INTRODUCTION:

Staphylococcus aureus is one of the leading pathogens in the world, causing a wide variety of infectious diseases; it is gram-positive bacteria that Staphylococcus aureus is a major bacterium that causes a wide range of infectious diseases around the world. Gram-positive bacteria are the have non-motile, cocci-shaped and non-spore-forming bacteria^1,2.This bacterium has multiple virulence factors as Staphylokinase (SaK), SaK is a small protein molecule, comprised of 136 amino acids and about 15 KDa molecules. S. aureus SaK is largely synthesized in the late exponential stage of development, where it is required to penetrate host tissues by dissolving blood clots generated at the injury location^3,4.

SaK performs its effects in the blood primarily via forming a plasminogen activating complex with plasmin, this inhibits the production of biofilm^5,6 and, more critically, starts the fibrinolytic cascade, which allows the infecting bacterium to migrate further into the host tissues, the SaK can neutralize antimicrobial peptides (AMPs) in the host. All animals produce AMPs, which are a broad range of peptides and different species generate as a defense mechanism against infections that are invasive⁷. Thrombolytic agents in clinical applications are recombinant human tissue plasminogen activator (tPA), Staphylokinase (SaK), urokinase (UK), and streptokinase (SK). Staphylokinase (SaK) is indeed a bacterial plasminogen activator protein that is now being studied in clinical trials to treat myocardial infarction and peripheral thrombosis^8,9.

Immunomodulators are molecules that work on pathways that control the immunological response in many aspects of the immune system by lowering (immunosuppressives) or increasing (immunostimulators). Cytokines are small secreted proteins (<40 kDa), which are released by immune cells to regulate and influence the immune response, cytokine divided anti- and pro-inflammatory cytokines are mandatory in any immune response^10,11. Both IL-4 and IL-17 Cytokines mediate proinflammatory responses, with variations depending on the kind and location of the inflammation. The balance of pro-inflammatory and anti-inflammatory cytokines determines the overall outcome of the inflammatory response¹². IL-10 is an anti-inflammatory cytokine that plays a key function in reducing the host immune response to infections;^13,14. TLR2 is a member of a Toll-like receptor family that acts in the innate immune system immunity and pathogen recognition. It helps to mediate innate immune responses to bacterial lipoproteins and other microbial cell wall components^15,16.

MATERIAL AND METHODS:

Extraction and purification Staphylokinase from S. aureus:

The selected S. aureus was grown in the staphylokinase production Media (Satoh's medium) containing5g/L NaCl,3g/L yeast extract,10ml/L glycerol 10g/L nutrient broth was inoculated at 30°C with a shacking rate of 100 rpm for 24 hours and extraction according to the Tarek (2017)¹⁰, Staphylokinase was extracted by Precipitation of ammonium sulfate and crude enzyme dialysis and Purified by Ion exchange chromatography and gel filtration chromatography according to the Mohanasrinivasan et al. (2015)⁸.

Streptokinase:

The Streptokinase was obtained from a Samarth Life Sciences Pvt Ltd company as a substance utilized in the treatment of thrombus sufferers.

In vivo study:

Laboratory animals:

Forty male of albino mice, aged between 8-12 week and 26–38 g weight were used in this study. The mice were obtained from Iraqi National Center for Drug Control and Research and kept in plastic cages, housed under a standard condition in the animal house of the Mustansiriyah University/Department of Biology/College of Science. The mice left for 2 weeks for adaptation before the experiments began.

Animals groups and injection schedule:

The current study was included eight (8) groups, all BALB/C mice injected intramuscularly (i.m.) for 12 days as follows: In the negative control group, 50µl of normal saline was administered. Positive control group: injected with 50µl of Staphylococcus aureus suspension equal to 1.5×10⁸ CFU/ml adjusted to 0.5 MacFarland turbidity tube. Group A1: injected with 50 µl of staphylokinase (1µg/mouse) Group A2: injected with 50µl of staphylokinase (4 µg/mouse) Group A3: injected with 50µl of staphylokinase of (8µg/mouse) Group B1: injected with 50µl of streptokinase of 1µg/mouse Group B2: injected with 50µl of streptokinase (4µg/mouse) Group B3: injected with 50 µl of streptokinase (8µg/mouse). Also, all groups were challenged on days 4 and 8 of the injection schedule by 10% sheep RBCs (0.2ml/mouse. i.p.) the immune response assayed on day 11 and 12 of treatment by using in vivo and in vitro tests.

