*3.5. PLA-PEMA and PLA-PVA Inhibit NF-κB Activation, But Do So at Different Rates*

PAMPs engage their respective TLRs and initiate complex signaling cascades that eventually lead to the production of cytokines and chemokines and expression of costimulatory molecules that eventually lead to widespread inflammation [27]. Because these signaling cascades are dependent upon activation of key transcriptional nodes, we next investigated the impact of LPS-mediated TLR4 stimulation via activation of the NF-κB p65 transcription factor and p38 MAPK (Figure 4A). In both signaling pathways, phosphorylation of p65 and p38 signifies engagement and activation of the upstream TLRs. To investigate the effects of iNPs, we first incubated the BMMΦs with iNPs prior to LPS stimulation for 0.5, 1, and 4 h to assess the activation of these key signaling pathways. Figure 4B shows that both PLA-PEMA and PLA-PVA decrease the phosphorylation of p65 compared to no particle treatment. Importantly, the decrease in phosphorylation in the case of PLA-PEMA treatment occurs earlier than that seen in PLA-PVA, suggesting that the more extensive uptake of PLA-PEMA compared to PLA-PVA (Figure 3A,B) plays a role in mediating this protective effect against activation of proinflammatory signaling cascades. Along with this, incubation with either PLA-PEMA or PLA-PVA alone results in no alteration in phosphorylation of either p65 nor a decrease in the total amount of the protein (Supplemental Figure S4). We next probed for MAPKs (Figure 4C). MAPKs are key in that they are activated by different stimuli (including LPS), yet p38, ERK1/2, and SAPK/JNK all have the capacity to phosphorylate transcription factors that form the AP-1 complex, a key regulator of the transcription of inflammatory cytokines [28,29]. We can see that phosphorylation of p38 is decreased secondary to LPS stimulation when treated with iNPs and that this result is opposite to that seen with phospho-ERK1/2 and phospho-JNK. Interestingly, when we evaluate the effects of iNP treatment on MAPK activation alone, we see that iNPs stimulate phosphorylation of ERK1/2, an effect not seen with the other probed MAPKs (Supplemental Figure S4). Finally, an investigation of upstream signaling components shows no decrease in phosphorylation of MKK3, MKK6, and TAK1, nor total levels of IRAK4 suggesting that the iNP-mediated effects downstream of LPS stimulation are limited to NF-κB p65 and p38 MAPK (Figure 4D). These data suggest that the iNP-based modifications to the BMMΦs are inherent to their capacity to respond to an inflammatory trigger rather than some basal change to the BMMΦs.

Furthermore, to establish that these transcription changes result in functional changes to the BMMΦs, we used Luminex to establish that these changes in transcription factor activation also resulted in a decrease in cytokine secretions. Indeed, we confirmed this across a multitude of signaling pathways including NF-κB-dependent IL-6 (Figure 5A), IRF3 dependent IFNβ (Figure 5B) [30], and the transcriptionally complex IL-10 (Figure 5C) [31]. Similar experiments conducted with murine macrophage-derived RAW 264.7 cells confirmed iNP-dependent decreases in IL-6 with iNPs following LPS stimulation (Supplemental Figure S5). Interestingly, PLA-PVA treatment resulted in an increase in TNF-α secretion with iNP treatment, while the opposite effect was observed with PLA-PEMA. Additionally, another potential consequence of iNP treatment is the induction of cell death driving the decrease in transcriptional activation and proinflammatory cytokines. We used flow cytometry and cell exclusion dye to establish that our iNPs do not induce cell death and can increase cell survival in the setting of LPS stimulation of BMMΦs (Figure 5D). When taken together, iNPs can drive changes in the function of BMMΦs through reprogramming of transcriptional activation. This leads to decreased proinflammatory cytokine secretions and also aids in extending survival of this cell population.

