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. 2018 Oct 16;25(4):476–485. doi: 10.1111/cns.13074

Cathepsin B inhibition ameliorates leukocyte‐endothelial adhesion in the BTBR mouse model of autism

Huan Wang 1,2, Yi‐Xuan Yin 2,3, Dong‐Mei Gong 2,3, Ling‐Juan Hong 2, Gang Wu 2, Quan Jiang 2, Cheng‐Kun Wang 2, Pablo Blinder 4, Sen Long 2,5, Feng Han 2,6,✉, Ying‐Mei Lu 3,✉
PMCID: PMC6488924  PMID: 30328295

Summary

Aims

Autism spectrum disorder (ASD) is a wide range of neurodevelopmental disorders involving deficits in social interaction and communication. Unfortunately, autism remains a scientific and clinical challenge owing to the lack of understanding the cellular and molecular mechanisms underlying it. This study aimed to investigate the pathophysiological mechanism underlying leukocyte‐endothelial adhesion in autism‐related neurovascular inflammation.

Methods

Male BTBR T+tf/J mice were used as an autism model. The dynamic pattern of leukocyte‐endothelial adhesion in mouse cerebral vessels was detected by two‐photon laser scanning microscopy (TPLSM). Using FACS, RT‐PCR, and Western blotting, we explored the expression of cell adhesion molecules, the mRNA expression of endothelial chemokine, the protein levels of cathepsin B, and inflammatory mediators.

Results

We found a significant increase in leukocyte‐endothelial adhesion in BTBR mice, accompanied by elevated expression of the adhesion molecule neutrophils CD11b and endothelial ICAM‐1. Our data further indicate that elevated neutrophil cathepsin B levels contribute to elevated endothelial chemokine CXCL7 levels in BTBR mice. The pharmacological inhibition of cathepsin B reverses the enhanced leukocyte‐endothelial adhesion in the cerebral vessels of autistic mice.

Conclusion

Our results revealed the prominent role of cathepsin B in modulating leukocyte‐endothelial adhesion during autism‐related neurovascular inflammation and identified a promising novel approach for autism treatment.

Keywords: adhesion, autism, cathepsin B, inflammation, leukocyte‐endothelial

1. INTRODUCTION

Autism spectrum disorder (ASD) is a pervasive disorder that broadly affects cognitive, social, and motor functioning.1 To date, the lack of efficient tools for diagnosing autism (except for the checklist of autism‐specific behavioral evaluation)2, 3 and the lack of an effective approach for medical therapy in a clinical setting make investigating the field of autism‐related neurological diseases an enormous challenge.1, 4, 5, 6, 7 Autism is arguably caused by a multifaceted combination of genetic, epigenetic, and environmental neurotoxicants as well as in utero risk factors.8, 9 This has attracted surging interest in exploring the ongoing pathological events and the one or more mechanisms underlying it. Accumulating clinical evidence suggests that notable neuroinflammation and microglial activation are observed in individuals with autistic‐like behaviors,2, 10, 11, 12, 13 and who might be suffering from neuronal malfunction associated with autism. However, a molecular understanding of these pathological events and processes remains unknown.14

Leukocyte attachment to the vascular endothelium is a hallmark of the inflammatory process; it facilitates transendothelial migration of leukocytes into the CNS and thereby elicits neuroinflammation in different brain diseases, as previously reported, for example, multiple sclerosis,15 atherosclerosis,16 and septic encephalopathy.17 In particular, the modulation pattern of leukocyte‐endothelium adhesion in autistic patients has remained controversial. Several studies have found reduced levels of adhesion molecules including the soluble platelet‐endothelial adhesion molecule‐1 (sPECAM‐1) and soluble platelet selectin (sP‐selectin), which are thought to largely contribute to the attachment of leukocytes to the endothelium, which in adults will lead to high‐functioning autism.18, 19, 20 By contrast, elevated levels of another group of adhesion molecules, tissue plasminogen activator (tPA) and E‐selectin, have also been reported in children with ASD.21 Until now, the precise role and mechanism underlying brain leukocyte‐endothelium adhesion in ASD‐related neurovascular inflammation remain largely uncharted.