Blood sample collection:

After 12 days of treatment, the whole blood samples were collected in two eppendrof tubes (EDTA for WBC and ANC and plan tubes to obtained serum), and the serum stored at -20°C until used for immunological assays.

Arthus and DTH Reactions:

All mice in study groups were injected with 50μl of 10% washed SRBC in the right footpad and the left footpad injected with 50μl of normal saline as control the thickness was measured after 4 hrs. to determine Arthus reactions and after 24 hours to determine DTH Reactions^17,18,19,20.

Absolute Neutrophil Count (ANC):

Blood cell counts, including WBC, Absolute neutrophil count and neutrophil count, blood samples were collected and white blood cell counts were measured according to Tefferi et al. (2005)²¹.

White Blood Cell count = [Total Cells (4 squares) × Dilution factor (20)] ÷ 0.4

While the WBC differentiation was performed manually. The differential count was determined using the Giemsa technique; simply, a drop of blood was thinly distributed over a glass slide and air-dried. The neutrophil was calculated by multiplying the total WBC by the percent of each. In the meantime, ANC was computed as follows:

ANC = (total WBC × % [PMNs+ bands]) ÷ 100

Hemagglutination test:

It was prepared according to Al-Joofy, (1996)¹⁴ as following: made serial double-fold dilution of serum samples that obtained on day 12 in the 96 well round bottom microtiter plate, by mixed 50μl of serum in the first well with 50μl of phosphate buffer saline and transport 50μl to another wall which contains 50μl PBS, then added 50μl of 5% washed sheep red blood cells each well, and the plate was incubated at 37°C for 2 hours after the incubation period, read agglutination. Control for serum and SRBCs were made. Titer is used to express hemagglutination: it is the reverse of the latest positive dilution.

Measurement of serum cytokines and TLRs-2 levels:

ELISA was used to assess the levels of IL-4, IL-10, IL-17, and TLRs-2 in mouse serum. All procedures were carried out in accordance with Elab science's manufacturer's instructions.

Statistical Analysis:

The Statistical Analysis values of the studied variables were presented in terms of Mean Standard Error, as well as the differences between means were assessed using the computer program SPSS version 20 and the ANOVA, LSD test. So when probability value was equal to less or than 0.01 the differences were regarded significant.

RESULTS AND DISCUSSION:

Production, extraction, and purification of staphylokinase:

A crude enzyme from Staphylococcus aureus production medium (Satoh's medium) was purified using ammonium sulphate precipitation at a concentration of 85%, dialysis, Ion exchange chromatography, as well as gel filtration chromatography, yielding specific activities of 6.4- 11.4- 21.1-58.8-255.5U/mg, respectively.

MATERIAL AND METHODS:

Extraction and purification Staphylokinase from S. aureus:

Streptokinase:

The Streptokinase was obtained from a Samarth Life Sciences Pvt Ltd company as a substance utilized in the treatment of thrombus sufferers.

In vivo study:

Laboratory animals:

Animals groups and injection schedule:

The current study was included eight (8) groups, all BALB/C mice injected intramuscularly (i.m.) for 12 days as follows: In the negative control group, 50µl of normal saline was administered. Positive control group: injected with 50µl of Staphylococcus aureus suspension equal to 1.5×10⁸ CFU/ml adjusted to 0.5 MacFarland turbidity tube. Group A1: injected with 50 µl of staphylokinase (1µg/mouse) Group A2: injected with 50µl of staphylokinase (4µg/mouse) Group A3: injected with 50µl of staphylokinase of (8 µg/mouse) Group B1: injected with 50µl of streptokinase of 1µg/mouse Group B2: injected with 50µl of streptokinase (4µg/mouse) Group B3: injected with 50 µl of streptokinase (8 µg/mouse). Also, all groups were challenged on days 4 and 8 of the injection schedule by 10% sheep RBCs (0.2ml/mouse. i.p.) the immune response assayed on day 11 and 12 of treatment by using in vivo and in vitro tests.

