BMB Reports 2019; 52(4): 289-294  https://doi.org/10.5483/BMBRep.2019.52.4.310
Ahnak-knockout mice show susceptibility to Bartonella henselae infection because of CD4+ T cell inactivation and decreased cytokine secretion
Eun Wha Choi1,2,*, Hee Woo Lee3, Jun Sik Lee4, Il Yong Kim5, Jae Hoon Shin5 & Je Kyung Seong5,6,*
1Department of Veterinary Clinical Pathology, College of Veterinary Medicine & Institute of Veterinary Science, Kangwon National University, Chuncheon 24341, 2Laboratory Animal Research Center, Samsung Biomedical Research Institute, Samsung Medical Center, Seoul 06351, 3Institute of Research and Development, Chaon Corp., Seongnam 13493, 4Department of Biology, Immunology Research Lab., College of Natural Sciences, Chosun University, Gwangju 61452, 5Laboratory of Developmental Biology and Genomics, BK21 Plus Program for Advanced Veterinary Science, Research Institute for Veterinary Science, College of Veterinary Medicine, and Korea Mouse Phenotyping Center, Seoul National University, Seoul 08826, 6Interdiscplinary Program for Bioinformatics, Seoul National University, Seoul 08826, Korea
Correspondence to: *Eun Wha Choi, Tel: +82-33-250-8794; Fax: +82-33-259-5625; E-mail: ewchoi@kangwon.ac.kr; Je Kyung Seong, Tel: +82-2-880-1259; Fax: +82-2-875-8395; E-mail: snumouse@snu.ac.kr
Received: December 20, 2018; Revised: February 15, 2019; Accepted: March 20, 2019; Published online: April 30, 2019.
© Korean Society for Biochemistry and Molecular Biology. All rights reserved.

cc This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

The present study evaluated the role of AHNAK in Bartonella henselae infection. Mice were intraperitoneally inoculated with 2 × 108 colony-forming units of B. henselae Houston-1 on day 0 and subsequently on day 10. Blood and tissue samples of the mice were collected 8 days after the final B. henselae injection. B. henselae infection in the liver of Ahnak-knockout and wild-type mice was confirmed by performing polymerase chain reaction, with Bartonella adhesion A as a marker. The proportion of B. henselae-infected cells increased in the liver of the Ahnak-knockout mice. Granulomatous lesions, inflammatory cytokine levels, and liver enzyme levels were also higher in the liver of the Ahnak-knockout mice than in the liver of the wild-type mice, indicating that Ahnak deletion accelerated B. henselae infection. The proportion of CD4+interferon-γ (IFN-γ)+ and CD4+interleukin (IL)-4+ cells was significantly lower in the B. henselae-infected Ahnak-knockout mice than in the B. henselae-infected wild-type mice. In vitro stimulation with B. henselae significantly increased IFN-γ and IL-4 secretion in the splenocytes obtained from the B. henselae-infected wild-type mice, but did not increase IFN-γ and IL-4 secretion in the splenocytes obtained from the B. henselae-infected Ahnak-KO mice. In contrast, IL-1α, IL-1β, IL-6, IL-10, RANTES, and tumor necrosis factor-α secretion was significantly elevated in the splenocytes obtained from both B. henselae-infected wild-type and Ahnak-knockout mice. These results indicate that Ahnak deletion promotes B. henselae infection. Impaired IFN-γ and IL-4 secretion in the Ahnak-knockout mice suggests the impairment of Th1 and Th2 immunity in these mice.

Keywords: AHNAK, Bartonella henselae, Cat scratch disease, Knockout mouse, Zoonosis
INTRODUCTION

Cat scratch disease (CSD), which is commonly transmitted by scratches or bites of cats or kittens (1, 2), is a zoonosis affecting people worldwide and is caused by Bartonella henselae (B. henselae) or possibly by B. clarridgeiae. CSD caused by B. henselae usually manifests regional lymphadenopathy in immunocompetent individuals; however, bacillary peliosis hepatis, bacillary splenitis and bacillary angiomatosis are also found in immunocompromised individuals (3, 4).

The prognosis of CSD is generally excellent even in debilitating patients. CSD in healthy individuals usually resolves spontaneously within months without permanent sequelae. However, it may be potentially life threatening for an individual with immunocompromised.

B. henselae is an intracellular pathogen; therefore, immunity against B. henselae is mediated by Th1 cells in immunocompetent hosts (3, 5). Interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) play a key role in B. henselae elimination (3, 6). In B. henselae-infected cats, Th2 cells are predominantly activated and result in the incomplete elimination of B. henselae (2). Therefore, these cats show prolonged B. henselae infection and act as reservoirs (7, 8).

