Loss of NLRX1 Exacerbates Neural Tissue Damage and NF- κB Signaling following Brain Injury

This information is current as Michelle H. Theus, Thomas Brickler, Armand L. Meza, of October 3, 2021. Sheryl Coutermarsh-Ott, Amanda Hazy, Denis Gris and Irving C. Allen J Immunol 2017; 199:3547-3558; Prepublished online 9 October 2017; doi: 10.4049/jimmunol.1700251 http://www.jimmunol.org/content/199/10/3547 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Loss of NLRX1 Exacerbates Neural Tissue Damage and NF-kB Signaling following Brain Injury

Michelle H. Theus,*,1 Thomas Brickler,*,2 Armand L. Meza,*,†,2 Sheryl Coutermarsh-Ott,*,1 Amanda Hazy,* Denis Gris,‡ and Irving C. Allen*,1

Traumatic and nontraumatic brain injury results from severe disruptions in the cellular microenvironment leading to massive loss of neuronal populations and increased neuroinflammation. The progressive cascade of secondary events, including ischemia, inflammation, excitotoxicity, and free-radical release, contribute to neural tissue damage. NLRX1 is a member of the NLR family of pattern recognition receptors and is a potent negative regulator of several pathways that significantly modulate many of these events. Thus, we hypothesized that NLRX1 limits signaling in the brain following trauma. To evaluate this hypothesis, we used Nlrx12/2 mice in a controlled cortical impact (CCI) injury murine model of traumatic brain injury (TBI). In this article, we show that Nlrx12/2 mice exhibited significantly larger brain lesions and increased motor deficits following CCI Downloaded from injury. Mechanistically, our data indicate that the NF-kB signaling cascade is significantly upregulated in Nlrx12/2 animals. This upregulation is associated with increased microglia and macrophage populations in the cortical lesion. Using a mouse neuroblas- toma line (N2A), we also found that NLRX1 significantly reduced under hypoxic conditions. In human patients, we identify 15 NLRs that are significantly dysregulated, including significant downregulation of NLRX1 in brain injury following aneurysm. We further demonstrate a concurrent increase in NF-kB signaling that is correlated with aneurysm severity in these human subjects. Together, our data extend the function of NLRX1 beyond its currently characterized role in host–pathogen http://www.jimmunol.org/ defense and identify this highly novel NLR as a significant modulator of brain injury progression. The Journal of Immunology, 2017, 199: 3547–3558.

raumatic brain injury (TBI) is a complex neurologic con- brain. These events may or may not involve injury to the skull or dition that has emerged as an important cause of morbidity overlying tissues. Conversely, nontraumatic brain injuries have a T and mortality in the adolescent and young adult pop- wide range of causes but are not directly associated with physical ulations. It is defined differently throughout the literature but is trauma. Examples of nontraumatic brain injury can include brain generally accepted as any external force that causes injury to the tumors, meningitis, hypoxic/anoxic brain injury, stroke, or aneu-

rysm. In traumatic and nontraumatic brain injury, the resulting by guest on October 3, 2021 morbidity and mortality seen clinically are not typically due to the *Department of Biomedical Sciences and Pathobiology, Virginia–Maryland College actual primary injury itself, but rather the secondary changes that † of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061; Department of Neu- occur in the brain as a result of the injury. These secondary roscience, Virginia Tech, Blacksburg, VA 24061; and ‡Programme d’Immunologie, Faculte´ de Me´decine et des Sciences de la Sante´,Universite´ de Sherbrooke, Sherbrooke, changes are associated with the activation of the innate and Quebec J1H 5N4, Canada adaptive immune system and include inflammation, infiltration of 1M.H.T., S.C.-O., and I.C.A. contributed equally to this work. immune cells, release of excitatory neurotransmitters, cerebral 2T.B. and A.L.M. contributed equally to this work. edema, vasospasm, ischemia, hypoxia, free-radical damage, and ORCIDs: 0000-0001-6485-2104 (M.H.T.); 0000-0002-1580-4808 (T.B.); 0000-0001- others (1–3). A great deal of research has looked into the role of 9573-5250 (I.C.A.). the adaptive immune system in brain injury and how it can mod- Received for publication February 21, 2017. Accepted for publication September 7, ulate these various secondary processes. However, only relatively 2017. recently have researchers begun to focus on the role of the innate This work was supported by National Institute of Neurological Disease and Stroke Awards NS096281 and NS081623. Student work was supported by National Institute immune system (1). The majority of studies have been focused on of Allergy and Infectious Diseases Animal Model Research for Veterinarians Train- the role of microglial cells in the progression of the injury, because ing Grant T32-OD010430 (to S.C.-O.). Student support throughout this project was they are the predominant innate immune cell type in the brain (2). also provided by the Virginia Tech Initiative for Maximizing Student Development program (GM0727-09) and the Virginia Tech Post-Baccalaureate Research and Ed- Somewhat similar to macrophages, microglial cells express a di- ucation Program (GM066534-14). The content is solely the responsibility of the verse variety of pattern recognition receptors (PRRs) that modulate authors and does not necessarily represent the official views of the National Institutes of Health or any other funding agency. their response to injury and drive many of the critical secondary Address correspondence and reprint requests to Dr. Michelle H. Theus and Dr. Irving changes seen in the brain following injury (2). C. Allen, Virginia Tech, Virginia–Maryland College of Veterinary Medicine, Depart- PRRs are associated with plasma and endosomal ment of Biomedical Sciences, IDRF 140, Duckpond Drive, Blacksburg, VA 24061. membranes, as well as the itself. These receptors and sen- E-mail addresses: [email protected] (M.H.T.) and [email protected] (I.C.A.) sors are responsible for recognition of various foreign and host mo- The online version of this article contains supplemental material. lecular motifs (known as damage-associated molecular patterns or Abbreviations used in this article: CCI, controlled cortical impact; EAE, experimen- pathogen-associated molecular patterns). Once these proteins bind tal autoimmune encephalomyelitis; GFAP, glial