Neonates and infants, due to the immaturity in their adaptive immunity, are thought to depend largely on the innate immune system for protection against bacterial infection. However, the innate immunity-mediated antimicrobial response in neonates and infants is incompletely characterized. Here, we report that infant mice were more susceptible to microbial sepsis than adult mice, with significantly reduced bacterial clearance from the circulation and visceral organs. Infant PMNs exhibited less constitutive expression of the chemokine receptor CXCR2, and bacterial infection caused further reduction of PMN CXCR2 in infant mice compared with adult mice. This correlates with diminished in vitro chemotaxis of infant PMNs toward the chemoattractant CXCL2 and impaired in vivo recruitment of infant PMNs into the infectious site. Furthermore, consistent with the reduced antimicrobial response in vivo, infant macrophages displayed an impaired bactericidal activity with a defect in phagosome maturation after ingestion of either gram-positive or gram-negative bacteria. Thus, infant mice exhibit an increased vulnerability to microbial infection with delayed bacterial clearance, which is associated with the inefficiency in their innate phagocyte-associated antimicrobial functions characterized by defects in PMN recruitment and macrophage phagosome maturation during microbial sepsis.
Despite advances in medicine and the best available supportive care, death associated with neonatal and infant sepsis has remained largely unchanged over the last two decades and approximately four million children under the age of 6 months die from infections each year worldwide [1-4]. Mortality rates from microbial sepsis in premature-birth and very low-birth-weight infants continue to increase and the incidence could be as high as 50% [5, 6]. Even in infants born in term, the inefficient response of their immune system to a variety of pathogens not only pre-disposes but makes them more vulnerable to microbial infection [4, 7]. Furthermore, neonates and infants who survive severe sepsis may suffer from developmental and growth impairment, which undoubtedly leads to long-term social and economic consequences [8-10].
Neonates and infants are generally more susceptible to a wider range of microbial infection than adults and are especially vulnerable to intracellular pathogen-associated infection [11-13]. This increased susceptibility to microbial infection has been attributed to deficiencies in both innate and adaptive immunity, in particular to the immature state of the adaptive immune system in neonates and infants compared with adults [4, 14, 15]. Neonates have limited exposure to antigens in utero, thus leaving them with the immaturity in adaptive immunity during infancy [4, 13]. Mounting evidence has shown several deficiencies of adaptive immunity in neonates and infants for both cell- and antibody-mediated responses. For example, in addition to low numbers of effector-memory T cells (CD45RA−CD45RO+) and memory-effector B cells (CD27+), large numbers of both recent thymic emigrants of T cells recently produced by the thymus and transitional B cells produced recently in the BM are present in the circulation during infancy [4, 16, 17]. These recent thymic emigrants exhibit a defect in their acquisition of the Th1 function, whereas transitional B cells are less functionally effective than mature naive B cells. Thus, the predominance of both transitional T cells and B cells may contribute to the vulnerability of neonates and infants to infection with intracellular pathogens. Furthermore, the adaptive immune system of neonates and infants is also characterized with defective NK cell activities, slow development of the CD4+ T-cell response, delayed, shortened, and reduced antibody responses, less efficient in the production of Th1-polarizing cytokines including type I IFN (or bias to the Th2-type response), and decreased MHC class II expression on APCs [13, 15, 18-20].
Due to the immature state of the adaptive immune system, neonates and infants are thought to rely more heavily on their innate immunity against microbial infection [4, 21]. Furthermore, a recent study revealed that survival from polymicrobial sepsis in murine neonates was neither dependent on an intact adaptive immune system nor affected by the T-cell–directed adaptive immune modulation , which highlights the increased importance of the innate immune response during microbial sepsis in neonates and infants. Both macrophages and PMNs, the professional phagocytes, are highly specialized innate effector cells and have evolved for the killing of microbial pathogens. The innate immunity-mediated antimicrobial response to bacterial infection is initiated by the receptor-associated recognition of invading pathogens, and subsequently, these invaded pathogens are engulfed by the professional phagocytes including macrophages and PMNs via phagocytic receptors and killed within the phagocyte through a process of phagosome/lysosome fusion, which is essential for host innate immunity to limit microbial infection [23-25]. However, the innate immunity-mediated antimicrobial response during microbial sepsis remains poorly defined in neonates and infants. Here, we show that infant PMNs, characterized with reduced expression of the chemokine receptor CXCR2, exhibit diminished in vitro chemotaxis and in vivo recruitment, whereas infant macrophages display impaired phagosome maturation and reduced killing of the ingested bacteria. These defects in innate phagocyte-associated antimicrobial functions render infant mice more susceptible to microbial sepsis.