The lysosomal cysteine protease cathepsin B has been implicated in pathological conditions such as tumor progression, neurodegenerative diseases, and autoimmune disorders.22 Cathepsin B plays one of the intricate roles in the NOD‐like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation; it represents a potential target for therapeutic intervention to ameliorate inflammation.23 Previous research indicated that the specific inhibition of cathepsin B successfully reduced aberrant inflammatory responses in a mouse model for autoimmunity and ischemia.24, 25, 26, 27 It would be interesting to better understand the regulatory pattern of cathepsin B activity during the inflammatory process involving leukocyte and endothelium interactions in autism.

We therefore investigated the pathophysiological relevance of induced autism‐related leukocyte‐endothelial cell adhesion and neurovascular inflammation in a mouse model of ASD, using an inbred mouse strain, BTBR T+tf/J (herein referred to as BTBR). The BTBR mice recapitulated well the key features of autistic‐like behaviors including relatively more grooming, less reciprocal social interaction, and different ultrasonic vocalizations, which may be relevant to autism.28, 29, 30, 31 Here, we defined the regulatory role of cathepsin B in the neurovascular inflammation of BTBR mice in vivo, whereby blockade of cathepsin B abolished enhanced leukocyte‐endothelial adhesion during autism‐related neurovascular inflammation, thus providing a rational framework for developing new anti‐autism drugs.

2. METHODS

2.1. Animals

Eight‐ to ten‐week‐old male adult BTBR T+tf/J (BTBR) mice were obtained from the Model Animal Research Center of Nanjing University.32 Additionally, adult male wild‐type C57BL/6J (C57) mice were used as controls in the present study. A total of 83 mice (n = 4‐5 animals/group/experiment) with 22‐25 g average body weight were divided into four groups: (a) C57 (n = 35 mice), (b) BTBR (n = 22 mice), (c) BTBR+saline (n = 13 mice), and (d) BTBR+CA‐074Me (n = 13 mice). All animals were group‐housed under standard conditions with a 12/12‐hours light/dark cycle and supplied with commercial food pellets and water ad libitum. The animal studies were approved by the Zhejiang University Committees for Animal Experiments in China and conformed to the NIH Guide for the Care and Use of Laboratory Animals (NIH publication no. 85‐23, revised 1996).

2.2. Drug treatment

Adult male BTBR mice were treated with cathepsin B inhibitor (CA‐074Me) or saline via osmotic minipumps (Alzet model 1002, small pumps: SP). An osmotic minipump was inserted into the lateral ventricle of the brain (icv) of each animal as reported.33, 34 CA‐074Me was administered at 1 mg/mL in saline with 1.5% Me2SO (Sigma) at a rate of 0.2 μL/h, equivalent to an estimated dose rate of 0.013mg/day‐g brain weight.33 Animals received a continuous infusion of CA‐074Me or carrier solution (saline with 1.5% Me2SO) for 7 days and were then evaluated in all subsequent experiments.

2.3. Preparation of mice for TPLSM in vivo

Mice were anesthetized using chloral hydrate (400 mg/kg body weight ip) and subjected to in vivo imaging. A craniotomy window, centered by stereotaxic coordinates (2.5 mm later bregma, 2.5 mm lateral),17, 35 was prepared for studying leukocyte adhesion. The custom‐made metal frame (1 cm diameter), with a removable GOMN glass lid, was carefully fixed to the skull using glue and dental cement.17 Low‐melting agarose (1.5%, w/v) in an artificial cerebrospinal fluid configuration was used to fill the exposed brain surface and the glass cover.

