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Relationship: 2834

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Tissue resident cell activation leads to Increase, Pro-Inflammatory Mediators

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Tissue resident cell activation

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Increase, Pro-Inflammatory Mediators

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Deposition of Energy Leading to Learning and Memory Impairment	adjacent	Moderate	Low	Vinita Chauhan (send email)	Open for citation & comment	Under Review

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Term	Scientific Term	Evidence	Link
human	Homo sapiens	Low	NCBI
mouse	Mus musculus	Moderate	NCBI
rat	Rattus norvegicus	Moderate	NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Unspecific	Moderate

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
All life stages	Moderate

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

Tissue-resident cell activation refers to the stimulation of resident cells in organ systems. Tissue-resident immune cells can be found throughout the body, each tissue and organ containing specific resident immune cells (Chen et al., 2018; Gray & Farber, 2022). Monocytes, found in the blood, and macrophages, found in all tissues in the body, are the main components of the immune system (Ivanova & Orekhov, 2016). In the brain, the primary tissue-resident macrophages are microglia, while astrocytes are also important cells found in the brain (Bourgognon & Cavanagh, 2020; Greene-Schloesser et al., 2012; Wang et al., 2020). Activated tissue-resident cells can undergo gliosis, whereby they adopt a hypertrophic morphology and proliferate, exhibiting rounding of the cell body and retraction of cell processes (Greene-Schloesser et al., 2012; Phatnani & Maniatis, 2015). It is well-characterized that activated tissue-resident cells can increase expression of pro-inflammatory mediators (Hladik & Tapio, 2016; Lumniczky, Szatmari & Safrany, 2017; Kaur et al., 2019). Acute inflammation from controlled biosynthesis of pro-inflammatory mediators protects tissue and promotes healing (Kim & Joh, 2006; Vezzani & Viviani, 2015). Prolonged tissue-resident cell activation leads to dysregulation in production or secretion of pro-inflammatory mediators, which results in chronic inflammation and damage to tissue (Kim & Joh, 2006; Vezzani & Viviani, 2015). Additionally, activated tissue-resident cells can show increased levels of transcription factor nuclear factor κB (NF-κB) and activated protein 1 (AP-1) DNA binding due to increased oxidative stress or DNA damage (Betlazar et al., 2016; Lumniczky, Szatmari & Safrany, 2017). Through the activity of NF-κB, AP-1 and other signaling pathways, activated immune cells can together produce/secrete a variety of cytokines and chemokines (Betlazar et al., 2016; Chen et al., 2018; Greene-Schloesser et al., 2012; Kim & Joh, 2006; Phatnani & Maniatis, 2015; Smith et al., 2012; Wang et al., 2020). Chronic secretion of these inflammatory proteins can lead to downstream detriments, such as in the brain, altering blood-brain barrier permeability (Lumniczky, Szatmari & Safrany, 2017).

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

The strategy for collating the evidence to support the relationship is described in Kozbenko et al 2022. Briefly, a scoping review methodology was used to prioritize studies based on a population, exposure, outcome, endpoint statement.

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

Overall Weight of Evidence: Moderate

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

Tissue-resident cells recognize pathogens or molecules released by injured or activated cells (Vezzani & Viviani, 2015). In response, resident cells become activated and release various pro-inflammatory mediators (Chen et al., 2018; Gray & Farber, 2022). There is an abundance of studies which explore this relationship using the brain microenvironment, where astrocytes and microglia are the primary tissue-resident cells. After activation, these cells increase in number (whether through proliferation or recruitment), undergo morphological changes and release cytokines (Greene-Schloesser et al., 2012; Kim & Joh, 2006).

Several pathways and molecules are involved in the inflammatory response to activate tissue-resident cells. These molecules include certain pro-inflammatory cytokines and various inflammatory stimuli, such as lipopolysaccharide (LPS), thrombin (a protease), β-amyloid (Aβ), interferon (IFN)-γ, CD40 and gangliosides (Dheen, Kaur & Ling, 2007; Kim & Joh, 2006). For example, LPS can diffuse into brain parenchyma and activate microglia, which then expresses inflammatory mediators and ROS to initiate inflammation (Dheen, Kaur & Ling, 2007; Kim & Joh, 2006). As well, pattern recognition receptor activation, through molecules such as damage-associated molecular pattern molecules (DAMPs), can activate tissue-resident cells and, in turn, lead to pro-inflammatory mediator secretion (Chen et al., 2018).

