Intranasal Vaccination with rePcrV Protects against <i>Pseudomonas aeruginosa</i> and Generates Lung Tissue-Resident Memory T Cells

Ou, Yangxue; Wang, Ying; Yu, Ting; Cui, Zhiyuan; Chen, Xin; Zhang, Weijun; Zou, Quanming; Gu, Jiang; Zuo, Qianfei

doi:https://doi.org/10.1155/2022/1403788

Journal of Immunology Research

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Abstract Introduction Materials and Methods Results Discussion Data Availability Ethical Approval Conflicts of Interest Authors’ Contributions Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2022 | Article ID 1403788 | https://doi.org/10.1155/2022/1403788

Intranasal Vaccination with rePcrV Protects against Pseudomonas aeruginosa and Generates Lung Tissue-Resident Memory T Cells

Yangxue Ou,¹Ying Wang,²Ting Yu,¹Zhiyuan Cui,¹Xin Chen,¹Weijun Zhang,¹Quanming Zou,¹Jiang Gu,¹and Qianfei Zuo¹

Academic Editor: Lele Zhu

Received20 Jun 2022

Revised09 Oct 2022

Accepted11 Nov 2022

Published26 Nov 2022

Abstract

Tissue-resident memory T (T_RM) cells are immune sentinels that bear a key role in the local immune system and rapidly respond to infection. Our previous studies showed that mucosal immunization via intranasal pathways was more effective than intramuscular route. However, the mechanism of enhanced protective immunity remains unclear. Here, we formulated a Pseudomonas aeruginosa vaccine composed of type III secretion protein PcrV from P. aeruginosa and curdlan adjuvant and then administered by the intranasal route. Flow cytometry and immunofluorescence staining showed that the ratio of CD44⁺CD62L^-CD69⁺CD4⁺ T_RM cells induced by this vaccine was significantly increased, and IL-17A production was notably enhanced. Further analysis revealed that vaccinated mice can protect against the P. aeruginosa challenge even after administration with FTY720 treatment. What is more, our results showed that CD4⁺ T_RM might be involved in the recruitment of neutrophils and provided partial protection against Pseudomonas aeruginosa. Taken together, these data demonstrated that CD4⁺ T_RM cells were elicited in lung tissues after immunization with rePcrV and contributed to protective immunity. Furthermore, it provided novel strategies for the development of vaccines for P. aeruginosa and other respiratory-targeted vaccines.

1. Introduction

Pseudomonas aeruginosa (P. aeruginosa), a prevalent opportunistic pathogen and Gram-negative bacteria, tends to cause acute and chronic severe pulmonary infections [1–3]. P. aeruginosa infections are particularly problematic in mechanically ventilated patients, chronic obstructive pulmonary disease (COPD) patients, and cystic fibrosis (CF) patients [4–9]. Recently, the emergence of multidrug-resistant (MDR) P. aeruginosa has become a serious clinical challenge, posing a serious threat to effective infection control in clinical [9–11]. Over the past decades, enormous efforts have been focused on P. aeruginosa vaccines. Regrettably, no approved vaccines are available for treatment of P. aeruginosa infections [12], because of its high diversity and variability.

Tissue-resident memory (T_RM) cells are a new subpopulation of memory T cells recently identified, which embedded within peripheral tissues [13–15]. T_RM cells serve as immune sentinels at the respiratory tract and provide rapid and broad-spectrum protective effects against a variety of respiratory infection pathogens [15–17]. Induction of memory T and B cells has now been widely accepted as the principal disciplines for effective vaccine design which could provide robust protective immunity against pathogens caused by prior infection [18–20]. Both CD4 and CD8 T_RM reside in mucosal could be produced by natural infection [21]; however, natural infection could be lethal. Thus, finding an effective way to induce highly protective T_RM cells could be an ideal choice especially for the prevention of P. aeruginosa. Previous study showed the type of vaccines and adjuvants, and the route of vaccination could influence the efficacy of T_RM. For pulmonary infectious diseases, mucosal immunization via the intranasal pathways is more effective than intramuscular route in inducing and stimulating immune protection of T_RM [20, 22].

Th17 has been regarded as a major player in the anti-P. aeruginosa immunity; indeed, in our previous study, we identified a soluble P. aeruginosa antigen called rePcrV which could induce Th17 response and provide protection against P. aeruginosa by intranasal immunization [23]. Another substrate, 1,3-β-glucan, derived from Alcaligenes faecalis, has also been reported to prompt a Th1/Th17 response [24, 25]. Therefore, we combined rePcrV and 1,3-β-glucan supplemented with curdlan as an adjuvant. After immunization with the vaccine by intranasal administration, we observed that the ratio of CD44⁺CD62L^-CD69⁺CD4⁺ T_RM cells induced by this vaccine was significantly increased, and IL-17A production of this subpopulation was notably enhanced after in vitro stimulation. Vaccinated mice infected with P. aeruginosa showed a sharp reduction in the bacterial burden. What is more, our results showed that CD4⁺ T_RM may involve the recruitment of neutrophils and provide partial protection against P. aeruginosa. Better understanding the underline mechanism could provide new strategies for the development of vaccines for P. aeruginosa and other respiratory-targeted vaccines.

2. Materials and Methods

2.1. Animals and Strains

Adult female C57BL/6 mice (6-8 weeks) were purchased from Beijing HFK Bioscience Limited Company. Adult female CD8 KO (Cd8a^tm1Mak) mice and adult female μMT mice were obtained from Army Medical University. Adult female CB-17 SCID mice (CB17/Icr-Prkdc^scid/IcrlcoCrl) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were bred in-house under specific pathogen-free (SPF) conditions at Army Medical University, Department of Microbiology and Biochemical Pharmacy. All animal studies were approved by the Animal Ethical and Experimental Committee of the Army Medical University. P. aeruginosa XN-1 was isolated in the Southwest Hospital of Army Medical University.