2.4. TPLSM imaging and analysis

A two‐photon confocal microscope (Olympus, Ltd., Tokyo, Japan, BX61W1‐FV1000), equipped with a femtosecond Ti:Sa laser excitation source and Spectra‐Physics MaiTai HP DeepSee, was used to acquire a stacked or single‐focal‐plane two‐photon image. A long working distance (2 mm) water immersion objective (×25, NA 1.05) was used to measure leukocyte movement in mouse brain vessels. Intravenous bolus injection of rhodamine 6G solution (50 μL, 0.01%, Sigma‐Aldrich) was used for the in vivo leukocyte adhesion assay.17 By adjusting the XY axis manually and the optimal observation area containing rhodamine 6G labeling, adherent leukocytes were detected for each animal group. Subsequently, each area was scanned at a magnification (water immersion objective 25 × with or without 2 × zoom) by manually setting the XYT stacks. XYT stacks were typically collected within a scan field of 12‐bit depth at 1024 × 1024 pixel resolution, a scanning speed of 2 μs/pixel, and an image acquisition time of 1 minute, in total.17, 36 We defined “adherent” leukocytes as those leukocytes firmly attached to the endothelium and those remain in place for more than 1 minute. We selected 4‐5 cortical vessels (at least five areas) with a diameter of 20‐40 μm and a depth of 150 μm below the cortical surface for two‐photon imaging for each mouse. The blood vessel imaging and leukocyte adhesion quantitative analysis were carried out as previously described.17, 36 We measured the leukocyte rolling velocity for leukocytes entering the distance of view by transillumination intravital microscopy. The diameter of cortical brain vessels was measured or determined manually using ImageJ (NIH, Bethesda, MD).

2.5. Neutrophil isolation

The neutrophils were isolated from the peripheral blood of mice following a protocol previously described.37 Briefly, blood was collected from the retro‐orbital vein and diluted in pre‐cooled K2‐ethylenediaminetetraacetic acid (EDTA) with an equal volume of PBS. Diluted blood (5 mL) was plated on a discontinuous gradient of 3 mL Histopaque 1119 and 3 mL Histopaque 1077 (Sigma‐Aldrich). Following centrifugation (35 minutes at 700 g), the neutrophil layer between the Histopaque 1077 and Histopaque 1119 was collected and washed in PBS. RBC lysis (BD Biosciences) was performed as above. Neutrophils were washed, counted, and resuspended according to their experimental condition.

2.6. Cathepsin B activity assays

The cathepsin B activity of neutrophil lysates was measured using an assay kit from BioVision (Mountain View, CA).38 This kit is a fluorescence‐based assay that utilizes the preferred cathepsin B substrate sequence labeled with amino‐4‐trifluoromethylcoumarin (AFC). Neutrophil lysates were incubated with the cathepsin B substrate of the assay kit for 1 hour at 37°C, and the fluorescence intensity at 505 nm was measured by a fluorescence microplate reader (Beckman Coulter Inc., Brea, CA, USA).

2.7. Cell preparation and flow cytometry analyses

We collected peripheral blood from mice before decapitation and used it for flow cytometry as described previously.17 Endothelial cells of mouse brain were isolated as described.17 Briefly, the cortex of the mouse brain was digested with freshly prepared Dulbecco's modified Eagle's medium (DMEM) containing 5 mmol/L Ca2+, 10 mmol/L HEPES, 10 mg/mL DNase I (Roche), and 400 U/mL collagenase (Sigma). The solution was centrifuged (1000 × g, 5 minutes); the pellet was then resuspended in DMEM solution containing bovine serum albumin (20% w/v, BSA, Roche), followed by further centrifugation (1000 g for 20 minutes). Cell suspensions from each digestion were pooled and filtered through a 100‐μm strainer (BD Falcon, San Jose, CA,). The enriched endothelial cells at the bottom were collected for further experiments. For the flow cytometry experiments,17 cells were resuspended in buffer containing 1% BSA in PBS and incubated for 30 minutes in the dark at 4°C with antibodies (eBioscience and BD Pharmingen) including PE‐CD11b(M1/70), APC‐Ly6G (RB6‐8c5), PE‐CD54 (KAT‐1), and APC‐CD31(390). We used isotype‐matched control antibodies as negative controls. Flow data were analyzed by FlowJo 7.6 (Tree Star Inc., Ashland, OR, USA) software.

2.8. Western blotting

Neutrophils were isolated as described previously in Section 2.5. The total brain and microvessel protein extracts from the mouse cerebral cortex were prepared for Western blotting.39 Protein samples were loaded onto 10% acrylamide denaturing gels and transferred to PVDF membrane for 1 hour at 50 V. Membranes were incubated with antibodies against Caspase‐1 (1:2000; sc‐514, Santa Cruz Biotechnology), Cleaved IL‐1β (1:2000; sc‐23460, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Cathepsin B (1:2000; ab58802, Abcam), and β‐actin (1:5000; Sigma Chemical Co., USA). Bands were visualized by enhanced chemiluminescence (Biological Industries, Beit HaEmek, Israel). We analyzed the band intensities with ImageJ and normalized them against the β‐actin band used as a loading control.

