frosty Cryo 1 &#176;C Freezing Container</td>
			<td>Nalgene</td>
			<td>5100-0001</td>
			<td>cooling device (containing Propanol-2)</td>
		</tr>
		<tr>
			<td>PBS 1x pH 7.4 [-] CaCl2 [-] MgCl2</td>
			<td>Gibco</td>
			<td>10010</td>
			<td>Phosphate-buffered saline</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140</td>
			<td>Pen/Strep</td>
		</tr>
		<tr>
			<td>Potassium bicarbonate - KHCO3</td>
			<td>Sigma</td>
			<td>P9144</td>
			<td></td>
		</tr>
		<tr>
			<td>PrestoBlue</td>
			<td>Invitrogen</td>
			<td>A13262</td>
			<td>Resazurin solution for viability assessment</td>
		</tr>
		<tr>
			<td>Propanol-2</td>
			<td>Merck</td>
			<td>1.09634</td>
			<td></td>
		</tr>
		<tr>
			<td>Read buffer</td>
			<td>MesoScale Discovery</td>
			<td>R92TC-3</td>
			<td>Tris-based buffer containing tripropylamine</td>
		</tr>
		<tr>
			<td>Recovery cell culture freezing medium</td>
			<td>Gibco</td>
			<td>12648-010</td>
			<td>freezing medium</td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute Medium (RPMI) with Glutamax</td>
			<td>Gibco</td>
			<td>61870</td>
			<td>+ 10% FBS for iMoDCs 
			+ 10% FBS + 1 mM Sodium Pyruvate + 100 U/mL Pen/Strep + 5 &#181;M &#946;-mercaptoethanol for human PBMCs and monocytes 
			+ 10% FBS + Pen/Strep + 5 &#181;M &#946;-mercaptoethanol for murine splenocytes</td>
		</tr>
		<tr>
			<td>Separation buffer</td>
			<td>Self-made</td>
			<td>-</td>
			<td>PBS pH 7.4 + 2% FBS + 1 mM EDTA pH 8.0</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Gibco</td>
			<td>11360</td>
			<td></td>
		</tr>
		<tr>
			<td>Trehalose-6,6-dibehenate</td>
			<td>Invivogen</td>
			<td>tlrl-tdb</td>
			<td>TDB</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P7949</td>
			<td>Polysorbate 20</td>
		</tr>
		<tr>
			<td>UltraPure 0.5 M EDTA pH 8.0</td>
			<td>Invitrogen</td>
			<td>15675</td>
			<td>Ethylenediaminetetraacetic acid</td>
		</tr>
		<tr>
			<td>Viewseal sealer</td>
			<td>Greiner BioOne</td>
			<td>676070</td>
			<td></td>
		</tr>
		<tr>
			<td>V-PLEX Proinflammatory Panel 1 Human Kit</td>
			<td>MesoScale Discovery</td>
			<td>K15049D</td>
			<td>electrochemiluminescent multiplex assay (IL-1&#946;, TNF-&#945;, IL-6, IL-8)</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>31350</td>
			<td></td>
		</tr>
	</tbody>
</table>

myeloid innate signaling pathway regulation by malt1 paracaspase activity

Besides its function in lymphoid cells, which has been addressed by numerous studies, the paracaspase MALT1 also plays an important role in innate cells downstream of pattern recognition receptors. Best studied are the Dectin-1 and Dectin-2 members of the C-type lectin-like receptor family that induce a SYK- and CARD9-dependent signaling cascade leading to NF-&#954;B activation, in a MALT1-dependent manner. By contrast, Toll-like receptors (TLR), such as TLR-4, propagate NF-&#954;B activation but signal via an MYD88/IRAK-dependent cascade. Nonetheless, whether MALT1 might contribute to TLR-4 signaling has remained unclear. Recent evidence with MLT-827, a potent and selective inhibitor of MALT1 paracaspase activity, indicates that TNF- production downstream of TLR-4 in human myeloid cells is independent of MALT1, as opposed to TNF- production downstream of Dectin-1, which is MALT1 dependent. Here, we addressed the selective involvement of MALT1 in pattern recognition sensing further, using a variety of human and mouse cellular preparations, and stimulation of Dectin-1, MINCLE or TLR-4 pathways. We also provided additional insights by exploring cytokines beyond TNF-, and by comparing MLT-827 to a SYK inhibitor (Cpd11) and to an IKK inhibitor (AFN700). Collectively, the data provided further evidence for the MALT1-dependency of C-type lectin-like receptor &#8212;signaling by contrast to TLR-signaling.

