Altered Inflammasome and Immune activation in Paediatric Traumatic Brain Injury

Altered Inflammasome and Immune activation in Paediatric Traumatic Brain Injury | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Altered Inflammasome and Immune activation in Paediatric Traumatic Brain Injury Emer Ryan, Lynne Kelly, Ashanty M Melo, Cian P Morgan, Mark Bates, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4172622/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction : Systemic Inflammation is associated with Traumatic Brain Injury (TBI) and therefore is a potential target for immunomodulation. Dysregulated immune function post-TBI increased susceptibility to infection and post-concussive syndrome. The inflammasome is a protein complex associated with an amplified proinflammatory response and is a potential target for immunomodulation that preserves antimicrobial immunity. Methods : Samples from children with mild TBI (mTBI; Glasgow coma scale (GCS) 14/15), severe TBI (sTBI; GCS < 8) and control children were collected at baseline and two week follow up and were treated with endotoxin and melatonin. Toll-like receptor (TLR4; marker of endotoxin responses) and CD11b (activation marker) expression on neutrophils and monocytes were evaluated by flow cytometry. Inflammasome-related genes and cytokines were assessed using TaqMan RT-PCR samples ELISA sandwich immunoassay, respectively. Results : A total of 214 children were enrolled including: TBI (n = 116), with mild TBI (mTBI; Glasgow coma scale (GCS) 14/15) and severe TBI (sTBI; GCS < 8), and (n = 98) control patients collected at baseline and two week follow up. Total monocyte and intermediate monocyte populations were reduced in mTBI at baseline. Neutrophil CD11b and TLR4 expression was decreased in mTBI at 10–14 days. NLRP3 and NLRP1 were downregulated at 10–14 days while IL-1β was increased at baseline at 0–4 days and further elevated by 10–14 days and significantly higher in those with no previous mTBI. Serum cytokines showed lower IL-18 and raised IL-33 in those with mTBI. Prior concussion did not influence serum cytokine levels. In addition, LPS did not stimulate an IL-18 and IL-1β response in the mTBI group at 10–14 days. Conclusions : Children with mTBI had reduced CD11b and TLR4 expression and NLRP3 inflammasome activation. IL-1β mRNA was raised and continued to rise after injury implicating the innate immune system in the subacute phase of injury. Immune dysregulation post-TBI in children may be a target for immunomodulation following further exploration in vitro of potential mechanisms and therapies. Traumatic Brain injury Concussion Inflammation Cytokines Innate Immunity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Childhood Traumatic Brain Injury (TBI) is an acquired brain injury defined as an insult to the brain from an external force that leads to temporary or permanent impairment of cognitive, physical, or psychosocial function. TBI can be divided by severity as mild, moderate, or severe and may be open (penetrating) or closed (non-penetrating) [ 1 ]. Mild TBI (mTBI) results in chronic and debilitating symptoms for a number of children with post concussive syndrome affecting almost a third of children, resulting in a significant number of missed days from school [ 2 ]. In children, TBI has longer-term effects associated with injury to a developing brain. Neuroinflammation and immune dysfunction are known to persist following TBI. Changes in the cell populations after TBI have been described in both animal [ 3 ] and human populations [ 4 ]. Severe TBI (sTBI) is known to induce leukocytosis, microglial activation and cytokine release. The immune profile is less well described in mTBI and the extent to which the mediators of inflammation involved in sTBI are involved in mTBI is not known. Mild TBI may also activate immune pathways with persistent immune dysfunction contributing to ongoing symptoms. This may also be involved in the phenomenon whereby a repeat mTBI tends towards prolonged symptoms relative to a first injury. Mild Traumatic Brain injuries have been associated with long-term cognitive deficits and neurodegeneration [ 5 ]. Various animal models have been developed which mimic moderate and sTBI with controlled cortical impact and fluid percussion injury to mimic blast injury. These models are reproducible and supply a fixed and measurable force in genetically identical breeds of mice with or without gene knock-out as comparators. Mild TBI in the human model is much more complex. The age and gender of the patient, the presence of prior injuries, the type of injury and the presence of prior systemic inflammation all vary. The burden of symptoms from a ‘simple’ school yard fall may be great, the area of the injury and the torque of the fall will also vary from injury to injury. Neutrophils and monocytes play a key role in severe TBI and infiltrate the brain early. Neutrophil populations expand in response to sTBI, and peak neutrophil count has been associated with unfavourable outcomes [ 6 ]. CD11b (a β 2 integrin) is a surface receptor that aids adherence of polymorphonuclear cells to the endothelial cell wall, facilitating their migration to the site of injury. It is well described that those with prior mTBI have worse symptoms for longer and are at higher risk of post concussive syndrome [ 7 ]. Experimental models show ongoing microglial activation, spreading of lesion volumes and neurodegeneration a year from the injury [ 8 ] and animal models, have altered microglial activation in the wake of injury with reduced oxidative damage [ 9 ]. A second injury during the ‘healing phase’ may result in worse outcomes especially if repeat mTBI is within a year of the first injury [ 7 ]. The inflammasome has been implicated in mTBI. The inflammasome complex is a self-perpetuating canonical structure that can be triggered by a number of pathways including the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3), NLR Family Pyrin Domain Containing 1 (NLRP1) and Absent in Melanoma − 2 (AIM2). Downstream nuclear factor–κB (NF-κB) triggers the expression of IL-1β. The inflammasome proteins have recently been used as biomarkers and prognosticators in sTBI [ 10 ]. NLRP3 has been targeted to modulate oedema in animal models of sTBI by administration of the drug pioglitazone which reduces NLRP3 expression [ 11 ]. Modulating NLRP3 shows better cognitive and motor outcomes in mouse models [ 12 ]. Dietary omega three fatty acids had beneficial effects in mTBI and attenuated NLRP3 [ 13 ] with a resultant decrease in IL-1β, IL-18 and IL-6 expression. We hypothesised that there is an altered innate immune phenotype in children with mTBI when compared to healthy control children which may be associated with inflammasome immune complex activation and may alter the systemic immune response following mTBI. We aimed to examine neutrophil, monocyte and inflammasome activation in paediatric TBI. Methods Study Population Children with mTBI were eligible if they had sustained either a direct blow to the head or an acceleration/deceleration movement of the head and had a GCS score of either 14 or 15 on presentation to the emergency department, as well as any one of the following: (i) loss of consciousness < 30 minutes, (ii) amnesia, (iii) any alteration in mental state at the time of the injury (i.e. agitation, irritability, sleepiness, lethargy, slow to respond, dazed or asking repetitive questions) or (iv) physically symptomatic of head injury with vision disturbance, ataxia, nausea, headache, dizziness, hearing disturbance. All children in the mild cohort had a GCS of 14–15. Children with extracranial injuries or bruising > 5cm were excluded. The severe TBI cohort had a lowest GCS of less than 8 and required intubation, ventilation and an Intensive Care stay. Paediatric controls included children attending for phlebotomy or day case procedures with normal results and clinical outcomes. Children in all groups were excluded if they had recent fever or evidence of infection [ 14 , 15 ]. Demographics recorded included birth history, developmental history including diagnoses of learning disorders, parental or self-reported anxiety, previous hospital presentations of head injury or self-reported prior ‘concussion’ or mTBI naive. A history of travel sickness, need for visual aids in the form of glasses and migraine was reported. Family history in first degree relatives of concussion, depression or migraine was recorded. The cause of the injury was recorded. The Ethics Committees of Children’s Health Ireland (CHI) at Tallaght and CHI at Temple Street Dublin Ireland approved the study. Experimental design: Blood samples (1-3mL) were collected in sodium citrate tubes and analysed within two hours of phlebotomy. Whole blood was incubated for 1 hour with vehicle, LPS (10ng/mL) (E Coli 0111:B4 Sigma Life Science Wicklow) and/or melatonin (10 − 3 M) (SIGMA, Ireland). Antibodies and Flow Cytometry: Blood samples were incubated with a dead cell stain (Fixable Viability Dye eFluor 506, Invitrogen, California, USA), diluted to working concentration in phosphate-buffered saline (PBS). The following fluorochrome-labelled monoclonal antibodies (mAb) were added to each sample: CD14-PerCP, CD15-PECy7, CD16-FITC, CD66b-Pacific Blue, and TLR4-APC (BioLegend®, California, USA) and PE-labelled CD11b (BD Biosciences, Oxford, UK). PBA buffer (PBS containing 1% bovine serum albumin and 0.02% sodium azide), prepared in house, was used to make up the antibody cocktail. Samples were incubated in the dark for 15 minutes before the addition of 1 mL FACS lysing solution (BD Biosciences, Oxford, UK), and the samples were then incubated for 15 minutes in the dark in order to lyse contaminating red blood cells. Cells were pelleted by centrifugation at 450g for 7 minutes at room temperature, washed twice with PBA buffer, and fixed in 300 µL of 1% paraformaldehyde. The final cell pellet was resuspended in 100 µL PBA buffer and analysed on a BD FACSCanto II flow cytometer. The expression of TLR4 and CD11b antigens on the surface of neutrophils and monocytes was evaluated by flow cytometry. Neutrophils were delineated based on SSC-A and CD66b + positivity as previously described [ 16 ], and monocytes were defined based on SSC-A and CD66b negativity and their subsets based on CD14 and CD16 expression: classical (CD14+/CD16-), intermediate (CD14+/CD16+) and nonclassical (CD14dim/CD16+) [ 17 ]. A minimum of 10,000 events were collected, and relative expression of TLR4 and CD11b was expressed as mean fluorescence intensity (MFI). Flow cytometry data was analysed using FlowJo software (Oregon, USA) [ 18 ]. PCR : Following incubation of samples, 1mL of TRIzol™ (Thermo Fisher) was added to 0.3mL of whole blood. Chloroform was added and the samples were incubated for 5mins at room temperatures. Following lysis, the aqueous