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. 2024 Feb 6;10(3):988–999. doi: 10.1021/acsinfecdis.3c00668

Interleukin-22 Contributes to Blood–Brain Barrier Disruption via STAT3/VEGFA Activation in Escherichia coli Meningitis

Ruicheng Yang †,‡, Jiaqi Chen †,‡, Xinyi Qu †,‡, Hulin Liu †,‡, Xinyi Wang †,‡, Chen Tan †,‡,§,∥, Huanchun Chen †,‡,§,∥, Xiangru Wang †,‡,§,∥,*
PMCID: PMC10928716  PMID: 38317607

Abstract

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Escherichia coli continues to be the predominant Gram-negative pathogen causing neonatal meningitis worldwide. Inflammatory mediators have been implicated in the pathogenesis of meningitis and are key therapeutic targets. The role of interleukin-22 (IL-22) in various diseases is diverse, with both protective and pathogenic effects. However, little is understood about the mechanisms underlying the damaging effects of IL-22 on the blood–brain barrier (BBB) in E. coli meningitis. We observed that meningitic E. coli infection induced IL-22 expression in the serum and brain of mice. The tight junction proteins (TJPs) components ZO-1, Occludin, and Claudin-5 were degraded in the mouse brain and human brain microvascular endothelial cells (hBMEC) following IL-22 administration. Moreover, the meningitic E. coli-caused increase in BBB permeability in wild-type mice was restored by knocking out IL-22. Mechanistically, IL-22 activated the STAT3-VEGFA signaling cascade in E. coli meningitis, thus eliciting the degradation of TJPs to induce BBB disruption. Our data indicated that IL-22 is an essential host accomplice during E. coli-caused BBB disruption and could be targeted for the therapy of bacterial meningitis.

Keywords: IL-22, Escherichia coli meningitis, blood–brain barrier, permeability, STAT3, VEGFA

Bacterial infections of the central nervous system (CNS) are serious and lead to significant morbidity as well as mortality. Patients often experience neurological sequelae, including hearing loss, developmental delay, and cognitive impairment.1Escherichia coli is a well-adapted and pathogenically versatile bacterial organism and is the leading cause of meningitis in newborns worldwide.2 Meningitis occurs when meningitic E. coli penetrates the blood–brain barrier (BBB), elicits inflammation in the brain, and enhances BBB permeability and pleocytosis.3 The BBB, which mainly comprises brain microvascular endothelial cells (BMECs), astrocytes, and microglial cells,4 plays a vital role in providing additional mechanisms for regulating transport in and out of the CNS.5 The highly organized expression of endothelial tight junction proteins (TJPs) creates exceedingly tight connections between adjacent BMECs, which promote high trans-endothelial electrical resistance (TEER) and therefore limits the paracellular flux of solutes.6 These TJPs include Occludin, Claudins (especially Claudin-5), and junctional adhesion molecules, which attach to the cellular actin cytoskeleton via zonula occludens proteins (especially ZO-1).7 Intercellular junctions between BMECs play a critical role in maintaining vascular integrity and permeability for barrier functions.8 Several factors have been reported to contribute to BBB disruption in bacterial meningitis, including direct cellular damage caused by bacterial virulence factors (e.g., lipopolysaccharide, capsule, peptidoglycan, enolase, hyaluronidase, and hemolysin),9 as well as host-specific proteins [e.g., matrix metalloproteinases (MMP)-3, MMP-8, MMP-9, vascular endothelial growth factor A (VEGFA), and SNAI1] or proinflammatory cytokines [e.g., interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α].10−12

IL-22 is a cytokine that belongs to the interleukin-10 (IL-10) family and is one of the most studied cytokines.13 IL-22R is a heterodimer complex comprising IL-22R1 and IL-10R2 subunits; however, IL-22 has a high affinity for only IL-22R1 and cannot bind to IL-10R2.14 IL-22 is mainly generated by immune cells, including innate lymphocytes and a fraction of T helper cells.15 IL-22 exhibits opposing effects in diverse disease models, ranging from protective to pathological effects.16 On the one hand, numerous studies have suggested that IL-22 is an essential signaling molecule with various beneficial effects, including antimicrobial immunity, promoting wound healing, inhibition of oxidative stress, and maintaining barrier integrity.17 On the other hand, IL-22 can activate glial cells and recruit chemokines to promote lymphocyte infiltration in the brain,18 and enhance inflammatory response alone or in combination with other cytokines and cause abnormal epithelial cell differentiation and proliferation.19 Notably, IL-22 has been linked to several CNS neuroinflammatory diseases including Alzheimer’s disease, multiple sclerosis, Parkinson’s disease, amyotrophic lateral sclerosis, and stroke.20 However, whether IL-22 is involved in BBB disruption in meningitic E. coli infections, and the detailed mechanism by which IL-22 disrupts the BBB, remains unclear.

In this work, to analyze the pathogenic processes of meningitic E. coli-induced BBB impairment, we characterized IL-22 as significantly increased in mouse brains in response to meningitic E. coli and investigated its potential role in mediating BBB permeability alteration. Our in vivo and in vitro findings strongly supported the contribution of IL-22 to BBB permeability via the activation of the signal transducer and activator of transcription 3 (STAT3)/VEGFA axis. The novelty of this study lies in the investigation of IL-22 mechanism in BBB permeability alteration, these observations will help to develop new approaches for the prevention and treatment of bacterial CNS infections.