fibrillary acid ; IHC, immu- nohistochemistry; IPA, Ingenuity Pathway Analysis; NLR, NOD-like receptor; PI, or sense their respective ligands, they are responsible for initiating propidium iodide; PRR, pattern recognition receptor; ROS, reactive oxygen species; a variety of cellular responses, including the activation of key in- shRNA, short hairpin RNA; TBI, traumatic brain injury. flammatory signaling pathways, such as the NF-kB signaling cas- Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 cade. NF-kB signaling has previously been shown to be important www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700251 3548 NLRX1 PROTECTS AGAINST BRAIN INJURY in the pathogenesis of brain injury. For example, Lian et al. (4) Materials and Methods showed that mice lacking IkBɑ,anNF-kB inhibitory protein, Animals showed significantly increased neuroinflammation when assessed in 2 2 The generation and characterization of Nlrx1 / mice have been previ- a model of TBI. Moreover, in another TBI model, suppressing the ously described (15). All mice were maintained on the C57BL/6J back- NF-kB signaling pathway through exogenous VEGI treatment at- ground. All animals used in experiments were male and between 2 and tenuated brain injury (5). Thus, these studies illustrate that unre- 4 mo of age. C57BL/6J and Nlrx12/2 mice were maintained as separate stricted NF-kB signaling is an important component in the colonies. All studies involving mice were repeated at least three inde- pathophysiology of brain injury. pendent times, with three to seven mice per genotype and treatment group. All experiments were conducted in accordance with the National Institutes NOD-like receptors (NLRs) are intracellular PRRs that are of Health’s Guide for the Care and Use of Laboratory Animals and were generally classified as either inflammasome-forming NLRs or conducted under the approval of the Virginia Tech Institutional Animal regulatory NLRs. The inflammasome-forming NLRs are well Care and Use Committee and the Virginia–Maryland College of Veterinary studied and are characterized by their capacity to initiate the Medicine. formation of the multiprotein inflammasome complex. Inflam- Cell culture and cell lines masome formation ultimately facilitates the maturation and ac- b All mouse neuroblastoma (N2A) cell lines were generated as previously tivation of the proinflammatory cytokines IL-18 and IL-1 . described (20). Briefly, we used OriGene TrueORF cDNA Clones with Several inflammasome-forming NLRs have been evaluated in the TurboFectin to generate Nlrx1 stable knock-in cells and OriGene short context of brain injury. For example, multiple studies have hairpin RNA (shRNA) plasmid against Nlrx1 to generate the knockdown suggested that NLRP1 and NLRP3 may play critical regulatory cells. Cell death was evaluated by flow cytometry using annexin V/propidium roles in TBI in humans and rodents (6–8). Moreover, NLRC4 and iodine (PI) staining. H2O2 was used to stimulate neuron cell death. All ex- periments were performed four independent times, and each experiment in- Downloaded from AIM2 have also been implicated in contributing to nontraumatic cluded all of the experimental groups. We evaluated 10,000 events, excluding brain injury in stroke models (9). However, although the cellular debris and cellular clumps, based on side and forward scatter. The inflammasome-forming NLRs have been well studied in multiple analysis was performed using automatic assignment of the set gates to all types of brain injury, there is a paucity of data pertaining to the samples within the FlowJo workspace. contribution of the regulatory NLRs. This subgroup includes CCI injury and behavioral assessment positive and negative regulatory NLRs that function through the direct regulation of inflammatory signaling pathways, such as Mice were anesthetized using i.p. injection of ketamine and xylazine. http://www.jimmunol.org/ k Following the loss of consciousness, mice were positioned in a stereotaxic NF- B, AKT, and IFN signaling (10–12). Three NLRs have been frame (21, 22). Body temperature was maintained at 37˚C and monitored shown to function as negative regulators, including NLRP12, via rectal probe throughout the surgery. During surgery, a skin incision was NLRC3, and NLRX1. NLRX1 is of particular interest in the made, followed by a 5-mm craniotomy using a portable drill over the right 2 context of brain injury because it has been shown to play a role parietal-temporal cortex ( 2.5 mm anterior/posterior and 2.0 mm lateral from bregma). Subsequently, CCI was induced by application of the eCCI- in a variety of cellular processes important to injury pathogen- 6.3 device (Custom Design & Fabrication; 4-mm impounder) at a velocity esis in a diverse range of cell types and tissues. For example, of 3.5 m/s, depth of 0.5 mm, and 150 ms impact duration (21, 22). Animals NLRX1 negatively regulates NF-kB signaling, type I IFN sig- designated as sham controls were placed under anesthesia, as described naling, and reactive oxygen species (ROS) production, as well as above, and following loss of consciousness, received a skin incision and by guest on October 3, 2021 acts as a positive regulator of autophagy in macrophages and closure only. Skin incisions were closed using Vetbond Tissue Adhesive (3M). Following surgery, all animals were placed in a heated cage to fibroblasts (11, 13–15). NLRX1 has been best characterized in maintain body temperature for 1 h. At 1, 3, or 14 d post–CCI injury, mice the context of pathogen recognition (13, 15, 16). However, recent were euthanized, and brain tissue was removed following decapitation. studies have extended the function of NLRX1 beyond this initial Fresh frozen tissue was embedded in OCT and coronally sectioned at 30 mm m role in modulating host–pathogen interactions and identified thickness. Serial sections were taken 300 m apart and stained for Nissl substance (22). Rotarod behavior assessment was performed as previously contributions to cancer, chronic obstructive pulmonary disease, described (22, 23). inflammatory bowel disease, and the modulation of cell death + (11, 17–19). Evaluation of contusion volume and CD11b cells The purpose of this study was to investigate the role of NLRX1 in Contusion volume was assessed by a blinded investigator using a Cavalieri the pathogenesis of brain injury. We hypothesized that loss of Estimator from Stereo Investigator (MBF Bioscience) and an Olympus NLRX1 would exacerbate NF-kB signaling and tissue