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. Author manuscript; available in PMC: 2020 Dec 10.
Published in final edited form as: Cell Rep. 2020 Nov 17;33(7):108407. doi: 10.1016/j.celrep.2020.108407

JAK1 Inhibition Blocks Lethal Immune Hypersensitivity in a Mouse Model of Down Syndrome

Kathryn D Tuttle 1, Katherine A Waugh 1, Paula Araya 1, Ross Minter 1, David J Orlicky 2, Michael Ludwig 1, Zdenek Andrysik 1,3, Matthew A Burchill 4, Beth AJ Tamburini 4,5, Colin Sempeck 1, Keith Smith 1, Ross Granrath 1, Dayna Tracy 1, Jessica Baxter 1, Joaquin M Espinosa 1,2,7,*, Kelly D Sullivan 1,6,*
PMCID: PMC7727402  NIHMSID: NIHMS1647637  PMID: 33207208

SUMMARY

Individuals with Down syndrome (DS; trisomy 21) display hyperactivation of interferon (IFN) signaling and chronic inflammation, which could potentially be explained by the extra copy of four IFN receptor (IFNR) genes encoded on chromosome 21. However, the clinical effects of IFN hyperactivity in DS remain undefined. Here, we report that a commonly used mouse model of DS overexpresses IFNR genes and shows hypersensitivity to IFN ligands in diverse immune cell types. When treated repeatedly with a TLR3 agonist to induce chronic inflammation, these animals overexpress key IFN-stimulated genes, induce cytokine production, exhibit liver pathology, and undergo rapid weight loss. Importantly, the lethal immune hypersensitivity and cytokine production and the ensuing pathology are ameliorated by JAK1 inhibition. These results indicate that individuals with DS may experience harmful hyperinflammation upon IFN-inducing immune stimuli, as observed during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, pointing to JAK1 inhibition as a strategy to restore immune homeostasis in DS.

Graphical Abstract

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In Brief

Individuals with Down syndrome (DS) display hyperactivation of interferon (IFN) signaling and chronic inflammation. Using a mouse model of DS, Tuttle et al. demonstrate a dysregulated antiviral response associated with heightened inflammation, liver pathology, and lethal weight loss. JAK inhibitors counteract this immune hypersensitivity and provide therapeutic benefits in mice.

INTRODUCTION

Trisomy 21 (T21), the chromosomal condition that causes Down syndrome (DS), predisposes affected individuals to a wide range of comorbidities that shorten their life expectancy and diminish their quality of life. People with DS experience a unique disease spectrum (Antonarakis et al., 2020) because they have lower rates of certain conditions, such as most solid malignancies (Hasle et al., 2000, 2016), while being strongly predisposed to others, such as Alzheimer’s disease (AD) (Hartley et al., 2015; Wiseman et al., 2015), leukemias (Maloney et al., 2015), autoimmune disorders (Chen et al., 2007; Mårild et al., 2013), some respiratory infections (Beckhaus and Castro-Rodriguez, 2018), and a range of neurological conditions, including autism and epilepsy (Startin et al., 2020). With the exception of their increased risk of developing AD, which has been linked to the presence of the APP gene on chromosome 21 (chr21) (Wiseman et al., 2015), little is known about the mechanisms underlying this differential clinical profile.

Recently, we discovered that T21 causes consistent activation of the interferon (IFN) response (Sullivan et al., 2016), a key component of the innate immune system, likely because of the fact that four IFN receptors (IFNRs) are encoded on chr21. Expansion of this study to characterize the proteome, metabolome, and immune cell repertoire of people with DS demonstrated that T21 causes (1) changes in the circulating proteome, indicative of chronic autoinflammation with clear upregulation of IFN-inducible cytokines (Sullivan et al., 2017); (2) activation of the IFN-inducible kynurenine pathway, leading to elevated production of neurotoxic tryptophan catabolites (Powers et al., 2019); and (3) widespread hypersensitivity to IFN simulation across all branches of the human immune system (Araya et al., 2019; Waugh et al., 2019).

Notably, the profile of comorbidities experienced by individuals with DS resembles that observed in individuals with interferonopathies, a class of genetic disorders in which errors in virus sensing, protein processing, or negative regulation of IFN lead to overproduction of type I IFNs (Crow, 2011; Crow and Manel, 2015). Individuals affected by interferonopathies also experience delayed development, widespread autoimmunity, and cognitive impairment (Crow, 2011; Crow and Manel, 2015), raising the possibility that DS could be understood, in part, as an interferonopathy. However, it is unclear at this point how increased IFN signaling may contribute to the clinical and developmental hallmarks of DS. To advance knowledge in this area, we evaluated the effect of chronic inflammation in mouse models of DS.

Genes on human chr21 are distributed in mice across three syntenic regions located on murine chromosomes 10, 16, and 17, and mouse models carrying an extra copy of each of these regions have been generated (Gupta et al., 2016). Here we report results obtained during study of the Dp16(1)/Yey mouse strain (hereafter referred to as Dp16; Li et al., 2007), a widely used mouse model of DS carrying a segmental duplication of the syntenic region on murine chromosome 16, including the four IFNR genes (Ifnar1, Ifnar2, Ifngr2, and Il10rb). We also evaluated Dp(10)1Yey/+ and Dp(17)Yey/+ mice (hereafter referred to as Dp10 and Dp17; Herault et al., 2017), which carry duplications of the syntenic regions on murine chromosomes 10 and 17, respectively, but are disomic for Ifnrs. We found that only Dp16 mice overexpress IFNRs in diverse immune cell types and are more responsive to type I and type II IFNs. Furthermore, only Dp16 mice display lethal immune hypersensitivity upon challenge with the TLR3 agonist polyinosinic:polycytidylic acid (P(I:C)), along with overexpression of key IFN-stimulated genes (ISGs) in diverse tissues. Dp16 mice exhibit liver inflammation even in the absence of stimulation, and this inflammation is exacerbated in response to P(I:C). The lethal immune phenotype, associated cytokine response, and liver pathology were blocked by treatment with a small-molecule inhibitor of the JAK1 kinase. Last, therapeutic doses of the US Food and Drug Administration (FDA)-approved JAK1/2 inhibitor baricitinib rescued the lethal phenotype without overt immunosuppression. These results support the hypothesis that IFNR copy number promotes immune dysregulation in DS and suggest that Dp16 mice are a suitable model for understanding why individuals with DS are predisposed to a variety of autoimmune and autoinflammatory conditions. Our results also call for a more thorough investigation of how altered immune responses in individuals with DS affect their response to viral pathogens. People with DS may be more susceptible to develop severe pathology in infections where robust inflammatory responses are pathological; the most recent example being severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) respiratory infection, where stronger inflammatory signatures correlate with adverse outcomes in coronavirus disease 2019 (COVID-19) (Espinosa, 2020; Mehta et al., 2020).