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 10 of 19

**Figure 4.** Both iNPs suppress NF-κB p65 and p38 MAPK activation, although at different rates. (**A**) TLR4 activation upon LPS binding triggers a complex signaling cascade where two key nodes of activity involve activation of NF-κB p65 and p38 MAPK to initiate downstream inflammatory cytokine expression. Following PLA-PVA (300 µg/mL) and PLA-PEMA (300 µg/mL) treatment, cells were then stimulated with LPS (100 ng/mL) for 0.5, 1, or 4 hrs. (**B**) Samples were then immunoblotted for key NF-κB proteins: phospho-NF-κB p65 (Ser536), total NF-κB p65, phospho-IκB (Ser32), and total IκB. (**C**) These samples were also immunoblotted for a variety of MAPKs: phsopho-p38 (Thr180/Tyr182), total p38, phospho-ERK1/2 (Thr202/Tyr204), total ERK2, phospho-SAPK/JNK (Thr183/Tyr185), and total JNK. (**D**) Finally, to address potential impacts of iNPs on upstream signaling proteins, these samples were immunoblotted for phospho-MKK3 (Ser189/)/MKK6 (Ser207), total MKK6, phospho-TAK1 (Thr184/187), total TAK1, IRAK4, and β-Actin. Schematic in (**A**) created with Bio-Render. Furthermore, to establish that these transcription changes result in functional **Figure 4.** Both iNPs suppress NF-κB p65 and p38 MAPK activation, although at different rates. (**A**) TLR4 activation upon LPS binding triggers a complex signaling cascade where two key nodes of activity involve activation of NF-κB p65 and p38 MAPK to initiate downstream inflammatory cytokine expression. Following PLA-PVA (300 µg/mL) and PLA-PEMA (300 µg/mL) treatment, cells were then stimulated with LPS (100 ng/mL) for 0.5, 1, or 4 h. (**B**) Samples were then immunoblotted for key NF-κB proteins: phospho-NF-κB p65 (Ser536), total NF-κB p65, phospho-IκB (Ser32), and total IκB. (**C**) These samples were also immunoblotted for a variety of MAPKs: phsopho-p38 (Thr180/Tyr182), total p38, phospho-ERK1/2 (Thr202/Tyr204), total ERK2, phospho-SAPK/JNK (Thr183/Tyr185), and total JNK. (**D**) Finally, to address potential impacts of iNPs on upstream signaling proteins, these samples were immunoblotted for phospho-MKK3 (Ser189/)/MKK6 (Ser207), total MKK6, phospho-TAK1 (Thr184/187), total TAK1, IRAK4, and β-Actin. Schematic in (**A**) created with BioRender.

#### changes to the BMMΦs, we used Luminex to establish that these changes in transcription *3.6. The PLA Polymer Composition of iNPs Drives the Suppression of NF-κB Signaling*

factor activation also resulted in a decrease in cytokine secretions. Indeed, we confirmed this across a multitude of signaling pathways including NF-κB-dependent IL-6 (Figure 5A), IRF3-dependent IFNβ (Figure 5B) [30], and the transcriptionally complex IL-10 (Figure 5C) [31]. Similar experiments conducted with murine macrophage-derived RAW 264.7 cells confirmed iNP-dependent decreases in IL-6 with iNPs following LPS stimulation (Supplemental Figure S5). Interestingly, PLA-PVA treatment resulted in an increase in TNF-α secretion with iNP treatment, while the opposite effect was observed with PLA-PEMA. Additionally, another potential consequence of iNP treatment is the induction of cell death driving the decrease in transcriptional activation and proinflammatory cytokines. We used flow cytometry and cell exclusion dye to establish that our iNPs do not induce cell death and can increase cell survival in the setting of LPS stimulation of BMMΦs (Figure 5D). When taken together, iNPs can drive changes in the function of BMMΦs through reprogramming of transcriptional activation. This leads to decreased proinflammatory cytokine secretions and also aids in extending survival of this cell population. Given that both of our iNP formulations produce an anti-inflammatory immunomodulatory effect, but PLA-PEMA does so more effectively, we next focused our efforts on understanding how these iNPs work by comparing our PLA-PEMA (denoted as PLA in Figures 6 and 7) to commercially available nanoparticle formulations composed of different polymer materials [polystyrene-COOH and poly(methyl methacrylate), herein referred to as PS and PMMA] (Figure 6A). Although both PS and PMMA are non-biodegradable, PS is of particular interest in that it has previously been investigated to be immunomodulatory in studies of inflammatory monocytes via a separate splenic sequestration mechanism [12]. To control for some of the physicochemical properties described as being key to this study (Figure 1), we ensured that the diameter and PDI of the commercial nanoparticles were within range of our iNPs (Supplemental Figure S6). Additionally, because we hypothesize that the lactic acid in our PLA-based iNPs plays a role in mitigating proinflammatory signaling, we also used soluble lactic acid (sLA) as a control to compare its activity to the iNPs. With LPS stimulation (Figure 6B), similarly to sLA, PLA particles suppress NF-κB p65 phosphorylation while PS and PMMA formulations did not, which confirms that the immunomodulatory activity of iNPs was dependent upon the polymers. Of note, when we