damage BX51TRF motorized microscope (Olympus America). Contusion volume following TBI. To test this, we used an Nlrx12/2 mouse in a (cubic millimeters) was determined as previously described (22). Briefly, volume analysis was performed by estimating the area of tissue loss in the model of controlled cortical impact (CCI) and evaluated quanti- ipsilateral cortical hemisphere for five coronal serial sections at or around tative and qualitative measures of brain injury. Consistent with our the epicenter (21.1 to 22.6 mm posterior from bregma) of injury. Nissl- hypothesis, mice lacking NLRX1 demonstrated increased patho- stained serial sections were viewed under brightfield illumination (original 3 physiological features consistent with increased brain injury magnification 4). A random sampling scheme was used that estimates every tenth section from rostral to caudal, yielding five total sections to be compared with wild-type control mice. Increased lesion volume in m 2/2 analyzed. A randomly placed grid, with points spaced 100 m apart, was Nlrx1 mice was associated with increased NF-kB signaling placed over the ipsilateral hemisphere, and the area of contusion was and influx of CD11b+ microglia and/or macrophage populations in marked within each grid. Contusion boundaries were identified by loss of the lesion site. These findings correlate with NLRX1-dependent Nissl staining, pyknotic neurons, and tissue hemorrhage. The contoured effects identified in a relevant neuroblast cell line in which we area, using grid spacing, was used to estimate total tissue volume based on section thickness, section interval, and total number of sections within the performed cell death assays. -expression analysis from the Cavalieri program. Data are presented as volume of tissue loss or contusion 2/2 cortex of CCI-injured Nlrx1 mice show increased changes in volume (cubic millimeters) for wild-type and Nlrx12/2 mice. A Stereo mRNA expression levels of involved in NF-kB signaling. Investigator optical fractionator was used on serial coronal brain sections + m 2 Similar correlations were found in gene-expression data from to estimate the total number of CD11b cells within 1500 m( 0.2 to 22.5 anterior/posterior) of injured cortical tissue, as previously de- human patients following brain injury associated with ruptured scribed (22, 24). aneurysms. Collectively, these studies provide evidence of NLRX1’s role beyond host–pathogen interactions and further our knowl- Expression profiling edge regarding the underlying mechanisms involved in brain Total RNA was collected from brain specimens following mechanical injury. homogenization, lysis, and extraction using TRIzol and the manufacturers’ The Journal of Immunology 3549 protocols. The purified RNA was quantified, and 1 mg was pooled from three randomly chosen brains prior to the cDNA reaction. Expression profiles were assessed using the RT2 Profiler PCR Array Platform PAMM-025Z (QIAGEN), following the manufacturer’s protocols. Ingenuity Pathways Analysis (IPA) software was used to assess the array data. In addition to the profiling studies, RNA samples (5 mg) were archived using a cDNA Archive Kit (ABI) and evaluated via RT-PCR using specifically targeted commercially available primer/probe sets (ABI). Human metadata analysis Human NLRX1 expression was evaluated using a publicly accessible microarray meta-analysis search engine (http://www.nextbio.com/b/search/ ba.nb), as previously described (25). Gene-expression and pathway anal- yses in human subjects and rodents were conducted using the following array data series (available through the National Center for Biotechnol- ogy Information: https://www.ncbi.nlm.nih.gov/): GSE11686, GSE3307, GSE58294, GSE36233, GSE1767, GSE21079, GSE66573, GSE43591, and GSE20141. Statistical analysis Data were analyzed using GraphPad Prism, version 6 (GraphPad, San Diego, CA). A Student two-tailed t test was used for comparison of two experimental groups. Multiple comparisons were done using one-way and Downloaded from two-way ANOVA where appropriate, followed by the Tukey posttest for multiple pairwise examinations. Correlation was also computed using GraphPad Prism. Changes were identified as statistically significant at p ,0.05. Mean values are reported with SEM.

Results http://www.jimmunol.org/ NLRX1 attenuates damage and motor deficits following controlled TBI

NLRX1 functions as a negative regulator of inflammation and FIGURE 1. Increased lesion volume and motor deficits in Nlrx12/2 modulates a variety of pathways associated with brain injury (10, mice following CCI injury. Nissl staining of wild-type brains, shown as a 2 2 12, 26). Thus, we hypothesized that Nlrx1 / mice would dem- mosaic-tiled image at original magnification 34(A–C), at 3 d post-CCI onstrate significantly increased tissue damage following TBI. To compared with Nlrx12/2 brains (D–F) shows increased lesion volume evaluate this hypothesis, we used previously characterized Nlrx12/2 (denoted by red dotted line) (G) in the absence of NLRX1. (H) Motor 2/2 mice in a CCI model (21, 22). Wild-type and Nlrx12/2 animals deficits are also more prominent in Nlrx1 mice compared with wild- , by guest on October 3, 2021 were subjected to a moderate CCI using a 4-mm impounder at a type mice at 3–14 d post-CCI. n = 5–7 per group. *p 0.05. velocity of 3.5 m/s, depth of 0.5 mm, and 150 ms impact duration, as previously described (6, 21, 22). TBI progression was evaluated Nlrx12/2 and wild-type sham-operated mice (Nlrx12/2 110.70 6 3 d postinjury, which was empirically determined to be the peak 11.42%; wild-type 115.37 6 5.24%) (data not shown). However, time point for acute neural tissue damage and pathophysiology in following CCI injury, Nlrx12/2 mice performed significantly prior studies using wild-type C57BL/6J animals in our CCI model worse on the Rotarod (Fig. 1H). Wild-type and Nlrx12/2 mice 2/2 (6, 21, 22). Upon necropsy, Nlrx1 mice were observed to have demonstrated motor deficits following injury. However, at 3 d significantly larger gross lesions and hemorrhage compared with the post–CCI injury, Nlrx12/2 mice had a significant increase in wild-type animals (data not shown). Serial sections were generated motor deficits on the Rotarod compared with wild-type animals from the whole brain and subjected to Nissl staining. Contusion (Nlrx12/2, 32.76 6 1.0%; wild-type, 59.17 6 5.45%) (Fig. 1H). boundaries were identified based on the loss of Nissl stain, pyknotic This trend continued through day 7 (Nlrx12/2, 