EMBO/soda-nel huggingface dataset:
miR-132 suppresses transcription of ribosomal proteins to promote protective Th1 immunity (2019)
Figure 1

Figure 1: The miR-132/212 cluster regulates RP mRNA levels in CD4+ T cells from chronically infected spleens.

Expression of indicated miRNAs in sorted naïve (CD62L+ CD44-) CD4+ T cells and following in vitro stimulation with anti-CD3 / anti-CD28 (1-3 days), relative to levels in cells prior to stimulation. Data from 3 independent experiments each using T cells pooled from 4 WT mice. Significance determined by one-way ANOVA.

Expression of indicated miRNAs in purified spleen lymphocytes (B cells, CD4+ T cells and CD8+ T cells) from d0 naive (white) and day 28 L. donovani-infected (grey) mice. Expression of each miRNA normalized to levels in whole naïve spleen (dotted line). Data is mean + SEM of 2 experiments with cells purified from 3-5 pooled spleens.

Volcano plot of RNAseq gene expression in splenic WT and miR-132-/- CD4+ T cells from d28 L. donovani infected mice. Fold change determined as log2 mean FPKM (miR-132-/-/WT) from 4 WT and 5 miR-132-/- mice. Transcripts significantly different between WT and miR-132-/- (p<0.05) are shown in red. Dotted box indicates transcripts significantly up-regulated in miR-132-/- CD4+ T cells by more than 50%.

STRING network analysis of significantly up-regulated transcripts in CD4+ T cells from spleen of d28 L. donovani infected miR-132-/- mice compared to WT cells. Cluster of ribosomal proteins shown in green circle, with coding RP transcripts (black) and pseudogenes (red) indicated. Secondary clusters are shown in grey.

Top enriched molecular function Gene Ontology terms for genes significantly up-regulated in CD4+ T cells from spleens of infected miR-132-/- mice compared to WT mice.

Volcano plot of all RP genes in splenic WT and miR-132-/- CD4+ T cells from d28 L. donovani infected mice. RPL genes are shown as circles, RPS genes as triangles, and pseudogenes as squares. Red symbols indicate significant difference between WT and miR-132-/- cells (p<0.05) whereas black non-significant.

Expression of RP transcripts determined by qPCR from L. donovani infected d28 WT (blue) and miR-132-/- mice (red). N=9 for each WT and miR-132-/- from 2 independent infection experiments. Box extends from 25-75th percentile, whiskers are minimum and maximum values, and horizontal lines indicate median. Significance determined by unpaired t-test.

Fold change of all RP transcripts (grey) in Th1 cells compared to naïve CD4+ T cells. Data taken from RNA sequencing experiments described in reference 24. Fold changes in IL-10 (red) and IFNγ (blue) indicated for comparison. The statistical significance of the observed up-regulation of RP transcripts in Th1 cells is determined by Chi-squared test.

Data information: * p<0.05, ** p<0.01, *** p<0.001.
miR-132 suppresses transcription of ribosomal proteins to promote protective Th1 immunity (2019)
Figure 2

Figure 2: The B-TFIID cofactor BTAF1 is a direct miR-132 target in CD4+ T cells.

A. Volcano plot (Log2(Fold Change) vs -Log(P value)) of RNA gene expression in purified naïve CD62L+ CD44- WT and miR-132-/- CD4+ T cells. Fold change determined as log2 mean FPKM (miR-132-/-/WT) from 4 WT and 4 miR-132-/- mice. Transcripts significantly different between WT and miR-132-/- cells (p<0.05) shown in red.

B. Volcano plot of RNA gene expression in purified naïve CD62L+ CD44- WT and miR-132-/- CD4+ T cells following 18hr in vitro stimulation with anti-CD3/anti-CD28 under Th1 conditions. Fold change determined as log2 mean FPKM (miR-132-/-/WT) from 4 WT and 4 miR-132-/- mice. Transcripts significantly different between WT and miR-132-/- cells (p<0.05) shown in red.

C. Volcano plot of transcripts containing a conserved miR-212/132-3p target site in naïve CD4+ T cells from WT or miR-132-/- mice.