look at IκB degradation as a marker of NF-κB activation, we see that PLA particles show similar protein levels to PS and PMMA, all of which were lower than for LPS alone and LPS plus sLA. This suggests a PLA-mediated NF-κB suppression unique to the p65 subunit. We then compared the effects of the polymer on mitigating proinflammatory cytokine secretions in response to LPS (Supplementary Figure S7). Again, the PLA-based iNPs successfully suppressed inflammatory cytokine secretion while PS and PMMA showed little immunomodulatory activity as expected based on the NF-κB results. *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 11 of 19

**Figure 5.** iNP suppression of transcription activation of genes for inflammatory cytokines result in correlated decreases in cytokine production with an increase in BMMΦ survival. Supernatants from cells incubated with LPS for 48 h following iNP treatment were also collected to assess secretion of (**A**) IL-6, (**B**) IFNβ, and (**C**) IL-10, key cytokines produced downstream of TLR4 engagement. (**D**) Additionally, these cells were assessed for viability. Both iNPs, especially PLA-PEMA, result in increased survival based on flow cytometry with DAPI exclusion dye. Statistical differences between groups were determined by performing Student's *t*-test. Error bars represent SD. \* for *p* ≤ 0.05, \*\* for *p* ≤ 0.01, and \*\*\* for *p* ≤ 0.001. **Figure 5.** iNP suppression of transcription activation of genes for inflammatory cytokines result in correlated decreases in cytokine production with an increase in BMMΦ survival. Supernatants from cells incubated with LPS for 48 h following iNP treatment were also collected to assess secretion of (**A**) IL-6, (**B**) IFNβ, and (**C**) IL-10, key cytokines produced downstream of TLR4 engagement. (**D**) Additionally, these cells were assessed for viability. Both iNPs, especially PLA-PEMA, result in increased survival based on flow cytometry with DAPI exclusion dye. Statistical differences between groups were determined by performing Student's *t*-test. Error bars represent SD. \* for *p* ≤ 0.05, \*\* for *p* ≤ 0.01, and \*\*\* for *p* ≤ 0.001. *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 12 of 19

being key to this study (Figure 1), we ensured that the diameter and PDI of the commercial nanoparticles were within range of our iNPs (Supplemental Figure S6). Additionally, because we hypothesize that the lactic acid in our PLA-based iNPs plays a role in mitigating proinflammatory signaling, we also used soluble lactic acid (sLA) as a control to compare its activity to the iNPs. With LPS stimulation (Figure 6B), similarly to sLA, PLA particles suppress NF-κB p65 phosphorylation while PS and PMMA formulations did not, which **Figure 6.** Suppression of NF-κB p65 is dependent upon lactic acid polymer of the particles. (**A**) BMMΦ cells treated with soluble lactic acid (sLA), or nanoparticles composed of PLA-PEMA (PLA), polystyrene-COOH (PS), or poly(methyl methacrylate) (PMMA), for 3 h followed by 1 h LPS stimulation to show differential activation of p65 and greater baseline degradation of IκB compared to sLA and LPS alone following particle incubation and 1h LPS stimulation. (**B**) Samples were immunoblotted for phospho-NF-kB p65 (Ser536), total NF-kB p65, phospho-IκB (Ser32), total IκB, and β-actin. Schematic in (**A**) created with BioRender. **Figure 6.** Suppression of NF-κB p65 is dependent upon lactic acid polymer of the particles. (**A**) BMMΦ cells treated with soluble lactic acid (sLA), or nanoparticles composed of PLA-PEMA (PLA), polystyrene-COOH (PS), or poly(methyl methacrylate) (PMMA), for 3 h followed by 1 h LPS stimulation to show differential activation of p65 and greater baseline degradation of IκB compared to sLA and LPS alone following particle incubation and 1h LPS stimulation. (**B**) Samples were immunoblotted for phospho-NF-kB p65 (Ser536), total NF-kB p65, phospho-IκB (Ser32), total IκB, and β-actin. Schematic in (**A**) created with BioRender.

