An autocrine Vitamin D-driven Th1 shutdown program can be exploited for COVID-19
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Abstract
Pro-inflammatory immune responses are necessary for effective pathogen clearance, but cause severe tissue damage if not shut down in a timely manner 1,2 . Excessive complement and IFN-γ-associated responses are known drivers of immunopathogenesis 3 and are among the most highly induced immune programs in hyper-inflammatory SARS-CoV2 lung infection 4 . The molecular mechanisms that govern orderly shutdown and retraction of these responses remain poorly understood. Here, we show that complement triggers contraction of IFN-γ producing CD4 + T helper (Th) 1 cell responses by inducing expression of the vitamin D (VitD) receptor (VDR) and CYP27B1, the enzyme that activates VitD, permitting T cells to both activate and respond to VitD. VitD then initiates the transition from pro-inflammatory IFN-γ + Th1 cells to suppressive IL-10 + Th1 cells. This process is primed by dynamic changes in the epigenetic landscape of CD4 + T cells, generating superenhancers and recruiting c-JUN and BACH2, a key immunoregulatory transcription factor 5–7 . Accordingly, cells in psoriatic skin treated with VitD increased BACH2 expression, and BACH2 haplo-insufficient CD4 + T cells were defective in IL-10 production. As proof-of-concept, we show that CD4 + T cells in the bronchoalveolar lavage fluid (BALF) of patients with COVID-19 are Th1-skewed and that VDR is among the top regulators of genes induced by SARS-CoV2. Importantly, genes normally down-regulated by VitD were de-repressed in CD4 + BALF T cells of COVID-19, indicating that the VitD-driven shutdown program is impaired in this setting. The active metabolite of VitD, alfacalcidol, and cortico-steroids were among the top predicted pharmaceuticals that could normalize SARS-CoV2 induced genes. These data indicate that adjunct therapy with VitD in the context of other immunomodulatory drugs may be a beneficial strategy to dampen hyperinflammation in severe COVID-19.
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SciScore for 10.1101/2020.07.18.210161: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Ethics approvals: Human studies were conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Guy’s Hospital (reference 09/H0707/86), National Institutes of Health (approval numbers 7458, PACI, 13-H-0065 and 00-I-0159) and Imperial College London (approval number 12/WA/0196 ICHTB HTA license number 12275 to project R14098).
Consent: All patients provided informed written consent.Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Antibodies Sentences Resources Where indicated, cells were activated in 96 well plates coated with … SciScore for 10.1101/2020.07.18.210161: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Ethics approvals: Human studies were conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Guy’s Hospital (reference 09/H0707/86), National Institutes of Health (approval numbers 7458, PACI, 13-H-0065 and 00-I-0159) and Imperial College London (approval number 12/WA/0196 ICHTB HTA license number 12275 to project R14098).
Consent: All patients provided informed written consent.Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Antibodies Sentences Resources Where indicated, cells were activated in 96 well plates coated with antibodies against CD3 (OKT3, made inhouse by the Washington University hybridoma facility), CD3 + CD28 (CD28.2, Becton Dickinson) or CD3 + CD46 (TRA-2-10, a gift from John P Atkinson, Washington University, USA), all at 2μg/mL in sterile PBS overnight at 4C, in the presence and absence of a cell soluble cathepsin L inhibitor (ALX-260-133-M001 from Enzo Life Sciences, Exeter, UK) at 1μM final concentration. CD3suggested: (GeneTex Cat# GTX13464, RRID:AB_369206)OKT3suggested: NoneCD28suggested: NoneTRA-2-10suggested: NoneFor surface staining we used the following antibodies: CD4 (OKT4, Thermo Fisher Scientific), IL-6 (MQ2-13A5, Biologend) OKT4suggested: NoneIL-6suggested: NoneMQ2-13A5suggested: NoneFor FACS sorting of memory CD4+ T cells, bulk CD4+ T cells enriched as described using RosetteSep Human CD4+ enrichment cocktail were stained with antibodies against CD4 (OKT4, Thermo Fisher Scientific), CD45RA (HI100, Biolegend), CD45RO (UCLH1, BD Biosciences) and CD25 (2A3, BD Biosciences) in MACS buffer at 4C for 30 min. CD4suggested: (Thermo Fisher Scientific Cat# MA1-42140, RRID:AB_2537291)CD45RAsuggested: NoneHI100suggested: NoneCD45ROsuggested: NoneCD25suggested: NoneImmunoblotting was performed according to standard protocols with initial blocking in PBS with 10% w/v clotting grade blocker (BioRad) or 3% w/v BSA (Sigma-Aldrich) and 0.1% v/v Tween20 (Sigma-Aldrich), followed by overnight incubation in primary antibodies: anti-VDR (D-6, Santa Cruz biotechnology) pSTAT3 (D3A7, Cell Signaling Technologies), STAT3 (124H6, Cell Signaling Technologies), Phospho-c-Jun (S63, R&D systems) or c-Jun (L70B11, Cell Signaling Technologies), followed by blocking and two hours