phase was used to isolate RNA. Purity and concentration were evaluated using an NanoDrop ND- 8000 Spectrophotometer and analysed using ND-ver 2.3.3. software. Total RNA was reverse transcribed to single-stranded cDNA using the High-Capacity cDNA Archive Kit (Applied Biosystems) following the manufacturer’s protocol and stored at -80°C until use. The evaluation of gene expression was performed by TaqMan® RT-PCR. Commercially available TaqMan® primer and probe combinations were used to detect the expression of the following inflammasome genes, IL-1β (Hs00174097_m1), NLRP3 (Hs00918082_m1), NLRP1 (Hs00248187_m1) and AIM2 (Hs00915710_m1), and measured at baseline and at two weeks. NLRP3 and IL-1β were also measured on samples treated with LPS and melatonin. All samples were assayed in triplicate. Thermal cycling conditions were as follows: 2 minutes at 50°C, 10 minutes at 95°C, and, for 40 cycles, 24 seconds at 95°C and 1 minute at 60°C, using the 7900HT Fast Real-Time PCR System. Relative quantification (RQ) values were calculated using the 2-ΔΔCt method [ 19 ]. Multiplex ELISA A multiplex ELISA immunoassay from Meso Scale Diagnostics (Rockville, Maryland USA) was performed using a 10-spot human serum plasma U-plex ELISA plate (Meso Scale) which was customised for the study and plates were analysed SECTOR Imager. Cytokines analysed included IL-1α IL-1ß, IL-33, IL-18, and IL-1ra. The limits of detection for the individual assays were within expected ranges. Statistics: Where data was normally distributed unpaired t-tests were performed. The Shapiro-Wilk test was performed to test normality. When it failed the Shapiro-Wilk test the non-parametric Mann Whitney test were used. NLRP1, AIM2 and IL-1β were assessed with Mann Whitney tests. For cytokine analysis, where the sample was below the lower limit of detection, the lower limit of detection for that assay was substituted [ 20 ] and Mann Whitney tests employed. The assay reproducibility was calculated using the intra-variation of the standard curves which was within an acceptable range. The median of the lower limit of detection for all plate runs had a closely aligned average value, demonstrating the reproducibility of the assay. When LLOD were replaced, over 50% of the sample were undetected for the IL-1β cytokine. No upper limits of detection were reached. Results In total 214 children were recruited including 116 children with TBI (mTBI n = 110; sTBI n = 6) and 98 control children. Samples were available for 98 mTBI patients as 12 recruited patients declined phlebotomy. The mean (SD) age of mTBI recruited was 11.2 +/- 4.0 years, with an age range of 7 months − 16 years. There were six children with severe TBI recruited, mean age 7.9 +/- 5.0 years and 98 control children, mean age 8.04 +/-4.2 years. Neutrophil and activation: Flow data was available for n = 10 mTBI at 0–4 days, n = 7 mTBI at 10–14 days and 4 children with sTBI. There was a general suppression of CD11b expression in the mTBI group at 10–14 days in the unstimulated vehicle and in the melatonin treatment group, (p = 0.0026 and p = 0.0042, respectively; Fig. 1 a) compared to controls. Children with severe TBI did not have reduced expression of CD11b compared to controls. Lipopolysaccharide increased CD11b expression in all cohorts compared to baseline. Toll-like receptor-4 (TLR4) expression in the neutrophil was increased in the mTBI cohort versus controls at baseline, reaching statistical significance in the melatonin cohort (p = 0.02). TLR4 expression was decreased at 10–14 days compared to controls and did not respond to LPS stimulation in mTBI. TLR4 expression was increased in the severe TBI cohort reaching statistical significance in the vehicle and LPS groups (p = 0.006 and p = 0.0002, respectively) compared to controls (Fig. 1 b). Melatonin decreased the TLR4 expression in the mTBI cohort but did not reach significance compared to baseline. Monocyte Activation The total monocyte population was reduced in mTBI for LPS, melatonin and LPS + melatonin treatment groups compared to controls (p = 0.04, p = 0.006, p = 0.01 respectively) (Fig. 2 a). Intermediate monocytes were decreased in mTBI, across all treatments groups treatment groups compared to controls (p = 0.03, p = 0.017, p = 0.04 respectively; Fig. 2 c). There were no significant differences within classical or non-classical monocyte populations (Fig. 2 b and 2 d). Inflammasome Pathway The inflammasome is a key regulator of pro-inflammatory genes. Controls (n = 15) were compared to mTBI (n = 15). In this cohort of mTBI, both NLRP3 and NLRP1 were downregulated compared to controls at presentation and at 10–14 days (p = 0.0019 and p = 0.0082) for NLRP3 (Fig. 3 a) and for NLRP1 (p < 0.0001) (Fig. 3 b). AIM1 which is a key regulator was upregulated compared to controls at 0–4 days (p = 0.02) (Fig. 3 c), and no effect was seen with AIM2 or ASC (Fig. 3 d). IL-1β was increased at baseline at 0–4 days and further elevated by 10–14 days (p = 0.0004 and p = 0.0002 respectively (Fig. 3 f) in mTBI. Patients with prior mTBI Children with prior concussions tend to have more prolonged symptoms on subsequent presentations prompting exploration of the differences in gene expression in those with and without (naïve) prior self-report of concussion or hospitalisation for head injury. IL-1β was significantly higher in those who were mTBI naïve (p = 0.003) at baseline and two weeks (p = 0.003) (Fig. 4 f), whereas at two weeks ASC was significantly higher in those who had a repeat injury compared to those who were mTBI naïve (304.5 v 19.85%; p = 0.001) (Fig. 4 e). NLRP3 was reduced in mTBI naïve, and this reduction was significantly higher in those with a repeat injury (Fig. 4 a). NLRP1 was also reduced in mTBI compared to controls in naïve and repeat injury (Fig. 4 b), while no significant differences were seen in AIM or AIM2 (Fig. 4 c and 4 d, respectively). Inflammasome-related serum Cytokine responses Cytokine data was available for patients with mTBI (n = 98), patients with mTBI two weeks post-injury (n = 29), and patients with sTBI (n = 6) compared to controls (n = 104). When inflammasome related serum cytokines were examined, IL-18 was lower in mTBI compared to controls (3013 v 2309pg/mL; p = 0.0001) (Fig. 5 ). IL-33 was elevated in mTBI (4.55 v 2.97 pg/mL; p = 0.04; (Fig. 5 ). IL-1β was not increased, however over 50% of the samples had undetectable levels of expression. IL-1ra was decreased in the mTBI group compared to controls (1461 v 981.9. pg/mL; p = 0.02) (Fig. 5 ). Prior concussion had no effect on the cytokine levels either at baseline or 2 weeks post-TBI. IL-1α which acts on the IL-1 receptor was elevated at baseline (35.51 v 24.45pg/mL; p = 0.01). IL-18 was suppressed in the control cohort in response to LPS, p = 0.006 (Fig. 5 ). This effect was not seen in the mTBI or sTBI cohorts, demonstrating hypo-responsiveness. Only the sTBI group responded to LPS stimulation with increased IL-33 production. IL-1β showed LPS responsiveness in controls, mTBI at baseline and sTBI (p = 0.0005, p < 0.000001, p = 0.003, respectively) (Fig. 5 ). This response was not seen in the mTBI at 10–14 days, the subacute phase, demonstrating LPS hypo-responsiveness. IL-1ra showed LPS responsiveness only in the mTBI and sTBI cohorts, demonstrating pre-conditioning while the controls and mTBI in the subacute phase did not respond. IL-1α increased in response to LPS in the mTBI at baseline and at 10–14 days and also the severe cohort again demonstrating pre-conditioning. The control population did not respond to LPS challenge with an increase in IL-1α production. There was a significant difference in the level of response to LPS between the controls, who did not respond and mTBI (p < 0.00001). Discussion Children with mTBI had a reduction in activated neutrophils, evidenced by reduced CD11b expression in the mTBI cohort reaching a significant decrease at 10–14 days post-injury. In parallel, total monocytes were reduced acutely with the reduction seen in intermediate monocytes. The TLR4 expression on neutrophils was highly upregulated in the acute phase and downregulated by 10–14 days, below that of control levels. Activated CD11b neutrophils play an important role in blood-brain barrier disruption and cytokine release in traumatic brain injury [ 21 ]. Infiltrating CD11b + neutrophils contribute to the size of the lesion in severe TBI [ 22 ]. Although severe TBI in this cohort had modest increases in CD11b, in this study the response in mTBI is reduced systemic CD11b expression. The dynamics of neutrophil survival differ with age [ 23 ]. This is exemplified in a neonatal study which showed an increase in both CD11b and TLR4 expression in infants with neonatal encephalopathy [ 24 ] which persisted over the first week of life with dysfunctional immune responses in later childhood [ 25 ] [ 26 ]. Elderly people have decreased CD11b expression, a phenomenon contributing to immune senescence [ 27 ] compromising their immune response. Age differences may be a factor in this decreased responsiveness, or the type of injury presenting, as mild TBI is distinct from severe TBI. Our cytokine analysis shows trends of immune suppression, consistent with adult studies of blast TBI [ 28 ] rather than the typical elevation of IL-8 seen in severe TBI [ 29 ]. There may have been an initial peak in immune activation that was not captured in this cohort as the highest neutrophil count in severe TBI is in the first three hours [ 30 ]. Systemic TLR4 expression was activated in mTBI and sTBI, especially in the latter group. However, TLR4 expression was suppressed in the subacute phase of mild traumatic brain injury. The rise of TLR4 production in the severe cohort was significant. The TLR4/NF-κB pathway has been demonstrated to play an important role in traumatic brain injury [ 31 ]. The TLR4 complex is expressed on the cell surface of the neutrophil and plays an important role in targeting inflammatory cytokine gene transcription. Tang et al. found that IL-1β and IL-6 were significantly lower in the wounds of TLR4-deficient mice [ 32 ]. When the receptor for TLR4 is activated, immune mediators released from TLR4-expressing cells may have neurotoxic effects [ 33 ]. Total monocytes were depleted in the initial period following mTBI, within the intermediate subset, with a recovery seen by 10–14 days. This is opposite to the effect seen in severe TBI where the monocytes were increased in the intermediate population, not reaching statistical significance. Monocytes aid recovery in spinal cord decompression [ 34 ] and their depletion in the animal model results in poorer motor results [ 34 ]. There is discrepancy in the literature, as a murine model shows depleted monocytes in the first day following TBI that persists for a month [ 35 ], with human studies showing elevation of the monocyte population in those with