Once resident cells become activated, various pathways, including the NF-κB transcription factor pathway and the mitogen-activated protein kinase (MAPK)-AP-1 signaling pathway, can result in pro-inflammatory mediator production (Chen et al., 2018; Vezzani & Viviani, 2015; Wang et al., 2020). When activated, microglia and astrocytes are sources of cytokines in the central nervous system (CNS) (Bourgognon & Cavanagh, 2020; Kim & Joh, 2006; Smith et al., 2012; Vezzani & Viviani, 2015; Boyd et al., 2021). Furthermore, uniquely in the brain, astrocyte activation can lead to blood-brain barrier permeability through decreased astrocyte function, which can allow pro-inflammatory mediators to enter from the blood (Lumniczky, Szatmari & Safrany, 2017).

An abundance of studies supports the connectivity of the two key events using activated glial cells in the brain microenvironment. Activated astrocytes express high levels of glial fibrillary acidic protein (GFAP) (Greene-Schloesser et al., 2012), while microglia activation results in the expression of OX-42, Iba1 (activated and non-activated), Mac-1, CD68, F4-80, Glut-5 and CR3/43. These markers play an important role in the phagocytic activity and morphological changes of activated microglia (Jurga, Paleczna & Kuter, 2020). Upon expression of these markers, cells release pro-inflammatory mediators, these can further activate other glial cells (Bourgognon & Cavanagh, 2020). This results in an inflammatory state, that initiates the further release of cytokines including IL-1 and TNF-⍺, IL-6, Cox-2 (Betlazar et al., 2016; Kim & Joh, 2006; Smith et al., 2012) and chemokines MCP-1 and ICAM-1 (Greene-Schloesser et al., 2012; Kyrkanides et al., 2002). Chronic activation of these cells can result in neurodegenerative disease due to their continuous release and the overexpression of potentially cytotoxic molecules, which may eventually lead to cognitive decline (Dheen, Kaur & Ling, 2007; Kaur et al., 2019; Smith et al., 2012). To suppress this activity, anti-inflammatory proteins need to be released such as tumor growth factor (TGF)-β and IL-10; these act to reduce neuron activity (Kim & Joh, 2006; Phatnani & Maniatis, 2015). In a state of chronic inflammation, the production of pro-inflammatory proteins increases, while anti-inflammatory proteins decrease, and this imbalance results in a reduced stress response (Jeon & Kim, 2016).

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

The evidence supporting the relationship between tissue-resident cell activation leading to increased pro-inflammatory mediators was gathered from studies using human (Chen et al., 2016; Lodermann et al., 2012) and mouse (Dong et al., 2015; Komatsu et al., 2017; Liu et al., 2010; Ramanan et al., 2008; Scharpfenecker et al., 2012; van Neerven et al., 2010; Welser-Alves & Milner, 2013) in vitro cell cultures as well as rat (Lee et al., 2010; Liu et al., 2010; Zhou et al., 2017) and mouse (Dong et al., 2015; Kyrkanides et al. 2002; Parihar et al., 2018) in vivo models. The stressors used were gamma rays (Kyrkanides et al., 2002; Lee et al., 2010), X-rays (Liu et al., 2010; Lodermann et al., 2012; Scharpfenecker et al., 2012), 4He ions (Parihar et al., 2018), nominal photon energy (Zhou et al., 2017) and LPS treatment (Komatsu et al., 2017; van Neerven et al., 2010; Welser-Alves & Milner, 2013). Tissue-resident cell activation is assessed by examining the morphology of activated cells (Dong et al., 2015; Zhou et al., 2017) and the detection of activation markers (Chen et al., 2016; Dong et al., 2015; Liu et al., 2010; Parihar et al., 2018; Scharpfenecker et al., 2012; van Neerven et al., 2010; Welser-Alves & Milner, 2013; Zhou et al., 2017) and AP-1 and NF-κB DNA binding (Komatsu et al., 2017; Lee et al., 2010; Lodermann et al., 2012; Ramanan et al., 2008). Pro-inflammatory mediators, including cytokines, adhesion markers, and inflammatory enzymes were assessed at both the protein and mRNA level although transcriptional responses may not necessarily translate to changes in active signaling molecules. Such studies provide indirect evidence to support the relationship.

