Cytokine 69 (2014) 211–217
Contents lists available at ScienceDirect
Cytokine journal homepage: www.journals.elsevier.com/cytokine
TLR-mediated inflammatory response to neonatal pathogens and co-infection in neonatal immune cells V. Sugitharini a, K. Pavani a, A. Prema b, E. Berla Thangam a,⇑ a b
Department of Biotechnology, School of Bio-engineering, SRM University, Kattankulathur, Chennai 603203, India Department of Pediatrics, SRM Medical College Hospital and Research Centre, Kattankulathur, Chennai 603203, India
a r t i c l e
i n f o
Article history: Received 17 January 2014 Received in revised form 30 April 2014 Accepted 4 June 2014
Keywords: TLR Cytokines Co-infection LPS PGN
a b s t r a c t Neonates heavily depend on the innate immune system for defence against invading pathogens. Toll-like receptors (TLRs) represent a primary line of host defence and play an important role in orchestrating the inflammatory response to invading pathogens. The most commonly infecting pathogens in neonates are E. coli, Klebsiella pneumoniae and Staphylococcus aureus. Also, co-infection with more than one organism is common in neonatal sepsis. Therefore, we aimed to study the TLR2 and TLR4 mediated neonatal inflammatory response to these pathogens. For this, we stimulated mononuclear cells from cord blood with LPS, PGN, E. coli, K. pneumoniae and S. aureus and analyzed the surface expression of TLR2 and TLR4 on CD14+CD16+ and CD14dimCD16+ and its inflammatory response in comparison with peripheral blood. We found that the TLR2 and TLR4 were differentially expressed on both monocyte subpopulations. Cytokines such as IL-6, IL-1b, IL-23, IL-10, IL-13, MCP-1 and IL-8 were measured using ELISA and we observed that although, neonatal cells were able to produce similar levels of the classical pro-inflammatory (IL-6, IL-1b) and anti-inflammatory (IL-10, IL-13) cytokines as that of adult cells, the amounts of IL-23 and MCP-1 were lower in CBMCs while the chemokine IL-8 was higher in CBMCs when compared with PBMCs. In addition, using Human Inflammation Antibody array technique we found that multiple cytokine production was impaired in cord blood when cells were co-infected with LPS and PGN. In conclusion, the TLR-mediated inflammatory response to neonatal pathogens is differentially regulated by different pathogens. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Neonatal sepsis is a highly inflammatory disease that culminates in septic shock and multi-organ failure due to uncontrolled activation of the inflammatory system in response to pathogen [1,2]. In our previous study we have reported that the major pathogens causing neonatal sepsis in South Indian populations were Klebsiella spp. (38%), Staphylococcus aureus (11%) and E. coli (11.1%) [19]. Furthermore, co-infections with two or more organisms are found in 4 to 24% of all neonatal infections. Mortality due to infections with two or more organisms is greater than those due to infections with individual organism (70% versus 23%). To date, research on coinfection in the neonate has been entirely observational [3,4]. There is an urgent need for research to better ⇑ Corresponding author. Address: Department of Biotechnology, School of Bio-engineering, SRM University, Kattankulathur 603203, Tamil Nadu, India. Tel.: +91 9444681340. E-mail address: [email protected] (E. Berla Thangam). http://dx.doi.org/10.1016/j.cyto.2014.06.003 1043-4666/Ó 2014 Elsevier Ltd. All rights reserved.
understand coinfection and develop effective strategies for treatment and prevention. In neonates, the primary defense mechanism relies heavily on the innate immune system, because the adaptive immune system of newborn and particularly preterm infants is not fully developed. Previous reports have demonstrated the deficiency of the innate immune system such as the lack of effective antibodies, decreased complement activity and impaired capacity of polymorphonuclear cells to upregulate oxidative burst and bacterial killing [5,7,8–13]. Thus, many aspects of the innate immune response to various neonatal pathogens show significant difference between neonates and adults. For example, in the case of CoNS infection, neonates significantly display reduced complement activity of both classical and alternative pathways [5,7]. Though, the neonatal immune cells were found to be defective in clearing the pathogens, they were able to mount a powerful inflammatory response [56]. In the initial phase of inflammation, cells of the innate immune system particularly monocytes and granulocytes play an important role in host defence against
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V. Sugitharini et al. / Cytokine 69 (2014) 211–217
infection. Immaturity of monocytes may lead to reduced cytokine production and impaired pathogen clearance [3]. Recently, it was reported that CD14dimCD16+ monocyte subpopulations are the major producers of pro-inflammatory cytokines by the activation of Toll-like Receptors (TLRs) [14,15]. Among the 10 TLRs known in humans, TLR2 and TLR4 are critical for the propagation of inflammatory response to components of Gram positive and Gram negative bacteria respectively. Activation of these receptors triggers a cascade of signaling events involving the adapter protein (MyD88), IL-1 receptor associated kinase (IRAK), TNF-a receptor associated factor (TRAF) – 6 to activate Nuclear factor (NF)-jB which in turn leads to the production of inflammatory mediators such as IL-1b, IL-6 and TNF-a. Previous reports have shown that TLRs are differentially regulated in the course of neonatal sepsis and low levels of TLR2 expression on neonatal immune cells compared to adults [5,6,16–18,20]. The aim of the study is to determine the TLR function in neonatal mononuclear cells which will likely contribute to a better understanding of the host defence in neonates. Therefore, we investigated the TLR-mediated inflammatory response to common neonatal pathogens such as E. coli, Klebsiella pneumoniae and S. aureus and co-infection in neonatal immune cells.
