Leucine rich repeats and calponin homology domain containing 1 inhibits microglia-mediated neuroinflammation in a rat traumatic spinal cord injury model

Background Spinal cord injury (SCI) triggers the primary mechanical injury and secondary inflammation-mediated injury. Neuroinflammation-mediated insult causes secondary and extensive neurological damage after SCI. Microglia play a pivotal role in the initiation and progression of post-SCI neuroinflammation. Methods To elucidate the significance of LRCH1 to microglial functions, we applied lentivirus-induced LRCH1 knockdown in primary microglia culture, and tested the role of LRCH1 in microglia-mediated inflammatory reaction both in vitro and in a rat SCI model. Results We found that LRCH1 was down-regulated in microglia after traumatic SCI. LRCH1 knockdown increased the production of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6 after in vitro priming with lipopolysaccharide and adenosine triphosphate. Furthermore, LRCH1 knockdown promoted the priming-induced microglial polarization towards the pro-inflammatory M1 type, as demonstrated by increased differentiation into inducible nitric oxide synthase (iNOS)+ microglia. LRCH1 knockdown also enhanced microglia-mediated N27 neuron death after priming. Further analysis revealed that LRCH1 knockdown increased priming-induced activation of p38 mitogen-activated protein kinase (MAPK) and Erk1/2 signaling, which are crucial for M1 polarization of microglia. When LRCH1-knockdown microglia were adoptively injected into rat spinal cords, they enhanced post-SCI production of pro-inflammatory cytokines, increased SCI-induced


Methods
To elucidate the significance of LRCH1 to microglial functions, we applied lentivirusinduced LRCH1 knockdown in primary microglia culture, and tested the role of LRCH1 in microglia-mediated inflammatory reaction both in vitro and in a rat SCI model.

Results
We found that LRCH1 was down-regulated in microglia after traumatic SCI. LRCH1 knockdown increased the production of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6 after in vitro priming with lipopolysaccharide and adenosine triphosphate. Furthermore, LRCH1 knockdown promoted the priming-induced microglial polarization towards the pro-inflammatory M1 type, as demonstrated by increased differentiation into inducible nitric oxide synthase (iNOS)+ microglia. LRCH1 knockdown also enhanced microglia-mediated N27 neuron death after priming. Further analysis revealed that LRCH1 knockdown increased priminginduced activation of p38 mitogen-activated protein kinase (MAPK) and Erk1/2 signaling, which are crucial for M1 polarization of microglia. When LRCH1knockdown microglia were adoptively injected into rat spinal cords, they enhanced post-SCI production of pro-inflammatory cytokines, increased SCI-induced 4 recruitment of leukocytes, aggravated SCI-induced tissue damage and neuronal death, and worsened the locomotor function.

Conclusion
Our study reveals for the first time that LRCH1 serves as a negative regulator of microglia-mediated neuroinflammation after SCI, and provides clues for developing novel therapeutic approaches against SCI.

Background
Spinal cord injury (SCI) triggers the primary mechanical injury and secondary inflammation-mediated injury [1]. The mechanical trauma of the spinal cord tissue initiates the primary injury, while the secondary neuroinflammatory reactions, which mediate additional and extensive neurological injury, takes place following the primary injury. Controlling the detrimental acute neuroinflammation could be a therapeutic strategy to confine the injury and promote functional recovery.
Microglia, a spinal cord-resident immune cell type, play a pivotal role in the post-SCI secondary injury. Microglia are usually located in the margins of the lesion core [2].
It is considered that classical activation microglia are neurotoxic and growth inhibitory through producing high levels of pro-inflammatory molecules [2,3].
However, microglia may exert anti-inflammatory and neuroprotective effects on injured spinal cords [4,5]. The exact role of microglia might change over time in different stages of SCI. Carefully tuning the microglia function is necessary to treat SCI to achieve better recovery.
Leucine rich repeats and calponin homology domain containing 1 (LRCH1) is a relatively novel gene encodes a protein with a leucine-rich repeat and a calponin homology domain. The significance of LRCH1 protein remains a mystery. Previous 5 studies suggest that a genetic variant in LRCH1 is associated with knee osteoarthritis [6,7]. A recent study indicates that LRCH1 act to restrain PKCαdedicator of cytokinesis 8 (DOCK8)-Cdc42 module-mediated T cell migration in experimental autoimmune encephalomyelitis, through competing with Cdc42 for interaction with DOCK8. However, no further study has been conducted to show the functions of LRCH1 in other cell types or on other signal pathways.
In the current research, we found that LRCH1 was down-regulated in microglia after traumatic SCI. To elucidate the significance of LRCH1 in microglial functions, we applied lentivirus-induced LRCH1 knockdown in primary microglia. Our study reveals for the first time that LRCH1 serves as a negative regulator of microglia-mediated neuroinflammation after SCI, and provides clues for developing novel therapeutic approaches against SCI.