2.2. Immunization Procedure

For active immunization, adult female mice were vaccinated intranasally (i.n.) with 20 μL of curdlan (10 mg/mL, Sigma) or purified proteins (25 μg/mouse) plus curdlan (10 mg/mL, Sigma), on days 0, 14, and 21. Mice were challenged at day 35 and were anesthetized with isoflurane or pentobarbital sodium followed by the intratracheal injection of P. aeruginosa XN-1. The lethal dose of P. aeruginosa XN-1 was per mouse. The sublethal dose of P. aeruginosa XN-1 was per mouse.

2.3. FTY720 Treatment

FTY720 (Cayman Chemical) dissolved in saline was continuously administered i.p. (0.5 mg/kg) to mice for a period of 7 d before infection [26].

2.4. Isolation of Lung Lymphocyte

At day 36 after treatment, mice were sacrificed under overdose isoflurane. The lungs were dissociated with collagenase D (150 UmL^-1, Gibco) and DNase I (1 unit/μL, Sigma) at 37°C on a rocker at 260 rpm for 1 hour. Then, lung tissues were transferred to a 70 μm cell strainer (Beyotime) to obtain cell suspensions. Monocytes were separated using by Percoll (Cytiva) [27].

2.5. Flow Cytometry

Mice were intravenously injected with 3 μg APC/Cy7 anti-mouse CD45 (BioLegend) diluted in 300 μL saline [28], 10 min before euthanasia. Then, lung mononuclear cells were stimulated with leukocyte activation cocktail, with BD GolgiPlug (BD Pharmingen™) for 4-6 h. PerCP/Cyanine5.5 anti-mouse CD4 (BioLegend), PE/Cy7 anti-mouse CD44 (BioLegend), FITC anti-mouse CD69 (BioLegend), and PE anti-mouse CD62L (BioLegend) were used for cell surface marker staining. APC anti-mouse IL-17A (BioLegend) and Brilliant Violet™510 anti-mouse IFN-γ (BioLegend) were used for intracellular staining. Zombie NIR™ Fixable Viability Kit (BioLegend) was used to distinguish between living and dead cells. For RNA-profiling, CD4⁺CD44⁺CD69⁺CD62L^- cells were sorted into DMEM (Gibco) with 20% fetal bovine serum (FBS, Gibco) on ice using BD FACS Aria II SORP before RNA extraction.

2.6. Real-Time PCR

RNA was extracted from sorted CD4⁺CD44⁺CD69⁺CD62L^- cells using MicroElute Total RNA Kit (OMEGA) according to the manual and stored at -80°C. Hobit, Blimp-1, RORγt, and T-bet were quantified using QuantiTect Probe RT-PCR Kit (200) (Qiagen) with SYBR-Green. The primers used were as follows: Hobit, forward: 5-CTCAGCCACTTGCAGACTCA-3, reverse: 5-CTGTCGGTGGAGGCTTTGTA-3; Blimp-1, forward: 5-TTCTCTTGGAAAAACGTGTGGG-3, reverse: 5-GGAGCCGGAGCTAGACTTG-3; RORγt, forward: 5-CAGAGGAAGTGTCAGAGGCT-3, reverse: 5-TGCAAATGTGAAGTGCCAGC-3; and T-bet, forward: 5-CATGCCAGGGAACCGCTTAT-3, reverse: 5-TTGGAAGCCCCCTTGTTGTT-3.

2.7. Histology and Immunofluorescence

The lungs were collected and fixed in 4% paraformaldehyde (Biosharp) and embedded in paraffin. Pathological changes were evaluated by hematoxylin and eosin stain (H&E stain) [29]. Anti-CD69 and anti-IL-17A were used for immunofluorescence staining of lung samples.

2.8. IL-7, IL-17A, and IFN-γ Neutralization

Mice were administrated 50 μg/mouse of an IL-7-neutralizing antibody (BioXCell, clone M25) at days 27, 30, 32, and 34 of the first immunization (day 0) [30]. IL-17A was blocked using 300 μg anti-mouse IL-17A mAb [31] (BioLegend, Clone TC11-18H10.1) administered i.v. into mice 2 d before P. aeruginosa XN-1 infection (at days 33 and 34). For neutralization of IFN-γ, mice were given intravenous injection 2 days of 300 μg anti-mouse IFN-γmAb [32] (BioLegend, Clone R4-6A2) before P. aeruginosa XN-1 infection (at days 33 and 34).

2.9. Neutrophil Depletion

Mice were daily injected intraperitoneally (i.p.) with anti-Ly6G antibody (BioXCell, clone 1A8, 50 μg/mouse) for a period of 7 d before challenge [33] (at days 28, 29, 30, 31, 32, 33, and 34).

2.10. Statistical Analysis

Data are presented as . Student’s -test and Mann–Whitney test were conducted, according to the data distribution. The survival rate was analyzed by the Kaplan-Meier survival curves. GraphPad Prism 8.0 (GraphPad Software) was used for data analyses. values less than 0.05 were considered significant.

