Immunomodulatory Effects of Helicobacter pylori on Pro and Anti-inflammatory Cytokines Production in Peripheral Whole Blood Cells Culture

1Department of Microbiology and Virology, Faculty of Medicine, Zanjan University of medical sciences, Zanjan, Iran. 2Professor of Biostatistics, Department of Biostatistics and Epidemiology, Faculty of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran. 3Department of Immunology & Cancer Gene Therapy Research Center, Zanjan University of Medical Sciences, Zanjan, Iran. 4Assistant professor of Microbiology, Department of Microbiology and Virology, Faculty of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran.

According to the previous studies, it is demonstrated that infectious diseases are one of the major challenges for Health System in which, alteration in the soluble mediators function fails to induce an adequate immune response and is accompanied by hypo/hyper activity of immune responses.In addition, some problems as: reappearance of invasive pathogens and changing nature of infectious disease manifestation, add more considerations up for scientific researchers and physicians.Accordingly, in light of these problems, sepsis has been continued to be a worldwide dilemma with the prevalence of high mortality 1 .
Also, indiscriminately usage of antibiotics has led to antibiotics resistance, multi drug resistant strains reappearance and chronicity of the diseases, recently.Overall, these issues, visualize several severe gaps in clinical trials which led scientists to look for novel directions toward immunobiology targeting 1 .Therefore, it is expected that an optimistic insight and supportive point of view on other therapeutic strategies should be created.This is where, it is rational to interface with some elements contributing to the reregulation of the dysregulated immune responses.
The term "Immunomodulation" refers to any intervention modifying the immune response with therapeutic intent."Immunomodulators" are considered as active substances in immunotherapy based therapeutic methods.These agents are approved by the International Organization for Food and Drug Administration (FDA) with fewer side effects and less potential for drug resistance advantages.There are a wide variety of immunomodulators from natural and organic compounds to synthetic and recombinant ones [2][3][4] .
In this strategy, activity of the host immune system, changes through the dynamic regulation of signaling molecules such as: antibodies, soluble receptors, bio drugs, hormones, acute or chronic stress, adhesion molecules, neurotransmitter peptides, cytokines and even bacterial extract (or cellular membrane fractions of bacteria).Generally, these changes include in immunostimulation, immunosuppression and immunological tolerance induction assisting to adjustment of the desired immunity level on the circumstances [1][2][3] .
Growing number of scientific investigations and documentations indicate that certain biological agents, specially a well-known group of microorganisms, also exert immunomodulatory properties by modulating leukocyte function and circulating concentrations of proinflammatory cytokines as extracellular (glyco) protein key regulators and messengers of the immune system 5,6 .
Anyway, our knowledge of the immunological communications about protection against pathogens is superficial.Subsequently, evaluation of immunopathophisiology of the pathogens, outstanding about cytokines activity and their receptors, cytokines cascades involved in microbial-host immune responses interactions, and recruitment of an appropriate immunomodulator to immunologic phase of diseases should be considered to accelerate the realization of overcoming to diseases rate 2, 3 .It seems that clinical applications of shifts in cytokines secretion due to the immunomodulators usage, may be a potentiated window for elimination of undesirable immunological reactions leading to improvement of immune levels for whom suffering from such immune related disorders.
Since that, there has been no study on whole blood cells with whole body of H.pylori in order to assess immunomodulation, the purpose of this research is conducting an in vitro study evaluating immunomodulatory properties of H.pylori on peripheral whole blood cells culture by cytokines assay.

Ethical considerations
This study accessed ethic's approval from the Zanjan University of Medical Sciences Research Ethic's Committee (no.ZUMS.REC.1394.143).Among the medical faculty students, seven young healthy (H.pylori-non infected, without chronic inflammatory diseases history) volunteer women (range 20-25 years old) were recruited for the purpose of this study.First, by giving them explanations about moral confidence and biosafety, oral consent was reached and then, a written informed consent was achieved.

Immunomodulatory compound preparation Bacterial strain culture
First, from a pure bacterial culture of an approved pathological positive biopsy endoscopy specimen (CagA + , VacA + ) belonged to a peptic ulcer patient, isolation of H. pylori was done.After transferring this bacterial culture petri dish into an equipped designated microbiology laboratory in a jar with gas pack C (Merck.KGaA.Germany), a loop of bacteria introduced into a Brain Heart Infusion (BHI) broth culture (Merck.Germany) as an enrichment/activator medium and incubated for 24 hours at 37°C in a microaerophilic condition.
Subsequently, Brucella Blood Agar (supplemented with 10% defibrinated sheep's blood (Provided by ROSHD, Med Biotech, IRAN), 10µg/L vancomycin (DANA Pharmaceutical Company, IRAN), 5µg/L trimethoprim (Amin Pharmaceutical Company, IRAN), 2.5 IU/L polymixin B and Brucella Agar (Merck.Germany)) was considered as a reference culture medium.A further streak cultured plate of bacteria was prepared and incubated for 5 days at 37°C at a candle jar to ensure bacterial characteristics (Main stock cultures were kept at -20°C with 18% glycerol in BHI broth).
Then, colonies with tiny, round, raised, flat, transparent features and 0.5-1 millimeter in diameter, were observed and microbial tests such as Gram staining for purity and bacterial morphology assessments were done.
Ultimately, the nature of bacterial strain was confirmed through positive results for biochemical tests such as tube Urease (Urea Agar culture), slide Catalase and oxidase.