Dose Concordance

Many studies demonstrate that activation of tissue-resident cells occurs at lower or the same doses as increased pro-inflammatory mediators. Although, at doses of ionizing radiation less than 1 Gy, evidence suggests anti-inflammatory activation of macrophages instead of towards inflammation (Wu et al., 2017). This was shown after irradiation of human monocytes from 0.1 to 0.7 Gy which resulted in decreased NF-κB nuclear translocation and decreased IL-1β levels (Lodermann et al., 2012). Dong et al. (2015) used various models and endpoints to study this relationship. In vitro mouse microglia showed an activated morphology, increased F4-80 (activated macrophage indicator), and increased levels and expression of TNF-α and IL-1β all after 16 Gy X-rays (Dong et al., 2015). With the same stressor and dose but in mice kidneys, F4-80 and various pro-inflammatory cytokines were increased (Scharpfenecker et al., 2012). In vivo mice with 10 Gy cranial X-ray irradiation showed a similar response (Dong et al., 2015). Activation of glial cells in rat brains as well as levels of IL-1β and TNF-α in BV2 murine microglial cells increased linearly after 2, 4, 6, 8 and 10 Gy X-rays (Liu et al., 2010). Multiple studies found that LPS treatment at 1 µg/mL resulted in both macrophage activation and TNF-α production (Lodermann et al., 2012; Welser-Alves & Milner, 2013). Rats with 10 Gy gamma ray irradiation showed increases in both NF-κB and AP-1 DNA binding activity, indicators of activated microglia. At the same dose, pro-inflammatory mediators TNF-α, IL-1β and MCP-1 also increased (Lee et al., 2010). When human microglia were irradiated with gamma rays, they showed a characteristic activated morphology after 8 Gy, while CR3/43 and Glut-5 (microglial activation markers) showed increased expression after 8 Gy, and IL-1α and TNF-α also showed increased expression after 8 Gy (Chen et al., 2016).

Time Concordance

Many studies observed that tissue-resident cell activation occurs earlier or at the same time as increased pro-inflammatory mediators. Mice irradiated with 4He particles showed increases in both activated microglia and pro-inflammatory chemokine CCL-3 after 1 year (Parihar et al., 2018). In vitro murine microglial cells and in vivo rat brains were irradiated with X-rays and showed increases in activated glial cells, as well as IL-1β and TNF-α pro-inflammatory mediators 24h post-irradiation (Liu et al., 2010). After mouse cranial X-ray irradiation, the number of F4-80 positive cells/mm2 was significantly increased from 3h to 2 weeks with a maximum after 24h, while TNF-α and IL-1β both showed significantly increased levels and expression at many times from 3h to 6 weeks post-irradiation, with maximum levels occurring after 24h (Dong et al., 2015). Gamma ray irradiation of rats showed increased NF-κB and AP-1 DNA binding at 4 and 8h after irradiation, with levels reduced to control after 24h, and mRNA and protein levels of pro-inflammatory mediators TNF-α, IL-1β and MCP-1 in the hippocampus and cerebellum showed a similar trend (Lee et al., 2010). Juvenile rats irradiated with 4MV photon energy showed that microglial density increased at 6 and 24 h post-irradiation in the external germinal layer of the cerebellum, while IL-1α, IL-1β, IL-6, IL-18, GRO/KC and CCL-2 were all significantly increased after 24h (Zhou et al., 2017). Human microglia irradiated with gamma rays showed a characteristic activated morphology after 7 days (Chen et al., 2016). In addition, activation markers CR3/43 and Glut-5 were expressed after 2 weeks and Glut-5 continued expression after 1 week and 10 days. IL-1α and TNF-α showed increased expression after 7 days, which was slightly lower after 2 weeks, but still significant (Chen et al., 2016).

