Is the Status of CXCL9-CXCL10-CXCL11/CXCR3 axes Altered in Brucellosis Patients?

Aim and objectives: Brucellosis is common in most parts of the world between humans and domestic animals. The causative agent is bacteria from the Brucella family, which are intracellular pathogens. Despite, Brucella spp. Is able to infect humans, their ability to induce production of chemokines by lung cells is unknown. In this study, we showed an association between brucellosis and CXCL9, 10 and 11chemokines Methods:We have totally recruited 71 brucella patients who have referred to the either local health centers or hospitals of kerman province. Diagnosis of acute brucellosis was made based on a brief history of the disease,clinical examinations results of serological tests,antibody titration in serums was performed by bacterial wright method, agglutination and 2ME with antigen. Brucellosis patients CXCL9, CXCL10 and CXCL11 serum levels were detected by sandwich ELISA using paired chemokine-specic mAbs, according to the manufacturer’s guidelines. Transcription levels of mRNA for the CXCR3 as the chemokine receptor for CXCL9, CXCL10 and CXCL11 was examined by QRT-PCR using primers. Results: We have found that there was a positive correlation between the level of chemokines and their receptor. Here ELISA test showed that B. abortus induces expression of some IFN-γ induced CXCL9, 10 and 11chemokines in brucellosis patients. We have also showed the increased expression of CXCR3 as their shared receptors in brucellosis patients with real time-PCR. Conclusion: According to these ndings there was a positive correlation between the level of chemokines and their receptor. We have found that there was a positive correlation between the level of chemokines and their receptor.


Introduction
Brucellosis is common in most parts of the world between humans and domestic animals (1,2). Domestic animals such as goat, sheep, and camel are well known as reservoirs of the disease. Cattle may also serve as the host of Brucella in some cases. The most common case of Brucella abortus is in cattle, wild cattle, buffalo, camels, and Asian cattle. Canines are producing the disease in dogs, and these types of Brucella are possible transmitted to humans and relative produce the related types of Brucellosis. In Iran, brucellosis could also be transmitted to the people via uncontrolled blood transfusions, animal contacts dairy products and meat. Various types of brucellosis are intracellular organisms that present inside the reticuloendothelial system cells including macrophages and monocytes. The causative agent is bacteria from the Brucella family, which are intracellular pathogens (3,4). The airways epithelial cells in addition to alveolar macrophages are of rst cells that are exposed to inhaled types of microorganisms and are therefore ready to run an immediate immune response. Inspite of, containing of anatomical barriers against processes of microbial invasion, the respiratory system epithelium is itself able to respond the arms of pathogens with a relative in ammatory response. The cytokines/chemokine network members are able to control infection and in ammation (5, 6). These types of epithelial responses might possibly further be elevated by the stimulated actions of cytokines that are secreted by alveolar macrophages (7,8). The respiratory epithelium generates reagents such as β-defensins, small antimicrobial peptides which normally present within uid lining of the respiratory tract alongside with other antimicrobial components e.g. lysozyme and cathelicidins, in response to the infection. The respiratory epithelium is also able to actively respond to infections throughout generating several other in ammatory mediators, like chemokines, especially CCL20. Alternatively, the so called liver and activation-regulated chemokine (LARC), the macrophage in ammatory protein-3 (MIP-3) or Exodus-1 and CCL20 as a named chemoattractant for the maturing dendritic cells as well as effector/ memory T-cells and B-cells. Although, CCL20 and its cognate receptor, the CCR6 have been indicated to regulate in vivo the chemo attraction of the dendritic cells and lymphocytes in various tissues, including lungs (9,10). Notably, the repertoire of CCR6 positive T cells and Th17 cells are speci cally recruited by CCL20 (11), a fact that could be relevant to the immune responses against infectious agents. CCL20 and β-defensins, especially hBD2, showed that share many similarities and both share CCR6 as a common membrane receptor. While reacting CCL20 to CCR6 was known as the mediator at chemotactic responses of the immature dendritic cells to the CCL20, more recent studies demonstrated that β-defensins also exhibits chemotactic activity for CCR6 (12,13). They both are able to act as chemoattractants for a wide variety of cell types involved in innate and adaptive immunity and can also stimulate various immune responses (including production of cytokines maturation of dendritic cells and etc.) (14,15). In particular, hBD2 induces several cell types memory T cells, immature dendritic cells, mast cells and neutrophils chemo taxis processes (16-18). In other words, despite the fact that, CCL20 was initially described as a chemokine but more recent studies have revealed that these types of molecules are also able to display antimicrobial activities against both the gram positive and gram negative bacteria (19,20). It has thus been proposed that the antimicrobial activity of CCL20 could possibly be due to the fact that this chemokine shares structural properties with β-defensins, including antiparallel β-pleated sheet core structure and charge distribution (19). The CCL20 and hBD2 expression have been demonstrated to be elevated in respiratory epithelial cells following exposure to infectious agents or antigens (21,22) as well as in response to members of pro-in ammatory cytokines network (23,24). The human brucellosis is mainly caused by Brucella melitensis, B. suis or B. abortus. The disease is as a zoonotic illness and affects over 500,000 people annually which is distributed, worldwide (25,26). These bacteria, inhalation of infected aerosols is frequently involved in contagion. Chemokines are a subclass of cytokines with the property of pro migratory and chemotactic feature for blood leukocytes. They themselves are divided in four sub-divisions of C, CC, CX3C.The studied chemokines in the (CXCL9, 10 and 12) present study belong to the CXC subdivision. In spite of the importance of the respiratory route of the entrance of Brucella within the organism, these bacteria reaction with the pulmonary cells has rarely been well examined, however, previous studies have reported that Brucella species have the ability to infect and replicate within the lung epithelial cells of human, and this can stimulate these cell, to produce CCL20 (27)(28)(29). Rewardingly, due to the both their chemotactic and antimicrobial activities, CXC chemokines have also been purposed to play fundamental parts in innate immune response of pulmonary system to the inhaled pathogensand compelling evidences have also indicated the enhancement of chemokines in lung tissues both following and during infection. Because (30,31), almost nothing is reported regarding the expression of these molecules in brucella infection or about their antimicrobial activity against this pathogen, the main goal of the present study was to address these issues.

