*3.3. Modulating Endodontic Pain*

While UV irradiation is not known to be directly anti-nociceptive, it does act on ERK, p38 MAPK, JNK, and NF-κB signaling pathways (Supplementary Figure S1) that produce AMPs and CCBMs that are involved in pain reduction (Supplementary Figure S2). Many of the molecules induced by UV irradiation (Table 1) can be pro-nociceptive, nociceptive, and anti-nociceptive stimuli. AMPs can be anti-nociceptive stimuli or pro-nociceptive stimuli, chemokines can be pro-nociceptive or nociceptive stimuli, and growth factors can be anti-nociceptive stimuli.

#### **4. Discussion**

We identified a list of 32 molecules expressed or secreted from cells and tissues after UV irradiation and used a bioinformatics approach to show that they were related to wound healing and innate and adaptive immune functions, including chemotaxis, movement, growth, and proliferation of cells. We then used a subset of 13 osteoinductive, angiogenic, proliferative, and proinflammatory molecules to show that HEPM cells and gingival fibroblasts treated with 255, 405, and 255/405 nm UV irradiation had different expression profiles. These results suggested that UV irradiation can activate innate and adaptive immune responses, which may supplement endodontic procedures to reduce infection, inflammation, and pain and assist tissues to heal.

Wound healing is a complex process, whereby secreted molecules from infected, injured, or damaged cells and tissues attract a variety of inflammatory cells to the injured site (inflammatory phase). Once present, inflammatory cells release additional CCBMs. These include growth factors that attract and transform fibroblasts and molecules that stimulate attracted cells to proliferate and stimulate other cells to begin forming new capillaries and blood vessels (proliferative phase). Tissue development, angiogenesis, and vasculogenesis occurs (maturation and remodeling phase). IPA identified 13/32 molecules (*<sup>p</sup>* = 1.53 × <sup>10</sup><sup>−</sup>20) in the IPA canonical pathway annotations associated with these phases in the wound healing signaling pathway (Table 2, Supplementary Table S3). Of the 13 molecules, TNFA, a proinflammatory cytokine, is secreted by activated monocytes, macrophages, B-cells, T-cells, and fibroblasts; IL6 regulates immune and inflammatory responses, including B-cell differentiation and antibody production; and IL10 inhibits the expression of pro-inflammatory cytokines, but enhances humoral immune responses and attenuates cell mediated immune reactions. CSF2 stimulates the development of neutrophils and macrophages. Growth factors including FGF1, FGF2, TGFA, TGFB1, and VEGFA are involved in cell motility; cell proliferation; cell growth and differentiation; redistribution of tissue; angiogenesis and vascular permeability of endothelial cells; and synthesis and deposition of the extracellular matrix. FN1 and collagen from fibroblasts allow tissues to contract [12], SMAD3 and SMAD4 play roles in the signaling of TGFB1 [13], and ICAM1 provides adhesion between endothelial cells and leukocytes after stress or injury.

Innate immunity is a type of nonspecific host resistance without memory, involving soluble molecules and cells [14]. Stimulation of receptors activates several cellular pathways, resulting in the production of AMPs and inflammatory cytokines. Stimulation also leads to changes in cellular metabolism, upregulation of numerous genes involved in cell defense and pathogen restriction, and the induction of regulated cell death [14].

AMPs are a large component of innate immune responses and UV irradiation induces their transcription and secretion (Supplementary Table S1). Their ability to modulate both innate immune responses, cellular immunity, and angiogenesis are very well known [15–18]. IPA identified 14/32 molecules involved in antimicrobial and antibacterial response annotations (Supplementary Table S4). These included CAMP (LL37), the defensin family (DEFB1, DEFB103A/DEFB103B, and DEFB4A/DEFB4B), the S100 family of calcium binding proteins (S100A7, S100A8, S100A9, and S100A12), and RNASE7. It also included the chemokine CCL20, which has antimicrobial activity [19] and cytokines IL6, IL10, TNFA, and TGFB1, which contribute to the antibacterial response. So far, AMP expression and secretion has been reported to occur in a narrow range from 280 to 400 nm (Supplementary

Table S1). AMP expression is increased after 280–313 nm irradiation [20,21], but not after 340 to 400 nm irradiation [22,23]. Thus, UV irradiation of an infected root canal would not have to kill 100% of microorganisms, but simply reduce the microbial infection burden to a level that could be managed by UV-induced innate immune mechanisms.