confirms that the immunomodulatory activity of iNPs was dependent upon the polymers. Of note, when we look at IκB degradation as a marker of NF-κB activation, we see that PLA particles show similar protein levels to PS and PMMA, all of which were lower than

proinflammatory cytokine secretions in response to LPS (Supplementary Figure S7). Again, the PLA-based iNPs successfully suppressed inflammatory cytokine secretion while PS and PMMA showed little immunomodulatory activity as expected based on the

**Figure 7.** GPR68 inhibition with OGM reverses lactate-mediated suppression of NF-κB p65 activation. BMMΦ cells were treated like previously with soluble lactic acid (sLA) or nanoparticles composed of PLA-PEMA (PLA) or polystyrene-COOH (PS). The addition of OGM, a GPR68 inhibitor, reverses the inhibition of p65 activation seen with PLA following particle incubation and 1-hr LPS stimulation. Samples were immunoblotted for phospho-NF-κB p65 (Ser536), total NF-κB p65, phos-

The inhibition of NF-κB p65 phosphorylation is dependent upon the PLA polymer of our iNPs. We next assessed the mechanism by which the lactic acid from the iNPs elicits its inhibition of inflammatory signaling pathways. Lactic acid is actively removed from the intracellular space [19], therefore we sought to identify the receptor through which the particle-mediated acidity is sensed. Previous work has shown that the GPR68 regulates intestinal inflammation and is a cellular pH sensor [32,33]. We hypothesized that the potential mechanism by which PLA-based iNPs work to inhibit LPS-induced inflammation is through the pH-sensing GPR68. In order to test this, we used OGM [22], a novel inhibitor of GPR68, to block the GPR68-mediated inhibition of inflammation (Figure 7). As expected, PLA iNPs alone showed less NF-κB p65 and p38 MAPK phosphorylation following LPS stimulation than LPS alone or OGM with LPS treatment alone. When we combined both PLA iNPs and OGM with LPS stimulation, not only did OGM increase the level of NF-κB p65 and p38 phosphorylation compared to just PLA iNPs, but it did so to a greater extent than the LPS only control. We further confirmed that the GPR68 inhibitor

pho-IκB (Ser32), total IκB, phospho-p38 (Thr180/Tyr182), total p38, and β-actin.

*3.7. The Protective Function of the PLA-Based iNPs Depends upon GPR68 Signaling* 

NF-κB results.

matic in (**A**) created with BioRender.

**Figure 6.** Suppression of NF-κB p65 is dependent upon lactic acid polymer of the particles. (**A**) BMMΦ cells treated with soluble lactic acid (sLA), or nanoparticles composed of PLA-PEMA (PLA), polystyrene-COOH (PS), or poly(methyl methacrylate) (PMMA), for 3 h followed by 1 h LPS stimulation to show differential activation of p65 and greater baseline

were immunoblotted for phospho-NF-kB p65 (Ser536), total NF-kB p65, phospho-IκB (Ser32), total IκB, and β-actin. Sche-