Results

IL-22 Was Significantly Upregulated in Meningitic E. coli-Challenged Mice and Promoted the Disruption of BBB Integrity In Vitro

Pathogen-induced cytokines and chemokines contribute to BBB impairment in various neuroinflammation models. Using enzyme-linked immunosorbent assay (ELISA), we confirmed that the expression of IL-22 in the serum and brain increased in a time-dependent manner in meningitic E. coli-challenged mice (Figure 1A). Because high levels of IL-22 were detected in the mouse serum and brain tissue after infection, we investigated the possible effect of IL-22 on the integrity of the human brain microvascular endothelial cells (hBMEC) monolayer in vitro. We first evaluated the effects of IL-22 on the TEER of hBMEC using the electrical cell-substrate impedance sensing (ECIS) system. Using different doses of recombinant human IL-22 (1, 5, 10, 20, and 100 ng/mL) to stimulate the hBMEC monolayer, we found that IL-22 dose-dependently downregulated the TEER of the hBMEC monolayer, suggesting that IL-22 is an important contributor to BBB dysfunction (Figure 1B). After treatment with different doses of IL-22, the protein levels of TJPs, including ZO-1, Occludin, and Claudin-5, decreased significantly (Figure 1C). Moreover, immunofluorescence analysis revealed that these TJPs were well arranged and distributed around hBMEC. In contrast, these proteins became inconsecutively and irregularly distributed, or scattered around the cells after treatment with 10 ng/mL IL-22, indicating a direct breakdown of TJPs between adjacent endothelial cells (Figure 1D). These observations indicated that IL-22 negatively affected the integrity of the hBMEC monolayer by downregulating TJPs.

Figure 1.

Figure 1

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Meningitic E. coli induction of IL-22 contributes to the degradation of TJPs in hBMEC. (A) ELISA analysis of IL-22 in serum and brain lysates from challenged mice (n = 3). Data were collected and presented as mean ± standard deviation (SD) from three replicates at each time point. *p < 0.05, **p < 0.01, ***p < 0.001. (B) TEER value changes in hBMEC when treated with multiple doses of IL-22 (0, 1, 5, 10, 20, and 100 ng/mL) monitored by the ECIS system. Data were collected and presented as mean ± SD from three replicated wells at each time point. (C) Western blot analysis of ZO-1, Occludin, and Claudin-5 in hBMEC in response to multiple doses of IL-22 (0, 1, 5, 10, 20, and 100 ng/mL). β-actin was used as the loading control. Densitometry was performed to analyze differences among the samples. *p < 0.05, **p < 0.01, ***p < 0.001. (D) Immunofluorescence analysis determining the distribution and expression of ZO-1, Occludin, and Claudin-5 in hBMEC after 10 ng/mL IL-22 treatment. ZO-1, Occludin, and Claudin-5 were stained in red. The cell nucleus was stained in blue with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 10 μm.

IL-22 Enhances BBB Permeability by Decreasing the Expression of TJPs In Vivo

To further confirm that IL-22 could effectively increase the permeability of BBB, the Evans blue infiltration was measured to evaluate the mouse BBB permeability. As shown in Figure 2A, tail vein injection of recombinant IL-22 (10 ng/mouse) increased the amount of Evans blue dye that leaked out of the blood vessels compared to the control group, which enhanced BBB permeability. Moreover, the expression levels of TJPs, including ZO-1, Occludin, and Claudin-5, were significantly lower than those of the control group (Figure 2B). Immunofluorescence analysis was performed to examine the distribution and expression of ZO-1, Occludin, and Claudin-5 in the mouse brain tissue following IL-22 administration. These TJPs were well organized and distributed around the blood vessels in the control mouse brain. In contrast, the vascular endothelial layer became inconsecutively distributed, irregular, or gapped in IL-22-treated mice, indicating the breakdown of TJPs between adjacent endothelial cells (Figure 2C).

Figure 2.

Figure 2

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IL-22 promotes BBB permeability enhancement in vivo. (A) Effects of the recombinant mouse IL-22 (10 ng/mouse) on the permeability of the mice brain evaluated using the Evans blue approach (n = 5). (B) Western blot analysis of ZO-1, Occludin, and Claudin-5 in the brains of IL-22-treated (10 ng/mouse) mice (n = 3). β-actin was used as the loading control. Densitometry was performed to analyze differences among the samples. **p < 0.01, ***p < 0.001. (C) Brain samples of both mice treated with and without IL-22 (10 ng/mouse) for 24 h were analyzed for vascular endothelium integrity by immunofluorescence. The figures represent the results of three mouse brains (three images per mouse). ZO-1, Occludin, and Claudin-5 were selected as the markers reflecting the vascular endothelium integrity. CD31 was specifically applied for labeling the microvessels in green. The cell nucleus was stained in blue with DAPI. Scale bar indicates 50 μm.