2. Materials and methods 2.1. Blood sample processing Cord blood was collected from healthy volunteers (n = 12) without complicated vaginal delivery from SRM hospital, Kattankulathur. Blood was collected in tubes containing 38% sodium citrate as anti-coagulant and processed within 4 h. Peripheral blood was collected from age-matched healthy non-pregnant volunteers by venupuncture without any type of antibiotic use or any medical intervention. Informed consent was obtained from all mothers and healthy volunteers.
2.2. Isolation of mononuclear cells and preparation of bacterial culture Peripheral blood mononuclear cells (PBMCs) and Cord blood mononuclear cells (CBMCs) were isolated by density gradient centrifugation using Lymphocyte separation Medium LSM (HiSep). Isolated cells were washed twice in phosphate buffered saline and resuspended in RPMI 1640 supplemented with 10% FBS and antibiotic solution. Cell viability was confirmed by trypan blue staining. Bacterial culture (E. coli, K. pneumoniae and S. aureus) was obtained from culture positive sepsis individuals and subcultured overnight. Culture from a single colony was inoculated in 10 ml of LB broth and incubated overnight at 37 °C. Serial dilutions were made and the concentration of bacteria was adjusted to 104 CFU/ml. The bacterial culture was heat-inactivated at 75 °C for 45 min. The culture was plated overnight to check for the nil growth of colonies.
2.4. Analysis of TLR2 and TLR4 expression by flow cytometry Four color flow cytometry was performed on freshly isolated PBMCs and CBMCs to analyze the surface expression of CD14, CD16, TLR2 and TLR4 on monocytes. The cells were washed with phosphate buffered saline containing 1% BSA (FACS buffer). 106/ml cells in 100 ll of FACS buffer were stained and incubated with fluorescent conjugated antibodies CD16-APC Invitrogen) and CD14-Percep (Invitogen), CD284 (TLR4) PE (eBioscience) and CD282 (TLR2) FITC (eBioscience) and their corresponding isotype controls for 30 min at 4 °C. The cells were washed and fixed in 500 ll of 4% paraformaldehyde. Analysis was performed using CellQuest pro software (BD Biosciences). The monocytes were gated based on the CD14 and CD16 staining properties and acquisition was performed on 5000 gated events. The TLR fluorescence was measured on a logarithmic scale in the FL1 channel (TLR2) and FL2 channel (TLR4). The mean fluorescence intensity (MFI) was used to determine the extent of cell surface TLR expression and expressed as the MFI of specific antibody subtracted from the MFI of isotype control. 2.5. Determination of various inflammatory cytokines and chemokines by ELISA The levels of cytokines in the culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA): IL-6 (eBioscience), IL-1b (eBioscience), IL-8 (eBioscience), MCP-1 (Antigenix America), IL-10 (eBioscience), IL-13 (R&D systems) and IL-23 (R&D systems)). Measurements were performed according to manufacturer’s instructions. 2.6. Inflammation protein profiling using Human Inflammation Antibody array Using Human Inflammation Antibody Array 3 (Raybiotech, USA), we analyzed the expression of inflammatory proteins in CBMC samples and compared with PBMCs. 106/ml of mononuclear cells from cord blood and peripheral blood were stimulated with 100 ng/ml each of LPS, PGN and LPS + PGN. Unstimulated cells were used as control. All steps were performed at room temperature. Briefly, the array membranes were incubated with blocking buffer at room temperature for 30 min, followed by incubation with 1 ml of culture supernatant for 2 h. After washing, the membranes were incubated with biotin-conjugated antibodies for 2 h followed by the addition of horseradish peroxidase–conjugated streptavidin for 2 h. The membranes were developed with chemiluminescence substrate; images were taken using fluorchem Q, and the spots were analyzed as per manufacturer’s instructions. 2.7. Statistical analysis All data were analyzed using Graphpad prism software version 6.0. Data between groups obtained from ELISA and flow cytometry were compared using one-way ANOVA (Dunnett test for comparison with control). p Values of
Contents lists available at ScienceDirect
Cytokine journal homepage: www.journals.elsevier.com/cytokine
TLR-mediated inflammatory response to neonatal pathogens and co-infection in neonatal immune cells V. Sugitharini a, K. Pavani a, A. Prema b, E. Berla Thangam a,⇑ a b
Department of Biotechnology, School of Bio-engineering, SRM University, Kattankulathur, Chennai 603203, India Department of Pediatrics, SRM Medical College Hospital and Research Centre, Kattankulathur, Chennai 603203, India
a r t i c l e
i n f o
Article history: Received 17 January 2014 Received in revised form 30 April 2014 Accepted 4 June 2014
Keywords: TLR Cytokines Co-infection LPS PGN