**Figure 7.** GPR68 inhibition with OGM reverses lactate-mediated suppression of NF-κB p65 activation. BMMΦ cells were treated like previously with soluble lactic acid (sLA) or nanoparticles composed of PLA-PEMA (PLA) or polystyrene-COOH (PS). The addition of OGM, a GPR68 inhibitor, reverses the inhibition of p65 activation seen with PLA following particle incubation and 1-hr LPS stimulation. Samples were immunoblotted for phospho-NF-κB p65 (Ser536), total NF-κB p65, phospho-IκB (Ser32), total IκB, phospho-p38 (Thr180/Tyr182), total p38, and β-actin. **Figure 7.** GPR68 inhibition with OGM reverses lactate-mediated suppression of NF-κB p65 activation. BMMΦ cells were treated like previously with soluble lactic acid (sLA) or nanoparticles composed of PLA-PEMA (PLA) or polystyrene-COOH (PS). The addition of OGM, a GPR68 inhibitor, reverses the inhibition of p65 activation seen with PLA following particle incubation and 1-hr LPS stimulation. Samples were immunoblotted for phospho-NF-κB p65 (Ser536), total NF-κB p65, phospho-IκB (Ser32), total IκB, phospho-p38 (Thr180/Tyr182), total p38, and β-actin.

#### *3.7. The Protective Function of the PLA-Based iNPs Depends upon GPR68 Signaling 3.7. The Protective Function of the PLA-Based iNPs Depends upon GPR68 Signaling*

The inhibition of NF-κB p65 phosphorylation is dependent upon the PLA polymer of our iNPs. We next assessed the mechanism by which the lactic acid from the iNPs elicits its inhibition of inflammatory signaling pathways. Lactic acid is actively removed from the intracellular space [19], therefore we sought to identify the receptor through which the particle-mediated acidity is sensed. Previous work has shown that the GPR68 regulates intestinal inflammation and is a cellular pH sensor [32,33]. We hypothesized that the potential mechanism by which PLA-based iNPs work to inhibit LPS-induced inflammation is through the pH-sensing GPR68. In order to test this, we used OGM [22], a novel inhibitor of GPR68, to block the GPR68-mediated inhibition of inflammation (Figure 7). As expected, PLA iNPs alone showed less NF-κB p65 and p38 MAPK phosphorylation following LPS stimulation than LPS alone or OGM with LPS treatment alone. When we combined both PLA iNPs and OGM with LPS stimulation, not only did OGM increase the level of NF-κB p65 and p38 phosphorylation compared to just PLA iNPs, but it did so to a greater extent than the LPS only control. We further confirmed that the GPR68 inhibitor The inhibition of NF-κB p65 phosphorylation is dependent upon the PLA polymer of our iNPs. We next assessed the mechanism by which the lactic acid from the iNPs elicits its inhibition of inflammatory signaling pathways. Lactic acid is actively removed from the intracellular space [19], therefore we sought to identify the receptor through which the particle-mediated acidity is sensed. Previous work has shown that the GPR68 regulates intestinal inflammation and is a cellular pH sensor [32,33]. We hypothesized that the potential mechanism by which PLA-based iNPs work to inhibit LPS-induced inflammation is through the pH-sensing GPR68. In order to test this, we used OGM [22], a novel inhibitor of GPR68, to block the GPR68-mediated inhibition of inflammation (Figure 7). As expected, PLA iNPs alone showed less NF-κB p65 and p38 MAPK phosphorylation following LPS stimulation than LPS alone or OGM with LPS treatment alone. When we combined both PLA iNPs and OGM with LPS stimulation, not only did OGM increase the level of NFκB p65 and p38 phosphorylation compared to just PLA iNPs, but it did so to a greater extent than the LPS only control. We further confirmed that the GPR68 inhibitor OGM or GPR81 inhibitor 3-OBA [34] (control) reversed the ability of PLA iNPs to mitigate proinflammatory cytokine secretions (Supplemental Figure S8). These results confirm that inhibition of inflammation is not only mediated by PLA-associated acidification of the microenvironment (negated with OGM) but is specific to sensing of the lactic acid byproduct (inhibited by 3-OBA) of PLA degradation.