2.9. RNA isolation and real‐time quantitative RT‐PCR

Mouse brain cortex endothelial cells were isolated as previously described.17 Tissues were homogenized, and RNA was extracted using RNAiso Plus (Takara, Shiga, Japan). We synthesized cDNA by reverse transcribing 500 ng of total RNA using a reagent (Kit PrimeScript™ RT) (Takara, Shiga, Japan) according to the manufacturer's instructions. mRNA expression of chemokine was determined by real‐time quantitative RT‐PCR. The qRT‐PCRs were measured in 96‐well plates with a Mastercycler® ep realplex (Eppendorf, Hamburger, Germany) with SYBR® Premix Ex Taq ™ (Takara, Shiga, Japan). PCR cycling conditions were 2 minutes at 95°C, followed by 40 cycles, each of which consisted of 15 seconds at 95°C, 15 seconds at 55°C, and 25 seconds at 68°C. The melting curve was analyzed using the default settings on the instrument, ranging from 50°C to 85°C. The Ct values were calculated using Mastercycler® ep realplex. The details of primer sequences are provided in Table S1.

2.10. Statistics

The two group data comparisons were analyzed with a two‐tailed unpaired t test (parametric data), and multi‐group comparisons were by one‐way analysis of variance (ANOVA), followed by a post hoc Dunnett's comparison of Tukey's test to the control. Data are shown as mean ± SEM, and significance was taken at P < 0.05.

3. RESULTS

3.1. Two‐photon imaging reveals increased leukocyte adhesion in the brain vessels of BTBR mice

Temporal and spatial fine‐tuning of the leukocyte adhesion cascade was successfully observed in BTBR mice (Figure 1A). Importantly, the number of adherent leukocytes (firm adhesion) from BTBR mice was significantly and robustly increased from 28 ± 10 per mm2 in wild‐type C57 mice to 779 ± 116 per mm2 in BTBR mice (28 ± 10 per mm2) (Figure 1B,C), indicating that the ongoing activation of inflammatory responses occurred in the brain vessels of autistic mice.

Figure 1.

Figure 1

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In vivo imaging to measure leukocyte adhesion changes in living mice. A, Rhodamine 6G‐positive cells were visualized for adhesion in brain vessel of living mice. Representative images of leukocytes define different states during a 1‐min observation period. Arrow indicates the images of leukocyte behaviors (rolling, loose adhesion, and firm adhesion) in vessels. Scale bar: 30 μm. B, The images show leukocyte adhesion of C57 and BTBR mice cortex vessels (white dashed lines). Scale bar: 50 μm. C, Firmly adhering leukocytes' quantitative measurements during an observation period of 1 min in C57 and BTBR mice. Adherent leukocytes were counted as the number of cells per mm2 of vascular surface area. The data are shown as the means ±SEM from at least three independent experiments. (n = 5, per group). **P < 0.01 vs C57

3.2. Changes in cell adhesion molecules and in endothelial chemokine in the brain of BTBR mice

To further investigate the components of adhesion‐related molecules that mediate leukocyte‐endothelial adhesion in BTBR mice, we assayed the changes in the adhesion molecules and the correlated integrins. The principal integrins that bind to ICAM‐1 are β2‐integrin lymphocyte function‐associated antigen 1 (LFA‐1) and CD11b in some diseases as previously reported.15, 16 Using BTBR mice, we observed neutrophil CD11b involvement in leukocyte adhesion to the vascular endothelium (Figure 2A). ICAM‐1‐positive endothelial cell counts were consistently increased in BTBR mice (Figure 2B). Next, we examined the chemokine profile of brain vessels, since it was reported that chemokines and chemokine receptors mediate the migration of neutrophils and monocytes/macrophages into damaged blood vessel walls.17, 40 mRNA analysis (Figure 3A‐F) revealed that in the brain blood vessel extracts of BTBR mice the chemokine CXCL7 was markedly increased (Figure 3B). These results indicate that CD11b and its endothelial ligand, ICAM‐1, as well as the endothelial chemokine CXCL7, contribute to leukocyte‐endothelial adhesion in the brain vessels of BTBR mice, further implicating autism‐related neurovascular inflammation.