Incidence Concordance

Two studies identified an incidence-concordant relationship between tissue resident cell activation and increase in pro-inflammatory mediators. Parihar et al. (2018) found increased microglial activation after mice were irradiated with 5 and 30 cGy 4He particles. Chemokine CCL-3 was increased slightly after both 5 and 30 cGy. Zhou et al. (2017) irradiated juvenile rats with a single dose of 6 Gy nominal photon energy (4MV) and showed microglial proliferation in the external germinal layer of the cerebellum, while cytokines (IL-6, IL-18) and chemokines (CCL-2, GRO/KC) increased significantly.

Essentiality

In the absence of tissue-resident cell activation, an increase in pro-inflammatory mediators is not expected. The activation of tissue-resident cells can be attenuated by tamoxifen. Tamoxifen, an estrogen receptor modulator that can serve as a radiosensitizer, attenuated microglial activation and significantly decreased inflammatory cytokine production, including IL-1β and TNF-α, compared to the X-ray irradiated samples (4, 6, 8, 10 Gy) in absence of tamoxifen (Liu et al., 2010). A study by van Neerven et al. (2010) pretreated cells of mice cerebral cortices with a transcriptional activator, retinoic acid (RA) containing anti-inflammatory effects, for 12 h, and then exposed the cells for another 12 h to RA and LPS, an endotoxin which induces production of IL-1β, IL-6, TNFα in astrocytes. RA, when used with LPS, led to a significant reduction of the LPS-induced release of IL-1β, IL-6, TNF-α (van Neerven et al., 2010). Similarly, N-acetyl-L-cysteine (NAC) at 40 and 80 µM was able to reduce the LPS-induced levels of IL-6, TNF-α while preventing NF-κB nuclear translocation (Komatsu et al., 2017). NF-κB and AP-1 transcription factors binding to DNA indicate activated microglia, which was observed post-irradiation in the study by Ramanan et al. (2008). Radiation led to an increase in TNFα and IL-1β expression in microglia cultures, as well as an increase in Cox-2 protein levels. Treatment with SP 600125 (SP), a specific c-jun kinase inhibitor, which prevented AP-1 binding to DNA, was found to inhibit the radiation-induced increase in TNF-α, IL-1β, and Cox-2 expression. In contrast, treating BV-2 cells with 6-amino-4-(4-phenoxyphenylethylamino) quinazoline (Q), NF-κB activation inhibitor, prevented the increase in NF-κB binding to DNA, thus blocking the activation of tissue-resident cells, which then inhibited the radiation-induced increase in IL-1β expression but allowed the radiation-induced increases in Cox-2 and TNF-α. (Ramanan et al., 2008). Proinflammatory marker, ICAM-1, increased by 2.2-fold 72 h following 35 Gy irradiation (Kyrkanides et al. 2002). However, inhibition of Cox-2, a microglial activator, by NS-398 led to a 0.6-fold decrease in ICAM-1 levels induced by radiation in the brain (Kyrkanides et al. 2002).

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

More work could be done to observe this relationship in human models.
Limited data is available to support an understanding of this relationship at low doses (<0.1 Gy).

