Expression of CXCR1 (IL-8 receptor A) in splenic, peritoneal macrophages and resident bone marrow cells after acute live or heat killed Staphylococcus aureus stimulation in mice

Microbial Pathogenesis 109 (2017) 131e150

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Expression of CXCR1 (IL-8 receptor A) in splenic, peritoneal macrophages and resident bone marrow cells after acute live or heat killed Staphylococcus aureus stimulation in mice Biswadev Bishayi*, Ajeya Nandi, Rajen Dey, Rana Adhikary Department of Physiology, Immunology Laboratory, University of Calcutta, University Colleges of Science and Technology, 92 APC Road, Calcutta 700009, West Bengal, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 March 2017 Received in revised form 15 May 2017 Accepted 19 May 2017 Available online 24 May 2017

Literature reveals that interaction with live Staphylococcus aureus (S. aureus) or heat killed S. aureus (HKSA) promotes secretion of CXCL-8 or interleukin-8 (IL-8) from leukocytes, however, the expressions of CXCR1 in murine splenic (SPM), peritoneal macrophages (PM) and resident fresh bone marrow cells (FBMC) have not been identified. Currently, very few studies are available on the functional characterization of CXCR1 in mouse macrophage subtypes and its modulation in relation to acute S. aureus infection. SPM, PM and FBMCs were infected with viable S. aureus or stimulated with HKSA in presence and absence of anti-CXCR1 antibody in this study. We reported here that CXCR1 was not constitutively expressed by macrophage subtypes and the receptor was induced only after S. aureus stimulation. The CXCR1 band was found specific as we compared with human polymorphonuclear neutrophils (PMNs) as a positive control (data not shown). Although, we did not show that secreted IL-8 from S. aureus-infected macrophages promotes migration of PMNs. Blocking of cell surface CXCR1 decreases the macrophage's ability to clear staphylococcal infection, attenuates proinflammatory cytokine production and the increased catalase and decreased superoxide dismutase (SOD) enzymes of the bacteria might indicate their role in scavenging macrophage derived hydrogen peroxide (H2O2). The decreased levels of cytokines due to CXCR1 blockade before S. aureus infection appear to regulate the killing of bacteria by destroying H2O2 and nitric oxide (NO). Moreover, functional importance of macrophage subpopulation heterogeneity might be important in designing new effective approaches to limit S. aureus infection induced inflammation and cytotoxicity. © 2017 Elsevier Ltd. All rights reserved.

Keywords: CXCR-1 CXCL8 Fresh bone marrow cells Inflammation Peritoneal macrophage

1. Introduction Interleukin-8 (IL-8) or CXCL8 is a pro-inflammatory ELRþ CXC chemokine, originally identified as a neutrophil chemoattractant [1]. CXCL8 makes an important contribution to the induction of innate immunity through its effect on neutrophil chemotaxis and activation. Accordingly, CXCL8 has been implicated in a number of inflammatory diseases [2,3]. CXCL8 mediates its effects via binding to two heterotrimeric G protein coupled receptors CXCR1 (IL-8RA) and CXCR2 (IL-8RB). These receptors have normally been found on the surface of human leukocytes (neutrophils, monocytes, macrophages, basophils, T lymphocytes) and endothelial cells [4]. In

* Corresponding author. E-mail addresses: [email protected], (B. Bishayi). http://dx.doi.org/10.1016/j.micpath.2017.05.028 0882-4010/© 2017 Elsevier Ltd. All rights reserved.

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human, two high affinity receptors for CXCL8 designated CXCR1 and CXCR2 [5e7] have been reported. However, functional characterization of CXCR1 in mouse macrophage subtypes of Swiss albino mice and its modulation in relation to acute S. aureus infection has not been reported. Recently, mice with targeted deletion of CXCR2 also known as murine CXCL8 homolog (CXCL8 RL) were constructed [8,9]. Murine CXCR1 share 64% and 89% homology at the amino acid level with the human CXCR1 and murine CXCR2 respectively [10]. Murine CXCR1 has been shown to bind many CXC chemokines, but it is unknown if the receptor is expressed in macrophages or bone marrow cells of Swiss albino mice [11]. CXCR1 expression in relation to mucosal and systemic candidiasis has been demonstrated in BALB/c and IL-8Rh/ mice [12]. Although chemokines and chemokine receptors probably evolved to coordinate leukocyte recruitment that supports an antimicrobial response, many have

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List of abbreviation CPCSEA Committee for the purpose of control and supervision of experiments on animal CXC-chemokine which have a single amino acid residue interposed between the first two canonical cysteines CXCL-8 interleukin-8 CXCR1-CXC chemokine receptor-1 EDTA ethylenediaminetetraacetic acid ELRþ glutamateeleucineearginine (ELR) motif near the N terminal FBS fetal bovine serum HBSS Hank's balanced salt solution

been exploited by infectious agents, like Staphylococcus aureus (S. aureus) to facilitate infection [13]. It has been reported that staphylococcal surface protein -A mediated CXCL8 secretion plays a central role in initiating the neutrophilic response against S. aureus infections. It is mediated by several signalling pathways which ultimately trigger NF-kB and other signalling molecules that induce proinflammatory cytokines and chemokines to promote neutrophil trafficking from the circulation into the infected tissue [14]. Thus, there has been intense interest in understanding how its production is triggered during acute S. aureus infection. Investigation on the functional expression of CXCR1 in macrophage and its regulation in respect to long term S. aureus infection in peritoneal macrophages of Swiss albino mice have been reported [15]. Studies evaluating the role of acute S. aureus infection induced CXCR1 expression in murine splenic, peritoneal macrophages and resident bone marrow cells of Swiss albino mice have not been described. Previously, bacterial infection was also shown to augment CXC chemokine receptor CXCR1 and CXCR2 expression by human epithelial cell lines, and anti-CXCR1 antibodies were shown to impair transmigration [16]. Based on their affinity for CXCL8 and on internalization and recycling characteristics, CXCR1 is thought to mediate CXCL8-induced chemotaxis at sites of inflammation (where CXCL8 concentration is high) [17]. Blockade of CXCR1/2 or blockade of chemokines, which act on these receptors, such as CXCL8 [interleukin-8 (IL-8)], have been shown to prevent neutrophil influx and tissue injury in several models of acute and chronic inflammation [18,19]. A blockade of CXCR1 or CXCR2 could inhibit excessive infiltration or activation of neutrophils during acute inflammatory processes [20]. There is an abundance of evidence supporting the validity of targeting CXCL8/CXCR1/2 signalling in cancer. Targeting of CXCR1 or CXCR2 receptors may be attempted using neutralizing antibodies, small molecule antagonists or peptide derived inhibitors [21,22]. Neutralizing antibodies may also be used to target CXCR1 and CXCR2 preventing ligand binding at the extracellular domain. Blockade of CXCR1 via neutralizing antibody has been shown to inhibit CXCL8 induced proliferation of cancer cells. There was an extensive body of evidence to support the use of CXCR1/2 targeted therapy in the treatment of cancer [23,24]. However, it has also been demonstrated that targeting of CXCR1 was likely to be more efficacious than neutralizing IL-8 alone [23]. Blockage of CXCR1 was considered a potential therapeutic approach for inflammation related malignancies [25e27]. It was reported that normal mice cleared infection within 3e7 days, but

IL-8RA iNOS JNK MAPK MHC-II NaNO3 NaOH NF-kB NOS2 PBMC PIP3 PMSF RIPA SDS

interleukin-8 receptor A inducible nitric oxide synthase c jun N-terminal kinase mitogen activated protein kinase major histocompatibility complex-II sodium nitrate sodium hydroxide nuclear transcription factor kappa beta nitric oxide synthase e2 peripheral blood mononuclear cells phospho inositol trisphosphate phenyl methyl sulfonyl fluoride radio immune precipitation assay buffer sodium dodecyl sulphate

the bacterial numbers increased in the CXCR1 knockout mice, which also developed symptoms of bacterimia [16,28] suggesting the involvement of CXCR1 in bacterial clearance. It was reported that CXCR2 is essential for protective innate host response in murine model [29]. Currently there is no study on the functional characterization of CXCR1 in mouse peritoneal, splenic macrophages and resident fresh bone marrow cells and its modulation in relation to acute S. aureus infection of wild type Swiss albino mice. The present study was performed in order to investigate the functional expression of CXCR1 and its regulation in respect to acute viable S. aureus infection and heat killed S. aureus stimulation in peritoneal, splenic macrophages and resident fresh bone marrow cells of Swiss albino mice. CXCR1 expression in different subtypes of macrophage in Swiss albino mice is not clearly known. Macrophages are remarkably versatile in their ability to recognize and respond to a wide range of stimuli, expressing a variety of surface and intracellular receptors, multiple signal transduction pathways and complex, adaptable arrays of gene expression [30]. For the present, the questions arise, how is the phenotype of macrophages influenced by different tissue environments, and what are the effects of macrophage activation on the particular tissue in which they reside? A further issue is whether organ-specific differences persist after macrophage activation by inflammation, infection, and malignancy [30]. In addition, macrophages have heterogeneous phenotypes and complex functions within both innate and adaptive immune responses [31]. Despite the toxic effects of reactive oxygen species (ROS) and reactive nitrogen species (RNS) of host macrophages S. aureus can survive and grow within macrophages. Upon stimulation by S. aureus and its products macrophages have been known to synthesize and release proinflammatory cytokines [32,33]. ROS and RNS are produced by macrophages as part of their antimicrobial response [34], whereas, several bacterial gene products have been associated with the detoxification of host derived ROS and RNS [35e37]. However, the role of CXCR1 in the intracellular survival of S. aureus and involvement of cytokines particularly in the peritoneal, splenic macrophages and resident fresh bone marrow cells of Swiss albino mice during acute bacterial infection was still unclear. So, this study was undertaken in Swiss Albino mice model to investigate the differential modulation of ROS and cytokines due to neutralization CXCR1 after acute infection with either live S. aureus (LSA) or heat killed S. aureus (HKSA) stimulation in splenic (SPM), peritoneal (PM) macrophages and fresh bone marrow cells (FBMC).