Blood sample collection:

After 12 days of treatment, the whole blood samples were collected in two eppendrof tubes (EDTA for WBC and ANC and plan tubes to obtained serum),and the serum stored at -20°C until used for immunological assays.

Arthus and DTH Reactions:

All mice in study groups were injected with 50μlof 10% washed SRBC in the right footpad and the left footpad injected with 50μl of normal saline as control the thickness was measured after 4 hrs. to determine Arthus reactions and after 24 hours to determine DTH Reactions^17,18,19,20.

Absolute Neutrophil Count (ANC):

Blood cell counts, including WBC, Absolute neutrophil count and neutrophil count, blood samples were collected and white blood cell counts were measured according to Tefferi et al. (2005)²¹.

White Blood Cell count = [Total Cells (4 squares) × Dilution factor (20)] ÷ 0.4

ANC = (total WBC × % [PMNs+ bands]) ÷ 100

Hemagglutination test:

It was prepared according to Al-Joofy, (1996)¹⁴ as following: made serial double-fold dilution of serum samples that obtained on day 12 in the 96 well round bottom microtiter plate, by mixed 50 μl of serum in the first well with 50 μl of phosphate buffer saline and transport 50 μl to another wall which contains 50 μl PBS, then added 50μl of 5% washed sheep red blood cells each well, and the plate was incubated at 37°C for 2 hours after the incubation period, read agglutination. Control for serum and SRBCs were made. Titer is used to express hemagglutination: it is the reverse of the latest positive dilution.

Measurement of serum cytokines and TLRs-2 levels:

ELISA was used to assess the levels of IL-4, IL-10, IL-17, and TLRs-2 in mouse serum. All procedures were carried out in accordance with Elab science's manufacturer's instructions.

Statistical Analysis:

RESULTS AND DISCUSSION:

Production, extraction, and purification of staphylokinase:

In vivo assays:

Arthus and DTH:

The study was revealed the Arthus reaction and Delayed type hypersensitivity, highest value recorded in the positive control group injected with S. aureus (3.53 ± 0.05) and (4.53± 0.04) respectively, while the groups treated with Streptokinase Group B3(3.45 ± 0.01) had the maximum level of Arthus reaction, regarding Delayed type hypersensitivity highest value was recorded in group B2 (4.45± 0.01), and the lowest value recorded in group A1(3.09 ± 0.02) and (3.25 ± 0.05) respectively, the significant difference at p ≤0.01 (Table:1 and figure:1). The obtained results showed that purified staphylokinase and streptokinase treated mice and sensitized with SRBCs have high levels of antibody and then these mice were injected subcutaneously with SRBCs (Arthus reaction). The Arthus reaction is the formation of antigen-antibody complexes. When a foreign molecule (usually a protein) is encountered for the first time by an organism's immune system, specialized cells including macrophages and dendritic cells seize the molecule and start breaking it down so these antigens can be presented to antigen-presenting cells (APC) such as dendritic cells, macrophages and B lymphocytes^{22, 23}. IL-6 is produced by B cells and functions as a powerful stimulant of B-cell proliferation, plasma cell cycle, and antibody production (Abs)^{24, 25}. This reaction is caused by medications and treatments such as streptokinase, antivenom, and various antitoxins^{26, 27}.

Table 1: The effect of Staphylokinase and Streptokinase on Arthus and delayed-type hypersensitivity in immunized albino mice

Study groups	Arthus reaction (4 h)	Delayed type hypersensitivity (24 h)
	Mean ± S.E (mm)	Mean ± S.E (mm)
Negative control	2.71± 0.21 F	3.08 ± 0.01 E
Positive control(bacteria)	3.53 ± 0.05 a b c	4.53± 0.04 A
A1(1µg)	3.09 ± 0.02 c d e	3.25 ± 0.05 d e
A2 (4µg)	3.29 ± 0.06 a b c d e	3.76 ± 0.10 B
A3 (8µg)	3.19 ± 0.01 b c d e	3.47 ± 0.04 c d
B1(1µg)	3.11 ± 0.027 c d e	3.43 ± 0.03 c d e
B2 (4µg)	3.18 ± 0.00 b c d e	4.45± 0.01 A
B3 (8µg)	3.45 ± 0.01 a b c d	3.49 ±0.02 c d
p-value	0	0
LSD	0.23	0.145

A; Staphylokinase; B; Streptokinase, S.E.: standard error, µg: microgram, ml: milliliter, P: probability. Different letters suggest a significant difference in means between columns (p ≤ 0.01), whereas the same letters denote non-significant variations for means between columns (p > 0.01).