AHNAK is the largest protein on our body and involved in the formation of cytoskeletal structure, muscular regeneration, and calcium homeostasis (9) and is involved in several biological processes. We previously reported that AHNAK is involved in obesity and cellular adipogenesis (1012). Moreover, AHNAK functions as a tumor suppressor protein to prevent the development of breast and lung cancers by inhibiting cancer cell growth through the potentiation of transforming growth factor-β (TGF-β) signaling pathway (13, 14). Immunologically, AHNAK is an essential component of calcium signaling during CD4+ T cell activation (15). Matza et al. reported that Ahnak-knockout (KO) mice showed impaired CD4+ T cell proliferation and decreased interleukin (IL)-2 production upon in vitro stimulation with an anti-CD3 antibody (15). However, the role of AHNAK in immune regulation and infections has not been completely understood. Therefore, we evaluated the immune responses of Ahnak-KO mice to B. henselae infection to elucidate whether these mice could be used as an animal model for CSD.

RESULTS

The four experimental groups were as follows: (i) control wild-type mice not infected with B. henselae (CW group), (ii) control Ahnak-KO mice not infected with B. henselae (CK group), (iii) wild-type mice infected with B. henselae (BW group), and (iv) Ahnak-KO mice infected with B. henselae (BK group).

Detection of B. henselae DNA in the liver tissues of the mice

PCR of badA indicated the presence of B. henselae DNA in the liver tissues of the mice in the BW and BK groups but not in the liver tissues of the mice in the CW and CK groups (Fig. 1A). badA PCR band density was not significantly different between the BW and BK groups (Fig. 1C).

Histopathological analysis of the liver tissues of the mice

B. henselae-induced histopathological changes were determined in the mice in the different study groups. A few B. henselae-infected cells were detected in the liver tissues of the mice in the BW group (Fig. 1B, D). However, many B. henselae-infected cells were detected in the liver tissues of the mice in the BK group (Mann-Whitney U test, P = 0.026; Fig. 1B, D). Granulomatous mononuclear cell infiltration was detected in the liver tissues of the B. henselae-infected mice on day 18 after initial infection with 2 × 108B. henselae, and the degree of this infiltration was significantly more severe in the mice in the BK group than in those in the BW group (Fig. 2A, B). Immunohistopathology for CD4, IFN-γ and TNF-α in the liver tissues are presented in Fig. S1.

Relative quantification of cytokine mRNA by using RNA extracted from the liver tissues

A significant difference was observed in the mRNA expression of the IFN-γ, TNF-α, and IL-10 genes relative to that of the β-actin gene in the liver tissues of the mice in the four study groups (Kruskal-Wallis test, P = 0.008, P = 0.023, and P = 0.009, respectively; Fig. 2C, D). The mRNA expression of the target genes was not significantly different between the liver tissues of the mice in the CW and CK groups. However, the mRNA expression of the IFN-γ gene was significantly higher in the mice in the BK group than in those in the BW group. The mRNA expression of the IL-10 gene was significantly higher in the mice in the BW group than in those in the CW group (Mann-Whitney U test, P = 0.024). Moreover, the mRNA expression of the IFN-γ and IL-10 genes was significantly higher in the mice in the BK group than in those in the CK group (P = 0.024 and P = 0.024, respectively).

Flow cytometric analysis of spleen cells

The proportion of B cells was not statistically significant among the groups (Fig. 3A). The proportion of CD4+ and CD8+ cells was significantly different among the mice in the different groups (Kruskal-Wallis test, P = 0.019 and P = 0.025, respectively). The proportion of CD4+ cells was significantly higher in the mice in the BK group than in those in the BW group (Mann-Whitney U test, P = 0.015; Fig. 3B). The proportion of CD8+ cells was significantly lower in the mice in the BK group than in those in the CK group (Mann-Whitney U test, P = 0.024; Fig. 3C). The proportion of IFN-γ+CD4+ and IL-4+CD4+ cells was significantly different among the mice in the different groups (Kruskal-Wallis test, P = 0.006 and P = 0.013, respectively). The proportion of IFN-γ+CD4+ cells was significantly lower in the mice in the BK group than in those in the BW group (Mann-Whitney U test, P = 0.004; Fig. 3D). The proportion of IL-4+CD4+ cells was also significantly lower in the mice in the BK group than in those in the BW group (Mann-Whitney U test, P = 0.026; Fig. 3E). Flow cytometric analysis profiles and gating strategies of Fig. 3C and D are presented in Fig. S2.

