IL-21 Enhances the Immune Protection Induced by the Vibrio vulnificus Hemolysin A Protein

We previously reported that the Vibrio vulnificus hemolysin A (VvhA) protein elicited good immune protection and could effectively control V. vulnificus infection in mice. However, its molecular mechanism remains unknown. We hypothesized that hemolysin A induces an immunoprotective response via IL-21 regulation. To demonstrate this, IL-21 expression in mice was regulated by injecting either specific antibodies or rIL-21, and the immune response was evaluated by flow cytometry. Our results suggested that IL-21 enhances immune protection by inducing a T follicular helper cell and germinal center B cell response. We used RNA-seq to explore molecular mechanisms and identified 10 upregulated and 32 downregulated genes involved in IL-21-upregulated protection. Gene Ontology analysis and pathway analysis of the differentially expressed genes were also performed. Our findings indicate that IL-21 can enhance the immune protection effect of the VvhA protein and may serve as a novel strategy for enhancing the immune protection effect of protein vaccines.


INTRODUCTION
Vibrio vulnificus is a marine gram-negative bacterium that is responsible for fatal septicemia and necrotizing wound infections through either the ingestion of contaminated seafood or wound infections in humans [1][2][3]. The high mortality and severity of infection emphasize the need for effective approaches to prevent V. vulnificus infection. Although the pathogenic mechanism of V. vulnificus infection has not been fully delineated, several of its potential virulence factors have been extensively described, such as hemolysins, cytotoxins, proteolytic enzymes, flagella, and phospholipase A [4][5][6]. Hemolysin is a very powerful virulence factor of V. vulnificus and is a potential toxin candidate involved in the pathogenesis of V. vulnificus infection [7]. V. vulnificus hemolysin A (VvhA) toxin has cytolytic and hemolytic activity and is responsible for the death of host cells during the infection [8,9]. This toxin attaches to the host cell membrane to induce hemolysis by forming small ion-permeable pores via colloidal osmotic shock [10,11]. Moreover, VvhA may IL-21 Enhances the Immune Protection Induced by the Vibrio vulnificus Hemolysin A Protein contribute to bacterial invasion from the intestine to the bloodstream and other organs; furthermore, deletion of VvhA from V. vulnificus has been shown to attenuate its virulence [12].
In our previous study, we expressed and purified recombinant VvhA protein. Our results revealed that the VvhA protein elicited a good immune protection effect and could effectively control V. vulnificus infection. We also found that the protective mechanism of VvhA protein after immunization is closely related to T follicular helper (Tfh) cells and germinal center (GC) B cells [13]. Tfh cells have recently emerged as a separate subset of CD4 + T helper cells [14]. The major function of Tfh cells is to aid in B cell activation and antibody production during humoral immune responses, specifically via interactions between molecules on the surface of Tfh cells and receptors or ligands located on the surface of B cells [15]. We also found that serum IL-21 levels were dramatically increased in VvhA-immunized mice. IL-21 is a cytokine that is preferentially expressed by Tfh cells and serves as an important regulator of humoral responses by directly regulating B cell proliferation and class switching [16][17][18].
Therefore, IL-21 likely plays an important role in the immune protection of the VvhA protein. In the present study, we aimed primarily to investigate the protective effects of VvhA protein-immunized mice via the regulation of IL-21. Next, we explored the possible regulatory mechanisms involved. We expected to find a more effective strategy to enhance the protective effect of the VvhA protein.

Bacterial Strains and Growth Conditions
The V. vulnificus M06-24/O strain, kindly provided by Professor Sang Ho Choi, Seoul National University, Seoul, South Korea, was originally isolated from a patient with septicemia. The strain was first grown on thiosulfate citrate bile salt agar plates and grown in alkaline peptone water (APW, pH 8.5) supplemented with 2% (w/v) NaCl at 37 °C for 16 h. The bacterial cells were centrifuged at 2500 × g for 5 min and then resuspended in APW. The concentration of bacteria suspended in APW was adjusted to 2 × 10 6 colony-forming units (CFU)/mL prior to infection. The dosage for each mouse was 0.5 mL to achieve V. vulnificus infection.

Expression and Purification of VvhA Protein
VvhA was expressed and purified as described previously [13]. Briefly, vvhA DNA and pET28a ( +) were digested with the same enzymes that were recovered for ligation. Then, E. coil Rosetta (DE3) transformed with pET28a ( +)-vvhA was cultured in Luria-Bertani medium containing ampicillin (100 mg/L) for 2 h and induced with isopropyl β-d-1-thiogalactopyranoside (1.0 mmol/L) at 30 °C for 4 h. Bacterial cells were harvested by centrifugation, and the total cell protein was obtained by boiling; next, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed. The protein bands were visualized by Coomassie blue staining and confirmed by western blot analysis using anti-His tag antibodies. Finally, recombinant VvhA fusion protein was affinity-purified from total cell protein using Ni-nitrilotriacetic acid resin.