RESULTS

Dp16 Mice Overexpress Ifnrs and Are Hypersensitive to IFNs

To model the effect of T21 on responses to chronic inflammation in mice, we employed the Dp16 mouse strain, which harbors triplication of a region syntenic to human chr21, including four Ifnrs (Li et al., 2007). Parallel analyses were done using Dp10 and Dp17 mice, which also have three copies of genes that are syntenic to chr21 but normal Ifnrs copy number (Herault et al., 2017). To define the relationship between gene dosage and IFNR protein expression, we evaluated surface expression of the four IFNRs encoded on murine chr16 in peripheral immune cells from all three strains of mice by flow cytometry (Figure S1A). Expression of IFNAR1 (type I receptor), IFNGR2 (type II receptor), and IL10RB (type III receptor) on the surface of CD45+ white blood cells was significantly higher in Dp16 mice, but not Dp10 or Dp17 animals, relative to wild-type (WT) littermates (Figure 1A). When different immune cell types were analyzed by flow cytometry, IFNAR1 expression was significantly higher in all Dp16 cell types tested, with expression of IFNGR2 and IL10RB also being elevated in numerous cell types (Figure 1B). This IFNR overexpression was not observed in any cell type tested for Dp10 mice, and Dp17 mice even showed lower expression of these receptors in several cell types (Figure S1B). These results are consistent with previous reports of mRNA overexpression for all Ifnrs in this gene cluster in various tissues of Dp16 mice (Aziz et al., 2018; Sullivan et al., 2016).

Figure 1. The Dp16 Mouse Strain Is Hypersensitive to IFN Stimulation.

Figure 1.

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(A) Flow cytometry analysis of IFNAR1, IFNGR2, and IL10RB cell surface expression on CD45+ white blood cells (WBCs) from the Dp10, Dp16, and Dp17 mouse strains; n = 5–9 animals per genotype. Data are presented as ratios of the geometric mean fluorescence intensity (RFI) of each IFNR on WBCs relative to WT littermates. Example histograms are shown in the top right corner, with light gray representing an isotype control antibody, dark gray/black representing values for WT littermates, and green representing values for Dp16.

(B) Volcano plots showing the fold change of IFNAR1, IFNGR2, and IL10RB surface expression levels (Dp16 versus WT) for 10 different immune cell types; n = 9 per genotype.

(C–E) Phosphorylation of STAT1 was analyzed via flow cytometry for nine cell types, using WBCs from Dp16 mice at baseline (C) or after simulation with IFN-α (10,000 U/mL, D) or IFN-γ (100 U/mL, E) for 30 min; n = 6–9 animals per genotype and condition.

Data are presented as ratios relative to WT littermates. All data are from at least two independent experiments and presented as mean ± SEM. Statistical significance was calculated using a Mann-Whitney test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S1.

To test whether upregulation of IFNRs conferred stronger responses to IFN ligands, we evaluated phosphorylation of STAT1 (pSTAT1) in WT and Dp16 blood samples by flow cytometry before and after stimulation with IFN-α and -γ. Before stimulation, we found that immune cells from Dp16 mice exhibited no significant differences in pSTAT1 basally (Figure 1C); however, all immune lineages examined responded more strongly to IFN-α, as defined by significantly higher levels of pSTAT1 relative to WT cells, except for bulk monocytes (Figure 1D). This widespread heightened response to IFN-α was not observed in Dp10 and Dp17 cells, although T cell subsets from the Dp17 strain exhibited more robust pSTAT1 compared with the WT (Figures S1C and S1D). Although Dp17 mice have only two copies of the Ifnr genes, this result suggests that some aspect of triplication of the region of chromosome 17 syntenic to human chr21 leads to differential regulation of type I IFN signaling specifically in T cells.

We next investigated the response to IFN-γ stimulation. Here we found that bulk monocytes, Ly6C+ monocytes, bulk T cells, CD4+ T cells, and CD8+ T cells from Dp16 mice responded more strongly to IFN-γ stimulation, whereas Ly6C− monocytes, B cells, DN (double-negative) T cells, and natural killer (NK) cells did not (Figure 1E). This heightened response to type II IFN simulation was not observed in Dp10 and Dp17 blood (Figures S1C and S1D). Overall, these results suggest that elevated expression of the Ifnrs in the Dp16 mouse strain confers increased responsiveness to type I and type II IFNs.

Dp16 Mice Display Lethal Responses to an IFN-Inducing TLR3 Agonist

Because Dp16 cells responded more strongly to IFN ligands, we next investigated the response of Dp16 mice to the TLR3 agonist P(I:C), an innate immune stimulus known to trigger an IFN response. First we measured levels of circulating IFN-α 6 h after the initial injection of P(I:C) (or sham injection). IFN-α was not detected in the plasma of sham-treated mice of either genotype; however, IFN-α was induced by P(I:C) in WT and Dp16 mice, with a trend toward a stronger response in Dp16 mice (Figure 2A). Elevated IFN ligand production in Dp16 mice is consistent with a predicted feedforward loop within type I IFN signaling in DS (Kirsammer and Crispino, 2016), which could potentially be explained by the fact that type I IFNRs and TLR3 are themselves IFN-inducible genes (see below). Next we elicited chronic systemic induction of IFN signaling in vivo by repeated administration of P(I:C). In WT C57BL/6J mice, this treatment regimen is known to produce an acute spike of type I IFN production (1–3 days), followed by low but persistent expression of type I IFN ligands and a robust cytokine response in the chronic phase (5–30 days) (Pietras et al., 2014). WT and Dp16 mice were given intraperitoneal injections of 10 mg/kg of P(I:C) or an equivalent volume of vehicle (sham injections) every other day for up to 16 days, with the experiment completed on day 17. Remarkably, Dp16 mice were profoundly sensitive to the treatment, which was largely tolerated by WT mice (Figure 2B). Body weight measurements during the course of the experiment revealed that Dp16 mice lost, on average, 5.3% of their body weight per dose of P(I:C) compared with 1.4% in WT controls (Figures 2C and S2A). Weight loss is often used as a metric for measuring severity of disease in infection models, and cachexia and anorexia have inflammatory components (Delano and Moldawer, 2006; Fung-Leung et al., 1991; Trammell and Toth, 2011). All but 1 of the 12 Dp16 mice had to be removed from the study prior to the pre-determined time point because of excessive weight loss (>15%). In contrast, WT animals did not lose as much weight, and 6 of 9 survived to the end of the experiment (Figures 2B and 2C). Notably, P(I:C)-induced weight loss was clearly dose dependent because mice receiving half the lethal dose (5 mg/kg) displayed only minor weight loss, with no significant differences between Dp16 and WT mice (Figure S2B). Last, Dp10 and Dp17 mice also tolerated chronic immune stimulation with P(I:C) (Figures S2C and S2D). Although P(I:C) treatment caused some weight loss in Dp10 and Dp17 mice, the percentage lost was comparable with WT levels (Figures S2E and S2F).

