Systemic Inflammation Leads to Changes in the Intracellular Localization of KLK6 in Oligodendrocytes in Spinal Cord White Matter

Axonal injury and demyelination occur in demyelinating diseases, such as multiple sclerosis, and the detachment of myelin from axons precedes its degradation. Paranodes are the areas at which each layer of the myelin sheath adheres tightly to axons. The destruction of nodal and paranodal structures during inflammation is an important pathophysiology of various neurological disorders. However, the underlying pathological changes in these structures remain unclear. Kallikrein 6 (KLK6), a serine protease produced by oligodendrocytes, is involved in demyelinating diseases. In the present study, we intraperitoneally injected mice with LPS for several days and examined changes in the localization of KLK6. Transient changes in the intracellular localization of KLK6 to paranodes in the spinal cord were observed during LPS-induced systemic inflammation. However, these changes were not detected in the upper part of brain white matter. LPS-induced changes were suppressed by minocycline, suggesting the involvement of microglia. Moreover, nodal lengths were elongated in LPS-treated wild-type mice, but not in LPS-treated KLK6-KO mice. These results demonstrate the potential involvement of KLK6 in the process of demyelination.


Introduction
Oligodendrocytes wrap around axons and produce myelin, which ensures the proper conduction of action potentials in the central nervous system (CNS). In myelinated axons in the CNS, oligodendrocytes also contribute to the production and maintenance of nodal regions, consisting of voltage-gated ion channels, cell adhesion molecules, the cytoskeleton, and scaffold proteins [1]. Since axons are insulated by the myelin sheath, an action potential is only regenerated at these nodes; therefore, conductance is quick and efficient.
Neuroinflammation has been implicated in a number of neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, dementia, and multiple sclerosis [2]. Autopsied brains from patients with neurodegenerative diseases showed marked astrogliosis, microglial activation, and high levels of proinflammatory cytokines [3]. Previous studies demonstrated that the clinical progression of Alzheimer's disease was related to common infections [4]. Moreover, the recurrence of multiple sclerosis was preceded by systemic infections in approximately one-third of cases [5]. These findings suggest that systemic infections and inflammation contribute to brain diseases by promoting the activation of the immune system. During systemic infections, pathogenassociated molecular pattern molecules, such as lipopolysaccharide (LPS), are released from bacteria and trigger the activation of the innate immune system [6]. Therefore, LPS is often used in various protocols to model neuroinflammation associated with neurodegeneration [7]. LPS induces the activation of innate immune responses in microglia, leading to oligodendrocyte damage and demyelination [8].
IL-1β, TNF-α, and nitric oxide from LPS-activated microglia induce the synthesis of matrix metalloproteases, which are capable of damaging axons [9,10]. The intraperitoneal (i.p.) administration of LPS was previously shown to induce demyelination and down-regulate the expression of synaptic proteins in the hippocampus, resulting in depressive behavior [11]. The risk of developing non-multiple sclerosis demyelinating syndromes was found to be 4.17-fold higher in patients with than in those without sepsis [12]. These findings support the hypothesis that systemic inflammation mediates demyelination.
Kallikrein (KLK) 6 is a member of the KLK serine protease family [13,14] and is closely associated with diverse neurodegenerative diseases. The hypothesis that KLK6 is involved in inflammatory CNS demyelination is supported by findings demonstrating the digestive activity of this protease against myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein. Moreover, in vitro studies show that KLK6 levels are associated with marked reductions in the differentiated oligodendrocyte process [15,16]. Previous studies reported that KLK6-neutralizing antibodies attenuated disease in experimental autoimmune encephalomyelitis (EAE) and a viral model of multiple sclerosis [17,18]. Moreover, KLK6 has been shown to activate protease-activated receptors in response to CNS injury, leading to the activation of several intracellular signaling pathways that may contribute to neurotoxicity [19]. Therefore, KLK6 may play a key role in the pathology of demyelinating diseases. However, the mechanisms by which systemic inflammation affects the expression of KLK6 and pathophysiology currently remain unclear. In the present study, we investigated changes in the expression of KLK6 using a LPS-induced systemic inflammation model in the CNS.