CCBMs are large components of innate immune responses and UV irradiation induces their transcription and secretion (Supplementary Table S2), CCBM expression and secretion has been reported to occur in a wider range from 254 to 404 nm (Supplementary Table S2). In irradiated cells, CCBM expression is increased for many CCBMs after 200 to 320 nm irradiation but decreased for others, such as BMP10 and FGF1 in HEPM cells and VEGF in fibroblasts [6]. At 340 to 405 nm irradiation, cells had increased levels of IL6, CXCL8, and CSF mRNA expression and secretion in keratinocytes [24,25] but decreased levels of secreted FGF1 in fibroblasts [6]. In irradiated human and murine skin at 2–3 MED (minimal erythemal dose), there were increased levels of immunostaining for CCL2, CCL20, CXCL1, CXCL8, ICAM1, IL1, IL10, SELE, SMAD3, SMAD4, TGFA, TGFB, TNFA, and VEGF 24–48 h after exposure [26–28].

UV-induced AMPs and CCBMs would have a variety of common functions (Figure 1). In addition to their potent antimicrobial activity mentioned above, these UV-induced molecules can chemoattract a variety of cells important to both immune protection and wound healing. Defensins attract keratinocytes, dendritic cells, and T-cells [29,30] and CAMP (LL37) attracts fibroblasts, microvascular endothelial cells, and human umbilical vein endothelial cells [31]. AMPs can regulate proinflammatory CCBM production [32]. At lower concentrations, defensins do not induce TNFA or IL1B expression in monocytes or macrophages [33,34]. However, at higher concentrations, defensins induce CCBM production in epithelial cells, keratinocytes, monocytes, and macrophages [30,35,36] and CAMP (LL37) induces CXCL8 in epithelial cells and macrophages [37]. Finally, AMPs and CCBMs play a direct role in wound healing, angiogenesis, and vasculogenesis [18]. DEFB4A (HBD2) increases keratinocyte proliferation [30,38] and CAMP (LL37) increases fibroblast proliferation, induces human microvascular endothelial cell and human umbilical vein endothelial cell proliferation and stimulates re-epithelialization [31,39,40].

UV irradiation also suppresses cellular immunity and acts primarily on T-cell-mediated immune reactions [41–43]. This application has been used to treat several T-cell-mediated diseases, including graft-versus-host disease and systemic scleroderma [44]. UV irradiation alters antigen specificity, alters antigen-presenting cell function, acts on effector and regulatory T-cells [41] and induces the production of CCBMs [42]. For example, UV irradiated dendritic cells do not present antigens effectively, and thus induce regulatory T-cells (CD4+CD25+), but not effector T-cells [41]. UV irradiation can lead to T-cell tolerance and prevents the priming of antigen-specific CD8+ T-cells (in models of contact hypersensitivity) independent of conventional CD4+ regulatory T-cells [44]. Tolerant CD8+ T-cells prevented migration of dendritic cells and prevented priming of other CD8<sup>+</sup> T-cells. TGFB and immunosuppressive IL10 are regulatory T-cell-associated cytokines [10,45].

There are differences based on the specific wavelength. UVB induces the infiltration of immature inflammatory myeloid CD11c+ bDCA1- dendritic cells, which may have a suppressive function [10]. UVA1 does not induce IL10, but does suppress the production of TNFA and IL12, and contributes to cis-UCA isomerization [10,46]. Immune suppression may be dependent upon the extent of UV irradiation-induced damage to DNA [41]. However, to what extent the secondary immunostimulatory effects of UV-induced AMPs and CCBMs offset the immunosuppressive effects of immune cells is not yet known.

Many oral related infections, inflammation, and tissue injury/peripheral nerve injury can be stimuli that activate receptors on the surface of cells, initiating signal transduction through MAPK and NF-κB signaling pathways (Supplementary Figure S2) [47]. The MAPK pathway regulates proinflammatory and pronociceptive molecules involved in inflammation and pain [48]. Nociceptive activity or nerve injury stimuli signal through raf and MEK1/2 to ERK in the cytoplasm and transcription factor CREB in the nucleus [48]. Chemokines (FKN), cytokines (TNFA), and nerve injury stimuli signal through TAK1

and MKK3,6 to p38 MAPK in the cytoplasm and on to transcription factor ATF-2 in the nucleus [48]. Cytokines (TNFA), growth factors BFGF (FGF2), and nerve injury stimuli signal through MLK3/MEKK1 and MKK4,7 to JNK in the cytoplasm and on to transcription factor c-Jun (AP-1) in the nucleus [48].