Patients and specimens
We have totally recruited 71 brucella patients who have referred to the either local health centers or hospitals of kerman province. Diagnosis of acute brucellosis was made based on a brief history of the disease,clinical examinations,results of serological tests,antibody titration in serums was performed by bacterial wright method, agglutination and 2ME with antigen and wright rate . The Elisa Juan behring device was used to read Elisa's results. Fifty (50) people of both sexes from the blood donors and students from kerman province of medical science who were normal without history of brucellosis or other types of bacterial infections control as participants in this study.

RNA isolation and preparation of cDNA
The expression of the CXCR3 mRNA was examined using quantitative real time PCR (qRT-PCR) for all of patients and controls. From the bu coat of blood samples total RNA was isolated employing Invitrogen Trizol according to the instructions of the manufacturer. Both quality and purity of the isolated RNA were examined by running on agarose gel and electrophoresis as well as measurement of optical density (A260/A280 ratio) by Nano Drop 1000 Spectrophotometer (Wilmington, DE, USA), respectively. The genomic DNA was removed from the RNA preparations, throughout DNase I, RNase-free kit from Thermo (Thermo Scienti c, USA), according to instructions to manufacturers. To achieve this, ; 1 µg RNA, 1 μl 10X Reaction Buffer with MgCl2, 1 μl DNase, RNase-free, water nuclease-free up to 10 μl and was incubated at 37°C for 30 min and then 1 µL 50 mM ETDA was added and the incubation was prolonged at 65°C for 10 min. The processes of reverse transcription (RT) was undertaken using the Revert Aid First Strand complementary DNA (cDNA) Synthesis kit from Thermo (Thermo Scienti c, USA). The RT reaction was made up 20μl and 11μl of solution that prepared in before staging for removal of genomic DNA, 1 μl Random Hexamer Primer, 4 μl 5x Reaction Buffer, 2 μl dNTP Mix (10 mM), 1 μl Ribo Lock RNase Inhibitor (20 U/μl), and 1 μl Revert Aid M-MuLV Reverse Transcriptase (200 U/μl) as a master mix. Adapted time scales and temperature pro les of RT reaction was as follow: incubation 5 min at 25 °C followed by 60 min at 42 °C. The reaction was terminated by heating at 70 °C for 5 min (13).

