INTRODUCTION:

Measles is one of the infectious diseases caused by the genus Morbillivirus^1,28. Measles virus that enters the body will attack the respiratory tract and lymphatic tract. And then the measles virus replicates and flows inside the blood. Viruses can infect erythrocytes or leukocytes. Leukocytes infected can cause a decrease in the number of CD4 T cells and causes inhibition of the immune system²⁹. In secondary infection, if not handled appropriately, it can cause complications such as pneumonia, blindness to death^2-3. Measles vaccination is given to toddlers with ages 9-12 months and a booster is given at 4-6 years old. The reaction that occurs after vaccination are mild fever, swelling, and soreness²⁵. Type vaccine that given is live attenuated vaccine derived from strain measles virus that devoid pathogenicity but can trigger an immune response inside the human body²⁷. Southeast Asia region has a large number of deaths due to measles virus infection, which is equivalent to 25% of total global deaths⁴. So far, vaccination is the most effective treatment but it has not completely eradicated the epidemic of measles. At least 10% of the total individuals who have received vaccinations are still not immune to the measles virus⁵. Other factors may contribute to the individual's resistance to the measles virus after receiving a vaccination.

External factors such as pollution and contamination of PAH are the biggest factors that occur for each individual, in particular, vaccine recipients. PAH contamination can occur through industrial emissions, agriculture, air, water, and grill food. One of the most PAH compounds contained is Benzo[a]pyrene (BaP)⁶. The previous study has shown that BaP can reduce cellular and humoral immune responses⁷. BaP can affect the immune cell such as Natural Killer (NK) cells and T cells which are involved in innate and adaptive immune responses^8-9. NK cells are an important component in defending against viral infections and contribute to regulating adaptive responses¹⁰, while T cells play a role in the formation of memory cells for secondary infection defense in the form of cellular responses and humoral mediators¹¹. However, the effect of BaP on immune cells after the measles vaccination is still unknown. Therefore, to uncover that, we exposed measles-vaccinated mice with BaP to analyze the BaP effect to immune cell response post-vaccination.

MATERIAL AND METHODS:

A total of 24 male Balb/C mice ( Mus musculus ) Aged 2 weeks obtained from pathogen free breeder state, Jember Regency. All mice used were fed and cared in a clean and proper animal room. The study protocol was following the Ethics Committee Brawijaya University with registration number 930-KEP-UB. Mice were divided into four equal experimental group as Normal mice (N), Benzo[a]pyrene injection (BaP) , Vaccine Injection (V), Vaccine and Benzo[a]pyrene (V + BaP).

Vaccination and Benzo[a]pyrene injection:

Measles Vaccine (PT Bio Farma, Bandung, Indonesia) was injected once subcutaneously to group V and V + BaP with single dose of 20 mg/kg BW Intraperitionally.. All Groups were injected intraperitoneally with a single dose of 20 mg/kg BW of BaP every 3 days for 5 weeks (9 total injections). For the V+BaP group, the vaccine was injected at the beginning and then followed by BaP injection 2 hours later.

Spleen cell isolation, Immunostaining, and flow cytometric analysis:

Mice were dislocated to obtained spleen organ after the last injection. The isolated spleen washed and homogenized using Phosphate Buffer Saline (PBS). Sample were centrifuged at 2500 rpm, 10^oC for 5 minutes. The supernatant was removed and pellet was resuspended with 1 mL PBS for Immunostaining and Flow cytometry Anlysis.. A total 50 µl of resuspended sample added with 50 µl extracellular antibodies FITC anti-mouse CD4 (BioLegend, San Diego), PE anti-mouse CD8a (BioLegend, San Diego), FITC anti-mouse NKp46 (BioLegend, San Diego). Sample incubated for 20 minutes at 4oC. Then, sample added 400 µl PBS and transferred to the cuvette for flow cytometry analysis (BD FACS Calibur, USA)

Data analysis:

The flow cytometry results were analyzed using the BD Cellquest ProTM software (BD Biosciences, San Jose, CA, USA). The data tabulated in Microsoft Excel 2010 program of windows. The data were then analyzed using one-way ANOVA and Tukey HSD test to determined the statistical difference among treatment by significance 95% using SPSS 22.0 For Windows (IBM Statistic, USA).

RESULT:

BaP induction after vaccination could reduce the relative number of Natural Killer cell (p<0.05). Vaccination could increased the number of NK cells in V group (Vaccine treatment) (P<0.05). But, BaP induction after vaccination caused decreased the relative number of NK cells (p<0.05). In addition, BaP itself could escalate the relative number of NK cells (P<0.05). Nevertheless, not as great as vaccine induced augmentation (p<0.05) (Figure 1).

Fig 1. BaP injection after vaccination reduces the number of NK cells shown in the flow cytometry (A) and statistical test (B). N: Normal; BaP: Benzo[a]pyrene; V: Vaccines; V + BaP: Vaccines + Benzo [a] pyrene. The displayed value is from the mean ± standard deviation, n = 6. The difference in notations showed significant differences (p <0.05).