Rat SCI model
The animal study was approved by the Animal Care and Use Committee of the Second Affiliated Hospital of Fujian Medical University and Shenzhen Pingle Orthopedic Hospital. The surgical procedures were conducted in compliance with the institutional guidelines for laboratory animal usage in neuroscience and behavioral research. Male Sprague-Dawley rats (10-week old, 250~300g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and were housed in the pathogen-free condition.
The SCI model was established by the following steps. Rats were anesthetized via inhaling 3% of isoflurane at the flow rate of 1 L/min. Midline skin incisions were cut and the T12 spinous processes were exposed. A laminectomy was performed at T12. 6 The compression was conducted by placing the base of a compression platform (area 2×5 mm 2 ) onto the exposed spinal cord. A 50-g weight was then placed steadily to the platform for 5 min. The platform was then removed and the muscles and skins were sutured. Rats were transferred to the cages after they regained the righting reflex. The urinary retention was relieved by twice-daily bladder expressions. The sham-operated rats received every surgical step except for the spinal cord compression.

Immune cell enrichment from spinal cords
Rats were anesthetized by inhalation of 3% isoflurane. Each rat was transcardially

Flow Cytometry and cell sorting
The following fluorophore-conjugated antibodies were purchased from Biolegend: Abcam. For cell surface marker staining, cells were stained with 2 µg/ml each antibody on ice for 30 minutes. Dead cells were excluded by staining with 1 µg/ml propidium iodide (PI, ebioscience). For intracellular staining, cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 90% ice-cold methanol for 30 minutes, followed by incubation with 1:100 diluted anti-Arginase-1 antibody and 10 µg/ml anti-iNOS antibody for 1 hour at room temperature. After three washes with PBS, cells were incubated with 1 µg/ml Alexa Fluor® 594 goat anti-rabbit IgG

Primary rat microglia culture
Primary microglia were obtained by isolation from mixed glial cell cultures of 1-day-8 old neonatal rat brains, according to previous publications [8][9][10]. Briefly, after removing the meninges, the cerebral cortices were incubated in 0.25% trypsin-EDTA (Invitrogen) for 30 minutes at 37 °C while shaking at 50 rpm on an orbital shaker.
The cortices were then mechanically dissociated in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS and 200 U/ml DNase I until no tissue clump was seen.
The whole cortical cells were passed through a 70-μm cell strainer and washed with DMEM once. The cells were cultured in DMEM supplemented with 10% FBS and the medium was replaced every 3 days. Two weeks later, the culture was mildly trypsinized with 1:4 diluted 0.25% trypsin-EDTA for 20 minutes at room temperature. The floating cells were carefully aspirated and microglia which remained attaching to the bottom were kept for further experiments.

Lentiviral packaging and transduction
Lrch1-set siRNA/shRNA/RNAi Lentivector (i050632) and the corresponding control lentivector piLenti-siRNA-GFP were purchased from abmgood Inc. The packaging was conducted using the Ecotropic Lentiviral Packaging System (VPK-205, Cell Biolabs, Inc.) according to the vendor's protocol. The lentiviruses were purified with Lenti-X™ Maxi Purification Kit (Clontech). The viral titer was determined by Neuronbiotech Company. The lentivirus containing the LRCH1 shRNA sequence was termed "LL" while the control virus was termed "LC".
Primary microglia were cultured at the density of 2×10 6 cells/ml in DMEM supplemented with 10% FBS, 4 mM L-glutamine, 50 µg/ml penicillin/streptomycin, and in the presence of 6 μg/ml polybrene (Thermo Fisher Scientific) in 48-well plates. Lentiviral particles were added into microglia culture at the MOI of 20 and incubated overnight. The next morning the medium was then replaced with fresh medium. Cells were incubated in fresh medium for 2 days, followed by incubation 9 with 2 μg/ml puromycin (Sigma-Aldrich) for 4 days.

Adoptive transfer of microglia
The adoptive transfer procedure was conducted based on established approaches with several modifications [5,11,12]. Briefly, immediately before SCI, 1×10 6 lentivirus-infected microglia in 5 μl of 0.9% NaCl solution was injected into the SCI area (epicenter) at a depth of 1 mm at the rate of 0.2 μl/min, using a 5-μl microsyringe with a 33-gauge Hamilton needle. Rats in the vehicle group received the same amount of saline. After injection, the injectors were removed and muscles and skins were sutured in separate layers. Hematoxylin and eosin (H&E) stainingRats were transcardially perfused with ice-cold PBS followed by cold 4% paraformaldehyde (PFA). Spinal cords were then fixed in 4% PFA for 16 hours. The next day, spinal cords were immersed in 30% sucrose-PBS for three days. Spinal cords were then embedded in paraffin. Five-micron thick cross sections were prepared and stained following the standard H&E staining protocol.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)
The spinal cord sections were labeled with the DeadEnd™ Fluorometric TUNEL System (Promega) following the manufacturer's manual.