In myeloid cells, MALT1 relays activation signals downstream of several C-type lectin-like receptors, such as Dectin-1, Dectin-2 and MINCLE6. These pathways rely on (hem)ITAM motif-containing receptors (e.g., Dectin-1) or ITAM motif-containing co-receptors (e.g., FcR&#947;, for Dectin-2 and MINCLE) that recruit and activate the SYK kinase (Figure 1). This leads to activation of a protein kinase C isoform, namely PKC&#...

Watch this Scientific Journal Video about Myeloid Innate Signaling Pathway Regulation by MALT1 Paracaspase Activity at JoVE.com

Myeloid Innate Signaling Pathway Regulation by MALT1 Paracaspase Activity

MALT1 regulates innate immunity but how this occurs remains ill-defined. We used the selective MALT1 paracaspase inhibitor MLT-827 to unravel the contribution of MALT1 to innate signaling downstream of Toll-like or C-type lectin-like receptors, demonstrating that MALT1 regulates the production of myeloid cytokines, and downstream of C-type lectin-like receptors, selectively.

Besides its function in lymphoid cells, which has been addressed by numerous studies, the paracaspase MALT1 also plays an important role in innate cells ...

So the aim of the method in our manuscript is to provide comparative analysis of signaling pathways in myeloid cells using human and mouse systems. It helps define which signaling pathway is involved downstream of selected pattern recognition receptors. So this method is simple to implement.It relies on primary cell preparations and validated reference compounds, which is essential for the elucidation of signaling mechanisms. Demonstrating the procedure will be Ratiba Touil and Adeline Unterreiner, respectively scientist and research associate in our group. In a laminar flow hood set out a sterile pair of scissors, a one liter beaker, and a plastic bag.Place the previously obtained buffy coat into the beaker and use the scissors to carefully open it. Using a 25 mm pipette add 100 ml of PBS supplemented with 2 millimolar of EDTA. Mix by slowly pipetting up and down.And then transfer 25 ml of the diluted buffy coat into six 50 ml conical centrifuge tubes that have each been prefilled with 15 ml of polysaccharide based density gradient. Centrifuge the tubes for 20 minutes at 800 g with moderate acceleration without the break to allow for separation of cells based on their densities. After centrifugation three layers will be visible, a pellet containing red blood cells and granulocytes, an upper layer made of plasma, and a white ring containing peripheral blood mononuclear cells.Use a 10 ml pipette to harvest the PBMC rings and transfer them into a new 50 ml tube. Top up with PBS and EDTA. Perform three successive washes with decreasing centrifugation time and speed as outlined in the text protocol.After the final wash, resuspend the pellet in 25 ml of ice cold lysis buffer to lyse the red blood cells by osmotic pressure. Incubate the solution at room temperature until it becomes clear. Then add 25 ml of separation buffer to stop the reaction.Centrifuge at 150 g for eight minutes to wash the solution. Pour off the supernatant and resuspend the pellet with separation buffer. After counting the cells, dilute them in culture medium down to 12, 500 cells per well.Distribute 30 ml of this cell suspension into each well of a 384 well plate. Next add 15 microliters of four times concentrated compound solutions to each well and preincubate the plate at 37 degrees Celsius with 5%carbon dioxide for one hour. If desired, add either LPS to final concentration of 1 ng per ml or depleted Zymosan to a final concentration of 100 mcg per ml.Then incubate the plate overnight at 37 degrees Celsius with 5%carbon dioxide. The next day take 10 microliters of the supernatant to measure the secreted TNF alpha levels. To begin the serial dilution, dilute the MLT-827 stock solution with medium to reach a concentration of 8 micromolar in one go.Perform a six-step 1:5 serial dilution using medium with 0.08%DMSO. To prepare for single dose testings, dilute the MLT-827, AFN700, and compound 11 stock solutions with medium to reach a concentration of 4 micromolar in one go. First, add 2 ml of sterile endotoxin-free water to 10 mg of depleted Zymosan.Vortex this stock solution to homogenize it. Aliquot the solution and then store the aliquots at 20 degrees Celsius. In this study human PBMCs and mouse spleen cells are stimulated with either depleted Zymosan, a Dectin-1 agonist, or lipopolysaccharide, a TL4 agonist.NTNF alpha release in the supernatant is measured after 20 hours. In both the human and mouse assays the MLT-827 compound selectively blocks TNF alpha production driven by the Dectin-1 pathway but not by the TLR4 pathway. In monocytes stimulated with LPS, production of TNF alpha is almost completely abrogated by AFN700 but it's not sensitive to Cpd11, which is consistent with the TLR4 pathway's dependency on NF-kappa B activity and its independency on SIC activity.In contrast TNF alpha production driven by Dectin-1 and MODCs shows sensitivity to MLT-827, AFN700, and Cpd11. This provides further evidence for the involvement of a SIC CBM signaling cascade in the Dectin-1 pathway. Production of IL-1 beta, IL-6, and IL-23 is also sensitive to the three inhibitors, indicating regulatory mechanisms similar to TNF alpha.However, the limited effect seen on the production of IL-8 suggests that this cytokine has a distinct regulatory mechanism. Raising Trehalose-dibehenate concentrations above 50 mcg per ml leads to production of TNF alpha, IL-6, and IL-1 beta, which relied on MALT1 paracaspase activity according to the blocking effect of MLT-827. Similar results were seen when challenging the MODCs with increasing concentrations of depleted Zymosan to stimulate Dectin-1.This method made it possible to show that the paracaspase MALT1 selectively regulates C-type lectin-like receptor signaling. It is essential to use well-characterized reference compounds. Pay attention to the procedure for the dilution of stock solutions.This is critical for compound with limited solubility.