Infant mice display an increased susceptibility to microbial infection
To explore whether infant mice are more susceptible to microbial infection than adult mice, we infected both infant and adult mice with live gram-positive Staphylococcus aureus (S. aureus) and monitored the survival rate for at least 14 days. In response to S. aureus challenge, adult mice had an overall survival of 72%, whereas infant mice showed a significantly reduced survival rate with 27% surviving to the end of the observation period (p = 0.0114 versus adult mice) (Fig. 1A). Blood samples were collected at different time points post S. aureus challenge from infant and adult mice for proinflammatory cytokine analysis. Although serum peak levels of TNF-α at 2 h and IL-6 at 6 h post S. aureus challenge were slightly lower in infant mice than those in adult mice, they did not reach statistical significances (Fig. 1B). Bacterial counts at 24 h post S. aureus challenge were significantly greater in the blood, liver, and spleen of infant mice compared with adult mice (p < 0.05) (Fig. 1C). At 48 h significantly higher bacterial counts were observed in the blood and all measured visceral organs of infant mice (p < 0.05 versus adult mice) (Fig. 1C).
Similar results were also observed in infant mice after being infected with live gram-negative Salmonella typhimurium (S. typhimurium), where a significantly higher mortality rate (p = 0.0062) (Fig. 1D) and substantial more bacterial counts in the blood and visceral organs (p < 0.05) (Fig. 1F) were evident in infant mice compared with adult mice, whereas serum TNF-α and IL-6 levels were comparable between infant and adult mice (Fig. 1E).
We further compared the antimicrobial response between infant and adult mice in a more clinically relevant model of polymicrobial sepsis induced by the cecal slurry method . Infant mice were more susceptible to polymicrobial sepsis with an overall mortality of 76% compared with a 42% mortality rate in adult mice (p = 0.0092) (Fig. 1G). There were no significant differences in the serum TNF-α and IL-6 levels post septic challenge between infant and adult mice (Fig. 1H); however, significantly higher bacterial counts were observed in the blood and visceral organs of infant mice at 12 and 24 h post polymicrobial infection (p < 0.05 versus adult mice) (Fig. 1I). These results indicate that, consistent with an enhanced mortality rate, infant mice exhibit impaired bacterial clearance in response to microbial infection.
Infant mice show impaired PMN influx and chemotaxis in response to bacterial challenge
PMN influx from the circulation into the infectious site during bacterial infection plays a key role in eradicating the invaded microbial pathogens . To ascertain the possible factors responsible for the delayed bacterial clearance observed in infant mice, we measured leukocyte populations in the peritoneal cavity of both infant and adult mice after being challenged with gram-positive or gram-negative bacteria. Infection of adult mice with either S. aureus (Fig. 2A) or S. typhimurium (Fig. 2B) resulted in markedly increased PMN accumulation in the peritoneal cavity at 12 and 24 h post septic challenge. By contrast, infant mice in response to bacterial infection recruited significantly fewer PMNs into the peritoneal cavity than adult mice (p < 0.05), albeit the population of peritoneal macrophages was identical between infant and adult mice (Fig. 2A and B).
