Effect Of Heavy Metals On Immunity Biology Essay

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Autoimmune disease is the state in which the immune system attacks self by some disorder. It is the third major cause of mortality.

Heavy metal induced auto immunity has been studied intensely using mice as the model organism. Sub toxic level of heavy metals like mercury or silver on susceptible mice can produce autoimmune responses. The aim of this study is to investigate the role of T regulatory cell in mercury treated mice. As the previous studies state, Tregs can suppress the autoimmune reaction. But here in the study the results are not significant, but the comparison of the results state that the previous studies are true.

Autoimmunity is the immune response against self-antigens in healthy individuals. These immune responses do not cause harm to the host due to the presence of mechanism that can control auto-reactivity and impede autoimmunity (1). Depending upon the target of attack, autoimmunity can produce a variety of clinical conditions like expansion of self reactive T and B Cells, production of auto-antibodies and tissue damage(2). Autoimmunity is not set off by a single cause and is triggered by a variety of agents and molecular and cellular pathways and events (3).

Some disorder in above mentioned mechanisms cause autoimmune diseases. Autoimmune diseases are developed when the last barrier is broken and the immune system attacks self antigens (3). Autoimmune diseases are an important cause of morbidity and mortality in the human population (4). It is the third major cause of morbidity and mortality (5). Autoimmune diseases are divided into types. They are systemic autoimmunity and organ specific autoimmunity. Systemic autoimmunity is heterogeneous group of diseases in which the pathology is evident in a number of organs. Systemic lupus erythematosus (SLE), scleroderma, Sjogren's syndrome, inflammatory myopathies, and overlap syndromes such as mixed connective tissue disease (MCTD) are some of the systemic autoimmune diseases. As the name mentions organ specific autoimmune diseases are those which affects the specific organ. Type 1 diabetes and thyroid diseases are main organ specific diseases.

1.2 Tregs

Tregs are T-regulatory cells which supress or regulates autoimmune responses. Failure of these cells results in autoimmune disease. Tregs CD4+CD25+ are two T-regulatory cells. CD4+CD25+ regulatory T (Treg) cells that express Forkhead box P3 (FoxP3) are the main factors for controlling autoimmune responses. Tregs are diverse and include at least three populations that differ by their phenotype, cytokines secretion profile and suppressive mechanism (6). The idea of tregs was relinquished due to the lack of specific markers which lead to the insufficient information of the treg cells. The name regulatory T cells were given to the new subpopulation of suppressor T cells which expressed CD4 (10).CD4+ T cells are divided into two categories. They are regulatory T (Treg) cells and conventional T helper (Th) cells. Th cells activate other effector immune cells which control adaptive immunity against pathogens and cancer. Treg cells are defined as CD4+ T cells responsible for suppressing harmful activities of Th cells (7). From all the studies it is clear that the depletion of the tregs cell barrier in animals lead to autoimmune disease. Various autoimmune diseases like gastritis, thyroiditis, and autoimmune diabetes are controlled or suppressed by T cells expressing CD25 which are suppressive and anergic (8). GITR, CD25, CD127, Foxp3 and CLTA-4 are some of the useful and most common markers (7). Foxp3 is a transcription factor and it regulates the activation of CD4+ T cells. In our study, Foxp3 is the most useful marker as it is more specific for CD4+. Some CD8+ cells also express Foxp3. In B.10.S mice strain, Foxp3 is expressed by CD4+CD25+ and CD4+CD25- .

Certain strains of mice develop Treg cells which suppress development of unspecified ANA autoantibodies induced by heavy metals like Hg. Susceptible mice pretreated with Hg showed resistance to induce systemic autoimmunity in the later stage. CD4+CD25+ Ts cells, can suppress the development of different autoimmune diseases, immunopathological conditions and responses to foreign antigens (9).

Effect of Mercury on Autoimmunity

The use of experimental models has increased the knowledge of autoimmune disease mechanisms (11). Heavy metals, especially mercury (Hg), generate autoimmune responses which are relevant with regard to human diseases (11), creating an opportunity to investigate the effects of immunomodulating agents on autoimmunity. The administration of subtoxic doses of heavy metals like mercury into the genetically susceptible mice, leads to systemic autoimmune diseases which are induced to develop antinucleolar antibodies (ANoA) and systemic immune complex (IC) deposits. (12). Rodents susceptibility to mercury induced autoimmunity is MHC dependent (13).