a b s t r a c t Neonates heavily depend on the innate immune system for defence against invading pathogens. Toll-like receptors (TLRs) represent a primary line of host defence and play an important role in orchestrating the inflammatory response to invading pathogens. The most commonly infecting pathogens in neonates are E. coli, Klebsiella pneumoniae and Staphylococcus aureus. Also, co-infection with more than one organism is common in neonatal sepsis. Therefore, we aimed to study the TLR2 and TLR4 mediated neonatal inflammatory response to these pathogens. For this, we stimulated mononuclear cells from cord blood with LPS, PGN, E. coli, K. pneumoniae and S. aureus and analyzed the surface expression of TLR2 and TLR4 on CD14+CD16+ and CD14dimCD16+ and its inflammatory response in comparison with peripheral blood. We found that the TLR2 and TLR4 were differentially expressed on both monocyte subpopulations. Cytokines such as IL-6, IL-1b, IL-23, IL-10, IL-13, MCP-1 and IL-8 were measured using ELISA and we observed that although, neonatal cells were able to produce similar levels of the classical pro-inflammatory (IL-6, IL-1b) and anti-inflammatory (IL-10, IL-13) cytokines as that of adult cells, the amounts of IL-23 and MCP-1 were lower in CBMCs while the chemokine IL-8 was higher in CBMCs when compared with PBMCs. In addition, using Human Inflammation Antibody array technique we found that multiple cytokine production was impaired in cord blood when cells were co-infected with LPS and PGN. In conclusion, the TLR-mediated inflammatory response to neonatal pathogens is differentially regulated by different pathogens. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Neonatal sepsis is a highly inflammatory disease that culminates in septic shock and multi-organ failure due to uncontrolled activation of the inflammatory system in response to pathogen [1,2]. In our previous study we have reported that the major pathogens causing neonatal sepsis in South Indian populations were Klebsiella spp. (38%), Staphylococcus aureus (11%) and E. coli (11.1%) [19]. Furthermore, co-infections with two or more organisms are found in 4 to 24% of all neonatal infections. Mortality due to infections with two or more organisms is greater than those due to infections with individual organism (70% versus 23%). To date, research on coinfection in the neonate has been entirely observational [3,4]. There is an urgent need for research to better ⇑ Corresponding author. Address: Department of Biotechnology, School of Bio-engineering, SRM University, Kattankulathur 603203, Tamil Nadu, India. Tel.: +91 9444681340. E-mail address: [email protected] (E. Berla Thangam). http://dx.doi.org/10.1016/j.cyto.2014.06.003 1043-4666/Ó 2014 Elsevier Ltd. All rights reserved.
understand coinfection and develop effective strategies for treatment and prevention. In neonates, the primary defense mechanism relies heavily on the innate immune system, because the adaptive immune system of newborn and particularly preterm infants is not fully developed. Previous reports have demonstrated the deficiency of the innate immune system such as the lack of effective antibodies, decreased complement activity and impaired capacity of polymorphonuclear cells to upregulate oxidative burst and bacterial killing [5,7,8–13]. Thus, many aspects of the innate immune response to various neonatal pathogens show significant difference between neonates and adults. For example, in the case of CoNS infection, neonates significantly display reduced complement activity of both classical and alternative pathways [5,7]. Though, the neonatal immune cells were found to be defective in clearing the pathogens, they were able to mount a powerful inflammatory response [56]. In the initial phase of inflammation, cells of the innate immune system particularly monocytes and granulocytes play an important role in host defence against
212
V. Sugitharini et al. / Cytokine 69 (2014) 211–217
infection. Immaturity of monocytes may lead to reduced cytokine production and impaired pathogen clearance [3]. Recently, it was reported that CD14dimCD16+ monocyte subpopulations are the major producers of pro-inflammatory cytokines by the activation of Toll-like Receptors (TLRs) [14,15]. Among the 10 TLRs known in humans, TLR2 and TLR4 are critical for the propagation of inflammatory response to components of Gram positive and Gram negative bacteria respectively. Activation of these receptors triggers a cascade of signaling events involving the adapter protein (MyD88), IL-1 receptor associated kinase (IRAK), TNF-a receptor associated factor (TRAF) – 6 to activate Nuclear factor (NF)-jB which in turn leads to the production of inflammatory mediators such as IL-1b, IL-6 and TNF-a. Previous reports have shown that TLRs are differentially regulated in the course of neonatal sepsis and low levels of TLR2 expression on neonatal immune cells compared to adults [5,6,16–18,20]. The aim of the study is to determine the TLR function in neonatal mononuclear cells which will likely contribute to a better understanding of the host defence in neonates. Therefore, we investigated the TLR-mediated inflammatory response to common neonatal pathogens such as E. coli, Klebsiella pneumoniae and S. aureus and co-infection in neonatal immune cells.
