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Generation of N18-12R2GFP clones. (A) The IL-12R2 cDNA was cloned into pcDNA3.1-CT-GFP plasmid. (B) The presence and orientation of the IL-12R2 cDNA was confirmed by restriction digestion digest. Lanes 1 and 4, unsuccessful cloning reactions; lanes 3 and 6, undigested plasmid control; lane 2, isolated clone with the insert in the reverse orientation; lane 3, isolated clone in the correct orientation. This clone was propagated and transfected into N18 cells. (C) Transfectants were selected based on G418 sulphate resistance and expression of the IL-12R2GFP fusion protein, which results in green fluorescent cells when compared to untransfected controls. Images were taken with a Kodak MDS-290 digital camera. Overlay of DAPI nuclear counter-stain and GFP signals prepared using Adobe Photoshop 7.01.
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Interleukin (IL)-12, a key cytokine bridging innate and acquired immunity, is efficacious in enhancing recovery from experimental vesicular stomatitis virus (VSV) infection of the mouse central nervous system (CNS). This response is associated with the upregulation of neuronal nitric oxide synthase (NOS-1), independent of IFN-gamma and TNF-alpha. W...
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Genetic deficiency of human IL-12 receptor beta1 chain (IL-12Rbeta1) results in increased vulnerability to weakly pathogenic strains of Mycobacteria and Salmonella. This phenotype results from the combined lack of IL-12 and IL-23 signaling as both cytokine receptors share IL-12Rbeta1. Such infections can be treated by administration of antibiotics...
We analyzed the expression of IL-12Rbeta1 and IL-12Rbeta2 and the role of IL-12 in the activation of monocyte-derived dendritic cells (DCs) via IL-12Rbeta1-mediated signaling events. Flow cytometric analysis revealed that IL-12Rbeta1 was expressed in T cells, Con A blasts, and monocyte-derived DCs, but not in monocytes, while its transcript was det...
IL-12 was recently shown to induce CCR5 on TCR-triggered mouse T cells. Considering that STAT4 is the most critical of IL-12 signaling molecules, this study investigated the role for STAT4 in the induction of CCR5 expression. IL-12R was induced by stimulation with anti-CD3 plus anti-CD28 mAb similarly on T cells from wild-type (WT) and STAT4-defici...
Two functionally distinct subsets of B cells that produce Th1- and Th2-like patterns of cytokines have recently been identified. Interleukin-12 (IL-12) is a critical immunoregulatory cytokine that promotes Th1 differentiation through activation of signal transducer and activator of transcription 4 (STAT4). IL-12 has been reported to induce interfer...
IL-12 promotes T(h)1 development/IFN-gamma expression by activating STAT4. However, it is still unclear how STAT4 elicits IFN-gamma promoter activation. Here, we investigated the mechanism by which IL-12-activated STAT4 functions for IFN-gamma induction in TCR-triggered T cells. TCR stimulation induced high levels of IFN-gamma production depending...
Citations
... Interestingly, while both IL12 and IL13 are expressed in the brain [53,54], IL12 is primarily known to regulate cell proliferation [53], whereas IL13 is mainly involved in differentiation-related processes [54]. Indeed, IL12 reduces cell proliferation and migration in several neuronal cultures via activation of distinct pro-inflammatory signalling pathways, including janus-activated kinase and signal transducers and activators of transcription (JAK-STAT) pathway [55,56], which we have also previously identified to be involved in the regulation of neuronal apoptosis upon exposure of hippocampal cell to IFN-α treatment [24]. Accordingly, previous evidence has reported an increase in the production of IL12 and IL13 in the CSF of COVID-19 patients with neurological manifestations [57,58]. ...