Next, the IL-22–/– mice were used to verify the disruptive effect of IL-22 on the BBB during meningitic E. coli infection. Evans blue entry into the brain was lower in the IL-22–/– group than in the wild-type (WT) group (Figure 3A). Western blotting and immunofluorescence assays were performed to examine the expression of TJPs in the brains of WT and IL-22–/– mice following meningitic E. coli infection. The expression levels of TJPs in the WT infection group were significantly decreased, whereas this reduction effect was significantly reversed by the IL-22 knockout (Figure 3B). Immunofluorescence results also indicated that these adverse effects of TJPs were reversed in the IL-22–/– infection group compared to the WT infection group (Figure 3C). Therefore, these findings largely support the idea that IL-22 is an important effector of BBB damage in E. coli meningitis.

Figure 3.

Figure 3

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IL-22 knockout in mice suppresses meningitic E. coli-induced BBB disruption. (A) Evans blue assay was used to evaluate the permeability of WT and IL-22–/– mice with or without meningitic E. coli infection (n = 5). (B) Brain lysates from the challenged WT and IL-22–/– mice were assessed using Western blot for the expression of TJPs (n = 3). β-Actin was used as the loading control. Densitometry was performed to analyze differences among the samples. *p < 0.05, **p < 0.01, ***p < 0.001. (C) Immunofluorescence analysis of vascular endothelium integrity in infected WT and IL-22–/– mice. The figures represent the results of three mouse brains (three images per mouse). ZO-1, Occludin, and Claudin-5 were stained in red. CD31 was specifically applied for labeling the microvessels in green. The cell nucleus was stained in blue with DAPI. Scale bar indicates 50 μm.

IL-22 Requires STAT3 Activation to Damage the BBB Integrity

After binding to the IL-22R complex, IL-22 activates tyrosine kinase 2 (TYK2) and Janus kinase (JAK), which trigger a series of downstream cascade reactions, including the activation of STAT1, STAT3, and STAT5 phosphorylation, among which STAT3 molecules play a pivotal role.21 We investigated whether IL-22-induced BBB damage during meningitic E. coli infection correlated with the activation of STAT3. We observed a dose-dependent increase in STAT3 phosphorylation in hBMEC after IL-22 administration (Figure 4A). Next, we blocked the effects of STAT3 using S3I-201, a specific inhibitor of STAT3. Expectedly, 50 μM S3I-201 treatment completely inhibited TEER in the hBMEC monolayer induced by 10 ng/mL IL-22 (Figure 4B). Western blot and immunofluorescence assays showed that the negative regulatory effects of IL-22 on BBB integrity were significantly reduced when 50 μM S3I-201 was administered. The downregulation and altered distribution of ZO-1, Occludin, and Claudin-5 in IL-22-treated hBMEC were significantly improved by inhibiting the STAT3 pathway (Figure 4C,D). Therefore, these results indicated that IL-22 functions by activating STAT3 signaling to negatively regulate BBB integrity.

Figure 4.

Figure 4

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IL-22 negatively regulates BBB integrity by activating STAT3. (A) Western blot analysis of STAT3 phosphorylation in hBMEC in response to multiple doses of IL-22 (0, 1, 5, 10, 20, and 100 ng/mL). β-Actin was used as the loading control. Densitometry was performed to analyze differences among the samples. ***p < 0.001. (B) ECIS system monitoring TEER changes of the IL-22-treated hBMEC with or without STAT3 specific inhibitor S3I-201 administration. Data were collected and presented as mean ± SD from three independently replicated wells at each time point. (C) Effects of the S3I-201 (50 μM) treatment on IL-22-induced (10 ng/mL) downregulation of ZO-1, Occludin, and Claudin-5 in hBMEC. β-Actin was used as the loading control. Densitometry was performed to analyze differences among the samples. **p < 0.01, ***p < 0.001. (D) Distribution of TJPs in IL-22-treated hBMEC with or without S3I-201 (50 μM) administration, as determined by immunofluorescence analysis. ZO-1, Occludin, and Claudin-5 were stained in red. The cell nucleus was stained in blue with DAPI. Scale bar, 10 μm.

IL-22/STAT3 Axis Contributes to VEGFA Expression

Our previous studies demonstrated that meningitic E. coli challenge can induce VEGFA expression, which exacerbates infection-dependent BBB disorder.22 Here, we examined the effect of IL-22 on VEGFA expression. After treatment with different doses of IL-22 (1, 2, 5, and 10 ng/mL) for 12 h, we observed that IL-22 stimulated the expression of VEGFA in hBMEC in a dose-dependent manner (Figure 5A). In vivo, we evaluated the contribution of IL-22 to the production of VEGFA in response to the infection using IL-22–/– mice. The results showed that the infection-induced upregulation of VEGFA in the serum and brain of IL-22–/– mice was significantly lower than that in WT mice (Figure 5B). Subsequently, we conducted a chromatin immunoprecipitation (ChIP)-polymerase chain reaction (PCR) assay to evaluate the possibility that transcription factor STAT3 regulates VEGFA expression via direct binding to the VEGF promoter. Protein–DNA complexes were immunoprecipitated with antibodies against p-STAT3 or normal immunoglobulin G (IgG) (negative control). An aliquot of nonimmunoprecipitated chromatin was used as an input control to demonstrate specificity. PCR results demonstrated a significant increase in STAT3 binding to the VEGF promoter after hBMEC were treated with 10 ng/mL IL-22 (Figure 5C). We found that the overexpression of VEGFA in hBMEC after IL-22 administration could be partly prevented by STAT3 specific inhibitor S3I-201 (50 μM) (Figure 5D). These results showed that IL-22 contributes to VEGFA expression by positively modulating STAT3 signaling.

