Increase in RONS leads to Tissue resident cell activation

Biological Plausibility is   Moderate. RONS can activate some inflammatory and anti-inflammatory pathways (TLR, TGF-β, NF-kB), and RONS are an essential part of multiple inflammatory and anti-inflammatory pathways (TLR4, TNF-a, TGF-β, NF-kB).

RONS activates or is essential to many inflammatory pathways including TGF-β  (Barcellos-Hoff and Dix 1996; Jobling, Mott et al. 2006), TNF (Blaser, Dostert et al. 2016), Toll-like receptor (TLR) (Park, Jung et al. 2004; Nakahira, Kim et al. 2006; Powers, Szaszi et al. 2006; Miller, Goodson et al. 2017; Cavaillon 2018), and NF-kB signaling (Gloire, Legrand-Poels et al. 2006; Morgan and Liu 2011). These interactions principally involve ROS, but RNS can indirectly activate TLRs and possibly NF-kB. Since inflammatory signaling and activated immune cells can also increase the production of RONS, positive feedback and feedforward loops can occur (Zhao and Robbins 2009; Ratikan, Micewicz et al. 2015; Blaser, Dostert et al. 2016).

Damage inflicted by RONS on cells activate TLRs and other receptors to promote release of cytokines (Ratikan, Micewicz et al. 2015). For example, oxidized lipids or oxidative stress-induced heat shock proteins can activate TLR4 (Miller, Goodson et al. 2017; Cavaillon 2018).

ROS is essential to TLR4 activation of downstream signals including NF-kB. Activation of TLR4 promotes the surface expression and movement of TLR4 into signal-promoting lipid rafts (Nakahira, Kim et al. 2006; Powers, Szaszi et al. 2006). This signal promotion requires NADPH-oxidase and ROS (Park, Jung et al. 2004; Nakahira, Kim et al. 2006; Powers, Szaszi et al. 2006). ROS is also required for the TLR4/TRAF6/ASK-1/p38 dependent activation of inflammatory cytokines (Matsuzawa, Saegusa et al. 2005). ROS therefore amplifies the inflammatory process.

RONS can also fail to activate or actively inhibit inflammatory pathways, and the circumstances determining response to RONS are not well known (Gloire, Legrand-Poels et al. 2006).

Empirical Evidence is Moderate. Both RONS and inflammation increase in response to agents that increase RONS or inflammation, and antioxidants reduce inflammation. Multiple studies show dose-dependent changes in both RONS and inflammation in response to stressors including ionizing radiation and antioxidants. RONS have been measured at the same or earlier time points as inflammatory markers, but additional studies are needed to characterize the inflammatory response at the earliest time points to support causation. Uncertainties come from the positive feedback from inflammation to RONS potentially interfering with attempts to establish causality, and from the large number of inflammation-related endpoints with differing responses to stressors and experimental variation.

Oxidative activity is required for or promotes the response to multiple inflammatory stressors, including ionizing radiation, UV radiation (particularly UVB), the endotoxin LPS and other pathogen associated immune activators, and hemorrhagic shock (Park, Jung et al. 2004; Nakahira, Kim et al. 2006; Powers, Szaszi et al. 2006; Zhao and Robbins 2009; Ha, Chung et al. 2010; Hiramoto, Kobayashi et al. 2012; Straub, New et al. 2015).

Both intracellular concentrations of RONS and a wide range of inflammatory markers increase in response to RONS stressors. This paired increase was observed in vivo in rodents in tissue from multiple internal organs following exposure to whole body or abdominal ionizing radiation (Berruyer, Martin et al. 2004; Ha, Chung et al. 2010; Sinha, Das et al. 2011; Sinha, Das et al. 2012; Das, Manna et al. 2014; Ozyurt, Cevik et al. 2014; Khan, Manna et al. 2015; Zetner, Andersen et al. 2016; Haddadi, Rezaeyan et al. 2017; Ezz, Ibrahim et al. 2018) or following UV skin irradiation (Sharma, Meeran et al. 2007; Hiramoto, Kobayashi et al. 2012; Martinez, Pinho-Ribeiro et al. 2016).  In vitro, the relationship has been reported in response to IR and UV in keratinocytes (Park, Ju et al. 2006; Kang, Kim et al. 2007; Martin, Sur et al. 2008; Lee, Jeon et al. 2010; Ren, Shi et al. 2016; Hung, Tang et al. 2017; Zhang, Zhu et al. 2017), immune cells (Matsuzawa, Saegusa et al. 2005; Nakahira, Kim et al. 2006; Manna, Das et al. 2015; Soltani, Ghaemi et al. 2016), as well as corneal and conjunctival epithelia, HEK cells, and vocal cord and foreskin fibroblasts (Narayanan, LaRue et al. 1999; Park, Jung et al. 2004; Saltman, Kraus et al. 2010; Black, Gordon et al. 2011; Han, Min et al. 2015). Direct application of micromolar concentrations of H2O2 in vitro also increases inflammatory markers in immune cells (Matsuzawa, Saegusa et al. 2005; Nakao, Kurokawa et al. 2008) and keratinocytes (Zhang, Zhu et al. 2017).