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2. Materials and methods

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2.4. Isolation of murine splenic macrophages, peritoneal macrophages and resident bone marrow cells

2.1. Maintenance of animals All experiments involving animals were conducted according to the protocols that had been approved by the Institutional Animal Ethics Committee (IAEC), Department of Physiology, University of Calcutta, under the guidance of CPCSEA [Approval Number: IAEC/ IV/Proposal/BB-1/2014, dated 26.08.2014], Ministry of Environment and Forest, Govt. of India. Wild type male Swiss albino mice were used throughout the study. Upon arrival, mice 6e8 weeks of age with body weight 20 ± 4 g were randomized into plastic cages with filter bonnets and saw dust bedding, followed by a one-week quarantine period. Mice were fed a normal rodent diet. Animal holding rooms were maintained at 21 to 24  C and 40e60% humidity with a 12 h light dark cycle. To minimize the feeling of hypoxia or discomfort before and during mouse dissection and tissue collection mice were anesthetized with ketamine hydrochloride (Sigma, Life Sciences) at a dose of 1 mg/kg body weight through the tail vein followed by cervical dislocation. 2.2. Preparation of bacteria The Staphylococcus aureus strain AG-789 was obtained from Apollo Gleneagles Hospital, Calcutta, West Bengal, India. S. aureus strain (AG-789) grown overnight in Luria Bertini broth (High media, Bombay, India) were diluted with fresh broth and cultured until mid-logarithmic phase of growth. Bacteria were harvested, washed twice with sterile saline and adjusted to the desired inoculum spectrophotometrically before infection (OD620 ¼ 0.2 for 5.0  107 cells/ml for S. aureus) and the colony forming unit (CFU) count of the desired inoculums was confirmed by serial dilution and culture on blood agar [38]. Among the three clinical isolates (AG-886, AG-591 and AG-789) used in the earlier studies from our laboratory it was observed that all of the three strains of S. aureus (AG-886, AG-591 and AG-789) showed the expression of both TSST1 and coagulase gene in all phases of their growth and induction was maximum at log and stationary phases of their growth. Furthermore, from the biochemical analysis and biotyping for characterization of the S. aureus (AG-789) isolate it was found that S. aureus (AG-789) was catalase-positive, coagulase-positive, can coagulate bovine plasma, was found to be resistant to methicillin. This MRSA was found to be sensitive to ciprofloxacin, chloramphenicol, and azithromycin. The results of the checker board assay showed no synergistic interaction between the antibiotics tested. Antagonism was observed with methicillin interacting with ciprofloxacin or azithromycin. Several other clinical isolates of S. aureus also have been extensively studied in the mouse model of arthritis with short term but nonlethal infection in our laboratory. We have also reported earlier that whether the differences in clinical outcome between infections with hospital isolates versus reference ATCC-25923 strain were due to different replication velocities of the bacteria. Although, we performed in vitro experiments only with one Staphylococcus aureus strain (AG-789) addition of several other clinical or ATCC Staphylococcus aureus strain could be helpful for the comparative analysis of acute S. aureus infection induced CXCR1 expression in different macrophage subtypes of Swiss albino mice. 2.3. Preparation of heat killed S. aureus (HKSA) Bacteria were grown in Luria Bertini broth for overnight under the same conditions. Overnight cultures were washed in 0.9% NaCl and used for heat-killed for 1 h at 90  C. 10 mg/ml HKSA was equivalent to 107 bacteria per ml [39].

Spleens were excised from the Swiss albino mice, immediately placed in Alsever's solution and macerated using frosted glass slides. Cells were repeatedly aspirated with a sterile Pasteur pipette until a single cell suspension was obtained. Suspension was then transferred to sterile tubes and kept in ice for cell debris to settle. The supernatant was then layered over 3 ml Histopaque-1077 and then centrifuged at 1500 rpm for 30 min. After centrifugation, the band of leukocyte enriched fraction at the interface was collected and washed with Dulbecco's phosphate buffered saline (DPBS) then the cell pellet was resuspended in RPMI-1640 containing 20 mM HEPES (pH-7.2) and 5% FBS and were allowed to adhere on plastic surface for 1 h at 37  C, 5% CO2 incubator. The non-adherent cells were removed and adherent cells were collected by repeated aspiration with Pasteur pipette. Cells were then washed and finally resuspended in culture media (RPMIþFBS) at a density of 5  106/ ml and more than 95% cells were found viable as determined by Trypan blue dye exclusion technique. The cells were stained with Giemsa and observed under oil-immersion microscope [40]. Murine splenic macrophages (5  106 cells/ml) were infected with S. aureus (5  106 CFU/ml) for 30, 60 and 90 min at 37  C. Mice were injected intraperitoneally with 2 ml of 4% sterile thioglycolate broth, and the resulting peritoneal exudate was harvested by lavage of the peritoneal cavities of mice with endotoxinfree Hanks' solution 4e5 days later. Peritoneal macrophages (PM) were suspended in 0.83% ammonium chloride solution containing 10% (v/v) Tris buffer (pH 7.65) to lyse erythrocytes. The cells were resuspended in RPMI 1640 medium supplemented with 10% FBS, 100 IU/ml penicillin, 100 mg/ml streptomycin, and then were allowed to adhere on plastic surface for 1 h at 37  C. Non-adherent cells were removed by aspiration and washing with RPMI 1640 medium before the addition of S. aureus. The adherent macrophages, more than 95% of which appeared to be typical macrophages by light microscopy, were used as peritoneal macrophages (PM) for each experiment [41]. Murine peritoneal macrophages (5  106 cells/ml) were infected with S. aureus (5  106 CFU/ml) for 30, 60 and 90 min at 37  C. For murine resident bone marrow cells were isolated in presence of growth factor M-CSF. Muscles connected to the bone were removed using clean gauze, and the femurs were placed into a polypropylene tube containing sterile PBS on ice. In a tissue culture hood, the bones were placed in 70% ethanol for 1 min, washed in sterile RPMI 1640 and then both epiphyses were removed using sterile scissors and forceps. The bones were flushed with a 24Gz syringe filled with RPMI 1640 to extrude bone marrow. A 5 ml plastic pipette was used to gently homogenize the bone marrow. The cell suspension generated thereafter is called fresh bone marrow cells. Fresh bone marrow cells (FBMC) were counted using a hemocytometer, centrifuged for 5 min at 200  g at 4  C and gently resuspended to obtain a solution containing from 4 to 6  106 cells/ml in RPMI-1640 media containing 10% fetal bovine serum [42]. All of these three isolated cells, murine SPMs, PMs and FBMCs (5  106 cells/ml) were infected separately with S. aureus (5  106 CFU/ml) for 30 min, 60 min and 90 min at 37  C. CD11b, known as the integrin alpha M chain, is implicated in different adhesive interactions of monocytes and macrophages. It has been shown that the spleen and peripheral blood monocyte progenitors share phenotypic markers with bone marrow progenitors, but differ in their expression of CD11b, which was low in bone marrow but high in periphery. It was also reported that in the bone marrow populations, these peripheral populations contain common progenitors for macrophages and dendritic cells. These progenitors share phenotypic markers with the bone marrow progenitor, but

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differ in their expression of CD11b. Therefore, characterization of these three macrophage subtypes (SPMs, PMs and FBMCs) by CD11b staining seems valid (data not shown). Further phenotypic studies based the expression of F4/80 and CD11b, or CD14 could suggest that they might present a monocyte/macrophage phenotype; however, we have not done it in our case. So, these SPMs, PMs and FBMCs are definitely monocyte/macrophages and devoid of neutrophils. Cells (SPMs, PMs and FBMCs) were used as macrophage subtypes, when more than 90% of cells were positive for CD11b, a monocyte/macrophage marker. The numbers of the three kinds of adherent cells were equal for each assay. 2.5. Blocking antibody reagents and culture conditions For CXCR1 blocking assays, rabbit polyclonal antibodies against CXCR1 (Biorbyt Limited, Cat No: orb 10487, Cambridge, UK) or control IgG isotype antibody (Abcam, Cat No: ab 37355, Cambridge, UK) were added at 10 mg/ml [16,43] to isolated peritoneal (PM), splenic (SPM) macrophages and fresh bone marrow cells (FBMC) and incubated for 1 h at 37  C in 5% CO2. Now all three types of cells were infected with live S. aureus and HKSA or medium alone, and the cells were incubated for an additional 30, 60 and 90 min at 37  C in 5% CO2. Cells were stimulated with 10 mg/ml of HKSA, which was equivalent to 107 bacteria per ml. This polyclonal antibody CXCR1 (Biorbyt Limited, Cat No: orb 10487, Cambridge, UK) was reactive against human (Swissprot: P25024), mouse, rat, cow, dog, rabbit and was recommended as actually quite homologous with murine CXCR1. 2.6. Western blot analysis for CXCR1 expression Isolated splenic, peritoneal macrophages and fresh bone marrow cells were lysed with RIPA-NP40 buffer containing 0.5 mM PMSF, 1 mM sodium orthovanadate, and 1 mL/mL protein inhibitor cocktail (1 mg/mL leupeptin, 1 mg/mL aprotonin, 10 mg/mL soybean trypsin inhibitor, 1 mg/mL pepstatin) and normalized to the protein content by Lowry method [44]. Samples containing equal amounts of protein (120 mg per lane) in equal volumes of sample buffer were separated in a denaturing 10% polyacrylamide gel and transferred to a 0.1 mm pore nitrocellulose membrane. Nonspecific binding sites were blocked with Tris-buffered saline (TBS; 40 mM Tris, pH 7.6, 300 mM NaCl) containing 5% nonfat dry milk for 1 h at room temperature. Membranes were then incubated with antibodies to CXCR1 (Biorbyt Limited, UK) in TBS with 0.1% Tween 20 (TBST). Blots were washed three times in TBST, incubated for 2 h with appropriate horseradish peroxidase-conjugated secondary antibodies, developed with the Super Signal chemiluminescent substrate (Thermo Scientific, USA) and exposed to X-Omat BT films (Kodak). We have used b-tubulin as loading control for Western blot to ensure equal loading throughout the gel as it is a housekeeping gene that exhibit high level, constitutive expression in the sample. It also has a different molecular mass than our protein of interest i.e. CXCR1, to help distinguish between both bands [15]. 2.7. Assay for intracellular killing The presence of a large number of S. aureus bacteria in some cells that had undergone in vitro culture observed in the bacterial plate count suggests that the bacteria grow intracellularly. In addition, it has been reported that S. aureus multiplies in the presence of intact macrophages in cell cultures and would not grow in macrophage-conditioned media. Therefore, the number of S. aureus bacteria grown in a petri dish after time-dependent phagocytosis indicates the number of S. aureus bacteria that have survived inside the macrophages after ingestion. For in vitro study,

