In-vitro evaluation of the immunomodulatory effects of Baricitinib: Implication for COVID-19 therapy

Abstract

Objective: Baricitinib seems a promising therapy for COVID-19. To fully-investigate its effects, we in-vitro evaluated the impact of baricitinib on the SARS-CoV-2-specific-response using the whole-blood platform.

Methods: We evaluated baricitinib effect on the IFN-γ-release and on a panel of soluble factors by multiplex-technology after stimulating whole-blood from 39 COVID-19 patients with SARS-CoV-2 antigens. Staphylococcal Enterotoxin B (SEB) antigen was used as a positive control.

Results: In-vitro exogenous addition of baricitinib significantly decreased IFN-γ response to spike- (median: 0.21, IQR: 0.01-1; spike+baricitinib 1000 nM median: 0.05, IQR: 0-0.18; p < 0.0001) and to the remainder-antigens (median: 0.08 IQR: 0-0.55; remainder-antigens+baricitinib 1000 nM median: 0.03, IQR: 0-0.14; p = 0.0013). Moreover, baricitinib significantly decreased SEB-induced response (median: 12.52, IQR: 9.7-15.2; SEB+baricitinib 1000 nM median: 8, IQR: 1.44-12.16; p < 0.0001). Baricitinib did modulate other soluble factors besides IFN-γ, significantly decreasing the spike-specific-response mediated by IL-17, IL-1β, IL-6, TNF-α, IL-4, IL-13, IL-1ra, IL-10, GM-CSF, FGF, IP-10, MCP-1, MIP-1β (p ≤ 0.0156). The baricitinib-decreased SARS-CoV-2-specific-response was observed mainly in mild/moderate COVID-19 and in those with lymphocyte count ≥1 × 10³/µl.

Conclusions: Exogenous addition of baricitinib decreases the in-vitro SARS-CoV-2-specific response in COVID-19 patients using a whole-blood platform. These results are the first to show the effects of this therapy on the immune-specific viral response.

Keywords: Baricitinib; COVID-19; IGRA; SARS-CoV-2; Specific immune-response.

Graphical abstract

Fig. 1

The exogenous addition of baricitinib decreases the in vitro IFN-γ response to SARS-CoV-2 peptides in COVID-19 patients. Baricitnib 10 nM or 1000 nM decreased the IFN-γ levels after stimulating whole-blood with spike- (A) or remainder-antigens-MPs (B) or SEB (C). IFN-γ was measured by ELISA in stimulated plasma. Statistical analysis was performed using the Wilcoxon test with Bonferroni correction, and p value was considered significant if ≤0.016. Number of patients analysed: A) n = 37; B) n = 36; C) n = 37. Footnotes: IFN: Interferon; SEB: Staphylococcal Enterotoxin B.

Fig. 2

The exogenous addition of baricitinib modulates the IFN-γ in vitro response to SARS-CoV-2 peptides mainly in patients with a lymphocytes count higher than 1 × 10³/µl.COVID-19 patients stratified based on the lymphocyte counts (A–C). Severity of each patient analysed is reported: black dots indicate mild disease, blue dots indicate moderate disease, red dots indicate severe and critical disease. Baricitinib at 10 nM and at 1000 nM significantly decreases the spike IFN-γ response in patients with ≥1<2 × 10³/µl lymphocytes and in patients with more than 2 × 10³/µl lymphocytes (A). Baricitinib 10 nM significantly decreases the IFN-γ response to the remainder-antigens in patients with ≥1<2 × 10³/µl lymphocytes (B). Baricitinib 1000 nM decreases SEB-response in patients with ≥1<2 × 10³/µl lymphocytes; baricitinib 10 nM and at 1000 nM decreases SEB-response in patients with ≥2 × 10³/µl lymphocytes (C). IFN-γ was measured by ELISA in stimulated plasma. The horizontal lines represent the median; statistical analysis was performed using the Friedman or Kruskall-Wallis tests, Wilcoxon or Mann-Whitney tests with Bonferroni correction and p ≤ 0.016 was considered significant. Number of patients analysed for each subgroup: A) n = 4, 13, 9; B) n = 3, 11, 6; C) n = 6, 18. Footnotes: IFN: Interferon; SEB: Staphylococcal Enterotoxin B.

Fig. 3

In COVID-19 patients, the exogenous addition of baricitinib decreases the in vitro levels of pro-inflammatory, Th1, Th17, Th2 cytokines, immunomodulatory factors, chemokines and growth factor in response to SARS-Cov-2 peptides. Evaluation of 27 analytes in response to spike and to remainder-antigens by multiplex technology. In COVID-19 patients baricitinib at 1000 nM significantly decreases the in vitro cytokine spike-specific response mediated by the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α (A-C), the Th1- and Th17-cytokines IFN-γ and IL-17 (D-E), the Th2-cytokines IL-4 and IL-13 (F-G), IL-10 and IL-1ra (H-I), the chemokines IP-10, MCP-1 and MIP-1β (J-L) and the growth factors FGF and GM-CSF (M-N). Baricitinib at 1000 nM decreased also IL-1β and IP-10 production in response to remainder-antigens (O-P). Analyte levels measured by luminex in stimulated plasma. The horizontal lines represent the median; statistical analysis was performed using the Wilcoxon test, and p value was considered significant if ≤0.016. Footnotes: IL: Interleukin; TNF: Tumor Necrosis Factor; IFN: Interferon; ra: receptor antagonist; IP: interferon-inducible protein; MCP: monocyte chemoattractant protein; MIP: macrophage inflammatory protein; FGF: fibroblast growth factor; GM-CSF: granulocyte-macrophage colony-stimulating factor.