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TLR-mediated inflammatory response to neonatal pathogens and co-infection in neonatal immune cells
Cytokine 69 (2014) 211–217
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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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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 <0.05 were considered statistically significant.
2.3. Activation of mononuclear cells with respective ligands
3. Results
The mononuclear cells were plated at a density of 106/ml and stimulated with 100 ng/ml each of LPS from E. coli (Invivogen, SanDiego) and PGN from S. aureus (Invivogen, SanDiego) and 104 CFU/ml of heat-inactivated bacteria for 24 h. The supernatant was collected and stored in 20 °C until further analysis.
3.1. Effect of LPS, PGN, E. coli, K. pneumoniae and S. aureus on CD14 and CD16 expression on human monocytes Freshly stained mononuclear cells showed two unique monocyte subpopulations. One population showed high expression of
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CD14 and CD16 (CD14+CD16+) while the other showed diminished CD14 and high CD16 expression (CD14dimCD16+). However, upon stimulation with LPS, PGN, E. coli, K. pneumoniae and S. aureus the CD14 + CD16 + populations differentiated more into CD14dimCD16 + populations as shown in Fig. 1.
the levels of TLR4 on both monocyte populations in PBMCs as shown in Fig. 3.
3.2. TLR2 and TLR4 expression on CD14+CD16+ and CD14dimCD16+ monocytes in CBMCs
During bacterial infection, there is an imbalance in the inflammatory network due to the combined effect of pro-inflammatory and anti-inflammatory cytokines and chemokines. Therefore, we determined the role of TLR2 and TLR4 in mediating the production of the key inflammatory cytokines and chemokines in neonatal bacterial infection. We measured the levels of the pro-inflammatory cytokines such as IL-6, IL-1b and IL-23, chemokines IL-8 and MCP-1 and anti-inflammatory cytokines IL-10 and IL-13 and found their levels to be highly upregulated in CBMCs and PBMCs compared to their respective control as shown in Fig. 4. The amounts of IL-8 were higher in CBMCs than PBMCs in stimulated cells whereas the levels of IL-23, and MCP-1 were less in CBMCs when compared to PBMCs in stimulated cells. There was no significant difference in the amounts of IL-6, IL-1b, IL-10 and IL-13 between CBMCs and PBMCs. Furthermore, co-stimulated cells produced
We determined the cell surface expression of TLR2 on monocyte subpopulations in response to LPS, PGN, E. coli, K. pneumoniae and S. aureus. As shown in Fig. 2, we found that in CBMCs, the expression of TLR2 was downregulated in all stimulated cells compared to unstimulated cells in both monocyte populations. There was no significant difference on both monocyte subpopulations when cells were stimulated with S. aureus whereas on PBMCs, TLR2 levels was upregulated in CD14+CD16+ and CD14dimCD16+ by LPS and PGN respectively. Although the expression of TLR4 was downregulated in all stimulated cells compared to unstimulated ones in CBMCs on both monocyte populations, LPS and E. coli significantly upregulated
3.3. TLR2 and TLR4 mediated production of inflammatory cytokines and chemokines in CBMCs
Fig. 1. Effect of LPS, PGN, E. coli, Klebsiella pneumoniae and Staphylococcus aureus on CD14 and CD16 expression on human monocytes. Mononuclear cells were stimulated with 100 ng/ml of LPS and PGN and 104 Cfu/ml of E. coli, Klebsiella pneumoniae (K.p) and Staphylococcus aureus (S.a) for 24 h. Cells were stained with CD14-APC and CD16PerCP for flow cytometry. Dot plots are gated on monocytes based on staining properties. Data shown are representative of six individual experiments.