Figure 2. The Dp16 Mouse Strain Displays Lethal Immune Activation in Response to a Virus Mimetic TLR3 Agonist.

Figure 2.

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(A) Circulating levels of IFN-α were measured 6 h after a single P(I:C) (or sham) injection in WT versus Dp16 mice; n = 5–7 animals per genotype and condition.

(B) Kaplan-Meier analysis of Dp16 mice and WT littermate controls treated with 10 mg/kg P(I:C) every 48 h for 16 days; n = 6–12 animals per treatment and condition.

(C) Percentage of weight lost was normalized to the number of injections to compare the effects of each dose in WT and Dp16 mice; n = 6–11 animals per treatment and condition.

(D) Expression of select IFN-stimulated genes (ISGs) was measured by qRT-PCR using RNA prepared from livers (top), lungs (center), and hearts (bottom) of WT and Dp16 mice treated chronically with P(I:C) (or sham); n = 6–10 animals per treatment and condition. Expression was normalized to 18S rRNA.

Data are presented as mean ± SEM and are from at least two independent experiments. Statistical significance for Kaplan-Meier analysis was calculated using the Mantel-Cox log rank test. Statistical significance for weight loss and qRT-PCR was calculated using a Mann-Whitney test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S2.

IFN signaling drives expression of a specific gene expression program that is commonly used as a readout for the magnitude of the IFN response (Mostafavi et al., 2016). We measured mRNA expression for several ISGs in the liver, lungs, and heart of WT and Dp16 mice at the time of sacrifice. When we measured endpoint mRNA levels of several ISGs from the organs of WT and Dp16 mice, we observed not only the expected induction upon P(I:C) treatment relative to sham-treated animals for both genotypes but also several instances of increased baseline expression and/or overinduction in Dp16 mice (Figure 2D). Interestingly, we found that, in addition to being elevated at baseline, levels of Ifnar1 mRNA were more strongly induced by P(I:C) in Dp16 mice in all three tissue types. Furthermore, levels of Tlr3, the pattern recognition receptor activated by P(I:C), were elevated in P(I:C)-treated animals of both genotypes, with a trend toward greater induction in Dp16 mice. Levels of the canonical ISGs Mx1 and Eif2ak2 (Pkr) were also induced by P(I:C) in both genotypes, with greater induction in Dp16 mice in the lungs and heart. These data are consistent with increased IFN signaling in the numerous tissues of Dp16 mice in response to P(I:C) treatment.

Dp16 Mice Undergo Hyperinflammation upon Activation of Toll-like Receptor (TLR) Signaling

Because Dp16 mice exhibited more robust responses to IFN ligands and P(I:C), we sought to determine how systemic inflammation affects specific organs. We first examined the mRNA expression of inflammatory genes in WT and Dp16 mice treated chronically with P(I:C) in the liver, lungs, and heart. In all tissues, we observed induction of Ccl2 (encoding monocyte chemoattractant protein 1 [MCP-1]), Cxcl10 (encoding IFN-inducible protein 10 [IP-10]), Ifng (encoding the type II IFN ligand [IFN-γ]), and Tnf (encoding tumor necrosis factor alpha [TNF-α]) in WT and Dp16 mice in response to P(I:C) (Figure 3A), with trends toward greater induction of Ccl2, Cxcl10, and Tnf in Dp16 animals. In the lungs, Ccl2 and Cxcl10 were significantly more induced in Dp16 animals treated with P(I:C), whereas in the heart, Tnf was more strongly induced in Dp16 animals.

Figure 3. Characterization of Cytokine and Chemokine Responses during Chronic Activation of TLR Signaling.

Figure 3.

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(A) Expression of select cytokines and chemokines was measured by qRT-PCR using RNA prepared from livers (top), lungs (center), and hearts (bottom) of WT and Dp16 mice treated chronically with P(I:C) (or sham); n = 5–10 animals per treatment and condition. Expression was normalized to 18S rRNA.

(B) Levels of circulating cytokines were evaluated on day 7 of the chronic course of treatment in WT versus Dp16 mice.

Data are presented as mean ± SEM and are from at least two independent experiments. Statistical significance was calculated using a Mann-Whitney test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S3.

Next we measured the circulating levels of these cytokines and chemokines in the bloodstream on days 3 and 7 of the chronic treatment protocol after two and four P(I:C) injections, respectively. As expected, P(I:C)-treated animals showed increased levels of MCP-1, IP-10, IFN-γ, and TNF-α at both time points regardless of genotype (Figures 3B and S3). On average, Dp16 mice did not show significant differences in the levels of circulating cytokines relative to their WT littermates.

These results indicate that activation of TLR signaling leads to strong induction of cytokines and chemokines, with some of them being more elevated in Dp16 mice in specific tissues, which could potentially contribute to their exacerbated immune sensitivity. Because IFN production synergizes with TLR signaling to promote expression of pro-inflammatory cytokines, such as MCP-1, IP-10, and TNF-α, it is possible that the slight elevations in cytokine expression observed in Dp16 tissues are due to enhanced IFN sensitivity (Pietras et al., 2014).

Dp16 Mice Show Increased Liver Inflammation and Pathology

Next we investigated serum biomarkers of inflammation and injury that are not produced by immune cells. We found that serum levels of the enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST), two commonly used markers of hepatocyte injury (Cai et al., 2020; Huang et al., 2020; Zhang et al., 2020), were elevated significantly upon P(I:C) treatment in Dp16 mice, reaching concentrations nearly an order of magnitude higher than those observed in their WT littermates (Figure 4A). Prompted by these results, we investigated liver pathology. Liver tissue sections were stained with hematoxylin and eosin (H&E) and evaluated by a trained histologist blinded against treatment group and genotype. Scoring of liver pathology was done as described previously (Kleiner et al., 2005; Lanaspa et al., 2018), using a system that included the parameters cell injury, inflammation, and reactive changes, which were summed to assign an overall pathology score.