3. Results

3.1. Intranasal Vaccination with rePcrV Enhanced Protection against P. aeruginosa Compared with Intramuscular Vaccination

PcrV has been proved to have immune protective effect by intramuscular or intraperitoneal immunization [34, 35]. In our study, we firstly compared the immune protective effects of these two different vaccination routes, intramuscular (i.m.) vaccination with the rePcrV protein formulated with aluminum adjuvant and intranasal (i.n.) immunization with curdlan. As expected, intranasal immunization route improved the efficacy of vaccine. The survival of the i.n. was higher () than the rate of i.m. at day 14 postinfection (Figure 1(a)). Then, mice were administrated a sublethal dose of P. aeruginosa. A histological analysis of lung tissues of rePcrV i.m. suggested a further increase in inflammatory cell infiltration. Meanwhile, the rePcrV i.n. showed significant reduction () in lung pathology score (Figure 1(b)). Furthermore, the bacterial burdens of the rePcrV i.n. were significantly decreased (rePcrV i.n. vs. rePcrV i.m. , Figure 1(c)). The mRNA expression of IL-6 (rePcrV i.n. vs. rePcrV i.m. , Figure 1(d)) and TNF-α (rePcrV i.n. vs. rePcrV i.m. , Figure 1(e)) was also reduced in rePcrV i.n. Thus, intranasal vaccination with rePcrV enhanced protection against P. aeruginosa compared with intramuscular vaccination.

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Figure 1

Intranasal vaccination (i.n.) with rePcrV enhanced protection compared with intramuscular vaccination (i.m.). (a) Schematic of the experimental protocol. The survival of immunized mice after challenge with the lethal dose () of P. aeruginosa XN-1 (). (b) H&E (hematoxylin and eosin) stain and histology scoring of pathology in lung tissues of immunized rePcrV + curdlan mice, immunized rePcrV + Al(OH)₃ mice, immunized curdlan mice, and vaccinated Al(OH)₃ mice (). (c) Lung CFU in vaccinated rePcrV + curdlan mice, vaccinated rePcrV + Al(OH)₃ mice, vaccinated curdlan mice, and vaccinated Al(OH)₃ mice (). (d) IL-6 mRNA level in the lungs of immunized mice (). (e) TNF-α mRNA level in the lungs of immunized mice (). Data are shown as . Significant differences were calculated with Student’s -test. ; ; ; .

3.2. CD4 T Cells Were Essential for rePcrV-Mediated Protection in P. aeruginosa Pneumonia

To inquire the role of lymphocyte-mediated immune responses during rePcrV-induced protection, adult female CB-17 SCID mice were vaccinated with rePcrV plus curdlan or rePcrV plus aluminum. Mice were challenged with P. aeruginosa XN-1 and were observed to survive for 14 days. As shown in Figure 2(a), there was no statistical difference () in survival rate between rePcrV-immunized SCID mice and -unimmunized mice, indicating that a complete lymphocyte system was required for protection after rePcrV immunization in P. aeruginosa pneumonia. In order to determine the relative requirements for humoral immunity and cellular immunity, rePcrV vaccine tested the protection in μMT mice (which lack mature B cells), CD8 T cell KO mice, and CD4-depleted mice (by intraperitoneal injection of anti-CD4 antibody GK1.5). As shown in Figure 2(b), rePcrV-immunized μMT mice were significantly protected () after P. aeruginosa XN-1 challenge, compared with unimmunized mice which were not protected. The result of CD8 T cell KO mice was the same (, Figure 2(c)). However, the rePcrV-immunized CD4-depleted mice (, Figure 2(d)) were not protected after P. aeruginosa XN-1 challenge. These data suggested the key role for CD4 T cells in mediating protection after immunization with rePcrV.

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Figure 2

CD4 T cells are essential for rePcrV-mediated protection in Pseudomonas aeruginosa pulmonary infection. (a) Survival in the rePcrV + curdlan vaccinated mice (immunized SCID i.n.), rePcrV + Al(OH)₃ mice (immunized SCID i.m.), and unimmunized SCID mice (). (b) Survival in the rePcrV + curdlan vaccinated mice (immunized μMT i.n.), rePcrV + Al(OH)₃ mice (immunized μMT i.m.), and unimmunized μMT mice (). (c) Survival in the rePcrV + curdlan vaccinated mice (immunized CD8^-/^- i.n.), rePcrV + Al(OH)₃ mice (immunized CD8^-/^- i.m.), and unimmunized CD8^-/^- mice (). (d) Survival in the rePcrV + curdlan vaccinated mice (immunized i.n. + anti-CD4), rePcrV + Al(OH)₃ mice (immunized i.m. + anti-CD4), rePcrV + curdlan vaccinated mice (immunized i.n. + control IgG), and unimmunized + control IgG mice (). Immunized 6-8-week-old female C57 mice were intraperitoneally (i.p.) treated with 200 μg of anti-GK1.5 Ab (BioXCell, to deplete CD4⁺ T cells) or isotype control (Rat IgG2b, κ, BioXCell) 2 days before vaccination and were administered weekly throughout the whole experiment to maintain CD4⁺ T cell depletion. All mice were challenged with of the P. aeruginosa XN-1 strain i.n. (in the nose) and i.m. (in the muscle). by log-rank test.

3.3. Intranasal Vaccination with rePcrV Initiates the CD4⁺ T_RM Cell Response

The result above showed that CD4⁺T cells are essential for the anti-P. aeruginosa immunity. However, it is still unknown whether circulating or resident CD4⁺ T cell is the major player. To this end, the lungs were dissociated into a single cell suspension and detected by flow cytometry. A dramatic increase in CD4⁺CD44⁺CD62L^-CD69⁺T_RM cells was observed in vaccinated mice compared with unimmunized mice (, Figure 3(a)). Transcriptional analysis of T_RM cells showed that they expressed a unique transcription factor profile. Since Hobit together with Blimp-1 regulates the differentiation and maintenance of T_RM cells [36], we purified T_RM cells from immunized or unimmunized mice and determined the level of Hobit, Blimp-1, RORγt, and T-bet mRNA. As shown in Figure 3(b), the level of Hobit, Blimp-1, and RORγt was increased in mice immunized with rePcrV compared with unimmunized (, respectively). To examine the expression of IL-17A production in CD4⁺ T_RM cells, we employed immunofluorescence staining. The result revealed that the IL-17A expression was enhanced in immunized mice (Figures 3(a) and 3(c)). Representative gating strategies were shown in figure S1.