severe TBI [ 30 ]. The monocyte population has not been documented in those with mTBI. The innate immune response is a significant contributor to inflammation after TBI regulated by the inflammasomes. Activation of IL-1β, the end product of inflammasome activation, includes pathways via TLR4/NF-κB, via Nod-like receptor pathways including NLRP3 and NLRP1 and via AIM1 pathways. These pathways activate the inflammasome via caspase driving cytosolic NF-κB transcription and IL-1β production, driving pyroptosis. The interleukin-1 family has autocrine properties and can self- propagate its own signal with IL-1α having local autocrine effects and IL-1β having paracrine effects [ 36 ]. Our study did not demonstrate an upregulation of the NLRP3 pathway in the mild form of TBI in children. The pathway was downregulated, and more so if there was a prior concussion. NLRP1 was also downregulated, which persisted into week two. The AIM pathway was upregulated on presentation. ASC was not statistically higher and was low in those previously suffering mTBI. ASC and IL-18 biomarker has previously proven to be a useful in determining outcome in severe TBI [ 37 ]. In our study IL-18 was depressed however, not raised. This may be accounted for in the mild and different nature of an mTBI to sTBI or by age. IL-1β mRNA was significantly upregulated at presentation with injury. At 10–14 days from injury, it was higher still showing an important role of inflammasome at the time of injury, but especially in the subacute phase, indicating it may have a long therapeutic window and mechanistic role in prolonged symptoms. Human tissue samples around brain contusion show an IL-1α acute spike following brain injury, while IL-1β shows a much more gradual increase that is thought to represent a portion of the delayed cytokine response to TBI [ 38 ]. It is a potent stimulus for other cytokine release including TNF-α and IL-6. IL-1β is a target for therapies to prevent secondary injury in TBI. A mouse model of chronic repetitive mTBI in adolescence demonstrated IL-1β and IL-18 production with prominent inflammasome activation. Mice with deficiencies in caspase and interleukin receptors in that study had better outcomes at a year [ 39 ]. Prior concussion carries higher risk of prolonged recovery time [ 40 ]. Increased inflammasome activation did not account for this in our study, however there were low numbers in our groups with heterogenous injuries. Further evaluation of these groupings is warranted. Overall there is a hyper-responsiveness of inflammasome-related cytokine release in response to LPS stimulation in the TBI population, but the interleukins do not respond in the subacute phase. This may represent priming by the injury event [ 41 ] which has been suggested to last one week [ 42 ]. Previous animal studies of severe TBI have demonstrated that pre-conditioning with LPS increased both pro- and anti-inflammatory cytokines, and their effect attenuated the extent of histological injury [ 42 ]. These models of injury have contusions with a penumbra of reduced cerebral blood flow. These brain injuries are different to mTBI, further studies looking at the preconditioning response are warranted in mTBI models and patients. IL-18 plays a role in downregulating TNF-α [ 43 ]. At low doses, LPS causes a decrease in IL-18 production and enhances the innate immune response to sepsis, at higher doses of LPS there is an increase of IL-18 production which influences death from sepsis [ 44 ]. IL-18, an end-product of the inflammasome, was significantly decreased in mTBI and the normal decrease of IL-18 in response to LPS not seen in the TBI cohorts. There were similarities with IL-33 with reduced expression in the controls and a marked response seen with the same LPS stimulus in severe TBI. IL-33 does not respond by priming and has enhanced secretion in response to attempt to prime [ 45 ]. The response of the mTBI group in the subacute phase fits that of priming described in the literature for the cytokines associated with the inflammasome complex. The inflammasome is a potential safe target of therapy. Numerous safe therapies modify NLRP3 including pioglitazone [ 12 ], rapamycin [ 46 ] and Omega three [ 14 ] and are under consideration as therapeutics. In our cohort this was not a dominant pathway. AIM1 was the only pathway upstream of IL-1β in our study that was upregulated. One small trial in adults with sTBI showed demonstrated the effect of interleukin-1 antagonist Anakinra in adults but was not powered to show clinical differences in outcomes [ 47 ]. Mild traumatic brain injury induces changes in the innate immune system. These effect of mTBI on the immune system is prolonged. This is evidenced by the modified response of the inflammasome complex to LPS stimulation at two weeks. Mild traumatic brain injury, like burns, are sterile injuries that induce systemic immune changes. It would be judicious to profile this response in these children at a later time- point out from injury to see if the immune activation quiesces. The clinical period of a year was noted by Eisenberg and colleagues to be the period within which it was critical not to get a repeat concussion [ 8 ] Treating serum with LPS and measuring the responsiveness of a number of key cytokines may demonstrate the window for which the injury persists. The current literature is based on animal models of more severe injury. Our findings are inconsistent with some severe TBI studies. Severe TBI usually involves gross intracranial bleeding. Furthermore, there are immune differences between adults and children. Finnerty and colleagues described how children after burn injury had a depressed response of important pro- and anti-inflammatory relative to adults [ 48 ] which persist for up to 3 years [ 49 ]. Animal studies demonstrate persistent immune dysfunction in sTBI [ 4 ]. Numerous studies have demonstrated the chronic changes associated with repeated head injury [ 9 ], that support the hypothesis of inflamma-aging [ 50 ]. The injuries in our study population were heterogenous. Only 40% had vomiting and some of the children only had isolated symptoms[ 16 ]. In the clinical setting it is impossible to quantify the volume of trauma that was sustained. The numbers in the study measuring the effect of previous mTBI on inflammatory cascade were small. A limitation of this study was the absence of data on analgesia and quantifying if there was an effect from paracetamol and ibuprofen. This study showed that the pathway in mild TBI includes a concerted response by the innate immune system with TLR4 pathway signalling, inflammasome activation and alterations in neutrophil activation and monocyte populations. The changes are sustained and result in altered systemic immune responses at two weeks from injury supported by the changes in LPS responsiveness. Abbreviations GCS Glasgow coma scale IL Interleukin LPS lipopolysaccharide mTBI mild traumatic brain injury PCSI post concussive symptom inventory SCAT Sports concussion assessment tool sTBI severe traumatic brain injury TBI Traumatic brain injury TNF-α Tissue necrosis factor-α Declarations Ethics approval and consent to participate This study was approved by the Ethics Committees of Children’s Health Ireland (CHI) at Tallaght (Ref: 2016-03 (21); approved 28.03.16) and CHI at Temple Street Dublin (Ref: 16.019; approved 23.03.16), Ireland. Informed consent was obtained from patients and/or parents. Consent for publication Not applicable Competing interests The authors have no conflict of interest to declare. Availability of data and materials The experimental data used to support the findings of this study are available from the corresponding author upon request. Funding This study was funded by the National Children’s Hospital Fund [206965], Tallaght, Dublin, Ireland Authors’ contributions The manuscript is being submitted on behalf of all the authors and is the original work of all authors. ER was responsible for recruitment, sample acquistion, lab experiments, analysis and was responsible for writing the main draft of the manuscript. LK, AMM, CS and MB added expert laboratory assisstance. LK, CS, ED, DD, AL, GB, DC, CM were responsible for patient recruitment, sample acquisition, reviewing and editing the manuscript. LK TB and EM were key in study design, supervising the research and its outcomes and providing crucial editorial assistance. All authors had editorial license to review and re-draft the manuscript, and all contributors had to approve the final edit. All authors are accountable for the accuracy and scientific integrity of this work. 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Zareen Z, Strickland T, Eneaney VM, Kelly LA, McDonald D, Sweetman D, et al. Cytokine dysregulation persists in childhood post Neonatal Encephalopathy. BMC Neurol. 2020;20(1):115. Lopes AB, Lopes LB, da Silveira Antunes RN, Fukasawa JT, de Calamita ACD. Effects of Immunosenescence on the Lower Expression of Surface Molecules in Neutrophils and Lymphocytes. Curr Aging Sci. 2018;11:118–25. Rusiecki J, Levin LI, Wang L, Byrne C, Krishnamurthy J, Chen L, et al. Blast traumatic brain injury and serum inflammatory cytokines: a repeated measures case-control study among U.S. military service members. J Neuroinflammation. 2020;17:20. Gopcevic A, Mazul-Sunko B, Marout J, Sekulic A, Antoljak N, Siranovic M, et al. Plasma interleukin-8 as a potential predictor of mortality in adult patients with severe traumatic brain injury. Tohoku J Exp Med. 2007;211:387–93. Rhind SG, Crnko NT, Baker AJ, Morrison LJ, Shek PN, Scarpelini S, Rizoli SB. Prehospital resuscitation with hypertonic saline-dextran modulates inflammatory, coagulation and endothelial activation marker profiles in severe traumatic brain injured patients. J Neuroinflammation. 2010;7:5. Tang R, Lin YM, Liu HX, Wang ES. Neuroprotective effect of docosahexaenoic acid in rat traumatic brain injury model via regulation of TLR4/NF-Kappa B signaling pathway. Int J Biochem Cell Biol. 2018;99:64–71. Chen L, Guo S, Ranzer MJ, DiPietro LA. Toll-like receptor 4 has an essential role in early skin wound healing. J Invest Dermatol. 2013;133:258–67. Tang SC, Arumugam TV, Xu X, Cheng A, Mughal MR, Jo DG, et al. Pivotal role for neuronal Toll-like receptors in ischemic brain injury and functional deficits. Proc Natl Acad Sci U S A. 2007;104:13798–803. Vidal PM, Ulndreaj A, Tetreault L, Hong J, Fehlings MG. The changes in systemic monocytes in humans undergoing surgical decompression for degenerative cervical myelopathy may influence clinical neurological recovery. 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Trends Neurosci. 2003;26:248–54. Longhi L, Gesuete R, Perego C, Ortolano F, Sacchi N, Villa P, et al. Long-lasting protection in brain trauma by endotoxin preconditioning. J Cereb Blood Flow Metab. 