Fig. 2. Expression of TLR2 on CD14+CD16+ and CD14dimCD16+ cells from CBMCs and PBMCs. Isolated mononuclear cells (106/ml) were incubated with LPS E. coli (100 ng/ml), PGN Staphylococcus aureus (100 ng/ml) and 104 CFU/ml of heat inactivated whole bacteria (E. coli, Klebsiella pneumoniae (K.p), Staphylococcus aureus (S.a)) for 24 h. The Mean Fluorescence Intensity (MFI) of TLR2 were determined by flow cytometry. Results were expressed as Mean ± SD as relative values with respect to the control. p Values of < 0.05 were considered statistically significant.
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Fig. 3. Expression of TLR4 on CD14+CD16+ and CD14dimCD16+ cells from CBMCs and PBMCs. Isolated mononuclear cells (106/ml) were incubated with LPS E. coli (100 ng/ml), PGN Staphylococcus aureus (100 ng/ml) and 104 CFU/ml of heat inactivated whole bacteria (E. coli, Klebsiella pneumoniae (K.p), Staphylococcus aureus (S.a)) for 24 h. The Mean Fluorescence Intensity of TLR2 were determined by flow cytometry. Results were expressed as Mean ± SD as relative values with respect to the control. p Values of <0.05 were considered statistically significant.
Fig. 4. Measurement of various pro-inflammatory and anti-inflammatory cytokines and chemokines. 106 mononuclear cells were stimulated with LPS E. coli (100 ng/ml), PGN Staphylococcus aureus (100 ng/ml) and 104 CFU/ml of heat inactivated whole bacteria (E. coli, Klebsiella pneumoniae, Staphylococcus aureus) for 24 h. The supernatant was collected and measured for cytokines IL-6 (A), IL-1b (B), IL-10 (C), IL-13 (D), IL-8 (E), MCP-1 (F) and IL-23 (G) using ELISA. Bars are representative of mean values of 6 different samples from different donors and the error bars represent the Standard deviation. p Values of <0.05 are considered statistically significant. K.p = Klebsiella pneumoniae, S.a = Staphylococcus aureus, ns = non significant.
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Fig. 5. Representative figure showing the expression of inflammatory proteins in (A) CBMCs and (B) PBMCs using Human Inflammatory Antibody Array technique when stimulated with 100 ng/ml of Gram positive (PGN) and Gram negative ligands (LPS) and co-stimulation (LPS + PGN) and compared with unstimulated cells (Control). Multiple cytokines such as GM-CSF, MIP-1a, MIP-1b, IL-10, TNF-a and RANTES along with IL-6, IL-8 and MCP-1 were expressed in co-stimulated cells in PBMCs than CBMCs.
higher amounts of IL-23 and IL-13 than the cells that were stimulated with individual organisms.
MIP-1b, IL-10, TNF-a and RANTES, IL-6, IL-8 and MCP-1 were also detected in PBMCs upon co-stimulation with LPS and PGN. 4. Discussion
3.4. TLR2 and TLR4 mediated expression of inflammatory proteins in CBMCs In order to find out the expression of the key inflammatory proteins mediated by TLR2 and TLR4 activation in CBMCs, we employed the Human Inflammatory Antibody Array technique that can detect up to 40 different inflammatory proteins in a single sample. As shown in Fig. 5, the expression of IL-6, IL-8 and MCP1 was much higher in co-stimulated cells when compared to the expression in cells stimulated with individual ligands in cord blood. Interestingly, multiple cytokines such as GM-CSF, MIP-1a,
Pattern Recognition Receptors (PRRs) such as TLRs play an important role in recognition and responding to invading pathogens. Here in this study, we demonstrated the differential cell surface expression of TLR2 and TLR4 on CD14+CD16+ and CD14dimCD16+ cells in response to common neonatal pathogens such as E. coli, K. pneumoniae and S. aureus and their respective ligands (LPS and PGN) in neonatal immune cells. Accumulating evidence on neonatal cord blood suggested that neonatal responses to TLR agonists such as LPS and PGN are impaired at birth due to a defective TLR-MyD88-IRAK4 pathway thereby contributing to