Isolation of PBMCs (peripheral blood mononuclear cells) by using LeucoSep tubes.
PBMCs were puri ed using LeucoSep tubes according to the instructions of the manufacturer (Greiner Bio-One). In brief, 3 ml of Ficoll-Paque was preloaded in a 14 ml LeucoSep tube by centrifugation for 30 s at 1,000 × g. The heparinized whole-blood samples were diluted with equal volumes of PBS, and 6 ml of the diluted blood was added to a LeucoSep tube. The cell separation tubes were centrifuged for 15 min at 800 × g without braking at room temperature. The cell suspension was collected, and the cells were washed twice in PBS (for 10 min at 640 and 470 × g, respectively, for the two successive washes) and resuspended in complete RPMI medium before counting (14,31).
Cell yield/viability. The both PBMCs yield and viability were determined using a NucleoCounter (ChemoMetec A/S, Allerød, Denmark). The NucleoCounter is able to detect nonviable cells using propidium iodide to stain cell nucleus and determines cellular viability of a blood sample employing total cell count and counting dead cells.
Quantitative-real-time-polymerase-chain-reaction (QRT-PCR) Transcription levels of mRNA for the CXCR3 as the chemokine receptor for CXCL9, CXCL10 and CXCL11 was examined by QRT-PCR using previously described primers [table 1]. As stated earlier, further isolation PBMCs were twice rinsed with phosphate-buffered saline and their total RNA content was isolated by TRIzol reagent (Invitrogen, Life Technologies). The total RNA (2 μg) was then reversely transcribed by standard reagents (Invitrogen). The complementary DNA (cDNA) corresponding to 200 ng RNA was ampli ed in a PCR containing 0.1 mM of CXCR3 speci c intron spanning primers (forward primer:5′-CTCTGCTGGACCCCCTATCA-3′; reverse primer:5′-GTCTCAGACCAGGATGAATC-3′, product: 255 bp) with an internal control for equal amounts of cDNA of a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)speci c intron spanning primer pair (5´-CCAGCCGAGCCACATCGCTC-3´; 5´-ATGAGCCCCAGCCTTCTCCAT-3´), which yielded a 360-bp ampli ed product. Ampli cation was performed using 25 to 40 cycles with denaturation at 94°C for 30s, primer annealing at 60°C for 30s, and extension at 72°C for 1 min. PCR products were subjected to electrophoresis on a 1% agarose gel and visualized by SYBR Safe staining (Invitrogen) (15).

Measurement of serum CXC chemokines by ELISA
Brucellosis patients CXCL9, CXCL10 and CXCL11 serum levels were detected by sandwich ELISA (R&D, Minneapolis or Peprotech respectively) using paired chemokine-speci c mAbs, according to the manufacturer's guidelines.
Following clinical examinations by the infectious disease specialists, intravenous blood samples were collected from patients and serum samples were then isolated from the patients and harvested samples have been kept at -80 °c for further chemokines (CXCL1 , CXCL9, CXCL10 and CXCL12) measurment.

Statistical Analysis:
Following of the collection the desired data, statistical evaluations on the mentioned variables with SPSS computer software (version18) of tukey test were used to analyze the ndings.

Analysis of our real time-PCR data
Statistical analysis of our real time-PCR data showed that the mRNA expression of CXCR3 was signi cantly increased in in acute brucellosis patients with 2ME rate of 1/80,1/160 and 1/320, respectively in compare to control subjects(P < 0.05) (Fig. 5) and the expression level of CXCR3 gene increased in was signi cantly increased in in acute brucellosis patients with wright rate of 1/80,1/160 and 1/320, respectively in compare to control subjects (P < 0.05) (Fig. 5).

Discussion
Brucellosis is de ned as a worldwide disease which is transmitted to human from the products of domestic animals such as sheep and goat milk. We have designed the current study to examine the serum concentrations of IFN-γ induced CXC chemokines along with their receptor (CXCR3) in brucellosis patients. There has been informed a lot of information regarding generation of antibodies against brucella within the body during brucellosis. With this regard, because the bacteria that cause brucellosis are basically intracellular, thus the cellular immunity plays a key part in destroying the intracellular bacteria and in turn improving the pathogen (30).