Interventions to reduce oxidative activity also reduce inflammation, further implicating RONS in the inflammatory process. Reduction of inflammation by these interventions has been documented in animals in response to IR (Berruyer, Martin et al. 2004; Sinha, Das et al. 2011; Sinha, Das et al. 2012; Das, Manna et al. 2014; Ozyurt, Cevik et al. 2014; Khan, Manna et al. 2015; Zetner, Andersen et al. 2016; Haddadi, Rezaeyan et al. 2017; Ezz, Ibrahim et al. 2018), UV (Sharma, Meeran et al. 2007; Lee, Jeon et al. 2010; Hiramoto, Kobayashi et al. 2012; Han, Min et al. 2015; Martinez, Pinho-Ribeiro et al. 2016; Ren, Shi et al. 2016; Hung, Tang et al. 2017) and hemorrhagic shock (Powers, Szaszi et al. 2006). In vitro, multiple studies in immune cells (Matsuzawa, Saegusa et al. 2005; Nakahira, Kim et al. 2006; Manna, Das et al. 2015; Soltani, Ghaemi et al. 2016)and keratinocytes (Park, Ju et al. 2006; Kang, Kim et al. 2007; Martin, Sur et al. 2008; Lee, Jeon et al. 2010; Ren, Shi et al. 2016; Hung, Tang et al. 2017; Zhang, Zhu et al. 2017) as well as HEK293, fibroblasts, and epithelial cells (Lee, Dimtchev et al. 1998; Narayanan, LaRue et al. 1999; Park, Jung et al. 2004; Han, Min et al. 2015) provide further evidence for reduction in various inflammatory markers with interventions to reduce RONS. Interventions include antioxidants such as propyl gallate, n-acetylcysteine, or naringin, as well as reduction in the function of NADPH oxidases (NOX/DUOX) via DPI or knockdown of gene expression. In studies using multiple doses of antioxidant, inflammation was reduced dose-dependently with the antioxidant dose (Nakahira, Kim et al. 2006; Manna, Das et al. 2015; Ren, Shi et al. 2016). Interventions reducing nitric oxide were not common, but in one study inhibiting iNOS did not reduce activation of NF-kB by IR (Lee, Dimtchev et al. 1998). The treatment to reduce RONS is administered before, or occasionally immediately after the inflammatory stressor, but experiments often continue treatment or don’t explicitly report changing media in vitro, so the exact time point at which RONS are required is difficult to pinpoint.

IR and RONS decrease endogenous antioxidant activity (glutathione, superoxide dismutase, and catalase), and antioxidants rescue this suppression in antioxidant activity (Sharma, Meeran et al. 2007; Das, Manna et al. 2014). Mice with more endogenous glutathione have a lower inflammatory response to IR (Berruyer, Martin et al. 2004), suggesting that IR increases inflammation in part by decreasing antioxidants.

In response to inflammatory stressors, RONS has been measured at the same (Nakao, Kurokawa et al. 2008; Ha, Chung et al. 2010; Saltman, Kraus et al. 2010; Azimzadeh, Scherthan et al. 2011; Ameziane-El-Hassani, Talbot et al. 2015; Azimzadeh, Sievert et al. 2015; Zhang, Zhu et al. 2017) or earlier time points as inflammatory markers (Nakahira, Kim et al. 2006; Black, Gordon et al. 2011), This suggests that RONS precedes the generation of inflammatory markers, consistent with a role for RONS in promoting inflammation. However, inflammatory markers are not typically measured at the earliest time points, and a more comprehensive survey of the appearance of these events at early time points would help to clarify the timeline and confirm the temporal evidence for causation.

A relatively small number of studies in a variety of cell types have examined both RONS and inflammatory markers across multiple doses. Three of these report dose-dependent increases in both RONS and inflammatory markers; one in which the key events are evaluated immediately after H2O2 application (Nakao, Kurokawa et al. 2008), and two others evaluating them 24 hours or 8-16 weeks after IR (Ha, Chung et al. 2010; Azimzadeh, Sievert et al. 2015). A fourth study reports a dose-dependent reduction in inflammation in response to treatment with antioxidants (Nakahira, Kim et al. 2006). In three other studies, some or all markers of inflammation increase at lower doses but decrease at higher doses (Saltman, Kraus et al. 2010; Black, Gordon et al. 2011; Zhang, Zhu et al. 2017). In two of these studies, RONS is also not consistently increasing with dose (Saltman, Kraus et al. 2010; Zhang, Zhu et al. 2017), however, this finding is consistent with findings from other studies about lack of dose-dependence of ROS measured at intermediate time points after IR. Similarly, 30 minutes after low dose, IR IL8 increases with dose while ROS does not (Narayanan, LaRue et al. 1999). The mixed inflammatory response at higher doses suggests that additional factors such as negative and positive feedback and crosstalk between pathways are also involved in the relationship between RONS and IR.

Although ROS can activate NF-KB (Gloire, Legrand-Poels et al. 2006), not all studies consistently show NF-kB activation after RONS stressor IR. It is possible that the link between ROS and NF-kB depends on the local environmental context, with different studies not adequately controlling all influential variables. One study offers a possible explanation based on temporal response: in macrophages, NF-kB was activated by shorter exposures to H2O2 (30 min), but the response disappeared with longer exposures (Nakao, Kurokawa et al. 2008).

While many models in vivo and in vitro showed a decreased inflammatory response to RONS stressors IR in combination with antioxidants, in endothelial cells in culture the increase in IL6 and IL8 after IR was not reduced by antioxidants, although a synergistic increase in those cytokines occurring with combined TNF-a and IR treatment was reduced by antioxidants (Meeren, Bertho et al. 1997). This is a reminder that multiple mechanisms can increase inflammation, that inflammatory factors participate in positive feedback loops, and that responses to stimuli vary between cells.

Since inflammatory signaling and activated immune cells can also increase the production of RONS, positive feedback and feedforward loops can occur (Zhao and Robbins 2009; Ratikan, Micewicz et al. 2015; Blaser, Dostert et al. 2016).