51.36 6 7.6%; neurons, and tissue hemorrhage. Consistent with the gross lesion wild-type, 79.35 6 7.8%) and day 14 (Nlrx12/2, 55.8 6 16.8%; 2/2 observations, Nlrx1 mice were found to have more expansive wild-type, 81.73 6 5.21%) (Fig. 1H). Together, these data indicate contusion boundaries, which radiated out significantly further from that Nlrx12/2 mice experience increased motor impairments fol- the impact epicenter compared with the injuries observed in the lowing CCI injury, and recovery is attenuated compared with wild-type animals (Fig. 1A–F). Using the Cavalieri Estimator, we wild-type animals. The Rotarod data correlate with the contusion 2/2 found significant differences in contusion volume between Nlrx1 volume estimates and confirm that loss of NLRX1 significantly 3 3 mice (10.28 6 1.15 mm ) and wild-type animals (4.55 6 0.47 mm ) impacts neural tissue damage and subsequent motor function re- post–CCI injury (Fig. 1G). These data support a role for NLRX1 in covery following CCI injury. limiting neural tissue loss and TBI pathogenesis in the cortex fol- + lowing CCI injury. NLRX1 regulates CD11b cell proliferation and influx We next sought to determine whether the increased damage following TBI following TBI in Nlrx12/2 mice resulted in any broad behavioral NLRX1 is broadly expressed in the brain and in a variety of cells effects. We used Rotarod behavioral analysis to determine whether known to influence TBI pathogenesis, including neurons, micro- motor deficit and recovery were affected following CCI injury. glia, and astrocytes (Supplemental Fig. 1). To better characterize Mice were trained on the Rotarod 4 d prior to CCI injury, and the the increased CCI injury observed in Nlrx12/2 mice, we next animals were subjected to motor assessments at 3, 7, and 14 d sought to determine the effects of NLRX1 loss on microglia and post–CCI injury or post–sham operation. The time to fall was astrocyte populations in the lesion following TBI. Frozen sections determined and then normalized to the average baseline time for were prepared for immunohistochemistry (IHC), and specimens each animal. No significant differences were observed between were evaluated for CD11b and glial fibrillary acid protein (GFAP) 3550 NLRX1 PROTECTS AGAINST BRAIN INJURY Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 2. Analysis of microglia and astrocyte activation 3 d post–CCI injury in wild-type and Nlrx12/2 mice. (A and A1–A3) Confocal images show CD11b+ microglia (green) in the lesioned cortex of wild-type mice. (B and B1–B3) Increased numbers of CD11b+ cells are seen in Nlrx12/2 mice. (C and D) Activated GFAP+ astrocytes are found in the peri-lesion site following CCI injury. No differences in astrocytes are observed between wild-type mice (C) and Nlrx12/2 mice (D). Scale bars, 1 mm (A–D), 20 mm(A1–A3) and (B1–B3). (E–L) Ortho view from confocal imaging shows dividing CD11b+ cells colabeled with anti-Ki67 staining (red) and nuclear DAPI staining (blue). There is greatly enhanced staining in Nlrx12/2 mice (I–L) compared with wild- type mice (E–H). Scale bars, 20 mm(E, I). Original magnification 340 (F–H and J–L). (M) Stereo Investigator optical fractionator analysis revealed a significant increase in CD11b+ cells in the cortex of Nlrx12/2 mice compared with wild-type mice. Data are mean 6 SEM. *p , 0.05. expression at 3 d post–CCI injury (Fig. 2A–D). In the intact brain, increase in CD11b+ cells was observed in Nlrx12/2 mice com- the microglia should be the only CD11b+ cells. However, fol- pared with wild-type animals following CCI injury (Fig. 2A, 2B). lowing injury, disruption to the blood–brain barrier can allow an In addition, astrocytes can also significantly impact TBI patho- increased influx of macrophages and into the lesion genesis (reviewed in Ref. 27). We used the prototypical astrocyte that will also contribute to the CD11b+ cell population. Increased marker GFAP to evaluate astrogliosis in the peri-lesion cortex CD11b+ cells were observed in all of the mice following CCI after CCI injury. No significant differences were observed in injury compared with the sham-operated animals. However, an density or distribution of GFAP+ cells between Nlrx12/2 mice and The Journal of Immunology 3551 wild-type animals following CCI injury (Fig. 2C, 2D). Together, Nlrx12/2 lesions compared with those in wild-type animals these data suggest that NLRX1 functions directly or indirectly to (Fig. 3, Supplemental Fig. 3). This evaluation revealed that 47 modulate microglia proliferation and/or macrophage influx, with genes associated with inflammation and NF-kB signaling were no effects on astrocyte populations following CCI injury. significantly upregulated, defined as $2-fold increase in gene Prior studies evaluating NLRX1 in cancer have found a role expression over wild-type (Supplemental Fig. 3). The genes with for NLRX1 in the regulation of macrophage proliferation (11). the greatest increase in expression in Nlrx12/2 brains included Specifically, macrophages harvested from Nlrx12/2 mice dem- Card10, Relb, and Il10, all of which were upregulated .7-fold onstrated a significant increase in proliferation under specific compared with wild-type. Conversely, the following three genes physiological conditions (11). Following TBI, microglia in the were significantly downregulated in Nlrx12/2 brains: Slc20a1, lesion area undergo proliferation in response to injury and play Tnf, and Ccl2 (Supplemental Fig. 3). IPA of the global changes in vital roles in the modulation of secondary injury and recovery (27, revealed a significant increase in NF-kB signaling 28). Although microglia are essential for proper subacute resolu- in Nlrx12/2 lesions (Fig. 3). This increase was observed in genes tion of damage in the CNS, acutely these cells can also negatively categorized across each functional group in the NF-kB signaling impact pathogenesis by propagating inflammation to tissues ad- cascade, including ligands and receptors, downstream signaling jacent to the site of injury and instigating significant collateral mediators, kinases, cytoplasmic sequestering and release factors, damage if dysregulated (29). Thus, we next sought to determine and four of the five NF-kB transcription factors (Fig. 3). Signifi- whether the increase in CD11b+ cells was associated with in- cant differences were also observed in NF-kB–responsive genes creased microglia proliferation or macrophage influx following associated with the immune response. Although, generally, these CCI