Plasmacytoid dendritic cells regulate megakaryocyte and platelet homeostasis

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   Jun 3, 2022

   SciScore for 10.1101/2022.05.31.494147: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IRB: The study was approved by and conducted according to requirements of the ethics committees at the Ludwig Maximilians University of Munich (20-1039). IACUC: All experimental animal procedures were approved by the Institutional Animal Committee of the San Raffaele Scientific Institute and all infectious work was performed in designed BSL-3 workspaces. Euthanasia Agents: After imaging the mice were euthanized by cervical dislocation.
   Sex as a biological variable	Both male and female mice were used in this study.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Platelet depleting antibodies (R300, anti-GPIbα) and isotype …

   SciScore for 10.1101/2022.05.31.494147: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IRB: The study was approved by and conducted according to requirements of the ethics committees at the Ludwig Maximilians University of Munich (20-1039). IACUC: All experimental animal procedures were approved by the Institutional Animal Committee of the San Raffaele Scientific Institute and all infectious work was performed in designed BSL-3 workspaces. Euthanasia Agents: After imaging the mice were euthanized by cervical dislocation.
   Sex as a biological variable	Both male and female mice were used in this study.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Platelet depleting antibodies (R300, anti-GPIbα) and isotype control (C301) were purchased from Emfret (Eibelstadt, Germany) and used according to the manufacturer’s protocol.	anti-GPIbα suggested: None C301 suggested: None
   pDC depleting antibody (ultra-LEAFTM purified anti-PDCA-1, clone 927, BioLegend) was injected i.p. for 9 consecutive days with a concentration of 150 µg per mouse at day 1 and 100 µg per mouse the following days.	anti-PDCA-1 suggested: None
   To monitor pDC activation and IFN alpha production, the following primary antibodies were used: CD69 mouse anti-human (#MA5-15612 Thermo Fisher)	CD69 mouse anti-human suggested: None anti-human suggested: (Millipore Cat# 4700-0360, RRID:AB_11210063)
   Secondary antibodies (1:200): donkey anti-rabbit-AF488, donkey anti-mouse-AF 555, and donkey anti-gost-AF647.	anti-rabbit-AF488 suggested: None anti-mouse-AF suggested: None anti-gost-AF647 suggested: None
   Lineage-biotin antibodies (Ter-119, CD3e, CD45R, CD11b, Ly-6G,) and streptavidin-PE were used with dilution of 1:200, all antibodies were purchased from eBioscience (San Diego, USA)	Lineage-biotin suggested: None CD3e suggested: (Thermo Fisher Scientific Cat# 88-7774-75, RRID:AB_476399) CD45R suggested: (Thermo Fisher Scientific Cat# 88-7774-75, RRID:AB_476399) CD11b suggested: (BD Biosciences Cat# 558074, RRID:AB_1645213) Ly-6G , suggested: None
   The following antibodies were used to identify MKs: 1:100 anti-mouse CD41-FITC+ and anti-mouse CD42d-APC+ (BioLegend); and MKPs: anti-mouse CD41-FITC+, Pacific blue lineage negative (Ter-119-, CD3e-, CD45R-, CD11b-, Ly-6G-)	anti-mouse CD42d-APC+ suggested: None anti-mouse CD41-FITC+ suggested: None Ter-119- , CD3e- suggested: None Ly-6G- suggested: None
   We identified pDCs using the following antibodies: anti-mouse SiglecH-FITC+, CD11b-PE-Cy7-, B220-APC+ from BioLegend (San Diego, USA) (	anti-mouse suggested: None
   1:200). pDC activation: anti-mouse CD69-FITC, CD86-PE, CD11b-APC-Cy7, CD317-APC, SiglecH-PercCy5.5 antibodies all from BioLegend and Life/Dead marker (405 nm excitation; ThermoFisher).	anti-mouse CD69-FITC suggested: (SouthernBiotech Cat# 1715-02, RRID:AB_2795177)
   IFN expression by pDCs: Following staining with pDC surface markers (see above), cells were fixed with PFA and methanol and stained with monoclonal anti-mouse p-IRF7 antibody (cellsignaling, clone D6M2I) in Perm buffer III (BD) as previously described31.	anti-mouse p-IRF7 suggested: None
   Briefly, cells were stained with surface marker antibodies (CD41, CD42) for 30 min RT in dark, followed by fixation for 15 min (4% PFA, provided in the kit) and permeabilization 15 min (saponin-based permeabilization and wash reagent, provided in the kit).	CD41 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Materials: Mouse strains: C57BL/6J, PF4-Cre (C57BL/6-Tg(Pf4-icre)Q3Rsko/J)44, Rosa26-iDTRflox (C57BL/6Gt(ROSA)26Sortm1(HBEGF)Awai/J)45, IFNαR-/- (B6.129S2-Ifnar1tm1Agt/Mmjax)46, IFNαR1flox (B6(Cg)-Ifnar1tm1.1Ees/J)47 and BDCA2-DTR (C57BL/6-Tg(CLEC4C-HBEGF)956Cln/J)(referred to as pDC-DTR)48 mice were purchased from The Jackson Laboratory. vWF-Cre mice were generated by W.	C57BL/6J suggested: None PF4-Cre suggested: None C57BL/6-Tg(Pf4-icre)Q3Rsko/J)44 suggested: None Rosa26-iDTRflox suggested: None C57BL/6Gt(ROSA)26Sortm1 suggested: None B6.129S2-Ifnar1tm1Agt/Mmjax)46, IFNαR1flox suggested: None C57BL/6-Tg(CLEC4C-HBEGF)956Cln/J) suggested: RRID:IMSR_JAX:014176)
