Baricitinib treatment resolves lower airway inflammation and neutrophil recruitment in SARS-CoV-2-infected rhesus macaques

Abstract

Effective therapeutics aimed at mitigating COVID-19 symptoms are urgently needed. SARS-CoV-2 induced hypercytokinemia and systemic inflammation are associated with disease severity. Baricitinib, a clinically approved JAK1/2 inhibitor with potent anti-inflammatory properties is currently being investigated in COVID-19 human clinical trials. Recent reports suggest that baricitinib may also have antiviral activity in limiting viral endocytosis. Here, we investigated the immunologic and virologic efficacy of baricitinib in a rhesus macaque model of SARS-CoV-2 infection. Viral shedding measured from nasal and throat swabs, bronchoalveolar lavages and tissues was not reduced with baricitinib. Type I IFN antiviral responses and SARS-CoV-2 specific T cell responses remained similar between the two groups. Importantly, however, animals treated with baricitinib showed reduced immune activation, decreased infiltration of neutrophils into the lung, reduced NETosis activity, and more limited lung pathology. Moreover, baricitinib treated animals had a rapid and remarkably potent suppression of alveolar macrophage derived production of cytokines and chemokines responsible for inflammation and neutrophil recruitment. These data support a beneficial role for, and elucidate the immunological mechanisms underlying, the use of baricitinib as a frontline treatment for severe inflammation induced by SARS-CoV-2 infection.

Figure 1.. Baricitinib is detectable in plasma and tissues from SARS-CoV-2 infected RMs, but has not impact viral kinetics.

(a) Study design; 8 RMs were infected intranasally and intratracheally with SARS-CoV-2 and at 2 days post-infection (DPI), 4 RMs began daily baricitinib administration (4 mg). Longitudinal collections performed are indicated in circles. (b) Concentration of baricitinib 24 hours post-dosing in plasma (6 DPI closed symbol; and 8 DPI open symbol) and (c) at necropsy in upper and lower lungs of baricitinib treated SARS-CoV-2 infected RMs. (d) Daily cage-side assessment and physical examination scores and (e) changes in body weight from baseline in baricitinib treated (blue symbols; n = 4) and untreated (red symbols; n = 4) SARS-CoV-2 infected RMs. (f-g) After SARS-CoV-2 inoculation, nose and throat swabs, and bronchoalveolar lavages (BAL) were collected and viral loads were quantified by qRT-PCR. (i) Viral loads in tissues measured at necropsy (10–11 DPI). Ct, cycle threshold. Different symbols represent individual animals. Thick lines represent the average of the baricitinib treated (blue lines), and untreated groups (red lines). Bars in b, c, and j, represent the average of the treated and untreated groups. Statistical analysis was performed using a non-parametric Mann-Whitney Test.

Figure 2.. Reduced respiratory disease and lower levels of lung pathology in baricitinib treated RMs.

(a) Representative ventrodorsal radiograph of an untreated RM before SARS-CoV-2 infection (−5 DPI), and at 4, and 7 DPI. Red squares indicate regions of pulmonary infiltrates and opacity. (b) Daily and (c) cumulative radiograph scores; ventrodorsal and lateral radiographs were scored for the presence of pulmonary infiltration by a clinical radiologist according to a standard scoring system (0: normal; 1: mild interstitial pulmonary infiltrates; 2: moderate pulmonary infiltrates with partial cardiac border effacement and small areas of pulmonary consolidation; 3: severe interstitial infiltrates, large areas of pulmonary consolidation, alveolar patterns and air bronchograms). Fold change of coagulation markers to 2 DPI for ferritin (d) and C reactive protein (CRP) levels (e). Panel (f) and (g) show representative lung lesions in an untreated SARS-Cov2 infected rhesus macaque with focally extensive interstitial pneumonia, type 2 pneumocytes hyperplasia, alveolar septal thickening, syncytia formation (arrow), neutrophils and macrophages infiltrations (arrowhead). Panel (h) shows Thyroid Transcription Factor-1 (TTF- 1) staining with prominent type 2 pneumocyte hyperplasia (brown) in a control SARS-Cov2 infected rhesus macaque. Panel (i) and (j) shows treatment effects of baricitinib in COVID-19 infected rhesus macaques with a reduction in pulmonary lesions (affected area marked by arrows), lesser inflammatory infiltrates (arrowhead) and reduced type 2 pneumocyte hyperplasia. Panel (k) shows TTF-1 staining with lesser type 2 pneumocyte hyperplasia (brown) after Baricitinib treatment. (l) Average pathology score per lobe. (m) Total Pathology Score. (n) Pathology scores for individual parameters. Magnification, Panels (g) and (i): 100 ×; Panels (g), (h), (j), (k): 200 ×. Scale bar, panels (f) and (i): 200μM; Panels (g), (h), (j) and (k): 50μM. Pathology scoring. Bars in (d), (e), (l), (m) and (n) indicate mean values for baricitinib treated (blue), and untreated (red) SARS-CoV-2 infected RMs. Each symbol represents individual animals. Statistical analysis in b, and j were performed using non-parametric Mann-Whitney Test.