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Figure 3

Intranasal vaccination with rePcrV primes the CD4⁺ T_RM cells response. (a) Schematic of the experimental protocol. Representative intracellular staining profiles and pooled data of IL-17A and IFN-γ in CD4⁺CD44⁺CD69⁺CD62L^- T cells in the lungs of immunized mice (rePcrV + curdlan i.n.) or unimmunized mice (). (b) Hobit, Blimp-1, ROR-γt, and T-bet expressions of CD4⁺ T cells in the lung tissues of immunized mice (rePcrV + curdlan i.n.) or unimmunized mice (). (c) Representative immunofluorescence images of the lung tissues stained with DAPI (blue), anti-CD69 (green), and anti-IL-17A (red) from immunized mice (rePcrV + curdlan i.n.) or unimmunized mice (). Data are presented as . values were calculated by Student’s -test. ; ; ; .

3.4. CD4⁺ T_RM Cells Partially Protected against Pseudomonas aeruginosa Pulmonary Infection

To exclude the contribution of circulating memory cells to the recall responses, we administered FTY720 [37, 38] (a S1P inhibitor that blocks the egress of T cells from repositioning from secondary lymphoid organs to the tissue). We found that FTY720 treatment followed by a P. aeruginosa XN-1 challenge induced higher survival in immunized mice () but not in unimmunized mice (Figure 4(a)). Vaccine efficacy was maintained in vaccinated mice with FTY720 treatment, as measured indirectly by global disease score (Figure 4(b)) and weight loss (Figure 4(c)). Furthermore, the bacterial load of immunized mice treated with FTY720 decreased significantly (, Figure 4(d)). In contrast, immunized mice significantly alleviated pathological damage (, Figure 4(e)). It should be noted that, compared with immunized mice without FTY720 treatment, immunized mice with FTY720 treatment diminished partial protection, which suggested that circulating T cells also played a role in preventing P. aeruginosa infection.

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Figure 4

CD4⁺ T_RM cells protect against Pseudomonas aeruginosa pulmonary infection. (a) The survival of naïve C57BL/6 mice with or without FTY720 treatment and rePcrV-immunized mice with or without FTY720 treatment (). by log-rank test. (b) Global disease score in 4 groups of mice after challenge with of P. aeruginosa XN-1 (). (c) Weight loss in the naïve mice with or without FTY720 treatment and rePcrV-immunized mice with or without FTY720 treatment after challenge with of P. aeruginosa XN-1 (). (d) Lung CFU 24 hours after infection in the naïve mice with or without FTY720 treatment and rePcrV-immunized mice with or without FTY720 treatment (). (e) Representative H and E stains in naïve mice with or without FTY720 treatment and rePcrV-immunized mice with or without FTY720 treatment lung 24 hours post-P. aeruginosa XN-1 challenge are shown () (). Significant differences are designated by using Student’s -test. ; ; ; .

3.5. rePcrV Vaccine Efficacy Depended on IL-17A Expression by CD4⁺ T_RM Cells and Remained Independent of IL-7

Lung CD4⁺ T_RM cells in vaccinated mice with FTY720 treatment showed higher level of IL-17A secretion compared with cells from FTY720-treated unimmunized mice (, Figures 5(a) and 5(b)). We treated mice with anti-IL-17A antibody and anti-IFN-γ antibody before and during vaccination to determine whether IL-17A or IFN-γ was required for rePcrV vaccine efficacy in the lungs. Anti-IL-17A-immunized mice were not protected against the challenge of P. aeruginosa XN-1 (Figure 5(c)). In line with this, the histological analysis of the lung tissues of anti-IL-17A-immunized mice revealed a further increase of peribronchial inflammatory cell infiltration (, Figure 5(d)).

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Figure 5

rePcrV vaccine efficacy depends on the IL-17A expression by CD4⁺ T_RM cells. (a) The representative dot plots showed CD4⁺CD44⁺CD69⁺CD62L^- T cells in the lungs of rePcrV-immunized mice with FTY720 treatment or naïve C57BL/6 mice with FTY720 treatment. The graph indicates the number of CD4⁺CD44⁺CD69⁺ CD62L^- T cells per mouse () found in lung tissues of naive and immunized mice with FTY720 treatment. Significant differences were calculated with Mann–Whitney test. (b) Representative immunofluorescence images of the lung stained with DAPI (blue), anti-CD69 (green), and anti-IL-17A (red) from naive and immunized mice with FTY720 treatment (). (c) Schematic of the experimental protocol. Survival in the FTY720 treatment-immunized mice with anti-IL-17A or IFN-γ Ab treatment (). by log-rank test. (d) Representative H and E stains in the FTY720 treatment-immunized mice with anti-IL-17A or IFN-γ Ab treatment (). Significant differences are designated by using Student’s -test. ; ; ; .

IL-7 signaling is regarded as a key mediator for homeostatic proliferation of CD4 T cells, which could explain the long-term and circulatory independent maintenance of T_RM cells. To assess whether IL-7 mediated the population expansion of T_RM cells and contributed to its survival, we applied a neutralizing antibody to IL-7 (anti-IL-7) at days 27, 30, 32, and 34 of the first immunization (day 0). The results showed that neutralization of IL-7 did not increase the bacterial load (, Figure 6(a)), and there was no statistical difference in histopathological examination between groups (, Figure 6(b)).