Figure 2.

Figure 2

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The adhesion molecule expression is increasing only in BTBR mice. A, Peripheral blood was collected from C57 and BTBR mice, and neutrophils were characterized as CD11b+ Ly6G+ cells by flow cytometry analysis. The dot plots represent the percentages of CD11b+ Ly6G+ cells. Bar graphs represent the percentage of CD11b+ Ly6G+ population. The results are the means ± SEM, n = 5, each group. All data are representative of at least three independent experiments. *P < 0.05 vs C57 mice. B, Flow cytometry analysis shows the number of ICAM+cells (labeled by CD31), which were estimated in isolated endothelial cells of C57 and BTBR mice. The results are shown as the means ± SEM, n = 5, each group. All data are representative of at least three independent experiments. *P < 0.05 vs C57 mice

Figure 3.

Figure 3

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Endothelial chemokine expression is increasing only in BTBR mice. The mRNA levels of chemokine CXCL1(A), CXCL7(B), CXCL10(C), CCL2(D), CCL3(E), and CX3CL1(F) from endothelial in cortex were measured. The chemokine mRNA was quantified by real‐time PCR analysis. Data are compared to C57 mice (means ± SEM from three independent experiments, n = 4, each group). **P < 0.01 vs C57 mice. NS, no significant

3.3. Upregulation of neutrophil cathepsin B during neuroinflammation in BTBR mice

Given the inflammatory response of leukocytes and the endothelium in brain vessels, we investigated the effect of vascular inflammation on the neighboring neurons in BTBR mice. Neuroinflammation is manifested by NLRP3‐inflammasome activation, resulting in increased cleavage of interleukin‐1β (IL‐1β).23, 41 Therefore, we measured the expression levels of cleaved IL‐1β and caspase‐1 in the cortex of C57 and BTBR mice to uncover the neuronal response to the inflammatory signal in the brain. The data indicated that the neuroinflammation indicators cleaved IL‐1β and caspase‐1, which were markedly increased in the cortex of BTBR mice, which coincide with enhanced leukocyte adhesion to the endothelium in BTBR mice (Figure 4A,B).

Figure 4.

Figure 4

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Neuroinflammatory response in brain cerebral cortex of BTBR mice. (A) Western blot detection of cleaved IL‐1β and caspase‐1 expression in cerebral cortex of C57 and BTBR mice. (B) Quantitative analysis of protein levels for (A) was used by densitometry. The data are compared to C57 mice (mean ± SEM from three independent experiments, n = 4, each group). *P < 0.05; **P < 0.01 vs C57. (C) Cathepsin B protein levels in neutrophils were assessed with immunoblotting in C57 and BTBR mice. (D) Densitometry of protein in (C) expressed as densitometry ratio of C57 (mean ± SEM from three independent experiments, n = 4, each group). *P < 0.05 vs C57. (E) Cathepsin B activities of neutrophils were analyzed using a cathepsin B activity assay kit. High cathepsin B activity of neutrophils was observed in BTBR mice. Proteins are expressed as densitometry ratio of C57 (mean ± SEM from three independent experiments, n = 4, each group). **P < 0.01 vs C57

To obtain a deeper insight into the cause governing neurovascular inflammation in autism, we monitored neutrophil cathepsin B levels in BTBR mice. Compared to C57 mice, we found a profound elevation in both cathepsin B protein expression and cathepsin B activity in the neutrophils of BTBR mice (Figure 4C‐E). This suggests that neutrophil cathepsin B activation could be closely related to leukocyte‐endothelial adhesion and neuroinflammation response during ASD.

3.4. Inhibition of cathepsin B suppresses leukocyte‐endothelial cell adhesion in BTBR mice

Inhibition of specific leukocyte adhesion molecules could be of therapeutic value for treating brain diseases.17 Herein, we investigated the role of neutrophil cathepsin B activation in leukocyte behavior (leukocyte‐endothelial cell adhesion, rolling velocity) using CA‐074Me, a specific inhibitor of cathepsin B in BTBR mice. As shown in Figure 4, CA‐074Me treatment significantly suppressed the number of adherent leukocytes by 69% compared with the saline‐treated group (Figure 5A,B) and increased their rolling velocities (Figure 5C,D).