D. Volcano plot of transcripts containing a conserved miR-212/132-3p target site in in vitro polarised (Th1 condtions, 18h post stimulation) CD4+ T cells from WT or miR-132-/- mice.

E. Volcano plot of transcripts containing a conserved miR-212/132-3p target site in spleen CD4+ T cells from d28 L. donovani infected WT or miR-132-/- mice.

F. BTAF1 transcript levels determined by qRTPCR in WT (blue) or miR-132-/- (red) in naïve (d0) and Th1 polarised for 18h (d1) CD4+ T cells, and CD4+ T cells from d28 L. donovani infected WT or miR-132-/- mice. N=8-9 for each WT and miR-132-/-.

G. Expression of BTAF1 protein in d0 naïve and d1 (18hr) Th1-polarised WT and miR-132-/- CD4+ T cells, as determined by Western blot. Each lane from individual mouse, and representative of two independent experiments.

H. Relative luciferase activity in HeLa transfected with plasmid containing WT BTAF1 3'UTR (white) or BTAF1 3'UTR on which the miR-132 binding site is mutated (grey) downstream of renilla luciferase, in the presence of miR-132-3p or miR-212-3p mimics. Error bars indicate SEM from eight replicate treatments.

Data information: Significance in (F) and (H) determined by unpaired t-test. * p<0.05, ** p<0.01.
miR-132 suppresses transcription of ribosomal proteins to promote protective Th1 immunity (2019)
Figure 3

Figure 3: miR-132 and its targets p300 and BTAF1 control RP expression.

A. mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with Non-targeting control (NTC) mimics (white) or miR-132-3p mimics (grey).

B. p300 and BTAF1 protein levels in MEF transfected with NTC mimics or miR-132-3p mimics determined by Western blot. GAPDH was used as a loading control. Right panel indicates mean + SEM of 4 experiments.

C. mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC siRNA (white) or p300 siRNA (grey).

D. mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC siRNA (white) or BTAF1 siRNA (grey).

E. mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC or miR-132-3p mimics and NTC siRNA or p300 or BTAF1 siRNAs for 48h. Levels are normalised to cells transfected with NTC siRNA and NTC mimic.

F. Puromycin incorporation (following 10-minute pulse and 50-minute chase) determined by western blot in MEFs transfected with NTC or miR-132-3p or miR-212-3p mimics.

Data information: Statistical significance is determined by unpaired t-test from 4-6 experiments. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
miR-132 suppresses transcription of ribosomal proteins to promote protective Th1 immunity (2019)
Figure 4

Figure 4: miR-132 controls the balance between IL-10 and IFNγ production in CD4+ T cells.

A. Percentage of IFNγ+ live TCRβ+ CD4+ cells from L. donovani infected WT (blue) or miR-132-/- (red) mice, determined by intracellular cytokine staining. Data representative of 3 independent experiments with 3-5 mice per group.

B. Percentage of IFNγ+/IL-10+ live TCRβ+ CD4+ cells from L. donovani infected WT (blue) or miR-132-/- (red) mice, determined by intracellular cytokine staining. Data representative of 3 independent experiments with 3-5 mice per group.

C. IL-10 mRNA levels, determined by RNA-sequencing, in TCRβ+ CD4+ cells purified from spleens of L. donovani infected WT (blue) or miR-132-/- (red) mice (n=5 per group).

D. Percentage of IFNγ+ WT (blue) or miR-132-/- (red) in vitro polarised Th1 cells (6 days) in the presence or absence of phenylephrine (PE), determined by intracellular cytokine staining.

E. Percentage of IL10+ WT (blue) or miR-132-/- (red) in vitro polarised Th1 cells (6 days) in the presence or absence of phenylephrine (PE), determined by intracellular cytokine staining.

F. Total cell counts following in vitro Th1 polarisation (6 days) in the presence or absence of phenylephrine (PE). For (D-E), cells were purified from 3 mice per group and 6 replicates performed.