cells were pooled from a minimum of 6 mice to obtain the requisite amount of individual cells (5  106/ml), counted using hemocytometer and were used for each experiments [45]. Murine SPMs, PMs, and FBMCs (5  106 cells/ml) were mixed with live S. aureus (5  106 CFU/ml) in a 1:1 cell: bacterium ratio [15], in RPMI-FBS (5%) and incubated at 37  C cell culture incubator for different times in presence and absence of anti CXCR1 antibody (10 mg/mL) as well IgG isotype control antibody (10 mg/mL). Extracellular S. aureus were removed by washing the suspension four times in RPMI by centrifugation at 250  g at 4  C for 5 min, note that we were unable to use antibiotics to kill bacteria present outside of macrophages because engulfed bacteria died quickly during the period necessary for the action of antibiotics. After centrifugation, cell culture supernatants were collected and stored at 80  C for further assay. Phagocytosis was stopped by adding cold (4  C) RPMI-1640 and extracellular S. aureus were removed by washing the suspension in RPMI, note that we were unable to use antibiotics to kill bacteria present outside of macrophages because engulfed bacteria died quickly during the period necessary for the action of antibiotics. At various time intervals, intracellular killing was terminated by transferring the tubes in crushed ice and spinning the cells at 4  C. The macrophages were disrupted by adding 1 ml of distilled water containing 0.01% BSA, to the cell pellet and vigorously shaking the suspension on a vortex mixture for 1 min. The number of viable bacteria was then determined by plating 10 fold serial dilutions of the suspensions into agar plates. Serial 10 fold dilutions in saline was made over a range assuming that at least one dilutions will contain between 100 and 1000 viable bacteria per ml. Aliquots (0.1 ml) of the 3 highest dilutions were pipetted onto each of 2 agar plates, the plates were incubated at 37  C for 18e24 h, and the number of colonies were counted. The number of viable bacteria per ml was calculated from the means of the colony counts of duplicate plates of the two highest dilutions, providing the plates contained <500 colonies [46]. 2.8. Assay for quantification of hydrogen peroxide (H2O2) production Activation of leukocytes by inflammatory stimuli results in the local release of ROS and induces hydrogen-peroxide (H2O2) production. Studies in animals support a critical role for phagocyte oxidative burst in controlling S. aureus infection. Therefore, the quantification of H2O2 in this experimental setup was quite relevant. After time-dependent phagocytosis, supernatants were collected and cell lysates were prepared from the pellet. H2O2 assay of the supernatant and lysate was performed according to the method as described earlier with slight modification [45]. Briefly 70 ml of supernatant or lysate, 20 ml Horse Raddish peroxidase (HRP) (500 mg/ml), 70 ml of Phenol red (500 mg/ml) and 40 ml medium were added and was allowed for incubation for 2 h at 37  C. The reaction was stopped by adding 25 ml of 2 N NaOH and the absorbance reading was taken at 620 nm. Control set received 40 ml of HBSS in place of supernatant/lysate. A standard H2O2 curve was plotted and H2O2 release in supernatants and lysate was evaluated and expressed in mM/106 cells. 2.9. Assay for quantification of superoxide anion (O2-) production Because intracellular killing is dependent on oxidants, it was expected that the amount of superoxide anion released by the host macrophages also contributes to oxidant-dependent killing of S. aureus. Since superoxide anion production has been implicated in many physiological and pathological processes, including host innate immune and inflammatory responses to pathogens, we were also interested in determining the amount of superoxide anion

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released in this experimental setup. Superoxide anion release assay measures the change in colour of cytochrome C (cyt C), when reduced by O-2 released from the stimulated cells infected with S. aureus pre-treated with or without exogenous SOD (catalogue no SC-11407) (2.78 mg/ml). The difference between the amounts of cytC reduced in the presence and absence of SOD represents the amount of superoxide anion generated during the incubation. The instrument was blanked on the samples containing cytC plus SOD and instructed sequentially to read the absorbance of all the samples at 550 nm in reference to the blank. The absorbance read in this manner is closest to the true SOD inhibitable cytC reduction. Cell supernatant and lysate obtained after the time-dependent phagocytosis by the cells tested, infected with S. aureus were incubated in presence of cytC (100 ml at 2 mg/ml). The production of superoxide anion was monitored spectrophotometrically at 550 nm with reference to the blank. The amount of superoxide anion production was calculated by the following formula: micromoles of superoxide anion ¼ (mean absorbance at 550 nm  15.87) [41]. 2.10. Assay for quantification of nitric oxide (NO) production Reactive nitrogen intermediates (RNI), including nitric oxide, are involved in the antimicrobial activity of activated macrophages against a variety of intracellular microorganisms. When macrophages are stimulated with IFN-g and other cytokines, ROS production is enhanced and RNI production is induced. However, whether NO are involved in the killing during primary and secondary infection has not been completely elucidated. In particular, the involvement of nitric oxide seems to be controversial. Therefore, the quantification of NO in this experimental setup was quite relevant. NO release was determined by the Griess assay. 50 ml of supernatant and lysate was incubated separately in 40 mM Tris (pH 7.9) containing 40 mM of the reduced form of b-nicotinamide adenine dinucleotide phosphate, 40 mM flavine adenine dinucleotide and 0.05 U/ml nitrate reductase at 37  C for 15 min. Reduced samples were incubated with an equal volume of Griess reagent consisting of sulphanilamide (0.25% (w/v)) and N-1naphthylethylenediamine (0.025% (w/v)), and the mixture was incubated for 10 min and the absorbance at 550 nm was measured. The total nitrate/nitrite concentration was determined by comparison to a reduced NaNO3 standard curve [41]. 2.11. Preparation of whole staphylococcal cells recovered after time dependent phagocytosis in presence or absence of anti-CXCR1 antibody from SPM, PM and FBMC The bacteria that survived after time-dependent phagocytosis, as obtained from the plates of respective phagocytic time were used to estimate the whole staphylococcal cell catalase/SOD activity as described previously [15,41]. The bacterial cells were grown overnight and washed twice in sterile distilled water and once in sterile 50 mM potassium phosphate buffer (pH 7.0). The washed bacterial cells were dispersed in phosphate buffer (pH 7.0) by shaking glass beads and diluting to give an absorbance of 0.62 at 600 nm. A 1 ml amount of the suspension of culture with an absorbance of 0.62 contained approximately 1.1  108 -1.5  109 CFU/ml by standard plate counts. Based on preliminary experiments 5  106 to 1.5  108 CFU/ml was used to determine catalase activity of cells in presence of 15 mM of H2O2/ml phosphate buffer. After 10 min of incubation, bacterial cells were centrifuged and supernatants were collected and stored at 20  C. The pellet so obtained after centrifugation were washed twice with lysis buffer containing 1 mM disodium EDTA, pH 7.2, 0.5 mM PMSF and finally resuspended in 2 ml buffer. Suspensions were sonicated on ice at 8 mm for 90 s (six 15 s bursts with 15 s cooling periods) and then

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centrifuged at 16,000  g for 45 min at 4  C. Supernatants (lysates) were filtered and stored at 20  C. Protein content of this crude bacterial lysate was determined using Bradford method. This cell free lysate was also used for determination of SOD and Catalase enzyme activity. 2.12. Assay of superoxide dismutase (SOD) enzyme activity In addition to blocking cell surface CXCR1, subsequent treatment of macrophages S. aureus or HKSA might regulate intracellular ROS and can also protect bacteria from oxidative stress by modulating bacterial superoxide dismutase (SOD) expression or catalase activity. So, estimation of SOD enzyme activity in this experimental setup was seemed helpful to get an idea of ROS level. 100 ml of the cell free lysate was mixed separately with 1.5 ml of a Tris-EDTA-HCl buffer (pH 8.5), then 100 ml of 7.2 mM/L pyrogallol was added and the reaction mixture was incubated at 25  C for 10 min. The reaction was terminated by the addition of 50 ml of 1 M HCl and measured at 420 nm. One unit was determined as the amount of enzyme that inhibited the oxidation of pyrogallol by 50%. The activity of SOD enzyme was expressed as U/mg protein [42]. 2.13. Assay of catalase enzyme activity Typically, S. aureus-stimulated leukocytes produce proinflammatory cytokines, which trigger ROS production in the tissues through NADPH oxidase activation. Thus, the level of antioxidant enzymes or their activity displays the intracellular complex mechanisms of the host's defense. Therefore, to determine the activity of these antioxidant enzymes in neutralizing the ROS molecules produced, we estimated the catalase enzyme activity in the cell-free lysate of macrophages after infection in the presence or absence of CXCR1 antibody. Catalase enzyme activity in the cell free lysate was determined spectrophotometrically by measuring the decrease in H2O2 concentration at 240 nm. At time zero, 100 ml of the cell free lysate was added separately to 2.89 ml of potassium phosphate buffer (pH 7.4) taken in a quartz cuvette. To it 0.1 ml of 300 mM H2O2 was added and absorbance was taken at 240 nm for 5 min at 1 min intervals. Catalase enzyme activity was expressed in terms of mM/min mg protein [42]. 2.14. Tumor necrosis factor alpha (TNF-a), interferon gamma (IFNg), interleukin 1 beta (IL-1b), interleukin-6 (IL-6), CXCL8 (IL-8) and interleukin-10 (IL-10) ELISA assays As high ratio of IL-10 to TNF-a was associated with fatal outcome in patients with infection, we focused our study on the production of pro-inflammatory cytokines and an anti-inflammatory cytokine. Cytokines such as IFN-g, IL-1b and IL-6 reportedly play a protective role in host resistance to facultative intracellularly growing bacteria. IL-10 is a potent anti-inflammatory cytokine and inhibits the synthesis of proinflammatory cytokines from TH1 cells, which have a suppressive effect on TNF-a, IFN-g and IL-12 production. Cytokine concentrations from cell culture supernatants were determined by sandwiched ELISA. Supernatants from different groups were normalized to the protein content by Lowry method [44] before the assay and determined the levels of cytokines TNF-a, IFN-g, IL-1b, IL6, IL-10 as per manufacturer's guidelines of Raybiotech, Inc, USA and CXCL8 as per manufacturer's guidelines of MyBioSource, Inc. USA in a BioRad ELISA Reader at 450 nm. The minimum detectable value of TNF-a was <60 pg/mL, IFN-g was <5 pg/mL, IL-6<2 pg/mL, IL-10 < 45 pg/mL, IL-1b < 5 pg/ml and CXCL8 was <3 pg/ml as given in the manual. The reproducibility of cytokine kits are intra-assay: CV < 10%, interassay: CV < 12%. For the MBS814587-mouse IL-8 ELISA kit, the gene information that is listed on the datasheet is