injury. Frozen sections were prepared for IHC, and specimens NF-kB–responsive genes were significantly upregulated, notable were evaluated for CD11b and Ki67 reactivity 3 d post–CCI in- exceptions included Tnf and Ccl2, which were significantly Downloaded from jury. Interestingly, we observed a significant increase in CD11b+ downregulated following CCI injury in Nlrx12/2 mice (Fig. 3, and Ki67+ cells in the injured cortex of Nlrx12/2 and wild-type Supplemental Fig. 3). Finally, several genes in the NF-kB sig- mice (Fig. 2E–L). However, we observed a greater density of these naling cascade and associated with apoptosis were also found to cells in Nlrx12/2 mice compared with wild-type animals (Fig. 2E– be significantly upregulated in lesions from Nlrx12/2 brains com- L). Indeed, in wild-type mice, very few double-positive cells were pared with wild-type lesions (Fig. 3, Supplemental Fig. 3). observed (Fig. 2E–H). Quantitative assessment of CD11b+ cells http://www.jimmunol.org/ NLRX1 ablation results in increased NF-kB signaling in using an optical fractionator showed an increase in the number of + CD11b+ cells present in the injured cortex of Nlrx12/2 mice CD11b cell populations following TBI (22,133 6 3,458 cells) compared with wild-type mice (7,966 6 NLRX1 is broadly expressed in the brain, and NF-kB is a master 1,270 cells) at 3 d post–CCI injury (Fig. 2M). Consistent with the regulator of gene transcription in cells throughout the CNS increased CD11b immunoreactivity, these Ki67 findings suggest (Supplemental Fig. 1) (31, 32). Thus, we next sought to identify that NLRX1 regulates microglia proliferation following TBI. It is the cell types associated with the increased NF-kB signaling in the also possible that infiltrating macrophages contribute to this cell CCI-injured cortical tissue of Nlrx12/2 mice. To identify these population. However, in either case, these findings are consistent cells, we used IHC targeting p-p65, which is a direct assessment of by guest on October 3, 2021 with increased TBI pathogenesis and could contribute directly or the activation of the canonical NF-kB signaling cascade. Like- indirectly to enhanced injury progression and motor deficits. wise, p65 was found to be significantly upregulated in the gene- expression and pathway analysis studies in Nlrx12/2 mice (Fig. k NLRX1 negatively regulates NF- B signaling following TBI 3). IHC analysis revealed increased immunoreactivity for p-p65 in NLRX1 has been previously shown to negatively regulate NF-kB the cortical lesions following CCI injury in Nlrx12/2 and wild- signaling and this attenuation is correlated with increased prolif- type animals (Fig. 4). In both genotypes, the p-p65 staining was eration and tumorigenesis in cancer models (11, 30). Thus, we localized to CD11b+ cells (Fig. 4A, 4D). Indeed, despite their next profiled gene expression to evaluate NF-kB signaling fol- lower numbers compared with Nlrx12/2 mice, the overwhelming lowing TBI in the Nlrx12/2 and wild-type brain following CCI majority of p-p65+ cells in wild-type animals were also double injury. Fresh 4 3 4 mm cortical tissue was dissected from positive for CD11b (Fig. 4A–C). Consistent with the gene- the ipsilateral and contralateral hemispheres of each animal at expression findings, we observed significantly increased num- 3 d postinjury. Total RNA was extracted from three randomly bers of CD11b+ p-p65+ double-positive cells in Nlrx12/2 mice chosen wild-type or Nlrx12/2 brains and the RNA from each compared with wild-type animals (Fig. 4D–F). These data suggest genotype was pooled in equal concentrations for cDNA synthesis that the CD11b+ cell population is a major source of NF-kB (Supplemental Fig. 2). Changes in gene expression were evaluated signaling, which may be suppressed by NLRX1 in the cortex using a panel of superarrays (QIAGEN) chosen to evaluate path- following CCI injury. Together, these findings are consistent with ways associated with inflammatory signaling, cell death, and NLRX1’s role as a negative regulator of canonical NF-kB sig- proliferation, as previously described (11, 15). Gene expression naling observed in other diseases and model systems and may was determined using the DD cycle threshold method to calculate underlie the increased proliferation, recruitment, and function of fold change, as described by the manufacturer. A panel of eight these cells in the brain following trauma (11, 15). housekeeping genes was used to normalize the expression of each gene on the array, and the fold change in gene expression between NLRX1 protects cultured neurons from oxidative damage the respective CCI-injured and uninjured Nlrx12/2 and wild-type In addition to regulating NF-kB signaling and CD11b+ cell pro- brains was determined (Supplemental Fig. 3). Gene-expression data liferation and recruitment, NLRX1 has been shown to modulate were further analyzed using IPA to better define and visualize the cell death in neurons (20). Neuronal death is a critical compo- signaling pathways influenced by the loss of NLRX1 (Fig. 3). nent of the lesion outcome observed in the CCI injury model, The gene-expression studies and pathway analysis revealed which was exacerbated in Nlrx12/2 mice. Likewise, we observed a significant increase in NF-kB signaling in the cortical brain significantly increased expression of several genes associated tissue from Nlrx12/2 and wild-type mice following injury. How- with cell death and apoptosis in Nlrx12/2 lesions (i.e., Fadd, Fasl, ever, canonical NF-kB signaling was significantly upregulated in and Agt) compared with those in wild-type animals (Fig. 3, 3552 NLRX1 PROTECTS AGAINST BRAIN INJURY Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 3. NLRX1 negatively regulates NF-kB signaling following TBI. Heat map schematic diagram illustrating the fold change in expression of all genes associated with NF-kB signaling that were identified as being significantly upregulated or downregulated in the brain following TBI in Nlrx12/2 mice compared with TBI in wild-type animals. Analysis was based on the DD cycle threshold method, for which all data were standardized to the average gene expression for a panel of eight housekeeping genes and normalized to the respective nonlesion contralateral region of each animal. A .2-fold change in gene expression was considered significant. Three randomly selected brains from each genotype and treatment group were selected and pooled for profiling studies. Pathway assessments were based on IPA (n = 3 per group).