Figure 5.

Figure 5

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Effects of the IL-22/STAT3 axis on VEGFA expression. (A) Western blot analysis of VEGFA expression in hBMEC in response to multiple doses of IL-22 (0, 1, 2, 5, and 10 ng/mL). β-actin was used as the loading control. Densitometry was performed to analyze differences among the samples. **p < 0.01. (B) ELISA analysis of VEGFA in serum and brain lysates from challenged WT and IL-22–/– mice. Data were collected and presented as mean ± SD *p < 0.05, ***p < 0.001. (C) ChIP-PCR validation of the STAT3 binding to the VEGFA promoter in hBMEC treated with 10 ng/mL IL-22. The nuclear extracts were immunoprecipitated with anti-p-STAT3 antibody or normal IgG and a ChIP assay was performed. The PCR primers were amplified in the −913 to −783 region of the VEGF promoter. Input refers to the same dose of nuclear extract administered prior to immunoprecipitation. (D) Western blot analysis of the effect of S3I-201 (50 μM) treatment on the IL-22-increased (10 ng/mL) VEGFA levels in hBMEC. β-actin was used as the loading control. Densitometry was performed to analyze differences among the samples. **p < 0.01.

IL-22-Induced BBB Breakdown Depends on the Expression of VEGFA

Considering that VEGFA has been implicated in the regulation of BBB permeability and that the IL-22/STAT3 axis can positively regulate VEGFA expression, we examined whether BBB dysfunction induced by the IL-22/STAT3 axis was dependent on the expression of VEGFA. In this study, VEGFA was genetically destroyed in hBMEC using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 approach, and its deletion was validated by PCR amplification and Western blot analysis (Figure 6A). As shown in Figure 6B, IL-22-induced decreases in TEER values of hBMEC were completely abolished in VEGFA-knockout cells. VEGFA knockout significantly inhibited the IL-22-reduced TJPs expression, including that of ZO-1, Occludin, and Claudin-5 (Figure 6C). Moreover, we used an immunofluorescence assay to evaluate the effect of IL-22 on TJPs distribution in VEGFA knockout hBMEC. The levels of ZO-1, Occludin, and Claudin-5 in adjacent endothelial cells were significantly decreased in hBMEC after IL-22 treatment; however, this effect was attenuated by VEGFA knockout (Figure 6D). Collectively, these results demonstrated that IL-22 promoted the disruption of BBB integrity, depending on VEGFA expression.

Figure 6.

Figure 6

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BBB damage caused by IL-22 depends on the upregulation of VEGFA. (A) VEGFA deleted in hBMEC through CRISPR/Cas9, and VEGFA knockout in hBMEC was identified by PCR amplification and Western blot analysis. (B) ECIS assay analyzing the barrier resistance of the WT and VEGFA knockout in hBMEC with the addition of IL-22 (10 ng/mL). (C) Western blot analysis of ZO-1, Occludin, and Claudin-5 expression in the WT and VEGFA knockout in hBMEC in response to IL-22 (10 ng/mL). β-Actin was used as the loading control. Densitometry was performed to analyze differences among the samples. *p < 0.05, ***p < 0.001. (D) Immunofluorescence analysis determining the distribution and the expression of TJPs in the WT and VEGFA knockout in hBMEC after 10 ng/mL IL-22 treatment. ZO-1, Occludin, and Claudin-5 were stained in red. The cell nucleus was stained in blue with DAPI. Scale bar, 10 μm.

Discussion

Increased BBB permeability was well recognized as a key feature of meningitic E. coli causing BBB dysfunction, which could be mediated by the degradation of TJPs like ZO-1, Occludin, and Claudin-5.23 It has been observed that E. coli LPS disrupt BBB integrity by affecting the crosstalk between protein kinase C and RhoA signals.24 The α-hemolysin in meningitic E. coli K1 strain has been demonstrated to reduce ZO-1 expression of hBMEC by inhibiting the TGFβ1-TGFBRII-Smad2/3-Gli1/2 axis.25 OmpA-mediated meningitic E. coli adhesion of hBMEC activated PKCα signaling, resulting in β-catenin dissociation from Cadherins, eventually leading to BBB breakdown.26 In addition, several host molecules have been reported to mediate the enhancement of BBB permeability during E. coli meningitis, such as platelet-derived growth factor B (PDGFB), IL-17A, Snail-1, IL-1β, IL-6, and TNF-α. In vitro and in vivo data showed that meningitic E. coli induced the upregulation of PDGFB, which dose-dependently increased BBB permeability by decreasing TJPs expression.9 A recent study found that meningitic E. coli-induced IL-17A significantly reduced hBMEC TJPs expression at the post-transcriptional level by inhibiting the proteinase 3/protease-activated receptor 2 axis, thus augmenting endothelial permeability and disrupting BBB integrity.27 It has been demonstrated that overexpression of transcription factor Snail-1 destroys the top complex of vascular endothelial cells.28 Meningitic E. coli-infected hBMEC were found to increase the expression of Snail-1 that mediated the degradation of ZO-1, Occludin, and Claudin-5, and disrupted endothelial barrier integrity.22 Understanding how meningitic E. coli affect BBB permeability will provide novel perspectives for investigating the pathogenesis, prevention, and treatment of E. coli meningitis.