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Modulating factor	Details	Effects on the KER	References
Drug	Flavonoids	Flavonoids can inhibit NF-κB, preventing transcription of pro-inflammatory mediators in active glial cells.	Wang et al., 2020
Drug	Tamoxifen (estrogen receptor modulator commonly used in breast cancer treatment)	Treatment with Tamoxifen decreased the radiation-induced activation of glial cells. It also consistently decreased the amount of TNF-α and IL-1β and blood-brain barrier permeability after irradiation at various doses.	Liu et al., 2010
Drug	RA (modulates inflammatory effects in different cell types)	RA treatment completely inhibited the increase in pro-inflammatory mediators after LPS-induced glial activation.	van Neerven et al., 2010
Drug	SP (JNK, c-jun N-terminal kinase, inhibitor)	AP-1 DNA binding (glial activation) was reduced by SP treatment after irradiation. TNF-α, Cox-2 and IL-1β were reduced by SP treatment after irradiation or viral infection.	Ramanan et al., 2008
Drug	Q (NF-κB inhibitor)	NF-κB DNA binding (glial activation) was reduced by Q treatment after irradiation. IL-1β was also reduced by Q treatment after irradiation.	Ramanan et al., 2008
Drug	NS-398 (Cox-2 inhibitor)	Treatment with NS-398 reduced TNF-α, IL-1β, IL-6, ICAM-1 and MCP-1 expression after irradiation.	Kyrkanides et al., 2002
Age	Increased age	Aging tissue becomes more sensitive to immune signals and increases inflammation. In the aging brain, microglia will produce more pro-inflammatory mediators.	Patterson, 2015
Drug	NAC	NAC treatment inhibited pro-inflammatory mediator production in macrophages.	Komatsu et al., 2017

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

The table below provides some representative examples of quantitative linkages between the two key events. It was difficult to identify a general trend across all the studies due to differences in experimental design and reporting of the data. All data is statistically significant unless otherwise stated.