To examine whether the reduced PMN recruitment observed in infant mice after septic challenge is due to a diminished number of circulating PMNs, we assessed systemic granulocytes and monocytes in infant and adult mice before and after bacterial infection. The percentage of granulocytes (Gr-1+CD11b+ cells) (Fig. 2C) and monocytes (F4/80+CD11b+ cells) (Fig. 2D) in the circulation of infant and adult mice increased substantially in response to either S. aureus or S. typhimurium challenge; however, there were no significant differences in circulating granulocytes and monocytes seen between infant and adult mice (Fig. 2C and D). We further assessed the percentage of monocytes (Gr-1+ CD11b+F4/80+ cells) and immature cells (Gr-1+CD11b+CD31+ cells) in the circulating granulocyte population. Both Gr-1+CD11b+F4/80+ cells (Fig. 2E) and Gr-1+CD11b+CD31+ cells (Fig. 2F) had slightly increases post septic challenge, but they were comparable between infant and adult mice (Fig. 2E and F).
The chemokine receptor CXCR2 is essential for the recruitment of PMNs, and reduced CXCR2 expression correlates closely with an inability of PMNs to migrate from the circulation into the infectious site during microbial sepsis [28, 29]. Therefore, we assessed surface expression of CXCR2 on circulating PMNs in infant and adult mice before and after bacterial infection. Circulating infant PMNs exhibited less constitutive expression of CXCR2 than circulating adult PMNs (p < 0.05) (Fig. 3A and B). S. aureus or S. typhimurium challenge downregulated CXCR2 expression on circulating adult PMNs, and caused further reduction of CXCR2 in circulating infant PMNs (p < 0.05 versus adult PMNs) (Fig. 3A and B). Consistent with the diminished CXCR2 expression, infant PMNs showed considerable less chemotaxis toward the chemoattractant CXCL2 than adult PMNs in the presence or absence of bacterial challenges (p < 0.05) (Fig. 3C).
G protein-coupled receptor kinase 2 (GRK2), a serine-threonine kinase, participates in phosphorylation and internalization of chemokine receptors and thus downregulates the expression of chemokine receptors including CXCR2 [30-32]. It is possible that infant PMNs may express more GRK2, which in turn leads to the downregulation of CXCR2. However, there were no significant differences in constitutive and bacteria-stimulated GRK2 expression found between infant and adult PMNs (Fig. 3D and E). To test this further, a GRK2 inhibitor (methyl 5-(2-(5-nitro-2-furyl)vinyl)-2-furoate) was used to attenuate the inhibitory effect of LPS, a TLR4 agonist and bacterial lipoprotein (BLP), a TLR2 agonist on PMN chemotaxis. However, the chemotaxis of infant PMNs toward CXCL2 was still significantly lower than that of adult PMNs after the blockage of GRK2 (p < 0.05) (Fig. 3F), indicating that GRK2 is not responsible for the reduced CXCR2 and chemotaxis in infant PMNs.
To further clarify the mechanism underlying the enhanced susceptibility to microbial infection and delayed bacterial clearance in infant mice, we measured the surface expression of two phagocytic receptors, complement receptor type 3 (CR3) and FcγIII/II receptor (FcγR) on macrophages from infant and adult mice. Significantly reduced constitutive expression of CR3, but not FcγR, was observed in infant macrophages (p < 0.05 versus adult macrophages) (Fig. 4A). Stimulation with LPS or BLP resulted in diminished upregulation of CR3 expression on infant macrophages compared with adult macrophages (p < 0.05) (Fig. 4A).
Although both constitutive and stimulated CR3 expression was reduced on infant macrophages, phagocytosis of either S. aureus or S. typhimurium by infant and adult macrophages was comparable (Fig. 4B). However, intracellular killing of the ingested live S. aureus and S. typhimurium by infant macrophages was markedly reduced compared with adult macrophages (p < 0.05) (Fig. 4C). Thus, infant macrophages display an impaired bactericidal activity after ingestion of gram-positive and gram-negative bacteria.
Phagosome maturation of professional phagocytes after ingestion of microbial bacteria is characterized by phagosomal acidification and phagosome/lysosome fusion [23, 25]. A significantly delayed and reduced phagosomal acidification after ingestion of S. aureus was observed in infant macrophages compared with adult macrophages (p < 0.05) (Fig. 5A). A similar defect in phagosomal acidification was also found in infant macrophages after ingestion of S. typhimurium (p < 0.05 versus adult macrophages) (Fig. 5B).
