IL7 and IL7 Flt3L co-expressing CAR T cells improve therapeutic efficacy in mouse EGFRvIII heterogeneous glioblastoma.

Chimeric antigen receptor (CAR) T cell therapy in glioblastoma faces many challenges including insufficient CAR T cell abundance and antigen-negative tumor cells evading targeting. Unfortunately, preclinical studies evaluating CAR T cells in glioblastoma focus on tumor models that express a single antigen, use immunocompromised animals, and/or pre-treat with lymphodepleting agents. While lymphodepletion enhances CAR T cell efficacy, it diminishes the endogenous immune system that has the potential for tumor eradication. Here, we engineered CAR T cells to express IL7 and/or Flt3L in 50% EGFRvIII-positive and -negative orthotopic tumors pre-conditioned with non-lymphodepleting irradiation. IL7 and IL7 Flt3L CAR T cells increased intratumoral CAR T cell abundance seven days after treatment. IL7 co-expression with Flt3L modestly increased conventional dendritic cells as well as the CD103+XCR1+ population known to have migratory and antigen cross-presenting capabilities. Treatment with IL7 or IL7 Flt3L CAR T cells improved overall survival to 67% and 50%, respectively, compared to 9% survival with conventional or Flt3L CAR T cells. We concluded that CAR T cells modified to express IL7 enhanced CAR T cell abundance and improved overall survival in EGFRvIII heterogeneous tumors pre-conditioned with non-lymphodepleting irradiation. Potentially IL7 or IL7 Flt3L CAR T cells can provide new opportunities to combine CAR T cells with other immunotherapies for the treatment of glioblastoma.

Cytostatic hypothermia and its impact on glioblastoma and survival.

Patients with glioblastoma (GBM) have limited options and require novel approaches to treatment. Here, we studied and deployed nonfreezing "cytostatic" hypothermia to stunt GBM growth. This growth-halting method contrasts with ablative, cryogenic hypothermia that kills both neoplastic and infiltrated healthy tissue. We investigated degrees of hypothermia in vitro and identified a cytostatic window of 20° to 25°C. For some lines, 18 hours/day of cytostatic hypothermia was sufficient to halt division in vitro. Next, we fabricated an experimental tool to test local cytostatic hypothermia in two rodent GBM models. Hypothermia more than doubled median survival, and all rats that successfully received cytostatic hypothermia survived their study period. Unlike targeted therapeutics that are successful in preclinical models but fail in clinical trials, cytostatic hypothermia leverages fundamental physics that influences biology broadly. It is a previously unexplored approach that could provide an additional option to patients with GBM by halting tumor growth.

Neuromechanobiology: An Expanding Field Driven by the Force of Greater Focus.

The brain processes information by transmitting signals through highly connected and dynamic networks of neurons. Neurons use specific cellular structures, including axons, dendrites and synapses, and specific molecules, including cell adhesion molecules, ion channels and chemical receptors to form, maintain and communicate among cells in the networks. These cellular and molecular processes take place in environments rich of mechanical cues, thus offering ample opportunities for mechanical regulation of neural development and function. Recent studies have suggested the importance of mechanical cues and their potential regulatory roles in the development and maintenance of these neuronal structures. Also suggested are the importance of mechanical cues and their potential regulatory roles in the interaction and function of molecules mediating the interneuronal communications. In this review, the current understanding is integrated and promising future directions of neuromechanobiology are suggested at the cellular and molecular levels. Several neuronal processes where mechanics likely plays a role are examined and how forces affect ligand binding, conformational change, and signal induction of molecules key to these neuronal processes are indicated, especially at the synapse. The disease relevance of neuromechanobiology as well as therapies and engineering solutions to neurological disorders stemmed from this emergent field of study are also discussed.

Engineered glycomaterial implants orchestrate large-scale functional repair of brain tissue chronically after severe traumatic brain injury.

Severe traumatic brain injury (sTBI) survivors experience permanent functional disabilities due to significant volume loss and the brain's poor capacity to regenerate. Chondroitin sulfate glycosaminoglycans (CS-GAGs) are key regulators of growth factor signaling and neural stem cell homeostasis in the brain. However, the efficacy of engineered CS (eCS) matrices in mediating structural and functional recovery chronically after sTBI has not been investigated. We report that neurotrophic factor functionalized acellular eCS matrices implanted into the rat M1 region acutely after sTBI significantly enhanced cellular repair and gross motor function recovery when compared to controls 20 weeks after sTBI. Animals subjected to M2 region injuries followed by eCS matrix implantations demonstrated the significant recovery of "reach-to-grasp" function. This was attributed to enhanced volumetric vascularization, activity-regulated cytoskeleton (Arc) protein expression, and perilesional sensorimotor connectivity. These findings indicate that eCS matrices implanted acutely after sTBI can support complex cellular, vascular, and neuronal circuit repair chronically after sTBI.