Figure 4. Dp16 Mice Display Increased Liver Inflammation and Pathology that Is Exacerbated by P(I:C) Treatment.

Figure 4.

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(A) Comparison of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the serum of WT and Dp16 mice treated with vehicle (sham) or 10 mg/kg of P(I:C) for up to 16 days; n = 6–9 animals per genotype and treatment.

(B) Representative images of H&E staining of liver sections from WT and Dp16 mice sham-treated or treated with P(I:C) as in (A). Arrows indicate the following structures: red, necrotic cells; blue, inflammatory infiltrates; green, apoptotic hepatocytes; purple, circulating apoptotic cells; yellow, ballooning hepatocytes; black, mitotic bodies. PT indicates the portal triad, and CV indicates the central vein. Scale bar = 100 μm.

(C) Metrics of liver pathology in Dp16 mice relative to their WT littermates; n = 10–12 animals per genotype and treatment.

Data are presented as mean ± SEM and are from at least two independent experiments. Statistical significance was calculated using a Mann-Whitney test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S4.

Dp16 mice have altered histopathology even before P(I:C) treatment, as demonstrated by significantly higher levels of cell injury, inflammation, and reactive changes relative to their WT littermates (Figures 4B and 4C). In particular, there is an increase in the number, diameter, and wall thickness of the hepatic arteries. Dp16 livers also have larger cross-sectional areas of the sinusoids that closely surround the portal triad. Furthermore, low-grade periportal inflammation is also present in Dp16 livers. Although the histopathology was significantly altered in Dp16 mice before P(I:C) treatment, the baseline levels of ALT and AST did not deviate from WT levels, suggesting that the liver damage is subclinical.

Upon P(I:C) treatment, all metrics of liver pathology increased in WT and Dp16 mice, with significantly higher overall pathology scores in Dp16 (Figures 4B and 4C). As observed previously for the weight loss phenotype, the effect of P(I:C) on increased liver pathology was dose dependent (Figure S4A). The livers from WT animals treated with P(I:C) developed a different pathology than what was observed at baseline in Dp16 mice, as evidenced by more inflammation in the periportal area, and extended in a limited fashion to the adjacent tissue. When Dp16 mice were treated with P(I:C), however, the inflammation was much more extensive and included apoptotic hepatocytes, necrotic cells, and ballooning hepatocytes, resulting in an increased overall damage score (Figure 4C and S4B). The presence of mitotic figures could be interpreted as compensatory cell division to repair the damage observed in Dp16 mice. Importantly, Dp10 and Dp17 animals did not show elevated liver pathology at baseline or after P(I:C) treatment relative to their WT littermates, indicating that the exacerbated liver pathology is unique to the Dp16 strain (Figure S4C).

Overall, these results indicate that Dp16 mice display increased liver pathology at baseline and upon immune activation, which could contribute to the poor health Dp16 mice experienced in response to P(I:C) treatment.

JAK1 Inhibition Blocks Lethal Responses to P(I:C)

All three types of IFN signaling employ the JAK1 kinase for signal transduction in combination with either JAK2 or TYK2 (Schwartz et al., 2016). Therefore, we sought to determine whether inhibiting JAK1 could attenuate the lethality and pathology caused by P(I:C) treatment in Dp16 mice. To this end, we used the JAK1-specific inhibitor INCB54707 (hereafter referred to as JAKi). Animals received a dose of 60 mg/ kg of JAKi (or an equivalent volume of methylcellulose vehicle) twice daily via oral gavage, beginning 24 h prior to the first P(I:C) injection, and every day during the course of the experiment. Remarkably, all Dp16 mice that received JAKi survived P(I:C) treatment (Figure 5A), whereas Dp16 animals that received vehicle and P(I:C) experienced weight loss and had to be euthanized (Figure 5B; see Figure S5A for additional controls).

Figure 5. JAK1 Inhibition Blocks Lethal Immune Responses, Cytokine Induction, and Increased Liver Pathology in Dp16 Mice.

Figure 5.

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(A) Kaplan-Meier analysis of Dp16 mice treated twice daily with INCB54707 (JAKi) or vehicle beginning 24 h prior to treatment with P(I:C) (10 mg/kg) every 48 h for 16 days; n = 3–6 animals per genotype and treatment.

(B) Weight loss was measured at the indicated time points for WT and Dp16 mice treated as described in (A).

(C) Levels of circulating cytokines and chemokines were evaluated at the time of sacrifice in WT and Dp16 mice treated with P(I:C) or sham treated; n = 5–7 animals per genotype and treatment.

(D) ALT and AST levels in the serum of WT and Dp16 mice treated as indicated; n = 3–7 animals per genotype and treatment.

(E) Representative images of H&E staining of liver sections from WT and Dp16 mice sham-treated or treated with P(I:C). Scale bar = 100 μm.

(F) Overall liver pathology scores for WT and Dp16 mice treated as indicated; n = 3–7 animals per genotype and treatment.

Data are presented as mean ± SEM and are from at least two independent experiments. Statistical significance for Kaplan-Meier analysis was calculated using the Mantel-Cox log rank test and by a Mann-Whitney test for all other results. *p < 0.05, **p < 0.01. See also Figure S5.

We then analyzed the effect of JAK1 inhibition on cytokine and chemokine production by comparing Dp16 mice treated with P(I:C) with and without co-treatment with JAKi. Serum levels of MCP-1, IP-10, IFN-γ, and TNF-α were clearly reduced by JAK1 inhibition (Figure 5C). Finally, we investigated whether JAK1 inhibition prevented development of liver inflammation and injury induced by P(I:C). Indeed, JAKi led to a reduction of circulating levels of ALT as well as trends toward reduction of AST (Figure 5D) and a decrease in overall liver pathology scores (Figures 5E and 5F). JAK1 inhibition alone had no effect on viability, liver pathology, or cytokine induction (Figures S5A-S5C).

These results indicate that blocking the catalytic activity of the JAK1 kinase prevents immune lethality in the Dp16 mouse model. The efficacy of this treatment likely relies on a combination of inter-related effects, which may include reduced cytokine production and improved liver function.