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3.6. Depletion of Neutrophils Impaired the Clearance of Pseudomonas aeruginosa from the Lung

Neutrophils are main orchestrators of lung inflammation and play a unique role in the connection between innate and adaptive immunity [39]. In order to investigate whether neutrophils play a role in CD4⁺ T_RM cells mediated protection against P. aeruginosa, neutrophils were deleted before challenge. The results showed that in the neutrophil depletion mice, CFU counts were increased in the lungs of mice treated with anti-Ly6G (, Figure 7(a)), and lung damage was worse (, Figure 7(b)). Flow cytometry showed that neutrophil depletion did not impact the CD4⁺ T_RM cell population (, Figure S2). These data indicated that CD4⁺ T_RM may be involved in recruitment of neutrophils and provided partial protection against P. aeruginosa.

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Figure 7

Depletion of neutrophils impairs clearance of Pseudomonas aeruginosa from the lung. (a) Experimental timeline. Lung CFU 24 hours after infection in FTY720 treatment mice with or without anti-Ly6G Ab and FTY720 treatment-immunized mice with or without anti-Ly6G Ab (). (b) Representative H and E stains in FTY720 treatment mice with or without anti-Ly6G Ab and FTY720 treatment-immunized mice with or without anti-Ly6G Ab (). Significant differences were calculated by using Student’s -test. ; ; ; .

4. Discussion

According to the different types of cytokines secreted, CD4⁺ T_RM cells are divided into Th1, Th2, or Th17 subtypes. Generally, CD4⁺ T_RM cells in viral infection and tumors mainly secreted IFN-γ, while CD4⁺ T_RM cells induced by bacterial or fungal infection mainly expressed IL-17A. The study indicated that dermal Candida albicans infection preferentially produces CD4⁺ IL-17A⁺ T_RM cells. When reinfected with Candida albicans, T_RM cells could rapidly clear infection challenges [40]. Previous work showed that lung T_RM cells were elicited by heat-killed K. pneumoniae [41]. By using IL-17A tracking-fate mouse models [42], CD4⁺ T_RM cells were found derived from effector Th17 cells [27]. Our previous study found that rePcrV could induce Th17 response and enhanced protection [23]. The results of this study initially demonstrate that rePcrV intranasal immunization could induce the generation of CD4⁺ T_RM cells secreting IL-17A in lung tissues of mice, and these cells produced a protective immune response after P. aeruginosa infection. Therefore, the origin of CD4⁺ IL-17A⁺ T_RM cells and their relationship with Th17 cells need to be further investigated in subsequent experiments.

FTY720 not only blocks the egress of T cells but also prevents migration of B cells from lymph nodes to the circulation [15, 43]. Indeed, FTY720 treatment appeared to affect bacterial burdens and survival in the immunized group, suggesting that circulating T cells or antibody-producing cells were also required in preventing P. aeruginosa infection. However, treatment with FTY720 did not affect T_RM cell expansion in the lungs. Our data showed that there was no statistical significance between mice with FTY720 treatment and mice without FTY720 treatment (Figure S3).

Long-term survival in peripheral tissues is another important characteristic of T_RM cells [13, 14, 44]. Furthermore, researches showed that the survival and expansion of T_RM cells in peripheral tissues were mainly regulated by the local immune microenvironment. The formation of the local microenvironment was associated with the involvement of multiple cytokines, such as IL-2, IL-7, IL-15, and TGF-β [45–47]. It also showed that multiple correlated signaling pathways may be involved in the maintenance induction of T_RM cells in peripheral tissues, including PI3K/Akt, JAK/STAT5, and Notch signal pathways [48]. In our research, we found that neutralization of IL-7 did not affect rePcrV vaccine efficacy, and there was no significance in the bacterial load (Figure 6). Regrettably, our work did not yet clarify the mechanisms of T_RM cell survival and amplification. We will continue to explore them in the future.

Studies have reported that the acellular pertussis vaccine vaccinated with intramuscular injection has a relatively short immunoprotection period and has no obvious effect on the colonization and transmission of B. pertussis in the nasal cavity [49]. On the contrary, nasal inoculation of attenuated pertussis vaccine BPZE1 can resist the infection caused B. pertussis [50]. Comparing the intranasal or injected influenza vaccines, we found that the route of administration and the type of vaccines (inactivated vaccine and live vaccine) also affect the production of CD4⁺ T_RM cells. Nasal vaccination with a live attenuated influenza vaccine (FluMist) induced antigen-specific CD4⁺ T_RM cells in the lung, mediating long-term protection against heterologous influenza virus strains. However, inactivated influenza virus vaccine (Fluzone) did not elicit T_RM cell production after nasal inoculation but induced strain-specific neutralizing antibody production [50]. Hence, choosing the appropriate vaccination route and vaccine type is an important means to induce respiratory T_RM cell production.

Data Availability

All the data are included in this paper.

Ethical Approval

The animal study was reviewed and approved by the Animal Ethical and Experimental Committee of the Army Medical University.

Conflicts of Interest

The authors declare no competing interests.

Authors’ Contributions

Y.X.O., Y.W., and Q.F.Z. designed the experiment and are responsible for data integrity and accuracy. T.Y., X.C., and Z.Y.C. contributed to the data generation and analysis. W.J.Z. and J.G. supervised the experiments. Y.X.O., Q.F.Z., J.G., and Q.M.Z. contributed to manuscript writing and revision. Yangxue Ou and Ying Wang contributed equally to this work and shared first authorship.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (31970877 and 32200771) and Chongqing Municipal Natural Science Foundation (cstc2019jcyj-msxmX0496, cstc2020jcyj-msxmX0296, 2022NSCQ-MSX4822, and 2021XZL02).

