In the present study, we used a well-established SCI contusion mouse model to provide evidence that Lcn2 is upregulated after SCI throughout the whole SC and not only in the primarily injured region. Beyond SC, we observed a Lcn2-induction in the cerebral cortex at both protein and mRNA level. Interestingly, we show a marked increase of Lcn2 in systemic circulation and also in liver in the early phase post SCI. Various studies have found a correlation between increased Lcn2 levels and CNS disorders, such as multiple sclerosis and stroke [28, 30, 43, 44]. Therefore, using Lcn2−/− mice, we investigate the effect of Lcn2 deficiency on astrogliosis as a hallmark of SCI. Since the results show a significant reduction of GFAP, a decrease of astrogliosis in Lcn2 deficient mice might be concluded.
Post SCI, astrocytes proliferate and undergo morphological changes which include hypertrophy and the development of extended processes [6, 45]. Through the release of neurotrophic factors, astrocytes support neurons in SC and thus, impaired astrocytic function has major consequences for neuronal function [17, 46]. In brain injury, the ablation of reactive astrocytes was found to lead to substantial neuronal degeneration . Moreover, astrocytes limit the spread of inflammation after SCI, since they are one of the dominant cell types of the glial scar which forms after injury [21, 45]. Furthermore, activated astrocytes can express a variety of cytokines, chemokines, and the respective receptors, and therefore play a pivotal role in the neuroinflammatory processes in SCI [45, 47]. Further, axonal regeneration is inhibited by the glial scar and chondroitin sulfate proteoglycans which are produced by reactive glial cells, including astrocytes [19, 21]. In addition, these proteoglycans impede process outgrowth of oligodendrocytes and thereby disturb remyelination [48, 49]. Based on the dual character of astrocytes, it has been suggested that they can be classified into a neurotoxic A1 and neuroprotective A2 phenotype [24, 25]. Different factors, such as chemokines and cytokines, e.g. IL-1β, TNF-α and IL-10, have been found to control the development of astrocytes in the direction of either phenotype [25, 50, 51]. One of the regulators of astrocyte polarization is Lcn2 which supports the pro-inflammatory A1 phenotype and decreases the polarization in the direction of A2 in vitro by inhibiting IL-4–STAT6 signaling . The influence of Lcn2 on astrocyte polarization, morphology and migration is an important aspect of its regulatory function in neuroinflammation [27, 52]. Lcn2 is involved in various pathological processes, such as stroke, metabolic inflammation, diabetes and nonalcoholic steatohepatitis [30, 31, 53, 54]. It promotes inflammation through induction of pro-inflammatory cytokines via release of high mobility group box 1, which binds to toll-like receptor 4 and induces oxidative stress by activation of NOX-2 signaling . Furthermore, beyond its effect on activation and polarization of microglia, Lcn2 supports the recruitment of inflammatory cells by the induction of CXCL10 secretion and release of the neutrophil-recruitment signal IL-8 [31, 34, 55–57].
In the present study, we could demonstrate that SCI induces an increase of Lcn2 expression throughout the whole SC. As the cellular source of Lcn2 in the CNS, previous studies have identified astrocytes and endothelial cells, which we confirm by our studies [26, 58]. However, we could not prove the production of Lcn2 by microglia in our animal model . The triggers of Lcn2 production in this context are, besides others, cytokines such as IL-6 and NF-kappa B activation [58, 60].
