A novel in vitro cell culture model of human foreskin to study the immunogenicity of live attenuated influenza vaccine

INTRODUCTION

Circumcision is a cultural, medical, and religious process in which the human foreskin (HFS) is either partially or entirely removed by surgery. Cells segregated from the circumcised tissues are called foreskin cells. These cells can serve as feeder cell lines for embryonic stem cells used in research.[1] Circumcision is beneficial in decreasing the risk of infection [2-4]. For example, a recent study confirmed that circumcision lowers HIV infection rate in heterosexual men by 50-60%.[5] Therefore, WHO and UNAIDS have recommended male circumcision as a vital measure of a broad HIV prevention bundle for males. [6] In addition, it has been proven that among infants <1 year of age with urinary tract infections (UTIs), about 86% were uncircumcised boys. [7] Based on this evidence, the American Academy of Pediatrics (AAP) and the CDC strongly recommend male circumcision .[8] In the USA, the CDC estimates that 81% of males aged 14 to 59 years are circumcised. [9] In Saudi Arabia, circumcision for boys is obligatory according to Sharia Law [10], which ensures a supply of foreskin for scientific use.

Both humoral and cellular immune responses in the genital mucosa have a vital role in the prevention of sexually transmitted infections (STIs), including infection with HIV-1.[11] Mucosal immunity is an important compartment of the immune system and the foreskin is an important component of the mucosal immune system. However, its role in mucosal immunity has not been fully characterized. [12] Cell cultures initiated from HFS tissues have been used for isolation of viruses from diagnostic specimens because the tissues are readily available, and preparation is not difficult. Investigators have recommended greater use in diagnosis and research.[13] In this study, we investigated the potential use of foreskin-derived cell culture as a novel human model to study immune response to live attenuated influenza vaccine (LAIV) as an alternative to currently used animal models. [2, 14, 15] Foreskin-derived cell culture has been shown to be useful in characterizing the levels of CD4+ and CD8+ T cells, memory T cells, and the production of pro-inflammatory cytokines in HIV infection. [16]

In this study, we developed an HFS cell culture model to observe the immunogenicity of LAIV. We isolated a single cell suspension from fresh foreskin tissues and measured the frequency of foreskin T cell subsets using multiparameter flow cytometry and ELISA for antibody detection. By studying immune cells from HFS, we aimed to determine if HFS could serve as an in vitro human model to assess immune response to different influenza viruses.

Materials and methods

Foreskin samples

A total of twenty-one foreskin tissues were obtained from male infants (6 -30 months of age) undergoing circumcision at local hospitals in the Madinah region after signed consent obtained from parents. Foreskins were immediately placed in HANKS transport medium, containing glutamine (2mM), penicillin (50 U/mL), and streptomycin (50μg/mL) (Sigma-Aldrich) and transferred to the laboratory for processing and isolation of MNCs.

Ethical statement: the study has been approved by Taibah University Ethical Committee (Ref No. MLT 2018050).

Isolation of foreskin mononuclear cells

Cell suspensions were prepared using a modified cell suspensions protocol from a previously described method that was used to isolate MNCs from the nose-associated lymphoid tissues (NALT). [17, 18] Foreskin samples were processed within one hour of surgery. The tissues were cut into small pieces, then transferred to an 8 cm-diameter sterile Petri dish and checked grossly. Each sample was mechanically disrupted and minced using a single sterile scalpel. The samples were then enzymatically digested with dispase II (Sigma-Aldrich) to release cells into the medium. The tissue–dispase II mixture was then kept in a water bath shaker for 2 hours at 37°C at 100 rpm. The remaining tissues were then cut, minced and incubated in cell release medium (RPMI 1640 medium with HEPES supplemented with 10% fetal bovine serum [FBS], glutamine [2mM], penicillin [50U/mL], and streptomycin [50μg/mL], Sigma-Aldrich). The cell suspension was then passed through a 70-μm sterile nylon mesh to eliminate debris and then centrifuged at 400 g for 10 min. The cells were washed twice in sterile phosphate-buffered saline (PBS) and divided into two portions. One was cultured in 3 mL of RPMI complete medium for antibody production and the second was processed for T cell proliferation assay Carboxyfluorescein succinimidyl ester (CFSE) staining.

Intranasal vaccine FluMist (LAIV)

FluMist vaccine LAIV Formula (2010-11) includes the following influenza strains; A/H1N1/2009; A/H3N2 and B influenza strains (BEI Resources, American Type Culture Collection(ATCC)).

Cell culture and foreskin MNCs stimulation for antibody production

Following foreskin MNCs isolation, each cell suspension was adjusted to contain 4 x105 cells/mL. Foreskin MNCs were co-cultured in RPMI complete medium in the presence and absence of LAIV. Unstimulated cells were used as a negative control. After preparation, a volume of 250 µL of cell suspension were cultured in a sterile 96 well cell culture plate (Costar) and placed in the incubator in a humidified, 5% CO2 atmosphere at 37°C. Cell culture supernatants were then collected at different time points and assayed by ELISA for measuring antibodies induced in response to LAIV stimulation.