Dose Concordance

Reference	Experiment Description	Result
Liu et al., 2010	In vivo. BV2 murine microglia were irradiated with 0, 2, 4, 6, 8 or 10 Gy X-rays to measure microglial activation, and OX-42 and GFAP staining was performed on 15 Gy irradiated rat brains to measure microglial and astrocyte activation, respectively. In vitro. BV-2 murine microglia were irradiated with 0, 2, 4, 6, 8 or 10 Gy X-rays to determine cytokines IL-1β and TNF-α production. Glial activation was identified by light microscopy and immunohistochemistry. ELISA was used to assess cytokine levels of IL-1β and TNF-α.	Irradiation of in vitro microglia cultures caused a dose-dependent increase in microglial activation from 0 to 10 Gy. At 15 Gy, in vivo astrocyte activation increased 5-fold, while microglial activation increased 3-fold. Levels of IL-1β and TNF-α production were also dose-dependently increased following 0 to 10 Gy irradiated microglia, resulting in an 8.6-fold increase for IL-1β and a 6.8-fold increase for TNF-α after 10 Gy.
Welser-Alves & Milner, 2013	In vitro. Cultures of microglia and astrocytes from postnatal mouse central nervous system were stimulated with 1 µg/mL LPS. ELISA and immunocytochemistry were used to measure glial cytokine (TNF-α) production with Mac-1 as a microglial marker.	Microglia activation with 1 µg/mL LPS led to increased TNF-α production from 40.6 pg/ml to 1875.0 pg/ml, and TNF-α showed co-localization with Mac-1 positive microglia. TNF-α was not present in astrocytes.
Lee et al., 2010	In vivo. Rats received whole-brain gamma ray irradiation at 10 Gy. Levels of AP-1 and NF-κB (microglial activation) as well as pro-inflammatory mediators TNF-α, IL-1β, IL-6, and MCP-1 were determined in the hippocampus and cortex. AP-1 and NF-κB DNA binding was determined through electrophoretic mobility shift assay (EMSA), and pro-inflammatory mediator levels were determined using enzyme-linked immunosorbent assay (ELISA).	After 10 Gy, DNA binding of NF-κB and AP-1 increased a maximum of 3.6-fold and 2.8-fold, respectively. Hippocampus: After 10 Gy at maximum, TNF-α was increased 23-fold, IL-1β increased 10-fold, IL-6 did not significantly change, and MCP-1 increased 1.6-fold. Cortex: After 10 Gy at maximum, TNF-α increased 30-fold, IL-1β increased 7-fold. IL-6 did not significantly change, and MCP-1 increased 2.2-fold.
Chen et al., 2016	In vitro. Human CHME5 microglia were irradiated with various doses of 137Cs gamma radiation delivered acutely over 1-3 min. Microglial activation markers CR3/43 and Glut-5 were determined by Western blot, morphology of microglia was determined through fluorescence microscopy, and expression of cytokines IL-1α and TNF-α were determined through RT-PCR.	After 8 Gy, microglia showed a characteristic activated morphology, but not after 0.5 Gy. CR3/43 and Glut-5 were both expressed after 8 Gy, but not 0.5 Gy. mRNA levels measured at 8 Gy were found to increase a maximum of 7.8-fold for IL-1α and 5.8-fold for TNF-α.
Dong et al., 2015	In vivo and in vitro. BV2 mouse microglial cells and C57BL/6J mice brains were irradiated with various doses of X-rays. Iba1 staining was performed to determine cell morphology, while anti-F4-80 antibodies were used to determine microglial activation. TNF-α and IL-1β levels were determined through RT-PCR, ELISA (in vitro), and Western blot (in vivo).	In vitro: At 16 Gy, microglia adopted a characteristic activated morphology while F4-80 was greatly upregulated. Also at 16 Gy, IL-1β expression increased a maximum of 23-fold, TNF-α expression increased a maximum of 13-fold, IL-1β increased from 0 to a maximum of 530 pg/mL, and TNF-α increased from almost 0 to a maximum of 115 pg/mL. In vivo: The number of F4-80 positive cells/mm2 increased from 9 to a maximum of 40 after 10 Gy. Also at 10 Gy, IL-1β expression increased a maximum of 7-fold, TNF-α expression increased a maximum of 5-fold, IL-1β levels increased a maximum of 10-fold, and TNF-α levels increased a maximum of 5-fold compared to controls.
Komatsu et al., 2017	In vitro. Murine macrophage RAW264 cell line activation was induced by 1 µg/mL LPS treatment. Activation was determined through NF-κB nuclear translocation measured by western blot. Pro-inflammatory mediators TNF-α and IL-6 were measured by ELISA.	After LPS treatment, cytosolic NF-κB decreased 0.64-fold, nuclear NF-κB increased 7.2-fold and IκB (NF-κB inhibitor) decreased 0.21-fold. Both TNF-α and IL-6 were increased from around 0 ng/mL to about 70 and 30 ng/mL, respectively.
Lodermann et al., 2012	In vitro. Human monocytic leukemia cell lines were irradiated with X-rays at 0.1, 0.3, 0.5, 0.7 and 1 Gy. Activation was determined through NF-κB nuclear translocation by western blot. IL-1β was measured by ELISA.	NF-κB and IL-1β both showed linear decreases from 0.1 to 0.7 Gy, resulting in maximum decreases of 0.6- to 0.7-fold. Although, the decrease for NF-κB was nonsignificant. No changes were observed at 1 Gy.
Scharpfenecker et al., 2012	In vitro. Mice kidneys were irradiated with 16 Gy of X-rays. Immunofluorescence staining was performed for IL-6, IL-1β and macrophage marker F4-80.	F4-80 positive area increased by 6.7 and 3.8-fold in Eng+/+ and Eng+/- irradiated mice respectively. Macrophages in irradiated mouse kidney led to IL-6 and IL-1β production.