ANA and ANoA

ANA is antinuclear antibody and ANoA is antinucleolar antibody. Literally ANA should include antibodies that react to the components of the nucleus like its membrane, nucleoli, nucleoplasm and nuclear organelles, but it normally ANA also react with other cell components like mitochondria and ribosomes (14)

The antibodies that only target the nucleolar components are known as ANoA. These ANoA level increases if there is some sort of autoimmune response occurs. So they stand as an indicator for autoimmune diseases. Heavy metal intake can induce the production of ANoA in healthy individual.

1.5 Aim of the project

Aim of the project is to study the effect of induced T-regulatory cells (Tregs) in mercury induced autoimmunity (HgIA).

1.6 Hypothesis

Testing if Treg cells developed in susceptible mercury treated mice is able to suppress induction of ANoA in Hg-treated mice.

2. Materials and Methods

2.1Mice

For the experimental purpose, the model organism used was female mice. Both knock out and wild type mice are used in this experiment. Interferon gamma is knocked out in the knockout mice. These animals used in this study were 8 to 14 weeks old at the beginning of the treatment. The animals were housed in steel- wire cages in a high - barrier unit at the Animal Facilities of the Faculty of Health Science, under maintained optimal conditions, 12 hours dark and 12 hours of light cycles and sterile food pellets (type R36; Lactamin, Vadstena ,Sweden ) and water are given. Mice were kept in a group of 3 to 5 in each cage. The study was approved by the Laboratory Animal Ethics Committee, Linkoping, Sweden.

2.2Mercury

Mercury is the heavy metal used here. These mice are treated with mercury in the form of mercuric chloride (HgCl2) in sub toxic level which induces the production of ANoA. 8mg of mercuric chloride is dissolved in 1 litre of tap water. Mercury is administered orally by the mice.

2.3Serum Collection

Blood is collected from the blood vessel bellow the eye region using a pastuer pipette. Blood is collected under painless and calm condition. Mice were made unconscious by isofluron. Blood was collected and serum was separated and stored in -20 refrigerators for further analysis.

2.4Treatment and study design

2.4.1Donor Mice

Forty interferon gamma knockout B 10.S mice were used as the donor. These mice were eight to twelve weeks aged at the onset of treatment. In this twenty mice were treated with 8mg/l mercuric chloride and the rest twenty were control and they were given only normal drinking water. They were treated for 5 weeks. Blood were collected from all the mice and they were sacrificed by cervical dislocation and spleen and kidney were collected. Kidney is preserved for Immunofluorescence.

2.4.2 Tregs cell isolation

Treg cells, CD4+CD25+ and CD4+CD25- cells were isolated from spleen. Single cell suspension of spleen from Hg treated and untreated mice were prepared by mechanically disrupting it. The procedure as described by Johansson 1997, silver IA. CD4+CD25+ and CD4+CD25- were purified from splenic single cell suspension by using the magnetic cell separating kits by Miltenyi Biotec. Cells were counted by flow cytometer analysis. Purified CD4+CD25+ and CD4+CD25- cell from both hg treated and untreated mice were sorted in four groups each and transferred to new set of mice.

2.4.3Recipient Mice

Recipient mice used were B 10.S wild type mice. Purified Treg cells from the treated and untreated mice were totally divided into 8 set and injected in the recipient mice. 105 cells per mouse in 0.1 ml PBS were injected via intraperitoneal injection. 39 mice were used for the purpose in which 19 were water treated and 20 were mercury treated. Here also 8mg mercuric chloride in 1 litre water was used for treatment. Control mice received tap water. Blood samples were collected from the mice at 0 weeks (just taken before the onset of treatment), 5 weeks, 7,9, 11,13,15 and 18th week after treatment. The mice were sacrificed by cervical dislocation after 18th week and kidney and spleen were collected and stored at -70 freezers which will be preserved for at least 10 years according to the rules. These kidney and spleen can also be used for immunofluorescence.

Recipient group

Donor mice treatment / tregs type

Number of mice

Treatment after injecting in

Recipient mice

A

H2O/ CD4+CD25+

4

Water

B

H2O /CD4+CD25-

5

Water

C

Hg/CD4+CD25+

5

Mercury

D

Hg/CD4+CD25-

5

Mercury

E

H2O/ CD4+CD25+

5

Water

F

H2O /CD4+CD25-

5

Water

G

Hg/CD4+CD25+

5

Mercury

H

Hg/CD4+CD25-

5

Mercury

Table 1: This table shows the type of treatment received by each mice and the frequency of the group.

