The present study implies that the accelerating effect of TBI on development of kindled seizures is inhibited by single i.c.v. injection of IL-4 shortly after TBI. IL-4 could reduce lesion size, and significantly rescue neurons from death in traumatic rats. IL-4 also suppressed expression of the M1 cytokine TNF-α. Moreover, IL-4 amplified overexpression of the M2 cytokines IL-10, and TGF-β, and the M2 marker Arg-1 in the damaged area. Therefore, directing TBI neuroinflammation toward an anti-inflammatory tone seems to be mainly involved in the blunting effect of IL-4 on the accelerated epileptogenesis in TBI context.
It is reported that intraperitoneal administration of IL-4 to mice 5 h before and 4 days after induction of epilepsy by pilocarpine could decrease frequency of seizures and inhibits development of epilepsy (Li et al., 2017). Therefore, we first assessed the ineffective dose of IL-4 on kindling rate in non-traumatic rats. In addition, we intended to exclude potential effect of IL-4 on peripheral immune system. Therefore, the i.c.v. route of administration was selected. Given that antiepileptogenic interventions are necessary after the presumed epileptogenic insult, in order to translate the drug-therapy protocol into practical clinical application, we administered IL-4 shortly after TBI. IL-4, 100ng/rat, slightly decreased the rate of kindling epileptogenesis. However, this effect was not statistically significant. Therefore, we examined the effect of this dose on development of kindled seizures in traumatic rats. IL-4 could successfully inhibit acceleration of kindling development in TBI state. The present study is the first report indicating that IL-4 if is administered for a while after trauma is able to prevent acceleration of epileptogenesis in traumatic rats. There are indirect studies that support finding of the present study. It is reported that the bacterial endotoxin LPS and its derivative monophosphoryl lipid A inhibit the increased potential for acquisition of seizures in traumatic rats (Eslami et al., 2015; Hesam et al., 2018). LPS can stimulate de novo IL-4 gene expression in murine microglia both in vitro and in vivo (Mukherjee et al., 2009; Lively and Schlichter, 2018). Moreover, LPS is able to induce M2 polarization through a time-dependent (Zhang et al., 2019) and species-dependent manner (Orecchioni et al., 2019). However, LPS is too toxic to be introduced in human clinical trials. In contrast to LPS, clinical studies suggest that IL-4 has acceptable safety. Clinical trials state recombinant IL-4 is safe and well tolerated in humans at the doses of 0.25–5 µg/kg/day and up to 10 µg/kg once administered 3 times/week (Leach et al., 1997; Vokes et al., 1998). Therefore, IL-4 may have significant clinical relevance for prevention of PTE.
There is strong evidence on the beneficial impacts of IL-4 on tissue repair following brain injury (Mantovani et al., 2013; Salmon-Her et al., 2000; Xiong et al., 2011). It is shown that administration of IL-4 48 h after spinal cord injury (SCI) markedly improves functional outcomes and reduces tissue damage after contusion injury (Francos-Quijorna et al., 2016). Therefore, we first examined the possible impact of IL-4 on TBI size as the primary mechanism of anti-epileptogenic effect of IL-4 in the TBI state. In our study, TTC staining showed a remarkable cortical injury 24 h after CCI. The size of the injured tissue did not expand at 48h post-TBI period. Then, 5 days after TBI, the injured area showed a size reduction. Our results are in agreement with the study by Başkaya et al., (2000). They showed that CCI with moderate severity (2 mm deformation depth) induces maximum cortical injury in rats after 1 and 2 days. Similar to our results, they found a gradual decrease in the lesion volume starting from day 4 and then a constant injury volume was observed from day 5 till 7. In our study, treatment of traumatic rats with IL-4 caused significant reduction in injured size at 48h post-TBI period. This finding is in line with the beneficial preventive effects of IL-4 on lesion development after SCI (Francos-Quijorna et al., 2016). Some research groups suggested that TTC staining has low power and cannot detect the tissue viability below a limit because the cells that are between death and survival cannot be detected by this method (Benedek et al., 2006). Moreover, this technique is only able to detect irreversible brain damage (Liszczak et al., 1984). At the same time, it is reported that despite protective effect on both neuronal and oligodendrocyte populations, IL-4 could not reduce the size of the injured tissue in SCI (Lima et al., 2017). In order to further verify the potential protective effect of IL-4 against TBI-induced tissue injury, we examined cell