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more susceptibility to bacterial infection [6,21–26]. Recently, Pedraza et al., reported that a reduced CD14+CD16+ activated/differentiated monocyte subset and a correspondingly lower level of functional TLR4 on monocytes contributes to the relatively lower response to LPS in neonates compared to their response in adults [27]. It has been shown previously that CD14+CD16+ cells differentiate more into CD14dimCD16+ cells and these cells were the major producers of pro-inflammatory cytokines [14,15]. In accordance with these reports, we also found that CD14dimCD16+ cells were increased upon stimulation with LPS, PGN, E. coli, K. pneumoniae and S. aureus. Furthermore, TLR2 and TLR4 were differentially expressed on both monocyte populations. We also observed that the levels of TLR4 expression varied significantly when stimulated with K. pneumoniae and E. coli. The observed difference of TLR4 expression may be due to the difference in PAMPs between the two organisms as K. pneumoniae recognition by TLRs takes place both due to LPS and CPS (Capsular polysaccharide). Also, TLR4 has been shown to play a crucial role in the early response to K. pneumoniae infections rather than in the late phase [58,59]. The activation of TLR2 and TLR4 by bacterial ligands initiate signaling cascades that result in the production of a range of pro-inflammatory cytokines and chemokines such as IL-6, TNF-a, IL-8 and IL-1b [51,52]. Though studies on neonatal cord blood demonstrated that TLR-induced cytokine responses were significantly different from adult TLR responses [48–55], we found that CBMCs had similar capacity to produce the early response cytokines IL-6 and IL-1b as that of PBMCs. Our data also confirms previous findings showing that infections with PGN and S. aureus elicited higher levels of IL-1b than LPS and E. coli in CBMCs. [28–33] Helen Karlson et al., showed that IL-6 production was not affected by combining different bacterial strains. We also found no significant difference in the levels of IL-6 in co-stimulated cells. In addition, neonatal immune cells were less potent in producing the pro-inflammatory cytokine IL-23 [35,36]. However, adult cells possess a greater capacity to produce IL-23 in cells co-stimulated with LPS and PGN than cells that were stimulated with individual pathogens. The immunomodulatory cytokines IL-10 and IL-13 were found to be highly upregulated by LPS when compared with PGN in CBMCs which is consistent with previous reports [34,37–39,47]. Furthermore, previous studies suggested that chemotaxis is deficient in neonates, particularly those that are delivered prematurely and this may contribute to their increased susceptibility to infection [40–45]. Our data shows that the powerful chemoattractants such as IL-8 and MCP-1 for neutrophil and monocyte respectively were differentially regulated by all the tested stimulants in CBMCs indicating that neonatal immune cells were capable of initiating the inflammatory response but were deficit in the recruitment of monocytes. Using the Human Inflammation Antibody array technique we have found that PGN elicited a lower response than LPS in both CBMCs and PBMCs which is consistent with previous reports proving that Gram negative infections show a high pro-inflammatory response than Gram positive infections [28–32]. Furthermore, studies on cord blood cells have suggested that these cells have been found to possess a greater capacity to produce IL-6 when stimulated with LPS than adult cells. We also found an enhanced expression of IL-6 in LPS stimulated CBMCs than PBMCs [49]. Co-infection or polymicrobial infection is known to impair or alter the host immune mechanisms [60]. Our data on PBMCs showed an enhanced effect of co-stimulated cells to produce multiple cytokines simultaneously than cells that were stimulated with individual ligands. Previous reports on polymicrobial sepsis suggested that there is impairment in leukocyte migration [61], defect in PMN function [62] and altered cytokine and chemokine expression [63]. In addition, in a neonatal rat model of co-infection, there was