Neurologic evaluation
The hindlimb locomotor function was evaluated before and 1, 3, 7, 14 and 21 days after SCI using the BBB locomotor test developed by Basso et al. [13]. The hindlimb movements during locomotion were quantified using a scale ranging from 0 to 21.
The rats were observed for 5 minutes at each time point by two observers who were blinded to the study.

Statistical analysis
Experiments were independently conducted three times unless specified, with 6 to 10 different samples in each group. Data were shown as mean ± standard deviation and were analyzed by GraphPad Prism 7.0. Student's t-test or one-way ANOVA was used to compare the mean values among the groups. P-value <0.05 was considered significant.

LRCH1 is down-regulated in post-SCI microglia
To determine LRCH1 expression in microglia after SCI, we enriched Granulocyte -CD45 low CD11b + spinal cord microglia from sham-operated rats and SCI rats ( Figure   1A). The LRCH1 mRNA quantity was determined by qRT-PCR and we found that LRCH1 mRNA was decreased in microglia in a time-dependent manner after SCI ( Figure 1B). LRCH1 protein underwent similar decreases on day 2, day 4 and day 7 after SCI ( Figure 1C).

LRCH1 knockdown elevates the production of pro-inflammatory cytokines
One of the crucial roles of post-SCI microglia is to produce pro-inflammatory cytokines in the acute stage [2]. To ascertain whether LRCH1 is involved in microglia-mediated neuroinflammation, we infected rat primary spinal cord microglia with a lentivirus that expresses both LRCH1 shRNA and GFP, followed by puromycin selection. The infection efficiency reached approximately 90% after puromycin selection, as indicated by the proportion of GFP + microglia on day 6 after infection ( Figure 2A). As shown in Figure 2B, LRCH1 protein was drastically reduced in microglia infected with shRNA-encoding lentivirus (hereinafter LL), in comparison to microglia infected with the control lentivirus (hereinafter LC). LRCH1 knockdown had no remarkable impact on microglia survival or cytokine transcription under the in vitro steady state ( Figure 2C and 2D). These microglia were then primed with LPS plus ATP into a pro-inflammatory state. The priming did not alter LRCH1 transcription in either LC-infected or LL-infected microglia ( Figure 2E). The analysis of cytokines in the culture supernatants revealed that without priming, LL-infected microglia and LC-infected microglia secreted comparable traces of IL-1β and TNF-α ( Figure 2F and 2G). After priming, both groups had dramatically high levels of IL-1β and TNF-α in the supernatants, whereas LL-infected microglia produced even more IL-1β and TNF-α than LC-infected microglia ( Figure 2F and 2G). With regard to supernatant IL-6, no matter the priming was conducted or not, LL-infected microglia always secreted more IL-6 than LC-infected microglia ( Figure 2H). To investigate whether the above changes of supernatant cytokines were attributed to an overall down-regulation of pro-inflammatory cytokine expression or deficient cytokine exocytosis, the microglia were lysed in a non-denaturing lysis buffer, and the lysates were mixed with the supernatants. The cytokines, including both mature and immature forms, were determined in the lysate-supernatant mixtures. It turned out that LL-infected microglia produced significantly more pro-inflammatory cytokines than LC-infected microglia ( Figure 2I).

LRCH1 knockdown facilitates microglial M1 polarization in vitro
To further understand the significance of LRCH1, we analyzed the microglial polarization towards M1 or M2 type by staining the M1 marker iNOS and M2 marker Arginase-1. As indicated in Figure 3, without priming, Arginase-1 + population was predominant in both LC-infected microglia and LL-infected microglia. However, there were more iNOS + cells in unprimed LL-infected microglia than that in unprimed LCinfected microglia. After priming, iNOS + cells were increased and Arginase-1 + cells were decreased in both LC-infected microglia and LL-infected microglia. However, primed LL-infected microglia possessed more iNOS + cells and less Arginase-1 + cells than primed LC-infected microglia. Therefore, LRCH1 is a negative regulator for microglial M1 polarization.
To test if LRCH1 is crucial for this process, we co-cultured unprimed or primed microglia with the N27 rat dopaminergic neural cell line for 24 hours. As indicated in Figure 4, primed microglia substantially induced N27 cell apoptosis and necrosis, and primed LL-infected microglia induced more apoptosis and necrosis of N27 cells than primed LC-infected microglia.

