Lidocaine reinforces the anti-inflammatory action of dexamethasone on myeloid and epithelial cells activated by inflammatory cytokines or SARS-CoV-2 infection

Abstract

Background: Severe cases of Coronavirus Disease 2019 (COVID-19) that require admission to the Intensive Care Unit (ICU) and mechanical ventilation assistance show a high mortality rate with currently few therapeutic options available. Severe COVID-19 is characterized by a systemic inflammatory condition, also called "cytokine storm", which can lead to various multi-organ complications and ultimately death. Lidocaine, a safe local anesthetic that given intravenously is used to treat arrhythmias, has long been reported to have an anti-inflammatory and pro-homeostatic activity.

Methods: We studied the capacity of lidocaine to modulate cytokine secretion of mouse and human myeloid cell lines activated by different cytokines or Toll Like Receptor (TLR) ligands (flagellin (FliC), Lipopolysaccharide (LPS), Polyinosinic:polycytidylic acid (Poly I:C) and N-Palmitoyl-S- [2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-(S)-seryl-(S)-lysyl-(S)-lysyl-(S)-lysyl-(S)-lysine x 3HCl (Pam3Cys-SKKKK)) or by Severe acute respiratory syndromecoronavirus 2 (SARS-CoV-2) infection to epithelial cells. Reporter cell lines were used to study modulation of lidocaine of specific signaling pathways.

Results: Lidocaine used in combination with dexamethasone, had an additive effect in the modulation of cellular inflammatory response triggered by Tumoral Necrosis Factor alpha (TNFα), Interleukin 1 beta (IL-1β) as well as different TLR ligands. We also found that lidocaine in combination with dexamethasone modulates the Nuclear factor kappa B (NF-κB) pathway, inflammasome activation as well as interferon gamma receptor (IFNγR) signaling without affecting the type I interferons (Type I IFNs) pathway. Furthermore, we showed that lidocaine and dexamethasone treatment of epithelial cells infected with SARS-CoV-2 modulated the expression of chemokines that contribute to pro-inflammatory effects in severe COVID.

Conclusions: We reported for the first time in vitro anti-inflammatory capacity of lidocaine on SARS-CoV-2 triggered immune pathways. These results indicated the potential of lidocaine to treat COVID-19 patients and add tools to the therapeutic options available for these concerning cases.

Keywords: Dexamethasone; Inflammation; Interferon; Lidocaine; Modulation; SARS-CoV-2.

Fig. 1

Dose-dependent immunomodulatory capacity of lidocaine on different monocytic-macrophage cell lines stimulated with a TLR3 agonist. RAW 264.7 (A), J774 (B) and THP-1 (C) cells were stimulated ON with poly I:C in presence of different concentrations of lidocaine or not. Pro-inflammatory cytokines, mIL-6 and hIL-8, were measured in supernatants. ∗(p < 0.05), ∗∗(p < 0.01), ∗∗∗(p < 0.001), a result significantly different from the stimulated unmodulated condition. Data are represented as mean ± SEM of three replicates. Graphs are representative of at least two independent assays.

Fig. 2

Lidocaine in combination with dexamethasone has an additive modulatory effect on TLR3, TLR5, TLR4 and TLR2 signaling pathways. RAW 264.7 cells were stimulated ON with poly I:C (A) and FliC (B), modulated or not with lidocaine and/or dexamethasone, and mIL-6 was measured in culture supernatants. THP-1 cells were stimulated ON with EcLPS (C) and Pam3Cys-SKKKK (D) in presence or not of different concentrations of lidocaine and/or dexamethasone, and hIL-8 was measured in supernatants. ∗ (p < 0.05), ∗∗ (p < 0.01), ∗∗∗ (p < 0.001), ∗∗∗∗ (p < 0.0001), a result significantly different from the stimulated unmodulated condition. # (p < 0.05), ## (p < 0.01), ### (p < 0.001), #### (p < 0.0001), a result significantly different from its counterpart stimulated and modulated with only lidocaine at the same concentration. Φ (p < 0.05) a result different from its counterpart stimulated and modulated with only dexamethasone at the same concentration. Data are represented as mean ± SEM of three replicates. Graphs are representative of at least three independent assays.

Fig. 3

Lidocaine in combination with dexamethasone has an additive modulatory effect different signaling pathways. TNFR signaling (A): THP1-XBlueTM-defMyD cells were stimulated ON with hTNFα and modulated or not with lidocaine and/or dexamethasone, and hIL-8 was measured in culture supernatants. IFNγR signaling (B): B16 IFNγ SEAP reporter cells were stimulated ON with mIFNγ in presence or not of different concentrations of lidocaine and/or dexamethasone, and SEAP activity was measured in supernatants. NF-κB signaling (C): HEK-hTLR4 SEAP reporter cells were stimulated ON with EcLPS and modulated or not with lidocaine and/or dexamethasone, and relative SEAP activity was measured in culture supernatants. Lidocaine and dexamethasone additively modulate poly I:C TLR3 signaling, but do not affect IFNAR signaling (D–E). B16 type I IFNs SEAP reporter cells were stimulated ON with poly I:C (D) or with supernatant from the same cell line previously stimulated with poly I:C (E) and modulated or not with lidocaine and/or dexamethasone. SEAP activity was measured in culture supernatants. ∗ (p < 0.05), ∗∗ (p < 0.01), ∗∗∗ (p < 0.001), ∗∗∗∗(p < 0.0001), a result significantly different from the stimulated unmodulated condition. # (p < 0.05), ## (p < 0.01), a result significantly different from its counterpart stimulated and modulated with only lidocaine at the same concentration. Data are represented as mean ± SEM of three replicates. Graphs are representative of at least two independent assays.

Fig. 4

Lidocaine in combination with dexamethasone has an additive modulatory effect on the innate inflammatory pathway of inflammasome activation. THP1-ASC-GFP cells were primed with EcLPS for 3 h, stimulated ON with transfected pcDNA and modulated or not with lidocaine and/or dexamethasone. Cells were prepared for fluorescent microscopy observation (A, 20×), intense green specks and cells were counted and ASC + cells/total cells ratio calculated (B) and hIL-1β was measured in culture supernatants (C). ∗ (p < 0.05), ∗∗ (p < 0.01), ∗∗∗ (p < 0.001), ∗∗∗∗ (p < 0.0001), a result significantly different from the stimulated unmodulated condition. # (p < 0.05), ## (p < 0.01), ### (p < 0.001), a result significantly different from its counterpart stimulated and modulated with only lidocaine at the same concentration. Data are represented as mean ± SEM of three replicates. Graphs are representative of at least two independent assays.

Fig. 5

Lidocaine and dexamethasone modulate pro-inflammatory gene expression in epithelial cells infected by SARS-CoV-2. Calu-3 cells were infected ON with the virus SARS-CoV-2 Wuhan type strain at a MOI of 1 and modulated or not with lidocaine and/or dexamethasone. Expression levels of CXCL10, CCL20, CXCL2 and Mx-1 mRNA were measured. ∗(p < 0.05), ∗∗(p < 0.01), ∗∗∗(p < 0.001), ∗∗∗∗(p < 0.0001), a result significantly different from the stimulated unmodulated condition. Data are represented as mean ± SEM of three replicates. Graphs are representative of at least two independent assays.