Figure 1: Delayed type hypersensitivity after 24 hours; A: right footpad control. B: left footpad test injection S.C by 50 µl of 10% SRBCs

The immunoglobulins involved in Arthus reaction are complement-fixing IgG or IgM and antibodies do not mediate DTH; This DTH to exogenous antigens includes T cells and APCs in a sensitized individual they all create cytokines that induce a local inflammatory response. Delayed type hypersensitivity has been widely used as one of the parameters to measure cell-mediated immune response of the animal^{28, 29}.

Table 2: Antibodies titer and absolute neutrophil count (cell/mm³) in study groups

Study groups	Antibodies titer *	Absolute Neutrophil count
	Mean ± S.E	Mean ± S.E cell/mm³
Negative control	50 ± 10 d e	26.58 ±2.67 c
Positive control (bacteria)	560± 80 a b	371.47±21.85 a
A1(1µg)	140± 20.00 c d e	71.53 ±11.59 c
A2 (4µg)	480 ± 92.37 a b c	181.21± 21.13 b
A3 (8µg)	240± 46.18 c d e	168.31 ±28.03 B
B1(1µg)	120 ± 23.09 c d e	72.32± 9.42 C
B2 (4µg)	280± 40.00 b c	200.19± 6.37 B
B3 (8µg)	200 ± 40.00 c d e	150.28 ± 16.96 b
p-value	0	0
LSD	150.615	49.167

*Titer is used to express hemagglutination: it is the inverse of the latest positive dilution

In vitro assays:

Absolute neutrophil count:

The ANC results reported that the maximum value was recorded in the positive control group that was injected with S. aureus (371.47±21.85) while the groups which were treated with Streptokinase the highest value was in group B2 (200.19±6.37) and the lowest value in group A1(71.53±11.59) treated with 1µgofstaphylokinase with a significant difference at p ≤0.01. As illustrated in table 2 and figure 2.

This increasing and decreasing in value between the groups in Neutrophil counts were associated with the initial signal of the immune response which was represented via a variety of pattern recognition receptors, including Toll-like -2in the current study, the control group injected with A2 and B2 groups recorded high level of TLR-2, TLR2 displayed in presenting cells such as (macrophages, neutrophils, dendritic cells) and phagocytes which are the major innate cells which respond to agents¹⁹. Thus, when the Neutrophils cells are stimulated by TLR-2, they produce chemotaxis and cytokines that attract more Neutrophil cells are transported to site of infection and thus the greater the volume in TLR2is the higher of signals produced from the neutrophil that high lead to an increase in several neutrophils³⁰.

Some studied showed the absence of TLR2 reduced the chemotaxis rate by murine neutrophils, as well as their killing mechanisms and survival, this was noticed in the groups that treated with Staphylokinase and streptokinase the neutrophils were showed a lower number because the poor immunogenicity and low MW of these enzymes that not able to be recognized by immune system components such as TLR-2, low level of TLR-2 will produce weak signals to activated neutrophil and lead to attracting other neutrophils and other cells^31,32. The absence or decrease of TLR2 influences neutrophil and macrophage anti-microbial activity mainly through an effect on TNF-α and other cytokines production³³.

Figure 2: Absolute Neutrophil Count in the blood of albino mice that were treated with the purified Staphylokinase, streptokinase, and immunized with 10% SRBCs

Antibodies titers:

The results showed the highest value of antibodies titers in the positive control group (560± 80), while in group A2 was 480±92.37 and the lowest value in group B1(120±23.09) that treated with (1µg) of Streptokinase, with a p-value ≤ 0.01 indicating a significant difference (table 2 and figure 3). The hemagglutination antibody titer assay is one of the parameters used to evaluate the humoral immune response of the animal. In the present work, it has been found that sensitization of albino mice with 10% SRBCs stimulate the immune system of mice for the formation of antibody and higher antibody titer found in a concentration of 4 µg/mouse of purified staphylokinase and streptokinase where at this concentration they have immune stimulant activity on the immune system of the albino mice^34,35.

