The effect of local anesthetic on pro-inflammatory macrophage modulation by mesenchymal stromal cells

Abstract

Administering local anesthetics (LAs) peri- and post-operatively aims to prevent or mitigate pain in surgical procedures and after tissue injury in cases of osteoarthritis (OA) and other degenerative diseases. Innovative tissue protective and reparative therapeutic interventions such as mesenchymal stromal cells (MSCs) are likely to be exposed to co-administered drugs such as LAs. Therefore, it is important to determine how this exposure affects the therapeutic functions of MSCs and other cells in their target microenvironment. In these studies, we measured the effect of LAs, lidocaine and bupivacaine, on macrophage viability and pro-inflammatory secretion. We also examined their effect on modulation of the macrophage pro-inflammatory phenotype in an in vitro co-culture system with MSCs, by quantifying macrophage tumor necrosis factor (TNF)-α secretion and MSC prostaglandin E2 (PGE2) production. Our studies indicate that both LAs directly attenuated macrophage TNF-α secretion, without significantly affecting viability, in a concentration- and potency-dependent manner. LA-mediated attenuation of macrophage TNF-α was sustained in co-culture with MSCs, but MSCs did not further enhance this anti-inflammatory effect. Concentration- and potency-dependent reductions in macrophage TNF-α were concurrent with decreased PGE2 levels in the co-cultures further indicating MSC-independent macrophage attenuation. MSC functional recovery from LA exposure was assessed by pre-treating MSCs with LAs prior to co-culture with macrophages. Both MSC attenuation of TNF-α and PGE2 secretion were impaired by pre-exposure to the more potent bupivacaine and high dose of lidocaine in a concentration-dependent manner. Therefore, LAs can affect anti-inflammatory function by both directly attenuating macrophage inflammation and MSC secretion and possibly by altering the local microenvironment which can secondarily reduce MSC function. Furthermore, the LA effect on MSC function may persist even after LA removal.

Keywords: Inflammation; Local anesthetics; Macrophages; Mesenchymal stromal cells.

Figure 1. Effects of LAs on macrophage viability

The bar heights represent the relative fluorescence intensities of reduced alamar blue reagent at 4 hours of incubation on unstimulated (−LPS, grey bars) or LPS-stimulated (+LPS, white bars) macrophages treated for (A) 1, (B) 6, or (C) 24 hours with medium (0 mM) or medium containing 0.5 or 1 mM of lidocaine or bupivacaine. The data are the mean ± SEM of n = 3 biological replicates, relative to 0 mM/−LPS. ^*Statistically significant difference compared to the basal medium control (0 mM). ^†Statistically significant difference compared 0.5 mM. ^#Statistically significant difference between corresponding −LPS and +LPS conditions.

Figure 2. Effects of LAs on macrophage TNF-α secretion

The bar heights represent the level of TNF-α (relative to 0 mM) secreted from LPS-stimulated macrophages treated for 24 hours with medium (0 mM) or medium containing 0.5 or 1 mM of lidocaine or bupivacaine. Data points on the overlaid line graph (grey circles, broken line) are alamar blue fluorescence values (relative to 0 mM) associated with the corresponding 24 hour time point. The data are the mean ± SEM of n = 3 biological replicates. ^*Statistically significant difference compared to the basal medium control (0 mM). ^†Statistically significant difference compared 0.5 mM.

Figure 3. MSC modulation of macrophage secretion in the presence of LAs

The bar heights represent the level of TNF-α secreted from LPS-stimulated macrophages co-cultured for 24 hours with (white bars) or without (grey bars) MSCs in LPS-supplemented medium (0 mM) or LPS-supplemented medium containing 0.5 or 1 mM of lidocaine or bupivacaine. Values are presented as cytokine level relative to the LPS control without MSCs (0 mM, − MSCs). The data are the mean ± SEM of n = 3 biological replicates. ^*Statistically significant difference compared to the LPS control (0 mM, −MSCs). ^#Statistically significant difference compared to the −MSC controls. ^†Statistically significant difference compared to 0.5 mM. ^ΔStatistically significant difference compared to no anesthetic MSC control (0 mM, +MSCs).

Figure 4. Secreted PGE2 levels in MSC/macrophage co-culture supernatants

The bar heights represent the level of secreted PGE2 present after co-culture of macrophages with MSCs for 24 hours in LPS-supplemented medium (0 mM) or LPS-supplemented medium containing 0.5 or 1 mM of lidocaine or bupivacaine. Values are presented as cytokine level relative to the LPS control (0 mM). The data are the mean ± SEM of n = 3 biological replicates. ^*Statistically significant difference compared to 0 mM. ^†Statistically significant difference compared to 0.5 mM.

Figure 5. Effects of LA pre-treatment on MSC modulation of macrophage secretion

The bar heights represent the level of TNF-α secreted from LPS-stimulated macrophages co-cultured for 24 hours with MSCs pre-treated for 6 hours with medium (0 mM) or medium containing 0.5 or 1 mM of lidocaine or bupivacaine. Values are presented as cytokine level relative to the LPS control (0 mM). The data are the mean ± SEM of n = 3 biological replicates. *Statistically significant difference compared to LPS-stimulated control. ^ΔStatistically significant difference compared to medium (0 mM) pre-treated MSC control. ^†Statistically significant difference compared to 0.5 mM.

Figure 6. Secreted PGE2 levels in pre-treated MSC/macrophage co-culture supernatants

The bar heights represent the level of secreted PGE2 present after 24 hour co-culture of LPS-activated macrophages with MSCs that were pre-treated for 6 hours with medium without (0 mM) or with 0.5 or 1 mM of lidocaine or bupivacaine. Values are presented as PGE2 level relative to 0 mM. The data are the mean ± SEM of n = 3 biological replicates. ^*Statistically significant difference compared to 0 mM. ^†Statistically significant difference compared to 0.5 mM.