Supplemental Fig. 3). To evaluate the contribution of NLRX1 to cell types (33, 34). NLRX1 was either knocked down or overex- neuronal cell death, we next used an in vitro model based on pressed, and cell death was evaluated following H2O2 treatment. mouse N2A cells (20). N2A cells are a murine neuroblastoma cell ROS has been suggested to be an important mediator of acute line that was shown in previous studies to derive multiple neuronal brain injury following TBI. In this study, we demonstrate that The Journal of Immunology 3553

a significant increase in apoptosis after treatment with H2O2 in all cell lines with the exception of N2ANlrx1 (Fig. 5A). Our data in- dicate that apoptosis is significantly reduced in N2ANlrx1 cells and significantly increased in N2Ash cells (Fig. 5A). Thus, we can conclude that NLRX1 plays a key antiapoptotic role in neuroblasts under oxidative stress like that occurring acutely following TBI, and our data suggest that NLRX1 may function to attenuate neuron loss in vivo. However, it should be noted that future in vivo and ex vivo studies using primary neurons are needed to provide additional insights necessary to confirm this hypothesis. NF-kB signaling is significantly dysregulated following nontraumatic brain injury in human subjects Our findings that NF-kB signaling is significantly upregulated in Nlrx12/2 mice following TBI are consistent with prior studies linking NLRX1 as a negative regulator of inflammation with its FIGURE 4. Microglia expression of p-p65 at 3 d post–CCI injury in the protective role in attenuating neuronal cell death (20). In human 2/2 A C cortex of wild-type and Nlrx1 mice. ( – ) High-magnification fluo- patients, increased NF-kB signaling has been shown to promote rescence images of CD11b (green) and p-p65 (red) in wild-type injured neurodegenerative mechanisms during Parkinson’s disease and cortex. (D–F) Increased numbers of CD11b/p-p65+ cells are seen in Downloaded from Nlrx12/2 mice. Scale bars, 100 mm. Alzheimer’s disease (35–39). However, there have been few hu- man studies directly evaluating NF-kB signaling following trauma and/or TBI. Likewise, there is a paucity of data associated with treatment of N2A cells with 100 mM H2O2 for 18 h resulted in a noninflammasome-forming NLRs, such as NLRX1, in human significant decrease in cell viability in all cell lines with the patients following brain injury. To address these shortcomings and exception of N2ANlrx1 cells, which overexpress Nlrx1 (Fig. 5). evaluate the relevance of our findings in human patients, we

Increased NLRX1 in N2A cells led to a significant reduction in conducted a retrospective analysis of gene-expression data ar- http://www.jimmunol.org/ H2O2-induced cell death compared with control cells or cells with chived as National Institutes of Health Gene Expression Omnibus shRNA knockdown of Nlrx1 (N2Ash), which conversely display datasets from human patients following ruptured brain aneurysms enhanced cytotoxicity (Fig. 5B, 5C). Annexin V/PI staining allows (GSE26969 and GSE54083) (25). Ruptured brain aneurysms are for the quantification of cells in the early stages of apoptosis, when considered nontraumatic brain injuries that have several clinical phosphatidylserine is flipped and faces outside of the cell, in- features in common with traumatic contusion injury and our creasing its availability for annexin V binding. To exclude necrotic CCI injury model, including massive hemorrhage, infiltration of cells and cells in the late stages of apoptosis, we used PI stain, peripheral-derived immune cells, neuronal and glial apoptosis, which penetrates all dead cells. Using this technique, we observed reduced cerebral blood flow, and blood–brain barrier permeability by guest on October 3, 2021

FIGURE 5. NLRX1 protects neurons from oxidative damage. (A) Representative flow cytometry plots of N2Asc, N2ANlrx1, and N2Ash cells stained with annexin V/PI before and after H2O2 treatment. (B) Quantification of flow cytometry data [lower left quadrant in (A)] showing the percentage of live cells. (C) Quantification of flow cytometry data [lower right quadrant in (A)]) showing the percentage of apoptotic cells. Cells transfected with scrambled control sc Nlrx1 sh (N2A ), with (N2A ), and with shRNA for nlrx1 (N2A ) were treated with 100 mM H2O2 and stained with annexin V/PI. These cells were characterized previously (20). Cells were analyzed by flow cytometry using a CytoFLEX 20 (Beckman Coulter). Data were analyzed using FlowJo software. n = 4 experiments were repeated four times; for analysis, 10,000 events were collected, excluding cellular debris and cell clumps based on size. Means of different groups were compared using two-way ANOVA, followed by the Tukey test. Significance was established at p , 0.05. *different from untreated control, #different from treated control. 3554 NLRX1 PROTECTS AGAINST BRAIN INJURY Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 6. Genes associated with NF-kB signaling were significantly dysregulated following nontraumatic brain injury in human subjects. Retrospective metadata analysis of gene-expression changes in specimens collected from the aneurysmal dome following ruptured, unruptured, or superficial intracranial aneurysms in human subjects were analyzed (GSE26969 and GSE54083). (A) Negative regulatory NLR expression in ruptured and superficial lesions. (B and C) Genes associated with NF-kB signaling were evaluated. (B) The 10 genes with the largest changes in gene expression compared with the superficial specimens. (C) Genes that were downregulated compared with specimens from superficial injuries. n = 5–10 specimens per group. *p , 0.05.

(40–42). Also, there are several well-characterized and controlled evaluating gene-expression changes in specimens collected from human brain aneurysm data sets publicly available to evaluate the aneurysmal dome following ruptured, superficial, or unrup- our hypotheses. We mined data from several independent studies tured intracranial aneurysms in up to 10 human subjects per The Journal of Immunology 3555 condition (43, 44). Each study evaluated gene expression on subjects but was significantly upregulated in the mouse CCI model .41,000 transcripts from 3 to 10 patients in each group. Ex- in the absence of NLRX1 (Figs. 3, 6C). However, despite these pression data were normalized to GAPDH, and the fold change in relatively few discrepancies, the sum of these data reveal that NF-kB expression was compared against the superficial aneurysm data. signaling is significantly upregulated in mouse and human and As mentioned above, several studies have evaluated the identifies a diverse range of mediators associated with this signaling contribution of inflammasome-forming NLRs in TBI in human cascade that impact the pathogenesis of brain injury. and rodent models (6–9, 45). However, data pertaining to noninflammasome-forming regulatory NLR family members have yet to be evaluated. This group of NLRs includes NLRX1, which Discussion has functional characteristics similar to two additional family In this article, we provide evidence that NLRX1 negatively reg- members: NLRP12 and NLRC3. All three of these NLRs have ulates NF-kB signaling, apoptosis and infiltration of CD11b+ cells been suggested to exert negative-regulatory pressure on signaling following TBI. Genetic ablation of Nlrx1 in mice results in sig- pathways associated with the activation of other families of PRRs, nificantly increased brain pathophysiology following CCI. such as TLRs and Rig-I–like helicase receptors (12, 15, 46, 47). Mechanistically, our findings suggest that NLRX1 attenuates brain We evaluated the expression of these three NLRs in the human injury through the regulation of NF-kB signaling, the recruitment aneurysm