Therapeutic Treatment of Lethal Immune Hypersensitivity with Baricitinib

To further explore the pre-clinical value of JAK inhibition on the immune hypersensitivity phenotype, we tested the effect of administering the FDA-approved JAK1/2 inhibitor baricitinib after eliciting hyperinflammation in the Dp16 strain and before onset of terminal weight loss. Toward this end, we started the P(I:C) time course and, 24 h after the first injection, we treated animals with baricitinib at 10 mg/kg daily, a therapeutic dose commonly used to combat inflammation, including in a mouse model of rheumatoid arthritis (Fridman et al., 2010). We found that this “therapeutic treatment” strategy was also able to rescue the lethal weight loss observed in the Dp16 strain (>15% weight loss) (Figure 6A). When we examined weight loss over time, we found that, on day 1, after the first dose of P(I:C), Dp16 mice lost more weight than WT littermates (Figure 6B). Immediately after starting baricitinib treatment, WT animals displayed a remarkable benefit: weight loss in WT mice that received baricitinib was equivalent to sham-treated animals for the remainder of the experiment, whereas animals that received P(I:C) and vehicle continued to lose weight until weight loss plateaued around day 9. In Dp16 mice, the therapeutic effects of baricitinib took longer to manifest, but by day 5, the weight loss of Dp16 mice that received P(I:C) and baricitinib stabilized compared with animals that received P(I:C) and vehicle. This difference was most prominent at 5 and 7 days. After 7 days, the animals that lost the most weight, Dp16 mice treated with P(I:C) and vehicle, were removed from the study, and there was no longer a difference between Dp16 mice that received the drug or the ones that received vehicle. Thus, although this milder regime of immune modulation with baricitinib after onset of the inflammatory insult did not prevent Dp16 mice from losing more weight than their WT littermates, it nonetheless was beneficial enough to prevent their sacrifice at the terminal endpoint of 15% total weight loss.

Figure 6. JAK1/2 Inhibition Rescues Lethal Immune Responses and Cytokine Induction in Dp16 Mice.

Figure 6.

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(A) Kaplan-Meier analysis of Dp16 mice treated daily with baricitinib (Bari) or vehicle beginning 24 h after initial treatment with P(I:C) (10 mg/kg) every 48 h for 16 days; n = 6–10 animals per genotype and treatment.

(B) Weight loss was measured at the indicated time points for WT and Dp16 mice treated as described in (A).

(C) Evaluation of circulating cytokine and chemokine levels in WT and Dp16 mice administered Bari and/or P(I:C), as indicated, for 4 and 8 h; n = 3–6 animals per genotype and treatment.

Data are presented as mean ± SEM and are from at least two independent experiments. Statistical significance for Kaplan-Meier analysis was calculated using the Mantel-Cox log rank test and by a Mann-Whitney test for all other results. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S6.

Next we evaluated the effects of baricitinib treatment on cytokine production. Serum was harvested from animals at terminal endpoints, 18 h after the last dose of P(I:C). We found that, consistent with what we observed at 3 and 7 days (Figures 3B and S3), and in previous endpoint analyses (Figure 5C), there was no significant difference in circulating levels of pro-inflammatory factors in WT and Dp16 mice treated with P(I:C) (Figure S6A). Interestingly, in contrast to what we observed when JAK1 inhibitors were administered preventively, we found that therapeutic administration of baricitinib did not significantly decrease the amounts of pro-inflammatory markers in the blood in WT or Dp16 mice at these late time points (Figure S6A).

A previous study of baricitinib in rodent models of arthritis demonstrated that, despite effectively ameliorating histological disease severity, the effect of baricitinib on circulating levels of pSTAT3 was transient and no longer observed by 24 h after administration (Fridman et al., 2010). Therefore, to test whether baricitinib transiently suppressed cytokine production at earlier time points, we co-administered a single dose of P(I:C) and baricitinib and evaluated cytokine production at 4 and 8 h (Figure 6C). MCP-1 levels were significantly lower upon baricitinib treatment in WT animals at 4 h and in Dp16 mice at 8 h. IP-10 levels were also higher at 4 h than at 8 h for both genotypes; however, baricitinib treatment had only modest effects on the levels of IP-10 for either genotype. IFN-γ production was similar among WT and Dp16 animals at 4 and 8 h and inhibited significantly by baricitinib at these time points. TNF-α was induced to higher levels at 4 h, with levels decreasing substantially by 8 h in both genotypes; however, the effect of baricitinib on TNF-α levels was quite modest.

We next examined the effect of baricitinib on ISG expression in the livers of the chronically treated mice shown in Figure 6A and found that levels of Ifnar1, Tlr3, and Eif2ak2 were decreased modestly but not significantly in WT and Dp16 animals treated with p(I:C) and baricitinib relative to those treated with p(I:C) alone (Figure S6B). Mx1 expression was decreased by baricitinib only in WT animals. Finally, we performed histology on the livers from this experiment and found that, although baricitinib improved liver inflammation in WT mice, this was not the case for Dp16 animals at the histological level (Figure S6C). These data are consistent with the notion that transient inhibition of cytokine signaling is sufficient to rescue some inflammatory phenotypes, such as the wasting observed in Dp16 mice, but not all. This observation has potentially profound implications for the use of JAK inhibitors in individuals with DS during episodes of hyperinflammation, even those caused by pathogens, because it indicates that clinical benefits could be achieved without fully impairing immune function.

DISCUSSION

Here, we report that the Dp16 mouse model of DS, which carries triplication of four of the six IFNRs, is hypersensitive to innate immune activation. We demonstrate that type I, II, and III IFNRs are overexpressed on the surface of numerous cell types and that ex vivo stimulation of these cells with type I or type II IFNs results in increased levels of active STAT1. ISG expression is elevated in multiple tissues of Dp16 mice in response to a TLR3 agonist, suggesting that quantitative upregulation of the receptors and downstream signaling lead to stronger activation of antiviral and pro-inflammatory programs. We also show that induction of chronic inflammation via treatment with the viral mimetic P(I:C) results in rapid weight loss in Dp16 mice, significantly more than in WT animals or the two other models of DS, Dp10 and Dp17 mice. These effects could be explained by the fact that IFN hyperactivity is associated with anorexic and cachexic phenotypes during inflammation (Matthys et al., 1991). TNF-α, which is induced significantly in Dp16 animals treated with P(I:C), may also contribute to inflammatory weight loss (Espat et al., 1994). Dp16 mice also display exacerbated liver inflammation and pathology, which, together with stronger production of some cytokines, may converge into a pathological cascade leading to the wasting phenotype. Additional studies will be required to mechanistically link IFN hypersensitivity because of increased IFNR gene copy number with heightened systemic, multi-organ inflammation and broader immune hypersensitivity in the Dp16 mouse model of DS. These follow-up experiments will necessarily include crossing Dp16 mice with IFNR knockout (KO) models, such as Ifnar1−/−, to definitively establish the role of increased IFNR copy number in immune hypersensitivity and other phenotypes described here as well as bone marrow chimeras to define the role of the hematopoietic compartment in these processes.