Several cellular safety based invastigations in laboratory rodents have been performed and researchers reported that TNF (tumor necrosis factor alpha), IFN-γ and interleukin 12 play key parts in resistance to
Brucella abortus, during brucellosis infection, as a pro le of pre-in ammatory cytokines. Animal model studies showed that in brucellosis patients, the IFN-γ concentration tends to be sharply elevated and if patients failed to produce adequate amounts of IFN-γ the infection continuously stay in the patient's body (32,33). As previously mentioned studies of cytokine, especially chemokine family production is very limited in brucellosis patients. Studies which was performed by some researchers have shown that IFN-γ and interleukin-12 levels in patients are higher than in normal subjects, however, they did not indicate in the report the clinical type (acute or chronic) of the studied patients (32).In accordance with the Ahmed K et al, we have also shown the elevated levels of IFN-γ-induced patients in response to both TNF-α and IFN-γ in several cell types and in ammatory disease (34). In addition to our group multiple other research teams also con rmed elevation of these chemokines in response to in ammation(34) and thus one possible mechanism which can explain the induced levels of these IFN-γ-induced mediators could possibly be related to the enhancement levels of in ammation-related mediators that are known to be well related to brucellosis in these patients.As an example, the so called CCL20 as a cc chemokine subfamily member has well been established to be produced following being infected with brucella infection by lung epithelial cells (19,22,35). CCL20 is elevated in the lungs by some types of various infections (22,35). [In line with these ndings, various studies have also reported that pulmonary derived CCL20 is able to both exert chemo active activity for lymphocytes and dendritic cells (36) as well as directly act against pathogens (19). Some other studies have claimed that in addition to CCL20, the pulmonary β-defenses are also able to act against infections, including Brucella spp. to infect hosts via inhalation.Here we have showed that the expression of some members of the cxc chemokine subfamily including IFN-γ-induced CXCL9,10,11 were elevated in serum samples which were obtained from Brucellosis patients, in parallel with the CXCR3 as their respective receptors. Although, there were not found a related investigation to do compare,but we have already shown the elevated measures of these chemokines in some other infectious diseases including, post-transfusion-transmitted hepatitis C virus infection, following hepatocyte isolation and in response to heat shock (34). As the brucellosis is developed the titer of antibodies are reduced and becomes unmeasurable following 2-3 years. Continuous high level of IgG is of the causes of chronic infection and the disease recurrence. Brucellosis is frequently associated with extensive tissue necrosis and eventually abscess production. Tissue granulomatous within organs such as liver and spleen is also achieved via abortion of Ca. Therefore, brucella immunopathological features depends on the type of organism and thus the clinical symptoms, laboratory changes and thus immunology system and its related arms (cytokine and chemokines) are involved its pathogenesis. Results of the present study are novel and authors could not address a homologue set of data to compare with, but, there exist few articles that have referred to the upstream signals that are involved in production of these in ammatory mediators, following infection/in ammation as an example, CCL20 has well been found to be generated by lung epithelial cells in response to injury (22,30,31). CCL20 is elevated in the lungs by some types of infections (30,31). In line with us various studies reported that pulmonary derived CCL20 is able to both exert chemotactive activity for lymphocytes and dendritic cells as well as directly act against pathogens (22). Again, investigations claimed that in addition to the β-defensins, CCL20 also defense against infections (including Brucella spp. that infect hosts) via inhalation. Studies reported that the elevate levels of cytokines mostly depends on the severity of the patient's symptoms. Nitric Oxide has also been slightly increased in patients, which in away may con rm that septic shock does not occur in brucellosis Researchers have demonstrated that in addition to the PAF (Platelet activating factor), and lL-12 aboard spectrum of anti-in ammatory molecules such as interleukin 4, interleukin 10 and interleukin I and soluble TNF receptors are also enhanced in the blood of patients with severe toxic septicemia.
IL-6 and IFN-γ as pro-in ammatory proteins, are also produced by many cell types in the body in response to cellular lesions and hence it appears that these are involved in running of the acute systemic response of the acute phase and serves as a members of this response.

Conclusion
According to these ndings there was a positive correlation between the level of chemokines and their receptor. We have found that there was a positive correlation between the level of chemokines and their receptor.

Declarations
-Ethical Approval and Consent to participate: The research and its tool were approved by Research Ethics Committee of Rafsanjan University of Medical Sciences, and ethical principles were observed during the -Informed consent: All subjects participating in the study and/or their respective legal representatives were informed about the objectives and signed the Free and Informed Consent Form.
-Consent for publication: All subjects participating in the study a signed the Free and Informed Consent for publication.
-Availability of data and materials: Database will be made available if required.
-Competing interests: The authors declare no con ct of interest.  Demonstrates that the mean serum measures of CXCL9 in patients with the wright rate of 1/80 , 1/160 and 1/320 , in compare to control subjects (P<0.05).

Figure 2
Demonstrates that the mean serum measures of CXCL10 in patients with the wright rate of 1/80 , 1/160 and 1/320 , in compare to control subjects (P < 0.05) Figure 3 Demonstrates that the mean serum measures of CXCL11 in patients with the wright rate of 1/80 , 1/160 and 1/320 , in compare to control subjects (P < 0.05) Figure 5 Demonstrates that that the mRNA expression of CXCR3 was signi cantly increased in in acute brucellosis patients with 2ME rate of 1/80,1/160 and 1/320, respectively in compare to control subjects(P < 0.05) and the expression level of CXCR3 gene increased in was signi cantly increased in in acute brucellosis patients with wright rate of 1/80,1/160 and 1/320, respectively in compare to control subjects (P < 0.05)