Coronavirus disease 2019 (COVID-19), represents an enormous new threat to our healthcare system and particularly to the health of older adults. Although the respiratory symptoms of COVID-19 are well recognized, the neurological manifestations, and their underlying cellular and molecular mechanisms, have not been extensively studied yet. Our study is the first one to test the direct effect of serum from hospitalised COVID-19 patients on human hippocampal neurogenesis using a unique in vitro experimental assay with human hippocampal progenitor cells (HPC0A07/03 C). We identify the different molecular pathways activated by serum from COVID-19 patients with and without neurological symptoms (i.e., delirium), and their effects on neuronal proliferation, neurogenesis, and apoptosis. We collected serum sample twice, at time of hospital admission and approximately 5 days after hospitalization. We found that treatment with serum samples from COVID-19 patients with delirium (n = 18) decreased cell proliferation and neurogenesis, and increases apoptosis, when compared with serum samples of sex- and age-matched COVID-19 patients without delirium (n = 18). This effect was due to a higher concentration of interleukin 6 (IL6) in serum samples of patients with delirium (mean ± SD: 229.9 ± 79.1 pg/ml, vs. 32.5 ± 9.5 pg/ml in patients without delirium). Indeed, treatment of cells with an antibody against IL6 prevented the decreased cell proliferation and neurogenesis and the increased apoptosis. Moreover, increased concentration of IL6 in serum samples from delirium patients stimulated the hippocampal cells to produce IL12 and IL13, and treatment with an antibody against IL12 or IL13 also prevented the decreased cell proliferation and neurogenesis, and the increased apoptosis. Interestingly, treatment with the compounds commonly administered to acute COVID-19 patients (the Janus kinase inhibitors, baricitinib, ruxolitinib and tofacitinib) were able to restore normal cell viability, proliferation and neurogenesis by targeting the effects of IL12 and IL13. Overall, our results show that serum from COVID-19 patients with delirium can negatively affect hippocampal-dependent neurogenic processes, and that this effect is mediated by IL6-induced production of the downstream inflammatory cytokines IL12 and IL13, which are ultimately responsible for the detrimental cellular outcomes.
... C. Lee, Liu, Brosnan, & Dickson, 1994;Peterson, Hu, Salak-Johnson, Molitor, & Chao, 1997;Vogel et al., 2015). Female Mcoln1 -/mice showed a similar trend, with top correlates at 1 month of age including IL-12p70, IP-10, IL-15, MIG, all of which are chemotactic or pro-inflammatory (Carter et al., 2007;Hanisch et al., 1997;Ireland & Reiss, 2004;Jana, Dasgupta, Pal, & Pahan, 2009;Y. B. Lee, Satoh, Walker, & Kim, 1996). ...
Mucolipidosis IV (MLIV) is an autosomal-recessive pediatric disease that leads to motor and cognitive deficits and loss of vision. It is caused by loss-of-function of the lysosomal channel transient receptor potential mucolipin-1 and is associated with an early pro-inflammatory brain phenotype, including increased cytokine expression. We thus hypothesized that peripheral blood cytokines would reflect inflammatory changes in the brain and would be linked to motor dysfunction. To test this, we collected plasma from MLIV patients and parental controls concomitantly with assessment of motor function using the Brief Assessment of Motor Function and Modified Ashworth scores. We found that MLIV patients had prominently increased cytokine levels compared to familial controls and identified profiles of cytokines correlated with motor dysfunction, including IFN-γ, IFN-α2, IL-17, IP-10. We found that IP-10 was a key differentiating factor separating MLIV cases from controls based on data from human plasma, mouse plasma, and mouse brain. Like MLIV patients, IL-17 and IP-10 were up-regulated in blood of symptomatic mice. Together, our data indicate that MLIV is characterized by increased blood cytokines, which are strongly related to underlying neurological and functional deficits in MLIV patients. Moreover, our data identify the interferon pro-inflammatory axis in both human and mouse signatures, suggesting an importance for interferon signaling in MLIV.
... infection of the CNS, enhanced recovery has been found to be mediated by neurons expressing the IL-12 receptor [177]. In this respect, also microglia have been shown to express the IL-12 receptor in primary culture conditions upon stimulation with IL-12 [178,179]. ...
... Our systematic analyses of the responding cell types required for IL-12 mediated glioma rejection showed that IL-12 tumor rejection relies on BM-derived cells responding to IL-12 ( Figure 7). Thus, these findings excluded the involvement of CNS-resident cells, previously reported to mediate recovery in an experimental model of VSV infection of the CNS [177]. Moreover, the requirement of IL-12 receptor-expressing microglia was also discarded, despite the reported upregulation of this receptor in these cells in primary culture conditions upon stimulation with IL-12 [178,179]. ...