Animals
All procedures involving animals and their care were performed in accordance with the Guidelines laid down by the NIH and the Guidelines for Proper Conduct of Animal Experiments, the Science Council of Japan to minimize the number of animals used and their suffering. The experimental protocol was approved by the Animal Ethics Experimental Committee of the Asahikawa Medical Center and Asahikawa Medical University. Male C57BL/6J mice and KLK6 knockout (KO) mice (C57BL/6J background) were used at 8-10 weeks old in experiments. Animals from our in-house colony were kept under pathogen-free standard conditions (25.0 ± 1.0 °C, 12-h light/dark cycle; lights on at 0700 h) and received commercial chow and water ad libitum.

Administration of LPS and Minocycline
To induce systemic inflammation by LPS, animals were i.p. injected once a day with 1 mg/kg LPS (Sigma-Aldrich, serotype 055:B5) from Escherichia coli or saline according to our previously reported method [20]. We used minocycline (Sigma-Aldrich, St. Louis, MO), a secondgeneration semisynthetic tetracycline antibiotic, to suppress microglial activity. Minocycline is a lipid-soluble chemical that crosses the blood-brain barrier (BBB) into the brain parenchyma and exerts anti-inflammatory effects [21]. Mice were i.p. injected with minocycline (50 mg/ kg dissolved in saline) or saline only as a vehicle control twice daily for two days, and from the next day, mice were i.p. injected with saline, minocycline (50 mg/kg), LPS (1 mg/kg), or a mixture of minocycline and LPS.

Confocal Observations and Quantification
Images were obtained using the laser-scanning FV1000-D confocal microscope (Olympus, Tokyo, Japan). We used 3-4 animals to quantify the areas of Iba1-positive microglia and KLK6-positive oligodendrocytes in each group. At least 5 sections were selected from the thoracic spinal cord. Confocal images were obtained under the same pinhole size, brightness, and contrast setting for a quantitative analysis. In quantitative analyses, the total area of Iba1-positive microglia and KLK6-positive oligodendrocytes was calculated using WinRoof, an image analyzing system (Mitani Corporation, Fukui, Japan). The significance of differences was assessed using a significance level of P < 0.05 with the Student's t-test or an ANOVA with Tukey's post hoc test.

Quantification of the Perinodal Pathology
Immunohistochemistry was performed on sagittal sections of the thoracic spinal cord for Caspr and Kv1.2., markers of the paranodes and juxtaparanodes, respectively. We examined at least three different animals in each group. Each area was defined as follows: paranodes are Caspr-positive regions, juxtaparanodes are Kv1.2positive regions, and nodes are areas located between Caspr. Nodal, paranodal, and juxtaparanodal lengths were measured using the line tool in ImageJ. At least 10 sections were selected from each mouse and measured for a subset of 10 randomly selected nodal regions. Data are expressed as the mean ± SEM. The significance of differences was assessed using a significance level of P < 0.05 with the Student's t-test or an ANOVA with Tukey's post hoc test.