Fig. 1. Up-regulated expression of CXCR1 (interleukin-8 receptor) by live S. aureus (SA) or heat killed S. aureus (HKSA) stimulation in murine splenic, peritoneal macrophages and fresh bone marrow cells. The splenic (SPM, Fig. 1A), peritoneal macrophages (PM, Fig. 1B) and fresh bone marrow cells (FBMC, Fig. 1C) of Swiss albino mice were isolated. Murine macrophages and FBMCs (5  106 cells/ml) were infected with S. aureus (5  106 CFU/ml) or stimulated with HKSA (10 mg/ml, equivalent to 107 bacteria/ml) for 90 min at 37  C in presence or absence of anti CXCR1 antibody. Whole cell lysates were prepared for analysis of CXCR1 by western blot. All the samples were probed with b-tubulin to show equal protein loading. ‘g’ significant difference with respect to uninfected control macrophages,‘#’ significant difference with respect to LSAM. ‘4’ significant difference between uninfected control and HKSAM and ‘$’ significant difference with respect to HKSAM at p < 0.05 significance level. ‘a’ significant difference between splenic and peritoneal macrophages, ‘b’ significant difference between peritoneal macrophages and FBMC and ‘c’ significant difference between splenic macrophages and FBMC at p < 0.05 significance level.

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just general gene information for CXCL8 and does not apply to this product. MBS814587 is indeed designed to detect mouse CXCL8. The UniProt and NCBI information referring to the pig sequence does not apply specifically to this product. This kit is designed to detect CXCL8 (IL-8) in mouse samples. The sequences for the antibodies and standards provided in MBS814587 are considered as per manufacturer. The standard is synthetic. The protein sequence is the N terminus. This kit has been reported for cross-reactivity with human CXCL8. 2.15. Statistical analysis Isolated SPMs, PMs, and FBMCs from mice were separately pooled together to obtain the requisite amount of cells (5  106 cells/ml) and the different parameters were measured. This was repeated for three times for each parameter (for e. g., H2O2 production) then the mean value of these triplicate experiments were taken for calculation. Data was expressed as mean ± S.D. Oneway model 1 ANOVA (Analysis of Variance) was performed between the groups. In ANOVA observed variance is partitioned into components due to different explanatory variables. A level of P < 0.05 was considered significant. Significant differences of the means between the groups were performed by One-Way ANOVA. Scheffe's F-test had been done as post hoc test for multiple comparisons of means of different groups when significant F value was found. A Scheffe's F-test post-hoc test for multiple comparisons of the different groups was done when significant P-values were obtained. A P-value < 0.01 was considered significant. All analyses were done using OriginPro 8 software (Origin Lab Corporation, Northampton, MA).

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post infection to get a wider overview of the end product. Although, we did not perform the time kinetics study for CXCR1 expression except 90 min, either for LSA or HKSA since form our earlier study on TLR2 expression in those cell types (except SPM) the optimum effects were observed at 90 min (data not shown). The data presented in Fig. 1A-C showed that stimulation of macrophages or FBMC with either LSA or HKSA induced expression of CXCR1 in SPM (Fig. 1A), PM (Fig. 1B) and FBMC (Fig. 1C) whereas, the expression was down regulated after anti CXCR1 antibody administration to all of the infected cells. Additionally, no such reduction in CXCR1expression was observed in IgG isotype antibody treated and S. aureus infected macrophages or fresh bone marrow cells. However, the presence of this receptor was not found either in control macrophages or FBMCs (Fig. 1A, B and 1C). 3.2. Phagocytic activity of murine SPM, PM and FBMC after blocking of CXCR1 during LSA infection Live S. aureus was used to investigate whether CXCR1 blocking alters the phagocytic activity of three different macrophages. Our results showed that the intracellularly viable bacterial count (CFU) was significantly (p < 0.05) elevated for 30, 60 and 90 min post infection when the murine PM and SPM were pre-incubated with anti-CXCR1 antibody for 1 h prior to live S. aureus infection, whereas the number of bacteria survived inside CXCR1 neutralized FBMCs, 90 min of phagocytic time was significantly higher than that of CXCR1 unblocked S. aureus infected macrophages (p < 0.05). Moreover, the number of engulfed bacteria was not the same for all three different macrophage populations (Table 1). 3.3. Alteration in H2O2 release by murine SPM, PM and FBMC after infection with LSA or HKSA stimulation due to CXCR1 blocking

3. Results 3.1. Effect of LSA infection and HKSA stimulation on the expression of CXCR1 in SPM, PM and FBMC of Swiss albino mice Since live S. aureus is known to activate macrophages and induce release of interleukin-8, the question arises whether acute viable S. aureus infection or heat killed S. aureus (HKSA) stimulation has any role in the expression of CXCR1 in peritoneal (PM), splenic (SPM) macrophages and fresh bone marrow cells (FBMC) of Swiss albino mice concomitant with the release of cytokines from the same source. The receptor expression was studied only at 90 min of

We tested whether hydrogen peroxide produced by SPM, PM and FBMC in culture or in lysate was CXCR1 dependent during acute live S. aureus infection and heat killed S. aureus stimulation. Both anti-CXCR1antibody untreated and anti-CXCR1 antibody treated SPM, PM and FBMCs were exposed to live S. aureus infection or HKSA stimulation, and incubated for 30, 60 and 90 min at 37  C. Our results showed (Fig. 2A and B) that the decrease in the H2O2 released in supernatant and lysate by live S. aureus infected SPM, PM and FBMC for 60 min and 90 min of phagocytic time in presence of anti-CXCR1 antibody were found to be significant (p < 0.05)

Table 1 Colony forming unit of the engulfed S. aureus recovered after time-dependent phagocytosis with murine splenic, peritoneal macrophages and fresh bone marrow cells in presence or absence of anti-CXCR1 antibody. Group

SPM (CFU/ml of lysate) (Mean ± SD)

Bacteria recovered after 30 min of phagocytosis LSAM 1000 ± 162.23 IgG isotype AbþLSAM 999 ± 155.35 AIL8RAbþ LSAM 1120 ± 290.28 Bacteria recovered after 60 min of phagocytosis LSAM 880 ± 121.91 IgG isotype AbþLSAM 878 ± 114.25 AIL8RAbþ LSAM 1680 ± 202.12 *# Bacteria recovered after 90 min of phagocytosis LSAM 1320 ± 182.50 IgG isotype AbþLSAM 1314 ± 168.74 AIL8RAbþ LSAM 2000 ± 283.36* #

PM (CFU/ml of lysate) (Mean ± SD)

FBMC (CFU/ml of lysate) (Mean ± SD)

1280 ± 128.27 1275 ± 115.78 1560 ± 183.32

1800 ± 160.30 1794 ± 160.12 2320 ± 272.21

1120 ± 165.25a 1114 ± 148.37 2120 ± 240.5*#a

1720 ± 246.63 bc 1718 ± 230.33 3040 ± 294.42*# bc

1920 ± 431.63a 1916 ± 424.98 2760 ± 314.42*#a

2880 ± 335.19 bc 2872 ± 331.31 4840 ± 507.76*# bc

bc

bc

S. aureus (5  106 CFU/ml) were allowed to interact with murine splenic, peritoneal macrophages and fresh bone marrow cells (5  106 CFU/ml) in presence or absence of antiCXCR1 antibody and incubated for different times at 37  C. Macrophages/FBMCs were lysed, plated, and incubated overnight to obtain the CFU of the intracellularly survived bacteria next day. Results were shown as mean ± SD of three independent experiments. ‘#’ significant difference with respect to LSAM. ‘*’ significant difference with respect to IgG isotype antibody treated LSAM, ‘a’ significant difference between splenic and peritoneal macrophages, ‘b’ significant difference between peritoneal macrophages and FBMC and ‘c’ significant difference between splenic macrophages and FBMC at p < 0.05 significance level.