Animal Experiments
All animal experiments were approved by the Animal Ethics and Experimental Committee of the 960 th Hospital of the PLA Joint Logistics Support Force, Ji'nan, China. Six-to eight-week-old specific-pathogenfree female BALB/c mice weighing approximately 20 g were purchased from the Center of Experimental Animals at Shandong University (Ji'nan, China). The immunization protocol used here is as previously described [13]. Female BALB/c mice were immunized by subcutaneous injection with 100 μg of purified recombinant VvhA fusion protein mixed with Freund's complete adjuvant on day 1, followed by subcutaneous injections on days 7, 14, and 21 with 100 μg of purified recombinant VvhA fusion protein emulsified in 0.5 mL of Freund's incomplete adjuvant. Next, 50 μg of purified recombinant VvhA fusion protein solution was administered by the intraperitoneal route on day 25. On day 29, the mice were intraperitoneally injected with V. vulnificus M06-24/O strain and observed for 72 h. Six mice per group were used for experiments.

Treatment of Mice with IL-21 or IL-21 mAb
The method used to block IL-21 was as previously described [19]. Briefly, mice were intraperitoneally injected with 10 µg of mouse IL-21 mAb (Clone 149,204, rat IgG2b, R&D Systems, Minneapolis, MN, USA) as the IL-21-downregulated group or with 0.5 µg of recombinant mouse IL-21 (R & D Systems) as the IL-21-upregulated group on days 3, 9, 16, 23, and 27. VvhA protein-immunized mice in the control group received an equal volume of phosphate-buffered saline alone.

Histopathological Evaluation
The treated mice were dissected at 72 h after intraperitoneal injection of V. vulnificus, and the liver and lung tissues were collected for hematoxylin and eosin (H&E) staining. The intensity of inflammation was evaluated in a blinded fashion by two pathologists according to established criteria [20]. Each section was scored from 0-5.

RNA Sequencing
Total RNA of the lymph node cells was isolated using an RNeasy Mini kit (Qiagen, Germany). Pairedend libraries were synthesized using a TruSeq™ RNA sample preparation kit (Illumina, USA) according to the manufacturer's instructions. Purified libraries were quantified by a Qubit® 2.0 Fluorometer (Life Technologies, USA) and validated by an Agilent 2100 bioanalyzer (Agilent Technologies, USA) to confirm the insert size and calculate the molar concentration. The library construction and sequencing were performed at the Shanghai Genminix Information Technology Co., Ltd (Shanghai, China). The IL-21-upregulated group served as the experimental group and the VvhA-immunized group served as the control group. Ballgown was used to compare the two groups, and fold change between the genes was determined [21]. Differentially expressed genes were selected on the basis of a fold change threshold set to > 2. Upregulated and downregulated genes could then be classified by the fold change intensities.

RT-qPCR
Total RNA was isolated from lymph node cells using TRIzol (Invitrogen, Carlsbad, CA, USA). RNA samples were subjected to DNase treatment (Promega, Madison, WI, USA) at 37 °C for 15 min and reversedtranscribed to cDNA in a PrimeScript™ RT reagent kit (TaKaRa, Japan). Expression levels of CXCR5, TLR7, CCR5, and CCR2 were analyzed using the SYBR green RT-qPCR method on a LightCycler instrument (Light-Cycler 480, Roche Diagnostics, Mannheim, Germany). Table 1 lists the primers used for the RT-qPCR. The thermal cycling conditions were as follows: an initial denaturation step at 94 ºC for 1 min; followed by 40 cycles of denaturation at 95 ºC (5 s), annealing at 60 °C (5 s), and extension at 72 °C (30 s). Fluorescence data were collected at the end of the 60 °C primer annealing step for 40 amplification cycles. The specificity of the amplification  1498 IL-21 Enhances the Immune Protection Induced by the Vibrio vulnificus Hemolysin A Protein product was verified by melting curve analysis after the process was complete. Gene expression was normalized against that of mouse GAPDH, and the relative gene expression was expressed as "fold change" using the 2 -ΔΔCt method [22].

Statistical Analysis
Data were expressed as means ± standard deviation (SD). Student's t test was used to analyze the differences between the two groups, but when the variances differed, the Mann-Whitney U test was used. A P value < 0.05 was considered statistically significant. Survival analysis was calculated with the Kaplan-Meier method with a log-rank test for statistical significance.