Systemic Inflammation Induces KLK6 Expression in Oligodendrocyte Paranodes in Spinal Cord White Matter
To investigate changes in the expression of KLK6 in systemic inflammation, mice were i.p. injected daily with LPS at a dose of 1 mg/kg or saline for 4 consecutive days. The experimental paradigm is shown in Fig. 1A. Low powermagnification observations revealed that KLK6-positive structures mainly localized to white matter in the spinal cord in both the vehicle and LPS groups. LPS injections induced denser KLK6-positive structures in the surface regions of white matter (Fig. 1B, C). High power-magnification observations revealed that KLK6 localized to the cell body and thick protrusions in APC-positive mature oligodendrocytes in the anterior funiculus of the thoracic spinal cord in the vehicle group. In contrast, many fine circular-shaped KLK6positive structures other than cell bodies were observed in the LPS group ( Fig. 1D-D″, E-E″). Moreover, these KLK6-positive fine circular-shaped structures in the LPS group encircled neurofilament H-positive axons ( Fig. 1F-F″, G-G″). Fine oligodendrocyte processes are myelin and uncompacted membranes, namely, the inner-and outermost tongues of myelin membranes and the paranodes [23]. We hypothesized that KLK6 may be expressed in the paranodes in addition to the cell bodies and, thus, performed immunostaining with the paranode marker Caspr. In the vehicle group, KLK6-positive structures were devoid of Casprpositive paranodes. In the LPS group, KLK6-positive scattered structures were also positive for Caspr, indicating that they were paranodes ( Fig. 1H-H″, I-I″). Three-dimensional analysis validated the presence of KLK6 in the Caspr-positive paranodes in the LPS group (right panels in Fig. 1H″, I″). The ratio of KLK6-expressing paranodes significantly increased in the spinal cord of the LPS group (17.1% paranodes were positive for KLK6 in the control group and 84.2 ± 5.5% in the LPS group) (Fig. 1J).

Changes in the Intracellular Localization of KLK6 in Oligodendrocytes and the Microglial Morphology Induced by Systemic Inflammation are Transient in the Spinal Cord
We examined the time course of KLK6 expression in the spinal cord. Since KLK6-positive areas appeared to increase as KLK6 was expressed in the paranodes, we measured KLK6-positive areas. The experimental paradigm is shown in Fig. 2A. Fine circular-shaped KLK6-positive structures were observed in the anterior funiculus of the spinal cord on the 3rd and 4th days of the LPS injections ( Fig. 2B-D), and disappeared by the 9th day (Fig. 2E). The quantitative analysis revealed that the daily injections of LPS significantly increased KLK6-positive areas on the 3rd and 4th days, with a return to control levels after the 9th day (Fig. 2F). LPS is known to activate microglia. We therefore hypothesized that activated microglia induces changes in the localization of KLK6. We examined the time course of microglial changes. Iba1 immunohistochemistry showed that the 3rd and 4th days of the LPS injections induced morphological changes in microglia, namely, round and larger cell bodies with shorter processes, suggesting that LPS induced the activation of microglia in the spinal cord, as previously reported [24] ( Fig. 2G-I). Changes in the microglial morphology returned to normal on the 9th day (Fig. 2J). The quantitative analysis revealed that the daily LPS injections significantly increased Iba1-positive areas on the 3rd and 4th days, with a return to control levels after the 9th day (Fig. 2K).

Changes in the Intracellular Localization of KLK6 in Oligodendrocytes During Systemic Inflammation in Each Segment of the Spinal Cord
To establish whether changes in the intracellular localization of KLK6 in oligodendrocytes and microglial activation by LPS also occurred in segments besides the thoracic spinal cord, we examined other segments. Animals were i.p. injected daily with 1 mg/kg LPS or saline for 4 consecutive days. In the vehicle group, KLK6 immunoreactivity was observed in cell bodies at all anterior and posterior funiculi of spinal cord segments, namely, the cervical, thoracic, lumbar, and sacral regions (Fig. 3A, C). On the other hand, KLK6-positive structures localized to more widely scattered areas in the thoracic, lumbar, and sacral regions, but not in the cervical region in the LPS group (Fig. 3B, D). The quantitative analysis revealed that the i.p. injections of LPS significantly increased KLK6positive areas in the anterior and posterior funiculi of the thoracic, lumbar, and sacral cords. Similar results were observed for the anterior funiculus of the cervical cord without a significant difference. LPS-induced changes in the localization of KLK6 were not observed in the posterior funiculus of the cervical cord, indicating the region-dependent heterogeneity of oligodendrocyte reactivity (Fig. 3E). Iba1-positive areas significantly increased in the anterior and posterior funiculi of all segments, including the cervical segment of the spinal cord (Fig. 3F).