Enriching neural stem cell and anti-inflammatory glial phenotypes with electrical stimulation after traumatic brain injury in male rats.

Traumatic brain injury (TBI) by an external physical impact results in compromised brain function via undesired neuronal death. Following the injury, resident and peripheral immune cells, astrocytes, and neural stem cells (NSCs) cooperatively contribute to the recovery of the neuronal function after TBI. However, excessive pro-inflammatory responses of immune cells, and the disappearance of endogenous NSCs at the injury site during the acute phase of TBI, can exacerbate TBI progression leading to incomplete healing. Therefore, positive outcomes may depend on early interventions to control the injury-associated cellular milieu in the early phase of injury. Here, we explore electrical stimulation (ES) of the injury site in a rodent model (male Sprague-Dawley rats) to investigate its overall effect on the constituent brain cell phenotype and composition during the acute phase of TBI. Our data showed that a brief ES for 1 hr on day 2 of TBI promoted anti-inflammatory phenotypes of microglia as assessed by CD206 expression and increased the population of NSCs and Nestin+ astrocytes at 7 days post-TBI. Also, ES effectively increased the number of viable neurons when compared to the unstimulated control group. Given the salience of microglia and neural stem cells for healing after TBI, our results strongly support the potential benefit of the therapeutic use of ES during the acute phase of TBI to regulate neuroinflammation and to enhance neuroregeneration.

Immuno-suppressive hydrogels enhance allogeneic MSC survival after transplantation in the injured brain.

Traumatic brain injury (TBI) triggers multiple biochemical and cellular processes that exacerbate brain tissue damage through a secondary injury. Therapies that prevent or limit the evolution of secondary injury could significantly reduce the neurological deficits associated with TBI. Mesenchymal stem cell (MSC) transplantation after TBI can ameliorate neurological deficits by modulating inflammation and enhancing the expression of neurotrophic factors. However, transplanted MSCs can be actively rejected by host immune responses, such as those mediated by cytotoxic CD8+ T cells, thereby limiting their therapeutic efficacy. Here, we designed an agarose hydrogel that releases Fas ligand (FasL), a protein that can induce apoptosis of cytotoxic CD8+ T cells. We studied the immunosuppressive effect of this hydrogel near the allogeneic MSC transplantation site and its impact on the survival of transplanted MSCs in the injured brain. Agarose-FasL hydrogels locally reduced the host cytotoxic CD8+ T cell population and enhanced the survival of allogeneic MSCs transplanted near the injury site. Furthermore, the expression of crucial neurotrophic factors was elevated in the injury penumbra, suggesting an enhanced therapeutic effect of MSCs. These results suggest that the development of immunosuppressive hydrogels for stem cell delivery can enhance the benefits of stem cell therapy for TBI.

The impact of modulating the blood-brain barrier on the electrophysiological and histological outcomes of intracortical electrodes.

OBJECTIVE: Successful application of chronic intracortical electrodes remains highly variable. The biological mechanisms leading to electrode failure are still being explored. Recent work has shown a correlation between blood-brain barrier (BBB) integrity and long-term recordings. Here we proposed to modulate the BBB healing after intracortical electrode implantation, while evaluating the functional electrophysiology. The CCL2/CCR2 pathway was chosen based on previous work demonstrating the positive histological effects in an intracortical electrode model, as well as in other neurodegenerative models. By disrupting this pathway, recruitment of pro-inflammatory monocytes (a result of a breached BBB) is potentially reduced at the electrode interface. APPROACH: Michigan electrodes were implanted for 2 and 12 weeks in rats, and a CCR2 antagonist (RS 102895) was administered daily to the treatment group. Functional electrodes were used for the 12 week cohort, and weekly electrophysiological recordings were taken. At 2 and 12 weeks, histology was analyzed. MAIN RESULTS: At 12 weeks, the CCR2-antagonist group had significantly higher signal-to-noise ratios (SNRs) than control. CCR2-antagonism at 2 weeks significantly increased the neural population and decreased BBB breach. At 12 weeks, CCR2-antagonism significantly increased number of neurons and BBB + vasculature within 100 µm of the electrode interface. SIGNIFICANCE: This work demonstrates that for intracortical electrodes, disruption of the CCL2/CCR2 pathway improves chronic outcomes in electrophysiology and histology.

