**4. Discussion**

Developing improved treatments for severe inflammation and sepsis is a burgeoning area where nanotechnology-based approaches hold significant promise. Current strategies under development have focused on single-target small molecules and biologics where the failure of these therapeutics in clinical trials suggests a need for strategies with broad activity against proinflammatory immune responses [3,5].

iNPs invoke multiple physical and biological mechanisms to accomplish their protective effects (Figure 8). As shown, both types of iNPs lack an ability to directly bind PAMPs including LPS and CpG ODN (Figure 2) but they do alter the ability of BMMΦs to interact with both PAMPs (Figure 3). With this change in BMMΦ-PAMP interaction, it is important to note that although a physical mechanism inhibiting BMMΦ-PAMP interactions is oc-

curring, we cannot yet formally conclude whether cell surface receptor downregulation is the sole response leading to this change (Supplemental Figure S3) or if iNPs serve to directly prevent the interaction of PAMPs with TLRs. Additionally, PLA (but not PLGA) nanoparticles have been shown to downregulate cell surface expression of CD80, CD86, and MHC class II [11,13], suggesting a mechanism by which there is global downregulation of a multitude of key immune cell surface receptors unique to PLA-based nanoparticles. Indeed, engagement of iNPs may trigger endocytosis of these receptors [35], thus making BMMΦs "blind" to PAMP stimulation and perhaps arrested from engaging T cells through the T cell receptor complex [36]. *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 14 of 19

**Figure 8.** iNPs invoke multiple physical and biological mechanisms to elicit a protective effect in BMMΦs. iNPs interact with BMMΦs to interrupt the engagement of PAMPs on TLRs at both the cell surface and endosomal surface, thus limiting the activation of TLR signaling networks. However, if LPS engages TLR4, the iNPs function via a secondary mechanism whereby their degradation triggers BMMΦ transcriptional reprogramming in response to LPS. This reduces the overall activation and production of inflammation mediators resulting in an overall protective effect to PAMP challenge. Schematic created with BioRender. **Figure 8.** iNPs invoke multiple physical and biological mechanisms to elicit a protective effect in BMMΦs. iNPs interact with BMMΦs to interrupt the engagement of PAMPs on TLRs at both the cell surface and endosomal surface, thus limiting the activation of TLR signaling networks. However, if LPS engages TLR4, the iNPs function via a secondary mechanism whereby their degradation triggers BMMΦ transcriptional reprogramming in response to LPS. This reduces the overall activation and production of inflammation mediators resulting in an overall protective effect to PAMP challenge. Schematic created with BioRender.

This proposed mechanism whereby iNPs suppress BMMΦ-PAMP compliments a similar strategy to one employed by Thamphiwatana et al. [44], where macrophage-like NPs served as a sponge for LPS and proinflammatory cytokines. Rather than induce a competition for LPS binding, our experiments show that our iNPs prevented BMMΦ-LPS and BMMΦ-CpG ODN interactions. In combination, these nanoparticle strategies could be combined to further reduce the overall interactions between BMMΦs and stimulating PAMPs. Alone, our iNPs eliminate the need for any cellular material to generate macrophage-like nanoparticles and simplify the synthesis process for the platform since it only requires off-the-shelf chemical components. This potentially avoids regulatory roadblocks in the future with any putative anti-inflammatory therapeutic containing biological components. Additionally, through usage of strictly polymer-based nanoparticles without the need of chemotherapeutic or biologic payloads, we have shown the inherent immunomodulatory capabilities of iNPs that also lend themselves to further modification to suit the needs of other potential therapeutic applications. As noted, this physical inhibitory iNP activity is assisted by the additional action of Of note, the uptake of iNPs and subsequent cellular transcriptional changes (Figure 4) appear to be independent of this iNP-mediated disruption of BMMΦ-PAMP interaction (Figure 3). Of particular interest is that these iNPs successfully mediate this disruption at two different cellular compartments given that LPS initially binds to TLR4 at the cell surface and TLR4 is then rapidly endocytosed for further LPS binding at the endosomal surface [37,38]. As a further validation of this multi-compartmental activity of iNPs compared to the stimulation of TLR4 at the cell surface prior to endocytosis, TLR9 is endosomal when it is stimulated by CpG ODN [39–41]. This suggests that the iNPs serve to disrupt multiple PAMP recognition pathways at different, distinct locations within the cell that lead to proinflammatory cytokine secretion [11], and further emphasizes their potential to serve as a broad-based therapeutic for inflammation. One curiosity that was encountered when evaluating NF-κB- and MAPK-mediated inflammatory signaling is that of all the analytes probed, iNPs drove downregulation of cytokine secretions independent of the formulation; however, TNF-α secretion was increased with PLA-PVA. TNF-α is

reprogramming the functional phenotype of these BMMΦs (Figures 4 and 5). Through alteration of BMMΦ effector activity secondary to LPS challenge, these iNPs take advantage of the inherent plasticity of BMMΦs to modify their activity at the location of

mechanism at play to synergize with the initial inhibition of BMMΦ-PAMP interactions (Figure 8). Reports of similar nanoparticle-driven innate cell reprogramming has been shown in models of spinal cord injury [45], experimental autoimmune encephalitis [46], and allergic airway inflammation [47]. The culmination of these studies aids in the idea that the iNP-mediated effects on immunomodulation alter the inherent responses of the produced downstream of NF-κB and MAPK activation, but it also has the additional characteristic of further MAPK activation downstream of engagement of its receptor TNFR1 [42]. Additionally, TNF-α exists preformed as pro-TNF-α at the cell membrane until cleavage to the activated form [43], suggesting the possibility that the PLA-PVA iNPs are less effective at inhibiting this cleavage activity.