ELISA for determining the levels of multiple cytokines in the spleen cell culture supernatants and serum samples

IFN-γ and IL-4 levels were significantly higher in the culture supernatant of spleen cells obtained from the mice in the BW group and cultured in the presence of B. henselae than in that of spleen cells obtained from these mice and cultured in the absence of B. henselae (Wilcoxon signed-rank test, P = 0.043 and P = 0.028, respectively; Fig. 4). However, IFN-γ and IL-4 levels were not significantly different in the culture supernatants of spleen cells obtained from the mice in the BK group and cultured in the presence or absence of B. henselae (P = 0.068 and P = 0.068, respectively). IL-1α, IL-1β, IL-6, IL-10, RANTES, and TNF-α levels were significantly higher in the culture supernatants of spleen cells obtained from the mice in the BW and BK groups and cultured in the presence of B. henselae than in those of spleen cells of obtained from these mice and cultured in the absence of B. henselae. However, MCP-1 and VEGF levels were not significantly different between the culture supernatants of spleen cells obtained from the mice in the BW and BK groups and cultured in the presence of B. henselae than those of spleen cells obtained from these mice cultured in the absence of B. henselae. Furthermore, IFN-γ, IL-1β, IL-4, RANTES, and VEGF levels were significantly higher in the culture supernatant of spleen cells obtained from the mice in the BW group and cultured in the presence of B. henselae than in that of spleen cells obtained from the mice in the CW group and cultured in the presence of B. henselae (Mann-Whitney U test, P = 0.048, P = 0.048, P = 0.024, P = 0.048, and P = 0.048, respectively; Fig. 4). However, no significant difference was observed in the levels of these cytokines between the culture supernatants of spleen cells obtained from the mice in the CK and BK groups and cultured in the presence of B. henselae.

IL-10, MCP-1, and VEGF levels were significantly higher in the culture supernatant of spleen cells obtained from the mice in the BW group and cultured in the absence of B. henselae than in that of spleen cells obtained from the mice the CW group and cultured in the absence of B. henselae (Mann-Whitney U test, P = 0.048, P = 0.048, and P = 0.048, respectively; Fig. 4). However, no significant difference was observed in the levels of these cytokines between the culture supernatants of spleen cells obtained from the mice in the CK and BK groups and cultured in the absence of B. henselae.

TNF-α level was significantly different in the serum samples of the mice in the different groups (Kruskal-Wallis test, P = 0.046). The highest IFN-γ, IL-4, IL-6, IL-10, MCP-1, RANTES, TNF-α, and VEGF levels were detected in the serum samples of the mice in the BW group. However, the increase in the levels of these cytokines was not statistically significant, except for the increase in TNF-α level. TNF-α level was significantly higher in the serum samples of the mice in the BK group than in those of the mice in the CK group. However, no significant differences were observed in the levels of other cytokines between the serum samples of the mice in the CW and BW groups (Fig. S3).

Serum chemistry

ALT, AST, and globulin levels were significantly higher and albumin levels were significantly lower in the mice in the BK group than in those in the CK group (Fig. S4). ALT levels were significantly higher in the mice in the BW group than in those in the CW group.

DISCUSSION

A previous study reported that the liver was the most exclusively affected organ in B. henselae-infected C57BL/6 mice and that cultivatable organisms cleared in the spleen and liver homogenates of C57BL/6 mice 6 days after B. henselae inoculation (4). BadA is a trimeric autotransporter adhesion molecule expressed by B. henselae and is implicated in B. henselae infection (16). Therefore, we used badA as a marker to determine B. henselae infection in the liver tissues of the Ahnak-KO and wild-type mice. The number of B. henselae-infected cells increased in the liver tissues of the Ahnak-KO mice. Granulomatous lesions and inflammatory cytokine levels were higher in the liver tissues of the Ahnak-KO mice than in those of the wild-type mice. Moreover, B. henselae infection significantly increased liver enzyme levels, indicating that B. henselae infection induced slight liver damage. Moreover, the degree of the increase in the liver enzyme levels was higher in the Ahnak-KO mice than in the wild-type mice. Total globulin levels increase in the presence of an infection, chronic inflammation, low albumin levels, and other disorders. Therefore, the result obtained for globulin levels in the present study indicate higher degree of B. henselae infection in the Ahnak-KO mice than in the wild-type mice.