Supplementary Materials

Figure S1: representative gating strategies for CD44⁺CD62L^-CD69⁺CD4⁺ T_RM cells in the lungs. CD44⁺CD62L^-CD69⁺CD4⁺ T_RM cells were gated on live CD45^- cells. Figure S2: the graph indicates the number of CD4⁺CD44⁺CD69⁺ CD62L^- T cells per mouse () found in the lungs of FTY720 treatment-immunized mice with or without anti-Ly6G Ab. Figure S3: CD4⁺ T_RM cells protect against Pseudomonas aeruginosa pulmonary infection. (A) The representative dot plots showed CD4⁺CD44⁺CD69⁺CD62L^- T cells in the lungs of rePcrV-immunized mice with or without FTY720 treatment. (B) The graph indicates the number of CD4⁺CD44⁺CD69⁺ CD62L^- T cells per mouse () found in the lungs of immunized mice with or without FTY720 treatment. (Supplementary Materials)

References

H. Huang, X. Shao, Y. Xie et al., “An integrated genomic regulatory network of virulence-related transcriptional factors in Pseudomonas aeruginosa,” Nature Communications, vol. 10, no. 1, p. 2931, 2019.
View at: Publisher Site | Google Scholar
C. Ma, X. Ma, B. Jiang et al., “A novel inactivated whole-cell Pseudomonas aeruginosa vaccine that acts through the cGAS-STING pathway,” Signal Transduction and Targeted Therapy, vol. 6, no. 1, p. 353, 2021.
View at: Publisher Site | Google Scholar
E. Sen-Kilic, C. B. Blackwood, A. B. Huckaby et al., “Defining the mechanistic correlates of protection conferred by whole-cell vaccination against Pseudomonas aeruginosa acute murine pneumonia,” Infection and Immunity, vol. 89, no. 2, 2021.
View at: Publisher Site | Google Scholar
M. Bassetti, A. Vena, A. Croxatto, E. Righi, and B. Guery, “How to manage Pseudomonas aeruginosa infections,” Drugs Context, vol. 7, article 212527, pp. 1–18, 2018.
View at: Publisher Site | Google Scholar
L. L. Burrows, “The therapeutic pipeline for Pseudomonas aeruginosa infections,” ACS Infectious Diseases, vol. 4, no. 7, pp. 1041–1047, 2018.
View at: Publisher Site | Google Scholar
L. Nguyen, J. Garcia, K. Gruenberg, and C. MacDougall, “Multidrug-resistant pseudomonas infections: hard to treat, but hope on the horizon?” Current Infectious Disease Reports, vol. 20, no. 8, p. 23, 2018.
View at: Publisher Site | Google Scholar
M. D. Parkins, R. Somayaji, and V. J. Waters, “Epidemiology, biology, and impact of clonal Pseudomonas aeruginosa infections in cystic fibrosis,” Clinical Microbiology Reviews, vol. 31, no. 4, 2018.
View at: Publisher Site | Google Scholar
C. Rezzoagli, E. T. Granato, and R. Kummerli, “Harnessing bacterial interactions to manage infections: a review on the opportunistic pathogen Pseudomonas aeruginosa as a case example,” Journal of Medical Microbiology, vol. 69, no. 2, pp. 147–161, 2020.
View at: Publisher Site | Google Scholar
D. Shortridge, M. A. Pfaller, J. M. Streit, and R. K. Flamm, “Antimicrobial activity of ceftolozane/tazobactam tested against contemporary (2015-2017) Pseudomonas aeruginosa isolates from a global surveillance programme,” Journal of Global Antimicrobial Resistance, vol. 21, pp. 60–64, 2020.
View at: Publisher Site | Google Scholar
Z. J. Zhao, Z. P. Xu, Y. Y. Ma, J. D. Ma, and G. Hong, “Photodynamic antimicrobial chemotherapy in mice with Pseudomonas aeruginosa-infected wounds,” PLoS One, vol. 15, no. 9, article e0237851, 2020.
View at: Publisher Site | Google Scholar
C. J. L. Murray, K. S. Ikuta, F. Sharara et al., “Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis,” The Lancet, vol. 399, no. 10325, pp. 629–655, 2022.
View at: Publisher Site | Google Scholar
C. Merakou, M. M. Schaefers, and G. P. Priebe, “Progress toward the elusive Pseudomonas aeruginosa vaccine,” Surgical Infections, vol. 19, no. 8, pp. 757–768, 2018.
View at: Publisher Site | Google Scholar
S. N. Mueller, T. Gebhardt, F. R. Carbone, and W. R. Heath, “Memory T cell subsets, migration patterns, and tissue residence,” Annual Review of Immunology, vol. 31, no. 1, pp. 137–161, 2013.
View at: Publisher Site | Google Scholar
Q. P. Nguyen, T. Z. Deng, D. A. Witherden, and A. W. Goldrath, “Origins of CD4+ circulating and tissue-resident memory T-cells,” Immunology, vol. 157, no. 1, pp. 3–12, 2019.
View at: Publisher Site | Google Scholar
M. M. Wilk, A. Misiak, R. M. McManus, A. C. Allen, M. A. Lynch, and K. H. G. Mills, “Lung CD4 tissue-resident memory T cells mediate adaptive immunity induced by previous infection of mice with Bordetella pertussis,” Journal of Immunology, vol. 199, no. 1, pp. 233–243, 2017.
View at: Publisher Site | Google Scholar
K. Hirahara, K. Kokubo, A. Aoki, M. Kiuchi, and T. Nakayama, “The role of CD4⁺ resident memory T cells in local immunity in the mucosal tissue-protection versus pathology,” Frontiers in Immunology, vol. 12, article 616309, 2021.
View at: Publisher Site | Google Scholar
N. M. Smith, G. A. Wasserman, F. T. Coleman et al., “Regionally compartmentalized resident memory T cells mediate naturally acquired protection against pneumococcal pneumonia,” Mucosal Immunology, vol. 11, no. 1, pp. 220–235, 2018.
View at: Publisher Site | Google Scholar
P. Roychoudhury, D. A. Swan, E. Duke et al., “Tissue-resident T cell-derived cytokines eliminate herpes simplex virus-2-infected cells,” The Journal of Clinical Investigation, vol. 130, no. 6, pp. 2903–2919, 2020.
View at: Publisher Site | Google Scholar
H. Sun, C. Sun, W. Xiao, and R. Sun, “Tissue-resident lymphocytes: from adaptive to innate immunity,” Cellular & Molecular Immunology, vol. 16, no. 3, pp. 205–215, 2019.
View at: Publisher Site | Google Scholar
K. D. Zens, J. K. Chen, and D. L. Farber, “Vaccine-generated lung tissue-resident memory T cells provide heterosubtypic protection to influenza infection,” JCI Insight, vol. 1, no. 10, 2016.
View at: Publisher Site | Google Scholar
P. Ogongo, J. Z. Porterfield, and A. Leslie, “Lung tissue resident memory T-cells in the immune response to Mycobacterium tuberculosis,” Frontiers in Immunology, vol. 10, p. 992, 2019.
View at: Publisher Site | Google Scholar
D. H. Paik and D. L. Farber, “Anti-viral protective capacity of tissue resident memory T cells,” Current Opinion in Virology, vol. 46, pp. 20–26, 2021.
View at: Publisher Site | Google Scholar
Y. Wang, X. Cheng, C. Wan et al., “Development of a chimeric vaccine against Pseudomonas aeruginosa based on the Th17-stimulating epitopes of PcrV and AmpC,” Frontiers in Immunology, vol. 11, article 601601, 2020.
View at: Publisher Site | Google Scholar
S. I. Gringhuis, J. den Dunnen, M. Litjens et al., “Dectin-1 directs T helper cell differentiation by controlling noncanonical NF-κB activation through Raf-1 and Syk,” Nature Immunology, vol. 10, no. 2, pp. 203–213, 2009.
View at: Publisher Site | Google Scholar