an increase in the rate of mortality and morbidity when compared to single infections [57]. Thus, the overproduction of cytokines and chemokines leads to tissue damage and multi-organ failure and eventually death in the case of polymicrobial sepsis. Kollmann et al., demonstrated that neonatal immune cells were less polyfunctional in response to TLR stimulation compared to adult cells [49]. We also observed an impairment of multiple cytokine production by CBMCs in response to co-stimulation with LPS and PGN. In addition, our data supports the notion that cord blood cells have a decreased ability to produce the chemokine RANTES than adult cells [46]. In summary, neonatal immune cells responded differently to different pathogens. Though neonatal mononuclear cells were able to mount a powerful inflammatory response through TLR2 and TLR4 activation in response to different pathogens, they were less capable of producing multiple cytokines upon co-stimulation showing the functional immaturity of the neonatal mononuclear cells. Acknowledgements This study is supported by the grant from Department of Biotechnology (DBT), Government of India (No. BT/PR12666/BRB/ 10/723/2009). The authors are thankful to SRM University for their support. The authors also thank Dr. M.R. Ganesh, ISISM, for his guidance in flow cytometry. Authors are also thankful to staffs of SRM hospital for assistance in sample collection. References [1] Kenzel S, Henneke L. The innate immune system and its relevance to neonatal sepsis. Curr Opin Infect Dis 2006;19(3):264–70. [2] Philbin Victoria Jane, Levy Ofer. Development of the innate immune response: implications for neonatal and infant vaccine development. Pediatr Res 2009;65(5 Pt 2):98R–105R. [3] Mohan Pammi Venkatesh, Don Pham, Mindy Fein, Lingkun Kong, Weisman Leonard E. Neonatal coinfection model of coagulase-negative Staphylococcus (Staphylococcus epidermidis) and Candida albicans: fluconazole prophylaxis enhances survival and growth. Antimicrob Agents Chemother 2007;51(4):1240. [4] Stoll BJ, Hansen N, Fanaroff AA. Late onset sepsis in very low-birth weight neonates: the experience of the national institute of child-health and human development neonatal research network. Pediatrics 2002;110(2 Pt 1):285–91. [5] Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004;4:499–511. [6] Sadeghi Kambis, Berger Angelika, Langgartner Michaela, Prusa AndreaRomana, Hayde Michael, Herkner Kurt, et al. Immaturity of infection control in preterm and term newborns is associated with impaired toll-like receptor signaling. J Infect Dis 2007;195(2):296–302. [7] Stoll BJ, Gordon T, Korones SB, Shankaran S, Tyson JE, Bauer CR, et al. Earlyonset sepsis in very low birth weight neonates: a report from the national institute of child health and human development neonatal research network. J Pediatr 1996;129(1):72–80. [8] Stoll BJ, Gordon T, Korones SB, Shankaran S, Tyson JE, Bauer CR, et al. Late-onset sepsis in very low birth weight neonates: a report from the national institute of child health and human development neonatal research network. J Pediatr 1996;129(1):63–71. [9] Moore MR, Schrag SJ, Schuchat A. Effects of intrapartum antimicrobial prophylaxis for prevention of group-B-streptococcal disease on the incidence and ecology of early-onset neonatal sepsis. Lancet Infect Dis 2003;3(4):201–13. [10] Carr R, Modi N, Dore C. G-CSF and GM-CSF for treating or preventing neonatal infections. Cochrane Database Syst Rev 2003(3):CD003066. [11] Mohan P, Brocklehurst P. Granulocyte transfusions for neonates with confirmed or suspected sepsis and neutropaenia. Cochrane Database Syst Rev 2011;5(10):CD003956. [12] Ohlsson A, Lacy JB. Intravenous immunoglobulin for suspected or subsequently proven infection in neonates. Cochrane Database Syst Rev. 2010; 17 (3): CD001239. [13] Suri M, Harrison L, Van de Ven C, Cairo MS. Immunotherapy in the prophylaxis and treatment of neonatal sepsis. Curr Opin Pediatr 2003;15(2):155–60. [14] Skrzeczyñska J, Kobylarz K, Hartwich Z, Zembala M, Pryjma J. CD14+CD16+ monocytes in the course of sepsis in neonates and small children: monitoring and functional studies. Scand J Immunol 2002;55(6):629–38. [15] Ziegler-Heitbrock L. The CD14+CD16+ blood monocytes: their role in infection and inflammation. J Leukoc Biol 2007;81(3):584–92.