The result that Dp16 mice have abnormal liver histopathology is, to our knowledge, the first report of this phenotype in animal models of DS. The unique histopathology in Dp16 mice suggests that there are structural and inflammatory changes to the liver in the absence of any experimentally induced immune activation. Thickening of the tunica media of the hepatic vessels combined with huge dilation of the peri-portal sinusoids suggests increased arterial pressure (Martinez-Quinones et al., 2018). The vascular aspect of liver pathology in Dp16 mice is fixed and largely unresponsive to P(I:C), suggesting that the underlying vascular pathology is unrelated to inflammation. Furthermore, although many pro-inflammatory cytokines that contribute to liver pathology are induced in WT and Dp16, a more comprehensive analysis of the molecular and cellular inflammatory phenotypes of the Dp16 liver is warranted to provide insight into the causes and consequences of the liver phenotype.

The finding of baseline hepatic inflammation and injury in Dp16 mice is potentially relevant to many aspects of liver biology in DS. Indeed, an increased incidence of fatty liver in individuals with DS has been documented previously (Adelekan et al., 2012; Roosen-Runge, 1947; Valentini et al., 2017). A recent study of 280 children with DS demonstrated that individuals with T21 are more likely to have non-alcoholic fatty liver disease (NAFLD), even in the absence metabolic syndrome (Valentini et al., 2017). Interestingly, lean NAFLD is often associated with inflammatory states and elevated levels of ALT (Das and Chowdhury, 2013). Liver failure also occurs in children with DS who experience myeloproliferative disorders, even after hematologic remission. In some cases, the liver disease is so severe that it is fatal, and poor outcome is associated with inflammatory markers (Bahr et al., 2020). The liver is also a site of induction of tolerance and a potential target for autoimmunity, and individuals with DS are more likely to develop autoimmune disorders such as autoimmune thyroid disease, celiac disease, and type I diabetes, and these conditions are linked to development of autoimmune hepatitis (AIH) (Zheng and Tian, 2019). Individuals with DS who develop AIH require longer immunosuppressive treatment and are likely to experience relapses compared with typical individuals (Goldacre et al., 2004). All of these pathologies should be considered in the context of higher baseline inflammation and detrimental inflammatory responses in the liver. Therefore, the Dp16 strain could be a good model for understanding whether and how inflammation contributes to more severe liver consequences in DS.

In this experimental paradigm, lethal immune hypersensitivity and the accompanying inflammation and cytokine production are blocked by administration of a small-molecule JAK1-specific inhibitor, INCB054707. These results demonstrate that, although TLR3 agonism induces several signaling cascades, including activation of IKK/nuclear factor κB (NF-κB) signaling, IKK/TBK1 signaling, and mitogen-activated protein kinase (MAPK) signaling (Kawasaki and Kawai, 2014), JAK1-dependent signaling is the major driver of the hyperinflammatory and wasting phenotypes in this setting. Ourfindings may be of critical significance to individuals with T21 because they point to dysregulated antiviral responses and identify therapeutic strategies for comorbidities more prevalent in this population, where IFN signaling could drive pathology, such as autoimmune skin conditions (Gholijani et al., 2020), arthropathy (Foley et al., 2019; Nehmar et al., 2018), and even AD (Mastrangelo et al., 2009; Roy et al., 2020; Taylor et al., 2014). The therapeutic potential of JAK inhibition in DS is already under investigation in a clinical trial (NCT04246372), and we showed recently that the JAK1/ JAK3 inhibitor tofacitinib has therapeutic benefits for alopecia areata in DS (Rachubinski et al., 2019).

In the context of the current COVID-19 pandemic, our results indicate that people with DS, who carry an extra copy of the four IFNRs encoded on chr21, should be considered a high-risk population for complications driven by SARS-CoV-2-induced hyperinflammation. As observed with RSV (Beckhaus and Castro-Rodriguez, 2018) and H1N1 (Pérez-Padilla et al., 2010) infection, people with DS are likely to develop more severe complications upon SARS-CoV-2 infection, including higher rates of hospitalization, mortality, and secondary bacterial infection (De Cauwer and Spaepen, 2020; Espinosa, 2020; Villani et al., 2020). A recent evaluation of 8 million medical records revealed a 4-fold higher rate of hospitalization and 10-fold higher rate of death for adults with DS affected by COVID-19 (Clift et al., 2020). We hope these results will encourage special attention for individuals with DS, including closer monitoring of hyperinflammation during COVID-19 and inclusion in clinical trials for immune-modulatory strategies. Finally, our data lend support to the notion that JAK inhibitors may be a valid therapeutic approach for treating a range of autoinflammatory conditions in individuals with DS.

STAR★METHODS

RESOURCE AVAILABILITY

Lead Contact

Further information and requests for resources and reagents should be directed and will be fulfilled by the Lead Contact, Joaquin Espinosa (joaquin.espinosa@cuanschutz.edu).

Materials Availability

This study did not generate new unique reagents.

Data and Code Availability

This study did not generate or analyze large datasets or code.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Study design

This study originated from our interest in understanding immune responses and IFN hyperactivity in mouse models of DS. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Colorado Anschutz Medical Campus. All molecular and cellular biology experiments were performed with validated reagents by trained personnel.

Mouse Models of Down syndrome

The Dp(10Prmt2-Pdxk)1Yey/J (Dp(10)1Yey/ +), Dp(16Lipi-Zbtb21)1Yey/J (Dp(16)1Yey/ + ), and Dp(17Abcg1-Rrp1b)1(Yey)/J (Dp(17) 1Yey/ +) strains have been previously described (Herault et al., 2017; Li et al., 2007; Yu et al., 2010). Dp10 and Dp17 mice were originally provided by Drs. Kathleen Gardiner and Santos Franco respectively, also located at the University of Colorado Anschutz Medical Campus. Dp16 mice were purchased from Jackson Laboratories or originally provided by Drs. Faycal Guedj and Diana Bianchi at the Eunice Kennedy Shriver National Institute of Child Health and Human Development. Animals were maintained on the C57BL/6J background and housed in specific pathogen-free conditions. All experiments used both male and female animals between 12-26 weeks old. In all cases, models of Down syndrome and their littermate controls were randomly assigned to experimental groups.