Time Concordance

Reference	Experiment Description	Result
Parihar et al., 2018	In vivo. C57BL/6 J mice were irradiated with 4He particles 1 year after irradiation. The level of CCL-3 was determined in the brain, and microglial activation was determined by immunohistochemistry in the perirhinal cortex.	At maximum, there was a 3.8-fold increase in activated microglia, CCL-3 increased 2.2-fold.
Liu et al., 2010	In vitro. BV2 murine microglia were irradiated with X-rays to measure microglial activation after 24h, and OX-42 and GFAP staining was performed on irradiated rat brains to measure microglial and astrocyte activation, respectively, after 3 days. BV-2 murine microglia were irradiated with X-rays to determine cytokines IL-1β and TNF-α production after 24h. Glial activation was identified by light microscopy and immunohistochemistry. ELISA was used to assess cytokine levels of IL-1β and TNF-α.	10 Gy irradiation of microglia cultures caused an increase in microglial activation after 24h. At 15 Gy after 3 days, astrocyte activation increased 5-fold, while microglial activation increased 3-fold. Levels of IL-1β and TNF-α were also increased in 10 Gy irradiated microglia, resulting in an 8.6-fold increase for IL-1β and a 6.8-fold increase for TNF-α after 24h.
Lee et al., 2010	In vivo. Rats received whole-brain gamma ray irradiation at 10 Gy. Levels of AP-1 and NF-κB (microglial activation) as well as pro-inflammatory mediators TNF-α, IL-1β, IL-6, and MCP-1 were determined 4, 8, and 24h after irradiation in the hippocampus and cortex. AP-1 and NF-κB DNA binding was determined through electrophoretic mobility shift assay (EMSA), and pro-inflammatory mediator levels were determined using enzyme-linked immunosorbent assay (ELISA).	DNA binding of NF-κB and AP-1 increased a maximum of 3.6-fold and 2.8-fold, respectively, after 8h. Binding activity returned to control levels after 24h. Hippocampus: At control, TNF-α was 3.6 pg/mg protein, which increased 23-fold after 4 hours, 8.3-fold after 8h, and 3.6-fold after 24h. IL-1β increased linearly from 4 to 24h, reaching a 10-fold maximum increase. IL-6 did not significantly change other than a non-significant decrease over 24h, even though an increase in IL-6 mRNA was observed at 4h with RT-qPCR. MCP-1 showed a maximum increase of 1.6-fold after 8h. Cortex: At control, TNF-α was 4.4 pg/mg protein, which increased 30-fold after 4 hours, 13-fold after 8h, and 4.1-fold after 24h. IL-1β showed a maximum increase of 7-fold after 4h. IL-6 did not significantly change other than a non-significant decrease over 24h, even though an increase in IL-6 mRNA was observed at 4h with RT-qPCR. MCP-1 showed a maximum increase of 2.2-fold after 8h.
Chen et al., 2016	In vitro. Human CHME5 microglia were irradiated with 8 Gy gamma radiation (137Cs source) delivered acutely over 1-3 min. Microglial activation markers CR3/43 and Glut-5 were determined by Western blot, morphology of microglia was determined through fluorescence microscopy, and expression of cytokines IL-1α and TNF-α were determined through RT-PCR.	Beginning after 7 days, microglia showed a characteristic activated morphology. CR3/43 was expressed after 2 weeks, while Glut-5 was expressed after 1 week, 10 days, and 2 weeks. After 7 days, mRNA levels were found to increase a maximum of 7.8-fold for IL-1α and 5.8-fold for TNF-α. mRNA levels dropped slightly but were still above controls after 2 weeks.
Zhou et al., 2017	In vivo. Juvenile rats were irradiated with 4MV nominal photon energy and a single dose of 6 Gy (2.3 Gy/min). At 6 or 24h, the molecular and cellular changes in the EGL of the cerebellum was studied. Immunohistochemistry staining was used to measure Iba1 (microglia marker) with morphometry analysis performed on microglia. Luminex assay measured cytokines, chemokines and growth factors for the inflammatory response.	Microglia density increased by 2.3-fold after 6 h and 6.77-fold 24 h post-irradiation. Most of the iba1-positive cells had a bushy or amoeboid morphology, signifying an activated state. At 6 h of irradiation, IL-1α and CCL-2 increased by 2-2.8-fold. IL-1β decreased at 6 h then slightly increased at 24 h post-irradiation. IL-6, IL-18, GRO/KC, VEGF, and GM-CSF all increased significantly at 24 h compared to the control group.
Dong et al., 2015	In vivo and in vitro. BV2 mouse microglial cells and C57BL/6J mice brains were irradiated with X-rays. Iba1 staining was performed to determine cell morphology, while anti-F4-80 antibodies were used to determine microglial activation. TNF-α and IL-1β levels were determined through RT-PCR, ELISA (in vitro), and Western blot (in vivo) from 3h to 6 weeks after irradiation.	In vivo, the number of F4-80 positive cells/mm2 increased from 9 to a maximum of 40 after 24h, decreased over 2 weeks, and returned to control levels at 4 and 6 weeks. IL-1β expression increased to a maximum after 72h and was significantly increased from 6h to 6 weeks. TNF-α expression was a maximum after 3 and 6h, was at controls from 24h to 1 week, and was increased again from 2 to 6 weeks. IL-1β levels were high after 3h, lower at 6h, increased to a maximum at 2 weeks, then decreased at 4 and 6 weeks. TNF-α levels increased to a maximum after 72h, then decreased until 4 weeks, where they increased again after 6 weeks. In vitro. IL-1β levels reached a maximum at 3h, but then decreased at 6h, before rising again at 12h. TNF-α levels remained elevated up to 24h, although its peak was at 6h.