Survival Rate of Mice After IL-21 Regulation
To investigate the function of IL-21 in VvhA protein immunization, immunized mice were intraperitoneally injected with IL-21 mAb or recombinant IL-21. At 72 h after infection with V. vulnificus, the survival rates of the mice were monitored. As shown in Fig. 1, four mice died within 36 h of treatment with IL-21 mAb, and the survival rate of the mice was 33.3%. Two mice died within 30 h after V. vulnificus infection in the VvhA immunization group, and the survival rate was 66.7%, whereas all the mice in the IL-21-upregulated group survived, and the survival rate in this group was 100%. These results suggested that IL-21 enhanced the protective effect after recombinant VvhA protein immunization.

Histopathological Evaluation
To explore the effect of IL-21 regulation on tissues and organs, liver and lung tissues with severe injury after V. vulnificus infection were sectioned to perform H&E staining. The results showed that liver injury was the most serious in mice after they were injected with IL-21 mAb, and this liver injury was accompanied with central venous congestion, severe osteoporosis, swelling, necrosis, and inflammatory cell infiltration in hepatocytes (Fig. 2a). Simultaneously, the alveolar walls were severely thickened, the alveolar cavities disappeared, and a large number of inflammatory cells were infiltrated in the lungs (Fig. 2b). The VvhA immunization group showed only slight changes in inflammation, whereas there were no significant changes in the IL-21-upregulated group both in liver and lung tissues. Histological scores of liver and lung sections are shown in Fig. 2c and d. These data implied that improving the function of IL-21 can enhance the immune protection effect of VvhA protein and reduce the damage caused by V. vulnificus infection in mice.

IL-21 Can Activate Tfh Cells and GC B Cells in Mice
To further explore the immune protective mechanism of IL-21, the ratio of Tfh cells and GC B cells was analyzed using flow cytometry in mouse lymph nodes treated with IL-21. The results revealed a higher ratio of Tfh cell and GC B cells in the IL-21-upregulated group compared to the other two groups (P < 0.05, Fig. 3), suggesting that IL-21 could activate Tfh cells and GC B cells in VvhA-immunized mice.

Differential Genes Regulated by IL-21
To gain insights into the mechanism of IL-21-induced protection, we performed RNA-seq to identify transcriptional targets of IL-21 upregulated in VvhA-immunized mice. After data analysis, the mRNA expression was clustered using Cluster 3.0 software. IL-21 treatment was found to promote the upregulation of 1145 genes and the downregulation of 2844 genes compared with the control group. The differentially expressed genes were screened by the threshold of 2.0-fold change and a P value lower than 0.05. All the differentially expressed genes have been submitted as supplementary material. Among these regulated genes, 42 were further screened out, including 10 upregulated genes, such as CXCR5, TLR7, CCR2, and CCR5, and 32 downregulated genes, such as CCR7, JAK1, and PKN (Fig. 4).

Gene Ontology (GO) Analysis and Pathway Analysis of Differential Genes
To further explore the function of IL-21 in gene regulation, we conducted GO analysis to identify general functional features implemented by the IL-21-upregulated group using the above-mentioned 42 genes. The threshold of significance was defined by a P value < 0.05. The highly significant GOs of 10 differentially upregulated genes included immune response, T cell differentiation, and chemotaxis (Fig. 5a). The highly significant GOs of 32 differentially downregulated genes included regulation of transcription, phosphorylation, and protein phosphorylation (Fig. 5b). Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that the 10 upregulated genes were significantly associated with cytokine-cytokine receptor interaction, chemokine signaling pathway, and the NF-kappa B signaling pathway (Fig. 6a). The 32 downregulated genes were significantly associated with cancer, the MAPK signaling pathway, and the PI3K-Akt signaling pathway (Fig. 6b).

Gene Expression Validation by RT-qPCR
To confirm the results of the RNA-seq, RT-qPCR was applied to further examine the expressions of target genes. The CXCR5, TLR7, CCR5, and CCR2 genes were chosen for further analysis using GAPDH to normalize gene expression. The results showed significantly higher CXCR5, TLR7, CCR5, and CCR2 mRNA expression in the IL-21-treated group compared to the VvhAimmunized group (P < 0.05, Fig. 7).