2011;31:1919–29. Sakao Y, Takeda K, Tsutsui H, Kaisho T, Nomura F, Okamura H, et al. IL-18-deficient mice are resistant to endotoxin-induced liver injury but highly susceptible to endotoxin shock. Int Immunol. 1999;11:471–80. Joshi VD, Kalvakolanu DV, Hasday JD, Hebel RJ, Cross AS. IL-18 levels and the outcome of innate immune response to lipopolysaccharide: importance of a positive feedback loop with caspase-1 in IL-18 expression. J Immunol. 2002;169:2536–44. Espinassous Q, Garcia-de-Paco E, Garcia-Verdugo I, Synguelakis M, von Aulock S, Sallenave JM, et al. IL-33 enhances lipopolysaccharide-induced inflammatory cytokine production from mouse macrophages by regulating lipopolysaccharide receptor complex. J Immunol. 2009;183:1446–55. Chen Y, Meng J, Xu Q, Long T, Bi F, Chang C, et al. Rapamycin improves the neuroprotection effect of inhibition of NLRP3 inflammasome activation after TBI. Brain Res. 2019;1710:163–72. Helmy A, Guilfoyle MR, Carpenter KL, Pickard JD, Menon DK, Hutchinson PJ. Recombinant human interleukin-1 receptor antagonist in severe traumatic brain injury: a phase II randomized control trial. J Cereb Blood Flow Metab. 2014;34(5):845–51. Finnerty CC, Jeschke MG, Herndon DN, Gamelli R, Gibran N, Klein M, et al. Temporal cytokine profiles in severely burned patients: a comparison of adults and children. Mol Med. 2008;14:553–60. Jeschke MG, Gauglitz GG, Kulp GA, Finnerty CC, Williams FN, Kraft R, et al. Long-term persistance of the pathophysiologic response to severe burn injury. PLoS ONE. 2011;6:e21245. Marcos-Pérez D, Sánchez-Flores M, Proietti S, Bonassi S, Costa S, Teixeira JP, et al. Association of inflammatory mediators with frailty status in older adults: results from a systematic review and meta-analysis. Geroscience. 2020;42:1451–73. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4172622","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":284544564,"identity":"970536be-ffcd-4439-b92c-0f09682374fc","order_by":0,"name":"Emer Ryan","email":"","orcid":"","institution":"Discipline of Paediatrics, School of Medicine, Trinity Translational Medicine Institute (TTMI) \u0026 Trinity Research in Childhood Centre (TRiCC), Trinity College Dublin","correspondingAuthor":false,"prefix":"","firstName":"Emer","middleName":"","lastName":"Ryan","suffix":""},{"id":284544567,"identity":"42b5fdd1-d233-431d-8531-4244a4d434aa","order_by":1,"name":"Lynne Kelly","email":"","orcid":"","institution":"Discipline of Paediatrics, School of Medicine, Trinity Translational Medicine Institute (TTMI) \u0026 Trinity Research in Childhood Centre (TRiCC), Trinity College Dublin","correspondingAuthor":false,"prefix":"","firstName":"Lynne","middleName":"","lastName":"Kelly","suffix":""},{"id":284544569,"identity":"0db66eeb-509e-4f52-842e-b4b0fa98b52e","order_by":2,"name":"Ashanty M Melo","email":"","orcid":"","institution":"Discipline of Paediatrics, School of Medicine, Trinity Translational Medicine Institute (TTMI) \u0026 Trinity Research in Childhood Centre (TRiCC), Trinity College Dublin","correspondingAuthor":false,"prefix":"","firstName":"Ashanty","middleName":"M","lastName":"Melo","suffix":""},{"id":284544571,"identity":"69d55116-f80c-4b43-9c6d-0c668baf6c2e","order_by":3,"name":"Cian P Morgan","email":"","orcid":"","institution":"Discipline of Paediatrics, School of Medicine, Trinity Translational Medicine Institute (TTMI) \u0026 Trinity Research in Childhood Centre (TRiCC), Trinity College Dublin","correspondingAuthor":false,"prefix":"","firstName":"Cian","middleName":"P","lastName":"Morgan","suffix":""},{"id":284544574,"identity":"a88a243e-a225-4501-85fd-1b8a28a2efa5","order_by":4,"name":"Mark Bates","email":"","orcid":"","institution":"Discipline of Paediatrics, School of Medicine, Trinity Translational Medicine Institute (TTMI) \u0026 Trinity Research in Childhood Centre (TRiCC), Trinity College 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Dublin","correspondingAuthor":false,"prefix":"","firstName":"Catherine","middleName":"","lastName":"Stacey","suffix":""},{"id":284544579,"identity":"7911d238-54a5-4eda-a963-114dbe902680","order_by":7,"name":"Eimear Duff","email":"","orcid":"","institution":"Discipline of Paediatrics, School of Medicine, Trinity Translational Medicine Institute (TTMI) \u0026 Trinity Research in Childhood Centre (TRiCC), Trinity College Dublin","correspondingAuthor":false,"prefix":"","firstName":"Eimear","middleName":"","lastName":"Duff","suffix":""},{"id":284544580,"identity":"c3ea13ba-4693-4898-bd22-e565a98d85c5","order_by":8,"name":"Ann Leonard","email":"","orcid":"","institution":"Department of Biochemistry, Tallaght University Hospital, Dublin, Ireland","correspondingAuthor":false,"prefix":"","firstName":"Ann","middleName":"","lastName":"Leonard","suffix":""},{"id":284544581,"identity":"d15958f0-8010-4361-b6f9-e22acb62d705","order_by":9,"name":"Gerard Boran","email":"","orcid":"","institution":"Department of Biochemistry, Tallaght University Hospital, Dublin, Ireland","correspondingAuthor":false,"prefix":"","firstName":"Gerard","middleName":"","lastName":"Boran","suffix":""},{"id":284544582,"identity":"3d9ae95d-813b-454c-9854-7c10876d2266","order_by":10,"name":"Dermot R Doherty","email":"","orcid":"","institution":"Department of Paediatric Critical Care, Children’s Hospital Ireland (CHI) at Tallaght, Crumlin \u0026 Temple St.","correspondingAuthor":false,"prefix":"","firstName":"Dermot","middleName":"R","lastName":"Doherty","suffix":""},{"id":284544584,"identity":"c755fde0-0b8b-4d8d-aea7-0445af172111","order_by":11,"name":"Darach Crimmins","email":"","orcid":"","institution":"Discipline of Paediatrics, School of Medicine, Trinity Translational Medicine Institute (TTMI) \u0026 Trinity Research in Childhood Centre (TRiCC), Trinity College Dublin","correspondingAuthor":false,"prefix":"","firstName":"Darach","middleName":"","lastName":"Crimmins","suffix":""},{"id":284544586,"identity":"17f74c11-5cf5-47ca-a313-8510f3077020","order_by":12,"name":"Turlough Bolger","email":"","orcid":"","institution":"Discipline of Paediatrics, School of Medicine, Trinity Translational Medicine Institute (TTMI) \u0026 Trinity Research in Childhood Centre (TRiCC), Trinity College Dublin","correspondingAuthor":false,"prefix":"","firstName":"Turlough","middleName":"","lastName":"Bolger","suffix":""},{"id":284544592,"identity":"65fbc818-aca8-4156-9efe-b0acb1c1397a","order_by":13,"name":"Eleanor J Molloy","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYDACdjiLufEAA4MNkJHAcACvFmY4i7EBqDJNgmQth8Fa8AJ+ZuaDnwsq7jHotjc2HPjYdr6Ovz2B8cAHPFokm9mSpWecKWYwO3Ow4eDMttsSEmceMBycgUeLwWEeA2netgQGsxuJDYd5gVoMJBIYDvPg0WJ/mP/zbyQt5yBa/uCzhZmHDdmWAxAt+LwvcZjNzJrnTAIP2C8zziVLzjjzsOFgDx4t/O3Nj2/zVCTImR1vPvjgQ5kdP3978uEPP/BZAwUQ/zKygckGIjTAAT5vj4JRMApGwYgFAPd5UVx2leP1AAAAAElFTkSuQmCC","orcid":"","institution":"Discipline of Paediatrics, School of Medicine, Trinity Translational Medicine Institute (TTMI) \u0026 Trinity Research in Childhood Centre (TRiCC), Trinity College Dublin","correspondingAuthor":true,"prefix":"","firstName":"Eleanor","middleName":"J","lastName":"Molloy","suffix":""}],"badges":[],"createdAt":"2024-03-27 00:44:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4172622/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4172622/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53756536,"identity":"25c2686a-2614-413b-acbb-8f768c19a654","added_by":"auto","created_at":"2024-03-29 19:03:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":91053,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) Granulocyte activation stained with CD11b (b) TLR4 expression on granulocytes in Paediatric Traumatic brain injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole blood was analysed by Flow Cytometry in children with mild TBI (mTBI) at time of presentation from injury (0-4 days), and at 2 weeks (10-14 days). Values expressed as mean channel florescence, displayed as mean and standard error of the mean. Samples size: (n = 4 controls, n = 6 mTBI at 0-4 days, n = 7 mTBI at 10-14 days, n = 4 severe; (*p ≤ 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001). Fig 1(a). p = 0.0026 controls compared to mTBI at 10-14 days unstimulated, p = 0.0042 control compared to mTBI with melatonin treatment. Fig1(b) p = 0.02 controls compared to mTBI at 0-4 days, p = 0.006, p = 0.0002 controls compared to sTBI unstimulated, and with LPS.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4172622/v1/0c21ceb7cfd87f7792aea188.jpg"},{"id":53757994,"identity":"c0125485-121b-46f5-8e39-f4733240479a","added_by":"auto","created_at":"2024-03-29 19:11:06","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":136793,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) Total Monocyte population and (b) Classical monocyte population (c) Intermediate monocyte population and (d) Non-classical monocyte population in Paediatric Traumatic brain injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole blood was analysed by Flow Cytometry in children with mild TBI (mTBI) at time of presentation from injury (0-4 days), and at 2 weeks (10-14 days) from injury as well as children with severe TBI (sTBI). Values expressed as % of total cells, displayed as mean and standard deviation. Samples size: (n = 4 controls, n = 10 mTBI at 0-4 days, n = 5 mTBI at 10-14 days, n = 3 severe) *p \u0026lt; 0.05, **p\u0026lt; 0.01. (a) p = 0.04, p = 0.006, p = 0.01 across LPS, melatonin and LPS + melatonin treatment groups in controls compared to mTBI. (b) No significant difference seen between controls and patients. (c) p = 0.03, p = 0.017, p = 0.04 across LPS, melatonin and LPS + melatonin treatment groups in controls compared to mTBI. (d) No significant difference seen between controls and patients.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4172622/v1/e98b6b1dcb306c10a7728ea4.jpg"},{"id":53756538,"identity":"72ea079b-4d82-4edc-a79c-c40907260109","added_by":"auto","created_at":"2024-03-29 19:03:06","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":77976,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) Nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) and (b) NLR Family Pyrin Domain Containing 1 (NLRP1) (c) Absent in Melanoma (AIM)1 (d) AIM2 in Paediatric mild Traumatic brain injury (mTBI)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene transcription was analysed by quantitative polymerase chain reaction in children with mTBI at 0-4 days and at 10-14 days: Gene transcription was analysed by quantitative polymerase chain reaction in children with first reported mild TBI (mTBI) and repeat reported mTBI. Values expressed as fold change, displayed as mean and standard deviation. Samples size: n = 15 controls, n = 15 mTBI at 0-4 days, n = 8 mTBI at 10-14 days (*p ≤ 0.05*, **p \u0026lt; 0.01, ***p \u0026lt; 0.001****p \u0026lt; 0.0001) (a) p = 0.0019 controls compared to mTBI at 0-4 days, p = 0.0082 controls compared to mTBI 10-14 days, (b) p \u0026lt; 0.0001 controls compared to mTBI at 0-4 days and compared to mTBI at 10-14 days. (c) p = 0.02 controls compared to mTBI at 0-4 days. No significant difference at 10-14 days. (d) No significant difference seen between controls and patients.