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 power-magnification 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 Klk6-positive 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 [24]. We hypothesized that Klk6 may be expressed in the paranodes and, thus, performed immunostaining with the paranode marker Caspr. In the vehicle group, Klk6-positive structures were devoid of Caspr-positive paranodes. In the LPS group, Klk6-positive scattered structures were also positive for Caspr, indicating that they were paranodes (Fig. 1H-H”, I-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, which may then induce changes in the localization of Klk6. Therefore, 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 [25] (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 Klk6-positive 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 [26]. 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 i.p. injections of saline, minocycline, 1 mg/kg LPS, or a mixture of minocycline and LPS for 4 consecutive days. 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 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 Iba1-positive area (Fig. 5E).
Systemic inflammation elongates node lengths in spinal cord white matter, which is rescued in Klk6-KO mice
LPS induces oligodendrocyte damage through the activation of innate immune responses in microglia and decreases in MBP expression in oligodendrocytes, leading to demyelination [8, 11]. Therefore, to investigate the effects of Klk6 expression in paranodes during systemic inflammation in nodal regions, we used double fluorescent immunohistochemistry with paranode and juxtaparanode markers to examine structural changes around the nodes. 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). 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. 6B). Paranode lengths were significantly longer in Klk6-KO mice treated with LPS than in WT mice treated with LPS (Fig. 6C). No significant differences were noted in juxtaparanode lengths between the groups examined (Fig. 6D).