Several in vivo and in vitro studies have demonstrated that IL-22 has protective and pathogenic effects in different disease models.18,29 In chronic kidney injury, IL-22 plays a protective role by promoting tissue repair and regeneration, inhibiting oxidative stress, and producing antimicrobial peptides.17 In some inflammatory conditions, such as psoriasis, allergic airway inflammation, and collagen-induced arthritis, IL-22 could accelerate the course of the disease.30,31 However, the role of IL-22 in E. coli-caused meningitis remains unknown. In this study, we reported that IL-22 played a profound and previously undiscovered role in the negative regulation of BBB integrity in E. coli-caused meningitis. IL-22 protein expression was upregulated in the serum and brain tissue following meningitic E. coli infection. IL-22 induced significant changes associated with the loss of BBB integrity, including the degradation and rearrangement of the permeability-associated TJPs ZO-1, Occludin, and Claudin-5, as well as decreased TEER. IL-22 depletion nearly abolished meningitic E. coli-induced BBB permeability enhancement. Our results also indicated that high levels of IL-22 induced VEGFA overexpression by activating STAT3 signaling, which is involved in the pathogenic process of BBB disruption. Therefore, we presented strong evidence that IL-22, an inflammatory molecule, could be an effective target for reducing BBB damage in bacterial meningitis.

IL-22 levels markedly increased under inflammatory conditions such as tissue damage or infection.32 IL-22 affects various barrier function-related cell types, including tubular epithelial cells, vascular endothelial cells, keratinocytes, bronchial and intestinal epithelial cells, and dermal fibroblasts.33 Both the pathological and protective effects on tissue barrier have been attributed to IL-22 in different disease conditions. IL-22 plays a protective role by maintaining the epithelial integrity. In inflammatory bowel disease, IL-22 promotes epithelial regeneration, innate defense, and membrane mucus production to maintain homeostasis and repair the epithelial barrier integrity.34 IL-22 preserves gut epithelial integrity and promotes disease remission by limiting infection-induced gut immunopathology during chronic Salmonella infection.35 Furthermore, IL-22 regulates endometrial regeneration by enhancing TJPs Claudin-2 and Claudin-10,36 modulates tubular atrophy by augmenting tubular epithelial integrity and epithelial barrier function,37 and promotes epithelial integrity and repair following infectious pathogen challenge in the lung.38 In this study, our data supported the essential role of IL-22 in increasing BBB permeability by degrading TJPs in BMEC (Figures 2 and 3). Similar results were obtained in a previous study on multiple sclerosis. The combination of IL-22 and IL-17 has been shown to destroy BBB integrity by breaking the tight connection.39,40 However, the precise mechanism underlying IL-22-induced BBB permeability remains unknown.

Once IL-22 binds to its receptor complex, it activates JAK and TYK2 phosphorylation, which initiates a series of downstream cascade reactions, including the phosphorylation of STAT1, STAT3, and STAT5. In addition to the JAK/STAT pathway, IL-22 activates phosphoinositide 3-kinase, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38.17 In this study, we discovered that IL-22 negatively affected BBB integrity by increasing STAT3 signaling (Figure 4). These findings were consistent with the known roles of IL-22 in Alzheimer’s disease, stroke, cerebral malaria, and sepsis-associated encephalopathy. STAT3 activation has been linked to BBB destruction owing to its promotion of the production of multiple proinflammatory cytokines, peroxidase, and hydrolase matrix metalloproteinases, such as IL-6, TNF-α, myeloperoxidase, and MMP-2 and -9.41 Previous studies found that exposing BMEC to Aβ peptides activated forms of STAT3 and caused STAT3-mediated vascular oxidative stress, resulting in cerebrovascular dysfunction.42 Findings showed that inhibiting absent in melanoma 2 (AIM2) preserved BBB integrity after ischemic stroke, at least in part, by modulating STAT3 activation.43 Furthermore, STAT3 signaling induced the expression of miR-181b, which promoted BBB impairment by decreasing BBB cell adhesion in sepsis-associated encephalopathy.44 In a study of human cerebral malaria, neuregulin 1 protected hBMEC against cell death/apoptosis by stimulating ErbB4 phosphorylation, followed by the inactivation of STAT3.45 The aforementioned research supported our finding that blocking STAT3 activation is an efficient way to maintain BBB integrity.