Data information: For (A and B) statistical significance was determined by unpaired t-test. For (D-F), significance was determined with 1-way ANOVA followed by Bonferroni's multiple comparison test. * p<0.05, ** p<0.01, *** p<0.001. NS: not significant.
miR-132 suppresses transcription of ribosomal proteins to promote protective Th1 immunity (2019)
Figure 5

Figure 5: miR-132 promotes protective immunity to L. donovani.

Liver LDU (Leishman Donovan units) at day 28 in infected WT mice treated with anti-IL-10R antibody or isotype control antibody (left panel, n=5 mice per group), or at day 21 and day 28 from WT (blue), IL-10+/- (open green circles) and IL-10-/- (filled green circles) mice (right panel n= 3-6 mice per group)

Day 28 splenic parasite burdens expressed as LDU with each data point representing an individual mouse in WT (blue) and miR-132-/- (miR-132-/-; red) mice. Data from 4 independent infection experiments.

Mean WT and miR-132-/- spleen parasite burdens from the 4 independent experiments shown in (B). Lines link individual experiments.

Splenic parasite burdens relative to WT group (WT mean = 1) for each of the 4 experiments shown in (B), with each data point representing individual mouse.

Spleen size expressed as % body weight for d0 (naïve) or day 28 L. donovani infected WT (blue) and miR-132-/- (miR-132-/-; red) mice.

Liver size expressed as % body weight for d0 (naïve) or day 28 L. donovani infected WT (blue) and miR-132-/- (red) mice.

Data information: Significance determined by unpaired t-test, and in (C) by paired t-test of mean values. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

EXPANDED VIEW FIGURE LEGENDS
Ribonuclease H2 mutations induce a cGAS/STING-dependent innate immune response (2016)
Figure 1

Figure 1. RNase H2 complex levels and enzymatic activity are reduced in Rnaseh2bA174T/A174T mouse and RNASEH2BA177T/A177T AGS patient cells

A. Targeted mutagenesis of the Rnaseh2b gene. Top: A 7 kb region of the Rnaseh2b genomic locus; black boxes, exons 6 (ex6) and 7 (ex7). Middle: NotI/SalI restriction fragment of the targeting construct, comprising 4.5 kb of genomic DNA and a Neomycin selection cassette (Neo) flanked by loxP sites (triangles). (Bottom) Targeted locus containing exon 7 with the c.520G>A mutation (ex7*). Red arrowheads, primers used to confirm correct targeting. Red bar, 400 bp probe for Southern blotting.

B. Southern blotting confirms successful targeting. Introduction of an additional EcoRI site results in a 4.1 kb restriction fragment detectable by Southern for targeted ES cells (A174T/+) but not for parental DNA (+/+).

C. Capillary sequencing for Rnaseh2b+/+, Rnaseh2bA174T/+, and Rnaseh2bA174T/A174T DNA confirmed the presence of the introduced missense mutation.

D. Mouse genotyping by multiplex PCR. Top: A 221 bp PCR product is present in wild-type mice (+/+); the Rnaseh2bA174T allele (also) give a 460 bp product. Bottom: Position of forward (x) and reverse primers (y, z).

E. Immunoblotting demonstrates depletion of all three RNase H2 protein subunits in Rnaseh2bA174T/A174T MEFs and RNASEH2BA177T/A177T LCLs. Representative of three independent experiments.

F. Schematic showing enzyme activities attributed to RNase H1 and RNase H2 (DNA blue, RNA red),

G, H. RNase H2 enzyme activity is reduced in mouse and patient cells. (G) Enzyme activity for Rnaseh2bA174T/A174T MEFs and passage-matched Rnaseh2b+/+ controls, against RNase H substrate (RNA:DNA heteroduplex) and RNase H2-specific substrate, double stranded DNA with a single embedded ribonucleotide (DRD:DNA). Mean activity for three independent cell lines, error bars, SEM. Enzymatic activity expressed relative to the average value of control MEFs. *** = p<0.001, two-tailed t-test (n=3 Rnaseh2bA174T/A174T and n=3 Rnaseh2b+/+ control MEF lines). (H) RNase H2 activity in LCLs from two independent healthy controls and an AGS patient homozygous for the RNASEH2B-A177T mutation. Enzyme activity normalised to average activity of control lines. Three independent experiments, error bars SEM. *** = p<0.001 versus either control, two-tailed t-test.
Ribonuclease H2 mutations induce a cGAS/STING-dependent innate immune response (2016)
Figure 2