patient subsets and found that NLRX1 (0.33 6 0.16 and expansion of CD11b+ cell populations in the lesion area, and aneurysm versus 1.00 6 0.03 superficial) and NLRP12 (0.26 6 potentially by limiting apoptosis in neuronal cell populations. In 0.13 aneurysm versus 1.01 6 0.05 superficial) were significantly human patients, the downregulation of NLRX1 expression and downregulated in these human patient populations compared with concomitant upregulation of NF-kB signaling are correlated with NLRC3 (1.46 6 0.90 aneurysm versus 0.72 6 0.53 superficial) injury severity following aneurysm. Thus, the findings from the Downloaded from (Fig. 6A). In addition to these three negative-regulatory NLRs, we mouse and human models appear consistent and further support evaluated expression patterns for the remaining 19 NLR family the importance of NLRX1 and the pathways regulated by this members (Supplemental Fig. 4). We found significant dysregu- unique protein in the brain following injury. lation among the majority of NLRs evaluated. Among the pyrin There is a growing appreciation that NLR family members have family NLRs, we found that NLRP1, NLRP2, NLRP3, NLRP4, and diverse functions in diseases beyond those directly associated with NLRP13 were significantly downregulated following aneurysm, pathogens. Indeed, studies using human clinical samples and/or http://www.jimmunol.org/ whereas NLRP6, NLRP7, NLRP10,andNLRP11 were significantly mouse models have shown a role for these unique proteins in upregulated in these human patients (Supplemental Fig. 4A). the modulation of neuroinflammation (48, 49). The vast majority Likewise, among the card family NLRs, NOD1 and NLRC5 were of studies have focused on inflammasome-forming NLRs in mice significantly upregulated, whereas NLRC4 was significantly down- and their contribution to the pathophysiology of neurodegenera- regulated (Supplemental Fig. 4B). Finally, assessment of CIITA and tive diseases, including Alzheimer’s disease, Parkinson’s disease, NAIP revealed that both of these NLRs were significantly down- and multiple sclerosis (49–51). Beyond neurodegenerative dis- regulated following aneurysm (Supplemental Fig. 4C). Together, eases, inflammasome-forming NLRs have also been found to these data show broad dysregulation among the NLR family significantly modulate various pathologic features of traumatic by guest on October 3, 2021 members in the context of brain injury in human subjects. However, and nontraumatic brain injury. Of the inflammasome-forming consistent with the findings from Nlrx12/2 mice in the CCI injury NLRs in brain injury, the NLRP1 and NLRP3 inflammasomes model, it appears that loss or attenuation of NLRX1 is associated are the best characterized in mice, and the majority of studies have with increased brain injury and pathogenesis in both human and focused on mechanisms associated with regulating IL-1b/IL-18 and rodent models. (6–8, 45, 50). However, aside from the inflammasome, In addition to the NLR genes, we analyzed the expression of the there is a paucity of data pertaining to the role of noninflammasome- genes associated with inflammation and NF-kB signaling in the forming regulatory NLRs in brain injury. The regulatory NLR mouse CCI injury model (shown in Supplemental Fig. 3) in the subgroups can be divided into positive regulators that promote human aneurysm datasets. Similar to the mouse findings, the NF-kB inflammation (i.e., NOD1/NLRC1 and NOD2/NLRC2) and neg- signaling cascade was significantly upregulated in the human ative regulators (i.e., NLRP12, NLRC3, and NLRX1) (52). NOD2 subjects. Indeed, there was a positive correlation between in- is the only regulatory NLR that has been evaluated in the context creased NF-kB gene expression and aneurysm severity (superficial of brain injury (53–55). In models of ischemia-reperfusion injury, versus unruptured: r =+0.405,p = 0.002; superficial versus rup- NOD2 was found to be significantly upregulated in microglia tured: r = +0.439, p = 0.0008). The 10 genes with the largest and astrocyte populations following focal cerebral ischemia- differences in expression between the two aneurysm groups are reperfusion injury; pretreatment with NOD2 agonists signifi- shown in Fig. 6B. Similar to the mouse TBI model, the majority of cantly increased infarct volume and neurologic dysfunction in genes associated with NF-kB signaling were either significantly wild-type mice and rats (53–55). Furthermore, genetic ablation upregulated or unchanged in the human subjects (Fig. 6B, data not of Nod2 significantly improved stroke outcomes and attenuated shown). However, we did observe a few notable differences in the neuroinflammation (53). Nod22/2 mice exhibited significantly human subject data. Specifically, in human subjects, CCL2 ex- reduced levels of IL-1b, IL-6, and TNF associated with atten- pression is significantly upregulated following ruptured aneurysm uated NF-kB, MAPK, and JNK signaling (53). All of these (2.80-fold increase), even though NLRX1 is significantly down- findings are consistent with the role of NOD2 as a positive regulated (Fig. 6A, 6B). Conversely, in the mouse, when NLRX1 regulator of inflammation signaling. is ablated, Ccl2 was significantly downregulated following CCI Because of these early studies associated with inflammasome- (8.61-fold decrease) (Fig. 3). In total, seven genes were found to forming NLRs and NOD2 modulation of ischemia–reperfusion be significantly downregulated in the human subjects. These in- injury and stroke pathophysiology, we sought to expand upon the cluded TNF expression, which was significantly downregulated in clinical relevance of these findings and evaluate the expression Nlrx12/2 mice following CCI and in the human aneurysm subjects patterns of all 22 NLR family members in human patients (Fig. 6, (Figs. 3, 6C). Conversely, expression of IL-10, EGFR, TNFSF14, Supplemental Fig. 4). We took advantage of the robust publicly CD40,andTLR2 was significantly downregulated in the human available gene-expression data sets for ruptured, unruptured, or 3556 NLRX1 PROTECTS AGAINST BRAIN INJURY superficial intracranial aneurysms in human subjects and used NLRX1 has been predominantly studied in the context of in- bioinformatics-based approaches to mine these data (43, 44). We flammation associated with or exposure (52, 60, 61). chose aneurysm because of our joint interests in traumatic and However, recent studies have emerged that reveal a more dynamic nontraumatic (ruptured aneurysm or therapeutic anticoagulation) role for NLRX1 in modulating pathological conditions that extend progressive hemorrhagic brain injury. Aneurysms have similar beyond specific host–pathogen interactions (11, 18, 20). There pathophysiological features that are relevant to TBI and the re- have been very few studies evaluating NLRX1 function in the spective animal models, including the CCI injury model used in CNS. To our knowledge, the only prior in vivo study of NLRX1 in the current study and previously to evaluate NLR function in mice the CNS characterized its role in modulating experimental auto- (6, 9). As in severe cases of traumatic intracranial hemorrhage immune encephalomyelitis (EAE) (62). EAE is a common auto- following TBI, intercerebral hemorrhage in patients with ruptured immune mouse model of multiple sclerosis and is associated with aneurysms results in increased cranial pressure, hypoxia/ischemia, the loss of immunological tolerance to CNS-derived Ags. NLRX1 necrosis and infiltration of the immune-derived compartment, was found to play a robust protective role in the EAE model (62). blood–brain barrier disruption, and oxidative stress. These com- Nlrx12/2 mice were shown to have significantly increased clinical mon pathological features are also well established in animal parameters associated with disease progression and corresponding models of brain contusion injury (oxidative stress, brain edema, increases in tissue damage during EAE (62). Nlrx12/2 animals blood–brain barrier permeability, and autonomic dysfunction from were also more susceptible to myelin-reactive T cells following TBI). Our data demonstrate that a large number of NLRs, 15 of adoptive transfer (62). The mechanism associated with NLRX1 the 22 identified in humans, are significantly dysregulated in hu- protection in the EAE model was further associated with the at- man aneurysm specimens, with the majority (9 of 15) upregulated

