Fig 1: Baricitinib inhibits cytokine-mediated increased infectivity of SARS-CoV-2 in organotypic primary human liver culture.(A) Immunofluorescence confocal imaging of a liver spheroid 48 hours after infection with SARS-CoV-2. Viral spike protein is shown in red, ACE2 in green, and DAPI (4′,6-diamidino-2-phenylindole) in blue. Arrows indicate examples of where spike protein and ACE2 signals are in close proximity. (B) Liver spheroids were treated with different cytokines (10 ng/ml), and the fold increase in ACE2 transcript levels are shown relative to controls (indicated by the solid line). Note that IFN-α2 and IFN-β significantly induce ACE2 levels. N = 2 technical replicates. (C) Combinatorial cytokine exposure does not result in increased ACE2 induction compared to IFN-α2 alone. “Other cytokines” corresponds to IFN-β, IFN-γ, TNFα, IL-1β, IL-6, IL-10, and IL-18. (D) IFN-α2 increases viral load in hepatocyte spheroids, and this effect is fully inhibited by baricitinib. N = 2 to 3 biological replicates. (E) IFN-α2–mediated induction of ACE2 is fully prevented by baricitinib. N = 3 biological replicates. All cytokine concentrations were 10 ng/ml unless stated otherwise. (F) ACE2 in lung organoids is not induced even by very high concentrations of IFN-α2 (50 ng/ml). N = 3 biological replicates. (G) By contrast, IFN-α2 slightly reduces viral load in lung organoids. N = 3 biological replicates. Error bars indicate SEM. A.U., arbitrary units; DMSO, dimethyl sulfoxide.
Fig 2: CD56-CD16+ NK cells are enriched in the NK cell compartment of KSHV-infected huNSG mice, as well as in Kenyan KSHV-sero-positive children(A) Representative flow cytometry dot plots of human CD45+NKp46+Lin- splenocytes of EBVzko and EBVzko+KSHV mice.(B) Frequency of peripheral blood CD56-and CD16-stained NK cell subsets over time in mock (n = 5), EBVzko (n = 5), and EBVzko+KSHV (n = 6) mice. REML followed by Fisher’s LSD test, q values, mean ± SEM. (C and D) Frequency of CD56-and CD16-stained NK cell subsets among splenic (C) or hepatic (D) CD45+CD3-NKp46+ cells of mice from four and two independent experiments, respectively, relative to the mean of the respective EBVzko group. Mean ± SEM, MWU.(E) Correlations between splenic KSHV DNA load and the frequency of CD56+CD16- (red) and CD56-CD16+ (yellow) NK cells are depicted for EBVzko+KSHV co-infected mice from four independent experiments. Solid lines represent trend lines obtained by linear regression, and shaded areas indicate 95% confidence interval (CI) of each trend line. Mice with values below 0.1 ORF26 copies per 106 cells were excluded.(F) Cytokine concentration was measured in the serum of EBVzko (n = 15) and EBVzko+KSHV (n = 16) mice at 4 weeks p.i. The median (IQR) is shown with minimum and maximum ranges as whiskers. Composite data are from four independent experiments, MWU. Dashed lines indicate lower level of quantification (LLOQ). (G) Frequency of CD56-CD16+ NK cells in KSHV-seropositive and KSHV-negative (KSHVab + and -) pediatric healthy donors (HDs). Mean ± SD. MWU.(H) Correlation between CD56-CD16+ NK cell frequencies and serum IL-18 in pediatric HDs and eBL patients.*p < 0.05, **p < 0.01, ***p < 0.001. rS, Spearman’s correlation. See also Figure S1.
Fig 3: SIOs promote ILCP stemness and maturation(A) Representative flow contour plots of ILCs yielded from ILCP + SIO or ILCP only in Matrigel (“NEG” black = ILCP + SIO; “NEG” magenta = ILCP without SIO).(B) Relative frequency of a4ß7+PD-1+CD25+c-KIT+ of all Lineage- cells.(C) Relative frequency of ROR?t-CCR6-NK1.1-NKp46-Klrg1-ST2- (NEG) within the Lineage- population, count of NEG, % of CD25+, c-KIT+, a4ß7+, and PD-1+ within the negative population (frequency of parent, SD).(D) Frequency and count of ILC1 (Live, EpCAM-, CD45+, Lin-, ROR?t-, Klrg1-, NK1.1+, NKp46+, Eomes-, T-bet+) and NK cells (Live, EpCAM-, CD45+, Lin-, ROR?t-, NK1.1+, NKp46+, T-bet+, Eomes+) depicted in (E) derived from PD-1+ ILCP co-cultures +/- SIO and compared with putative ILC3 (Live EpCAM-CD45+Lin-ROR?t+Klrg1-NK1.1+/-NKp46+/-Eomes-T-bet+/-) and ILC2 (Live EpCAM-CD45+Lin-ROR?t-Klrg1+NK1.1-NKp46-Eomes-T-bet-) (N = 7 animals, two experiments).(F) Frequency of ILC1 and NK cells expressing IFN-? (FMO, blue) in (E) and granzyme B after 4-h stimulation with PMA/Ionomycin and IL-18 compared with SI-LP ILC1s and NK cells depicted in (G).(H) Frequency and count of ILC3 (Live EpCAM-CD45+Lin-NK1.1+/-NKp46+/-Klrg1-ST2-ROR?t+CCR6-) and CCR6+ LTi-like cells (Live EpCAM-CD45+Lin-NK1.1-NKp46-Klrg1-ST2-ROR?t+CCR6+) depicted in (I) derived from PD-1+ ILCP co-cultures +/- SIOs and compared with primary ILC3s (N = 5 animals, two experiments).(J) Frequency of ILC3 and CCR6+ LTi-like cells expressing IL-22 (FMO, blue) and IL-17A (FMO, magenta) in (I) after 4-h stimulation with PMA/Ionomycin and IL-23 when compared with SI-LP ILC3 and Lti depicted in (K).Error bars represent SEM; p values are from unpaired Student’s t tests.