Electrotaxis of Glioblastoma and Medulloblastoma Spheroidal Aggregates.

Treatment of neuroepithelial cancers remains a daunting clinical challenge, particularly due to an inability to address rampant invasion deep into eloquent regions of the brain. Given the lack of access, and the dispersed nature of brain tumor cells, we explore the possibility of electric fields inducing directed tumor cell migration. In this study we investigate the properties of populations of brain cancer undergoing electrotaxis, a phenomenon whereby cells are directed to migrate under control of an electrical field. We investigate two cell lines for glioblastoma and medulloblastoma (U87mg & DAOY, respectively), plated as spheroidal aggregates in Matrigel-filled electrotaxis channels, and report opposing electrotactic responses. To further understand electrotactic migration of tumor cells, we performed RNA-sequencing for pathway discovery to identify signaling that is differentially affected by the exposure of direct-current electrical fields. Further, using selective pharmacological inhibition assays, focused on the PI3K/mTOR/AKT signaling axis, we validate whether there is a causal relationship to electrotaxis and these mechanisms of action. We find that U87 mg electrotaxis is abolished under pharmacological inhibition of PI3Kγ, mTOR, AKT and ErbB2 signaling, whereas DAOY cell electrotaxis was not attenuated by these or other pathways evaluated.

Engineering Controlled Peritumoral Inflammation to Constrain Brain Tumor Growth.

Brain tumors remain a great clinical challenge, in part due to their capacity to invade into eloquent, inoperable regions of the brain. In contrast, inflammation in the central nervous system (CNS) due to injuries activates microglia and astrocytes culminating in an astroglial scar that typically "walls-off" the injury site. Here, the hypothesis is tested that targeting peritumoral cells surrounding tumors to activate them via an inflammatory stimulus that recapitulates the sequelae of a traumatic CNS injury, could generate an environment that would wall-off and contain invasive tumors in the brain. Gold nanoparticles coated with inflammatory polypeptides to target stromal cells in close vicinity to glioblastoma (GBM) tumors, in order to activate these cells and stimulate stromal CNS inflammation, are engineered. It is reported that this approach significantly contains tumors in rodent models of GBM relative to control treatments (reduction in tumor volume by over 300% in comparison to controls), by the activation of the innate and adaptive immune response, and by triggering pathways related to cell clustering. Overall, this report outlines an approach to contain invasive tumors that can complement adjuvant interventions for invasive GBM such as radiation and chemotherapy.

In Vitro Transcribed mRNA Vaccines with Programmable Stimulation of Innate Immunity.

In vitro transcribed (IVT) mRNA is an appealing platform for next generation vaccines, as it can be manufactured rapidly at large scale to meet emerging pathogens. However, its performance as a robust vaccine is strengthened by supplemental immune stimulation, which is typically provided by adjuvant formulations that facilitate delivery and stimulate immune responses. Here, we present a strategy for increasing translation of a model IVT mRNA vaccine while simultaneously modulating its immune-stimulatory properties in a programmable fashion, without relying on delivery vehicle formulations. Substitution of uridine with the modified base N1-methylpseudouridine reduces the intrinsic immune stimulation of the IVT mRNA and enhances antigen translation. Tethering adjuvants to naked IVT mRNA through antisense nucleotides boosts the immunostimulatory properties of adjuvants in vitro, without impairing transgene production or adjuvant activity. In vivo, intramuscular injection of tethered IVT mRNA-TLR7 agonists leads to enhanced local immune responses, and to antigen-specific cell-mediated and humoral responses. We believe this system represents a potential platform compatible with any adjuvant of interest to enable specific programmable stimulation of immune responses.

Discovery of Lipidome Alterations Following Traumatic Brain Injury via High-Resolution Metabolomics.