Warmerdam et al. (2002)³⁶ showed that after staphylokinase injection, the majority of patients acquire neutralizing Ab. Many antigenic sites identified by these Ab have been found, and staphylokinase is a T cell-dependent Ag, suggesting that immunological memory can be developed. Additional thrombolysis with staphylokinase stimulates the growth of staphylokinase-specific T cells³⁷. The apparent response to bacterial antigens, such as streptokinase, is confirmed by an increase in the rate of interleukin-4 and interleukin-5. Th2 cell production of IL4 may be one of the immunological changes responsible for increased IgE antibody production³⁸. A high antibody titer is linked to streptokinase hypersensitivity responses. This has significant ramifications for reinfarction thrombolysis. Avoidance of streptokinase-related thrombolytic medicines should be considered in patients whose anti-streptokinase antibody titers are expected to be elevated³⁹.

Figure 3: Serum antibody titer comparison between purified staphylokinase and streptokinase

The mice treated (im) with different concentration of staphylokinase and streptokinase for 12 days, and 10% SRBCs (ip) in day 4 and 8 of the schedule.

Toll-like receptors:

The results of TLR-2 showed the highest value in the positive control group (21.343 ± 1.55) followed by Group A2 (15.06± .58) & the lowest value was observed in group B1 with a significant difference (p ≤0.01), but the other groups had various values, as indicated in table -3 and figure-4.

Figure 4: SerumTLR-2 level in of albino mice treated with the purified Staphylokinase, streptokinase, and immunized with 10% SRBCs

The findings of the current investigation showed that the highest value TLR-2 in group A2 and B2 in comparison with other groups this indicates the role of TLR-2 induces the production of pro-inflammatory cytokines IL-4 and IL-17. Toll-like receptors are important for bacterial clearance because they allow the innate immune system to recognize infections early. Many pattern recognition receptors (PRRs) have been implicated in the early sensing of bacterial components, including Toll-like and (NOD)-like (NLRs) receptors²⁷. Toll-like receptors are the most important class of innate pattern recognition receptor by which host immune cells can recognize pathogen-associated molecular PRRs that may also recognize endogenous signals generated by host cells after stress or death, such as heat shock proteins or extracellular matrix protein fragments. TLRs are expressed by numerous types of cells, including dendritic cells, macrophages, B and T lymphocytes, mucosal epithelial cells& endothelial cells²⁸, and cell engagement often results in the release of cytokines and chemokines, which facilitates the recruitment and activation of APCs as well as the stimulation of adaptive B- and T-cell responses⁴⁰.

Interleukin-4:

In contrast, this study discovered that results of a IL-4, which was the highest value recorded in the positive control group (141.03 ± 6.74) while the highest value in group B2 (85.07 ± 2.73) which treated with streptokinase and the lowest value in group A1 (60.30 ± 3.18) treated with (1µg) of staphylokinase with the a significant difference at p ≤0.01 (table 3 and figure 5). This means that the purified staphylokinase and streptokinase at a concentration of 4µg (A2 and B2) stimulate the APCs that affect CD4⁺ T cell response directly and stimulate production of cytokine IL-4⁴¹, interleukin 4 effects on immune cells including differentiation of naïve T cells to type- 2 T helper and T follicular helper cells, B cell antibody production and isotype switching to immunoglobulin E (IgE), expansion of basophils, mast cell activation, eosinophil's, and macrophages⁴². Interleukin 4 associated with activation B lymphocytes and differentiation to IgE producing cells, IgE has a role in evoking allergy, the decline in IL-4 is a good marker that staphylokinase (8µg) could be used in the treatment of thrombosis.

Figure 5: SerumIL-4level in of albino mice treated with the purified Staphylokinase, streptokinase, and immunized with 10% SRBCs

Figure 6: Serum IL-10 level in of albino mice treated with the purified Staphylokinase, streptokinase, and immunized with 10% SRBCs

Interleukin-10:

According to the findings of this investigation, IL-10 has the highest value recorded in the positive control group injected with S. aureus (89.53 ± 1.69) and group A3 (72.52± 1.10), while the lowest value of IL 10 recorded in group B1(66.15 ±1.18) treated with 1µgof Streptokinase with the significant difference at p ≤0.01 as shown in table -3and figure 6.

