A blood-based biomarker panel (NIS4) for non-invasive diagnosis of non-alcoholic steatohepatitis and liver fibrosis: a prospective derivation and global validation study.

BACKGROUND: Non-invasive tests that can identify patients with non-alcoholic steatohepatitis (NASH) at higher risk of disease progression are lacking. We report the development and validation of a blood-based diagnostic test to non-invasively rule in and rule out at-risk NASH (defined as non-alcoholic fatty liver disease [NAFLD] activity score [NAS] ≥4 and fibrosis stage ≥2). METHODS: In this prospective derivation and global validation study, blood samples, clinical data, and liver biopsy results from three independent cohorts with suspected NAFLD were used to develop and validate a non-invasive blood-based diagnostic test, called NIS4. Derivation was done in the discovery cohort, which comprised 239 prospectively recruited patients with biopsy-confirmed NASH (NAFLD NAS ≥3; fibrosis stage 0-3) from the international GOLDEN-505 phase 2b clinical trial. A complete matrix based on 23 variables selected for univariate association with the presence of at-risk NASH and avoiding high multi-collinearity was used to derive the model in a bootstrap-based process that minimised the Akaike information criterion. The overall diagnostic performance of NIS4 was externally validated in two independent cohorts: RESOLVE-IT diag and Angers. The RESOLVE-IT diag cohort comprised the first 475 patients screened for potential inclusion into the RESOLVE-IT phase 3 clinical trial. Angers was a retrospective cohort of 227 prospectively recruited patients with suspected NAFLD and clinical risk factors for NASH or fibrosis stage 2 or more according to abnormal elastography results or abnormal liver biochemistry. Both external validation cohorts were independently analysed and were combined into a pooled validation cohort (n=702) to assess clinical performance of NIS4 and other non-invasive tests. FINDINGS: The derived NIS4 algorithm comprised four independent NASH-associated biomarkers (miR-34a-5p, alpha-2 macroglobulin, YKL-40, and glycated haemoglobin; area under the receiver operating characteristics curve [AUROC] 0·80, 95% CI 0·73-0·85), and did not require adjustment for age, sex, body-mass index (BMI), or aminotransferase concentrations. Clinical cutoffs were established within the discovery cohort to optimise both rule out and rule in clinical performance while minimising indeterminate results. NIS4 was validated in the RESOLVE-IT diag cohort (AUROC 0·83, 95% CI 0·79-0·86) and the Angers cohort (0·76, 0·69-0·82). In the pooled validation cohort, patients with a NIS4 value less than 0·36 were classified as not having at-risk NASH (ruled out) with 81·5% (95% CI 76·9-85·3) sensitivity, 63·0% (57·8-68·0) specificity, and a negative predictive value of 77·9% (72·5-82·4), whereas those with a NIS4 value of more than 0·63 were classified as having at-risk NASH (ruled in) with 87·1% (83·1-90·3) specificity, 50·7% (45·3-56·1) sensitivity, and a positive predictive value of 79·2% (73·1-84·2). The diagnostic performance of NIS4 within the external validation cohorts was not influenced by age, sex, BMI, or aminotransferase concentrations. INTERPRETATION: NIS4 is a novel blood-based diagnostic that provides an effective way to non-invasively rule in or rule out at-risk NASH in patients with metabolic risk factors and suspected disease. Use of NIS4 in clinical trials or in the clinic has the potential to greatly reduce unnecessary liver biopsies in patients with lower risk of disease progression. FUNDING: Genfit.

Toll-like receptor 4 deficiency impairs microglial phagocytosis of degenerating axons.