T. Higashi, K. Hashimoto, R. Takagi et al., “Curdlan induces DC-mediated Th17 polarization via Jagged1 activation in human dendritic cells,” Allergology International, vol. 59, no. 2, pp. 161–166, 2010.
View at: Publisher Site | Google Scholar
T. Ichikawa, K. Hirahara, K. Kokubo et al., “CD103^hi T_reg cells constrain lung fibrosis induced by CD103^lo tissue-resident pathogenic CD4 T cells,” Nature Immunology, vol. 20, no. 11, pp. 1469–1480, 2019.
View at: Publisher Site | Google Scholar
M. C. Amezcua Vesely, P. Pallis, P. Bielecki et al., “Effector TH17 cells give rise to long-lived TRM cells that are essential for an immediate response against bacterial infection,” Cell, vol. 178, no. 5, pp. 1176–1188, 2019.
View at: Publisher Site | Google Scholar
N. Mato, K. Hirahara, T. Ichikawa et al., “Memory-type ST2⁺CD4⁺ T cells participate in the steroid-resistant pathology of eosinophilic pneumonia,” Scientific Reports, vol. 7, no. 1, p. 6805, 2017.
View at: Publisher Site | Google Scholar
K. Hirahara, M. Yamashita, C. Iwamura et al., “Repressor of GATA regulates T_H2-driven allergic airway inflammation and airway hyperresponsiveness,” The Journal of Allergy and Clinical Immunology, vol. 122, no. 3, pp. 512–520.e11, 2008.
View at: Publisher Site | Google Scholar
C. Le Saout, R. B. Hasley, H. Imamichi et al., “Chronic exposure to type-I IFN under lymphopenic conditions alters CD4 T cell homeostasis,” PLoS Pathogens, vol. 10, no. 3, article e1003976, 2014.
View at: Publisher Site | Google Scholar
E. Smith, M. A. Stark, A. Zarbock et al., “IL-17A inhibits the expansion of IL-17A-producing T cells in mice through “short-loop” inhibition via IL-17 receptor,” Journal of Immunology, vol. 181, no. 2, pp. 1357–1364, 2008.
View at: Publisher Site | Google Scholar
L. I. Terrazas, D. Montero, C. A. Terrazas, J. L. Reyes, and M. Rodriguez-Sosa, “Role of the programmed Death-1 pathway in the suppressive activity of alternatively activated macrophages in experimental cysticercosis,” International Journal for Parasitology, vol. 35, no. 13, pp. 1349–1358, 2005.
View at: Publisher Site | Google Scholar
A. T. Shenoy, G. A. Wasserman, E. I. Arafa et al., “Lung CD4⁺ resident memory T cells remodel epithelial responses to accelerate neutrophil recruitment during pneumonia,” Mucosal Immunology, vol. 13, no. 2, pp. 334–343, 2020.
View at: Publisher Site | Google Scholar
S. Hamaoka, Y. Naito, H. Katoh et al., “Efficacy comparison of adjuvants in PcrV vaccine against Pseudomonas aeruginosa pneumonia,” Microbiology and Immunology, vol. 61, no. 2, pp. 64–74, 2017.
View at: Publisher Site | Google Scholar
C. Wan, J. Zhang, L. Zhao et al., “Rational design of a chimeric derivative of PcrV as a subunit vaccine against Pseudomonas aeruginosa,” Frontiers in Immunology, vol. 10, p. 781, 2019.
View at: Publisher Site | Google Scholar
L. K. Mackay, M. Minnich, N. A. M. Kragten et al., “Hobit and Blimp1 instruct a universal transcriptional program of tissue residency in lymphocytes,” Science, vol. 352, no. 6284, pp. 459–463, 2016.
View at: Publisher Site | Google Scholar
B. D. Hondowicz, D. An, J. M. Schenkel et al., “Interleukin-2-dependent allergen-specific tissue-resident memory cells drive asthma,” Immunity, vol. 44, no. 1, pp. 155–166, 2016.
View at: Publisher Site | Google Scholar
J. Ryu, J. Jhun, M. J. Park et al., “FTY720 ameliorates GvHD by blocking T lymphocyte migration to target organs and by skin fibrosis inhibition,” Journal of Translational Medicine, vol. 18, no. 1, p. 225, 2020.
View at: Publisher Site | Google Scholar
G. K. Aulakh, “Neutrophils in the lung: “the first responders”,” Cell and Tissue Research, vol. 371, no. 3, pp. 577–588, 2018.
View at: Publisher Site | Google Scholar
C. O. Park, X. Fu, X. Jiang et al., “Staged development of long-lived T-cell receptor αβ T_H17 resident memory T-cell population to Candida albicans after skin infection,” The Journal of Allergy and Clinical Immunology, vol. 142, no. 2, pp. 647–662, 2018.
View at: Publisher Site | Google Scholar
K. Chen, J. P. McAleer, Y. Lin et al., “Th17 cells mediate clade-specific, serotype-independent mucosal immunity,” Immunity, vol. 35, no. 6, pp. 997–1009, 2011.
View at: Publisher Site | Google Scholar
N. Gagliani, M. C. A. Vesely, A. Iseppon et al., “Th17 cells transdifferentiate into regulatory T cells during resolution of inflammation,” Nature, vol. 523, no. 7559, pp. 221–225, 2015.
View at: Publisher Site | Google Scholar
K. Adachi and K. Chiba, “FTY720 story. Its discovery and the following accelerated development of sphingosine 1-phosphate receptor agonists as immunomodulators based on reverse pharmacology,” Perspectives in Medicinal Chemistry, vol. 1, pp. 11–23, 2007.
View at: Publisher Site | Google Scholar
P. A. Szabo, M. Miron, and D. L. Farber, “Location, location, location: tissue resident memory T cells in mice and humans,” Science Immunology, vol. 4, no. 34, 2019.
View at: Publisher Site | Google Scholar
T. Adachi, T. Kobayashi, E. Sugihara et al., “Hair follicle-derived IL-7 and IL-15 mediate skin-resident memory T cell homeostasis and lymphoma,” Nature Medicine, vol. 21, no. 11, pp. 1272–1279, 2015.
View at: Publisher Site | Google Scholar
T. Hirai, Y. Yang, Y. Zenke et al., “Competition for active TGFβ cytokine allows for selective retention of antigen-specific tissue- resident memory T cells in the epidermal niche,” Immunity, vol. 54, no. 1, pp. 84–98.e5, 2021.
View at: Publisher Site | Google Scholar
B. D. Hondowicz, K. S. Kim, M. J. Ruterbusch, G. J. Keitany, and M. Pepper, “IL-2 is required for the generation of viral-specific CD4+ Th1 tissue-resident memory cells and B cells are essential for maintenance in the lung,” European Journal of Immunology, vol. 48, no. 1, pp. 80–86, 2018.
View at: Publisher Site | Google Scholar
P. Hombrink, C. Helbig, R. A. Backer et al., “Programs for the persistence, vigilance and control of human CD8⁺ lung-resident memory T cells,” Nature Immunology, vol. 17, no. 12, pp. 1467–1478, 2016.
View at: Publisher Site | Google Scholar
J. M. Warfel, L. I. Zimmerman, and T. J. Merkel, “Acellular pertussis vaccines protect against disease but fail to prevent infection and transmission in a nonhuman primate model,” Proceedings of the National Academy of Sciences of the United States of America, vol. 111, no. 2, pp. 787–792, 2014.
View at: Publisher Site | Google Scholar
C. Locht, J. F. Papin, S. Lecher et al., “Live attenuated pertussis vaccine BPZE1 protects baboons against Bordetella pertussis disease and infection,” The Journal of Infectious Diseases, vol. 216, no. 1, pp. 117–124, 2017.
View at: Publisher Site | Google Scholar