Immunotoxic effect of BaP after vaccination could diminish the ratio of helper T-cell that stained with anti-CD4 and cytotoxic T-cells that stained with anti-CD8 compared to normal conditions (p<0.05). Vaccine administration did not increase the ratio of Helper T-cell (CD 4+) that stained with anti-mouse CD4 and cytotoxic T-cell (CD 8+) that stained anti-mouse CD8 compared to normal condition (p<0.05). Vaccine administration did not increase the CD 4+ : CD 8⁺ ratio, nor did BaP exposure (Figure 2b.) The difference in the ratio was due to an increased in CD 8⁺counts in each group. CD 8⁺ count increased significantly in the Vaccine and Benzo[a]pyrene group (V + BaP) (p<0.05). Vaccine group (V), Benzo[a]pyrene group (BaP) or Vaccine and Benzo[a]pyrene group (V + BaP) had reduce CD 4⁺cells when compared to normal group (N) (p<0.05). Accordingly, BaP induction after vaccination was more influential on CD 8⁺ cells compared to CD 4⁺ cells (Figure 2a and 2c).

Fig 2. BaP injection after vaccination gives the opposite effect to CD4⁺ and CD8⁺ T cells count thus lead to CD4⁺:CD8⁺ ratio declining. Data displayed in the histogram of the results of flow cytometry (A) and statistical tests for the ratio of CD4⁺:CD8⁺ (B) and comparison of the number of CD4⁺ and CD8⁺ T cells (C). N: Normal; BaP: Benzo[a]pyrene; V: Vaccines; V + BaP: Vaccines + Benzo [a] pyrene. The displayed value is from the mean ± standard deviation, n = 6. The difference in notations showed significant differences (p <0.05).

DISCUSSION:

BaP induction after vaccination has been shown to reduce the number of NK cells (fig 1). Moreover, vaccination without intervention from BaP could increase innate immune responses, particularly NK cells, through IFN type I which can act as an activator and initiator of proliferation during pathogen infection¹⁰. Exposure to pollutants such as BaP after vaccination can induce NK cell death due to the toxic nature of BaP¹². The measles vaccine is a type of live attenuated vaccine developed from weakened living organisms. The vaccine can be bacteria or viruses that have been lost pathogenicity. However, these organisms can still live in the host and able to trigger an immune response³⁰. The role of NK is crucial in responding to antigens especially viruses¹³. NK cells play a key role in the preclusion of viral infections during the process of adaptive immune response formation¹⁴. In addition, NK cells can also form memory cells that play a role in warding off secondary infections^15-16. NK cells can express various combinations of receptors that make each cell clone different from the others so that they can be definite for certain pathogens¹⁶. Therefore, a decrease in the number of NK cells has many consequences for the susceptibility of individuals to measles infections so that no resistance is formed even after vaccination performed.

The decline in the number of NK cells has a big influence on the process of adaptive immune response activation. Stimulated NK cells by several cytokines such as IL-12 and IL-18 can express IFN-γ then augment macrophage response to the pathogen¹⁷. Moreover, IFN-γ with some NK cells-derived chemokines can also activate dendritic cells (DC)^18-19. DC also related to humoral immunity systems directly interact with B cell and indirectly induce the development and differentiation of helper T cell²⁶ Activated DC can orchestrate the adaptive immune response through T cell differentiation into a Th1 subset that provides an inflammatory response¹⁸. Activated Th1 cells will induce B cells to proliferate into plasma cells which will produce IgG as well as memory cells for the defense of secondary infection²⁰. In this regard, the decrease in the number of NK cells in the vaccination process can certainly have an impact on the formation of adaptive immune memory cells, especially in the formation of specific antibodies against the measles virus.

The influence of BaP exposure after vaccination on the adaptive immune system, specifically those related to the cellular immune response, was denoted by reduced CD4⁺ T cells and increased CD8⁺ T cells (fig 2). Although the effect of BaP has more effect on CD8⁺ T cells counts, its immunotoxicity also seen in CD4⁺ T cells counts when compared to normal conditions (fig 2C). This can disrupt the formation of the adaptive immune system to form memory cells, given the crucial role of CD4⁺T cells in modulating the activation of a cellular and humoral immune response²¹. The important role of CD4⁺T cells in the formation of memory cells begins with the activation of specific antigens from T cells by direct binding with Antigen Presenting Cell (APC) such as macrophage and DC. Activated CD4⁺will produce cytokines that are important for the proliferation and differentiation of T cells into Th1 and Th17 also B cells into plasma cells and memory cells²².

The CD8⁺ T cells subset is the most affected adaptive immune cell by the presence of BaP after vaccination (fig 2C). The increase in CD8⁺ T cells counts in the V + BaP group is a form of the immune response to BaP exposure⁷. BaP will be metabolized by cells in the endoplasmic reticulum and then presented through MHC I as an immunogenic substance²³. The association of antigens with MHC I can activate CD8⁺T cells²⁴. This makes the immune response majorly mediated by CD8 represented by augmentation of CD8⁺ T cells count in the V + BaP group. Hence, the effectiveness of measles vaccination in priming long-term immune systems will be distressed by the toxicity of BaP to innate immune systems and cellular immune responses and the individual becomes less resistant to measles infection.

ACKNOWLEDGMENT:

We would like to thanks Mr. Pristiwanto for expert assistance during the experiment and also acknowledge Mr. Suwoyo for funding this research.

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Received on 21.10.2019 Modified on 13.01.2020

Research J. Pharm. and Tech. 2020; 13(12):6128-6131.

DOI: 10.5958/0974-360X.2020.01069.0