Glioblastoma is a highly destructive primary brain cancer with poor prognosis despite aggressive treatment consisting of surgical resection, radiation, and chemotherapy. Hence, development of novel treatment strategies represents a continuous need. One treatment approach aims to revert the suppressive tumor microenvironment into a pro- inflammatory microenvironment leading to tumor rejection. Multiple clinical trials are currently ongoing using intratumoral delivery of the pro-inflammatory cytokine interleukin (IL)-12 with the aim of minimizing toxicity while maximizing efficacy. However, improvement of IL-12 based immunotherapy requires an immunological understanding of the mechanistic underpinnings. Using a syngeneic mouse model for glioblastoma, we previously showed that intratumoral delivery of IL-12 leads to tumor rejection. Here we show that tumor control is dependent on IL-12 signaling in tumor- invading (cluster of differentiation 8) CD8+ T cells, driving local expansion. We observed a concomitant influx of myeloid cells into the tumor microenvironment. However, chemokine-receptor 2 (CCR2)-dependent monocyte-derived cells were dispensable for IL-12-mediated tumor rejection. Tumor control was supported by CD103+ dendritic cells (DCs) found within the tumor microenvironment. However, in the absence of CD103+ DCs mice seem to control, but are not able to reject IL-12 expressing tumors. Our findings provide new insights into the mechanism of IL-12 mediated glioma rejection, including requirement of directly and indirectly responsive cell types, to consider for the development of novel treatment strategies. Das Glioblastom ist ein bösartiger Hirntumor, der trotz aggressiver Behandlung bestehend aus chirurgischer Resektion, Strahlentherapie und Chemotherapie schlechte Prognosen aufzeigt. Daher ist es essenziell kontinuierlich neue Behandlungsstrategien zu entwickeln. Ein Behandlungsansatz zielt darauf ab di suppressive Tumormikroumgebung in eine proinflammatorische Mikroumgebung umzuwandeln, die zur Abstossung des Tumors führt. Aktuelle klinische Studien basieren auf der intratumoralen Verabreichung des pro-inflammatorischen Zytokin Interleukin (IL) -12. Das Ziel ist, die Toxizität zu minimieren und gleichzeitig die therapeutische Wirksamkeit zu maximieren. Voraussetzung für die Entwicklung der IL-12-basierten Immuntherapie ist das Verständnis der grundlegenden immunologischen Mechanismen. Mit einem Mausmodell für das Glioblastom konnten wir nachweisen, dass die intratumorale Verabreichung von IL-12 zur Tumorabstossung führt. In der vorliegenden Studie zeigen wir, dass die IL-12 induzierte Tumorkontrolle von der Wirkung auf tumor-infiltrierende CD8+ T-Zellen basiert und zu deren Expansion führt. Gleichzeitig war eine Anhäufung von myeloiden Zellen in der Tumormikroumgebung zu beobachten, wobei CCR2-abbhängige monozytische Zellen für die IL-12 vermittelte Tumorabstossung entbehrlich waren. Die Tumorkontrolle wird von CD103+ dendritischen Zellen innerhalb der Tumormikroumgebung unterstützt, wobei in Abwesenheit von CD103+ dendritischen Zellen eine Tumorkontrolle, aber keine Abstossung von IL-12 überexprimierenden Tumoren stattfindet. Unsere Ergebnisse liefern neue Erkenntnisse über den Mechanismus der IL-12- vermittelten Gliomabstossung, einschliesslich der direkt und indirekt involvierten Zelltypen, die für die Entwicklung neuer Therapiestrategien Berücksichtigtung finden sollten.
... In the biofunctional studies, IL12p70 has been shown to regulate both innate and adaptive immunity in the immune response (Trinchieri, 1995) and to promote neurite outgrowth in mouse ganglion neurons in vitro (Lin et al., 2000). Neurons also express the IL12 receptor (Ireland and Reiss, 2004); and IL12p80 is an antagonist of IL12p70 (Gillessen et al., 1995) in that IL12p80 binds to IL12 receptor but does not elicit biological activity in human T cells . However, no report thus far indicates the function of IL12p80 in neurons or NSCs. ...