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Fig. 2. Evaluation of hydrogen peroxide (H2O2) release by the murine splenic, peritoneal macrophages and fresh bone marrow cells in presence or absence of anti- CXCR1 antibody after infection with live S. aureus (SA) or heat killed S. aureus (HKSA) stimulation. The splenic (SPM), peritoneal macrophages (PM) and fresh bone marrow cells (FBMC) (5  106 cells/ml) of Swiss albino mice were allowed to interact with S. aureus (5  106 CFU/ml) in a 1:1 cell/bacterium ratio or stimulated with HKSA (10 mg/ml, equivalent

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when compared to the H2O2 release after time-dependent phagocytosis with no antibody. In addition to it, the amount of H2O2 (mM/ 106 cells) released in supernatant and lysate by HKSA stimulated SPM, PM and FBMC at only 90 min of phagocytic time in presence of antibody were found significantly lower than that of released after phagocytosis with antibody untreated only infected macrophage group. However, amount of H2O2 release was significantly different among SPM, PM and FBMCs. However, no such differences in H2O2 production were observed in S. aureus infected or HKSA stimulated macrophages and FBMCs with or without IgG isotype control antibody treatment. Although, in the 90 min lysate higher H2O2 level was found than all other time points, we are unable to clarify it from this study. But S. aureus infected or HKSA stimulated macrophages and FBMCs, pretreated with anti CXCR-1antibody showed significant decrease (p < 0.05) in H2O2 production with respect to IgG isotype treated groups. 3.4. Alteration in O 2 release by murine SPM, PM and FBMC after infection with LSA or HKSA stimulation due to CXCR1 blocking Murine SPM, PM and FBMC were infected in vitro with LSA or stimulated with HKSA as described or left uninfected. On 30, 60 or 90 min after infection, the amount of superoxide anion produced was evaluated. As shown in Fig. 3A and B that the decrease in the O 2 released in supernatant and lysate by live S. aureus infected SPM, PM and FBMC for 60 min and 90 min of phagocytic time in presence of anti-CXCR1 antibody were found to be significant (p < 0.05) when compared to the O 2 release after time-dependent phagocytosis with no antibody. In addition to it, the amount of O 2 released in supernatant and lysate by HKSA challenged SPM, PM and FBMC at only 90 min of phagocytic time in presence of antibody were found significantly lower than that of released after phagocytosis with antibody untreated only infected macrophage group. However, amount of O 2 release was significantly different among SPM, PM and FBMCs. However, no such differences in O 2 production were observed in S. aureus infected or HKSA stimulated macrophages and FBMCs with or without IgG isotype control antibody treatment. But S. aureus infected or HKSA stimulated macrophages and FBMCs, pretreated with anti CXCR-1antibody showed significant decrease (p < 0.05) in O 2 production with respect to IgG isotype treated groups. 3.5. Alteration in NO release by murine SPM, PM and FBMC after infection with LSA or HKSA stimulation due to CXCR1 blocking To investigate the involvement of CXCR1 in acute in-vitro infection/stimulation of SPM, PM and FBMC with live and heat killed S. aureus we determined the NO release in the supernatant and lysate respectively at 30, 60 and 90 min in presence or absence of anti CXCR1 antibody. A significant decrease in NO production in the supernatant and lysate by LSA infected SPM and PM for 60 min and 90 min of phagocytic time in presence of anti-CXCR1 antibody were found to be significant (p < 0.05) when compared to the NO release after time-dependent phagocytosis with no antibody. Identical results were observed in case of HKSA stimulation. Whereas, there was a significant decrease (p < 0.05) in NO production only in lysate for 90 min of phagocytic time by anti CXCR1

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treated as well as LSA infected FBMC compared to antibody untreated only LSA infected counterpart (Fig. 4A and B). However, no such differences in nitric oxide production were observed in S. aureus infected or HKSA stimulated macrophages and FBMCs with or without IgG isotype control antibody treatment. But S. aureus infected or HKSA stimulated macrophages and FBMCs, pretreated with anti CXCR-1antibody showed significant decrease (p < 0.05) in NO production with respect to IgG isotype treated groups.

3.6. Alteration in SOD enzyme activity from the recovered S. aureus after time-dependent phagocytosis in presence or absence of CXCR1 antibody of SPM, PM and FBMC Crude bacterial lysates of the recovered bacteria after time dependent phagocytosis in presence and absence of anti-CXCR1 mediated receptor blocking were prepared as mentioned previously. A marked decrease in the bacterial SOD enzyme activity has been observed in the cell free lysate of recovered bacteria at 30, 60 and 90 min of phagocytosis when macrophages and FBMCs were incubated with anti-CXCR1 antibody prior to S. aureus infection than that of macrophages infected with S. aureus alone (Table 2). Similarly, there was a significant decrease in SOD enzyme activity in cell free lysate of bacteria at all three time points of phagocytosis when macrophages and FBMCs were incubated with anti-CXCR1 antibody prior to S. aureus infection than that of macrophages and FBMCs incubated with IgG isotype control antibody prior to S. aureus infection (Table 2). Anti CXCR1 antibody significantly (p < 0.05) inhibited SOD enzyme activity by LSA infected SPM, PM and FBMC at 60 and 90 min after infection, compared with the antibody untreated and infected PM, SPM and FBMC (Table 2).

3.7. Alteration in catalase enzyme activity from the recovered S. aureus after time-dependent phagocytosis in presence or absence of CXCR1 antibody of SPM, PM and FBMC As several lines of report suggested that bacterial catalase enzymes played an important role in the intracellular survival of S. aureus in SPM, PM and FBMC, we became interested to find out whether there occur any changes in catalase enzyme activity of the recovered S. aureus from SPM, PM and FBMC after different times of phagocytosis in presence or absence of antiCXCR1 antibody. A marked increase (p < 0.05) in the catalase enzyme activity has been observed by murine SPM, PM and FBMC for 30min, 60 min and 90 min of phagocytosis when macrophages and FBMCs were incubated with anti CXCR1 antibody prior to LSA infection than that of macrophages infected with LSA alone. Similarly, there was a significant increase in catalase enzyme activity in cell free lysate of bacteria at all three time points of phagocytosis when macrophages and FBMCs were incubated with anti-CXCR1 antibody prior to S. aureus infection than that of macrophages and FBMCs incubated with IgG isotype control antibody prior to S. aureus infection (Table 3).

to 107 bacteria/ml) and incubated at 37  C cell culture incubator for different times in presence and absence of anti CXCR1 antibody. Supernatants (A) and lysates (B) were prepared as mentioned earlier. H2O2 content was expressed in terms of mM/106 cells. Results are shown as mean ± SD of three independent experiments. ‘g’ significant difference with respect to uninfected control macrophages,‘#’ significant difference with respect to LSAM, ‘*’ significant difference with respect to IgG isotype antibody treated LSAM ‘4’ significant difference between uninfected control and HKSAM and ‘$’ significant difference with respect to HKSAM, ‘%’ significant difference with respect to IgG isotype antibody treated HKSAM at p < 0.05 significance level. ‘a’ significant difference between splenic and peritoneal macrophages, ‘b’ significant difference between peritoneal macrophages and FBMC and ‘c’ significant difference between splenic macrophages and FBMC at p < 0.05 significance level.

Fig. 3. Evaluation of superoxide anion (O2.¡) production by the murine splenic, peritoneal macrophages and fresh bone marrow cells in presence or absence of anti- CXCR1 antibody after infection with live S. aureus (SA) or heat killed S. aureus (HKSA) stimulation. Superoxide anion release assay was determined by measuring the changes in color of cytochrome C, when reduced by O2. released from the stimulated macrophages/FBMCs. Supernatants (A) and lysates (B) were prepared as mentioned earlier. Superoxide anion content was expressed in terms of nM/106 cells. Results are shown as mean ± SD of three independent experiments. ‘g’ significant difference with respect to uninfected control macrophages,‘#’ significant difference with respect to LSAM, ‘*’ significant difference with respect to IgG isotype antibody treated LSAM, ‘4’ significant difference between uninfected control and HKSAM and ‘$’ significant difference with respect to HKSAM, ‘%’ significant difference with respect to IgG isotype antibody treated HKSAM at p < 0.05 significance level. ‘a’ significant difference between splenic and peritoneal macrophages, ‘b’ significant difference between peritoneal macrophages and FBMC and ‘c’ significant difference between splenic macrophages and FBMC at p < 0.05 significance level.

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3.8. Neutralization of CXCR1 significantly alters cytokine (TNFalpha, IFN- gamma, IL-6, IL-1 beta, IL-8 and IL-10) production during infection of SPM, PM and FBMC with LSA and HKSA stimulation Our results showed a gradual decrease in the TNF-a (Fig. 5A), IFN- g (Fig. 5B), IL-6 (Fig. 6A) and IL-1b (Fig. 6B), CXCL8 (Fig. 7A) and IL-10 (Fig. 7B) level in the media (culture supernatant) by SPM, PM and FBMC at all time points after phagocytosis in presence of antiCXCR1 antibody, prior to LSA infection and also HKSA stimulation significantly (p < 0.05) when compared to the cytokine production after timeedependent phagocytosis in LSA infected as well as HKSA challenged different macrophages/FBMC in absence of CXCR1 antibody. However IL-10 levels (Fig. 7B) have been found to be significantly (p < 0.05) decreased only by BM at all time points but increased by PM and SPM at also 30 min, 60 min and 90 min due to CXCR1 blocking before LSA infection than that of macrophages infected only with LSA whereas HKSA stimulation significantly (p < 0.05) decrease IL-10 level by BM for all of three time dependent phagocytosis and increase IL-10 level by SPM and PM at only 60 min and 90 min of phagocytic time in presence of anti CXCR1 antibody in comparison with only HKSA stimulated macrophages/FBMC in absence of anti CXCR1 antibody. However, no such differences in cytokine production were observed in S. aureus infected macrophages and fresh bone marrow cells with or without IgG isotype control antibody treatment. 4. Discussion Although the functional characterization of CXCR-1 receptor has been widely studied in myeloid cells [47], the present study reported the expression of specific CXCR-1 receptor after acute S. aureus infection in peritoneal, splenic macrophages and resident bone marrow cells of Swiss albino mice. However, we found that under normal conditions, CXCR-1 is not expressed in any of the cell type tested (Fig. 1A, B and 1C). Blocking of cell surface CXCR-1 by antibody decreased the macrophages and FBMC's ability to clear staphylococcal infection, decreased production of H2O2 (Fig. 2A and B), superoxide anion (Fig. 3A and B), and nitric oxide (Fig. 4A and B) and also attenuates pro-inflammatory cytokine and CXCL8 (IL-8) production (Figs. 5e7). The neutralization of other receptors (like murine CXCR-2) by this antibody cannot be excluded as per recommendation of the manufacturer. We have speculated that altered macrophage/FBMC activation and/or phagocytosis after S. aureus infection could be responsible as macrophages/FBMCs from anti CXCR-1 antibody treated mice were different from antibody untreated cells in these parameters. The surface expression of CXCR1 upon S. aureus infection by flow cytometry as well as rate of transcription of CXCR1/CXCL8 mRNA by quantitative PCR, and the number of CXCR-1 molecules expressed on the respective cell surface might be helpful for better understanding of the role of CXCR-1 which required further study. It has already been established that effective blocking of cell surface CXCR-1 in PM significantly reduces phagocytosis of S. aureus [15] during long term infection study. Results of this study revealed that neutralization of cell surface CXCR-1 effectively increase the intracellular survival of S. aureus both in PM and SPM at all three time points (30 min, 60 min and 90 min after incubation) tested as evidenced by higher CFU counts when these host cells were infected with live S. aureus. But in FBMC, increased bacterial survival was evidenced only after 90 min of incubation. So, these observations led us to hypothesize that cell surface CXCR-1 was involved in the killing of S. aureus within PM, SPM and FBMC during acute staphylococcal infection. In addition, these results also