Traumatic brain injury (TBI) can occur across wide segments of the population, presenting in a heterogeneous manner that makes diagnosis inconsistent and management challenging. Biomarkers offer the potential to objectively identify injury status, severity, and phenotype by measuring the relative concentrations of endogenous molecules in readily accessible biofluids. Through a data-driven, discovery approach, novel biomarker candidates for TBI were identified in the serum lipidome of adult male Sprague-Dawley rats in the first week following moderate controlled cortical impact (CCI). Serum samples were analyzed in positive and negative modes by ultraperformance liquid chromatography-mass spectrometry (UPLC-MS). A predictive panel for the classification of injured and uninjured sera samples, consisting of 26 dysregulated species belonging to a variety of lipid classes, was developed with a cross-validated accuracy of 85.3% using omniClassifier software to optimize feature selection. Polyunsaturated fatty acids (PUFAs) and PUFA-containing diacylglycerols were found to be upregulated in sera from injured rats, while changes in sphingolipids and other membrane phospholipids were also observed, many of which map to known secondary injury pathways. Overall, the identified biomarker panel offers viable molecular candidates representing lipids that may readily cross the blood-brain barrier (BBB) and aid in the understanding of TBI pathophysiology.

Correlation of mRNA Expression and Signal Variability in Chronic Intracortical Electrodes.

OBJECTIVE: The goal for this research was to identify molecular mechanisms that explain animal-to-animal variability in chronic intracortical recordings. APPROACH: Microwire electrodes were implanted into Sprague Dawley rats at an acute (1 week) and a chronic (14 weeks) time point. Weekly recordings were conducted, and action potentials were evoked in the barrel cortex by deflecting the rat's whiskers. At 1 and 14 weeks, tissue was collected, and mRNA was extracted. mRNA expression was compared between 1 and 14 weeks using a high throughput multiplexed qRT-PCR. Pearson correlation coefficients were calculated between mRNA expression and signal-to-noise ratios at 14 weeks. MAIN RESULTS: At 14 weeks, a positive correlation between signal-to-noise ratio (SNR) and NeuN and GFAP mRNA expression was observed, indicating a relationship between recording strength and neuronal population, as well as reactive astrocyte activity. The inflammatory state around the electrode interface was evaluated using M1-like and M2-like markers. Expression for both M1-like and M2-like mRNA markers remained steady from 1 to 14 weeks. Anti-inflammatory markers, CD206 and CD163, however, demonstrated a significant positive correlation with SNR quality at 14 weeks. VE-cadherin, a marker for adherens junctions, and PDGFR-ß, a marker for pericytes, both partial representatives of blood-brain barrier health, had a positive correlation with SNR at 14 weeks. Endothelial adhesion markers revealed a significant increase in expression at 14 weeks, while CD45, a pan-leukocyte marker, significantly decreased at 14 weeks. No significant correlation was found for either the endothelial adhesion or pan-leukocyte markers. SIGNIFICANCE: A positive correlation between anti-inflammatory and blood-brain barrier health mRNA markers with electrophysiological efficacy of implanted intracortical electrodes has been demonstrated. These data reveal potential mechanisms for further evaluation to determine potential target mechanisms to improve consistency of intracortical electrodes recordings and reduce animal-to-animal/implant-to-implant variability.

Cerivastatin Nanoliposome as a Potential Disease Modifying Approach for the Treatment of Pulmonary Arterial Hypertension.

In this study we investigated nanoliposome as an approach to tailoring the pharmacology of cerivastatin as a disease-modifying drug for pulmonary arterial hypertension (PAH). Cerivastatin encapsulated liposomes with an average diameter of 98 ± 27 nm were generated by a thin film and freeze-thaw process. The nanoliposomes demonstrated sustained drug-release kinetics in vitro and inhibited proliferation of pulmonary artery (PA) smooth muscle cells with significantly less cellular cytotoxicity as compared with free cerivastatin. When delivered by inhalation to a rat model of monocrotaline-induced PAH, cerivastatin significantly reduced PA pressure from 55.13 ± 9.82 to 35.56 ± 6.59 mm Hg (P < 0.001) and diminished PA wall thickening. Echocardiography showed that cerivastatin significantly reduced right ventricle thickening (monocrotaline: 0.34 ± 0.02 cm vs. cerivastatin: 0.26 ± 0.02 cm; P < 0.001) and increased PA acceleration time (monocrotaline: 13.98 ± 1.14 milliseconds vs. cerivastatin: 21.07 ± 2.80 milliseconds; P < 0.001). Nanoliposomal cerivastatin was equally effective or slightly better than cerivastatin in reducing PA pressure (monocrotaline: 67.06 ± 13.64 mm Hg; cerivastatin: 46.31 ± 7.64 mm Hg vs. liposomal cerivastatin: 37.32 ± 9.50 mm Hg) and improving parameters of right ventricular function as measured by increasing PA acceleration time (monocrotaline: 24.68 ± 3.92 milliseconds; cerivastatin: 32.59 ± 6.10 milliseconds vs. liposomal cerivastatin: 34.96 ± 7.51 milliseconds). More importantly, the rate and magnitude of toxic cerivastatin metabolite lactone generation from the intratracheally administered nanoliposomes was significantly lower as compared with intravenously administered free cerivastatin. These studies show that nanoliposome encapsulation improved in vitro and in vivo pharmacologic and safety profile of cerivastatin and may represent a safer approach as a disease-modifying therapy for PAH.