Figure 2. Increased ISG expression in tissues from Rnaseh2bA174T/A174T mice

A. Transcript levels of multiple ISGs are significantly elevated in heart.

B. Transcript levels of a subset of ISGs are significantly increased in kidney.

C. No ISG upregulation is evident in the brain. ISG transcript levels determined by RT-qPCR normalised to transcript levels of the housekeeping gene HPRT (Oas1a was undetectable in brain). Each data point represents the mean of technical replicates of tissue RNA from a single mouse. n=9 nine-month old Rnaseh2bA174T/A174T mice, n=4 age-matched control wildtype C57BL/6J mice. Horizontal line, mean; error bars, SEM. * = p<0.05, ** = p<0.01, two-tailed t-test.
Ribonuclease H2 mutations induce a cGAS/STING-dependent innate immune response (2016)
Figure 3

Figure 3. A proinflammatory response is detectable in vitro in Rnaseh2b-/- MEFs

Rnaseh2b-/- MEFs have significantly elevated ISG transcript levels and secrete the proinflammatory cytokines CXCL10 and CCL5.

A. Validation of Rnaseh2b-/- MEF lines. RER activity (DRD:DNA) is undetectable in a Rnaseh2b-/- line consistent with complete inactivation of the Rnaseh2b gene (Reijns et al, 2012). Mean of three independent experiments; error bars, SEM, **** = p<0.0001, two-tailed t-test. Rnaseh2b-/- and Rnaseh2b+/+ control MEFs on a C57BL/6 p53-/- background.

B. ISG transcript levels are increased in Rnaseh2b-/- MEFs.

C. BeadArray transcript analysis (Illumina) detected induction of multiple ISGs, including the cytokines CXCL10 and CCL5, but not other cytokine transcripts in Rnaseh2b-/- MEFs. Plotted, average fold enrichment versus p-value of significantly upregulated transcripts (p<0.05, after multiple testing correction) comparing two Rnaseh2b-/- MEF lines versus 4 Rnaseh2b+/+ MEF lines. 17 out of 29 transcripts are ISGs.

D. CXCL10 and CCL5 transcripts (detected by RT-qPCR) are significantly elevated.

E. Increased CXCL10 and CCL5 protein (detected by ELISA) is secreted by Rnaseh2b-/- MEFs. (B, D, E) Mean data from three experiments for six independent Rnaseh2b-/- MEF lines versus four independent Rnaseh2b+/+ MEF lines. Error bars, SEM. * = p<0.05, ** = p<0.01, Mann-Whitney U test.
Ribonuclease H2 mutations induce a cGAS/STING-dependent innate immune response (2016)
Figure 4

Figure 4. ISG induction in RNase H2 deficiency is dependent on the cGAS-STING nucleic acid sensing pathway

A. ISG activation and cytokine secretion in Rnaseh2b-/- MEFs is markedly impaired by cGAS or STING siRNA depletion. Upper left and lower panels: RT-qPCR of IFIT1, cGAS and STING transcripts, 48 h after siRNA targeting luciferase (control), cGAS or STING. Upper right panel: CCL5 is significantly reduced in culture supernatants 48 h after cGAS or STING depletion. Concentration of CCL5 (ELISA), normalised to luciferase siRNA control levels in each experiment. Mean from three independent experiments using one Rnaseh2b-/- MEF line; error bars, SEM. * = p<0.05, ** = p<0.01, two-tailed t-test for RT-qPCR, one sample t-test for CCL5 ELISA.