Regeneration of injured peripheral nerves is a slow, complicated process that could be improved by implantation of neural stem cells (NSCs) or nerve conduit. Implantation of NSCs along with conduits promotes the regeneration of damaged nerve, likely because (i) conduit supports and guides axonal growth from one nerve stump to the other, while preventing fibrous tissue ingrowth and retaining neurotrophic factors; and (ii) implanted NSCs differentiate into Schwann cells and maintain a growth factor enriched microenvironment, which promotes nerve regeneration. In this study, we identified IL12p80 (homodimer of IL12p40) in the cell extracts of implanted nerve conduit combined with NSCs by using protein antibody array and Western blotting. Levels of IL12p80 in these conduits are 1.6-fold higher than those in conduits without NSCs. In the sciatic nerve injury mouse model, implantation of NSCs combined with nerve conduit and IL12p80 improves motor recovery and increases the diameter up to 4.5-fold, at the medial site of the regenerated nerve. In vitro study further revealed that IL12p80 stimulates the Schwann cell differentiation of mouse NSCs through the phosphorylation of signal transducer and activator of transcription 3 (Stat3). These results suggest that IL12p80 can trigger Schwann cell differentiation of mouse NSCs through Stat3 phosphorylation and enhance the functional recovery and the diameter of regenerated nerves in a mouse sciatic nerve injury model.
... Mechanistically, a possible scenario includes persistent Aβdriven release of p40 by microglia, which binds the IL12Rβ1 receptor in a paracrine fashion in neighboring astrocytes, as we found a substantial upregulation of Il12rb1 in the nonmyeloid Gfap mRNA-rich stromal CNS compartment accompanying the increase in p40 expression in microglia. Given that the IL12Rβ1 receptor has been shown to be expressed by neurons 38 and, accord ing to the distribution pattern of the receptor by in situ hybridization, in macroglia 39 , intrinsic neuroepithelial cells, probably astrocytes, may be a primary target and downstream effector cell of increased microglial p40 expression in APPPS1 mice. This may also explain why chimeric APPPS1 × Il12b −/− mice, irrespective of having received p40 −/− or p40 +/+ bone marrow, show reduced Aβ burden. ...
The pathology of Alzheimer's disease has an inflammatory component that is characterized by upregulation of proinflammatory cytokines, particularly in response to amyloid-β (Aβ). Using the APPPS1 Alzheimer's disease mouse model, we found increased production of the common interleukin-12 (IL-12) and IL-23 subunit p40 by microglia. Genetic ablation of the IL-12/IL-23 signaling molecules p40, p35 or p19, in which deficiency of p40 or its receptor complex had the strongest effect, resulted in decreased cerebral amyloid load. Although deletion of IL-12/IL-23 signaling from the radiation-resistant glial compartment of the brain was most efficient in mitigating cerebral amyloidosis, peripheral administration of a neutralizing p40-specific antibody likewise resulted in a reduction of cerebral amyloid load in APPPS1 mice. Furthermore, intracerebroventricular delivery of antibodies to p40 significantly reduced the concentration of soluble Aβ species and reversed cognitive deficits in aged APPPS1 mice. The concentration of p40 was also increased in the cerebrospinal fluid of subjects with Alzheimer's disease, which suggests that inhibition of the IL-12/IL-23 pathway may attenuate Alzheimer's disease pathology and cognitive deficits.
... NB41A3, HN9e, N18, L929, and RAW 264.7 cells were purchased from ATCC, and were maintained as previously described (37). All tissue culture reagents were obtained from Mediatech (Manassas, VA). ...
... NB41A3 cells, a mouse neuroblastoma line, has been used extensively for studies of VSV infection, of responses to cytokines, and of dynamics of NOS-1 activity (8,(13)(14)(15)36,37,44,78,79). To determine if the NB41A3 cell line was appropriate for our experimental system, the reverse transcription polymerase chain reaction (RT-PCR) was performed to detect CB 1 or CB 2 mRNA transcripts. ...
Interferon-gamma (IFN-gamma) has potent antiviral activity in neurons which is affected by the production of nitric oxide (NO). This study examines the interactions between cannabinoid receptor-1 (CB(1)), IFNgamma-induced pathways, and inhibition of vesicular stomatitis virus (VSV) replication in neuronal cells. CB(1) is abundantly expressed in neurons of the CNS and the NB41A3 neuroblastoma cell line. CB(1) activation of NB41A3 cells by the synthetic cannabinoid, WIN55,212-2, is associated with an inhibition of Ca(2+) mobilization, leading to diminished nitric oxide synthase (NOS)-1 activity and the production of NO, in vitro. This ultimately results in antagonism of IFN-gamma-mediated antiviral activity and enhanced viral replication. Therefore, activation of cells expressing CB(1) by endogenous (or exogenous) ligands may contribute to decreased inflammation and to increased viral replication in neurons and disease in the CNS.
