Down-regulation of spinal cord LRRC4 during EAE pathogenesis
To determine if LRRC4 has a role in CNS autoimmunity, we induced EAE in C57BL/6 mice using myelin oligodendrocyte glycoprotein (MOG35-55) and subsequently determined the mRNA and protein levels of LRRC4 before (naive) or 15 days after immunization. LRRC4 mRNA expression in the spinal cords of the immunized mice was significantly down-regulated relative to the healthy mice, while little difference was detected in the brains (Figure 1A). We also found that the LRRC4 protein level was significantly reduced during the development of EAE in the spinal cords (Figures 1B, C). The down-regulation of LRRC4 during EAE pathogenesis indicated that LRRC4 may play a role in the process.
LRRC4 deletion leads to exacerbated EAE progression
The down-regulation of LRRC4 during EAE pathogenesis led us to assess the effect of LRRC4 deletion on EAE. We constructed mice with LRRC4 deletion (LRRC4-/-) and subsequently induced EAE in LRRC4-/- mice and the wild type (WT) littermates (Figures S1A-C). Consistent with the earlier report by Zhang et al, LRRC4-/- mice showed decreased threshold of auditory brainstem response (ABR) (Figures S1D, E), suggesting reduction in synchronization of auditory neurons in the spiral ganglia [40]. In addition, LRRC4-/- mice exhibited much more exacerbated disease development than WT mice with the difference peaking at day 16 (Figure 2A). Furthermore, LRRC4-/- mice showed accelerated loss of body mass (Figure 2B). Experiments with hematoxylin-eosin (H&E) staining of the spinal cords collected at day 15 after immunization revealed increased lymphocyte infiltration in the spinal cords of LRRC4-/- mice compared with those of WT mice (Figure 2C). We also used luxol fast blue staining and found more severe demyelination in LRRC4-/- mice than in WT mice. Finally, as shown by immunohistochemistry analysis with anti-Iba1 and GFAP antibodies, there was an increased density of microglia and astrocytes around demyelinated lesion sites in LRRC4-/- mice compared with WT mice. Thus, the loss of LRRC4 leads to aggravated demyelination and inflammation in EAE, implying a protective role for LRRC4 in EAE.
LRRC4 deletion disrupts the balance between Th1 and Treg cells
It is well established that helper T cells have a strong influence on the progression of EAE. We therefore examined whether LRRC4 deletion causes any alterations in the cell populations during EAE development. To do so, we measured the proportion of cytokine-producing cells in the spleen and blood from LRRC4-/- and WT mice (both naïve mice and mice with EAE induction) 15 days after immunization by using flow cytometry. Intracellular staining of IL-4, IL-17A and IFN-γ showed that LRRC4 deletion did not change the proportion of Th2 (CD4+ IL-4+) cells or Th17 (CD4+ IL-17A+) cells in the spleen and blood (Figures 3A, B) whether or not EAE occur. However, although LRRC4 deletion failed to change the proportion of Th1 (CD4+ IFN-γ+) cells in the spleen and blood of naïve mice, it increased the proportion of Th1 cells in the spleen and blood of EAE mice (Figure 3C). We next assessed the effect of LRRC4 deletion on regulatory T (Treg) cells, which reportedly play a critical role in the regulation of immune processes during EAE. Little difference in Treg cells (CD4+CD25+FoxP3+) was found between naïve LRRC4-/- and WT mice in the spleen. However, a marked reduction in Treg cells was seen in the spleen of LRRC4-/- EAE mice compared with WT EAE mice (Figure 3D). Thus, LRRC4 deletion disrupts the balance between Th1 and Treg cells in EAE and causes a shift to Th1 cells, which may contribute to EAE progression.
RNA-seq analysis revealed a role of rab7b in EAE
To obtain a more comprehensive understanding of the distinct molecular programs between WT mice and LRRC4−/− mice, we isolated the spinal cords from WT and LRRC4−/− mice before or 15 days after immunization and analyzed them by using RNA sequencing (RNA-seq). The top 50 genes in WT versus LRRC4−/− mice were selected for cluster analysis (Figure 4A). We performed GO and KEGG analyses to investigate the molecular function and biological pathways of the differentially expressed genes (DEGs). The top 10 enriched GO terms and KEGG pathways of the up-regulated and down-regulated DEGs, according to the percentage of genes, were selected (Figure 4). For instance, Figure 4B showed that cellular response to IFN-γ was among the enriched GO terms of up-regulated DEGs, consistent with earlier findings that IFN-γ−/− and IFN-γR−/− mice show more severe and chronic-progressive course of EAE [41]. We also compared common DEGs between the up-regulated and down-regulated DEGs, and as shown in Figure 4D and 4E, the number of shared DEGs in GO terms between up-regulated and down-regulated DEG was small. Among these DEGs between WT and LRRC4−/− mice, Rab7b was of special interest, because it was recently shown that Rab7b is involved in the regulation of phorbol 12-myristate 13-acetate (PMA)-induced activation of NF-κB and enhances the production of IL-6 [42] . We examined Rab7b expression in the spinal cords of WT and LRRC4−/− mice and found that both the mRNA and protein levels were elevated in LRRC4−/− mice compared with WT mice (Figures 4F, G). We also assessed Rab7b mRNA expression in the spinal cords of naïve mice and EAE mice and detected increased Rab7b mRNA expression in the spinal cords of EAE mice (Figure 4H). Furthermore, Rab7b mRNA levels correlated inversely with LRRC4 expression (Figure 4I). Thus, Rab7b expression in the spinal cords of EAE mice is elevated upon LRRC4 deletion, raising the possibility that Rab7b may be involved in EAE pathogenesis.