Microglia are rapidly activated in the central nervous system (CNS) in response to a variety of injuries, including inflammation, trauma, and stroke. In addition to modulation of the innate immune response, a key function of microglia is the phagocytosis of dying cells and cellular debris, which can facilitate recovery. Despite emerging evidence that axonal debris can pose a barrier to regeneration of new axons in the CNS, little is known of the cellular and molecular mechanisms that underlie clearance of degenerating CNS axons. We utilize a custom micropatterned microfluidic system that enables robust microglial-axon co-culture to explore the role of Toll-like receptors (TLRs) in microglial phagocytosis of degenerating axons. We find that pharmacologic and genetic disruption of TLR4 blocks induction of the Type-1 interferon response and inhibits phagocytosis of axon debris in vitro. Moreover, TLR4-dependent microglial clearance of unmyelinated axon debris facilitates axon outgrowth. In vivo, microglial phagocytosis of CNS axons undergoing Wallerian degeneration in a dorsal root axotomy model is impaired in adult mice in which TLR4 has been deleted. Since purinergic receptors can influence TLR4-mediated signaling, we also explored a role for the microglia P2 receptors and found that the P2X7R contributes to microglial clearance of degenerating axons. Overall, we identify TLR4 as a key player in axonal debris clearance by microglia, thus creating a more permissive environment for axonal outgrowth. Our findings have significant implications for the development of protective and regenerative strategies for the many inflammatory, traumatic, and neurodegenerative conditions characterized by CNS axon degeneration.

Curcumin protects axons from degeneration in the setting of local neuroinflammation.

Axon degeneration is a hallmark of several central nervous system (CNS) disorders, including multiple sclerosis (MS), Alzheimer's disease (AD) and Parkinson's disease (PD). Previous neuroprotective approaches have mainly focused on reversal or prevention of neuronal cell body degeneration or death. However, experimental evidence suggests that mechanisms of axon degeneration may differ from cell death mechanisms, and that therapeutic agents that protect cell bodies may not protect axons. Moreover, axon degeneration underlies neurologic disability and may, in some cases, represent an important initial step that leads to neuronal death. Here, we develop a novel quantitative microfluidic-based methodology to assess mechanisms of axon degeneration caused by local neuroinflammation. We find that LPS-stimulated microglia release soluble factors that, when applied locally to axons, result in axon degeneration. This local axon degeneration is mediated by microglial MyD88/p38 MAPK signaling and concomitant production of nitric oxide (NO). Intra-axonal mechanisms of degeneration involve JNK phosphorylation. Curcumin, a compound with both anti-oxidant and JNK inhibitory properties, specifically protects axons, but not neuronal cell bodies, from NO-mediated degeneration. Overall, our platform provides mechanistic insights into local axon degeneration, identifies curcumin as a novel axon protectant in the setting of neuroinflammation, and allows for ready screening of axon protective drugs.

Toll/interleukin-1 receptor domain-containing adapter inducing interferon-ß mediates microglial phagocytosis of degenerating axons.

Following CNS injury, microglial phagocytosis of damaged endogenous tissue is thought to play an important role in recovery and regeneration. Previous work has focused on delineating mechanisms of clearance of neurons and myelin. Little, however, is known of the mechanisms underlying phagocytosis of axon debris. We have developed a novel microfluidic platform that enables coculture of microglia with bundles of CNS axons to investigate mechanisms of microglial phagocytosis of axons. Using this platform, we find that axon degeneration results in the induction of type-1 interferon genes within microglia. Pharmacologic and genetic disruption of Toll/interleukin-1 receptor domain-containing adapter inducing interferon-ß (TRIF), a Toll-like receptor adapter protein, blocks induction of the interferon response and inhibits microglial phagocytosis of axon debris in vitro. In vivo, microglial phagocytosis of axons following dorsal root axotomy is impaired in mice in which TRIF has been genetically deleted. Furthermore, we identify the p38 mitogen-activated protein kinase (MAPK) cascade as a signaling pathway downstream of TRIF following axon degeneration and find that inhibition of p38 MAPK by SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole) also blocked clearance of axon debris. Finally, we find that TRIF-dependent microglial clearance of unmyelinated axon debris facilitates axon outgrowth. Overall, we provide evidence that TRIF-mediated signaling plays an unexpected role in axonal debris clearance by microglia, thereby facilitating a more permissive environment for axonal outgrowth. Our study has significant implications for the development of novel regenerative and restorative strategies for the many traumatic, neuroinflammatory, and neurodegenerative conditions characterized by CNS axon degeneration.