Carboxyfluorescein succinimidyl ester (CFSE) staining

Foreskin MNCs were prepared and resuspended in sterile PBS in 45 ml sterile tube. CFSE was prepared at 5mM just before use by adding 5 µL of neat to 10 ml sterile PBS. After that, 3 ml of the preparation were added to the cells then kept into the incubator in 5% CO2 at 37°C for 8 min. Adding of 10 ml ice cooled RPMI 1640 complete medium to block the reaction and then centrifuged at 400 x g for 10 min and then the supernatant discarded. The pellet was resuspended into 2 ml RPMI complete and then cells number was calculated via a haemocytometer. After adjusting cells concentration to 4 x105 cells/ml, cells were stimulated with LAIV and then incubated with 5% CO2 at 37°C for four days. After incubation, cell culture supernatants were collected for cytokines analysis and cells collected for T cell proliferation assay by flow cytometer.

Measurement of LAIV induced antibody levels by ELISA

Antibodies induced by LAIV stimulation were analyzed following the ELISA procedure as previously described [18]. In brief, ELISA plates were coated with 100 µL of LAIV (2 µg/mL) and incubated overnight at 4°C. After 5 washings, plates were blocked with 200-µL of 10% FBS followed by incubation of cell culture supernatants at different dilutions for 1.5 hour and then discarded and blotted on dry tissues. Then 50 µL of alkaline phosphatase conjugate of anti-human IgG, IgM and IgA (Sigma) were added and incubated for 1.5 hour. Afterward 5 washings, 50 µL of p-nitrophenyl phosphate (PNPP) substrate was added. Optical density (OD) was measured at 405 nm.

T cells proliferation assay

Following foreskin MNCs stimulation with LAIV and incubation, percentage of proliferative T cell subpopulations including CD4+ and CD8+ T cells subsets was analyzed by CFSE (FITC) and T cell marker staining followed by flow cytometry (BD FACScalibur (BD Biosciences)). CellQuest software was used for flow cytometer data acquisition. Data analysis was performed using WinMDI 2.9 software. Subsequent stimulation, cells were harvested in 0.02% BSA-PBS buffer (Sigma-Aldrich). Cells were washed and centrifuged at 400xg for 8 min. The supernatants were thrown away then the cell pellet was resuspended in 50 μl 0.02% BSA for surface staining. A volume of 5 μl of mouse anti-human CD4-PE-Cy5 and 5 μl of mouse anti-human CD8-PE (BD) were added to stain CD4+ as well as CD8+ T cells. The cells with the antibodies were incubated at 4°C for 30 min. Then the cells were washed tow times with 500 μl 0.02% BSA and then centrifuged for 8 min at 400g at 4°C. The cells were then resuspended in 300 μl 0.02% BSA and transported to FACS tube (Falcon) for analysing flow cytometer.

Statistical analysis

All the calculations and statistical analysis were performed using GraphPad Prism statistical software (version 5, USA). Data were expressed as mean ± standard deviation (SD). Two-group comparisons were performed using paired two test. A P < 0.05 was considered significant for all tests.

RESULTS

LAIV elicits for stronger humoral immune response

LAIV primes for stronger humoral immune response in the foreskin cell culture model. ELISA was used to measure the antibody levels in cell culture supernatants at different time points. Significant antibody levels were detected at day 10 following stimulation of MNCs with LAIV when compared with unstimulated negative control. Interestingly, all antibody classes were detected. Antibody class IgA was dominant over the other two classes whereas IgM was higher than IgG class antibody; (Fig. 1, p <0.05, n=21 (a) IgG, (b) IgM and (c) IgA)

LAIV induces cellular immune response

The percentage of total T cell proliferation index using CFSE staining shows significant increase in total T cell responses following LAIV stimulation in CD4+ (Fig. (2 a), p <0.05, n=21) and CD8+(Fig. (2 b), p <0.05, n=21) when compared with unstimulated negative control. CD4+ T cell proliferation index was used to quantify the CD4+ T cell response following LAIV stimulation. Gating strategy illustrated in figure 3. Lymphocyte and CD4+ and CD8+ T cell gates were used in this analysis during flow cytometry. Lymphocytes were gated on the basis of their forward scatter and side scatter properties. As shown in figure (3 a) the lymphocytes were gated on region 1 (R1). CD4+ T cell gate was then set as region 2 (R2) as in (figure 3 b). Finally, R2-gated CD4+ T cells were shown in a new window as in (figure 3 c) with CD4-PE on y axis against CFSE (FITC) on x axis. The CD4+ T cell proliferative index was defined as the percentage of proliferating CD4+ T cell of total CD4 T cells = A/A+B, A= upper left quadrant, B=upper right quadrant). Representative proliferative index of CD4+ T cell following stimulation with LAIV (figure 3 d) was shown as compared to unstimulated negative control (3 c).

Moreover, stimulation with LAIV induced also significant increases in IFNγ in the foreskin-derived MNCs (p<0.01, n=21) (data not shown).

Discussion

To the best of our knowledge, the present study is the first to report immune cell densities in the HFS using LAIV as immunogen for stimulation. We also provided the first evidence that foreskin-derived MNCs can be used as a model for an in vitro human cell culture model for studying mucosal immune response to respiratory pathogens such as influenza viruses.