The Th phenotype of CD4+ T cells involved in infectious agent-induced cell-mediated immunity can be differentiated based on the cytokine secretion pattern of these cells (17); Th1 cells have a role in controlling intracellular pathogen infection by IL-2 and IFN-γ secretion, and macrophage activation induction. In contrast, Th2 cells have a role in helping B cells to elicit antibodies by IL-4, IL-5, and IL-13 secretion (18). Because B. henselae is an intracellular pathogen, immunity against this pathogen is mediated by Th1 cells in immunocompetent hosts (3, 5). B. henselae infection elicits a Th1-type immune response in humans and mice, which is characterized by increased IFN-γ and TNF-α secretion and subsequent nitric oxide production. Cellular immune responses are primarily mediated by CD4+ T cells and interfere with B. henselae pathogenesis. In the present study, we observed that B. henselae-induced systemic Th1 immune response decreased in the absence of Ahnak expression. Moreover, we observed that IFN-γ secretion was significantly elevated in the spleen cells obtained from the B. henselae-infected wild-type mice but was not elevated in the spleen cells obtained from the B. henselae-infected Ahnak-KO mice. B. henselae infection also significantly increased IL-4 secretion from Th2 cells in the culture supernatant of spleen cells obtained from the wild-type mice. Similar to IFN-γ, IL-4 production was not significantly increased in the spleen cells obtained from the Ahnak-KO mice infected with B. henselae. In contrast, TNF-α, IL-1β, and VEGF secretion from macrophages was not significantly different in spleen cells cultures in response to B. henselae infection irrespective of Ahnak deletion.

Calcium plays important roles in T cell activation and proliferation and cytokine production (19, 20). Matza et al. reported that 50% reduction in Cav1.1α1 membrane protein expression in TCR-stimulated Ahnak−/− T cells in the presence of IL-2 and AHNAK is critical for Cav1 channel-mediated calcium signaling in T cells (15). In the study by Matza et al., Ahnak-KO mice showed severely impaired IFN-γ production, which is required for Leishmania major clearance in vivo, and a dramatic increase in IL-4 production, suggesting that AHNAK is required for the induction of Th1 response against L. major (15). The pathogenesis of B. henselae infection is very similar with that of L. major infection because both B. henselae and L. major are intracellular pathogens. Therefore, we expected that the Ahnak-KO mice would show a severe impairment in IFN-γ production and a dramatic increase in IL-4 production in response to B. henselae infection and would show the same immune response as that observed in B. henselae-infected cats. However, in the present study, the Ahnak-KO mice showed an impairment of both IFN-γ and IL-4 production, suggesting the impairment of Th1 and Th2 immunity. Thus, the results of the present study suggest the potential of the Ahnak-KO mice to be used as a mild immunocompromised animal model or susceptible animal model for B. henselae infection.

MATERIALS AND METHODS

Experimental animals

This study included nine wild-type and nine Ahnak-KO C57BL/6 female mice, which were generated in our previous study (10, 21, 22). All the mice were aged 10–11 weeks at the time of use in the study. This study was reviewed and approved by the Institutional Animal Care and Use Committee of the Samsung Biomedical Research Institute (SBRI). SBRI is an Association for the Assessment and Accreditation of Laboratory Animal Care International accredited facility and abides by the Institute of Laboratory Animal Resources guidelines. All procedures used in this study were in compliance with the Animal Welfare Act Regulations and the Guide for the Care and Use of Laboratory Animals.

Experimental groups and inoculation of the mice with B. henselae

The mice were divided into the following four groups: (i) control wild-type mice not infected with B. henselae (CW group; n = 3), (ii) control Ahnak-KO mice not infected with B. henselae (CK group; n = 3), (iii) wild-type mice infected with B. henselae (BW group; n = 6), and (iv) Ahnak-KO mice infected with B. henselae (BK group; n = 6). The mice in the BW and BK groups were intraperitoneally inoculated with 100 μl bacterial suspension containing 2 × 108 colony-forming units (CFU) of B. henselae Houston-1 (ATCC 49882, American Type Culture Collection, Manassas, VA) suspended in phosphate-buffered saline (PBS) on day 0 and subsequently on day 10. The mice in the CW and CK groups were intraperitoneally injected with 100 μl saline on the same days. Blood and tissue samples of the mice were collected on day 8 after the final B. henselae injection (day 18 of the study).

Detailed procedures of experimental methods are described in the supplementary information.

Statistical analysis

All data are expressed as box-and-whisker plots. Data were analyzed using Kruskal-Wallis test followed by Mann-Whitney U test. Wilcoxon signed-rank test was used to compare means from two related samples. Confidence levels of 95% or higher were considered significant (P < 0.05). All statistical analyses were performed using SPSS software version 21.0 (IBM, Armonk, NY, USA).