Changes in the Intracellular Localization of KLK6 in Oligodendrocytes During Systemic Inflammation in Brain White Matter
We investigated whether systemic inflammation induced changes in the intracellular localization of KLK6 in brain white matter. Mice were i.p. injected with 1 mg/kg LPS daily for 4 consecutive days. KLK6 was detected in white matter regions, namely, the anterior part of the anterior commissure (aca), corpus callosum (cc), ventral hippocampal commissure (vhc), fimbria (fi), spinal trigeminal tract (sp5), and pyramidal tract (py), in the control group with weaker immunoreactivity in the aca, cc, and vhc, belonging to the higher brain (Fig. 4A). Significant changes in the intracellular localization of KLK6 were observed in sp5 after the LPS injections, but not in other brain regions (Fig. 4B, E). In contrast, microglial activation was induced in all of these brain regions (Fig. 4C, D). The quantitative analysis revealed that Iba1-positive areas significantly increased in all of the brain regions examined (Fig. 4F).

Effects of Minocycline on Changes in the Intracellular Localization of KLK6 in Oligodendrocytes During Systemic Inflammation
Minocycline has been shown to inhibit the activation of microglia and expression of inflammatory cytokines by microglia [25]. Minocycline was used in the present study to assess the effects of microglial activation on changes in KLK6 expression in the spinal cord. Mice were pretreated with minocycline or saline for two days, followed by daily The experimental paradigm is shown in Fig. 5A. The treatment with minocycline alone did not affect KLK6 or Iba1 immunoreactivity. The LPS injection alone affected changes in the intracellular localization of KLK6 and the morphology of microglia, as shown above. Minocycline suppressed both of these changes (Fig. 5B, C). The quantitative analysis revealed that the KLK6-positive area in the anterior funiculus of the spinal cord was significantly larger in the LPS  and Iba1-positive (F) areas in brain white matter after the vehicle or LPS injection (n = 4 mice per group). Data are expressed as the mean (± SE). * P < 0.05, ** P < 0.01, and *** P < 0.001 vs vehicle by the Student's t-test group than in the vehicle group. In contrast, the KLK6-positive area was significantly smaller in the minocycline and LPS group than in the LPS group (Fig. 5D). As expected, minocycline abolished the LPS-induced increase in the Iba1positive area (Fig. 5E).

Systemic Inflammation Elongates Node Lengths in Spinal Cord White Matter, Which is Rescued in KLK6-KO Mice
Previous studies reported that structural changes in the nodal region is associated with several pathological conditions, including MS, meningeal inflammation, spinal cord injury and ageing [26-28]. Although demyelination was not observed in the experimental period (up to 20 days), KLK6 may induce the structural changes of nodal regions, which have been shown to associate demyelination pathology [26-28]. To examine the changes of nodal regions, we performed double fluorescent immunohistochemistry with paranode and juxtaparanode markers. Each length was defined as follows: paranode lengths are Caspr-positive regions, juxtaparanode lengths are Kv1.2-positive regions, and node lengths are areas located between Caspr (Fig. 6A). To study the function of KLK6, we used KLK6-KO mice. We confirmed that expression of KLK6 protein was not detected in the spinal cord by immunohistochemistry in both the vehicle and LPS groups in the KLK6-KO mice (see Supplementary material). No significant differences in the Iba1 area were found in the KO LPS group in comparison with the WT LPS group (see Supplementary material). Typical images are shown in Fig. 6B. Node lengths were significantly longer in LPS-treated WT mice than in the vehicle group, whereas no significant changes were observed in KLK6-KO mice (Fig. 6C). Paranode lengths were significantly longer in KLK6-KO mice treated with LPS than in WT mice treated with LPS (Fig. 6D). No significant differences were noted in juxtaparanode lengths between the groups examined (Fig. 6E). To clarify whether microglia were involved in changes in node length, minocycline was used to assess the effects of microglial activation on changes in node length in the WT mice. The experimental paradigm is the same as in Fig. 5A. Typical images are shown in Fig. 6F. The treatment with minocycline alone did not affect node (Fig. 6G), paranode (Fig. 6H) or juxtaparanode length (Fig. 6I). The quantitative analysis revealed that the node length was significantly smaller in the minocycline and LPS group than in the LPS group (Fig. 5G).