viability in the trauma area by PI staining. Administration of IL-4 (100 ng/rat, i.c.v.) after TBI significantly prevented development of neuronal death 48h after TBI. This finding is correlated with reduction of the injured size by IL-4 at 48h post-TBI period. In contrast to our results, intrahippocampal injection of mesenchymal stromal cells transiently expressing 100 ng IL-4, 5 days after TBI could not reduce hippocampal and cortical neurodegeneration and improve functional outcomes in mice (Enam et al., 2020). The early times after TBI are critical in subsiding development of TBI pathogenesis. Regardless of the tool used to deliver IL-4 into the CNS, we injected IL-4 acutely, up to 15 min, after TBI. This period is quite early compared to the 5 days post-TBI period of IL-4 injection used by Enam et al. This might be the main reason of positive results obtained in our study. It is known that IL-4 inhibits cell death and apoptosis (Zamorano et al., 1996). IL-4 also enhances survival of murine basophils (Reinhart and Kaufmann, 2018) and mast cells (Burton et al., 2013). Various mechanisms are suggested for this attribute of IL-4 (Zamorano et al., 1996; Reinhart and Kaufmann, 2018; Chen et al., 2020). In cerebral ischemic-reperfusion injury, which is associated with hyperexcitablity of neurons and cell death, supplemental IL-4 has been able to increase viability of cortical neurons by reducing spontaneous neuronal firing and network burst activity (Chen et al., 2020). This mechanism seems to be also involved in the preserving viability of neurons by IL-4 in our study. This suggestion is supported by the behavioral finding of our study as IL-4 could decrease brain hyperexcitablity and the accelerated rate of kindling epileptogenesis induced by TBI.
TBI is obviously associated with acute neuroinflammation and release of inflammatory cytokines. Neuroinflammation is the main causative factor in PTE (Mukherjee et al., 2020; Sun et al., 2021). Therefore, modulation of neuroinflammation would have major role in preventing PTE. Hence, we measured impact of IL-4 on the TBI-induced brain expression of TNF-α, as the typical inflammatory cytokine, and IL-10 as well as TGF-β, as the classic anti-inflammatory cytokines. Our results showed that TNF-α protein level increased in the traumatic brain to a level more than twice of the sham level at 3 and 6 h after TBI. Then the TNF-α level returned to sham level 24 h after TBI. This finding is consistent with previous studies demonstrating the increase of TNF-α protein level in rat brain during a 3-12 h period after CCI (Eslami et al., 2015) and fluid percussion injury (Taupin et al., 1993; Clausen et al., 2019). We selected the 6 h post-CCI interval, and measured TNF-α brain expression after treatment of traumatic rats with IL-4. IL-4 by itself increased TNF-α level in the sham-operated rats. This is the first report indicating increase in TNF-α level in sham (control) group by IL-4. This finding is in contrast to the previous report indicating that IL4 does not change TNF-α level in control conditions such as unstimulated monocytes (Hart et al., 1988). The difference in doses of IL-4 and conditions of the two studies (in vivo versus in vitro) might be the underlying reason for the different findings. IL-4 is predominantly regarded as an anti-inflammatory cytokine. However, there are in vitro and in vivo evidence suggesting that IL-4 by itself can also promote the inflammatory response including TNF-α and IL-1β expression in parallel (Latta et al., 2015; Ratthé et al., 2009; Van Kampen et al., 2005). The target cell, the time of application, and concentration during various phases of immune responses are determinant of pro- or anti-inflammatory effects of IL-4. The TNF-α increase by IL-4 in the sham-operated rats, is in line with the kindling data of the present study. IL-4 increased the rate of epileptogenesis in sham-operated rats, which was marginally significant (p=0.05). Given the proconvulsant effect of TNF-α (Chong et al., 2018; Lagarde et al., 2016; Riazi et al., 2008), the increased expression of TNF-α (and other inflammatory cytokines such as IL-1β) seems to be mainly involved in the acceleration of kindling development by IL-4 in non-traumatic rats. On the other hand, IL-4 blocked the increase in TNF-α level after TBI. Anti-inflammatory activity of IL-4 is attributed to suppression of TNF-α and interleukin 1-β production and activity (Hart et al., 1989; Leving et al., 1999). Considering the positive impact of TNF-α on seizure threshold and epileptogenesis (Chong et al., 2018; Patel et al., 2017; Rana and Musto, 2018) the suppressing effect of IL-4 on the acceleration of kindling development by TBI is mediated (in part) via inhibiting TNF-α increase in the traumatic brain area.