SUPPLEMENTARY INFORMATION
ACKNOWLEDGEMENTS

We would like to thank the staff of the Laboratory Animal Research Center, SBRI, Samsung Medical Center for their technical support and assistance. This research was supported by the R&D Project for Korea Mouse Phenotyping Center (2013M3A9D5072550) of the National Research Foundation, which is funded by the Ministry of Science and ICT. This research was also supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, which is funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI13C2148), and was partially supported by the Brain Korea 21 Plus Program and the Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University to Je Kyung Seong. Moreover, this research was supported by a 2018 research grant from Kangwon National University (No. 520180048) to Eun Wha Choi.

CONFLICTS OF INTEREST

The authors have no conflicting interests.

Figures
Fig. 1. Detection of badA in the liver tissues of the B. henselae-infected mice PCR & liver tissues of the B. henselae-infected mice. (A) PCR was performed to amplify a part of badA with the specific primers. The reaction product was visualized by electrophoresing; M: 100-bp DNA size marker and C: control (DNA from B. henselae; badA PCR product size: 1,007 bp; template DNA: 10 μg). (B) Hematoxylin and eosin staining: Magnification of the marked area (yellow) shown in right panel. (C) badA PCR band density (D) Score of B. henselae-infected cell number. *P < 0.05 according to the Mann-Whitney U test. badA: Bartonella adhesion A, B. henselae: Bartonella henselae, KO: knockout, CW: control wild-type mice not infected with B. henselae, CK: control Ahnak-KO mice not infected with B. henselae, BW: wild-type mice infected with B. henselae, BK: Ahnak-KO mice infected with B. henselae.
Fig. 2. Mononuclear cell infiltration in the liver & relative quantification of cytokine mRNA by using RNA extracted from the liver tissues. (A) Mononuclear cell infiltration in the liver (mononuclear foci per 1.4 mm2). (B) Representative liver sections of the mice in the CW, CK, BW, and BK groups. (C) PCR amplification band. (D) Gene expression relative to the β-actin gene expression. *P < 0.05 according to the Mann-Whitney U test; P < 0.05 according to the Kruskal-Wallis test. B. henselae: Bartonella henselae, KO: knockout, CW: control wild-type mice not infected with B. henselae, CK: control Ahnak-KO mice not infected with B. henselae, BW: wild-type mice infected with B. henselae, BK: Ahnak-KO mice infected with B. henselae.
Fig. 3. Flow cytometric analysis of spleen cells. (A) The proportion of B cells, (B) the proportion of CD4+ T cells, (C) the proportion of CD8+ T cells, (D) the production of IFN-γ in CD4+ T cells, and (E) the production of IL-4 in CD4+ T cells. *P < 0.05 according to the Mann-Whitney U test; P < 0.05 according to the Kruskal-Wallis test. CW: control wild-type mice not infected with B. henselae, CK: control Ahnak-KO mice not infected with B. henselae, BW: wild-type mice infected with B. henselae, BK: Ahnak-KO mice infected with B. henselae.
Fig. 4. Cytokine levels in culture supernatants of spleen cells. In all, 5 × 105 spleen cells/well were cultured in the presence or absence of B. henselae (MOI: 10 [5 × 106 CFU/well]) in 96-well plates at 37°C in a humidified atmosphere containing 5% CO2. After 3 days, the culture supernatants were collected and stored at −70°C for further analysis. The supernatants were assayed for IFN-γ, IL-1α, IL-1β, IL-4, IL-6, IL-10, MCP-1, RANTES, TNF-α, and VEGF levels by using the Luminex multiplex murine cytokine analysis kit. *P < 0.05 according to the Wilcoxon signed-rank test; P < 0.05 according to the Mann-Whitney U test. B. henselae: Bartonella henselae, Bh: B. henselae-stimulated spleen cells, CW: control wild-type mice not infected with B. henselae, CK: control Ahnak-KO mice not infected with B. henselae, BW: wild-type mice infected with B. henselae, BK: Ahnak-KO mice infected with B. henselae, IFN-γ: interferon-γ, IL: interleukin, KO: knockout, MCP-1: monocyte chemoattractant protein-1, MOI: multiplicity of infection, RANTES: regulated on activation, normal T cell expressed and secreted, TNF-α: tumor necrosis factor-α, VEGF: vascular endothelial growth factor, and X: unstimulated spleen cells.
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