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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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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 <0.05 were considered statistically significant.
2.3. Activation of mononuclear cells with respective ligands
3. Results
The mononuclear cells were plated at a density of 106/ml and stimulated with 100 ng/ml each of LPS from E. coli (Invivogen, SanDiego) and PGN from S. aureus (Invivogen, SanDiego) and 104 CFU/ml of heat-inactivated bacteria for 24 h. The supernatant was collected and stored in 20 °C until further analysis.
3.1. Effect of LPS, PGN, E. coli, K. pneumoniae and S. aureus on CD14 and CD16 expression on human monocytes Freshly stained mononuclear cells showed two unique monocyte subpopulations. One population showed high expression of
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CD14 and CD16 (CD14+CD16+) while the other showed diminished CD14 and high CD16 expression (CD14dimCD16+). However, upon stimulation with LPS, PGN, E. coli, K. pneumoniae and S. aureus the CD14 + CD16 + populations differentiated more into CD14dimCD16 + populations as shown in Fig. 1.
the levels of TLR4 on both monocyte populations in PBMCs as shown in Fig. 3.
3.2. TLR2 and TLR4 expression on CD14+CD16+ and CD14dimCD16+ monocytes in CBMCs
During bacterial infection, there is an imbalance in the inflammatory network due to the combined effect of pro-inflammatory and anti-inflammatory cytokines and chemokines. Therefore, we determined the role of TLR2 and TLR4 in mediating the production of the key inflammatory cytokines and chemokines in neonatal bacterial infection. We measured the levels of the pro-inflammatory cytokines such as IL-6, IL-1b and IL-23, chemokines IL-8 and MCP-1 and anti-inflammatory cytokines IL-10 and IL-13 and found their levels to be highly upregulated in CBMCs and PBMCs compared to their respective control as shown in Fig. 4. The amounts of IL-8 were higher in CBMCs than PBMCs in stimulated cells whereas the levels of IL-23, and MCP-1 were less in CBMCs when compared to PBMCs in stimulated cells. There was no significant difference in the amounts of IL-6, IL-1b, IL-10 and IL-13 between CBMCs and PBMCs. Furthermore, co-stimulated cells produced
We determined the cell surface expression of TLR2 on monocyte subpopulations in response to LPS, PGN, E. coli, K. pneumoniae and S. aureus. As shown in Fig. 2, we found that in CBMCs, the expression of TLR2 was downregulated in all stimulated cells compared to unstimulated cells in both monocyte populations. There was no significant difference on both monocyte subpopulations when cells were stimulated with S. aureus whereas on PBMCs, TLR2 levels was upregulated in CD14+CD16+ and CD14dimCD16+ by LPS and PGN respectively. Although the expression of TLR4 was downregulated in all stimulated cells compared to unstimulated ones in CBMCs on both monocyte populations, LPS and E. coli significantly upregulated
3.3. TLR2 and TLR4 mediated production of inflammatory cytokines and chemokines in CBMCs
Fig. 1. Effect of LPS, PGN, E. coli, Klebsiella pneumoniae and Staphylococcus aureus on CD14 and CD16 expression on human monocytes. Mononuclear cells were stimulated with 100 ng/ml of LPS and PGN and 104 Cfu/ml of E. coli, Klebsiella pneumoniae (K.p) and Staphylococcus aureus (S.a) for 24 h. Cells were stained with CD14-APC and CD16PerCP for flow cytometry. Dot plots are gated on monocytes based on staining properties. Data shown are representative of six individual experiments.
Fig. 2. Expression of TLR2 on CD14+CD16+ and CD14dimCD16+ cells from CBMCs and PBMCs. Isolated mononuclear cells (106/ml) were incubated with LPS E. coli (100 ng/ml), PGN Staphylococcus aureus (100 ng/ml) and 104 CFU/ml of heat inactivated whole bacteria (E. coli, Klebsiella pneumoniae (K.p), Staphylococcus aureus (S.a)) for 24 h. The Mean Fluorescence Intensity (MFI) of TLR2 were determined by flow cytometry. Results were expressed as Mean ± SD as relative values with respect to the control. p Values of < 0.05 were considered statistically significant.