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pointed out to the findings that CXCR-1 is also involved in the late phagocytosis process only in FBMCs indicating differential response of the cells studied (Table 1). It has been reported that production of CXCL8 also correlated with intracellular S. aureus burden in THP1 cells and in human PBMC [48]. The cytokine response to S. aureus is enhanced by an increase in intracellular bacterial load and this coupled cytokine response is mainly attributable to S. aureus burden. The increased intracellular S. aureus observed after CXCR-1 blocking might not be sufficient to conclude to alteration of phagocytosis. Indeed the viability of S. aureus might be explained by the alterations of cytokines production and of H2O2 rather than by alterations of phagocytosis. Our model is supported by a recent study showing that cytokine responses to S. aureus are dependent on phagocytosis [49]. The importance of CXCR-1 has been shown in a urinary tract infection model in which mice deficient in the receptor was unable to clear bacterial infection [50] also supported our study where CXCR-1 blockade leading to less S. aureus killing by macrophages/FBMC. Both superoxide anion (O 2 ) and hydrogen peroxide (H2O2) release became elevated after live S. aureus (LSA) infection and heat killed S. aureus (HKSA) stimulation compared to control macrophages/FBMCs in all three time points irrespective of their subtypes. But the release of H2O2 (Fig. 2) was more pronounced in PM as compared to SPM and FBMC. This increase in H2O2 (Fig. 2) and O 2 (Fig. 3) release was significantly attenuated when different subtypes of macrophages/FBMCs were neutralized with CXCR-1 antibody prior to LSA infection or HKSA stimulation. In addition, NO (Fig. 4) produced by the LSA infected or HKSA stimulated macrophages combine with O 2 to generate peroxynitrite, a compound with more bactericidal activity, which might also be curtailed due to CXCR-1 neutralization favouring intracellular survival of S. aureus inside the PM, SPM and FBMC (Fig. 4). In this context, this is to further add that the degree of modulation of ROS generation due CXCR1 neutralization was higher in LSA infected macrophages as compared to HKSA stimulated macrophages and also was more pronounced in PM, compared to SPM and was less in FBMC. Thus, it indicates that not only the live cells but also the virulent components, released by the dead bacteria after heat killing, have also the potential to increase O 2 , H2O2 and NO release. Such pathway might also involve H2O2 which play key roles in cell signaling particularly in regulation of the production of cytokines [51e53]. CXCR-1 neutralization may reduce the release of H2O2 (Fig. 2) and superoxide anion (Fig. 3), subsequently the toxic effects of ROS to kill the bacteria is inhibited. In addition, NO produced by S. aureus stimulated macrophages (Fig. 4) combine with superoxide to generate additional product with enhanced toxicity, such as peroxynitrite, which might also be limiting due to CXCR-1 blocking, suggesting that S. aureus might survive and grow within macrophages/FBMCs [34,35]. Since the IFN-g has been reported to be a classical activator of macrophage antimicrobial activity and is an inducer of MHC-II and NOS-2, reduced IFN-g level due to CXCR-1 receptor blocking may support the IFN-g mediated reduction of NO in this case. Moreover, since CXCL8 is an oxidative stress responsive chemokine [54,55] its release is also decreased due to less ROS/RNS level after CXCR-1 blockade leading to diminished leukocyte influx and inflammation. After being ingested, S. aureus uses even more manifold weaponry of immune evasion molecules. Catalase and superoxide dismutase (SOD) enzymes eliminate harmful ROS. ROS also trigger a broad response of S. aureus to circumvent innate host defence mechanisms [56]. The increased bacterial SOD enzyme neutralizes the bactericidal activity of O 2 by converting it into H2O2. Catalase, a protein with known free radical scavenging activities, metabolizes H2O2, a toxic oxygen metabolite. Prior to CXCR-1 blocking, SOD enzyme activity was increased to counteract the increased

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Fig. 4. Evaluation of nitric oxide (NO) release by the murine splenic, peritoneal macrophages and fresh bone marrow cells in presence or absence of anti- CXCR1 antibody after infection with live S. aureus (SA) or heat killed S. aureus (HKSA) stimulation. Murine macrophages/FBMCs (5  106 cells/ml) were allowed to interact with S. aureus (5  106 CFU/ml) in a 1:1 cell/bacterium ratio or stimulated with HKSA (10 mg/ml, equivalent to 107 bacteria/ml) and incubated at 37  C cell culture incubator for different times in

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Table 2 Bacterial superoxide dismutase (SOD) enzyme activity from the recovered S. aureus after time-dependent phagocytosis in presence or absence of antibody. Groups

SPM

PM

FBMC

Bacterial SOD activity (units of SOD/mg bacterial protein) Mean ± SD After 30 min of phagocytosis S. aureus-infected macrophage for 30 min IgG isotype antibody-treated macrophageþ S. aureus infection for 30 min Anti-CXC-R1 antibody-treated macrophageþ S. aureus infection for 30 min After 60 min of phagocytosis S. aureus-infected macrophage for 60 min IgG isotype antibody-treated macrophageþ S. aureus infection for 60 min Anti-CXC-R1 antibody-treated macrophageþ S. aureus infection for 60 min After 90 min of phagocytosis S. aureus-infected macrophage for 90 min IgG isotype antibody-treated macrophageþ S. aureus infection for 90 min Anti-CXC-R1 antibody-treated macrophageþ S. aureus infection for 90 min

0.67 ± 0.11 0.64 ± 0.08

0.78 ± 0.11 0.72 ± 0.14

0.58 ± 0.06 0.54 ± 0.06

0.61 ± 0.10

0.72 ± 0.09

0.62 ± 0.12

0.88 ± 0.03 0.89 ± 0.04

0.89 ± 0.07 0.85 ± 0.05

0.66 ± 0.05bc 0.65 ± 0.04 bc

0.74 ± 0.09*#

0.64 ± 0.06*

1.06 ± 0.02 1.03 ± 0.03

1.63 ± 0.09a 1.60 ± 0.07

0.73 ± 0.07bc 0.72 ± 0.04 bc

0.89 ± 0.08 *#

1.11 ± 0.10 *#a

0.61 ± 0.04 *#

#

0.58 ± 0.02 *#

c

bc

The recovered S. aureus after time dependent phagocytosis in presence or absence of anti-CXCR1 antibody were used to determine SOD enzyme activity and was expressed in terms of unit of SOD/mg of bacterial protein. Results were presented as mean ± SD of three independent experiments. ‘#’ significant difference with respect to S. aureus infected macrophages (LSAM), ‘*’ significant difference with respect to IgG isotype antibody treated LSAM at p < 0.05 significance level. ‘a’ significant difference between splenic and peritoneal macrophages, ‘b’ significant difference between peritoneal macrophages and FBMC and ‘c’ significant difference between splenic macrophages and FBMC at p < 0.05 significance level.