METHOD DETAILS

Chronic P(I:C) treatment

Vaccigrade P(I:C) (high molecular weight) was purchased from Invivogen and reconstituted in saline according to the manufacturer’s instructions. For chronic experiments, mice were injected with P(I:C) at 10 mg/kg of body weight at 2-day intervals for up to 16 days. Weight was monitored throughout the duration of the experiment. Animals were sacrificed one day after the final dose (day 17) or when they lost more than 15% of their body weight. For the shorter time course in Figure 6, mice were injected with P(I:C) at 10 mg/kg one time and animals were sacrificed at four and eight hours after injection.

JAK inhibitor treatment

INCB54707 (JAKi)

Mice were treated with 60 mg/kg of INCB54707 (or an equivalent volume of 0.5% methylcellulose vehicle) twice daily via oral gavage. Treatment was administered 24 hours prior to the first P(I:C) injection, and every day during the course of the experiment.

Baricitinib treatment

Mice were treated with 10 mg/kg of Baricitinib (or an equivalent volume of 0.5% methylcellulose vehicle) once a day via oral gavage. Treatment was administered 24 hours after the first P(I:C) injection, and every day during the course of the experiment.

IFN stimulations and intracellular staining

Peripheral blood was collected from the submandibular vein into Lithium Heparin tubes. 25 μL of blood were transferred to a 96-well plate, briefly subjected to ammonium chloride-potassium (ACK) RBC lysis buffer, and then re-suspended in 30 μL of media containing 10,000 units/mL of recombinant IFN-α2A or 100 units/mL of recombinant IFN-γ (R&D Systems). Antibodies against SiglecF, Ly6C, CD115, NK1.1 and CD11b (conjugated to methanol-stable fluorophores) and FC-receptors (to block non-specific surface staining) were also included in the stimulation media. These fluorophore-conjugated antibodies were included at this step because the fixatives required to detect phospho-STAT1 destroys their epitopes, preventing identification of several myeloid populations. Cells were stimulated for 30 minutes at 37°C, washed once in cold FACS buffer (0.5% BSA, 1xPBS, 2mM EDTA, 0.05% NaN3), then fixed in 200 μL of BD Lyse-Fix buffer for 10 minutes at 37°C. Cells were permeabilized for 30 minutes in ice-cold permeabilization buffer III (BD Biosciences). Following permeabilization cells were stained with fluorophore-conjugated antibodies specific for the following: CD3, CD4, CD8, B220, Ly6C, MHCII, NK1.1 and phospho-STAT1 (Tyr701). Cells were washed twice in FACS buffer and analyzed on a five laser Cytek Aurora spectral cytometer. Antibody information is provided in the Key Resources Table. Flow cytometry data was analyzed with FlowJo Software (Becton, Dickinson & Company).

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
Rat monoclonal anti-mouse IA/IE (clone M5/114.115.2) conjugated to BV650 Biolegend Cat# 107641; RRID: AB_2565975
Rat monoclonal anti-mouse CD4 (clone RM4-5) conjugated to BV711 Biolegend Cat# 100557; RRID: AB_2562607
Rat monoclonal anti-mouse Ly6C (clone HK1.4) conjugated to BV605 Biolegend Cat# 128036; RRID: AB_2562353
Rat monoclonal anti-mouse CD8 (clone HK1.4) conjugated to BV510 Biolegend Cat# 100751; RRID: AB_2561389
Rat monoclonal anti-mouse CD115 (clone AFS98) conjugated to BV421 Biolegend Cat# 135513, RRID: AB_2562667
Mouse monoclonal anti-mouse CD45 (clone 104) conjugated to PacificBlue Biolegend Cat# 109820, RRID: AB_492872
Rat monoclonal anti-mouse SiglecF (clone E50-2440) conjugated to BB515 BD Biosciences Cat# 566211, RRID: AB_2739601
Rat monoclonal anti-mouse CD11b (clone M1/70) conjugated to AF532 Thermo Fisher Cat# 58-0112-82, RRID: AB_2811905
Rat monoclonal anti-mouse NK1.1 (clone PK136) conjugated to BB700 BD Biosciences Cat# 566502, RRID: AB_2744491
Hamster monoclonal anti-mouse CD3 (clone 145-2C11) conjugated to PE/Cy7 Biolegend Cat# 100319, RRID: AB_312684
Rat monoclonal anti-mouse Ly6G (clone 1A8) conjugated to APC/Cy7 Biolegend Cat# 127623, RRID: AB_10645331
Rat monoclonal anti-mouse B220 (clone RA3-6B2) conjugated to AF700 Thermo Fisher Cat# 56-0452-82, RRID: AB_897458
FcR block of“TruSTain FcX” rat monoclonal anti-mouse CD16/32 (clone 93) Biolegend Cat# 101320; RRID: AB_1574975
Mouse monoclonal anti-mouse IFNAR1 (clone MAR1-5A3) conjugated to PE Biolegend Cat# 127312; RRID: AB_2248000
Mouse monoclonal IgG1k isotype control for IFNAR1 (clone MOPC-21) conjugated to PE Biolegend Cat# 400112; RRID: AB_2847529
Goat polyclonal IgG anti-mouse IFNAR2 conjugated to PE R&D Systems Cat# FAB1083P; RRID: AB_10718996
Goat polyclonal IgG isotype control for IFNAR2 conjugated to PE R&D Systems Cat# IC108P; RRID: AB_10174792
Hamster monoclonal anti-mouse IFNGR2 (clone MOB-47) conjugated to PE Biolegend Cat# 113603; RRID: AB_313560
Hamster monoclonal IgG isotype control for IFNGR2 (clone HTK888) conjugated to PE Biolegend Cat# 400907; RRID: AB_326593
Recombinant human anti-mouse IL10RB (clone REA856) conjugated to PE Miltenyi Biotec Cat# 130-114-688; RRID: AB_2726752
Recombinant human IgG1 isotype control for IL10RB (clone REA293) conjugated to PE Miltenyi Biotec Cat# 130-113-438; RRID: AB_2733893
Mouse monoclonal anti-mouse STAT1 pY701 (clone 4a) conjugated to PE BD Biosciences Cat# 612564, RRID: AB_399855
Chemicals, Peptides, and Recombinant Proteins
Recombinant mouse IFNα A protein R&D Systems Cat# 12100-1
Recombinant mouse IFNγ protein R&D Systems Cat# 485-MI
Poly(I:C) (HMW) VacciGrade™ InvivoGen Cat# 31852-29-6
Baricitinib (INCB028050) SelleckChem Cat# S2851
INCB54707 Incyte Provided by company
Critical Commercial Assays
LEGENDplexTM mouse anti-virus response panel (13-plex) with V-bottom plate Biolegend Cat# 740622
V-PLEX Mouse Cytokine 19-Plex Kit Meso Scale Discovery Cat# K15255D
BD lyse/fix buffer, 5x BD Biosciences Cat# 5580249
BD perm buffer III BD Biosciences Cat# 558050
Experimental Models: Organisms/Strains
B6;129S7-Dp(10Prmt2-Pdxk)2Yey/J The Jackson Laboratory Cat# JAX:013529, RRID:IMSR_JAX013529
B6.129S7-Dp(16Lipi-Zbtb21)1Yey/J The Jackson Laboratory Cat# JAX:013530, RRID:IMSR_JAX013530
B6;129S7-Dp(17Abcg1-Rrp1b)3Yey/J The Jackson Laboratory Cat# JAX:013531, RRID:IMSR_JAX013531
Oligonucleotides
Table S1 This paper N/A
Software and Algorithms
FlowJo Tree Star v10.5.3, RRID:SCR_008520
Graphpad Prism Graphpad v.8.4.2, RRID:SCR_002798
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Flow cytometry for IFNR expression