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4172622/v1/2b8833aba24fe735563711aa.jpg"},{"id":53756541,"identity":"224760e3-6fcf-4efe-bff2-06435cf7cbf5","added_by":"auto","created_at":"2024-03-29 19:03:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":70475,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) NLRP3 nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) and (b) NLR Family Pyrin Domain Containing 1 (NLRP1) (c) Absent in Melanoma (AIM)1 (d) AIM2 in Paediatric mild Traumatic brain injury (mTBI) at 0-4 days from injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene transcription was analysed by quantitative polymerase chain reaction in children with first reported mild TBI (mTBI) and repeat reported mTBI. Values expressed as fold change, displayed as mean and standard deviation. Samples size: n = 15 controls, n = 8 previous mTBI n = 7 mTBI naïve (*p ≤ 0.05*, **p \u0026lt; 0.01, ***p \u0026lt; 0.001****p \u0026lt; 0.0001) Fig4(a) *p = 0.0228 controls compared to mTBI naïve (0.804-fold change), ***p = 0.0002 controls compared to repeat mTBI (0.558-fold change). (a) p = 0.02 mTBI naïve compared to controls, p = 0.002 repeat mTBI compared to control (b) p \u0026lt; 0.0001 control compared both cohorts, prior mTBI and mTBI naïve.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4172622/v1/9151f40884fffc8b966eed0c.jpg"},{"id":53756542,"identity":"2ca457c6-ac6f-4824-976c-5c36301195cb","added_by":"auto","created_at":"2024-03-29 19:03:06","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":91549,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytokines (a) Interleukin-18 (IL-18) (b) Interleukin-33 (IL-33) (c) Interleukin-1a (IL-1a) (d) Interleukin-1b (IL-1b) and (e) Interleukin-1ra (IL-1ra) response in Paediatric Traumatic brain injury responses in Paediatric Traumatic brain injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum was analysed using ELISA in children with mild TBI (mTBI) at time of presentation from injury (0-4 days), and at 2 weeks (10-14 days) from injury as well as children with severe TBI (sTBI). Values expressed as pg/mL, displayed as mean and standard deviation. Samples size (n = 104 controls, n = 98 mTBI at 0-4 days, n = 29 mTBI at 10-14 days, n = 6 severe) naïve, *p ≤ 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4172622/v1/5a7e308936e7e087b6d09a26.jpg"},{"id":53756540,"identity":"cbcad2e1-506b-4a69-9798-a70a5dc58aaa","added_by":"auto","created_at":"2024-03-29 19:03:06","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":116631,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytokines (a) Interleukin-18 (b) Interleukin-33 (c) Interleukin-1ra (IL-1ra) (d) Interleukin (IL)-1a (e) Interleukin (IL)-1b response in Paediatric Traumatic brain injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum was analysed using ELISA in children with mild TBI (mTBI) at time of presentation from injury (0-4 days), and at 2 weeks (10-14 days) from injury as well as children with severe TBI (sTBI). Values expressed as pg/mL, displayed as mean and standard error of mean. Samples size: (n = 104 controls, n = 98 mTBI at 0-4 days, n = 29 mTBI at 10-14 days, n = 6 severe, LPS: n = 11 controls, n = 18 mTBI at 0-4 days, n = 9 mTBI at 10-14 days, n = 4 severe). *p ≤ 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001) ; #### p \u0026lt; 0.00001 LPS response in controls compared to mTBI 0-4 days.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4172622/v1/12a9b58a66efa416310eff3d.jpg"},{"id":55264423,"identity":"be5cd6c3-449b-44cf-89e7-e9d68e0030b6","added_by":"auto","created_at":"2024-04-25 01:42:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1094992,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4172622/v1/c75e9694-1d17-4149-9768-2255061078d2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Altered Inflammasome and Immune activation in Paediatric Traumatic Brain Injury","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChildhood Traumatic Brain Injury (TBI) is an acquired brain injury defined as an insult to the brain from an external force that leads to temporary or permanent impairment of cognitive, physical, or psychosocial function. TBI can be divided by severity as mild, moderate, or severe and may be open (penetrating) or closed (non-penetrating) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Mild TBI (mTBI) results in chronic and debilitating symptoms for a number of children with post concussive syndrome affecting almost a third of children, resulting in a significant number of missed days from school [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In children, TBI has longer-term effects associated with injury to a developing brain. Neuroinflammation and immune dysfunction are known to persist following TBI. Changes in the cell populations after TBI have been described in both animal [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and human populations [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Severe TBI (sTBI) is known to induce leukocytosis, microglial activation and cytokine release. The immune profile is less well described in mTBI and the extent to which the mediators of inflammation involved in sTBI are involved in mTBI is not known. Mild TBI may also activate immune pathways with persistent immune dysfunction contributing to ongoing symptoms. This may also be involved in the phenomenon whereby a repeat mTBI tends towards prolonged symptoms relative to a first injury. Mild Traumatic Brain injuries have been associated with long-term cognitive deficits and neurodegeneration [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eVarious animal models have been developed which mimic moderate and sTBI with controlled cortical impact and fluid percussion injury to mimic blast injury. These models are reproducible and supply a fixed and measurable force in genetically identical breeds of mice with or without gene knock-out as comparators. Mild TBI in the human model is much more complex. The age and gender of the patient, the presence of prior injuries, the type of injury and the presence of prior systemic inflammation all vary. The burden of symptoms from a \u0026lsquo;simple\u0026rsquo; school yard fall may be great, the area of the injury and the torque of the fall will also vary from injury to injury.\u003c/p\u003e \u003cp\u003eNeutrophils and monocytes play a key role in severe TBI and infiltrate the brain early. Neutrophil populations expand in response to sTBI, and peak neutrophil count has been associated with unfavourable outcomes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. CD11b (a β\u003csub\u003e2\u003c/sub\u003e integrin) is a surface receptor that aids adherence of polymorphonuclear cells to the endothelial cell wall, facilitating their migration to the site of injury. It is well described that those with prior mTBI have worse symptoms for longer and are at higher risk of post concussive syndrome [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Experimental models show ongoing microglial activation, spreading of lesion volumes and neurodegeneration a year from the injury [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and animal models, have altered microglial activation in the wake of injury with reduced oxidative damage [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. A second injury during the \u0026lsquo;healing phase\u0026rsquo; may result in worse outcomes especially if repeat mTBI is within a year of the first injury [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe inflammasome has been implicated in mTBI. The inflammasome complex is a self-perpetuating canonical structure that can be triggered by a number of pathways including the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3), NLR Family Pyrin Domain Containing 1 (NLRP1) and Absent in Melanoma \u0026minus;\u0026thinsp;2 (AIM2). Downstream nuclear factor\u0026ndash;κB (NF-κB) triggers the expression of IL-1β. The inflammasome proteins have recently been used as biomarkers and prognosticators in sTBI [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. NLRP3 has been targeted to modulate oedema in animal models of sTBI by administration of the drug pioglitazone which reduces NLRP3 expression [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Modulating NLRP3 shows better cognitive and motor outcomes in mouse models [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Dietary omega three fatty acids had beneficial effects in mTBI and attenuated NLRP3 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] with a resultant decrease in IL-1β, IL-18 and IL-6 expression.\u003c/p\u003e \u003cp\u003eWe hypothesised that there is an altered innate immune phenotype in children with mTBI when compared to healthy control children which may be associated with inflammasome immune complex activation and may alter the systemic immune response following mTBI. We aimed to examine neutrophil, monocyte and inflammasome activation in paediatric TBI.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Population\u003c/h2\u003e \u003cp\u003eChildren with mTBI were eligible if they had sustained either a direct blow to the head or an acceleration/deceleration movement of the head and had a GCS score of either 14 or 15 on presentation to the emergency department, as well as any one of the following: (i) loss of consciousness\u0026thinsp;\u0026lt;\u0026thinsp;30 minutes, (ii) amnesia, (iii) any alteration in mental state at the time of the injury (i.e. agitation, irritability, sleepiness, lethargy, slow to respond, dazed or asking repetitive questions) or (iv) physically symptomatic of head injury with vision disturbance, ataxia, nausea, headache, dizziness, hearing disturbance. All children in the mild cohort had a GCS of 14\u0026ndash;15. Children with extracranial injuries or bruising\u0026thinsp;\u0026gt;\u0026thinsp;5cm were excluded. The severe TBI cohort had a lowest GCS of less than 8 and required intubation, ventilation and an Intensive Care stay. Paediatric controls included children attending for phlebotomy or day case procedures with normal results and clinical outcomes. Children in all groups were excluded if they had recent fever or evidence of infection [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDemographics recorded included birth history, developmental history including diagnoses of learning disorders, parental or self-reported anxiety, previous hospital presentations of head injury or self-reported prior \u0026lsquo;concussion\u0026rsquo; or mTBI naive. A history of travel sickness, need for visual aids in the form of glasses and migraine was reported. Family history in first degree relatives of concussion, depression or migraine was recorded. The cause of the injury was recorded. The Ethics Committees of Children\u0026rsquo;s Health Ireland (CHI) at Tallaght and CHI at Temple Street Dublin Ireland approved the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design:\u003c/h2\u003e \u003cp\u003eBlood samples (1-3mL) were collected in sodium citrate tubes and analysed within two hours of phlebotomy. Whole blood was incubated for 1 hour with vehicle, LPS (10ng/mL) (E Coli 0111:B4 Sigma Life Science Wicklow) and/or melatonin (10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003eM) (SIGMA, Ireland).