... The failure to elicit IFN in the CNS may be attributable to the lack of expression of TLR7 by neurons, and contributes to the disease pathogenesis (Trottier et al, in preparation). NB41A3 cells, a neuroblastoma cell line, have been shown to have many properties of neurons, including the ability to form neurotubules and neurofilaments and in the storage and release of catecholamines (Breakfield et al, 1975; Schubert et al, 1969); they constitutively express neuronal nitric oxide synthase (NOS-1), IFN-γ eceptor (IFN-γ R), interleukin-12 receptor (IL-12R), and the N-methyl-D-aspartate (NMDA) receptor ( Ireland and Reiss, 2004; Komatsu et al, 1996). The effects of type I and II IFN on these cells have also been studied; the inhibitory effects of these cytokines on VSV replication are similar in these cells and in primary murine neurons (Chesler et al, 2003; Trottier et al, 2005). ...
Type I interferon (IFN) is critical for resistance of mice to infection with vesicular stomatitis virus (VSV). Wild type (wt) VSV infection did not induce type I IFN production in vitro or in the central nervous system (CNS) of mice; however IFN-beta was detected in lungs, spleen, and serum within 24 h. The M protein mutant VSV, T1026R1 (also referred to as M51R), induced type I IFN production in vitro and in the CNS, with poor expression in spleens. In addition, VSV T1026R1 was not pathogenic to mice after intranasal infection, illustrating the importance of IFN in controlling VSV replication in the CNS. Experiments with chemical sympathectomy, sRAGE, and neutralizing antibody to VSV were performed to investigate the mechanism(s) utilized for induction of peripheral IFN; neither sRAGE infusion nor chemical sympathectomy had an effect on peripheral IFN production. In contrast, administration of neutralizing antibody (Ab) readily blocked the response. Infectious VSV was transiently present in lungs and spleens at 24 h post infection. The results are consistent with VSV traffic from the olfactory neuroepithelium to peripheral lymphoid organs hematogenously or via lymphatic circulation. These results suggest that VSV replicates to high titers in the brains of mice because of the lack of IFN production in the CNS after intranasal VSV infection. In contrast, replication of VSV in peripheral organs is controlled by the production of large amounts of IFN.
Objectives
The central nervous system disorder in systemic lupus erythematosus (SLE), called neuropsychiatric lupus (NPSLE), is one of the most severe phenotypes with various clinical symptoms, including mood disorder, psychosis and delirium as diffuse neuropsychological manifestations (dNPSLE). Although stress is one of the aggravating factors for neuropsychiatric symptoms, its role in the pathogenesis of dNPSLE remains to be elucidated. We aimed to investigate stress effects on the neuropsychiatric pathophysiology in SLE using lupus-prone mice and patients’ data.
Methods
Sleep disturbance stress (SDS) for 2 weeks was placed on 6–8-week-old female MRL/ lpr and control mice. Behavioural phenotyping, histopathological analyses and gene and protein expression analyses were performed to assess SDS-induced neuroimmunological alterations. We also evaluated cytokines of the cerebrospinal fluid and brain regional volumes in patients with dNPSLE and patients with non-dNPSLE.
Results
SDS-subjected MRL/ lpr mice exhibited less anxiety-like behaviour, whereas stressed control mice showed increased anxiety. Furthermore, stress strongly activated the medial prefrontal cortex (mPFC) in SDS-subjected MRL/ lpr . A transcriptome analysis of the PFC revealed the upregulation of microglial activation-related genes, including Il12b . We confirmed that stress-induced microglial activation and the upregulation of interleukin (IL) 12/23p40 proteins and increased dendritic spines in the mPFC of stressed MRL/ lpr mice. IL-12/23p40 neutralisation and tyrosine kinase 2 inhibition mitigated the stress-induced neuropsychiatric phenotypes of MRL/ lpr mice. We also found a higher level of cerebrospinal fluid IL-12/23p40 and more atrophy in the mPFC of patients with dNPSLE than those with non-dNPSLE.