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Fig. 3. Expression of TLR4 on CD14+CD16+ and CD14dimCD16+ cells from CBMCs and PBMCs. Isolated mononuclear cells (106/ml) were incubated with LPS E. coli (100 ng/ml), PGN Staphylococcus aureus (100 ng/ml) and 104 CFU/ml of heat inactivated whole bacteria (E. coli, Klebsiella pneumoniae (K.p), Staphylococcus aureus (S.a)) for 24 h. The Mean Fluorescence Intensity of TLR2 were determined by flow cytometry. Results were expressed as Mean ± SD as relative values with respect to the control. p Values of <0.05 were considered statistically significant.
Fig. 4. Measurement of various pro-inflammatory and anti-inflammatory cytokines and chemokines. 106 mononuclear cells were stimulated with LPS E. coli (100 ng/ml), PGN Staphylococcus aureus (100 ng/ml) and 104 CFU/ml of heat inactivated whole bacteria (E. coli, Klebsiella pneumoniae, Staphylococcus aureus) for 24 h. The supernatant was collected and measured for cytokines IL-6 (A), IL-1b (B), IL-10 (C), IL-13 (D), IL-8 (E), MCP-1 (F) and IL-23 (G) using ELISA. Bars are representative of mean values of 6 different samples from different donors and the error bars represent the Standard deviation. p Values of <0.05 are considered statistically significant. K.p = Klebsiella pneumoniae, S.a = Staphylococcus aureus, ns = non significant.
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Fig. 5. Representative figure showing the expression of inflammatory proteins in (A) CBMCs and (B) PBMCs using Human Inflammatory Antibody Array technique when stimulated with 100 ng/ml of Gram positive (PGN) and Gram negative ligands (LPS) and co-stimulation (LPS + PGN) and compared with unstimulated cells (Control). Multiple cytokines such as GM-CSF, MIP-1a, MIP-1b, IL-10, TNF-a and RANTES along with IL-6, IL-8 and MCP-1 were expressed in co-stimulated cells in PBMCs than CBMCs.
higher amounts of IL-23 and IL-13 than the cells that were stimulated with individual organisms.
MIP-1b, IL-10, TNF-a and RANTES, IL-6, IL-8 and MCP-1 were also detected in PBMCs upon co-stimulation with LPS and PGN. 4. Discussion
3.4. TLR2 and TLR4 mediated expression of inflammatory proteins in CBMCs In order to find out the expression of the key inflammatory proteins mediated by TLR2 and TLR4 activation in CBMCs, we employed the Human Inflammatory Antibody Array technique that can detect up to 40 different inflammatory proteins in a single sample. As shown in Fig. 5, the expression of IL-6, IL-8 and MCP1 was much higher in co-stimulated cells when compared to the expression in cells stimulated with individual ligands in cord blood. Interestingly, multiple cytokines such as GM-CSF, MIP-1a,
Pattern Recognition Receptors (PRRs) such as TLRs play an important role in recognition and responding to invading pathogens. Here in this study, we demonstrated the differential cell surface expression of TLR2 and TLR4 on CD14+CD16+ and CD14dimCD16+ cells in response to common neonatal pathogens such as E. coli, K. pneumoniae and S. aureus and their respective ligands (LPS and PGN) in neonatal immune cells. Accumulating evidence on neonatal cord blood suggested that neonatal responses to TLR agonists such as LPS and PGN are impaired at birth due to a defective TLR-MyD88-IRAK4 pathway thereby contributing to
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more susceptibility to bacterial infection [6,21–26]. Recently, Pedraza et al., reported that a reduced CD14+CD16+ activated/differentiated monocyte subset and a correspondingly lower level of functional TLR4 on monocytes contributes to the relatively lower response to LPS in neonates compared to their response in adults [27]. It has been shown previously that CD14+CD16+ cells differentiate more into CD14dimCD16+ cells and these cells were the major producers of pro-inflammatory cytokines [14,15]. In accordance with these reports, we also found that CD14dimCD16+ cells were increased