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies and Flow Cytometry:\u003c/h2\u003e \u003cp\u003eBlood samples were incubated with a dead cell stain (Fixable Viability Dye eFluor 506, Invitrogen, California, USA), diluted to working concentration in phosphate-buffered saline (PBS). The following fluorochrome-labelled monoclonal antibodies (mAb) were added to each sample: CD14-PerCP, CD15-PECy7, CD16-FITC, CD66b-Pacific Blue, and TLR4-APC (BioLegend\u0026reg;, California, USA) and PE-labelled CD11b (BD Biosciences, Oxford, UK). PBA buffer (PBS containing 1% bovine serum albumin and 0.02% sodium azide), prepared in house, was used to make up the antibody cocktail. Samples were incubated in the dark for 15 minutes before the addition of 1 mL FACS lysing solution (BD Biosciences, Oxford, UK), and the samples were then incubated for 15 minutes in the dark in order to lyse contaminating red blood cells. Cells were pelleted by centrifugation at 450g for 7 minutes at room temperature, washed twice with PBA buffer, and fixed in 300 \u0026micro;L of 1% paraformaldehyde. The final cell pellet was resuspended in 100 \u0026micro;L PBA buffer and analysed on a BD FACSCanto II flow cytometer.\u003c/p\u003e \u003cp\u003eThe expression of TLR4 and CD11b antigens on the surface of neutrophils and monocytes was evaluated by flow cytometry. Neutrophils were delineated based on SSC-A and CD66b\u0026thinsp;+\u0026thinsp;positivity as previously described [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and monocytes were defined based on SSC-A and CD66b negativity and their subsets based on CD14 and CD16 expression: classical (CD14+/CD16-), intermediate (CD14+/CD16+) and nonclassical (CD14dim/CD16+) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. A minimum of 10,000 events were collected, and relative expression of TLR4 and CD11b was expressed as mean fluorescence intensity (MFI). Flow cytometry data was analysed using FlowJo software (Oregon, USA) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003ePCR\u003c/em\u003e:\u003c/h2\u003e \u003cp\u003eFollowing incubation of samples, 1mL of TRIzol\u0026trade; (Thermo Fisher) was added to 0.3mL of whole blood. Chloroform was added and the samples were incubated for 5mins at room temperatures. Following lysis, the aqueous phase was used to isolate RNA. Purity and concentration were evaluated using an NanoDrop ND- 8000 Spectrophotometer and analysed using ND-ver 2.3.3. software. Total RNA was reverse transcribed to single-stranded cDNA using the High-Capacity cDNA Archive Kit (Applied Biosystems) following the manufacturer\u0026rsquo;s protocol and stored at -80\u0026deg;C until use. The evaluation of gene expression was performed by TaqMan\u0026reg; RT-PCR. Commercially available TaqMan\u0026reg; primer and probe combinations were used to detect the expression of the following inflammasome genes, IL-1β (Hs00174097_m1), NLRP3 (Hs00918082_m1), NLRP1 (Hs00248187_m1) and AIM2 (Hs00915710_m1), and measured at baseline and at two weeks. NLRP3 and IL-1β were also measured on samples treated with LPS and melatonin. All samples were assayed in triplicate. Thermal cycling conditions were as follows: 2 minutes at 50\u0026deg;C, 10 minutes at 95\u0026deg;C, and, for 40 cycles, 24 seconds at 95\u0026deg;C and 1 minute at 60\u0026deg;C, using the 7900HT Fast Real-Time PCR System. Relative quantification (RQ) values were calculated using the 2-ΔΔCt method [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMultiplex ELISA\u003c/h2\u003e \u003cp\u003eA multiplex ELISA immunoassay from Meso Scale Diagnostics (Rockville, Maryland USA) was performed using a 10-spot human serum plasma U-plex ELISA plate (Meso Scale) which was customised for the study and plates were analysed SECTOR Imager. Cytokines analysed included IL-1α IL-1\u0026szlig;, IL-33, IL-18, and IL-1ra. The limits of detection for the individual assays were within expected ranges.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistics:\u003c/h2\u003e \u003cp\u003eWhere data was normally distributed unpaired t-tests were performed. The Shapiro-Wilk test was performed to test normality. When it failed the Shapiro-Wilk test the non-parametric Mann Whitney test were used. NLRP1, AIM2 and IL-1β were assessed with Mann Whitney tests. For cytokine analysis, where the sample was below the lower limit of detection, the lower limit of detection for that assay was substituted [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and Mann Whitney tests employed. The assay reproducibility was calculated using the intra-variation of the standard curves which was within an acceptable range. The median of the lower limit of detection for all plate runs had a closely aligned average value, demonstrating the reproducibility of the assay. When LLOD were replaced, over 50% of the sample were undetected for the IL-1β cytokine. No upper limits of detection were reached.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eIn total 214 children were recruited including 116 children with TBI (mTBI n\u0026thinsp;=\u0026thinsp;110; sTBI n\u0026thinsp;=\u0026thinsp;6) and 98 control children. Samples were available for 98 mTBI patients as 12 recruited patients declined phlebotomy. The mean (SD) age of mTBI recruited was 11.2 +/- 4.0 years, with an age range of 7 months \u0026minus;\u0026thinsp;16 years. There were six children with severe TBI recruited, mean age 7.9 +/- 5.0 years and 98 control children, mean age 8.04 +/-4.2 years.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eNeutrophil and activation:\u003c/h2\u003e \u003cp\u003eFlow data was available for n\u0026thinsp;=\u0026thinsp;10 mTBI at 0\u0026ndash;4 days, n\u0026thinsp;=\u0026thinsp;7 mTBI at 10\u0026ndash;14 days and 4 children with sTBI. There was a general suppression of CD11b expression in the mTBI group at 10\u0026ndash;14 days in the unstimulated vehicle and in the melatonin treatment group, (p\u0026thinsp;=\u0026thinsp;0.0026 and p\u0026thinsp;=\u0026thinsp;0.0042, respectively; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) compared to controls. Children with severe TBI did not have reduced expression of CD11b compared to controls. Lipopolysaccharide increased CD11b expression in all cohorts compared to baseline.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eToll-like receptor-4 (TLR4) expression in the neutrophil was increased in the mTBI cohort versus controls at baseline, reaching statistical significance in the melatonin cohort (p\u0026thinsp;=\u0026thinsp;0.02). TLR4 expression was decreased at 10\u0026ndash;14 days compared to controls and did not respond to LPS stimulation in mTBI. TLR4 expression was increased in the severe TBI cohort reaching statistical significance in the vehicle and LPS groups (p\u0026thinsp;=\u0026thinsp;0.006 and p\u0026thinsp;=\u0026thinsp;0.0002, respectively) compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Melatonin decreased the TLR4 expression in the mTBI cohort but did not reach significance compared to baseline.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMonocyte Activation\u003c/h2\u003e \u003cp\u003eThe total monocyte population was reduced in mTBI for LPS, melatonin and LPS\u0026thinsp;+\u0026thinsp;melatonin treatment groups compared to controls (p\u0026thinsp;=\u0026thinsp;0.04, p\u0026thinsp;=\u0026thinsp;0.006, p\u0026thinsp;=\u0026thinsp;0.01 respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Intermediate monocytes were decreased in mTBI, across all treatments groups treatment groups compared to controls (p\u0026thinsp;=\u0026thinsp;0.03, p\u0026thinsp;=\u0026thinsp;0.017, p\u0026thinsp;=\u0026thinsp;0.04 respectively; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). There were no significant differences within classical or non-classical monocyte populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eInflammasome Pathway\u003c/h2\u003e \u003cp\u003eThe inflammasome is a key regulator of pro-inflammatory genes. Controls (n\u0026thinsp;=\u0026thinsp;15) were compared to mTBI (n\u0026thinsp;=\u0026thinsp;15). In this cohort of mTBI, both NLRP3 and NLRP1 were downregulated compared to controls at presentation and at 10\u0026ndash;14 days (p\u0026thinsp;=\u0026thinsp;0.0019 and p\u0026thinsp;=\u0026thinsp;0.0082) for NLRP3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) and for NLRP1 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). AIM1 which is a key regulator was upregulated compared to controls at 0\u0026ndash;4 days (p\u0026thinsp;=\u0026thinsp;0.02) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), and no effect was seen with AIM2 or ASC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). IL-1β was increased at baseline at 0\u0026ndash;4 days and further elevated by 10\u0026ndash;14 days (p\u0026thinsp;=\u0026thinsp;0.0004 and p\u0026thinsp;=\u0026thinsp;0.0002 respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef) in mTBI.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePatients with prior mTBI\u003c/h2\u003e \u003cp\u003eChildren with prior concussions tend to have more prolonged symptoms on subsequent presentations prompting exploration of the differences in gene expression in those with and without (na\u0026iuml;ve) prior self-report of concussion or hospitalisation for head injury. IL-1β was significantly higher in those who were mTBI na\u0026iuml;ve (p\u0026thinsp;=\u0026thinsp;0.003) at baseline and two weeks (p\u0026thinsp;=\u0026thinsp;0.003) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef), whereas at two weeks ASC was significantly higher in those who had a repeat injury compared to those who were mTBI na\u0026iuml;ve (304.5 v 19.85%; p\u0026thinsp;=\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). NLRP3 was reduced in mTBI na\u0026iuml;ve, and this reduction was significantly higher in those with a repeat injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). NLRP1 was also reduced in mTBI compared to controls in na\u0026iuml;ve and repeat injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), while no significant differences were seen in AIM or AIM2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, respectively).