Bacterial solution preparation
It should be noted that concentrations of whole bacteria used overall this experiment, were reached in our previous trials in order to induce immunomodulatory effects as follows: Here, confirmed colonies were scrapped from Brucella Blood Agar surface and transferred into BHI broth.Next, 1.5 ml of 24 hours harvested H.pylori in BHI broth media was collected into an eppendorf microtube and centrifuged (during log growth phase of bacteria).Supernatant fluid was removed and remained pellets were once suspended in 1.5 ml of 0.01M ice-cold sterile PBS (Phosphate Buffered Saline, pH=7.28)(MERCK.Germany).The eppendorf microtube was again centrifuged.Next to discarding supernatant, 1.5 ml of RPMI-1640 was added which followed by gently mixing to reach homogenous bacterial solution.Concentration was determined by colony forming unit (CFU) counting.

Whole blood cells preparation
To substructure TNF-alpha and TGF-beta expression manifestation in the response to antigenic stimulation (immunomodulator), human peripheral whole blood cells were chosen for our experiment's end.

Participants (blood donors)
Median age and BMI of participants were shown in table2.All donors had antigens A on their erythrocytes surface (blood group A).

Blood collection
Participants were pleased to come to the laboratory.Blood samples were withdrawn by an authorized supervisor with sterile gauge needles.Antecubital venous blood (7 ml) was collected into vacutainer blood collection tubes containing sodium heparin (Caspian Tamin Co. Iran).

Blood analysis
Blood samples which were collected into heparinized tubes, first inverted several times to mix properly with anticoagulant agent.Then, they were delivered to hematology ward for hematological analysis immediately to achieve the best unbiased results by an automated hematology analyzer (Nihan Koden.Cell Tac E. Japan).Each sample was measured twice and the average value was recorded for all donors (Table 2).

Study procedure
After preparation of reagents, procedure of study was defined as below:

Whole blood cells culture stimulation
After confirmation about study design, first of all, stimulant was reconstituted by bringing it to room temperature up.As shown in Table 1, required volumes of RPMI-1640 were added into the culture wells of a 48 flat bottomed wells sterilepolystyrene cell culture plate (Jet Biofil.Mainland) (1.55 ml and 0.19-0.29 ml, Max and working well volume respectively) followed by 0.25 ml of heparinized whole blood cells (final estimation: 2×10 6 cells per ml).Next, followed by adding the appropriate amount of bacteria solution (Table 1) in aseptic condition under biological safety cabinet (Laminar flow class II).Then, ingredient of each well was gently mixed by several times aspiration and ejection.

Incubation conditions of treated whole blood cells
Thereafter, antigen stimulated specimens (table1), were incubated for 22h at 37°C and 5% CO 2 atmosphere in a 90% humidified chamber (Time of incubation was set).After cultivation, and cell counting, viability assay was done for human whole blood cells.

Supernatant collection
After 22h incubation, supernatants of all samples were transferred into 1.5 ml microcentrifuge eppendorf tubes and spun for 5 min at 5000 rpm at room temperature (Sigma).Subsequently, supernatant phase of fluid for all samples, were collected into another new labelled eppendorf tubes and stored temporarily at -20°C freezer.

Measurement of cytokine secretion
First, supernatant fluids were removed from freezer and kept at room temperature for thermal equilibrium.Results of this study, were documented with scales of secreted cytokines (TGF-beta and TNF-alpha) profile of homogenated human peripheral whole blood cells stimulated by pathogenic isolate of H.pylori through an Enzyme Linked Immunosorbant Assay (ELISA) method.All assays were run in triplicate for all wells containing samples and the average scores were registered by a blind technologist.