VEGFA is an appealing target for modulating brain function at the neurovascular interface. On the one hand, VEGFA plays critical roles in angiogenesis, neuroprotection, and neurogenesis. On the other hand, pathologically elevated VEGFA levels increase vessel permeability and leakage, while compromising BBB integrity.46 Our data showed that IL-22 contributed to VEGFA expression by positively modulating STAT3 signaling (Figure 5). In agreement with our study, the activation of STAT3 promotes tumor angiogenesis in cancers such as colon tumor, lung adenocarcinoma, and gastric cancer by increasing VEGFA.47,48 Moreover, another study found that VEGFA released by Japanese encephalitis virus-infected astrocytes activated STAT3 signaling in BMEC.49 Therefore, the feedback loop formed between STAT3 and VEGFA is one of the factors contributing to BBB injury. In our previous study, we demonstrated that meningitic E. coli increased VEGFA production to increase BBB permeability.22 Furthermore, VEGFA also contributes to the breakdown of the BBB in meningitis caused by Haemophilus influenzae type a, Japanese encephalitis virus, and Mycobacterium tuberculosis.49−51 In stroke, increased VEGFA levels cause vascular leakage and BBB breakdown, which disrupts homeostasis and results in the invasion of peripheral immune cells and edema.52 A recent study showed that VEGFA overexpression in Alzheimer’s disease mice contributed to abnormal endothelial nitric oxide synthase/occludin-associated BBB permeability, increased the incidence of capillary stalls, and decreased cerebral blood flow.53 These findings show that inhibiting VEGFA function can effectively maintain BBB integrity in CNS diseases. Similar to our results, IL-22-induced BBB dysfunction was completely abolished in VEGFA-knockout hBMEC (Figure 6). This also revealed the importance of STAT3/VEGFA signaling for BBB homeostasis.

Conclusions

This study revealed that IL-22 is a crucial cytokine for the destruction of BBB integrity. High circulatory IL-22 in E. coli meningitis activated STAT3 signaling followed by the induction of VEGFA secretion in the brain and caused BBB dysfunction through the degradation of TJPs ZO-1, Occludin, and Claudin-5 in vascular endothelial cells. The novelty of this study lies in the investigation of IL-22 mechanism in BBB permeability alteration, these findings strongly suggest that targeting IL-22 may be a promising therapeutic strategy for maintaining BBB function.

Materials and Methods

Bacterial Strains and Cell Culture

The E. coli strain PCN033 (GenBank: CP006632.1) was a highly virulent cerebrospinal fluid isolate first isolated in 2006 in Hunan Province, China. This strain was able to invade the host CNS, increase BBB permeability, and cause severe neuroinflammation.22 The E. coli strain was consistently grown in Luria-Bertani medium at 37 °C under aerobic conditions. hBMEC were purchased from ScienCell (Carlsbad, CA). hBMEC were cultured in Roswell Park Memorial Institute 1640 medium with 10% fetal bovine serum, MEM essential and nonessential amino acids, sodium pyruvate (1 mM), l-glutamine (2 mM), penicillin-streptomycin (100 U/mL), and vitamins (Thermo Fisher Scientific, Waltham, MA).54 Cells were cultured using dishes and flasks purchased from Jet Biofil (Guangzhou, China). Cells were incubated at 37 °C under 5% CO2 until monolayer confluence was reached. Confluent cells were washed with phosphate-buffered saline (PBS; pH 7.4) and cultured in a serum-free medium for 12–14 h before the experiment.

ELISA

Pathogen-free C57BL/6 WT mice were obtained from the Laboratory Animal Services Center of Huazhong Agricultural University. Mice aged 25 days were challenged with meningitic E. coli through the tail vein at 1 × 107 CFUs suspended and diluted in 100 μL PBS. At 2, 4, 6, 8, 10, and 12 h postinoculation, the mice were anesthetized by intraperitoneal injection of tribromoethanol (Avertin; 250 mg/kg) and the serum and brains (by cardiac perfusion) were harvested. Control mice were injected via the tail vein with 100 μL of PBS, and the same anesthesia was used for sample collection. Mice brains were homogenized in radioimmunoprecipitation assay buffer (RIPA) (Epizyme, Shanghai, China) containing protease inhibitor cocktail (GlpBio, Montclair, CA) and centrifuged at 12,000 rpm for 10 min at 4 °C to remove the insoluble cell debris. Secretory IL-22 from serum and brain extracts were quantified using a mouse IL-22 ELISA kit (RayBiotech, Norcross, GA).

IL-22–/– mice (C57BL/6) were obtained from Model Animal Research Centre, Nanjing University. The WT mice used here were littermates of the IL-22–/– mice. WT mice and IL-22–/– mice aged 25 days were challenged with meningitic E. coli through the tail vein at 1 × 107 CFUs. At 6 h postinoculation, the mice were anesthetized and the serum and brains were harvested. Secretory VEGFA from serum and brain extracts were quantified using a mouse VEGFA ELISA kit (Neobioscience, Shenzhen, China).