Therapeutic efficacy of microtube-embedded chondroitinase ABC in a canine clinical model of spinal cord injury.

See Moon and Bradbury (doi:10.1093/brain/awy067) for a scientific commentary on this article.Many hundreds of thousands of people around the world are living with the long-term consequences of spinal cord injury and they need effective new therapies. Laboratory research in experimental animals has identified a large number of potentially translatable interventions but transition to the clinic is not straightforward. Further evidence of efficacy in more clinically-relevant lesions is required to gain sufficient confidence to commence human clinical trials. Of the many therapeutic candidates currently available, intraspinally applied chondroitinase ABC has particularly well documented efficacy in experimental animals. In this study we measured the effects of this intervention in a double-blinded randomized controlled trial in a cohort of dogs with naturally-occurring severe chronic spinal cord injuries that model the condition in humans. First, we collected baseline data on a series of outcomes: forelimb-hindlimb coordination (the prespecified primary outcome measure), skin sensitivity along the back, somatosensory evoked and transcranial magnetic motor evoked potentials and cystometry in 60 dogs with thoracolumbar lesions. Dogs were then randomized 1:1 to receive intraspinal injections of heat-stabilized, lipid microtube-embedded chondroitinase ABC or sham injections consisting of needle puncture of the skin. Outcome data were measured at 1, 3 and 6 months after intervention; skin sensitivity was also measured 24 h after injection (or sham). Forelimb-hindlimb coordination was affected by neither time nor chondroitinase treatment alone but there was a significant interaction between these variables such that coordination between forelimb and hindlimb stepping improved during the 6-month follow-up period in the chondroitinase-treated animals by a mean of 23%, but did not change in controls. Three dogs (10%) in the chondroitinase group also recovered the ability to ambulate without assistance. Sensitivity of the dorsal skin increased at 24 h after intervention in both groups but subsequently decreased to normal levels. Cystometry identified a non-significant improvement of bladder compliance at 1 month in the chondroitinase-injected dogs but this did not persist. There were no overall differences between groups in detection of sensory evoked potentials. Our results strongly support a beneficial effect of intraspinal injection of chondroitinase ABC on spinal cord function in this highly clinically-relevant model of chronic severe spinal cord injury. There was no evidence of long-term adverse effects associated with this intervention. We therefore conclude that this study provides strong evidence in support of initiation of clinical trials of chondroitinase ABC in humans with chronic spinal cord injury.

Enrichment of endogenous fractalkine and anti-inflammatory cells via aptamer-functionalized hydrogels.

Early recruitment of non-classical monocytes and their macrophage derivatives is associated with augmented tissue repair and improved integration of biomaterial constructs. A promising therapeutic approach to recruit these subpopulations is by elevating local concentrations of chemoattractants such as fractalkine (FKN, CX3CL1). However, delivering recombinant or purified proteins is not ideal due to their short half-lives, suboptimal efficacy, immunogenic potential, batch variabilities, and cost. Here we report an approach to enrich endogenous FKN, obviating the need for delivery of exogenous proteins. In this study, modified FKN-binding-aptamers are integrated with poly(ethylene glycol) diacrylate to form aptamer-functionalized hydrogels ("aptagels") that localize, dramatically enrich and passively release FKN in vitro for at least one week. Implantation in a mouse model of excisional skin injury demonstrates that aptagels enrich endogenous FKN and stimulate significant local increases in Ly6CloCX3CR1hi non-classical monocytes and CD206+ M2-like macrophages. The results demonstrate that orchestrators of inflammation can be manipulated without delivery of foreign proteins or cells and FKN-aptamer functionalized biomaterials may be a promising approach to recruit anti-inflammatory subpopulations to sites of injury. Aptagels are readily synthesized, highly customizable and could combine different aptamers to treat complex diseases in which regulation or enrichment of multiple proteins may be therapeutic.

Engineering challenges for brain tumor immunotherapy.