Since Lcn2 is secreted, and elevated concentrations can be found in the blood circulation under pathological conditions, like multiple sclerosis, intestinal inflammation and arthritic diseases, it has been described as a biomarker in several pathologies . In the present study, we show that the Lcn2 concentration is significantly increased in the serum as a direct consequence of SCI, which might suggest this molecule as a potential biomarker for traumatic SCI. Further, circulating Lcn2 could be considered as a part of the systemic inflammatory response (SIR) which affects the homeostasis of peripheral organs such as liver, kidney, lung and intestine. Thereby it contributes to the pathogenesis of multiple organ dysfunction after SCI and supports secondary injury to the SC [38, 62–65]. In addition, we were able to detect elevated Lcn2 levels in brain and liver. This can have at least two reasons: Lcn2 might be produced in the respective tissue. This is supported by the fact that we have found significantly increased Lcn2 mRNA in both, brain and liver. Additionally, the identification of Lcn2 + cells in both tissues after IHC staining indicates a production of Lcn2 by the resident cells. In the brain, we could identify endothelial cells as a cellular source of Lcn2 by double-immunofluorescence staining. One of the possible triggers of Lcn2 production in the brain are cytokines. For example, the i.p. application of IL-6 induces Lcn2 production by vascular cells in the brain in mice . In adipocytes also TNFα and IL-1β trigger Lcn2 production in vitro . Since various cytokines have been shown to be upregulated in the blood stream after SCI, they might lead to an increase in Lcn2 production in endothelial cells .
In the liver, hepatocytes and neutrophil granulocytes have been identified as cellular sources of Lcn2 [68, 69]. It has been demonstrated in vitro that the cytokine IL-1β induces Lcn2 production in a NF-kappa B-dependent manner in both cell types [70–72]. Due to the structure of the hepatic tissue, hepatocytes and recruited neutrophils come into close contact with cytokines, reaching the liver via the hepatic artery which might induce Lcn2 production . Since we have found elevated Lcn2 levels in serum post SCI, Lcn2 might also, besides its production by resident cells, reach the brain and the liver via the bloodstream.
In the brain, Lcn2 has different beneficial as well as harmful effects . In the ischemic brain, Lcn2 contributes to neuronal cell death by promoting neuroinflammation . However, in an experimental model of multiple sclerosis, Lcn2-deficient mice exhibited increased disease severity, suggesting a neuroprotective role of Lcn2 . In liver pathology, the effects of Lcn2 have been discussed controversially. In phases of acute liver injury, Lcn2 plays an essential role in liver homeostasis and lipid metabolism and protects hepatocytes, whereas it promotes liver injury and hepatic steatosis in a model of alcoholic steatohepatitis [76–78].
In our studies, the decrease of the astrogliosis marker GFAP in Lcn2−/− mice is a first, valuable hint at a possible promotion of astrogliosis by Lcn2 in SCI . In vitro, it has already been demonstrated that GFAP expression is promoted by Lcn2 . However, according to our results, Lcn2 does not affect the regulatory mechanism underlying the phenotypic polarization of activated astrocytes in our animal model. The promotion of the classical inflammatory activation of astrocytes by Lcn2 has, up to now, been only confirmed in vitro and in an animal model of transient middle cerebral artery occlusion [25, 81]. Eventually the effect of Lcn2 on astrocyte polarization depends on the underlying pathology.
So far, we base our conclusions regarding the influence of Lcn2 on astrogliosis and astrocyte polarization on qPCR studies. Therefore, possible posttranslational modifications cannot be taken into account. This limitation has to be addressed in further studies. Nevertheless, we confirm a general positive effect of Lcn2 deficiency on the functional outcome in SCI based on BBB locomotor scoring. It is assumed that the elevated level of Lcn2 after SCI may exacerbate axonal degeneration and contribute to poor neurological outcome by enhancing inflammatory cell infiltration and promoting neuronal apoptosis .
In summary, we found that SCI promotes the Lcn2-upregulation in SC, brain, blood circulation and peripheral organs such as the liver. Consequently, Lcn2 might play a role in systemic effects and multiple organ dysfunction in SCI pathology. The precise effect of Lcn2 on peripheral organs has to be examined thoroughly to understand the resulting SCI-induced impairment of these tissues. As a local consequence of SCI pathology, Lcn2 promotes specific aspects of astrogliosis, which suggests that Lcn2 can be therapeutically targeted to modulate the reaction of astrocytes in certain pathologies such as SCI. Further studies are needed to elucidate the precise mechanisms responsible for astrocyte activation and polarization to better understand the role played by Lcn2 in this process.