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eInflammasome-related serum Cytokine responses\u003c/h2\u003e \u003cp\u003eCytokine data was available for patients with mTBI (n\u0026thinsp;=\u0026thinsp;98), patients with mTBI two weeks post-injury (n\u0026thinsp;=\u0026thinsp;29), and patients with sTBI (n\u0026thinsp;=\u0026thinsp;6) compared to controls (n\u0026thinsp;=\u0026thinsp;104). When inflammasome related serum cytokines were examined, IL-18 was lower in mTBI compared to controls (3013 v 2309pg/mL; p\u0026thinsp;=\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). IL-33 was elevated in mTBI (4.55 v 2.97 pg/mL; p\u0026thinsp;=\u0026thinsp;0.04; (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). IL-1β was not increased, however over 50% of the samples had undetectable levels of expression. IL-1ra was decreased in the mTBI group compared to controls (1461 v 981.9. pg/mL; p\u0026thinsp;=\u0026thinsp;0.02) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Prior concussion had no effect on the cytokine levels either at baseline or 2 weeks post-TBI. IL-1α which acts on the IL-1 receptor was elevated at baseline (35.51 v 24.45pg/mL; p\u0026thinsp;=\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIL-18 was suppressed in the control cohort in response to LPS, p\u0026thinsp;=\u0026thinsp;0.006 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This effect was not seen in the mTBI or sTBI cohorts, demonstrating hypo-responsiveness. Only the sTBI group responded to LPS stimulation with increased IL-33 production. IL-1β showed LPS responsiveness in controls, mTBI at baseline and sTBI (p\u0026thinsp;=\u0026thinsp;0.0005, p\u0026thinsp;\u0026lt;\u0026thinsp;0.000001, p\u0026thinsp;=\u0026thinsp;0.003, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This response was not seen in the mTBI at 10\u0026ndash;14 days, the subacute phase, demonstrating LPS hypo-responsiveness. IL-1ra showed LPS responsiveness only in the mTBI and sTBI cohorts, demonstrating pre-conditioning while the controls and mTBI in the subacute phase did not respond. IL-1α increased in response to LPS in the mTBI at baseline and at 10\u0026ndash;14 days and also the severe cohort again demonstrating pre-conditioning. The control population did not respond to LPS challenge with an increase in IL-1α production. There was a significant difference in the level of response to LPS between the controls, who did not respond and mTBI (p\u0026thinsp;\u0026lt;\u0026thinsp;0.00001).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eChildren with mTBI had a reduction in activated neutrophils, evidenced by reduced CD11b expression in the mTBI cohort reaching a significant decrease at 10\u0026ndash;14 days post-injury. In parallel, total monocytes were reduced acutely with the reduction seen in intermediate monocytes. The TLR4 expression on neutrophils was highly upregulated in the acute phase and downregulated by 10\u0026ndash;14 days, below that of control levels.\u003c/p\u003e \u003cp\u003eActivated CD11b neutrophils play an important role in blood-brain barrier disruption and cytokine release in traumatic brain injury [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Infiltrating CD11b\u0026thinsp;+\u0026thinsp;neutrophils contribute to the size of the lesion in severe TBI [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Although severe TBI in this cohort had modest increases in CD11b, in this study the response in mTBI is reduced systemic CD11b expression. The dynamics of neutrophil survival differ with age [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This is exemplified in a neonatal study which showed an increase in both CD11b and TLR4 expression in infants with neonatal encephalopathy [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] which persisted over the first week of life with dysfunctional immune responses in later childhood [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Elderly people have decreased CD11b expression, a phenomenon contributing to immune senescence [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] compromising their immune response. Age differences may be a factor in this decreased responsiveness, or the type of injury presenting, as mild TBI is distinct from severe TBI. Our cytokine analysis shows trends of immune suppression, consistent with adult studies of blast TBI [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] rather than the typical elevation of IL-8 seen in severe TBI [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. There may have been an initial peak in immune activation that was not captured in this cohort as the highest neutrophil count in severe TBI is in the first three hours [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSystemic TLR4 expression was activated in mTBI and sTBI, especially in the latter group. However, TLR4 expression was suppressed in the subacute phase of mild traumatic brain injury. The rise of TLR4 production in the severe cohort was significant. The TLR4/NF-κB pathway has been demonstrated to play an important role in traumatic brain injury [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The TLR4 complex is expressed on the cell surface of the neutrophil and plays an important role in targeting inflammatory cytokine gene transcription. Tang et al. found that IL-1β and IL-6 were significantly lower in the wounds of TLR4-deficient mice [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. When the receptor for TLR4 is activated, immune mediators released from TLR4-expressing cells may have neurotoxic effects [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTotal monocytes were depleted in the initial period following mTBI, within the intermediate subset, with a recovery seen by 10\u0026ndash;14 days. This is opposite to the effect seen in severe TBI where the monocytes were increased in the intermediate population, not reaching statistical significance. Monocytes aid recovery in spinal cord decompression [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and their depletion in the animal model results in poorer motor results [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. There is discrepancy in the literature, as a murine model shows depleted monocytes in the first day following TBI that persists for a month [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], with human studies showing elevation of the monocyte population in those with severe TBI [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The monocyte population has not been documented in those with mTBI.\u003c/p\u003e \u003cp\u003eThe innate immune response is a significant contributor to inflammation after TBI regulated by the inflammasomes. Activation of IL-1β, the end product of inflammasome activation, includes pathways via TLR4/NF-κB, via Nod-like receptor pathways including NLRP3 and NLRP1 and via AIM1 pathways. These pathways activate the inflammasome via caspase driving cytosolic NF-κB transcription and IL-1β production, driving pyroptosis. The interleukin-1 family has autocrine properties and can self- propagate its own signal with IL-1α having local autocrine effects and IL-1β having paracrine effects [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Our study did not demonstrate an upregulation of the NLRP3 pathway in the mild form of TBI in children. The pathway was downregulated, and more so if there was a prior concussion. NLRP1 was also downregulated, which persisted into week two. The AIM pathway was upregulated on presentation. ASC was not statistically higher and was low in those previously suffering mTBI. ASC and IL-18 biomarker has previously proven to be a useful in determining outcome in severe TBI [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In our study IL-18 was depressed however, not raised. This may be accounted for in the mild and different nature of an mTBI to sTBI or by age.\u003c/p\u003e \u003cp\u003eIL-1β mRNA was significantly upregulated at presentation with injury. At 10\u0026ndash;14 days from injury, it was higher still showing an important role of inflammasome at the time of injury, but especially in the subacute phase, indicating it may have a long therapeutic window and mechanistic role in prolonged symptoms. Human tissue samples around brain contusion show an IL-1α acute spike following brain injury, while IL-1β shows a much more gradual increase that is thought to represent a portion of the delayed cytokine response to TBI [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. It is a potent stimulus for other cytokine release including TNF-α and IL-6. IL-1β is a target for therapies to prevent secondary injury in TBI.\u003c/p\u003e \u003cp\u003eA mouse model of chronic repetitive mTBI in adolescence demonstrated IL-1β and IL-18 production with prominent inflammasome activation. Mice with deficiencies in caspase and interleukin receptors in that study had better outcomes at a year [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Prior concussion carries higher risk of prolonged recovery time [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Increased inflammasome activation did not account for this in our study, however there were low numbers in our groups with heterogenous injuries. Further evaluation of these groupings is warranted.\u003c/p\u003e \u003cp\u003eOverall there is a hyper-responsiveness of inflammasome-related cytokine release in response to LPS stimulation in the TBI population, but the interleukins do not respond in the subacute phase. This may represent priming by the injury event [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] which has been suggested to last one week [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Previous animal studies of severe TBI have demonstrated that pre-conditioning with LPS increased both pro- and anti-inflammatory cytokines, and their effect attenuated the extent of histological injury [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. These models of injury have contusions with a penumbra of reduced cerebral blood flow. These brain injuries are different to mTBI, further studies looking at the preconditioning response are warranted in mTBI models and patients.\u003c/p\u003e \u003cp\u003eIL-18 plays a role in downregulating TNF-α [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. At low doses, LPS causes a decrease in IL-18 production and enhances the innate immune response to sepsis, at higher doses of LPS there is an increase of IL-18 production which influences death from sepsis [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. IL-18, an end-product of the inflammasome, was significantly decreased in mTBI and the normal decrease of IL-18 in response to LPS not seen in the TBI cohorts. There were similarities with IL-33 with reduced expression in the controls and a marked response seen with the same LPS stimulus in severe TBI. IL-33 does not respond by priming and has enhanced secretion in response to attempt to prime [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The response of the mTBI group in the subacute phase fits that of priming described in the literature for the cytokines associated with the inflammasome complex.