Fig. 4
Hierarchical clustering of 42 genes (fold change > 2.0, P < 0.05) altered by IL-21 in mouse lymph node cells. The horizontal axis represents the sample names between groups, blue represents the VvhA-immunized mice as the control group, and pink represents the upregulated IL-21 in VvhA-immunized mice as experimental group. The vertical axis represents the differentially expressed genes. Red represents the high expression of the differentially expressed genes, and green represents the low expression of differentially expressed genes. Statistically significant differences between the VvhA-immunized mice and upregulated IL-21 in VvhA-immunized mouse groups are indicated by P < 0.05. Con = control group; Exp = experimental group.

DISCUSSION
Vaccines are an effective, safe, and economical measure to prevent bacterial or viral infection. Our previous study proved that the VvhA protein elicited good immune protection, and the serum levels of IL-21 were dramatically increased in the VvhA-immunized mice [13]. IL-21 plays a key role in CD4 + Tfh cells in regulating B cell function, including antibody maturation and conversion between Ig isotypes and IgG isotypes [23,24]. A previous study reported that IL-21 as an immune adjuvant can significantly increase antibody quality induced by heterologous DNA-MVA primeboost vaccine [25]. Another study also reported that IL-21 enhanced influenza vaccine responses in aged macaques with suppressed SIV infection [26]; however, few reports about protein vaccines against bacterial infection have been published. In our study, IL-21 upregulation was found to increase the survival rate of mice and reduce liver and lung tissue injury, suggesting that IL-21 could enhance the immune protection of the VvhA vaccine.  IL-21 can promote the differentiation of CD4 + T cells into Tfh cells and is a critical regulator of Tfh development. In addition, the IL-21 receptor (IL-21R) is composed of heterogenous IL-21R α-and γ-chain subunits, which are expressed on the surfaces of several types of cells, including B cells, Tfh cells, Th17 cells, and NK T cells. However, B cells are its primary target [16,27,28]. Here, to explore the immune protective mechanism of IL-21, we detected changes in the percentages of Tfh and GC B cells in mouse lymph nodes using flow cytometry. The results revealed that IL-21 upregulation could significantly increase the expression of Tfh cells and GC B cells. However, the gene that is involved in IL-21-mediated immune protection remains to be unknown.
Next, we further detected the changes in gene expression profiles of lymphocytes in IL-21-regulated model mice by RNA sequencing. The results showed that 10 genes including CXCR5, TLR7, CCR2, and CCR5 were significantly upregulated, while 32 genes including CCR7, JAK1, and PKN1 were significantly downregulated. CXCR5 is a surface chemokine receptor that is stably and persistently expressed in Tfh cells. After antigen stimulation, T cells downregulated the expression of CCR7 and upregulated the expression of CXCR5 [29]. Upregulated CXCR5 expression promotes the migration and localization of Tfh cells in the GC, where Tfh cells interact with B cells to activate B cells [30,31].
Toll-like receptors are a class of pattern recognition receptors that play a pivotal role in bridging innate and adaptive immunity [32,33]. TLR7, which is primarily identified as the sensor for single-stranded RNA virus during viral infections, can also be triggered by immune modifiers that share a similar structure to nucleosides during bacterial infections [34,35]. Moreover, vaccines that are formulated with a Toll-like receptor 7-dependent adjuvant can induce a high level and broad range of protection against S. aureus [36]. These findings suggest that TLR7 signaling might play an important role in the host innate immune response during bacterial infection.
Chemokine receptor-2 (CCR2) is a specific receptor for monocyte chemotactic protein-1 (MCP-1), which belongs to the G-protein-coupled receptor superfamily. CCR2 is a major chemokine receptor that induces monocyte/macrophage recruitment to inflammatory sites [37]. CCR5 is a β-chemokine receptor that can trigger numerous types of JAK-STAT signal transduction pathways depending on cell types, and it plays a specific role in mediating antigen presentation of dendritic cells and T cell activation [38]. Here, we demonstrated that IL-21 could upregulate the expression of CCR2 and CCR5, perhaps by recruiting monocytes/macrophages to the inflammatory site or activating T cells to resist the invasion of V. vulnificus.
In conclusion, IL-21 can enhance the immune protection effect of the VvhA protein vaccine, which may effectively control the infection of V. vulnificus. Exploring the potential mechanism will open up new ideas and provide new evidence for enhancing the immune protection effect of protein vaccines.

SUPPLEMENTARY INFORMATION
The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s10753-022-01632-1. Fig. 7 RT-qPCR analysis of gene expression in mouse lymph node cells. VvhA-immunized mice were treated with IL-21, and the total RNA was extracted from the lymph node cell. The gene expression was analyzed using the SYBR green RT-qPCR method. Data (fold change) are expressed as mean ± SD (n = 3). *P < 0.05 compared with the control group.