ECIS

hBMEC were grown on collagen-coated gold-plated electrodes in 96-well chamber slides (8W1E+) linked to ECIS Zq equipment (Applied BioPhysics, Troy, NY), which was continuously monitored to detect any changes in the monolayer cell barrier. 250 μL of medium containing 3 × 105 hBMEC was added to each well, and a stable TEER of approximately 13,000–15,000 ohms is reached in approximately 16–18 h. After reaching a stable maximum resistance, recombinant human IL-22 protein (Novoprotein, Summit, NJ) was added to the cells at a specified dose (1, 5, 10, 20, and 100 ng/mL) and TEER changes were automatically monitored by the ECIS system. For inhibitor treatment, hBMEC were pretreated with STAT3 inhibitor 50 μM S3I-201 (HY-15146, MCE, Monmouth Junction, NJ) and dimethyl sulfoxide (DMSO) 9 h before 10 ng/mL IL-22 administration. For VEGFA-knockout cells, IL-22 (10 ng/mL) was added to wells after hBMEC and VEGFA-knockout hBMEC reaching a stable maximum resistance and TEER changes were automatically monitored by the ECIS system. All data recorded in the ECIS system were analyzed. Each treatment was performed in triplicate.

Western Blot

The confluent hBMEC were starved in serum-free medium, and then recombinant human IL-22 protein was added to the cells at a specified dose (1, 5, 10, 20, and 100 ng/mL) for 12 h. Cells were washed with PBS, and protein was extracted with RIPA. For inhibitor treatment, hBMEC were pretreated with 50 μM S3I-201 9 h (during the time of starvation treatment) before human 10 ng/mL IL-22 administration. For VEGFA-knockout cells, the confluent hBMEC and VEGFA-knockout hBMEC were starved in serum-free medium, and then recombinant human IL-22 (10 ng/mL) was added to the cells for 12 h. Recombinant mouse IL-22 was purchased from Novoprotein (Summit, NJ). The mice were injected intravenously with 100 μL of 100 ng/mL recombinant mouse IL-22 diluted in PBS, and control mice were injected with an equal volume of PBS. After 24 h, the mice were anesthetized and their brains were harvested after cardiac perfusion. For bacterial infection, WT mice and IL-22–/– mice were challenged with meningitic E. coli through the tail vein at 1 × 107 CFUs. At 8 h postinoculation, the mice were anesthetized and the brains (by cardiac perfusion) were harvested. Protein concentrations in the brain or cell lysates were determined using a BCA protein assay kit (Beyotime, Shanghai, China). Aliquots of each sample were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes. The blots were blocked with 5% bovine serum albumin in Tris-buffered saline containing Tween-20 at room temperature for 1 h, and then incubated overnight at 4 °C with primary antibodies. ZO-1 (ab216880, 1:1000) and Claudin-5 (ab131259, 1:2000) antibodies were obtained from Abcam (Cambridge, MA). β-actin (66009-1-Ig, 1:50000), Occludin (13409-1-AP, 1:2000), VEGFA (19003-1-AP, 1:1000), and STAT3 (60199-1-Ig, 1:5000) antibodies were obtained from Proteintech (Chicago, IL). p-STAT3 (9145S, 1:2000) was obtained from Cell Signaling Technology (Danvers, MA). Horseradish peroxidase-conjugated antirabbit immunoglobulin G (IgG) (BF03001, 1:5000) and anti-mouse IgG (BF03008, 1:5000) antibodies were obtained from Biodragon (Beijing, China). ECL substrates for Western blotting were obtained from Bio-Rad (Hercules, CA).

Immunofluorescence Analysis

hBMEC were grown in 35 mm glass-bottom dishes and treated with IL-22 or bacteria as described in the Western blot section. Cells were fixed with 4% paraformaldehyde for 30 min, followed by three washes in PBS. The cells were then incubated with the primary ZO-1 (Abcam, ab216880, 1:200), Occludin (Proteintech, 27260-1-AP, 1:400), or Claudin-5 (Abclonal, A10207, 1:100) antibodies for 12 h at 4 °C, and then incubated with goat antirabbit IgG Cy3 antibody (Proteintech, SA00009-2, 1:200) for 1 h. Cells were counterstained with DAPI (Beyotime, Shanghai, China) to visualize nuclear morphology and photographed using a BX41 fluorescence microscope (Olympus, Tokyo, Japan).

Mice were challenged with IL-22 or bacteria as described in the Western blot section. Brain samples were collected, fixed in 4% formaldehyde solution for over 24 h, and embedded in paraffin. Sections were incubated with primary ZO-1 (Abcam, ab216880, 1:200), Occludin (Proteintech, 27260-1-AP, 1:400), or Claudin-5 (Abclonal, A10207, 1:100) antibodies, followed by incubation with a secondary antibody conjugated with cyanine-3 (Proteintech, SA00009-2, 1:100). The sections were then incubated with a primary CD31 antibody (Proteintech, 66065-2-Ig, 1:100), followed by incubation with the appropriate secondary antibody, fluorescein isothiocyanate (Proteintech, SA00003-9, 1:100), prior to final nuclear staining with DAPI. The sections were photographed using a BX41 fluorescence microscope. The image shown is representative of nine images per three numbers of mice.