In order to verify impact of IL-4 on expression of the classic anti-inflammatory cytokines L-10 and TGF-β after TBI, the time course of expression of these cytokines was first determined. IL-10 level increased 12 h after TBI, and remained higher than basal level till 48 h. TGF-β showed different pattern of expression. The level increased 6 and 24h after TBI but then decreased to the level less than control at 48h after TBI. IL-10 is shown modest elevation in mice 24 h after CCI with the peak 3 days after CCI (Lagraoui et al., 2012). However, in a weight drop model of TBI in rats, IL-10 acutely raised in brain from 2 h after trauma followed by a progressive increase over 24 h (Kamm et al., 2006). These findings demonstrate variability in the degree of cytokine response based on the mechanism and severity of injury. Then, we determined the effect of IL-4 on brain expression of IL-10 and TGF-β at 24 h (the time of rise in both cytokines) and 48h (the time of decline in both cytokines) after TBI. At both 24 h and 48 h after sham operation, IL-4 had no effect on TGF-β brain level but increased IL-10 brain level. IL-4 has induced IL-10 expression from stimulated T helper 1 cells (Mitchell et al., 2017) as well as endotoxin-stimulated monocytes/microglia (Kambayashi et al., 1996; Cao et al., 2005) but not from unstimulated (control) cells. We report for the first time that IL-4 is able to induce IL-10 production in control condition in vivo. Administration of IL-4 to traumatic rats significantly intensified IL-10 expression at both 24 h and 48 h post-CCI periods. IL-10 is a well-known anti-inflammatory cytokine which is expressed following TBI. IL-10 inhibits development of epileptiform activity evoked by transient episodes of hypoxia in rat hippocampal slices (Levin and Godukhin, 2007), and focal convulsions in electrical kindling (Godukhin et al., 2009). IL-10 can also increase threshold of temperature-induced seizures in rats (Ishizaki et al., 2009). Thus, amplification of IL-10 expression by IL-4 plays a role in suppressing the TBI–induced acceleration of kindling development. With regard to TGF-β, IL-4 did not affect the increased TGF-β expression in traumatic rats 24 h after TBI but could significantly raise TGF-β expression at 48 h (the time of fall in the cytokine level after TBI) post-TBI period. TGF-β possesses both pro- and anti-inflammatory functions depending on the context. TGF-β is involved in epilepsy and PTE. Over-expression of cerebral TGF-β in transgenic mice causes development of a set of neuropathological complications including seizures (Wyss-Coray et al., 1995). Moreover, TGF-β and TGF-β signaling pathway are demonstrated among the main elements in development of epileptiform activity in rats after the brain injury induced by blood brain barrier disruption (Cacheaux et al., 2009). TGF-β is overexpressed 3 days after CCI in mice, and i.p. injection of TGF-β blocker is associated with a decrease in seizure behavior and EEG power spectrum (Wang et al., 2017). On the other hand, TGF-β has shown neuroprotective action against glutamate neurotoxicity and ischemic brain injury (Prehn et al., 1993). The genetic defects in the TGF-β pathway is accompanied by epilepsy in humans (Kotlarz et al., 2018). Moreover, inhibition of TGF-β signaling blocks the anti-seizure effects of the oligonucleotide miRNA inhibitors (antagomirs) in three different rodent models of temporal lobe epilepsy (Venø et al., 2020). Thus, strengthening the TGF-β expression by IL-4 also seems to play a role in suppression of TBI–induced acceleration of kindling development. Given the central role of neuroinflammation in pathogenesis of PTE (Mukherjee et al., 2020; Sun et al., 2021), amplification of anti-inflammatory response greatly contributes in the suppressing impact of IL-4 on TBI–induced acceleration of kindling development.
