The Dark Side of an Essential Amino Acid: L-Arginine in Spinal Cord Injury.

L-arginine is a semi-essential amino acid involved in a variety of physiological processes in the central nervous system (CNS). It is essential in the survival and functionality of neuronal cells. Nonetheless, L-arginine also has a dark side; it potentiates neuroinflammation and nitric oxide (NO) production, leading to secondary damage. Therefore, modulating the L-arginine metabolism is challenging because both detrimental and beneficial effects are dependent on this semi-essential amino acid. After spinal cord injury (SCI), L-arginine plays a crucial role in trauma-induced neuroinflammation and regenerative processes via the two key enzymes: nitric oxide synthase (NOS) and arginase (ARG). Studies on L-arginine metabolism using ARG and NOS inhibitors highlighted the conflicting role of this semi-essential amino acid. Similarly, L-arginine supplementation resulted in both negative and positive outcomes after SCI. However, new data indicate that arginine depletion substantially improves spinal cord regeneration after injury. Here, we review the challenging characteristics of L-arginine metabolism as a therapeutic target after SCI.

Macrophage-based delivery of interleukin-13 improves functional and histopathological outcomes following spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) elicits a robust neuroinflammatory reaction which, in turn, exacerbates the initial mechanical damage. Pivotal players orchestrating this response are macrophages (Mφs) and microglia. After SCI, the inflammatory environment is dominated by pro-inflammatory Mφs/microglia, which contribute to secondary cell death and prevent regeneration. Therefore, reprogramming Mφ/microglia towards a more anti-inflammatory and potentially neuroprotective phenotype has gained substantial therapeutic interest in recent years. Interleukin-13 (IL-13) is a potent inducer of such an anti-inflammatory phenotype. In this study, we used genetically modified Mφs as carriers to continuously secrete IL-13 (IL-13 Mφs) at the lesion site. METHODS: Mφs were genetically modified to secrete IL-13 (IL-13 Mφs) and were phenotypically characterized using qPCR, western blot, and ELISA. To analyze the therapeutic potential, the IL-13 Mφs were intraspinally injected at the perilesional area after hemisection SCI in female mice. Functional recovery and histopathological improvements were evaluated using the Basso Mouse Scale score and immunohistochemistry. Neuroprotective effects of IL-13 were investigated using different cell viability assays in murine and human neuroblastoma cell lines, human neurospheroids, as well as murine organotypic brain slice cultures. RESULTS: In contrast to Mφs prestimulated with recombinant IL-13, perilesional transplantation of IL-13 Mφs promoted functional recovery following SCI in mice. This improvement was accompanied by reduced lesion size and demyelinated area. The local anti-inflammatory shift induced by IL-13 Mφs resulted in reduced neuronal death and fewer contacts between dystrophic axons and Mφs/microglia, suggesting suppression of axonal dieback. Using IL-4Rα-deficient mice, we show that IL-13 signaling is required for these beneficial effects. Whereas direct neuroprotective effects of IL-13 on murine and human neuroblastoma cell lines or human neurospheroid cultures were absent, IL-13 rescued murine organotypic brain slices from cell death, probably by indirectly modulating the Mφ/microglia responses. CONCLUSIONS: Collectively, our data suggest that the IL-13-induced anti-inflammatory Mφ/microglia phenotype can preserve neuronal tissue and ameliorate axonal dieback, thereby promoting recovery after SCI.

L-Arginine Depletion Improves Spinal Cord Injury via Immunomodulation and Nitric Oxide Reduction.

BACKGROUND: Spinal cord injury (SCI) elicits robust neuroinflammation that eventually exacerbates the initial damage to the spinal cord. L-arginine is critical for the responsiveness of T cells, which are important contributors to neuroinflammation after SCI. Furthermore, L-arginine is the substrate for nitric oxide (NO) production, which is a known inducer of secondary damage. METHODS: To accomplish systemic L-arginine depletion, repetitive injections of recombinant arginase-1 (rArg-I) were performed. Functional recovery and histopathological parameters were analyzed. Splenic immune responses were evaluated by flow cytometry. Pro-inflammatory gene expression and nitrite concentrations were measured. RESULTS: We show for the first time that systemic L-arginine depletion improves locomotor recovery. Flow cytometry and immunohistological analysis showed that intraspinal T-cell infiltration was reduced by 65%, and peripheral numbers of Th1 and Th17 cells were suppressed. Moreover, rArg-I treatment reduced the intraspinal NO production by 40%. Histopathological analyses revealed a 37% and 36% decrease in the number of apoptotic neurons and neuron-macrophage/microglia contacts in the spinal cord, respectively. CONCLUSIONS: Targeting detrimental T-cell responses and NO-production via rArg-I led to a reduced neuronal cell death and an improved functional recovery. These findings indicate that L-arginine depletion holds promise as a therapeutic strategy after SCI.

CD36-mediated uptake of myelin debris by macrophages and microglia reduces neuroinflammation.

BACKGROUND: The presence of foamy macrophages and microglia containing intracellular myelin remnants is a pathological hallmark of neurodegenerative disorders such as multiple sclerosis (MS). Despite the importance of myelin internalization in affecting both central nervous system repair and neuroinflammation, the receptors involved in myelin clearance and their impact on the phagocyte phenotype and lesion progression remain to be clarified. METHODS: Flow cytometry, quantitative PCR, and immunohistochemistry were used to define the mRNA and protein abundance of CD36 in myelin-containing phagocytes. The impact of CD36 and nuclear factor erythroid 2-related factor 2 (NRF2) on the phagocytic and inflammatory features of macrophages and microglia was assessed using a pharmacological CD36 inhibitor (sulfo-N-succinimidyl oleate) and Nrf2-/- bone marrow-derived macrophages. Finally, the experimental autoimmune encephalomyelitis (EAE) model was used to establish the impact of CD36 inhibition on neuroinflammation and myelin phagocytosis in vivo. RESULTS: Here, we show that the fatty acid translocase CD36 is required for the uptake of myelin debris by macrophages and microglia, and that myelin internalization increased CD36 expression through NRF2. Pharmacological inhibition of CD36 promoted the inflammatory properties of myelin-containing macrophages and microglia in vitro, which was paralleled by a reduced activity of the anti-inflammatory lipid-sensing liver X receptors and peroxisome proliferator-activated receptors. By using the EAE model, we provide evidence that CD36 is essential for myelin debris clearance in vivo. Importantly, CD36 inhibition markedly increased the neuroinflammatory burden and disease severity in the EAE model. CONCLUSION: Altogether, we show for the first time that CD36 is crucial for clearing myelin debris and suppressing neuroinflammation in demyelinating disorders such as MS.