In Vivo BBB Permeability Assay

BBB permeability of C57BL/6 WT mice and IL-22–/– mice was evaluated using Evans blue dye (Sigma-Aldrich, St. Louis, MO). The mice were injected intravenously with 100 μL of 100 ng/mL recombinant mouse IL-22 diluted in PBS, and control mice were injected with an equal volume of PBS. After 24 h, the mice were anesthetized for Evans blue assay. For bacterial infection, WT mice and IL-22–/– mice were challenged with meningitic E. coli through the tail vein at 1 × 107 CFUs, and control mice were injected with an equal volume of PBS. When the WT-infected group of mice displayed severe neurological symptoms, all groups of mice were anesthetized for the Evans blue assay. Each mouse in all groups was injected with 500 μL of Evans blue (5 mg/mL) through the tail vein. After 15 min, the mice were anesthetized and cardiac perfusion was performed for 10 min with 20 mL of sterile PBS. The brains were collected and photographed for dye staining.22

ChIP

ChIP was performed to test the interaction between STAT3 and its target genes VEGFA by using SimpleChIP Plus Enzymatic Chromatin IP Kit (Cell Signaling Technology, Danvers, MA). Briefly, the hBMEC in the dishes were fixed in formaldehyde to cross-link the proteins with DNAs. Cells were digested with micrococcal nuclease and immunoprecipitated with antibodies against p-STAT3 (Cell Signaling Technology, 9145S, 1:100) or normal IgG (negative control).55 The products were treated with protease K and subjected to DNA isolation. Purified DNA was used for ChIP-PCR amplification with the following primers: 5′-CTGGCCTGCAGACATCAAAGTGAG-3′ (forward) and 5′-CTTCCCGTTCTCAGCTCCACAAAC-3′ (reverse).

CRISPR/Cas9 Genomic Editing

A CRISPR/Cas9 plasmid containing the puromycin resistance gene was obtained from YSY Biotech (Nanjing, China). Human VEGFA sgRNA1 (5′-AGATGTACTCGATCTCATCAGGG-3′) and sgRNA2 (5′-CCATCCAATCGAGACCCTGGTGG-3′) were synthesized and cloned into the CRISPR/Cas9 plasmid. Transfection was performed as previously described.22 The cells were transferred into 96-well plates using the limiting dilution method and incubated until a single-cell clone was formed. Genomic DNA was extracted from each clone. PCR was performed to amplify the target region with the following primers: 5′-ACTTGCCTGATTCGGAAG-3′ (forward) and 5′-GGAGAAGGAGATGGTTGG-3′ (reverse). PCR-positive edited cells were identified using sequencing and Western blot analysis. Negatively edited cells were selected as a control for VEGFA-knockout cells.

Statistical Analysis

Data are expressed as mean ± standard deviation (mean ± SD). The statistical significance of the differences between groups was analyzed by a one-way analysis of variance (ANOVA) or two-way ANOVA embedded in GraphPad Prism, version 6.0 (GraphPad Software, Inc., La Jolla, CA). p < 0.05 (*) was considered significant, and p < 0.01 (**) or p < 0.001 (***) was considered extremely significant.

Ethics Approval and Consent to Participate

All animal experiments in this study were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the China National Institutes of Health. All procedures and handling techniques were approved by the Committee for Protection, Supervision, and Control of Experiments on Animals guidelines at Huazhong Agricultural University (Permit No. SYXK2020-0084, Animal Welfare Assurance No. HZAUMO-2020-0087). All efforts were made to treat the experimental animals in this study ethically and to minimize suffering.

Acknowledgments

The National Natural Science Foundation of China (NSFC) (32102749), the National Key Research and Development Program of China (2021YFD1800800), the China Postdoctoral Science Foundation (2022M721277), the Natural Science Foundation of Hubei Province (2021CFA016), the Fundamental Research Funds for the Central Universities (2662023PY005), and the China Agriculture Research System of MOF and MARA (CARS-35) provided funding for this project.

Glossary

Abbreviations Used

E. coli

Escherichia coli

IL-22

interleukin-22

BBB

blood–brain barrier

hBMEC

human brain microvascular endothelial cells

CNS

central nervous system

TJPs

tight junction proteins

TEER

trans-endothelial electrical resistance

JAMs

junctional adhesion molecules

STAT3

signal transducer and activator of transcription 3

VEGFA

vascular endothelial growth factor A

IL-10

interleukin-10

DMSO

dimethyl sulfoxide

PBS

phosphate-buffered saline

CFUs

colony-forming units

ECIS

electrical cell-substrate impedance sensing

ChIP

chromatin immunoprecipitation

SD

standard deviation

ELISA

enzyme-linked immunosorbent assay

RIPA

radioimmunoprecipitation assay buffer

WT

wild-type

PCR

polymerase chain reaction

CRISPR

clustered regularly interspaced short palindromic repeats

JAK

Janus kinase

TYK2

tyrosine kinase 2

PDGFB

platelet-derived growth factor B

AIM2

absent in melanoma 2

Data Availability Statement

The data sets generated during the current study are available from the corresponding author upon reasonable request. The data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Author Contributions

X.W. and R.Y. designed the experiments. J.C., R.Y., X.Q., H.L., and X.W. performed the experiments. R.Y. and J.C. analyzed the data and performed the statistical analysis. R.Y. wrote the manuscript. X.W. revised the manuscript. H.C. and C.T. provided technical and administrative support. All authors read and approved the final manuscript.

Author Contributions

⊥ R.Y. and J.C. contributed equally to this work.

The authors declare no competing financial interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data sets generated during the current study are available from the corresponding author upon reasonable request. The data supporting the conclusions of this article will be made available by the authors, without undue reservation.

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