JAK inhibition reduces SARS-CoV-2 liver infectivity and modulates inflammatory responses to reduce morbidity and mortality

Abstract

Using AI, we identified baricitinib as having antiviral and anticytokine efficacy. We now show a 71% (95% CI 0.15 to 0.58) mortality benefit in 83 patients with moderate-severe SARS-CoV-2 pneumonia with few drug-induced adverse events, including a large elderly cohort (median age, 81 years). An additional 48 cases with mild-moderate pneumonia recovered uneventfully. Using organotypic 3D cultures of primary human liver cells, we demonstrate that interferon-α2 increases ACE2 expression and SARS-CoV-2 infectivity in parenchymal cells by greater than fivefold. RNA-seq reveals gene response signatures associated with platelet activation, fully inhibited by baricitinib. Using viral load quantifications and superresolution microscopy, we found that baricitinib exerts activity rapidly through the inhibition of host proteins (numb-associated kinases), uniquely among antivirals. This reveals mechanistic actions of a Janus kinase-1/2 inhibitor targeting viral entry, replication, and the cytokine storm and is associated with beneficial outcomes including in severely ill elderly patients, data that incentivize further randomized controlled trials.

Fig. 1. CONSORT flow diagram for University of Pisa and Albacete Hospital cohorts.

PS, propensity score.

Fig. 2. Kaplan-Meier analysis of the propensity score–matched cohorts from Pisa University and Albacete Hospital cohorts.

Fig. 3. 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. 4. Baricitinib reverses IFN-mediated gene expression signature alterations.

(A) Venn diagram depicting the overlap of differentially expressed genes upon IFN-α2 (10 ng/ml) in SARS-CoV-2–infected and uninfected liver spheroids. (B) Circle plots illustrating significantly deregulated genes falling into specific Reactome terminologies in infected and noninfected samples (FDR < 0.05). Circle diameter is indicative for the number of genes per category. (C) Heatmap representation of IFN-responsive genes (n = 832). z scores of normalized TPMs (transcripts per million mapped reads) are plotted (purple, high; white, low). (D) Volcano plot showing the differentially expressed genes in all infected samples upon treatment with baricitinib. Blue and red dots indicate genes that are significantly up- and down-regulated upon IFN treatment. Note that with the exception of SAA1/2, baricitinib results in inverse changes to gene expression compared to IFN, thus ameliorating IFN-induced gene expression alterations. (E) Heatmap visualization of genes for which significant effects on gene expression were detected for baricitinib and IFN.

Fig. 5. Baricitinib blocks viral entry of SARS-CoV-2.

Superresolution dSTORM microscopy of short-term (4 hours) infected liver spheroids stained for nucleocapsid treated with vehicle control (A) or baricitinib (100 nM) (B). (C) Relative mean fluorescence intensities (MFIs) for regions with dimensions of 20 μm by 20 μm of infected and treated organoids and secondary antibody–only controls; five regions per 3D tissue culture. Bars are means ± SD; ***P < 0.001 two-tailed Student’s t test. Ab, antibody; ns, not significant. (D) qPCR analysis of viral load in organotypic primary human liver culture following short-term (4 hours) infections corroborates inhibition of viral entry. (E) Suggested mechanism of dual baricitinib antiviral action on viral entry and inflammatory signaling. Baricitinib inhibits on viral entry by inhibition of the NAKs AAK1 and GAK. In addition, baricitinib blocks inflammatory JAK/STAT signaling, resulting in reduced expression of the IFN target gene and SARS-CoV-2 receptor ACE2. A plasmacytoid dendritic cell is shown on the left, and a hepatocyte is shown on the right.