LRRC4 deletion up-regulates NF-κB in EAE mice
After showing that LRRC4 deletion caused elevated inflammatory responses in the spinal cords and exacerbation of EAE pathogenesis, we sought to dissect the underlying molecular mechanisms. LRRC4 has been reported to inhibit NF-κB activation and regulate the ERK/MAPK and PI3K/AKT pathway [32]. NF-κB plays an important role in controlling expression of genes including pro-inflammatory cytokines, chemokines, nitric oxide synthases and cell adhesion molecules related to the pathogenesis of autoimmunity [43]. The ERK/MAPK and PI3K/AKT signaling pathways reportedly modulate Tregs and Th17 cells differentiation, while the PI3K/AKT signaling pathway promotes oligodendrocyte differentiation and myelination [44]. The well established role of NF-κB, ERK/MAPK and PI3K/AKT signaling in inflammation and EAE pathogenesis led us to investigate whether they might play a role in mediating the function of LRRC4 in EAE.
We first assessed the levels of NF-κB, p-AKT and p-ERK1/2 in LRRC4-/- and WT mice under naïve and EAE conditions. Under naïve conditions, NF-κB p65 was up-regulated, while the ratio of p-AKT/AKT was reduced in the spinal cords of LRRC4-/- mice compared with that of WT mice (Figures 5A). After immunization, LRRC4-/- mice expressed higher levels of NF-κB p65 than WT mice, at clinical score 2 and 3 in the spinal cords (Figures 5B). The ratio of p-AKT/AKT decreased in spinal cords of LRRC4-/- mice at score 2 and 3 compared with that of WT mice. In contrast, the ratio of p-ERK/ERK exhibited little difference between WT and LRRC4-/- mice, while increasing at score 2 and decreasing at score 3 in spinal cords of LRRC4-/- mice. We also examined the levels of various cytokines. We found that the levels of IL-6 mRNA were elevated in the spinal cords of LRRC4-/- mice with different scores of EAE when compared with WT mice (Figures 5C). In addition, IFN-γ mRNA levels were enhanced at score 2 and 3 of EAE in spinal cords of LRRC4-/- mice. In contrast, IL-10 mRNA levels were reduced at the score 1 and 2 of EAE in spinal cords, while increasing at the score 3 in LRRC4-/- mice. Meanwhile, TNF-α mRNA levels were reduced at the score 1 and 2 in spinal cords, while TGF-β levels decreased at the score 2 of EAE spinal cords in LRRC4-/- mice. In contrast, IL-17A mRNA levels exhibited no significant difference in spinal cords between LRRC4-/- and WT mice. Thus, LRRC4 deletion induces an up-regulation of NF-κB, causing alterations in the levels of inflammation-related cytokines in the spinal cords, which may contribute to accelerated progression of EAE.
Rab7b mediates NF-κB up-regulation
The elevated Rab7b expression in LRRC4-/- mice susceptible to EAE led us to determine the molecular link between LRRC4, Rab7b and NF-κB. To do so, we isolated mouse neurons of WT and LRRC4-/- mice and subsequently determined Rab7b and NF-κB p65 expression. As expected, Rab7b and NF-κB p65 were up-regulated in LRRC4-/- neurons compared with LRRC4+/+ neurons (Figure 6A). Ectopic expression of LRRC4 with the pcDNA3.1-LRRC4 expression vector induced a reduction of Rab7b and NF-κB p65 expression (Figure 6B). RNAi-mediated knockdown of Rab7b in LRRC4+/+ neurons caused NF-κB p65 expression to reduce but not in LRRC4-/- neurons (Figure 6C). Ectopic expression of Rab7b had little effect on NF-κB p65 expression but strongly inhibited NF-κB p65 expression when Rab7b was co-transfected with LRRC4 (Figure 6D). Thus, Rab7b regulates NF-κB in the presence of LRRC4 but plays no such role in LRRC4-deficient neurons. These results suggest that Rab7b might serve as a downstream effector of LRRC4 in the regulation of NF-κB.
Ectopic LRRC4 expression alleviates EAE progression
Having shown that LRRC4 is down-regulated in EAE mice and that LRRC4 deletion leads to aggravated EAE progression, we asked whether ectopic LRRC4 expression could rescue the pathological defects of EAE. We injected adeno-associated virus (AAV) vector intravenously to ectopically express LRRC4 (AAV-LRRC4) or control virus vector (AAV-CON) in the mice and subsequently induced EAE with MOG35-55 10 days after injection. Clinical scores of EAE were measured and documented daily, showing that AAV-LRRC4 injection alleviated the progression of EAE and body mass loss compared with AAV-CON injection (Figures 7A, B). At the tissue level, AAV-LRRC4 injection caused lymphocyte infiltration into spinal cords to decrease in EAE mice, as revealed by H&E staining (Figure 7C). In addition, experiments with luxol fast blue (LFB) staining showed that overexpression of LRRC4 decreased demyelination in the spinal cords of EAE mice, while the density of microglia and astrocytes were also reduced around demyelinated lesion sites in the spinal cords of mice injected with AAV-LRRC4, as illustrated by immunohistochemical analysis using anti-Iba1 and GFAP antibody (Figure 7C). Thus, LRRC4 ectopic expression alleviates the defects in demyelination and autoimmunity caused by EAE. As such, AAV-LRRC4 virus may be potentially used as a therapeutic tool for treating MS patients.
Consistent with our earlier findings, NF-κB p65 was down-regulated in the spinal cords of mice injected with AAV-LRRC4. In contrast, the levels of p-ERK1/2 and p-AKT were unaltered in the spinal cords with AAV-LRRC4 injection, indicating that the down-regulation of NF-κB p65 was specific (Figure 7D). These results again suggested that NF-κB serves as a key downstream signaling molecule to mediate the function of LRRC4 in protecting mice from CNS autoimmunity.