B-D. ISG induction and cytokine secretion is abolished in Rnaseh2b-/- cGAS-/- MEFs. cGAS was targeted by CRISPR/Cas9 genome editing of a Rnaseh2b-/- MEF line to inactivate cGAS/STING signalling. In addition to sequence validation, functional inactivation of cGAS was confirmed in Rnaseh2b-/- cGas-/- CRISPR lines by the absence of CCL5 secretion in response to dsDNA (Appendix Fig S4). CCL5 (B) and CXCL10 (C) production, as well as ISG expression (D) was abrogated in Rnaseh2b-/- cGAS-/- clones, assessed by ELISA and RT-qPCR respectively. Four independent experiments, n=2 Rnaseh2b-/- cGAS-/- clones, n=4 Rnaseh2b-/- cGAS+/+ clones, error bars, SEM of each experiment * = p<0.05, ** = p<0.01, *** = p<0.001, two-tailed t-test.

E. ISG induction in Rnaseh2bA174T/A174T mice is STING dependent. RT-qPCR of RNA extracted from hearts from Sting+/+ Rnaseh2bA174T/A174T (n=4) and Sting-/-Rnaseh2bA174T/A174T (n=6) three-month old mice.

F. Absence of STING does not significantly decrease basal ISG expression. RT-qPCR of RNA extracted from hearts from Sting+/+ (n=3) and Sting-/- (n=5) three-month old mice. (E, F) Each data point represents the mean of technical triplicates from one mouse. Error bars, SEM. * = p<0.05, Mann-Whitney U test.
Ribonuclease H2 mutations induce a cGAS/STING-dependent innate immune response (2016)
Figure 5

Figure 5. Cellular RER and not enzyme activity against RNA:DNA hybrids correlates with DNA damage and proinflammatory response

A. Overexpression of RNase H1 in Rnaseh2b-/- cells restores RNase H activity against RNA:DNA hybrids to 81 ± 10% of wildtype levels, while overexpression of RNase H2B restores cellular enzyme activity for cleavage of both RNA:DNA and DRD:DNA substrates (RER). Rnaseh2b-/- MEFs were complemented with Rnaseh1 (+H1), Rnaseh2b (+H2B) or EGFP by retroviral infection. Mean of n=3 independent experiments ± SEM.

B, C. DNA damage is reduced to wildtype levels by complementation with Rnaseh2b but not Rnaseh1, measured by 53BP1 foci formation in detergent-extracted fixed cells. (B) Representative images (scale bar, 10 µm). (C) At least 150 cells were counted for each cell line in three independent experiments. Mean ± SEM, **** = p<0.0001 two-tailed t-test.

D-F. CCL5 (D) and CXCL10 production (E), as well as ISG induction (F) in Rnaseh2b-/- MEFs are reduced close to wildtype levels (Rnaseh2b+/+), by complementation with Rnaseh2b but not Rnaseh1. Mean of n=6 independent experiments ± SEM for complemented cells; n=3 independent experiments for Rnaseh2b-/- parental and Rnaseh2b+/+ controls cells. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001 two-tailed t-test indicates significantly reduced expression compared to Rnaseh2b-/- parental cells.
Neutrophils impose strong immune pressure against PfEMP1 variants implicated in cerebral malaria (2022)
Figure 1

Figure 1. Human neutrophils interact with and phagocytose P. falciparum-infected red blood cells

(A). Human neutrophils stained for CD66b after incubation with RBCs infected with GFP+ P. falciparum parasites (white arrows). Nuclei were stained with DAPI (blue), neutrophils were stained with CD66b (red), GFP expressing parasites (green). Scale bar, 5 µm. The upper and lower panel show two different cells.

(B). Giemsa staining of freshly isolated human neutrophils from a healthy donor incubated with iRBCs harboring late stages P. falciparum parasites (black arrows). Scale bar, 10 µm. (C). Flow cytometry analysis of human neutrophils incubated with opsonized or non-opsonized GFP+-late-stage iRBCs. Free neutrophils were used as control.