An increase in the level of IL-10 provides evidence that this Interleukin 10 is a strong anti-inflammatory cytokine that plays a critical, and often essential, role in avoiding inflammatory and autoimmune diseases. IL-10 is an expression is associated immune regulatory cytokine that regulates the production of anti-inflammatory cytokines and is produced by a variety of innate and adaptive immune cells, including, macrophage, monocyte, T lymphocytes, and B-lymphocytes. IL-10 serves a crucial protective role & it is needed to inhibit bacterial propagation and host morbidity by regulating effector T cells and the related downstream hyper activation in inflammatory phagocytes⁴³.

Interleukin-17:

The results in table-3 and figure-7 showed the quantity of IL-17 in the serum in immunized BALB-c mice appeared the highest value recorded in the positive control group injected with S. aureus (448.59 ± 18.98) and groupB2 (409.04± 14.22), while the lowest value of IL 17 recorded in group A3 (223.35 ± 15.82) treated with 8µgof Staphylokinase with the significant difference at p ≤0.01. Increase the level of IL-17 provides evidence that this cytokine playsan essential role in immune and inflammatory response when Th17 cells produce interleukin-17, which is important for defense against extracellular pathogens on mucosal surfaces⁴⁴, and Interleukin-17 has emerged as an important role in the immune system of mammals. Although the cytokine plays a host-defensive function in very many infectious disorders, it promotes inflammatory disease in autoimmunity as well as host defenses against fungal and bacterial infections^{45, 46}.

CONCLUSION:

In the present study, we can conclude that staphylokinase had a positive immunomodulatory effect on immune response in immunized mice. As shown in the results of the level of IL-4, IL-10, IL-17, TLR-2, Abs titer, absolute neutrophil count, and both Arthus and DTH reactions. The best treatment efficiency was recorded in staphylokinase threat groups in different concentration compared with other groups because SaK stimulates the immune system, such as increased level of IL-10, the neutrophil which works to regulate the immune system, less appearance of hypersensitivity in staphylokinase than streptokinase treated mice, sodecline in IL-4 is a good marker that indicates the possibility to use in the treatment of thrombosis. Whereas act as an immunomodulator and staphylokinase is a small size plasminogen activator compared to streptokinase and not prevent the systemic degradation of fibrinogen., also, other studies need to investigate the effect of staphylokinase on tissue and organs.

ACKNOWLEDGMENT:

The authors would like to thank the Biology Department of Mustansiriyah University College of Science in Iraq's capital city Baghdad.

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Received on 15.04.2022 Modified on 01.06.2022

Research J. Pharm. and Tech 2023; 16(2):839-847.

DOI: 10.52711/0974-360X.2023.00143

Study groups	TLR-2	IL-4	IL-10	IL-17
	Mean± S.E pg/ml	Mean± S.E pg/ml	Mean± S.E pg/ml	Mean± S.E pg/ml
Negative control	1.76 ± 0.24 H	23.43 ± 2.04 F	60.01 ± 1.88 E	147.75 ± 3.92 c
Positive control (bacteria)	21.343 ± 1.55 A	141.03 ± 6.74 A	89.53 ± 1.69 A	448.59 ± 18.98 a
A1(1µg)	12.70± 1.42 b c d e	60.30 ± 3.18 d e	67.71± .72 b c d	247.91 ± 4.57 b
A2 (4µg)	15.06± .58 b c d	80.63 ±3.37 b c	69.74 ± 1.10 b c d	264.96± 3.14 b
A3 (8µg)	11.58± 1.61 b c d e f	57.84 ± 3.19 d e	72.52± 1.10 b c	223.35 ± 15.82 b
B1(1µg)	4.76± .38 f g	64.86 ± 5.37 c d e	66.15 ±1.18 c d	407.34± 12.27 a
B2 (4µg)	9.81± .55 c d e f	85.07 ± 2.73 b c	68.77 ±.73 b c d	409.04± 14.22 a
B3 (8µg)	8.68± .57 d e f g	79.20± 1.48 b c d	70.69±1.01 b c d	398.40 ± 29.79 a
p-value	0	0	0	0
LSD	2.966	11.305	3.624	44.961