2.5. Analysis

2.5.1Detection of ANoA

ANA test is an indirect immunofluorescence method for detection of serum anti nucleolar antibody detection. This method was obtained from (12). In this method serum is diluted in PBS at the ratio of 1:80. These samples are loaded on slides pre coated with monolayer of Hep-2 cells (Binding Site Ltd., Birmingham, UK). Then it is incubated for 30 minutes at room temperature which is followed by 10 minutes washing in PBS. Detection agent, goat anti mouse IgG (gamma - chain specific) FITC conjugate (Southern Biotechnology Associates Inc., Birmingham, UK), was diluted at 1:50 dilution in PBS. Detection agent helps in detecting the bound ANoA. Incubated for 30 minutes and cover slip was placed after washing for 10 mins. After 2hours the slide is viewed under fluorescence microscope (Nikon Instech Co.Ltd., Kanagawa, Japan) and graded according to the strength.

2.5.2 Detection by anti-DNP ELISA

The method used here was described by (12). ELISA plates (Nunc, Copenhagen, Denmark) were coated with DNP-albumin (Albumin Human Dinitrophenyl, Sigma). They are incubated overnight in refrigerator at 40C. These coated plates can be stored for5 days. Plates were washed with PBS pH7.35- tween20 (0.1%) BSA (1%). Serum was diluted in PBS pH7.35- tween20 (0.1%) BSA (1%) at 1:100 dilutions. Serum sample is loaded along with the strong positive and negative which was obtained from pooled sera of NMRI mice. The plate was incubated for 1.30 hours and washed as above. After was, goat anti-mouse-IgG-ALP(polyvalent IgG,IgA,IgM) (Sigma Aldrich chemie, Steinheim, Germany) was added. Again the plates were washed after incubation and the substrate was added to each well and after incubation for 20 mins, plates were read at OD 405nm. Then the plates were stopped when the positive value reached 1.5 with 3M NaOH.

2.5.3 Detection by ssDNA

The method used here was described by (12). ELISA plates (Nunc, Copenhagen, Denmark) were coated with single stranded DNA (ssDNA). They are incubated overnight in refrigerator at 40C. These coated plates can be stored for5 days. Plates were washed with PBSpH 7.35 and then they are blocked for 1 hour with PBS pH7.35- tween20 (0.2%) BSA (1%). Serum was diluted in PBS pH7.35- tween20 (0.2%) BSA (1%) at 1:150 dilutions. Serum sample is loaded along with the strong positive and negative which was obtained from pooled sera of NMRI mice. The plate was incubated for 1.30 hours and washed with PBS pH7.35- tween20 (0.2%) BSA (1%. After wash, goat anti-mouse-IgG-ALP (polyvalent IgG,IgA,IgM) (Sigma Aldrich chemie, Steinheim, Germany) was added and incubated for 2 hours. Again the plates were washed after incubation and the substrate was added to each well and after incubation for 20 mins, plates were read at OD 405nm. Then the plates were stopped when the positive value reached 1.5 with 3M NaOH.

2.5.4 Detection by IgG1 ELISA

The method used here was described by (13). ELISA plates (Nunc, Copenhagen, Denmark) were coated with Rat anti-mouse IgG1 Purified 1mg/ml (BD 559749Parmingen) and incubated overnight at 40C. These plates are washed 3 times with PBS pH7.35- tween20 (0.1%). These plates after washing were blocked with fat free milk solution 5%. It is then incubated for 2 hours at 370C. Meanwhile the sera were diluted in PBS at 1:200 dilutions. Plates are washed as above and the serum samples were loaded in double wells. It is again incubated for 2 hours at370C and washed as above. IgG1 was detected with Peroxidase conjugate (LO-MG1-2 HR, Belgium). Plates were again incubated at 370C and washed as above. Substrate were added in each well and the plates were incubated for 3 mins and read using ELISA plate reader at 450nm OD and stopped using 2M sulphuric acid. The concentrations of IgG1 in the serum were obtained by the standard curve using IGM standard (MADNP-1, Belgium).

2.6 Statistical Methods

All the data available were analysed statistically using the software Graphpad Prism. The results were compared using Kruskal Wallis statistical test and the significance was found using the non-parametric Mann Whitney Test.

3. Results

3.1 ANoA

When the serum sample of the donor mice (both treated and control) were analysed they didn't show any ANoA. This is because the mice were interferon gamma knockout. Interferon knockout mice never produce ANoA and thus do not show any response to mercury treatment.

In the recipient mice those received treg cells from the treated mice show low reaction to the treatment. But the level decreases with time. When groups C and D (donor treated with water) is compared with groups G and H (Donor treated with Hg), the ANoA level decreased in groups H and G. Even though the hypothesis is not completely correct in this case, it is clear that the Treg cells can suppress the autoimmune responses.