Fig 4: MAP4K1 enhances the expression of IL-18R and IL-6R and promotes the proliferation of glioblastoma multiforme cells.(A) Real-time quantitative PCR analyses of IL-18R and IL-6R mRNA levels in MAP4K1-knockdown (KD) U87 and T98G cells and their negative control (NC) cells (n = 9, the data were from three independent experiments). (B) IL-18R and IL-6R protein expression detected by immunohistochemistry in mouse glioma tissues constructed by MAP4K1-KD (n = 3) and NC (n = 8) of U87 cells. Scale bar, 50 μm. (C, D, E) Respective flow cytometry histograms and relative levels of the mean fluorescence intensity for membrane-bound IL-18R in MAP4K1-KD or knockout (MAP4K1−/−) U87, T98G, and respective control cells 48 h after seeding (n = 3, the data were from an independent experiment, and the same experiment was repeated three times). (F, G, H) CCK8 assay of cell viability. The data were from three independent experiments. (F) MAP4K1-KD and NC cells were stimulated with IL-18 (100 ng/ml) and detected at different time points (24, 48, and 72 h) (n = 11). (G) MAP4K1−/− and MAP4K1+/+ T98G cells were treated with different doses of IL-18 (0, 50, and 100 ng/ml). (H) IL-18R was blocked with a neutralizing antibody (0, 1, 5 μg/ml). (G, H) Cell viability was determined at 96 h (n = 15 in (G, H)). In (A, C, D, E, F, G, H), data are presented as the mean ± SD. In (A, C, D, E), the data were analyzed by t tests. In (F, G, H), data were analyzed by two-way ANOVA. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig 5: Calhm6 −/− macrophages and NK cells are capable of normal responses to activating signals when stimulated in isolation, in vitro A, B WT and Calhm6 −/− mice were injected i.p. with Poly(I:C) (200 μg/mouse) or PBS. On day 3 spleens were collected, stained with antibodies against NK cell‐maturation markers and analysed by flow cytometry. Representative contour plots (A) and pooled flow cytometry results (B) are shown (results from one experiment, Poly(I:C) WT mice = 5, Calhm6 −/− mice = 5, control WT mice = 2, Calhm6 −/− mice = 2 mice, One‐way ANOVA with multiple comparisons).C CD3−NK1.1+ cells were isolated from naïve mouse spleens and stimulated in vitro with PMA/ionomycin, IL‐12 (50 ng/ml), TNF (50 ng/ml), or IL‐18 (50 ng/ml). After 6 h stimulation, supernatant was collected and IFN‐γ was measured by ELISA (pooled results from two independent experiments, Untreated and PMA stimulation WT mice = 6, Calhm6 −/− mice = 6, TNF + IL‐12 stimulation: WT mice = 3, Calhm6 −/− mice = 3, otherwise WT mice = 3, Calhm6 −/− mice = 2, One‐way ANOVA with multiple comparisons).D, E BMDM with or without 24 h IFN‐γ priming were grown in antibiotic‐free medium, washed and cultured with Listeria monocytogenes (MOI 1) for 30 min, at which time gentamicin (5 ng/ml) was added to the medium to kill extracellular bacteria. Supernatant from BMDM infected with L. monocytogenes was collected after 6 h. IL‐1β (D, pooled results from five independent experiments, WT mice = 5, Calhm6 −/− mice = 5, Kruskal–Wallis test) and IL‐18 concentration (E, pooled results from three independent experiments, WT mice = 3, Calhm6 −/− mice = 3, Kruskal–Wallis test) were quantified by ELISA, and normalised to WT set to 100%, to allow pooling of independent experiments.F NO production in WT and Calhm6 −/− BMDM treated overnight with LPS or Poly(I:C) quantified with a Griess test on cell supernatant (results from one experiment, WT mice = 3, Calhm6 −/− mice = 3, Kruskal–Wallis test with multiple comparisons).G BMDM with or without IFN‐γ priming were grown overnight in antibiotic‐free medium. Cells were infected with L. monocytogenes (MOI 1) for 30 min. Gentamicin (5 ng/ml) was then added to the medium to kill extracellular bacteria. At 0, 4 and 8 h cells were harvested, lysed with 0.1% Triton X‐100 and the lysate was plated on antibiotic‐free BHI plates for 24 h, and CFU were quantified (pooled results from three independent experiments, WT mice = 3, Calhm6 −/− mice = 3, Two‐way ANOVA test). Data information: Error bars represent SD. Source data are available online for this figure.
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