Valve-based microfluidic compression platform: single axon injury and regrowth.

We describe a novel valve-based microfluidic axon injury micro-compression (AIM) platform that enables focal and graded compression of micron-scale segments of single central nervous system (CNS) axons. The device utilizes independently controlled "push-down" injury pads that descend upon pressure application and contact underlying axonal processes. Regulated compressed gas is input into the AIM system and pressure levels are modulated to specify the level of injury. Finite element modeling (FEM) is used to quantitatively characterize device performance and parameterize the extent of axonal injury by estimating the forces applied between the injury pad and glass substrate. In doing so, injuries are normalized across experiments to overcome small variations in device geometry. The AIM platform permits, for the first time, observation of axon deformation prior to, during, and immediately after focal mechanical injury. Single axons acutely compressed (~5 s) under varying compressive loads (0-250 kPa) were observed through phase time-lapse microscopy for up to 12 h post injury. Under mild injury conditions (< 55 kPa) ~73% of axons continued to grow, while at moderate (55-95 kPa) levels of injury, the number of growing axons dramatically reduced to 8%. At severe levels of injury (> 95 kPa), virtually all axons were instantaneously transected and nearly half (~46%) of these axons were able to regrow within the imaging period in the absence of exogenous stimulating factors.

Circular compartmentalized microfluidic platform: Study of axon-glia interactions.

We describe a compartmentalized circular microfluidic platform that enables directed cell placement within defined microenvironments for the study of axon-glia interactions. The multi-compartment platform consists of independent units of radial microchannel arrays that fluidically isolate somal from axonal compartments. Fluidic access ports punched near the microchannels allow for direct pipetting of cells into the device. Adjacent somal or axonal compartments can be readily merged so that independent groups of neurons or axons can be maintained in either separate or uniform microenvironments. We demonstrate three distinct modes of directed cell placement in this device, to suit varying experimental needs for the study of axon-glia interactions: (1) centrifugation of the circular platform can result in a two-fold increase in axonal throughput in microchannels and provides a new technique to establish axon-glia interactions; (2) microstencils can be utilized to directly place glial cells within areas of interest; and (3) intimate axon-glia co-culture can be attained via standard pipetting techniques. We take advantage of this microfluidic platform to demonstrate a two-fold preferential accumulation of microglia specifically near injured CNS axons, an event implicated in the maintenance and progression of a number of chronic neuroinflammatory and neurodegenerative diseases.

Compartmentalized microfluidic culture platform to study mechanism of paclitaxel-induced axonal degeneration.

Chemotherapy induced peripheral neuropathy is a common and dose-limiting side effect of anticancer drugs. Studies aimed at understanding the underlying mechanism of neurotoxicity of chemotherapeutic drugs have been hampered by lack of suitable culture systems that can differentiate between neuronal cell body, axon or associated glial cells. Here, we have developed an in vitro compartmentalized microfluidic culture system to examine the site of toxicity of chemotherapeutic drugs. To test the culture platform, we used paclitaxel, a widely used anticancer drug for breast cancer, because it causes sensory polyneuropathy in a large proportion of patients and there is no effective treatment. In previous in vitro studies, paclitaxel induced distal axonal degeneration but it was unclear if this was due to direct toxicity on the axon or a consequence of toxicity on the neuronal cell body. Using microfluidic channels that allow compartmentalized culturing of neurons and axons, we demonstrate that the axons are much more susceptible to toxic effects of paclitaxel. When paclitaxel was applied to the axonal side, there was clear degeneration of axons; but when paclitaxel was applied to the soma side, there was no change in axon length. Furthermore, we show that recombinant human erythropoietin, which had been shown to be neuroprotective against paclitaxel neurotoxicity, provides neuroprotection whether it is applied to the cell body or the axons directly. This observation has implications for development of neuroprotective drugs for chemotherapy induced peripheral neuropathies as dorsal root ganglia do not possess blood-nerve-barrier, eliminating one of the cardinal requirements of drug development for the nervous system. This compartmentalized microfluidic culture system can be used for studies aimed at understanding axon degeneration, neuroprotection and development of the nervous system.