LRCH1 knockdown promotes the activation of p38 MAPK and Erk1/2
To find out the signal pathways that are responsible for the effect of LRCH1, we analyzed the MAPK pathways because they are critical to LPS-mediated proinflammatory activation of microglia [18,19]. As shown in Figure 5, under unprimed condition, the activating phosphorylation of p38 MAPK, Erk1/2 and JNK was comparable in LC-infected microglia and LL-infected microglia. However, after priming, we observed higher phosphorylation of p38 MAPK and Erk1/2 in LL-infected microglia as compared with LC-infected microglia. The JNK phosphorylation showed no significant difference in LC-infected microglia and LL-infected microglia after priming.

LRCH1 knockdown in microglia increases post-SCI leukocyte recruitment in vivo
To elucidate the effect of LRCH1 in vivo, we microinjected LC-infected microglia or LL-infected microglia into the T12 spinal cord of each normal rat before SCI was conducted on these recipient rats. At indicated time points after SCI, the T12 spinal cord was taken and the injected microglia and infiltrating leukocytes were isolated. Figure 6A and 6C, on day 3 after SCI, GFP + transferred microglia were present in the spinal cords, and the frequencies of LC-infected microglia and LL-infected microglia were comparable in the recipient spinal cords. After SCI, in the spinal cords injected with LL-infected microglia, there were more infiltrating TCR αβ + αβT cells, TCR γδ + γδT cells, and Granulocyte + neutrophils as compared with the spinal cords injected with LC-infected microglia ( Figure 6A, 6B and 6D).

As indicated in
Furthermore, we retrieved the transferred microglia from the recipient spinal cords and evaluated the expression of pro-inflammatory cytokines. We found that after SCI, LL-infected microglia expressed higher IL-1β, TNF-α, and IL-6 than LC-infected microglia in the spinal cords ( Figure 6E).

LRCH1 knockdown in microglia aggravated spinal cord damage and function
About the post-SCI lesion, the H&E staining displayed more extensive and severe tissue damage in the spinal cords injected with LL-infected microglia, as compared with the spinal cords injected with LC-infected microglia ( Figure 7A). After SCI, the spinal cords injected with LL-infected microglia also had more apoptotic neurons than the spinal cords injected with LC-infected microglia ( Figure 7B and 7C).
Moreover, the rats receiving LL-infected microglia showed a lower BBB score than the rats receiving LC-infected microglia from day 7 to day 21 after SCI, suggesting that LRCH1 knockdown aggravated post-SCI locomotor function impairment ( Figure   7D). Furthermore, the overall levels of IL-1β, TNF-α, and IL-6 were remarkably higher in the spinal cords injected with LL-infected microglia, in comparison to those in the spinal cords injected with LC-infected microglia ( Figure 7E).

Discussion
Microglia are important effector cells in the injured spinal cord tissue. In the secondary phase of SCI, inflammation is the most important mechanism that directly or indirectly controls the sequelae after SCI. It is reported that microglia turn into M1 type (classical activation) shortly after SCI and secrete pro-inflammatory cytokines and chemokines which mediate secondary damage following mechanical injury [20]. However, depending on the spinal cord microenvironment, microglia can also be protective after SCI, through polarization towards M2 type (alternative activation) to secrete anti-inflammatory cytokines and chemokines lead to the suppression of excessive inflammatory responses [2]. Therefore, unveiling the mechanisms by which microglial polarization is regulated is important for understanding the pathogenesis of SCI, and for developing therapeutic interventions against post-SCI secondary damage.
Our study discloses for the first time the effect of LRCH1 on microglia function. We Our study demonstrates that LRCH1 is an inhibitory factor of microglia-mediated inflammation both in vitro and in vivo. Using the RNA interference method, we showed that LRCH1 alleviated the production of pro-inflammatory cytokines in LPSand-ATP-primed microglia, likely through inhibiting the polarization into M1 type.
It was recently reported that LRCH1 competes with Cdc42 for interaction with DOCK8 and restrains T cell migration in experimental autoimmune encephalomyelitis [7]. It is also reported that DOCK8 modulates macrophage migration through Cdc42 activation [30]. Hence, it is very likely that LRCH1 regulates microglia migration or motility under normal or pathological conditions.
Although in the present study we did not pay much attention to macroglia migration, it will be tempting to investigate the role of LRCH1 in microglia migration, phagocytosis, and secretion of other neurotoxic or neuroprotective mediators. Meanwhile, we will continue to explore the effect of LRCH1 in the M2 polarization of microglia in the chronic stage of SCI to elucidate the significance of LRCH1 for the resolution of neuroinflammation and tissue recovery after SCI.
Moreover, Cdc42 has been shown to activate MAPK signaling in certain cell types [31,32], so perhaps Cdc42 exerts the same effect on MAPK activation in microglia, and LRCH1 blocks Cdc42 activation and subsequently inhibits MAPK activation. Our future studies will test this hypothesis. Supplementary information.docx