(D). Quantification of GFP+ iRBCs phagocytosed by neutrophils. The phagocytosis was analysed by flow cytometry (FACS). Data information: Results represent the average of three biological replicates (n=3) ± standard error of the mean (SEM). Statistical significance was determined using student t test * p ˂0.05.
Neutrophils impose strong immune pressure against PfEMP1 variants implicated in cerebral malaria (2022)
Figure 2

Figure 2. Killing of P. falciparum-infected red blood cells by human neutrophils

(A). Interaction between neutrophils and opsonized (red) or non-opsonized (blue) GFP+ iRBCs by time in a "pulse-chase" experiment. The percentage of GFP+-neutrophils following 5 min of co-culture was set as 100%.

(B). Model of how a low number of GFP+ neutrophils may be explained by the escape of GFP+ iRBCs (upper part) and how a high number of GFP+ neutrophils might be explained by the elimination (phagocytosis or extracellular killing) of GFP+ iRBCs (lower part) by the neutrophils. The escape of GFP+ iRBCs will allow parasites to propagation, while elimination will impair parasite propagation and cause reduced parasitemia of the culture.

(C). Parasitemia (percentage %) of NF54 iRBCs cultures with or without daily addition of freshly isolated neutrophils.

(D). Parasitemia (percentage %) of cultures of RBC infected by different P. falciparum lines (NF54, Dd2, SL, PfEMP1-KO). The parasites were kept in culture for six days in the presence (Neut.) or absence (Cont.) of neutrophils.

(E). Percentage change in parasitemia following neutrophil challenge of RBCs infected by different parasite lines (NF54, Dd2, SL, PfEMP1-KO).

(F). Flow cytometric analysis of the effect of neutrophil challenge on parasite cell cycle progression. Neutrophils were stained with an anti-CD11b antibody, while iRBCs were labeled with SYBR green. Gating for iRBCs were performed by gating for the SYBR green and CD11b negative population, excluding the neutrophils that were SYBR green negative, but CD11b positive.

(G). Short term neutrophil killing of late-stage NF54-luciferase positive RBCs in the presence (red) or absence (blue) of catalase.

(H). Parasitemia of cultures of iRBCs challenged with neutrophils in the presence or absence of catalase.

(I). Short-term luciferase-based killing assay using neutrophils from three different donors in the presence or absence of 5 uM cytochalasin D.

Data information: Results represents the average of 3 biological replicates (n=3) ± standard error of the mean (SEM). Statistical significance was determined using student t test * = p˂0.05; ** = p<0.01.
Neutrophils impose strong immune pressure against PfEMP1 variants implicated in cerebral malaria (2022)
Figure 3

Figure 3. PfEMP1 is the major ligand mediating the interaction and killing of iRBCs by human neutrophils

(A). Representative flow cytometric analysis of human neutrophils cultured alone (Control), incubated with late-stage NF54 parasites (wild type) or PfEMP1 Knock out (PfEMP1 KO) GFP+ iRBC.

(B). Flow cytometric quantification of the effect of trypsin treatment on neutrophil interaction with wild type and PfEMP1 KO GFP+ iRBC.

(C). Luciferase-based killing assay and neutrophil elimination of late-stage wild type (wt) and PfEMP1ko iRBC.

Data information: Results represent the average of 3 biological replicates (n=3) ± standard error of the mean (SEM). Statistical significance was determined using student t test ** = p<0.01.
Ferroportin‐mediated mobilization of ferritin iron precedes ferritin degradation by the proteasome (2006)
Figure 1

Fpn‐mediated decrease in ferritin is not prevented by chloroquine or leupeptin. (A) HEK293T‐Fpn cells were incubated with FAC (10 μM Fe) for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A. Cells were then incubated with and without 100 μM chloroquine or 10 μM leupeptin for 10 h and harvested. The ferritin content was determined by ELISA. Induction of Fpn resulted in decreased ferritin levels and this was not prevented by treatment with chloroquine or leupeptin. The data are presented as the standard deviation from three different experiments. (B) Cells treated as in (A) were incubated in the presence of 1 μg/ml EGF for 2 h. Cells were fixed and processed for immunofluorescence using mouse anti‐EGF receptor and Alexa 594 conjugated goat anti‐mouse IgG. Chloroquine and leupeptin effectively inhibit degradation of EGF receptor.