Baricitinib restrains the immune dysregulation in patients with severe COVID-19

Abstract

BACKGROUNDPatients with coronavirus disease 2019 (COVID-19) develop pneumonia generally associated with lymphopenia and a severe inflammatory response due to uncontrolled cytokine release. These mediators are transcriptionally regulated by the JAK/STAT signaling pathways, which can be disabled by small molecules.METHODSWe treated a group of patients (n = 20) with baricitinib according to an off-label use of the drug. The study was designed as an observational, longitudinal trial and approved by the local ethics committee. The patients were treated with 4 mg baricitinib twice daily for 2 days, followed by 4 mg per day for the remaining 7 days. Changes in the immune phenotype and expression of phosphorylated STAT3 (p-STAT3) in blood cells were evaluated and correlated with serum-derived cytokine levels and antibodies against severe acute respiratory syndrome-coronavirus 2 (anti-SARS-CoV-2). In a single treated patient, we also evaluated the alteration of myeloid cell functional activity.RESULTSWe provide evidence that patients treated with baricitinib had a marked reduction in serum levels of IL-6, IL-1β, and TNF-α, a rapid recovery of circulating T and B cell frequencies, and increased antibody production against the SARS-CoV-2 spike protein, all of which were clinically associated with a reduction in the need for oxygen therapy and a progressive increase in the P/F (PaO2, oxygen partial pressure/FiO2, fraction of inspired oxygen) ratio.CONCLUSIONThese data suggest that baricitinib prevented the progression to a severe, extreme form of the viral disease by modulating the patients' immune landscape and that these changes were associated with a safer, more favorable clinical outcome for patients with COVID-19 pneumonia.TRIAL REGISTRATIONClinicalTrials.gov NCT04438629.FUNDINGThis work was supported by the Fondazione Cariverona (ENACT Project) and the Fondazione TIM.

Keywords: COVID-19; Immunology; Innate immunity.

Figure 1. Baricitinib treatment restores normal lymphocyte counts in the blood.

Peripheral blood from patients with COVID-19 enrolled in either the baricitinib (n = 12) or basic treatment (n = 8, Ctrl) arm was analyzed by flow cytometry at t0 (baseline) and t7 (7 days after treatment). The number of cells per microliter is reported for lymphocytes (A), T lymphocytes (B), B lymphocytes (C), CD4⁺ T lymphocytes (D), CD4⁺ T effector memory (TEM) cells (E), and CD8⁺ T lymphocytes (F). The normal reference range is shown in the light gray boxes. Data are reported as the mean ± SEM. Statistical analysis was determined by 1-way, repeated-measures ANOVA. (G) t-SNE analysis of peripheral blood from 12 baricitinib-treated patients at t0 (left) and t7 (right). The following identified clusters are shown in different colors: monocytes, CD16⁺ monocytes, residual polymorphonuclear neutrophils (PMNs) (CD16^hi), B lymphocytes (CD19⁺CD45RA⁺), CD4⁺ T central memory (TCM) cells (CD3⁺CD4⁺CD27⁺CD45RA^–), CD4⁺ TEM cells (CD3⁺CD4⁺CD57⁺CD27^–CD45RA^–), CD4⁺ naive T cells (CD3⁺CD4⁺CD27⁺CD45RA⁺), CD4⁺ TEM cells reexpressing CD45RA (TEMRA) (CD3⁺CD4⁺CD45RA⁺CD57⁺), CD8⁺ T memory (TM) cells (CD3⁺CD8⁺CD27⁺CD45RA^–), CD8⁺ TEM cells (CD3⁺CD8⁺CD45RA^–CD57⁺), CD8⁺ naive T cells (CD3⁺CD8⁺CD27⁺CD45RA⁺), CD8⁺ senescent T cells (CD3⁺CD8⁺CD57⁺CD45RA⁺), NK T (NKT) cells (CD3⁺CD16⁺CD56⁺CD45RA⁺), senescent NKT cells (CD3⁺CD16⁺CD56⁺CD45RA⁺CD57⁺), NK cells (CD16⁺CD56⁺CD45RA⁺), and senescent NK cells (CD16⁺CD56⁺CD45RA⁺CD57⁺). Ctrl, control; dim, dimensionality.

Figure 2. Baricitinib treatment affects IgG levels and the production of inflammatory cytokines that contribute to the cytokine storm.

Plasma from patients with COVID-19 enrolled in either the baricitinib (n = 20) or basic (n = 8) treatment arm was analyzed at t0 (baseline) and t7 (7 days after treatment) to measure the concentrations of IgA (A), IgG (B), IL-1β (C), IL-6 (D), TNF-α (E), and IL-8 (F). For serological data, the light gray boxes identify the range of Ab detection. The normal median value for cytokines is indicated in the light gray boxes. Data are presented as the mean ± SEM. Statistical analysis was determined by 1-way, repeated-measures ANOVA.

Figure 3. Baricitinib treatment alters the immune-suppressive abilities and distribution of myeloid cells during the recovery phase.

(A) Plasma from a patient with COVID-19 enrolled in the baricitinib treatment arm who was admitted to the ICU during treatment was analyzed at t0 (baseline), t4 (4 days after treatment), and t7 (7 days after treatment) to assess proinflammatory cytokine levels by automated immunoassay system. Peripheral blood from the same patient was tested while the patient was in the ICU and after leaving the ICU (No ICU). (B) Monocytes (CD14⁺), LDNs (CD66b⁺), and NDNs (CD66b⁺) were isolated from the peripheral blood. The immune-suppressive abilities of either enriched myeloid cells or their conditioned media were tested in functional assays to assess T cell activation and are shown as the percentage of suppression. Monocyte (C) and neutrophil (D) subsets were evaluated by flow cytometry and are reported as the number of cells per microliter. (E) Cytokines released in the conditioned media by CD14⁺ and CD66b⁺ LDNs were quantified by multiplex ELISA. (B–D) Data are reported as the mean ± SEM. P values in B–D were determined by 2-tailed Student’s t test. SN, supernatant.