Figure 5.

Figure 5

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Blocking cathepsin B improves leukocyte‐endothelial adhesion in BTBR mice. (A) TPLSM analysis of leukocyte adhesion to brain vessels. The BTBR mice were treated with either saline or CA‐074Me for 7 d. Adherent leukocytes were declined in CA‐074Me‐treated BTBR mice vs saline‐treated BTBR mice. Scale bar: 50 μm. (B) Adherent leukocyte quantitative analysis in (A). Results are shown as means ± SEM from at least three independent experiments. n = 4, each group. *P < 0.05; **P < 0.01. (C) In CA‐074Me‐treated group, the cumulative frequency of velocities of rolling leukocytes demonstrated a significant reduction in rolling velocity compared with saline‐treated group. Rolling velocities of 200 cells per group (n = 4 mice) were measured. (D) The histogram shows rolling velocities of leukocyte reduction in CA‐074Me‐treated mice compared with saline‐treated mice. Rolling velocities of 200 cells per group (means ± SEM from at least three independent experiments, n = 4, each group) were measured. **P < 0.01

Next, we examined the inhibitory effect of CA‐074Me on the levels of adhesion molecules and chemokine in BTBR mice. Flow cytometry showed that blocking cathepsin B activity by CA‐074Me reduced leukocyte CD11b and endothelial ICAM‐1 levels (Figure 6A,B). This finding is consistent with the fact that integrin ligand ICAM‐1 interacts with CD11b on leukocytes.42 Moreover, the treatment also inhibited the elevation of endothelial chemokine CXCL7 induced in BTBR mice (Figure 6C). Taken together, these results suggest that neutrophil cathepsin B activation initiates leukocyte‐endothelial cell adhesion by increasing adhesion molecules CD11b and ICAM‐1 in BTBR mice.

Figure 6.

Figure 6

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Cathepsin B inhibition reduces adhesion molecules and chemokine in BTBR mice. Flow cytometry analysis shows the effect of CA‐074Me treatment on CD11b+ cells in neutrophils (A) and ICAM‐1 + cells in endothelial cells of mice (B) at indicated groups. (A) Representative flow cytometry dot plots show neutrophils (Ly6G + CD11b +) and the percentage of neutrophils in peripheral blood of C57, BTBR+saline and BTBR+CA‐074Me mice. CA‐074Me treatment reduced the number of CD11b+ Ly6G+ cells in BTBR mice compared with saline‐treated BTBR mice. Data are expressed as the means ± SEM (n = 5, each group). *P < 0.05. (B) CA‐074Me treatment inhibits the number of expressing cells of ICAM+CD31+ cells in endothelial cells of BTBR mice compared with saline‐treated BTBR mice. Data are expressed as the means ± SEM (n = 5, each group). All data are representative of at least three independent experiments. *P < 0.05. (C) CA‐074Me treatment decreased CXCL7 mRNA levels in brain vasculature of BTBR mice. The upregulation of chemokine CXCL7 mRNA level was inhibited after CA‐074Me treatment in BTBR mice. Data are expressed as the means ± SEM from three independent experiments, n = 4, each group. *P < 0.05, **P < 0.01. (D) Schematic illustration of neutrophil cathepsin B activation contributes to leukocyte‐endothelial adhesion in autism

4. DISCUSSION

In this research, we describe the importance of neutrophil cathepsin B in regulating leukocyte‐endothelial cell adhesion and increasing neurovascular inflammation during autism development. Importantly, we found that overactivation of neutrophil cathepsin B initiates leukocyte‐endothelial cell adhesion, followed by leukocyte CD11b/endothelial ICAM‐1 activation, a pathological process that can be suppressed by the cathepsin B inhibitor CA‐074Me. Our results provide new mechanistic insights into the role of neutrophil cathepsin B in inducing neurovascular inflammation during autism.