Influenza is a very transmissible acute respiratory illness initiated by influenza virus. The virus infects the mucosa of the host respiratory tract. Intranasal immunization with activated vaccine is more potent than intravenous inactivated vaccine and is a more biologically appropriate means of vaccination against respiratory infections such as influenza since it imitates natural infection. [19] Our results show a significant antibody production following stimulation with LAIV. As presented, IgA class antibody shows to be dominant over the other two classes, which is consistence with other study, which showed higher antibody levels in mucosal system. [19] IgG class antibody level appears to be the lowest among all classes which is expected as all subjects are infants less than three years. Therefore, they were expectedly lees exposed to influenza viruses during their lives compared with adult. LAIV provokes secretory IgA, the major antibody secreted at mucosal surfaces, which plays an essential role in protecting against influenza and is a critical part of the immune system, being the second line of defense against pathogens. [20-22] . LAIV seems to be superior in eliciting nasal IgA and CD4 T cells when compared with natural infection and inactivated influenza vaccine. However, LAIV is about as strong as natural infection in providing cross-protective immunity. [19]

The local mucosal immune tissue critically influence intranasal vaccination. [23] Human adenoids and tonsils are the main mucosal immune organs, specifically the NALT in humans and are recognized as essential points of induction of mucosal (and systemic) immunity against upper respiratory tract pathogens. [24, 25]

Natural influenza infection elicits a wide-range of immune responses, which include humoral and cellular components. [26, 27] Mucosal antibodies are potent defenders of the upper respiratory airways and points of viral access. Serum antibodies mostly defend the lower respiratory tract Existing inactivated influenza vaccines induce strain-specific antibodies and not CD8+ T cells. [28] Our results also show significant increase in cellular immune responses after LAIV stimulation namely, CD4+ and CD8+ T cells. This result is in accordance with another study showed that LAIV mimics natural infection through viral replication in the upper respiratory tract and provokes many immune responses, comprising antibodies and CD4+ and CD8+ T cells. [29]

Several models have been used to study and evaluate infections caused by several influenza viruses. In a previous study, we showed that NALT from human adenoids and tonsils is one of the most successful models for that purpose. [18] Now we show that an HFS model is an alternative and novel in vitro cell human model. The present study demonstrates the ability of MNC-derived from human foreskin to be used as a model for studying humoral and cellular immune response. An advantage of HFS is that all immune cells are present in these tissues, which is well documented. [1, 30] Additionally, a previous study investigated mucosal response of tissues from the penises of transformed males following penal removal and found that the penis is a site of efficient mucosal response [31] . Studies in animal models have found that the presence of CD4 T cells in the foreskin of infected monkeys played a crucial role in fighting the HIV virus. [ 2] The role of IFNγ is very essential in fighting viral infection as influenza virus. IFNγ is released in large quantities in several immune compartments such as, macrophages, activated CD8 T cells, NK T cells, and Th1 CD4 T cells. [32, 33] Our data shows the ability of HFS model to produce IFNγ, which mimics the natural viral infection, and this is consistence with several studies that demonstrated the ability of LAIV to induce the secretion of IFNγ. [34-36]

Our data can be added to the accumulation of evidences to verify the appropriateness of foreskin to study the interactions between influenza virus and the immune system. This result is consistent with a study in children when T cells were produced abundantly, which might offer clinical protection. [26] In addition, another study reported similar results after intranasal LAIV. [28] The finding of significant antibody levels following stimulation of MNCs with LAIV means the model is valid for other studies to assess the immune responses to different pathogens that cause infection in humans such as MERS-CoV, pneumonia and many others. This result is also consistent with several studies of the NALT model. [37-40]

Conclusion

This study using an in vitro cell culture model of HFS to measure humoral as well as cellular immune responses to LAIV provides a new model to attest and study of intranasal vaccines. Use of human-derived cells is a more realistic model for investigation of the immune response against pathogens that cause human infection such as influenza viruses. Understanding mucosal immunity to respiratory tract infections such as influenza viruses may lead to meaningful improvements in effective intranasal vaccines against other respiratory infections.

Legends:

Figure 1: Significant antibody levels were detected (p <0.05, n=21) following stimulation of MNCs with LAIV when compared with unstimulated negative controls. Interestingly, all antibody classes were detected (IgG (a), IgM (b) and IgA(c)).

Figure 2: Significant increase in total T-cell responses after stimulation (p <0.05, n=21). Stimulation with LAIV induced significantly increases in total CD4+ (a) and CD8+ (b) T cells when compared with unstimulated negative control.

Figure 3: Representative CD4+ T of proliferative index following stimulation with LAIV 3(c) was shown as compared to unstimulated negative control 3(d)). Representative CD4+ T cells and gating strategy used in quantifying CD4+ and CD8+ proliferation index using CFSE staining.

Acknowledgement

We thank the BEI Resources company for providing us by FluMist vaccine for free.

Author Contributions

All authors having contributed equally to the work.

Disclosure of potential conflicts of interest

The authors claim no conflict of interest.

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