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7.3K ビュー.  Novartis Institutes for BioMedical Research. そこで、私たちの原稿の方法の目的は、ヒトとマウスのシステムを用いて骨髄細胞のシグナル経路の比較分析を提供することです。これは、選択されたパターン認識受容体の下流に関与するシグナル経路を定義するのに役立ちます。したがって、このメソッドは実装が簡単です。これは、シグナル伝達機構の解明に不可欠な一次細胞製剤および検証された基準化合?...

ビデオ: Myeloid Innate Signaling Pathway Regulation by MALT1 Paracaspase Activity

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

Reaggregate Thymus Cultures

Stromal cells within lymphoid tissues are organized into three-dimensional structures that provide a scaffold that is thought to control the migration and development of haemopoeitic cells. Importantly, the maintenance of this three-dimensional organization appears to be critical for normal stromal cell function, with two-dimensional monolayer cultures often being shown to be capable of supporting only individual fragments of lymphoid tissue function. In the thymus, complex networks of cortical and medullary epithelial cells act as a framework that controls the recruitment, proliferation, differentiation and survival of lymphoid progenitors as they undergo the multi-stage process of intrathymic T-cell development. Understanding the functional role of individual stromal compartments in the thymus is essential in determining how the thymus imposes self/non-self discrimination. Here we describe a technique in which we exploit the plasticity of fetal tissues to re-associate into intact three-dimensional structures in vitro, following their enzymatic disaggregation. The dissociation of fetal thymus lobes into heterogeneous cellular mixtures, followed by their separation into individual cellular components, is then combined with the in vitro re-association of these desired cell types into three-dimensional reaggregate structures at defined ratios, thereby providing an opportunity to investigate particular aspects of T-cell development under defined cellular conditions. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

In this video the preparation of 2-dGuo-treated reaggregate thymus cultures is demonstrated.

Stromal cells within lymphoid tissues are organized into three-dimensional structures that provide a scaffold that is thought to control the migration and ...

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2&bull;-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-&alpha; (TNF&alpha;) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2&bull;- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2&bull;- that are important in the host defense mechanism11,12. Although paracrine-derived O2&bull;- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2&bull;- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2&bull;- lead to calcium signaling in adjacent endothelial cells.

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...