Brain inflammation has long‐term consequences and could speculatively modulate the risk for a variety of neurological disorders including autism spectrum disorders.11, 43 Family members of patients with autism have a high risk for autoimmune disorders. BTBR mice partially recapitulate this phenotype and have abnormal immune responses, such as microglia activation and increased cytokine secretion.44, 45, 46, 47 Adhesive interactions between endothelial cells and leukocytes are key cellular mediators for inflammation under pathological conditions.48, 49, 50 We recorded here for the first time the dynamic pathogenesis of leukocyte‐endothelial cell adhesion in ASD animal models, suggesting the systemic inflammation and activation of the local vasculature in BTBR mice. We determined that CD11b in neutrophils and ICAM‐1 in endothelial cells were significantly higher in autistic mice, providing further support to previous reports that adhesion molecules may be essential in the pathophysiology of ASD, possibly by causing abnormalities in the immune system.38, 51

There is increasing evidence that different caspases and inflammatory cytokine IL‐1β upregulation are involved in mediating the function between inflammation and neuronal diseases including autism.52, 53, 54 In parallel, here we also observed that cleaved IL‐1β and caspase‐1 were elevated in the cortex of BTBR mice, indicating aberrant changes in ASD‐associated neuroinflammation. Chemokines have been identified as functional mediators of neuroinflammatory disorders and may be associated with the pathophysiology of autism.55, 56

In this study,our results are the same as a previous study showing that the elevation of endothelial chemokine CXCL7 plays a role in the development of neurobehavioral alterations that are triggered by various risk factors.57, 58 Taken together, these data suggest that high levels of inflammatory cytokine and endothelial chemokine are similar to those found in the autistic‐like mouse models.

More importantly, the inhibition of cathepsin B blocked NLRP3 inflammasome activation as well as effectively improved the neurological recovery of mice in brain disease,24, 25, 26, 27, 59, 60 which implicates a promising target of cathepsin B in modulating neurovascular inflammation. Consistently, we showed that treatment using cathepsin B inhibitor CA‐074Me prevented the interaction between CD11b and endothelial ICAM‐1. Thus, it abolished ASD‐induced leukocyte‐endothelial adhesion, following CA‐074Me treatment in BTBR mice. Similarly, it is reasonable to postulate that excessive or uncontrolled neutrophil cathepsin B activation can lead to pathological inflammation, as seen in BTBR mice. Therefore, we suggest that the increased adhesion was dependent on neutrophil cathepsin B activation and the augmented expression of ICAM‐1 and chemokine CXCL7 on endothelial cells (Figure 6D).

Although our study has provided multiple lines of evidence addressing the pivotal role of cathepsin B‐initiated neurovascular inflammation in autism, it remains an open question and more attention is required to better understand the intrinsic mechanisms. Activation of an upstream inflammatory molecule during inflammation triggers a series of complex molecular events that activate a cascade of multiple downstream effects. However, the exact mechanism underlying cathepsin B activation in neutrophils and its mode of action remain to be elucidated. It has been reported that neutrophil elastase up‐regulates the expression of cathepsin B in human macrophages in vitro. This would encourage the identification and therapeutic intervention of neutrophil elastase in ASD. Moreover, our data did not sufficiently elucidate the causal relationship between neuroinflammation and the leukocyte‐endothelial cell adhesion of ASD. Thus, more evidence is required to study the dynamic changes that occur in leukocytes, to further elucidate whether it resulting in endothelial adhesion in vessels and transfer into brain parenchyma during pathological process of ASD. We expect to enhance our understanding of the underlying mechanisms in order to provide new insights for treatment strategies to counteract these biochemical and autistic‐like behavioral abnormalities.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Supporting information

 

Click here for additional data file. (171.4KB, docx)

ACKNOWLEDGMENTS

This study was funded by the National Natural Science Foundation of China (81673417; 81473202) and Science and Technology Commission Foundation of Hangzhou (20172016A05).

Wang H, Yin Y‐X, Gong D‐M, et al. Cathepsin B inhibition ameliorates leukocyte‐endothelial adhesion in the BTBR mouse model of autism. CNS Neurosci Ther. 2019;25:476–485. 10.1111/cns.13074

The first two authors contributed equally to this work.

Contributor Information

Feng Han, Email: fenghan169@njmu.edu.cn.

Ying‐Mei Lu, Email: lufx@zju.edu.cn.

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