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Copyright © 2022 Yangxue Ou et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Journal of Immunology Research

Intranasal Vaccination with rePcrV Protects against Pseudomonas aeruginosa and Generates Lung Tissue-Resident Memory T Cells

Abstract

1. Introduction

2. Materials and Methods

2.1. Animals and Strains

2.2. Immunization Procedure

2.3. FTY720 Treatment

2.4. Isolation of Lung Lymphocyte

2.5. Flow Cytometry

2.6. Real-Time PCR

2.7. Histology and Immunofluorescence

2.8. IL-7, IL-17A, and IFN-γ Neutralization

2.9. Neutrophil Depletion

2.10. Statistical Analysis

3. Results

3.1. Intranasal Vaccination with rePcrV Enhanced Protection against P. aeruginosa Compared with Intramuscular Vaccination

3.2. CD4 T Cells Were Essential for rePcrV-Mediated Protection in P. aeruginosa Pneumonia

3.3. Intranasal Vaccination with rePcrV Initiates the CD4+ TRM Cell Response

3.4. CD4+ TRM Cells Partially Protected against Pseudomonas aeruginosa Pulmonary Infection

3.5. rePcrV Vaccine Efficacy Depended on IL-17A Expression by CD4+ TRM Cells and Remained Independent of IL-7

3.6. Depletion of Neutrophils Impaired the Clearance of Pseudomonas aeruginosa from the Lung

4. Discussion

Data Availability

Ethical Approval

Conflicts of Interest

Authors’ Contributions

Acknowledgments

Supplementary Materials

References

Copyright

3.3. Intranasal Vaccination with rePcrV Initiates the CD4⁺ T_RM Cell Response

3.4. CD4⁺ T_RM Cells Partially Protected against Pseudomonas aeruginosa Pulmonary Infection

3.5. rePcrV Vaccine Efficacy Depended on IL-17A Expression by CD4⁺ T_RM Cells and Remained Independent of IL-7