Table 3 Alteration in catalase enzyme activity of whole staphylococcal cells from the recovered S. aureus after time-dependent phagocytosis in presence or absence of antibody. Groups

SPM

PM

FBMC

Bacterial catalase enzyme activity (nM/min.mg bacterial protein) mean ± SD After 30 min of phagocytosis S. aureus-infected macrophage for 30 min IgG isotype antibody-treated macrophageþ S. aureus infection for 30 min Anti-CXC-R1 antibody-treated macrophageþ S. aureus infection for 30 min After 60 min of phagocytosis S. aureus-infected macrophage for 60 min IgG isotype antibody-treated macrophageþ S. aureus infection for 60 min Anti-CXC-R1 antibody-treated macrophageþ S. aureus infection for 60 min After 90 min of phagocytosis S. aureus-infected macrophage for 90 min IgG isotype antibody-treated macrophageþ S. aureus infection for 90 min Anti-CXC-R1 antibody-treated macrophageþ S. aureus infection for 90 min

42.63 ± 6.50 40.27 ± 6.14 54.33 ± 2.10

*#

59.31 ± 4.38 57.14 ± 4.12 76.44 ± 4.30

*#

55.16 ± 5.66 54.03 ± 5.12 80.29 ± 8.20

*#

33.13 ± 12.33 31.07 ± 11.02

29.42 ± 5.21c 27.34 ± 5.01c

71.18 ± 9.54*#a

38.75 ± 2.80*#bc

61.12 ± 8.51 60.17 ± 8.34

47.20 ± 5.13bc 46.37 ± 5.01 bc

77.39 ± 3.21*#

66.11 ± 5.28*#bc

62.36 ± 10.71 60.37 ± 10.87

41.02 ± 6.32bc 40.34 ± 5.97 bc

95.21 ± 6.42*#a

62.51 ± 8.21*#bc

The recovered S. aureus after time dependent phagocytosis in presence or absence of anti-CXCR1 antibody were used to determine catalase activity in presence of 15 mM of H2O2/ml of phosphate buffer. Catalase enzyme activity was expressed in terms of mM/min mg protein. Results were shown as mean ± SD of three independent experiments. ‘#’ significant difference with respect to S. aureus infected macrophages (LSAM), ‘*’ significant difference with respect to IgG isotype antibody treated LSAM, at p < 0.05 significance level. ‘a’ significant difference between splenic and peritoneal macrophages, ‘b’ significant difference between peritoneal macrophages and FBMC and ‘c’ significant difference between splenic macrophages and FBMC at p < 0.05 significance level.

generation of O 2 (Table 2). The increased bacterial SOD enzyme neutralizes the bactericidal activity of O 2 by converting it into H2O2, another oxygen metabolite having potential antimicrobial activities. Further, the bacteria recovered after time dependent phagocytosis from PM, SPM and FBMC neutralized with anti CXCR-1

antibody showed decreased SOD enzyme activity and increased catalase enzyme activity suggesting the reduced elaboration of ROS by the host cells leading to less killing of internalized S. aureus (Table 2). These results motivate us to hypothesize that after cell surface CXCR-1 blocking, macrophages have less phagocytic activity

the presence and absence of anti-CXCR1 antibody. Results in this figure represent mean nitric oxide content (mM) in the medium (A) or from cell-free lysate (B) of murine macrophages/FBMCs from different groups of triplicate experiments. ‘g’ significant difference with respect to uninfected control macrophages,‘#’ significant difference with respect to LSAM, ‘*’ significant difference with respect to IgG isotype antibody treated LSAM, ‘4’ significant difference between uninfected control and HKSAM and ‘$’ significant difference with respect to HKSAM, ‘%’ significant difference with respect to IgG isotype antibody treated HKSAM at p < 0.05 significance level. ‘a’ significant difference between splenic and peritoneal macrophages, ‘b’ significant difference between peritoneal macrophages and FBMC and ‘c’ significant difference between splenic macrophages and FBMC at p < 0.05 significance level.

Fig. 5. AeB Tumor necrosis factor a and interferon gamma released in the media after phagocytosis. Levels of TNF-a (Fig. 5A), IFN- g (Fig. 5B) in the supernatants collected after 30, 60, and 90 min of S. aureus-infected or HKSA stimulated macrophages/FBMCs in presence or absence of anti-CXCR1 antibody were determined by utilizing ELISA according to the manufacturer's recommendations and were expressed from triplicate experiments. ‘g’ significant difference with respect to uninfected control macrophages,‘#’ significant difference with respect to LSAM, ‘*’ significant difference with respect to IgG isotype antibody treated LSAM, ‘4’ significant difference between uninfected control and HKSAM and ‘$’ significant difference with respect to HKSAM, ‘%’ significant difference with respect to IgG isotype antibody treated HKSAM at p < 0.05 significance level. ‘a’ significant difference between splenic and peritoneal macrophages, ‘b’ significant difference between peritoneal macrophages and FBMC and ‘c’ significant difference between splenic macrophages and FBMC at p < 0.05 significance level.

Fig. 6. AeB Interleukin-6 and interleukin-1b released in the media after phagocytosis. Levels of IL-6 (Fig. 6A), IL-1b (Fig. 6B) in the supernatants collected after 30, 60, and 90 min of S. aureus-infected or HKSA stimulated macrophages/FBMCs in presence or absence of anti-CXCR1 antibody were determined by utilizing ELISA according to the manufacturer's recommendations and were expressed from triplicate experiments. ‘g’ significant difference with respect to uninfected control macrophages,‘#’ significant difference with respect to LSAM, ‘*’ significant difference with respect to IgG isotype antibody treated LSAM, ‘4’ significant difference between uninfected control and HKSAM and ‘$’ significant difference with respect to HKSAM, ‘%’ significant difference with respect to IgG isotype antibody treated HKSAM at p < 0.05 significance level. ‘a’ significant difference between splenic and peritoneal macrophages, ‘b’ significant difference between peritoneal macrophages and FBMC and ‘c’ significant difference between splenic macrophages and FBMC at p < 0.05 significance level.

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Fig. 7. AeB Interleukin-8 and interleukin-10 released in the media after phagocytosis. Levels of IL-8 (Fig. 7A) and IL-10 (Fig. 7B) in the supernatants collected after 30, 60, and 90 min of S. aureus-infected or HKSA stimulated macrophages/FBMCs in presence or absence of anti-CXCR1 antibody were determined by utilizing ELISA according to the manufacturer's recommendations and were expressed from triplicate experiments. ‘g’ significant difference with respect to uninfected control macrophages,‘#’ significant difference

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as evidenced by increased CFU count and the bacterial catalase might be the crucial factor for more survival of S. aureus inside the different subtypes of macrophages/FBMCs due to CXCR-1 blocking. Several studies have suggested that bacterial catalase serves as a virulence factor due to its ability to cleave hydrogen peroxide, a reactive oxygen intermediate (ROI) responsible for bactericidal activities of phagocytes. Catalase of pathogenic bacteria is important for optimal detoxification of H2O2 survival in macrophages. In our case, the increased catalase enzyme activity after timedependent phagocytosis in the presence of anti-CXCR-1 antibody (Table 3) might suggesting the degradation of macrophage-derived H2O2 by bacterial catalase, leading to reduced killing of S. aureus by H2O2. This might indicate that enhanced bacterial catalase enzyme activity is the major factor for less intracellular killing of S. aureus inside the macrophages due to CXCR-1 blocking. To understand whether catalase contributing in the intracellular survival was of bacterial origin or not, 3-amino, 1, 2, 4-triazole (ATZ) was used to inhibit specifically macrophage-derived catalase. Catalase enzyme activity from the whole staphylococcal cells in the presence of ATZ suggested that the released catalase were of extracellular origin (data not shown). Pretreatment of macrophages with ATZ specifically inhibited the macrophage-derived catalase; therefore, the enzyme activity obtained in the supernatant or lysate indicated that the released catalase was of bacterial origin (data not shown). In an ongoing study, catalase protein expression from the whole staphylococcal cells recovered after phagocytosis from CXCR-1 neutralized macrophages/FBMC also indicated that the catalase might be from S. aureus. Phagocytic cells modulate innate immune responses through the production of proinflammatory cytokines in response to invading microbes [48]. In this study, we have shown that when confronted with LSA or HKSA, macrophages of different subtypes/ FBMCs (Figs. 5e7) were extremely efficient producer of TNF-a, IFNg, IL-6, IL-1b and CXCL8. In this scenario, host cells do not produce significant levels of IL-10 (Fig. 7B) at least during acute staphylococcal infection. This overall cytokine profile could result in decreased bacterial burden inside the macrophages. S. aureus infections are characterized by a profound inflammatory response, which contributes significantly to pathogenesis but is also required for bacterial clearance. Among the proinflammatory cytokines induced, TNF-a and IFN-g has been shown to be crucial for the eradication of bacteria in several experimental models [57]. In this study TNF-a (Fig. 5A) and IL-b (Fig. 6B) level was significantly decreased due to blocking of CXCR-1 in SPM, SPM and FBMC infected with both LSA or HKSA stimulation, which was in line with the earlier finding that blocking of CXCR-1/CXCR-2 inhibits production of TNF-a. But the effect was more pronounced in PM and SPM only. Further TNF-a signalling is finely regulated by the availability of TNFR-1 at the cell surface. Also, shedding of TNFR1 was critical for the neutralization of TNF-a signaling and the arrest of inflammation. It was reported previously that CXCR-1 neutralization causes shedding of TNFR-1 [15] which might also be responsible for the decreased TNF-a after CXCR-1 neutralization in this study, although we have not tested this in acute infection study. These results reinforce the concept that, akin to anti-TNF-a treatment, CXCR-1 blockade may also be a useful therapeutic strategy in the setting of staphylococcal arthritis. Normally, CXCL8 is barely detectable in non-induced cells, but