Incidence Concordance

Reference

Experimental Description

Result

Parihar et al., 2018

In vivo. C57BL/6 J mice were irradiated with 4He particles at either 5 or 30 cGy (5 cGy/min). The level of CCL-3 was determined in the brain, and microglial activation was determined by ED-1 immunohistochemistry in the perirhinal cortex.

Microglial activation increased 3.5-fold after 5 cGy and 3.8-fold after 30 cGy. CCL-3 increased 1.4-fold (non-significant, ns) after 5 cGy and 2.2-fold after 30 cGy.

Zhou et al., 2017

In vivo. Juvenile rats were irradiated with 4 MV nominal photon energy and a single dose of 6 Gy (2.3 Gy/min). At 6 or 24h, the molecular and cellular changes in the EGL of the cerebellum was studied. Immunohistochemistry staining was used to measure Iba1 (microglia marker) with morphometry analysis performed on microglia. Luminex assay measured cytokines, chemokines and growth factors for the inflammatory response.

Microglia density increased by 2.3-fold after 6 h and 6.77-fold 24 h post-irradiation. Most of the iba1-positive cells had a bushy or amoeboid morphology, signifying an activated state.

At 6 h of irradiation, IL-1α and CCL-2 increased by 2-2.8-fold. IL-1β decreased at 6 h then slightly increased at 24 h post-irradiation. IL-6, IL-18, GRO/KC, VEGF, and GM-CSF all increased significantly at 24 h compared to the control group.

Response-response Relationship

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Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

Known Feedforward/Feedback loops influencing this KER

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It is well-characterized that activated tissue-resident cells can increase expression of pro-inflammatory mediators (Hladik & Tapio, 2016; Lumniczky, Szatmari & Safrany, 2017; Kaur et al., 2019). However, there exists a feedforward loop for this key event relationship as pro-inflammatory mediators can also activate tissue-resident cells within the brain and perpetuate the inflammatory response (Kim & Joh, 2006; Vezzani & Viviani, 2015). Thus, after stimulation by cytokines, chemokines or inflammogens such as from damaged neurons, microglia and astrocytes activate inflammatory signaling pathways, which result in increased expression and/or release of inflammatory mediators such as cytokines, eicosanoids, and metalloproteinases (Dong & Benveniste, 2001; Bourgognon & Cavanagh, 2020). Various studies have shown that overexpression of IL-1β in mouse models resulted in the appearance of inflammatory markers including activated glial cells and increased pro-inflammatory cytokine and chemokine mRNAs (Hein et al., 2010; Moore et al., 2009). Additionally, IL-6 plays a role in activating glial cells as mouse models with IL-6 knocked out showed reduced astrocytic population, as well as a reduced ability in activating microglia (Klein et al., 1997). Cytokines and chemokines can also increase the permeability of the blood-brain barrier, further increasing pro-inflammatory mediator levels (Lumniczky, Szatmari & Safrany, 2017).

Domain of Applicability

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Evidence for this relationship comes from in vitro mouse- and human-derived models, as well as in vivo mouse and rat models. The relationship is not sex or life stage specific.

References

List of the literature that was cited for this KER description. More help

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