   PF4-Cre were crossed with Rosa26-iDTR mice to induce megakaryocyte cell death in vivo (PF4-Cre; RS26-iDTR)18. PF4-Cre;RS26-iDTR were crossed with vWF-eGFP to visualize the megakaryocytic lineage following induction of MK cell death (referred to as MK-iDTR).	Rosa26-iDTR suggested: None
   vWF-Cre mice were crossed with IFNαR1floxto conditionally delete IFNaR in the megakaryocytic lineage. pDC-DTR and IFNαR-/- were cross bred to achieve pDC depletion in IFNaR-/- animals (pDC-DTR; IFNaR-/-).	vWF-Cre suggested: None
   Drug treatments: Diphtheria Toxin (DT) was purchased from Sigma-Aldrich (322326, Saint Louis, MO, USA) and was injected intraperitoneally into pDC-DTR- and pDC-DTR-IFNαR-/- mice with a dose of 8 ng/g per day for consecutive 3 days.	pDC-DTR-IFNαR-/- suggested: None
   Mouse model of SARS-CoV2 infection: B6.Cg-Tg(K18-ACE2)2Prlmn/J mice (on a C57BL/6 background) were purchased from The Jackson Laboratory and bred against FVB mice to obtain C57BL/6 x FVB F1 hybrids.	B6.Cg-Tg(K18-ACE2)2Prlmn/J suggested: RRID:IMSR_JAX:034860) C57BL/6 suggested: None FVB suggested: None
   Blood vessel were visualized by i.v. injection of Dextran Tetramethylrhodamine (TRITC-Dextran, 100µg in 100µl solution, ThermoFisher Scientific) or Dextran Cascade Blue 10.000 MW (D1976, ThermoFisher Scientific) before imaging. vWF-eGFP mice was used to visualize the megakaryocytic lineage; pDCs were labeled with SiglecH-PE antibody (eBioscience) injected intravenously 20 min before imaging (20 µl diluted with 100 µl NaCl).	vWF-eGFP suggested: None
   Recombinant DNA
   Sentences	Resources
   vWF-Cre mice were crossed with IFNαR1floxto conditionally delete IFNaR in the megakaryocytic lineage. pDC-DTR and IFNαR-/- were cross bred to achieve pDC depletion in IFNaR-/- animals (pDC-DTR; IFNaR-/-).	pDC-DTR suggested: None
   Drug treatments: Diphtheria Toxin (DT) was purchased from Sigma-Aldrich (322326, Saint Louis, MO, USA) and was injected intraperitoneally into pDC-DTR- and pDC-DTR-IFNαR-/- mice with a dose of 8 ng/g per day for consecutive 3 days.	pDC-DTR- suggested: None
   Software and Algorithms
   Sentences	Resources
   Animals were bred and maintained in the animal facilities of the Walter-Brendel Zentrum, the Zentrum für Neuropathologie und	Neuropathologie suggested: None
   Labelling of MKs/MKPs: primary: CD41-FITC+ and CD42-purified hamster anti-mouse (BioLegend); secondary: goat anti-hamster Alexa Fluor 647 (Abcam) (1:100).	BioLegend) suggested: (BioLegend, RRID:SCR_001134)
   Images were taken with step size of 2 µm, range in z-stack of 40 µm, and analyzed with Zen Blue software.	Zen Blue suggested: (ZEN Digital Imaging for Light Microscopy, RRID:SCR_013672)
   Cell volumes of 3D rendered bone marrow stacks were measured automatically in Imaris.	Imaris suggested: (Imaris, RRID:SCR_007370)
   Gene set enrichment analysis (GSEA): To prepare the data for GSEA, DESeq2 analysis was performed using Galaxy and default parameters51, 52.	Gene set enrichment analysis suggested: (Gene Set Enrichment Analysis, RRID:SCR_003199) DESeq2 suggested: (DESeq, RRID:SCR_000154)
   For further analysis, the tool GSEA (version 4.0.3) of UC San Diego and Broad Institute was used53 and 54, referring to their RNASeq manual pages for analysis.	GSEA suggested: (SeqGSEA, RRID:SCR_005724)
   Statistics: GraphPad Prism software (9.1.2, San Diego, USA) was used for all statistical analysis.	Statistics: GraphPad Prism suggested: None GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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   Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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   Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   Results from rtransparent:

   • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
   • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
   • No protocol registration statement was detected.

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   Results from scite Reference Check: We found no unreliable references.

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   Version published to 10.1101/2022.05.31.494147v1 on bioRxiv

   Jun 1, 2022