ELISA for cytokine assay TGF-beta1
The assay was performed in order to manufacturer's instruction commercial kit for quantative detection of TGF-beta1 cytokine (eBioscience.Bender Med System GmbH.Austria.REF BMS249/4/) in cell culture supernatants which was estimated as below: Briefly, after pretreatment of samples with assay buffer 1x, 1N HCl and 1N NaOH, 100 µl of assay buffer 1x was added to blank well.100 µl of each standard solutions was added to standard wells.Then, 60 µl of assay buffer 1x and 40 µl of vortexed pretreated samples were added to sample wells.After incubating for 2h at room temperature and 400 rpm (set on a microplate thermoshaker), the human anti TGF-beta1 coating antibodies which are adsorbed onto microwells, bind to human TGF-beta present in the samples and standards.
Next to washing, 100 µl of Biotin-Conjugated anti human TGF-beta detecting antibody was added to each microwell strips and incubated as clarified recently in order to bind to complex which was made in previous step.
After washing, unbound biotin conjugates were removed and 100 µl diluted Streptavidin-Horse Radish Peroxidase (HRP) was added to all wells and incubated as said before, to bind to 3-fraction complex which was created in previous step.
After last washing for removing unbound Streptavidin-HRP, 100 µl of Tetra Methyl Benzidine (TMB) Substrate (as cromogene), was added to each well to associate to last 4-fraction complex.It was incubated for about 30 minutes at room temperature for color development without exposure to direct intense light.Color intensity is proportional to the HRP activity.
After that, as soon as the highest standard was reached to dark blue color and an OD of 0.90-0.95for standard solution was recorded, enzyme-substrate reaction should be terminated by pipetting 100 µl of stop solution (Sulfuric Acid) into each well.This is where, changing pH of the medium by stop solution seems essential for inhibition of enzymatic reaction and stabilizing color development to reach an accurate intensity measurement.Absorbance of each well was read immediately on an ELISA MICROPLATE READER (Garni Company) using 450 nm (primary) and 620 nm (reference) wave lengths.Cytokines concentrations were calculated with a standard curve.

TNF-alpha
The main investigation procedure of TNF-alpha assay was done similarly to TGF-beta exception for sampling step (samples don't any need to pretreatment and only should be diluted by adding sample diluent buffer) in order to manufacturer's instruction (eBioscience.DX.Bender Med Systems GmbH.Austria.REF BMS223/ 4CE).
Two kits for mentioned cytokines were human specific with sensitivity of 8.6±2 pg/ml and 2.3±2 pg/ml for TGF-beta and TNF-alpha, respectively.

Statistical Analysis of data
The Statistical Package for the Social Sciences (SPSS) version 16.0 computer software (SPSS Inc., USA) was used for the statistical analysis of acquired data.Non-parametric statistical tests (Freidmann's and Mann Whitney) and descriptive tests were applied for information and data of the experiment.Results were expressed as Mean plus minus Standard Deviation (SD).Statistical significance was considered at p value<0.05.

Hematological parameters of blood samples
Table 2 indicates the Mean and SD scores recorded for hematological analysis of blood samples.All of these hematological parameters scores fell into the normal ranges.TGF-beta cytokine production by H.pylori stimulated whole blood cells culture Table 3, shows the average concentrations of the TGFbeta cytokines produced in the H.pylori stimulated whole blood cells culture meanwhile the nature of the wells were clarified after data statistical calculation.

Cytokines production by H.pylori stimulated whole blood cells culture
As shown in Table 3, H.pylori significantly upraised TGF-beta cytokines secretion by whole blood cells after 22h incubation (well no.3) compared to control well (well no.2) (p value=0.017).
TNF-alpha cytokine production by H.pylori stimulated whole blood cells culture Table 4, shows the average concentrations of the TNF-alpha cytokines produced in the H.pylori stimulated whole blood cells culture meanwhile the nature of the wells were clarified after data statistical calculation.
As shown in Table 4, H.pylori significantly upraised TNF-alpha cytokine production by whole blood cells after 22h incubation (well no.3) compared to control well (well no.2) (p value=0.018).