Figure 3.. Baricitinib treatment suppresses gene expression of inflammation and neutrophil degranulation in the BAL of SARS-CoV-2 infected NHPs.

Bulk RNA-Seq profiles of BAL cell suspensions from NHPs obtained at Day −5 prior to SARS-CoV-2 inoculation (Baseline), at 2 days post-inoculation, prior to baricitinib administration (2 DPI) treatment, and at 4 days post-inoculation, 2 days after initiation of baricitinib treatment (4 DPI). Both the untreated arm and baricitinib arm were comprised of n = 4 animals. (a) Venn diagrams indicating the number of DEGs detected at 2 DPI or 4 DPI relative to −5dpi in the untreated (blue) and baricitinib treated (red) experimental groups. The total DEGs for each comparison are shown in parentheses. (b) Bar plots showing enrichment of top scoring inflammatory and immunological gene signatures from the MSIGDB (Hallmark and Canonical Pathways) and databases, and custom genesets (ISGs, see below) ranked by GSEA comparisons of gene expression in the 4 DPI vs 2 DPI samples from the untreated animal group (pink bars), or 4 DPI vs 2 DPI in the baricitinib treated group (blue bars). The x-axis depicts the normalized enrichment score (NES); a positive enrichment score indicated higher expression at 4 DPI relative to 3 DPI, conversely, negative scores of a pathway indicate cumulatively higher expression in 2 DPI samples relative to 4 DPI. Nominal p-values are indicated; negative enrichment (2 DPI > 4 DPI) is indicated by bars facing left, positive enrichment (4 DPI > 2 DPI) denoted by bars facing right. (c-f) GSEA enrichment plots depicting pairwise comparison of gene expression of 2 DPI samples vs 4 dpi samples. 2 DPI vs 4 DPI comparisons for the untreated group are depicted by red symbols, and comparisons for the baricitinib treated group are shown in blue. The top-scoring (i.e. leading edge) genes are indicated by solid dots. The hash plot under GSEA curves indicate individual genes and their rank in the dataset. Left-leaning curves (i.e. positive enrichment scores) indicate enrichment, or higher expression, of pathways at 4 DPI, right-leaning curves (negative enrichment scores) indicate higher expression at 2 DPI. Sigmoidal curves indicate a lack of enrichment, i.e. equivalent expression between the groups being compared. The normalized enrichment scores and nominal p-values testing the significance of each comparison are indicated. (c) REACTOME_NEUTROPHIL_DEGRANULATION (MSIDB #M27620) (d) GSEA line plot of HALLMARK_TNFA_SIGNALING_VIA_NFKB pathway (MSIGDB # M5890). (e) GSEA line plot of HALLMARK_IL6_JAK_STAT3_SIGNALING (MSIGDB# M5897) (f) a custom geneset of ISGs from prior NHP studies (Nganou-Makamdop et al., 2018; Palesch et al., 2018; Sandler et al., 2014); (g-h) Heat maps of top-scoring (i.e. leading edge) from the untreated 4 DPI vs 2 DPI GSEA analyses. The color scale indicates the log2 expression relative to the median of all baseline samples.

Figure 4.. Baricitinib treatment abolishes inflammatory cytokine and neutrophil chemoattractant expression in bronchoalveolar macrophages.