2. Materials and methods 2.1. Blood sample processing Cord blood was collected from healthy volunteers (n = 12) without complicated vaginal delivery from SRM hospital, Kattankulathur. Blood was collected in tubes containing 38% sodium citrate as anti-coagulant and processed within 4 h. Peripheral blood was collected from age-matched healthy non-pregnant volunteers by venupuncture without any type of antibiotic use or any medical intervention. Informed consent was obtained from all mothers and healthy volunteers.
2.2. Isolation of mononuclear cells and preparation of bacterial culture Peripheral blood mononuclear cells (PBMCs) and Cord blood mononuclear cells (CBMCs) were isolated by density gradient centrifugation using Lymphocyte separation Medium LSM (HiSep). Isolated cells were washed twice in phosphate buffered saline and resuspended in RPMI 1640 supplemented with 10% FBS and antibiotic solution. Cell viability was confirmed by trypan blue staining. Bacterial culture (E. coli, K. pneumoniae and S. aureus) was obtained from culture positive sepsis individuals and subcultured overnight. Culture from a single colony was inoculated in 10 ml of LB broth and incubated overnight at 37 °C. Serial dilutions were made and the concentration of bacteria was adjusted to 104 CFU/ml. The bacterial culture was heat-inactivated at 75 °C for 45 min. The culture was plated overnight to check for the nil growth of colonies.
2.4. Analysis of TLR2 and TLR4 expression by flow cytometry Four color flow cytometry was performed on freshly isolated PBMCs and CBMCs to analyze the surface expression of CD14, CD16, TLR2 and TLR4 on monocytes. The cells were washed with phosphate buffered saline containing 1% BSA (FACS buffer). 106/ml cells in 100 ll of FACS buffer were stained and incubated with fluorescent conjugated antibodies CD16-APC Invitrogen) and CD14-Percep (Invitogen), CD284 (TLR4) PE (eBioscience) and CD282 (TLR2) FITC (eBioscience) and their corresponding isotype controls for 30 min at 4 °C. The cells were washed and fixed in 500 ll of 4% paraformaldehyde. Analysis was performed using CellQuest pro software (BD Biosciences). The monocytes were gated based on the CD14 and CD16 staining properties and acquisition was performed on 5000 gated events. The TLR fluorescence was measured on a logarithmic scale in the FL1 channel (TLR2) and FL2 channel (TLR4). The mean fluorescence intensity (MFI) was used to determine the extent of cell surface TLR expression and expressed as the MFI of specific antibody subtracted from the MFI of isotype control. 2.5. Determination of various inflammatory cytokines and chemokines by ELISA The levels of cytokines in the culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA): IL-6 (eBioscience), IL-1b (eBioscience), IL-8 (eBioscience), MCP-1 (Antigenix America), IL-10 (eBioscience), IL-13 (R&D systems) and IL-23 (R&D systems)). Measurements were performed according to manufacturer’s instructions. 2.6. Inflammation protein profiling using Human Inflammation Antibody array Using Human Inflammation Antibody Array 3 (Raybiotech, USA), we analyzed the expression of inflammatory proteins in CBMC samples and compared with PBMCs. 106/ml of mononuclear cells from cord blood and peripheral blood were stimulated with 100 ng/ml each of LPS, PGN and LPS + PGN. Unstimulated cells were used as control. All steps were performed at room temperature. Briefly, the array membranes were incubated with blocking buffer at room temperature for 30 min, followed by incubation with 1 ml of culture supernatant for 2 h. After washing, the membranes were incubated with biotin-conjugated antibodies for 2 h followed by the addition of horseradish peroxidase–conjugated streptavidin for 2 h. The membranes were developed with chemiluminescence substrate; images were taken using fluorchem Q, and the spots were analyzed as per manufacturer’s instructions. 2.7. Statistical analysis All data were analyzed using Graphpad prism software version 6.0. Data between groups obtained from ELISA and flow cytometry were compared using one-way ANOVA (Dunnett test for comparison with control). p Values of