Recipient Group

Donor mice treatment / Type of Treg

Treatment

No

Of Mice

0 weeks

5th week

7th

9th

11th

13th

15th

18th

C

H2O /CD4+CD25+

hg

5

0

5

3

3

2

2

2

2

D

H2O /CD4+CD25-

Hg

5

0

5

5

4

3

3

3

3

G

Hg/CD4+CD25+

Hg

5

0

2

2

1

1

1

1

1

H

Hg/CD4+CD25-

Hg

5

0

2

1

1

1

1

1

1

Table 2: This table gives the information about the treated mice. This table give an idea how the level of ANoA differs in each group treated with mercury. ANoA production decreased with time in all the cases.

Recipient Group

Donor mice treatment / Type of Treg

Treatment

No

Of Mice

0 weeks

5th week

7th

9th

11th

13th

15th

18th

A

Water/ CD4+CD25+

Water

4

0

0

0

0

0

0

0

0

B

Water /CD4+CD25-

Water

5

0

0

0

0

0

0

0

0

E

Hg/ CD4+CD25+

Water

5

0

0

0

0

0

0

0

0

F

Hg /CD4+CD25-

Water

5

0

0

0

0

0

0

0

0

Table 3: This table show the untreated group of mice. These mice produced any ANoA.

Fig 1: ANoA negative cells Fig 2: ANoA positive cells

3.2 Anti-DNP ELISA

Figure 2: This figure is the graphical representation of the data obtained from Anti DNP ELISA. Each graph denotes each group and the changes during the treatment time. Data from 0, 5, 7, 9, 11, 13, 15, and 18th week are recorded here.

In both the treated and untreated mice, there is no significant difference in the level of total anti-DNP antibody. In the case of treated mice, the groups which got CD25+ cell have more anti DNP antibody level in the serum than the mice which received CD25- cells. In this case the anti DNP level keeps on increasing till the 15th week and then it start to decrease. Even though the difference in the anti DNP antibody level of treated and untreated mice is not significant, we can see a considerable difference in the level of antibody.

3.3 Anti-ssDNA ELISA

Figure 3: This figure is the graphical representation of the data obtained from ssDNA ELISA. Each graph denotes each group and the changes during the treatment time. Data from 0, 5, 7, 9, 11, 13, 15, and 18th week are recorded here.

From the above graphs the, it can be observed that the concentration keeps on increasing till the 11th week and then it starts decreasing irrespective of the treatment. By the anti-ssDNA antibodies, polyclonal B-cell activation can be measured. Even though we can't see any significant difference between any group, when treated mice are compared with untreated once, we can see that the B-cell activation is more in activated mice that the untreated ones. In all groups irrelevant of the factor of treatment, 11th week show significant difference in activation when compared with the 5th week and the 18th week. 11th week is the maximum time that can initiate B-cell activation.

3.4 IgG1 ELISA Result

Figure 4: This figure is the graphical representation of the data obtained from IgG1 ELISA. Each graph denotes each group and the changes during the treatment time. Data from 0, 5, 7, 9, 11, 13, 15, and 18th week are recorded here.

Here also the levels are not significant in any case but we can observe that the treated mice have higher levels of IgG1. Here the IgG1 level increases till the 9th or 11th week and decreases at the same rate. In the treated mice, the groups which received CD25+ show higher level of IgG1 than the CD25-.

Discussion

As there is no ANoA in the donor mice it is clear that Interferon gamma is an important factor for the production of ANA. It was an expected result. In other case, if it were wild type mice that were used instead of knockout, it would surely produce ANoA.

But those mice which received the treg cell from the non- treated mice didn't show any auto immune response to the mercury treatment .In the case of recipient mice which received the Treg cells from the mercury treated mice showed less ANoA production than those which received treg cell from non -treated mice.

Different types of ELISA were done with the serum samples taken from the mice at different weeks. In all the cases the result supports the previous studies but there is no significant difference in any case. Thus the study cannot be considered significant. Lack of significance may be due some mistakes done while carrying out the ELISA experiments or may be because some mice in same cage received more mercury and other less due to the fight between them. One of the important reasons for this sort of result may be due to the group size. Here each group contain only 5 mice in which abnormal results obtained from 1 or 2 mice from each group can change the total result. Further experiments should be carried on with increased group size.

Even though the hypothesis is not completely correct in this case, it is clear that the Treg cells can suppress the autoimmune responses.

Conclusion

From different studies that have been done already, it was clear that Treg cells can suppress the immune response in the mice which is treated with mercury. Even though we couldn't find any significant result, our study also support the fact as there is some sort of favourable difference between the treated and untreated mice.

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