DISCUSSION
H.pylori is a bacterium with colonization in gastric mucosa, has tight association to several adverse clinical sequelae like peptic ulcers, gastric cancers (metaplasia and dysplasia of gastric mucosa) induction, non-Hodgkin lymphoma, bacteremia (as a rare complication) and amazing ability for co-existence with humans for decades with subsequent evasion of chronic gastric inflammation [21][22][23][24][25] .Persistence and severity of H.pylori infection, depends on bacterial components, host associated factors (such as susceptibility, immune responses and genetic factors) and environmental factors that determine bacterium switching to commensalism or pathogenicity variations 2,21,[23][24][25][26] .
In addition, it is demonstrated that H.pylori chronic infection may also be beneficial to host immune system for conferring protection against allergy, rhinitis, asthma, atopic dermatitis (Eczema) and Inflammatory Bowel Diseases (IBD) which is highly associated with suppressive Treg cells function 9, 12-14, 18, 20, 24, 35-47 .
Inducible Treg cells are considered to immune tolerance maintenance through induction of anergy and deletion of autoreactive T cells and believed to be generated by tolerogenic semi mature Dendritic Cells (DCs) which are highlighted for us with converting naïve T cells into Foxp3 Treg via antigen presentation in the absence of costimulatory molecules.Reprogramming of H.pylori experienced DCs to tolerogenic phenomenon, requires inflammasome and caspase-1 activation, secretion of IL-18, and a TGF-beta dependent manner 23,24,36,48 .
These already mentioned, had been clinical studies based on H.pylori immunomodulation properties.So far, whole body of H.pylori effects on whole blood cells in order to immunomodulation assessment has remained unclear.Overall, in the present study aimed to investigation of H.pylori immunomodulatory impact on peripheral whole blood cells culture, our results corroborated that H.pylori may trigger a strong modulation and upregulation on both TGFbeta and TNF-alpha cytokines secretion in whole blood cells compared to control (p value= 0.017, 0.018, respectively).The amounts of secreted TNFalpha by whole blood cells after exposure with H.pylori were more than TGF-beta.So, it seems that H.pylori can induce Th1 and Th2 responses which promote pro and anti-inflammatory actions.There are no such as this study and our results are in accordance with some partially similar previous studies.M.Wiese et.al in their study aimed to investigation of Immunomodulatory effects of live Lactobacillus Planatarum and Helicobacter Pylori on some cytokines and cell surface markers induction in whole blood culture, showed that both, H. pylori alone and in combination with L. plantarum, had a strong modulatory effect on the synthesis of IL-10 and an inhibitory effect on IFNgamma secretion 61 .
Jay Luther et.al showed that H pylori DNA has the ability to downregulate proinflammatory responses from DC and this may partly explain the inverse association between H pylori and IBD with lower systemic type 1 IFN 62 .
Frank Meyer et.al indicated that Th1 polarization of the gastric immune response may be due in part to the direct effects of multiple different H. pylori components that enhance IFNgamma and IL-12 production while inhibiting both IL-2 production and cell proliferation that may be necessary for Th2 responses 63 .
Songhua Zhang et.al observed that antigen-specific functional differences between the T cell repertoires of H. pylori positive andnegative subjects for a set of 90 immunoinformaticpredicted T-cell epitopes from seven genetically diverse H. pylori strains.Cytokine ELISA measurements performed using in vitro PBMC culture supernatants, demonstrated significantly higher levels of TNF-alpha, IL-2, IL-4, IL-6, IL-10, and TGF-beta1 in the H. pylori-infected subjects, whereas IL-17A expression was not related to the subjects H. pylori-infection status 22 .
Benedetta Romi et.al for finding if and how H. pylori could induce and modulate human T cell activation, studied direct interaction of live, but not killed bacteria with purified CD3+ T lymphocytes on lymphocytes freshly isolated from peripheral blood of H. pylori-negative donors.The results showed activation of CD3+ T lymphocytes, predominantly gamma-delta T cells, upregulation of CD69 and cytokines such as TNF-alpha and IFN-gamma favouring to inflammatory microenvironment leading to incompletely clearance of pathogen, local inflammation and eventually to the gastric and duodenal diseases 64 .
In conclusion, to attribute and generalize results of this study to clinical manifestations, it should be noticed that there is a complex immune response to H.pylori.In addition, this pathogen has ability to induce Th1 and Th2 immune responses.Induction of TNF-alpha production as a multifunctional cytokine has a key role in apoptosis, cell survival and antitumor activity as well as in inflammation and immunity.Upregulation of TGF-beta secretion, stimulates Th2 responses differentiation, Th1-mediated autoimmune disease reduction, and appears immunological tolerance leading to Treg cell proliferation, multi-organ inflammation and autoantibodies development diminution.

CONCLUSION
According to results of our study, H. pylori has a special modulatory impact on immune system responses with pro and anti-inflammatory cytokine induction.
Immune related anomalies have been still remained as a most common concerns for health care system.It appears that consideration of time for immune intervention, accurate understanding on immunopathophisiology of infectious related immune disorders, predomination of pro and/or anti-inflammatory cytokines and conditions of immune microenvironment which are pivotal to fulfill immunomodulation.
This study refers to an optimistic point of view provided to further investigation on clinical applications of alteration in cytokines secretion due to the immunomodulators usage.This may be a promising window in order to elimination of undesirable immunological reactions and improvement of immune related diseases.

Table 1 .
Consumed volumes of RPMI-1640 and immunostimulants solution for each well * were blank for culture medium and blood cells, respectively

Table 2 .
Descriptive Statistics results of hematological parameters recorded for blood samples at a glance

Table 3 .
TGF-beta cytokine production (pg/ml) in response to H.pylori stimulated whole blood cells culture

Table 4 .
TNF-alpha cytokine production (pg/ml) in response to H.pylori whole blood cells culture