UV irradiation also alters the expression of ERK, p38 MAPK, JNK, and NF-κB signaling pathways [49] (Supplementary Figure S1) and it is possible that treatment of endodontic infections, inflammation, and tissue injury/peripheral nerve injury can modulate downstream production of CCBMs and be a potential intervention at these nodes to mitigate acute or chronic pain. Phosphorylation of ERK in nerve injury is induced early, is long lasting, and is involved in the induction of pain (Supplementary Figure S2). Suppressing this step in ERK is thought to be a promising strategy for treatment of neuropathic pain [50]. Likewise, targeting the p38 MAPK pathway and its signaling is also thought to be a potential therapeutic strategy for pain management [51].

Our analysis and results support those of Ou and Peterson [9] and Vieyra-Garcia et al. [10] and also suggest that UV irradiation can induce the production of AMPs and CCBMs. Our results also suggest that the production of these molecules can induce the innate and adaptive immune responses involved in attenuating infection, inflammation, and pain and enhancing healing and regeneration of tissue. However, these results are based on bioinformatics analysis of molecules induced by UV irradiation reported in the literature and produced in culture from cells treated with UV irradiation. These concepts and results form a strong hypothesis for future studies and should be examined in detail.

#### **5. Conclusions**

In summary, UV irradiation has the ability to kill microorganisms, but could also be used to activate innate and adaptive immune mechanisms in endodontic root canals directly or through UV-induced molecules. UV irradiation-induced effects appear to be wavelength specific and could supplement procedures to reduce infection, to reduce inflammation, and to facilitate local tissue healing.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/pharmaceutics14091979/s1, Supplementary Figure S1: A schematic diagram of UV-induced MAPK signaling; Supplementary Figure S2: A schematic diagram of neuropathic pain signaling; Supplementary Table S1: Antimicrobial peptides (AMPs) expressed or secreted in cells, tissues, and tissue explants after irradiation with UVC, UVB, or UVA; Supplementary Table S2: Chemokines, cytokines, and biomarkers (CCBMs) expressed or secreted in cells, tissues, and tissue explants after irradiation with UVC, UVB, or UVA; Supplementary Table S3: Antimicrobial peptides and biomarkers in the literature dataset (*n* = 32) participating in the activation of innate and immune mechanisms, as determined using IPA canonical pathways annotations; and Supplementary Table S4: Antimicrobial peptides and biomarkers in the literature dataset (*n* = 32) participating in the activation of innate and immune mechanisms as determined using IPA diseases and function annotations. Refs. [52–71] are cited in Supplementary Materials.

**Author Contributions:** The authors contributed to the study in the following aspects: Conceptualization, K.A.M., R.H.S. and K.A.B.; Methodology, K.A.M., E.Z. and K.A.B.; Validation, E.Z.; Formal Analysis, K.A.M., E.Z. and K.A.B.; Data Curation, K.A.M. and K.A.B.; Writing—Original Draft Preparation, K.A.M., R.H.S. and K.A.B.; Writing—Review and Editing, K.A.M., R.H.S., E.Z. and K.A.B.; and Project Administration, K.A.M. and K.A.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Morio et al. reported the concentrations of 13 CCBMs in tissue culture media of HEPM cells and gingival fibroblasts at 0, 24, and 48 h after treatment with 255, 405, or 255/405 UV irradiation [6]. In the current study, we used these CCBM values as a subset of data to assess the ability of UV irradiation to activate or inhibit cellular pathways related to immune functions.

**Conflicts of Interest:** The authors declare no conflict of interest. We have no financial affiliation (e.g., employment, direct payment, stock holdings, retainers, consultantships, patent licensing arrangements, or honoraria), or involvement with any commercial organization with direct financial interest in the subject or materials discussed in this manuscript, nor have any such arrangements existed in the past 3 years. Kimberly A. Morio is an Endodontist at Apex Endodontics and an Adjunct Instructor at the University of Iowa; Robert H. Sternowski is the President of Softronics, Ltd.; Erliang Zeng is a Biostatistician and a Computational Biologist at the University of Iowa; and Kim A. Brogden is an Emeritus Professor at the University of Iowa. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. The company had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.