We found that administration of Il-4 shortly after TBI causes overexpression of IL-10 and TGF-β, and impedes expression of TNF-α. TNF-α is the typical proinflammatory cytokine released from M1 macrophage/microglia, whereas IL-10/TGF-β are the representative cytokines of M2 macrophage/microglia (Zhou et al., 2012, Laffer et al., 2019; Yao et al., 2019). Therefore, we assessed potential polarization of macrophage/microglia to M2 phenotype by measuring brain expression of the classic marker of M2 macrophage/microglia Arg-1. In our study, TBI itself was associated with Arg-1 overexpression in 24 h but not 48 h after TBI. Overexpression of Arg-1 24 h after TBI is comparable with the period of overexpression of M2 cytokines IL-10 and TGF-β after TBI as they also enhanced in our study during first 24 h after TBI. It is reported that in addition to M2 phenotype, Arg-1 is also upregulated in a fraction of murine M1 macrophage as well (Amici et al., 2017). Therefore, in addition to M2 phenotype, M1 inflammatory macrophage/microglia might be another source of Arg-1 expression in the brain of traumatic rats. In a study similar to ours, Turtzo et al., utilized CCI model of trauma and measured the cytokine expression at post-transcription level (Turtzo et al., 2014). However, they found a 5-7 days post-TBI period for microglia mixed M1 and M2 response in rats (Turtzo et al., 2014). In contrast to our study, they used female rats. Macrophage/microglia response in vivo is highly complex depending on many factors, of which gender differences is critical and seems to be the key reason for different macrophage/microglia time response between the two studies.
In our study, administration of IL-4 to sham-operated (non-traumatic) rats significantly increased expression of Arg-1 after 24h. This stimulatory effect on Arg-1 was not seen at 48h after administration, which is most likely due to short half-life of IL-4 (Conlon et al., 1989). Our finding is in line with previous study indicating temporal upregulation of Arg-1 in frontal cortex and striatum microglia of naïve control rats 8-16h after direct injection of IL-4 into the third ventricle (Pepe et al., 2014). In addition, lentiviral delivery of IL-4 into the fourth ventricle of naïve control mice has been associated with overexpression of Arg-1 of spinal cord microglia at posttranscriptional level (Rossi et al., 2018). In contrast to the time effect of IL-4 in control non-traumatic rats, we observed IL-4 administration to traumatic rats could increase Arg-1 expression 48 h but not 24 h after TBI. It is recently reported that injection of IL-4 to the hippocampus of mice 5 days after head-closed injury could not significantly increase M2-like macrophages after 48h (Enam et al., 2020). In our study overexpression of Arg-1 in traumatic rats 48h after IL-4 administration is associated with increased TGF-β expression at this time point. However, IL-10 was overexpressed both 24 h and 48 h after IL-4 administration to traumatic rats. There is a range of activation states for microglial cells that span from the M1 to the M2 phenotypes, and the phenotype of the activated microglia will fall somewhere along this spectrum depending on the signal encountered. Moreover, the in vivo effects of IL-4 are also complex, and the final response depends on the local environment, pathological state, and the doses used. These factors might be responsible for different pattern of expression of Arg-1, IL-10 and TGF-β in the traumatic rats after IL-4 administration.
Macrophages (both resident and the invaded blood borne) as well as microglia have critical role in TBI (Hirschberg and Schwartz, 1995; Kumar and Loane, 2012). Meanwhile, IL-4 directly or indirectly attracts monocytes/macrophages from the hematopoietic system to infiltrate the CNS (Rossi et al., 2018). Although macrophage may differ from microglia for their capability to respond to IL-4 signaling, they share common markers and secreted cytokines (Rossi et al., 2018). Therefore, we cannot discriminate between the extent of contribution from microglia and from macrophages to the finding of the present study. Though many methods have been developed for distinguishing between macrophages and microglia, they are challenging and inconclusive. Hence, we used the term macrophage/microglia throughout the text.
We conclude that a single i.c.v. administration of IL-4 suppresses accelerated rate of kindling epileptogenesis in traumatic rats. Various mechanisms are involved in this finding. IL-4 capability to polarize macrophage/microglia to the M2 anti-inflammatory phenotype, blocking inflammation cascade, initiating anti-inflammation processes, and protecting cells from death are supported by our study.