Discussion
The present study revealed that LPS-induced systemic inflammation induced the paranodal expression of KLK6 in oligodendrocytes, and is the first to report a demyelination-associated molecule that changes its localization from the cell body to the paranodes. In addition, we observed region-specific heterogeneity in the CNS with changes in the intracellular localization of KLK6 within oligodendrocytes during LPS-induced systemic inflammation. The expression of KLK6 in paranodes in the spinal cord white matter during systemic inflammation was observed except in the cervical segments. In the brain white matter, by contrast, the expression of KLK6 in paranodes during systemic inflammation was observed only in the sp5. Previous studies reported that anatomical differences exist between the cervical and other spinal cord segments resulting in different pathophysiological responses to inflammation. For instance, the cervical spine has increased central and peripheral vascular supply and flow and a higher gray-white matter ratio [29,30]. Besides, differences in tight junction proteins, adherens junction proteins, and transporter molecules between the BBB and BSCB of endothelial cells may make BSCB more permeable than BBB [31-33]. Another series of experiments showed regional differences in the permeability of the spinal cord to interferon-α, IFN-γ, and TNF-α between BBB and BSCB [34]. In EAE, a close relationship was observed between the degree of BBB permeability and the severity of the disease [35]. Therefore, differences in the molecular composition of barriers and permeability between BBB and BSCB may affect neuroinflammatory responses.
The sp5 (the spinal trigeminal tract) is a tract composed of neuronal axons rerated to pain, tactile sense and thermal sensations. Previous study reported that the trigeminal neurons are capable of detecting pathogenic bacterial components leading to sensitization of TRPV1, possibly contributing to the inflammatory pain often observed in bacterial infections [36]. Besides, in the sp5, the expression level of KLK6 in the control group may be higher than in other brain regions (see Fig. 4A). This indicates that the properties of oligodendrocytes in response to inflammation may differ from other brain white matter.
Developmental studies showed that oligodendrocyte progenitors arise from distinct ventral and dorsal domains within the ventricular germinal zones of the embryonic CNS. The responsiveness of oligodendrocyte progenitors to demyelination and its contribution to remyelination differ depending on their site of origin [37]. Furthermore, mature 1 3 oligodendrocyte subpopulations have a spatial preference in the CNS and differential susceptibility to traumatic spinal cord injury and EAE [38,39]. Thus, anatomical differences, differences in neuronal activity in response to inflammation, differences in the molecular composition of barriers and permeability between BBB and BSCB and mature oligodendrocyte subpopulations may result in region-heterogeneity of KLK6 expression dynamics.
In the present study, an analysis of time-dependent changes after daily i.p. injections with 1 mg/kg LPS showed that KLK6-positive areas in the spinal cord were 1.7-fold larger on day 3 and 1.9-fold larger on day 4 than those in the vehicle group. Similarly, Iba1-positive areas, which may represent a change in the activation state of microglia due to inflammation, were 3.4-fold larger on day 3 and 2.5-fold larger on day 4 in the LPS group than in the vehicle group. Furthermore, experiments using minocycline, an inhibitor of microglial activation, canceled LPS-induced increases in KLK6-positive areas.
In the normal condition, peripheral LPS cannot cross the BBB and BSCB [40,41]. Other studies demonstrating that systemic cytokines such as TNF-α and IL-1β mediate the effects of peripheral inflammation on the CNS. Blood TNFα, for example, cross the BBB and induce further release of TNF-α from other CNS sources such as microglia [24,42,43]. Thus, in present study, peripheral inflammation activates CNS microglia, which may result in change in the intracellular localization of KLK6 within oligodendrocytes.