upon stimulation with LPS, PGN, E. coli, K. pneumoniae and S. aureus. Furthermore, TLR2 and TLR4 were differentially expressed on both monocyte populations. We also observed that the levels of TLR4 expression varied significantly when stimulated with K. pneumoniae and E. coli. The observed difference of TLR4 expression may be due to the difference in PAMPs between the two organisms as K. pneumoniae recognition by TLRs takes place both due to LPS and CPS (Capsular polysaccharide). Also, TLR4 has been shown to play a crucial role in the early response to K. pneumoniae infections rather than in the late phase [58,59]. The activation of TLR2 and TLR4 by bacterial ligands initiate signaling cascades that result in the production of a range of pro-inflammatory cytokines and chemokines such as IL-6, TNF-a, IL-8 and IL-1b [51,52]. Though studies on neonatal cord blood demonstrated that TLR-induced cytokine responses were significantly different from adult TLR responses [48–55], we found that CBMCs had similar capacity to produce the early response cytokines IL-6 and IL-1b as that of PBMCs. Our data also confirms previous findings showing that infections with PGN and S. aureus elicited higher levels of IL-1b than LPS and E. coli in CBMCs. [28–33] Helen Karlson et al., showed that IL-6 production was not affected by combining different bacterial strains. We also found no significant difference in the levels of IL-6 in co-stimulated cells. In addition, neonatal immune cells were less potent in producing the pro-inflammatory cytokine IL-23 [35,36]. However, adult cells possess a greater capacity to produce IL-23 in cells co-stimulated with LPS and PGN than cells that were stimulated with individual pathogens. The immunomodulatory cytokines IL-10 and IL-13 were found to be highly upregulated by LPS when compared with PGN in CBMCs which is consistent with previous reports [34,37–39,47]. Furthermore, previous studies suggested that chemotaxis is deficient in neonates, particularly those that are delivered prematurely and this may contribute to their increased susceptibility to infection [40–45]. Our data shows that the powerful chemoattractants such as IL-8 and MCP-1 for neutrophil and monocyte respectively were differentially regulated by all the tested stimulants in CBMCs indicating that neonatal immune cells were capable of initiating the inflammatory response but were deficit in the recruitment of monocytes. Using the Human Inflammation Antibody array technique we have found that PGN elicited a lower response than LPS in both CBMCs and PBMCs which is consistent with previous reports proving that Gram negative infections show a high pro-inflammatory response than Gram positive infections [28–32]. Furthermore, studies on cord blood cells have suggested that these cells have been found to possess a greater capacity to produce IL-6 when stimulated with LPS than adult cells. We also found an enhanced expression of IL-6 in LPS stimulated CBMCs than PBMCs [49]. Co-infection or polymicrobial infection is known to impair or alter the host immune mechanisms [60]. Our data on PBMCs showed an enhanced effect of co-stimulated cells to produce multiple cytokines simultaneously than cells that were stimulated with individual ligands. Previous reports on polymicrobial sepsis suggested that there is impairment in leukocyte migration [61], defect in PMN function [62] and altered cytokine and chemokine expression [63]. In addition, in a neonatal rat model of co-infection, there was