tenuation of macrophage and microglia activation (62). Indeed, Downloaded from with increasing aneurysm severity (Fig. 6, Supplemental Fig. 4). microglia and macrophages cultured from Nlrx12/2 mice have Interestingly, we did not observe any significant differences in been previously shown to generate excessive amounts of various NOD2 expression in the aneurysm specimens (Supplemental Fig. proinflammatory cytokines, including IL-6 and CCL2, following 4B). This is counter to our anticipated results based on the pre- 2/2 pathogen-associated molecular pattern stimulation (10, 11, 15, vious preclinical mouse model data for Nod2 animals (53–55). 62). This excessive activation of the microglia and macrophages in However, we did observe a significant increase in NOD1 gene Nlrx12/2 mice resulted in chronic inflammatory signaling and, http://www.jimmunol.org/ expression in the aneurysm specimens (Supplemental Fig. 4B). ultimately, enhanced neurodegeneration. NOD1 NOD2 and are functionally related and modulate similar In general, the findings from prior EAE studies are consistent biochemical pathways. Thus, it is possible that NOD1 may have a with our findings for TBI. Similar to EAE, Nlrx12/2 mice have more prevalent role in human brain injury, and future studies using significantly enhanced injury progression, tissue damage, and Nod12/2 mice may shed insights into this intriguing observation. behavioral abnormalities consistent with increased brain lesion Although NOD1 and NOD2 are regulatory NLRs that function volume (Fig. 1). Also consistent with the prior EAE findings, we to promote inflammation, NLRP12, NLRC3, and NLRX1 are regu- observed increased CD11b+ cells in the lesion that is consistent latory NLRs that negatively regulate inflammatory signaling cascades with increased microglia proliferation and/or macrophage influx activated by other classes of PRRs. Interestingly, the gene-expression following damage (Fig. 2). These findings are consistent with the by guest on October 3, 2021 data analysis from the human aneurysm studies revealed that NLRX1 increased cell recruitment observed in the EAE studies, as well and NLRP12 are significantly downregulated in ruptured aneu- as prior observations that CD11b+ cells from Nlrx12/2 mice are rysms compared with the superficial aneurysms, whereas we did not hyperproliferative under controlled ex vivo conditions (11). CD11b+ observe a significant difference in NLRC3 expression (Fig. 6A). proliferative cells appear to be a major contributing source of the NLRX1 and NLRC3 negatively regulate canonical NF-kB signaling, k whereas NLRP12 attenuates canonical and noncanonical NF-kB increased NF- B signaling at the 3-d time point following TBI signaling pathways (15, 46, 47, 56). Thus, consistent with the (Fig. 4). The significant increase in gene expression associated with k findings that NLRX1 and NLRP12 were downregulated, our ex- NF- B signaling in the TBI mouse model corresponds with the panded analysis of genes associated with inflammation revealed findings from the human ruptured aneurysm specimens and the that a large number of genes associated with NF-kB signaling prior data associated with EAE, with a few notable exceptions. For were significantly upregulated in the human aneurysm specimens, example, in the mouse EAE model, IL-6 and CCL2 expression was and this upregulation was correlated with injury severity (Fig. 6B). significantly upregulated and correlated with disease severity in 2/2 The role of NF-kB signaling in ruptured aneurysm and TBI is Nlrx1 mice (62). However, 3 d following TBI, CCL2 is signif- 2/2 quite complex. In the CNS, NF-kB signaling regulates inflam- icantly downregulated in Nlrx1 animals, and expression of Il-6 mation and apoptotic cell death following nerve injury and dam- was not significantly changed in NLRX1-deficient animals com- age. This pathway has also been found to contribute to infarction pared with wild-type (Fig. 3, data not shown). Similarly, in the EAE and cell death in various stroke models and patients (57, 58). model, no differences were observed in TNF generation, whereas However, NF-kB signaling is also a critical component for neu- TNF was significantly downregulated in the TBI model (Fig. 3). ronal survival and the attenuation of neurodegeneration, and ac- These few discrepancies are likely associated with the fundamental tivation of this cascade also facilitates recovery postinjury (59). differences between the two models. Specifically, the EAE model is Ultimately, the impact of increased NF-kB signaling in brain in- considered a model of “sterile inflammation” and is inherently jury likely depends on which NF-kB factors are activated, where driven by immune system hyperactivation (62), whereas in the TBI the injury occurs, and what cell types are involved. Likewise, it model, the resultant inflammation is driven purely by acute me- is reasonable to speculate that the temporal dynamics of path- chanical damage recognition in the CNS and the resultant injury way activation is also a significant factor. The current data response and resolution. Despite the few discrepancies in specific demonstrate that NF-kB signaling is a common feature that is cytokine levels, the overall consistency between the phenotypes and dysregulated following ruptured aneurysm in humans and brain proposed mechanisms support the conclusion that NLRX1 limits contusion injury in the rodent, suggesting a potential common target inflammation in the CNS, and its downregulation following CCI based on known similarities in the pathological progression of these injury may contribute to cellular apoptosis and tissue loss. Fur- disorders. thermore, the two studies suggest that the attenuation in pathology The Journal of Immunology 3557 is associated through the modulation of inflammatory signaling Acknowledgments + cascades in CD11b cell populations. We thank Dr. Jenny P.Y. Ting (University of North Carolina Chapel Hill) for Beyond inflammatory signaling cascades and CD11b+ cells, it is kindly providing the Nlrx12/2 mice used in this study. We also would like critical to consider the impact of NLRX1 on neuronal cell death to acknowledge Dr. Andrea Bertke and Angela Ives (Virginia–Maryland following TBI. In addition to dysregulated inflammation, neuronal College of Veterinary Medicine) for technical assistance and support. cell death is a hallmark feature associated with the severity of injury. As our data indicate, we observed significantly increased Disclosures 2 2 cortical tissue loss in Nlrx1 / mice compared with wild-type The authors have no financial conflicts of interest. animals following TBI (Fig. 1). The increase in apoptotic gene expression in Nlrx12/2 mice partially correlates with increased NF-kB signaling in these animals (Fig. 3). A variety of genes References 1. Rivest, S. 2009. Regulation of innate immune responses in the brain. Nat. Rev. associated with cell death and apoptosis were significantly up- Immunol. 9: 429–439. regulated in our brain-profiling studies (Fig. 3, Supplemental Fig. 2. Loane, D. 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