incubation in appropriate HRP conjugated secondary anti-mouse or anti-rabbit antibodies (TrueBlot antibodies, Rockland). anti-VDRsuggested: NoneD-6suggested: NonepSTAT3suggested: (Fluidigm Cat# 3158005, RRID:AB_2661827)STAT3suggested: (Cell Signaling Technology Cat# 9139, RRID:AB_331757)Phospho-c-Jun ( S63suggested: Nonec-Jun ( L70B11suggested: (Cell Signaling Technology Cat# 2315, RRID:AB_490780)anti-mousesuggested: Noneanti-rabbitsuggested: NoneAntibody against BACH2 (D3T3G, Cell Signaling Technologies, 1:5000 dilution) was added in blocking buffer and incubated with membranes overnight at 4C with rotation. BACH2suggested: (Cell Signaling Technology Cat# 80775, RRID:AB_2799961)Human phospho-kinase antibody array: Array (Proteome Profiler Human Phospho-Kinase Array Kit, R&D systems) was carried out as per manufacturer’s protocol. Human phospho-kinasesuggested: NoneRabbit antibodies targeting H3K27Ac (ab4729, Abcam), c-JUN (60AB, Cell Signaling Technologies), and non-specific IgG (31235, Thermo Fisher Scientific) were used, together with pAG-MNase (123461, Addgene) for small DNA fragment generation. H3K27Acsuggested: (Abcam Cat# ab4729, RRID:AB_2118291)ab4729 , Abcam) ,suggested: Nonec-JUNsuggested: NonepAG-MNase ( 123461suggested: NoneThe samples were incubated with anti-CD3 (F7.2.38, Thermo Fisher Scientific) and anti-BACH2 (D3TG, Cell Signaling) primary antibodies overnight at 4C and washed in 0.01M PBS the next day. anti-BACH2 ( D3TG , Cell Signaling )suggested: NoneSoftware and Algorithms Sentences Resources Alternatively, we used LEGENDplex Human Inflammation Panel 1 (13plex) (740808, BioLegend) following manufacturers protocol using an Invitrogen Attune NxT Flow Cytometer and analyzed using FlowJo v9 software (BD Biosciences). FlowJosuggested: (FlowJo, RRID:SCR_008520)geNorm 6 gene kit, catalogue number ge-SY-6 from PrimerDesign UK) as housekeeping genes. geNormsuggested: (geNORM, RRID:SCR_006763)Hockey-stick plots of rank ordered stitched-enhancers plotted against VitD minus carrier H3K27Ac signal were produced in RStudio (ver. 1.2.5033 RStudiosuggested: (RStudio, RRID:SCR_000432)The bowtie index for RSEM alignment was generated by ‘rsem-prepare-reference’ on all RefSeq genes, downloaded from UCSC table browser in April 2017. RefSeqsuggested: (RefSeq, RRID:SCR_003496)UCSC table browsersuggested: NoneData were filtered to include only genes expressed at greater than 0.25 counts per million in at least 2 samples, TMM normalized within the edgeR package (v3.28.1), and differential expression performed using the glmQLFit and glmQLFTest functions in edgeR. edgeRsuggested: (edgeR, RRID:SCR_012802)Confocal images were collected on a Zeiss 780 inverted confocal microscope at 40X with oil-immersion and analyzed using ImageJ software. ImageJsuggested: (ImageJ, RRID:SCR_003070)Data presentation and statistical analysis: Figures were prepared using Adobe Illustrator (Adobe) Adobe Illustratorsuggested: (Adobe Illustrator, RRID:SCR_010279)Statistical analysis and graphical visualizations were carried out in GraphPad Prism (v.8.4.0), XLstat biomed (v2017.4), DataGraph 4.5.1 ( GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)DataGraphsuggested: NoneResults 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).
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.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 19. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
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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SciScore for 10.1101/2020.07.18.210161: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Antibodies Sentences Resources Accordingly, we stimulated Th cells with VitD in the presence or absence of Tocilizumab, an IL-6 receptor (IL-6R) blocking antibody used clinically for the management of IL-6dependent cytokine release syndrome, including the suggested cytokine storm in COVID-19. IL-6Rsuggested: NoneSoftware and Algorithms Sentences Resources We therefore focused our analyses of the scRNAseq datasets on CD4+ T cells. s…SciScore for 10.1101/2020.07.18.210161: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Antibodies Sentences Resources Accordingly, we stimulated Th cells with VitD in the presence or absence of Tocilizumab, an IL-6 receptor (IL-6R) blocking antibody used clinically for the management of IL-6dependent cytokine release syndrome, including the suggested cytokine storm in COVID-19. IL-6Rsuggested: NoneSoftware and Algorithms Sentences Resources We therefore focused our analyses of the scRNAseq datasets on CD4+ T cells. scRNAseqsuggested: NoneWe next used EnrichR to predict 461 significant drugs that could potentially be used to counteract the genes upregulated in COVID-19 BALF Th cells. EnrichRsuggested: (Enrichr, SCR_001575)Also shown is expression of CYP27B1 (highlighted in red). j, EnrichR-predicted ENCODE and ChEA ( ChEAsuggested: (ChEA, SCR_005403)Data from additional tools added to each annotation on a weekly basis.
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