Malignant brain tumors represent one of the most devastating forms of cancer with abject survival rates that have not changed in the past 60years. This is partly because the brain is a critical organ, and poses unique anatomical, physiological, and immunological barriers. The unique interplay of these barriers also provides an opportunity for creative engineering solutions. Cancer immunotherapy, a means of harnessing the host immune system for anti-tumor efficacy, is becoming a standard approach for treating many cancers. However, its use in brain tumors is not widespread. This review discusses the current approaches, and hurdles to these approaches in treating brain tumors, with a focus on immunotherapies. We identify critical barriers to immunoengineering brain tumor therapies and discuss possible solutions to these challenges.

Corrigendum: Kilohertz frequency nerve block enhances anti-inflammatory effects of vagus nerve stimulation.

This corrects the article DOI: 10.1038/srep39810.

Immunoengineering nerve repair.

Injuries to the peripheral nervous system are major sources of disability and often result in painful neuropathies or the impairment of muscle movement and/or normal sensations. For gaps smaller than 10 mm in rodents, nearly normal functional recovery can be achieved; for longer gaps, however, there are challenges that have remained insurmountable. The current clinical gold standard used to bridge long, nonhealing nerve gaps, the autologous nerve graft (autograft), has several drawbacks. Despite best efforts, engineering an alternative "nerve bridge" for peripheral nerve repair remains elusive; hence, there is a compelling need to design new approaches that match or exceed the performance of autografts across critically sized nerve gaps. Here an immunomodulatory approach to stimulating nerve repair in a nerve-guidance scaffold was used to explore the regenerative effect of reparative monocyte recruitment. Early modulation of the immune environment at the injury site via fractalkine delivery resulted in a dramatic increase in regeneration as evident from histological and electrophysiological analyses. This study suggests that biasing the infiltrating inflammatory/immune cellular milieu after injury toward a proregenerative population creates a permissive environment for repair. This approach is a shift from the current modes of clinical and laboratory methods for nerve repair, which potentially opens an alternative paradigm to stimulate endogenous peripheral nerve repair.

Bacterial Carriers for Glioblastoma Therapy.

Treatment of aggressive glioblastoma brain tumors is challenging, largely due to diffusion barriers preventing efficient drug dosing to tumors. To overcome these barriers, bacterial carriers that are actively motile and programmed to migrate and localize to tumor zones were designed. These carriers can induce apoptosis via hypoxia-controlled expression of a tumor suppressor protein p53 and a pro-apoptotic drug, Azurin. In a xenograft model of human glioblastoma in rats, bacterial carrier therapy conferred a significant survival benefit with 19% overall long-term survival of >100 days in treated animals relative to a median survival of 26 days in control untreated animals. Histological and proteomic analyses were performed to elucidate the safety and efficacy of these carriers, showing an absence of systemic toxicity and a restored neural environment in treated responders. In the treated non-responders, proteomic analysis revealed competing mechanisms of pro-apoptotic and drug-resistant activity. This bacterial carrier opens a versatile avenue to overcome diffusion barriers in glioblastoma by virtue of its active motility in extracellular space and can lead to tailored therapies via tumor-specific expression of tumoricidal proteins.

Kilohertz frequency nerve block enhances anti-inflammatory effects of vagus nerve stimulation.

Efferent activation of the cervical vagus nerve (cVN) dampens systemic inflammatory processes, potentially modulating a wide-range of inflammatory pathological conditions. In contrast, afferent cVN activation amplifies systemic inflammatory processes, leading to activation of the hypothalamic-pituitary-adrenal (HPA) axis, the sympathetic nervous system through the greater splanchnic nerve (GSN), and elevation of pro-inflammatory cytokines. Ideally, to clinically implement anti-inflammatory therapy via cervical vagus nerve stimulation (cVNS) one should selectively activate the efferent pathway. Unfortunately, current implementations, in animal and clinical investigations, activate both afferent and efferent pathways. We paired cVNS with kilohertz electrical stimulation (KES) nerve block to preferentially activate efferent pathways while blocking afferent pathways. Selective efferent cVNS enhanced the anti-inflammatory effects of cVNS. Our results demonstrate that: (i) afferent, but not efferent, cVNS synchronously activates the GSN in a dose-dependent manner; (ii) efferent cVNS enabled by complete afferent KES nerve block enhances the anti-inflammatory benefits of cVNS; and (iii) incomplete afferent KES nerve block exacerbates systemic inflammation. Overall, these data demonstrate the utility of paired efferent cVNS and afferent KES nerve block for achieving selective efferent cVNS, specifically as it relates to neuromodulation of systemic inflammation.