Single cell suspensions from BALs of SARS-CoV-2 infected RMs were subject to 10X Genomics capture and sequencing. (a) UMAP showing major cell types in BAL samples (n = 10 samples; untreated, baseline n = 3; untreated, 4 DPI n = 3; treated, baseline n = 2; treated, 4 DPI). (b) UMAP showing clusters in BAL samples by treatment days (n = 10). (c) UMAP projection of pro-inflammatory cytokines in macrophages. (d) UMAP projection of neutrophil chemoatttractants and pro-inflammatory chemokines. (e and f) Expression of chemokines and interferon stimulated genes (ISGs) in treated and untreated samples at baseline and 4 DPI. The colored expression scale of expression in UMAPs is depicted on a per gene basis: the scale represent the per cell reads for each gene divided by the total reads for of that cell, scaled to the factor shown and natural log-transformed.

Figure 5.. Baricitinib treated RMs have decreased infiltration of innate immune cells and lowered neutrophil NETosis.

(a) UMAP analysis of BAL in baricitinib treated (n= 4) and untreated (n= 4) SARS-CoV-2 infected RMs before infection (D −5 PI; baseline), and at 4, and 10 DPI. (b) Representative flow cytometry staining of neutrophil infiltration in BAL at baseline, and 4 and 10 DPI. Longitudinal levels of neutrophils within BAL samples depicted as a % of CD45+ cells (c) and fold change to 2 DPI (d) in treated (blue) and untreated (red) RMs. (e) Fold change to 2 DPI of neutrophils in blood of baricitinib treated and untreated SARS-CoV-2 infected RMs. (f) Representative microscopy images of NETS by Sytox green assay in baricitinib treated and untreated SARS-CoV-2 infected RMs. Scale bar: 200 μm. (g) Quantification of NETosis activity upon staining extracellular DNA with Sytox in isolated stimulated neutrophils from blood. Fold change of Sytox levels to −5 DPI. In c, d, e, and g each symbol represents individual animals. Thick lines represent the average of the baricitinib treated (blue line), and untreated groups (red line). Bars in d, e, and g represent the average of the treated and untreated groups. Statistical analysis was performed using a non-parametric Mann-Whitney Test.

Figure 6.. Decreased levels of T cell proliferation and activation in baricitinib treated RMs.

Longitudinal levels of (a) circulating CD4⁺ T cells and (b) CD4⁺ T_Reg cells measured by flow cytometry of baricitinib treated (blue) and untreated (red) SARS-CoV-2 infected RMs. (c) Fold changes to 2 DPI of circulating CD4⁺ T_Reg cells. (d) Levels of circulating CD8⁺ T cells and (e) proliferating (Ki-67⁺) memory CD8⁺ T cells. (f) Levels of CD4⁺ T cells, and (g) HLA-DR⁻CD38⁺ CD4⁺ T cells in bronchoalveolar lavages (BAL) measured by flow cytometry. (h) Levels of CD8⁺ T cells, (i) proliferating (Ki-67⁺) memory CD8⁺ T cells and (j) HLA-DR⁻CD38⁺ CD8⁺ T cells in BAL. Each symbol represents individual animals. Thick lines represent the average of the baricitinib treated (blue line), and untreated groups (red line). (k-m) Representative staining of Ki-67 and CD38 by HLA-DR. Bars in (c) represent the average of the treated and untreated groups. Statistical analysis in b was performed using non-parametric Mann-Whitney Test.

Figure 7.

(a) SARS-Cov-2 infection in rhesus macaques results in an accumulation of inflammatory macrophages and neutrophils in the lower airway. These airway macrophages produce high amounts of inflammatory cytokines and neutrophil-attracting chemokines and show upregulated Type I interferon signaling. Neutrophil NETs and the inflammation induced by SARSCoV-2 infection both contribute to lung pathology. (b) Baricitinib treatment reduced the levels of macrophages producing inflammatory cytokines and neutrophil-attracting chemokines, decreased the infiltration of neutrophils into the lung and reduced T cell activation. The Netosis activity of neutrophils was also reduced. In treated animals, the antiviral interferon response was maintained, viral replication was not impacted, and lung pathology was mild.