Whole blood was processed as described above in IFN stimulations and intracellular staining with the following alterations. Whole blood was pre-incubated with FcR block then spiked with stain for all surface markers at 4°C for 30 minutes before two immediate 2 and 5 minute incubations in 200 uL ACK lysis buffer. Cells were washed twice in FACS buffer and fixed in 4% PFA for 10 minutes at room temperature. Fixative was washed-out in FACS buffer and cells were analyzed by flow cytometry using a five laser Cytek Aurora spectral cytometer. Surface markers were the same as described above in IFN stimulations and intracellular staining, with the addition of antibodies against IFNAR1, IFNGR2, IL10RB, or isotype controls.

Q-RT-PCR

RNA was isolated using the QIAGEN AllPrep DNA/RNA/Protein mini kit. cDNA was generated using the Applied Biosystems High Capacity cDNA synthesis kit. Q-RT-PCR was performed using the Applied Biosystems Viia7 384-well block real time PCR system. Q-RT-PCR master mix was prepared with Applied Biosystems SYBR Select Master Mix for CFX. Standard curves were run for every primer pair in each Q-RT-PCR experiment to ensure efficient amplification of target transcripts within all experimental tissues. All samples were run in triplicate, averaged and normalized to 18 s rRNA. Primer sequences at provided in Table S1.

Cytokine measurements

Plasma cytokine levels were measured using Meso Scale Discovery Assays and/or Biolegend LEGENDplex assays per manufacturer’s instructions. All samples were analyzed in duplicate and the average used for statistical analysis. Missing values were set to the lower limit of detection. LEGENDplex assay was evaluated on an Accuri C6 flow cytometer. Data was analyzed using LEGENDplex data analysis software.

Liver Histopathology

Formalin-fixed paraffin-embedded pieces of liver were sectioned at 5 microns and stained with hematoxylin and eosin (H&E). Scoring of liver sections used procedures adapted for mice as described (Lanaspa et al., 2018) from the validated histological scoring system established by Kleiner et al. (2005).

QUANTIFICATION AND STATISTICAL ANALYSIS

All statistical analysis was done with the Prism software and the exact tests and sample sizes employed are described in the legends for each figure. Statistical significance for Kaplan-Meier analysis was calculated using the Mantel-Cox Log-rank test. All other p values were calculated using the Mann-Whitney test. In all cases, statistical significance is indicated as follows: *p < 0.05, **p <0.01, ***p < 0.001, ****p < 0.0001.

Supplementary Material

1

Highlights.

  • A mouse model of Down syndrome displays lethal immune hypersensitivity

  • A heightened inflammatory response is associated with rapid wasting

  • Liver pathology is revealed in this mouse model and exacerbated by immune activation

  • JAK1 and JAK1/2 inhibitors provide therapeutic benefits in this mouse model

ACKNOWLEDGMENTS

We are grateful to all staff members at the Linda Crnic Institute for Down Syndrome, especially Hannah Dougherty. We also thank the staff at the University of Colorado Cancer Center Flow Cytometry Shared Resource, the Human Immune Monitoring Shared Resource (HIMSR), and the Barbara Davis Center Flow Cytometry Core, who assisted with various aspects of this work. We are grateful to Incyte Corporation for providing INCB54707 and for expedited review of these results prior to publication. This work was supported by the NIH Office of the Director and the National Institute of Allergy and Infectious Diseases (NIAID) through grants R01AI145988 and R01AI150305 as part of the NIH INCLUDE Project. Additional funding was provided by NIH grant P30CA046934, the Linda Crnic Institute for Down Syndrome, the Global Down Syndrome Foundation,the Anna and John J. Sie Foundation, the Human Immunology and Immunotherapy Initiative (HI3), the GI & Liver Innate Immune Program, the University of Colorado School of Medicine, the Boettcher Foundation, and Fast Grants.

Footnotes

SUPPLEMENTAL INFORMATION

Supplemental Information can be found online at https://doi.org/10.1016/j.celrep.2020.108407.

DECLARATION OF INTERESTS

K. D.T., K.A.W., K.D.S., and J.M.E. are co-inventors on two patents related to JAK inhibition: U.S. Provisional Patent Application Serial No. 62/992,855 entitled “JAK1 Inhibition For Modulation Of Overdrive Anti-Viral Response To COVID-19” and U.S. Provisional Patent Application Serial No. 62/993,749 entitled “Compounds and Methods for Inhibition or Modulation of Viral Hypercytokinemia.” J.M.E. currently serves on the COVID Development Advisory Board for Elly Lilly, the manufacturer of baricitinib, and on the Cell Reports Advisory Board.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1647637-supplement-1.pdf (755.1KB, pdf)

Data Availability Statement

This study did not generate or analyze large datasets or code.

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