an increase in the rate of mortality and morbidity when compared to single infections [57]. Thus, the overproduction of cytokines and chemokines leads to tissue damage and multi-organ failure and eventually death in the case of polymicrobial sepsis. Kollmann et al., demonstrated that neonatal immune cells were less polyfunctional in response to TLR stimulation compared to adult cells [49]. We also observed an impairment of multiple cytokine production by CBMCs in response to co-stimulation with LPS and PGN. In addition, our data supports the notion that cord blood cells have a decreased ability to produce the chemokine RANTES than adult cells [46]. In summary, neonatal immune cells responded differently to different pathogens. Though neonatal mononuclear cells were able to mount a powerful inflammatory response through TLR2 and TLR4 activation in response to different pathogens, they were less capable of producing multiple cytokines upon co-stimulation showing the functional immaturity of the neonatal mononuclear cells. Acknowledgements This study is supported by the grant from Department of Biotechnology (DBT), Government of India (No. BT/PR12666/BRB/ 10/723/2009). The authors are thankful to SRM University for their support. The authors also thank Dr. M.R. Ganesh, ISISM, for his guidance in flow cytometry. Authors are also thankful to staffs of SRM hospital for assistance in sample collection. References [1] Kenzel S, Henneke L. The innate immune system and its relevance to neonatal sepsis. Curr Opin Infect Dis 2006;19(3):264–70. [2] Philbin Victoria Jane, Levy Ofer. Development of the innate immune response: implications for neonatal and infant vaccine development. Pediatr Res 2009;65(5 Pt 2):98R–105R. [3] Mohan Pammi Venkatesh, Don Pham, Mindy Fein, Lingkun Kong, Weisman Leonard E. Neonatal coinfection model of coagulase-negative Staphylococcus (Staphylococcus epidermidis) and Candida albicans: fluconazole prophylaxis enhances survival and growth. Antimicrob Agents Chemother 2007;51(4):1240. [4] Stoll BJ, Hansen N, Fanaroff AA. Late onset sepsis in very low-birth weight neonates: the experience of the national institute of child-health and human development neonatal research network. Pediatrics 2002;110(2 Pt 1):285–91. [5] Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004;4:499–511. [6] Sadeghi Kambis, Berger Angelika, Langgartner Michaela, Prusa AndreaRomana, Hayde Michael, Herkner Kurt, et al. Immaturity of infection control in preterm and term newborns is associated with impaired toll-like receptor signaling. J Infect Dis 2007;195(2):296–302. [7] Stoll BJ, Gordon T, Korones SB, Shankaran S, Tyson JE, Bauer CR, et al. Earlyonset sepsis in very low birth weight neonates: a report from the national institute of child health and human development neonatal research network. J Pediatr 1996;129(1):72–80. [8] Stoll BJ, Gordon T, Korones SB, Shankaran S, Tyson JE, Bauer CR, et al. Late-onset sepsis in very low birth weight neonates: a report from the national institute of child health and human development neonatal research network. J Pediatr 1996;129(1):63–71. [9] Moore MR, Schrag SJ, Schuchat A. Effects of intrapartum antimicrobial prophylaxis for prevention of group-B-streptococcal disease on the incidence and ecology of early-onset neonatal sepsis. Lancet Infect Dis 2003;3(4):201–13. [10] Carr R, Modi N, Dore C. G-CSF and GM-CSF for treating or preventing neonatal infections. Cochrane Database Syst Rev 2003(3):CD003066. [11] Mohan P, Brocklehurst P. Granulocyte transfusions for neonates with confirmed or suspected sepsis and neutropaenia. Cochrane Database Syst Rev 2011;5(10):CD003956. [12] Ohlsson A, Lacy JB. Intravenous immunoglobulin for suspected or subsequently proven infection in neonates. Cochrane Database Syst Rev. 2010; 17 (3): CD001239. [13] Suri M, Harrison L, Van de Ven C, Cairo MS. Immunotherapy in the prophylaxis and treatment of neonatal sepsis. Curr Opin Pediatr 2003;15(2):155–60. [14] Skrzeczyñska J, Kobylarz K, Hartwich Z, Zembala M, Pryjma J. CD14+CD16+ monocytes in the course of sepsis in neonates and small children: monitoring and functional studies. Scand J Immunol 2002;55(6):629–38. [15] Ziegler-Heitbrock L. The CD14+CD16+ blood monocytes: their role in infection and inflammation. J Leukoc Biol 2007;81(3):584–92.
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