Morphological change in microglia at 3-4 days after daily injections of LPS is consistent with previous reports that multiple intraperitoneal challenges with LPS showed microglial activation in mice and rats [24,44]. The microglial activation returned to normal by day 9 of the daily LPS administration reported here is similar to previous studies [44]. Microglia are active and dynamic cells that respond quickly to changes in the CNS microenvironment. The phenotype of microglia changes during de-and remyelination [4]. An i.p. injection of LPS elicits a rapid innate immune response. Activated microglia are associated with demyelination and axonal damage through the release of proinflammatory cytokines may be involved in oligodendrocyte damage and demyelination [45]. In cerebellar cultures, microglia activated by LPS released pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α), increased the expression of inducible nitric oxide synthase and production of reactive oxygen species, and were associated with demyelination and axonal damage [46]. In addition to pro-inflammatory factors, stimulated microglia also produce IL-10, which is a potent anti-inflammatory cytokine. During early stages of neuroinflammation, only small quantities of IL-10 can be detected. Microglial production of IL-10 increases over time, and plays a role in a resolution of inflammation [47]. Moreover, previous in vivo study reported that attenuation of fever and release of cytokines after repeated injections of LPS [48]. Therefore, the results of this study that the loss of changes of microglial morphology and the KLK6 expression in the paranode by day 9 suggest that a regulatory response due to the longer duration of continuous LPS administration. The mechanisms connecting changes in the intracellular localization of KLK6 and microglial activation remain unclear. Recent studies suggest that oligodendroglia respond to TNF and IFNγ and can exert immunomodulatory functions, which are relevant in the context of neurodegeneration and demyelinating diseases [49]. Thus, such cytokine-mediated inflammatory responses may have induced changes in the intracellular localization of KLK6.
Our experiments show that the nodal lengths were elongated in LPS-treated WT mice and the elongation was not observed after minocycline treatment. Moreover, LPS-treated KO mice did not elongate the node length. In addition, our experiments show that there is a difference in paranode length between LPS-treated WT and LPS-treated KO mice. These observations suggest an important role for KLK6 in node and paranode length changes.
Previous studies suggested that lengthening in node length is due to myelin retraction and a breakdown of the paranodal junction, leading to a redistribution of Kv channels [50,51]. The present study showed no change in paranode length after LPS administration in WT mice. It is not possible to determine from present study the exact mechanism involved in the elongation of nodal lengths. The paranode length did not show change, while the node was elongated by LPS in WT mice. In some of conditions, however, it is possible that the node was elongated while maintaining intact paranodal and juxtaparanodal structures because there is an insertion of more axonal membrane at the node [28]. In the paranodes, several proteins, including Caspr, contactin, NF155, αII-and βII-spectrin, and ankyrin B, are known to be enriched and mediate paranode-axon interactions [52,53]. Myelin thickness and nodal lengths were previously shown to be reversibly altered by the thrombin-dependent proteolysis of NF155, a cell adhesion molecule that attaches myelin to axons [54]. KLK6 has been reported to degrade MBP, myelin oligodendrocyte glycoprotein, and several per group). Data are expressed as the mean (± SE). Asterisks in all panels indicate a significant difference between groups (*P < 0.05 and ** P < 0.01, an ANOVA with Tukey's post hoc tests). F Typical fluorescent immunolabeling images in each experimental group in the WT mice. Scale bar: 5 µm. G-I Graph showing nodal (G), paranodal (H), and juxtaparanodal (I) lengths (n = 4 mice per group). Data are expressed as the mean (± SE). Asterisks in all panels indicate a significant difference between groups (* P < 0.05 and ** P < 0.01, an ANOVA with Tukey's post hoc tests) extracellular matrix proteins, including laminin, collagen I, and fibronectin [16,55]. Furthermore, KLK6 has the potential to destroy nodes and related structural proteins by activating other proteases, such as MMP-9 [56]. The substrates of KLK6 will be further elucidated in the future. Therefore, KLK6 may regulate nodal lengths through direct or indirect proteolysis.
Further studies are needed to establish whether KLK6 expressed in the nodal region directly or indirectly disrupts nodes or related structural proteins. A more detailed understanding of the functional role and mechanisms underlying the dynamics of changes in the intracellular localization of KLK6 will contribute to the development of new therapies for demyelinating diseases.