Conclusions
The microglial IL-12/23 axis in the mPFC might be associated with the pathogenesis and a promising therapeutic target for dNPSLE.
Innate immune responses to pathogens are evolutionarily ancient and are found in the most primitive organisms. These are highly conserved and are not pathogen-specific, but are in response to classes of molecular structures. Infections can be perceived both extracellularly and intracellularly by Pathogen Associated Molecular Patterns (PAMPs) and their host cell ligands, Pathogen Recognition Receptors (PRRs), among them, Toll-Like Receptors (TLRs). The innate immune response to infection includes the release of soluble preformed mediators, or synthesis of cytoplasmic enzymes, cytokines, chemokines, interferons (IFNs), lipid mediators, proteins of the complement cascade, neurotransmitters, nucleotides, and components of transcription factors (High Mobility Group B1, receptors for sex hormones/steroids). Directed cellular migration of parenchymal astrocytes and microglia, as well as recruitment across the blood brain barrier (BBB) of circulating neutrophils, natural killer, monocytes, macrophages, dendritic cells, and ultimately T lymphocytes to the site of infection are also hallmarks of innate responses to infections. These responding cells contribute their own secreted effector molecules and effector activities (such as phagocytosis). Distinct viruses are capable of infecting every cell type (endothelial cells, ependymal cells, perivascular macrophages and pericytes, astrocytes, microglia, oligodendrocytes, Schwann cells, and neurons) in the central nervous system (CNS). These CNS infections challenge the host with a different set of problems than do peripheral viral infections. Among the complications are (a) neurons that rarely express Class I or Class II Major Histocompatibility Complex (MHC) molecules and are thus not suitable targets for either CD4+ or CD8+ MHC-restricted T cells, (b) an enclosed volume that is constrained from swelling during inflammation, as well as poorly developed lymphatic drainage, and (c) the immunologic privilege of the CNS which leads to extremely limited immune surveillance for pathogens. Therefore, the role of innate immunity, both from CNS-resident cells and their products, and from circulating inflammatory cells and molecules which traverse the BBB are essential to “buy time,” inhibiting viral replication and dissemination, until the host can marshal an adaptive immune response. The immune responses are crucial for host survival from the infection. Consequently, successful pathogens, especially those that persist, have developed a wide variety of evasive approaches to limit the inhibition of replication. Many of these pathways are highlighted in individual chapters that precede this one. These evasive measures range from neutralizing host secreted molecules (cytokines and chemokines) with soluble receptors, encoding anti-inflammatory proteins in their genome, preventing signal transduction, blocking inhibition of protein synthesis, degradation of essential antiviral molecules, preventing apoptosis, and blockade of the nuclear pore complex. In this chapter, I will attempt to cover the breadth of the innate immune response to viral infection, but will also devote more space to generally under-considered aspects than to the well-known components. There are some caveats to consider, as most experiments have been performed in the murine model and not in man; further, conclusions from experiments using in vitro cultured cells (whether primary or established lines) may not reflect physiological conditions in an undisturbed CNS. Lastly, we now appreciate the complexities imposed on hosts by polymorphisms in genes of critical pathways, leading to increased susceptibility or resistance of that individual but not others.
Vesicular stomatitis virus (VSV), a negative-sense, single-stranded RNA rhabdovirus, causes acute viral encephalitis when administered intranasally to mice. Interleukin-12 (IL-12) is a key pro-inflammatory cytokine that is produced largely by the antigen presenting cells (APC) and that bridges the innate and acquired immune responses. IL-12 is efficacious in enhancing recovery from VSV infection of the murine central nervous system. This effect is mediated by nitric oxide (NO) produced by the neuronal isoform of nitric oxide synthase (NOS-1), and is independent of the pro-inflammatory cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). These data implied a link between IL-12 and NOS-1. Here we investigate the role of the IL-12R during VSV pathogenesis, using IL-12R beta2 and IL-12R beta1-deficient mice. We showed that a deficiency in either IL-12R beta2 or IL-12R beta1 had no effect on the outcome of VSV infection of the CNS or on the clearance of VSV from the CNS. Furthermore, these data indicate that IL-23 is not acting redundantly in the absence of IL-12 during VSV-induced encephalitis.