This proposed mechanism whereby iNPs suppress BMMΦ-PAMP compliments a similar strategy to one employed by Thamphiwatana et al. [44], where macrophage-like NPs served as a sponge for LPS and proinflammatory cytokines. Rather than induce a competition for LPS binding, our experiments show that our iNPs prevented BMMΦ-LPS and BMMΦ-CpG ODN interactions. In combination, these nanoparticle strategies could be combined to further reduce the overall interactions between BMMΦs and stimulating PAMPs. Alone, our iNPs eliminate the need for any cellular material to generate macrophage-like nanoparticles and simplify the synthesis process for the platform since it only requires off-the-shelf chemical components. This potentially avoids regulatory roadblocks in the future with any putative anti-inflammatory therapeutic containing biological components. Additionally, through usage of strictly polymer-based nanoparticles without the need of chemotherapeutic or biologic payloads, we have shown the inherent immunomodulatory capabilities of iNPs that also lend themselves to further modification to suit the needs of other potential therapeutic applications.

As noted, this physical inhibitory iNP activity is assisted by the additional action of reprogramming the functional phenotype of these BMMΦs (Figures 4 and 5). Through alteration of BMMΦ effector activity secondary to LPS challenge, these iNPs take advantage of the inherent plasticity of BMMΦs to modify their activity at the location of PAMP insult. This strategy is of additional benefit in that it serves as a redundant second mechanism at play to synergize with the initial inhibition of BMMΦ-PAMP interactions (Figure 8). Reports of similar nanoparticle-driven innate cell reprogramming has been shown in models of spinal cord injury [45], experimental autoimmune encephalitis [46], and allergic airway inflammation [47]. The culmination of these studies aids in the idea that the iNP-mediated effects on immunomodulation alter the inherent responses of the BMMΦs independent of potential sequestration mechanisms. Given this change in the effector phenotype of the BMMΦs, it remains to be fully elucidated how exactly iNPs elicit these functional responses. Recent work in bone marrow-derived dendritic cells (BMDCs) with PLGAand PLA-based particles argues that the released lactate from the degradation of these particles lock dendritic cells in an immature phenotype [13,36]. This further suggests that these presumed inert polymeric materials have inherent biological activities that has thus far been under-appreciated, especially the ability of these biomaterials to functionally reprogram the in-situ activity of a variety of immune cells when challenged by known activators of innate immune cells.

Interestingly, these earlier studies and the work described herein highlight the need for increased understanding of the crosstalk between nanoparticle degradation products and the burgeoning field of immunometabolism. PLA is first biodegraded via non-enzymatic random hydrolytic ester cleavage to form oligomers and monomers of lactic acid via surface and bulk erosion [48]. These oligomers and monomers are then free to interact with cells to interact with a variety of cellular processes including the Krebs cycle [14] and, more importantly for our interests, inflammatory pathways. Although degradation of synthetic polymers is better established via passive hydrolysis rather than enzymatic reactions [49], reports in the literature note the existence of fungal [50] and bacterial [50,51] enzymes that can degrade PLA into its monomeric form suggesting a role for infection to drive PLA degradation [52]. Additionally, macrophages and other innate cells secrete an array of enzymes such as lactate dehydrogenase and its coenzyme NADH-reductase during inflammation that can catalyze the degradation of PLA in the setting of PLA implants [53].

When we consider the converse—the role of lactate in modifying the inflammatory response—we see that lactate has been established to play a role in dampening the proinflammatory response within macrophages. An early study compared the role of lactic acid

and hydrochloric acid at inducing different inflammatory patterns in RAW 264.7 stimulated with LPS. In this work they showed that when cells were titrated to more acidic environments such as pH 6.5, HCl treatment essentially drove a proinflammatory response with LPS stimulation as measured by evolution of NO, IL-6-to-IL-10, and NF-κB DNA binding. In contrast, lactic acid treatment (controlled for pH) effectively inhibited LPS-induced NO, IL-6, IL-10, and NF-κB DNA binding [54]. This work is key because it establishes that the acidity of the environment alone does not alone drive the anti-inflammatory effects that we have also observed, but rather that lactate serves as a unique molecule driving the suppression of inflammatory responses in macrophages. Further work built upon this to establish a key role for GPR81, a cell-surface receptor for lactate, in mediating lactate suppression of proinflammatory responses in the GI tract using animal models for dextran sulfate-sodium-induced colitis [55] and acute hepatitis and pancreatitis [56]. Interestingly, in other inflammatory models utilizing macrophages from non-GI sources, the role of GPR81 in lactate-mediated responses remains controversial [20,21] suggesting the potential of other pH-sensing receptors, such as GPR68, to play a complementary role [57].