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its production is rapidly induced by a very wide range of stimuli encompassing proinflammatory cytokines such as TNF-a or IL-1b, bacterial or viral products and cellular stress [58]. Also, CXCL8 is an oxidative stress responsive chemokine [15,59]. Decreased TNF-a, IL-1b and reduced ROS production due to CXCR-1 blocking might be responsible for the lower CXCL8 production in different subtypes of macrophages/FBMCs (Fig. 7A) in this study, indicating inhibition of excessive infiltration or activation of macrophages during acute inflammatory responses. Cytokines including IFN-g reportedly regulate host resistance against S. aureus infection [60] and IL-12 p70 is a critical inducer of IFN-g in the innate immune response [61]. Further, it was earlier reported that IL-12p70 was modified by CXCR-1/2 inhibitor treatment [62]. IL-12 production was also reported to be impaired due to CXCR-1 neutralization in peritoneal macrophages during S. aureus infection [15]. Therefore, this may be the plausible explanation for reduced production of IFN-g in CXCR-1 neutralized SPM, PM and FBMC (Fig. 5B) infected with either LSA or HKSA stimulation in acute staphylococcal infection model. IL-6 (Fig. 6A) was found to be down regulated due to blocking of CXCR-1 in different subtypes of macrophages infected with LSA or stimulated with HKSA. This may be due to reduced TNF-a production or decreased ROS generation due to CXCR-1 neutralization as it was earlier reported that H2O2 and TNF-a induce an increase in IL-6 release with a corresponding increase in the activation of NF-kB in A549 cells. IL-10 is an immunoregulatory cytokine and its main biological function is limitation and termination of inflammatory responses. Absence of IL-10 significantly increases the severity of S. aureusinduced arthritis [63]. LSA or HKSA induced decrease in IL-10 production in different subtypes of macrophages/FBMCs (Fig. 7B) were found to be elevated due to neutralization of CXCR-1 in this study. Modulation of proinflammatory cytokines (TNF-a, CXCL8 and IFN-g) in LSA infected or HKSA stimulated macrophages due to blocking of CXCR-1 were prominent in both PM and SPM as compared to FBMC in this study. In contrast, differential production of IL-10 in macrophages neutralized with anti CXCR-1 antibody and infected with LSA or stimulated with HKSA was found to be maximum in FBMC when compared with PM and SPM. It was earlier reported that S. aureus failed to induce IL-12 directly, suggesting an initial bias away from Th1 differentiation. In the presence of IFN-g, IL-12 is induced by S. aureus in monocytes/ macrophages while IL-10 is suppressed, leading to expression of high levels of MHC class II and co-stimulatory molecules [64]. Therefore, we speculate that CXCR-1 neutralization causes down regulation in the induction of IL-12 or IFN-g and thereby increased production of IL-10 in this current set up of acute staphylococcal infection (Scheme 1). This increased production of IL-10 also justifies the result of decreased production of CXCL8 due to CXCR-1 neutralization as IL10 is a potent inhibitor of CXCL8 synthesis [65]. Previous report demonstrates that in normal PMN, CXC receptors are transiently internalized following in vitro stimulation by CXCL8 [64]. Subsequently, CXCR-1 is rapidly re-expressed on the cell surface, whereas CXCR2 is re-expressed at a considerably slower rate [66]. In this study, infection of different subtypes of macrophages with either LSA or stimulation with HKSA resulted in the increased expression of CXCR-1 in all the subtypes but the degree of expression was maximum in PM, than in SPM and was least in FBMC. Western blot

with respect to LSAM, ‘*’ significant difference with respect to IgG isotype antibody treated LSAM, ‘4’ significant difference between uninfected control and HKSAM and ‘$’ significant difference with respect to HKSAM, ‘%’ significant difference with respect to IgG isotype antibody treated HKSAM at p < 0.05 significance level. ‘a’ significant difference between splenic and peritoneal macrophages, ‘b’ significant difference between peritoneal macrophages and FBMC and ‘c’ significant difference between splenic macrophages and FBMC at p < 0.05 significance level.

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Scheme 1. A) Splenic and peritoneal macrophages or FBMC response during acute S. aureus infection in absence of anti CXCR-1 antibody: During acute staphylococcal infection, Internalization of S. aureus increases reactive oxygen/nitrogen species generation pathways along with the elaboration of Th1 pro-inflammatory cytokines like TNF-a, IFNg, CXCL-8 (IL-8) which leads to more oxidative burst and also simultaneous inhibition of Th2 cytokines like IL-10. These increased pro-inflammatory cytokines and decreased antiinflammatory cytokine profile as well as increased generation of ROS and RNS leads to enhance bacterial killing via the modulation of bacterial antioxidants. B) Splenic and peritoneal macrophages or FBMC response during acute S. aureus infection in presence of anti CXCR-1 antibody: Phagocytosis of S. aureus was decreased as activation of splenic and peritoneal macrophages or FBMC became altered due to the blockade of CXCR-1 which next down regulates ROS/RNS generation pathway by modulating bacterial anti oxidant enzymes. This condition facilitated up regulation of Th2 responses with simultaneous down regulation of Th 1 response. This Th2/Th 1 balance also favours less ROS and RNS generation and lower oxidative burst which ultimately inhibits CXCR-1 expression and also CXCL-8 (IL-8) production. Thus, due to surface CXCR-1 blocking unable to protect the host macrophages/FBMC derived H2O2 from degradation leading to less bacterial killing [Green arrows represent up-regulation or increment; red arrows indicate downregulation or decrement].

analysis of CXCR-1 presented evidence that CXCR-1 is not constitutively expressed in any subtypes of macrophages. Further, neutralization of CXCR1 in PM, SPM and in FBMC and infected with LSA or stimulated with HKSA leads to downregulation of CXCR-1 expression in these macrophages. We speculate that complex cytokine mediated mechanism in CXCR-1 neutralized PM, SPM and FBMC infected with LSA or HKSA stimulation may contribute to the differential expression of CXCR-1 in different subtypes of macrophages/FBMCs. Flow cytometric analysis of the surface expression of CXCR-1 upon S. aureus infection as well as rate of transcription of CXCR1/CXCL8 mRNA by quantitative PCR, and the number of CXCR1 molecule expressed on the cell surface might be helpful for better understanding of the role of CXCR-1. In a previous study from our laboratory we have reported expression of CXCR1 (Interleukin-8 Receptor) in murine macrophages after Staphylococcus aureus infection and its possible implication on intracellular survival correlating with cytokines and bacterial anti-oxidant enzymes; where we have studied the expression of CXCR1 (Interleukin-8 Receptor) by utilizing only peritoneal macrophages and cells were infected only with live Staphylococcus aureus for 24, 48 and 72 h. So this was completely a different study [15]. Current study reports on the functional characterization of CXCR1 in mouse macrophage subtypes and its modulation in relation to acute live S. aureus infection and the response of different subtypes of macrophages when surface CXCR1 were abrogated. We suggested that neutralization of cell

surface CXCR-1 in different subtypes of macrophages (SPM, PM) and FBMC infected with live S. aureus resulted in the increased survival of S. aureus inside the macrophages. Further, blockade of CXCR-1 and LSA infection or HKSA stimulation in macrophages modulates ROS generation and pro and anti-inflammatory cytokine level. It is apparently beneficial for the host and protecting against S. aureus induced oxidative tissue damage but simultaneously it increases bacterial SOD and catalase enzyme activity which is responsible for the diminution of phagocytic ability of macrophages. This study also provides powerful reference on the understanding of the functional importance of macrophage subpopulation heterogeneity which will be important in designing new and potentially more effective approaches to limit inflammation and cytotoxicity. In conclusion, this basic study might be a platform to further explore the potential therapeutic role for the modulation of CXCR-1 receptor signalling in the treatment chronic inflammatory diseases caused by S. aureus infection. Further, it can be suggested that, the neutralization of CXCR-1 along with a potent antibiotic should be the fruitful treatment strategy of acute S. aureus infection. This combined therapy not only reduces the bacterial burden in macrophages but also helps to minimize oxidative damage to the host. Further investigations demands to find out the most effective antibiotic combination along with anti CXCR-1 antibody. Because CXCL-8 recruit and activate leukocytes selectively via CXCR-1, their induction in direct response to S. aureus infection by macrophages in vitro may be important to

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toxic shock as well as the initiation of an inflammatory signal, such as in bacterial septic arthritis. Further studies are required to examine whether the S. aureus-induced expression of CXCR-1 protein is dependent on TLR signaling or autocrine cytokine signaling. To attend these problems, macrophages from different sources as tested here could be incubated with various TLR ligands followed by examination of the expression of CXCR-1. Furthermore, macrophages obtained from different sources could be pretreated with inhibitors of protein synthesis such as cycloheximide prior to S. aureus infection then studying the expression of CXCR-1 and CXCL8 production. Inflammation induced by reactive oxygen species has many of the features related to classical activation of the innate immune system and, as such, can resemble that seen after TLR activation with bacterial components. However, the interactive role of TLR-2 and CXCR1 network during gram positive infections could have a major impact on understanding the pathogenesis of these conditions, as well as on designing strategies to alter the disequilibrium in the cytokine production away from inflammation in the direction of host defense and elimination of S. aureus infection. Because CXCL8 recruit and activate the phagocytic cells selectively via CXCR1, their induction in response to acute S. aureus infection by macrophage subtypes in vitro may be important to toxic shock as well as the initiation of an inflammatory signal, such as in bacterial septic arthritis. Although this study reports on the functional characterization of CXCR1 in mouse macrophage subtypes and its modulation in relation to acute live S. aureus infection or heat killed S. aureus (HKSA) stimulation and the response of different subtypes of macrophages when surface CXCR1 were blocked, but for better understanding the detailed mechanisms further studies are warranted. Conflict of interest For the manuscript entitled “Expression of CXCR1 (IL-8 receptor A) in splenic, peritoneal macrophages and resident bone marrow cells after acute live or heat killed Staphylococcus aureus stimulation in mice” by Bishayi et al. authors declared that they have no conflict of interest for this manuscript towards submission in Microbial Pathogenesis. The authors also state that we do not have a direct financial relation with the commercial identities mentioned in this manuscript that might lead to a conflict of interest for any of the authors. Acknowledgements This work was supported by the Department of Science and Technology (DST), Science and Engineering Research Board (SERB), Ministry of Science and Technology, Government of India, New Delhi, India [grant number SR/SO/HS/0013/2012, dated 21 May 2013 to BB] for funding this project. The author (Biswadev Bishayi) is indebted to the Department of Science and Technology, Government of India for providing us with the instruments procured under the DST-PURSE program to the Department of Physiology, University of Calcutta. References [1] K. Matsushima, K. Morishita, T. Yoshimura, S. Lavu, Y. Kobayashi, W. Lew, et al., Molecular cloning of a human monocyte-derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor, J. Exp. Med. 167 (1988) 1883e1893. [2] F.M. Brennan, C.O. Zachariae, D. Chantry, C.G. Larcen, M. Turner, R.N. Maini, et al., Detection of interleukin 8 biological activity in synovial fluids from patients with rheumatoid arthritis and production of interleukin 8 mRNA by isolated synovial cells, Eur. J. Immunol. 20 (1990) 2141e2144. [3] I.F. Charo, R.M. Ransohoff, The many roles of chemokines and chemokine receptors in inflammation, N. Eng. J. Med. 354 (2006) 610e621.

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