\u003c/p\u003e \u003cp\u003eThe inflammasome is a potential safe target of therapy. Numerous safe therapies modify NLRP3 including pioglitazone [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], rapamycin [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and Omega three [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and are under consideration as therapeutics. In our cohort this was not a dominant pathway. AIM1 was the only pathway upstream of IL-1β in our study that was upregulated. One small trial in adults with sTBI showed demonstrated the effect of interleukin-1 antagonist Anakinra in adults but was not powered to show clinical differences in outcomes [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMild traumatic brain injury induces changes in the innate immune system. These effect of mTBI on the immune system is prolonged. This is evidenced by the modified response of the inflammasome complex to LPS stimulation at two weeks.\u003c/p\u003e \u003cp\u003eMild traumatic brain injury, like burns, are sterile injuries that induce systemic immune changes. It would be judicious to profile this response in these children at a later time- point out from injury to see if the immune activation quiesces. The clinical period of a year was noted by Eisenberg and colleagues to be the period within which it was critical not to get a repeat concussion [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] Treating serum with LPS and measuring the responsiveness of a number of key cytokines may demonstrate the window for which the injury persists.\u003c/p\u003e \u003cp\u003eThe current literature is based on animal models of more severe injury.\u003c/p\u003e \u003cp\u003eOur findings are inconsistent with some severe TBI studies. Severe TBI usually involves gross intracranial bleeding. Furthermore, there are immune differences between adults and children. Finnerty and colleagues described how children after burn injury had a depressed response of important pro- and anti-inflammatory relative to adults\u003c/p\u003e \u003cp\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] which persist for up to 3 years [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Animal studies demonstrate persistent immune dysfunction in sTBI [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Numerous studies have demonstrated the chronic changes associated with repeated head injury [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], that support the hypothesis of inflamma-aging [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe injuries in our study population were heterogenous. Only 40% had vomiting and some of the children only had isolated symptoms[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In the clinical setting it is impossible to quantify the volume of trauma that was sustained. The numbers in the study measuring the effect of previous mTBI on inflammatory cascade were small. A limitation of this study was the absence of data on analgesia and quantifying if there was an effect from paracetamol and ibuprofen.\u003c/p\u003e \u003cp\u003eThis study showed that the pathway in mild TBI includes a concerted response by the innate immune system with TLR4 pathway signalling, inflammasome activation and alterations in neutrophil activation and monocyte populations. The changes are sustained and result in altered systemic immune responses at two weeks from injury supported by the changes in LPS responsiveness.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGCS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlasgow coma scale\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterleukin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLPS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elipopolysaccharide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emTBI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emild traumatic brain injury\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCSI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epost concussive symptom inventory\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSCAT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSports concussion assessment tool\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003esTBI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esevere traumatic brain injury\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTBI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTraumatic brain injury\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTNF-α\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTissue necrosis factor-α\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committees of Children\u0026rsquo;s Health Ireland (CHI) at Tallaght (Ref: 2016-03 (21); approved 28.03.16) and CHI at Temple Street Dublin (Ref: 16.019; approved 23.03.16), Ireland. Informed consent was obtained from patients and/or parents.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflict of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental data used to support the findings of this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the National Children\u0026rsquo;s Hospital Fund [206965], Tallaght, Dublin, Ireland\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe manuscript is being submitted on behalf of all the authors and is the original work of all authors. ER was responsible for recruitment, sample acquistion, lab experiments, analysis and was responsible for writing the main draft of the manuscript. LK, AMM, CS and MB added expert laboratory assisstance. LK, CS, ED, DD, AL, GB, DC, CM\u0026nbsp;were responsible for patient recruitment, sample acquisition, reviewing and editing the manuscript. LK TB and EM were key in study design, supervising the research and its outcomes and providing crucial editorial assistance. All authors had editorial license to review and re-draft the manuscript, and all contributors had to approve the final edit. All authors are accountable for the accuracy and scientific integrity of this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThank you to all the families and children who participated in this study with the aim of helping others in the future. We are also grateful to the staff of the Paediatric emergency department in CHI at Tallaght in the PICU in CHI at Temple St. for their support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGennarelli TA, Graham DI. Neuropathology. In \u003cem\u003eTextbook of Traumatic Brain Injury\u003c/em\u003e. Edited by Silver JM, McAllister TW. Washington DC: American Psychiatric Publishing, Inc.; 2005:27\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBabcock L, Byczkowski T, Wade SL, Ho M, Bazarian JJ. Inability of S100B to predict postconcussion syndrome in children who present to the emergency department with mild traumatic brain injury: a brief report. Pediatr Emerg Care. 2013;29:458\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRitzel RM, Doran SJ, Barrett JP, Henry RJ, Ma EL, Faden AI, et al. Chronic Alterations in Systemic Immune Function after Traumatic Brain Injury. 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Geroscience. 2020;42:1451\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Traumatic Brain injury, Concussion, Inflammation, Cytokines, Innate Immunity","lastPublishedDoi":"10.21203/rs.3.rs-4172622/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4172622/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cb\u003eIntroduction\u003c/b\u003e: Systemic Inflammation is associated with Traumatic Brain Injury (TBI) and therefore is a potential target for immunomodulation. Dysregulated immune function post-TBI increased susceptibility to infection and post-concussive syndrome. The inflammasome is a protein complex associated with an amplified proinflammatory response and is a potential target for immunomodulation that preserves antimicrobial immunity.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMethods\u003c/b\u003e: Samples from children with mild TBI (mTBI; Glasgow coma scale (GCS) 14/15), severe TBI (sTBI; GCS\u0026thinsp;\u0026lt;\u0026thinsp;8) and control children were collected at baseline and two week follow up and were treated with endotoxin and melatonin. Toll-like receptor (TLR4; marker of endotoxin responses) and CD11b (activation marker) expression on neutrophils and monocytes were evaluated by flow cytometry. Inflammasome-related genes and cytokines were assessed using TaqMan RT-PCR samples ELISA sandwich immunoassay, respectively.\u003c/p\u003e \u003cp\u003e \u003cb\u003eResults\u003c/b\u003e: A total of 214 children were enrolled including: TBI (n\u0026thinsp;=\u0026thinsp;116), with mild TBI (mTBI; Glasgow coma scale (GCS) 14/15) and severe TBI (sTBI; GCS\u0026thinsp;\u0026lt;\u0026thinsp;8), and (n\u0026thinsp;=\u0026thinsp;98) control patients collected at baseline and two week follow up. Total monocyte and intermediate monocyte populations were reduced in mTBI at baseline. Neutrophil CD11b and TLR4 expression was decreased in mTBI at 10\u0026ndash;14 days. NLRP3 and NLRP1 were downregulated at 10\u0026ndash;14 days while IL-1β was increased at baseline at 0\u0026ndash;4 days and further elevated by 10\u0026ndash;14 days and significantly higher in those with no previous mTBI. Serum cytokines showed lower IL-18 and raised IL-33 in those with mTBI. Prior concussion did not influence serum cytokine levels. In addition, LPS did not stimulate an IL-18 and IL-1β response in the mTBI group at 10\u0026ndash;14 days.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConclusions\u003c/b\u003e: Children with mTBI had reduced CD11b and TLR4 expression and NLRP3 inflammasome activation. IL-1β mRNA was raised and continued to rise after injury implicating the innate immune system in the subacute phase of injury. Immune dysregulation post-TBI in children may be a target for immunomodulation following further exploration in vitro of potential mechanisms and therapies.\u003c/p\u003e","manuscriptTitle":"Altered Inflammasome and Immune activation in Paediatric Traumatic Brain Injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-29 19:03:01","doi":"10.21203/rs.3.rs-4172622/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aeb1725a-2c22-4980-ad2f-ce9ad8e0c659","owner":[],"postedDate":"March 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-22T04:47:23+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-29 19:03:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4172622","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4172622","identity":"rs-4172622","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}