We subsequently loaded peritoneal macrophages with LysoTracker red that selectively labels late endosomes/lysosomes and monitored the maturation of phagosomes that have ingested S. aureus--FITC by examining their ability to colocalize with LysoTraker red over time. Almost all the ingested S. aureus-FITC were colocalized with LysoTraker red in the adult macrophage at 60 min after macrophages were chased with S. aureus-FITC, whereas most S. aureus-FITC ingested by the infant macrophage at this time point did not colocalize with LysoTraker red (Fig. 5C). A substantially reduced colocalization of Escherichia coli-FITC with LysoTraker red was also found in the infant macrophage compared with the adult macrophage (Fig. 5D). These results indicate that, in contrast to adult macrophages, infant macrophages show a defect in phagosome maturation after ingestion of microbial bacteria.
Neonates and infants are featured with the immature state in both innate and adaptive immunity, which places them at risk for the development of severe sepsis in response to bacterial infection [4, 13-15]. In the present study, we demonstrated that infant mice were more susceptible to microbial sepsis. When infected with live bacteria or challenged with a clinically relevant, cecal slurry-induced polymicrobial sepsis, infant mice displayed a significantly higher mortality rate than adult mice. As one of the fundamental functions of the host innate immunity during microbial infection is to rapidly eradicate the invaded pathogens from the body , we further examined bacterial clearance in infant mice after septic challenges. Consistent with an increased susceptibility to microbial sepsis, infant mice showed delayed and reduced bacterial clearance from the circulation and visceral organs post septic challenges, with significantly higher bacterial counts in the blood, liver, spleen, and lungs compared with adult mice. This defect in bacterial clearance by infant mice is likely to have been underestimated when considering the total amount of bacteria or cecal contents injected between infant and adult mice. Infant mice in response to microbial infection; however, produced comparable proinflammatory cytokines to those of adult mice, which is somewhat discordant with studies in both murine and human neonates [26, 34-36] where significantly reduced inflammatory cytokines were observed in neonates compared with adults. This discordance might be due to a more matured ability of immune cells to produce inflammatory cytokines in infants compared with neonates. Indeed, other studies have revealed that stimulus-induced production of several inflammatory cytokines by neonatal monocytes and APCs is equal to or even exceeds that of adults [37, 38]. These results indicate that, despite an appropriate proinflammatory cytokine production in response to microbial infection in infant mice, the antimicrobial response of their host innate immunity is defective and thus less efficient.
Innate phagocytes including PMNs and macrophages form the first line in the host defense against microbial infection. However, in contrast to the well-described deficiencies in adaptive immunity, the innate immune response and in particular the innate phagocyte-associated antimicrobial function in neonates and infants during microbial sepsis remains poorly defined. PMN influx from the circulation into the infectious site plays a key role in eradicating the invaded microbial pathogens  and successful clearance of bacterial infection has been shown to rely on a rapid and efficient PMN migration into the infectious site such as peritoneal cavity in several experimentally established murine polymicrobial sepsis models [39-41]. Therefore, a defective and/or reduced recruitment of PMNs into the infectious site may account, at least in part, for the impaired bacterial clearance and increased susceptibility to microbial sepsis observed in infant mice. In the present study, we found that in response to bacterial challenge substantial amounts of PMNs were rapidly recruited into the peritoneal cavity in adult mice; however, infant mice recruited significantly fewer PMNs into the peritoneal cavity. This reduced PMN influx after septic challenge was not due to a diminished systemic PMN population in infant mice, as both infant and adult mice showed comparable increases in circulating granulocytes and monocytes in response to bacterial challenge. It has been demonstrated that PMN recruitment depends strongly on the chemokine receptor CXCR2, and reduced CXCR2 expression on circulating PMNs is associated with an inability of PMNs to migrate into the infectious site during microbial sepsis [28, 29]. We demonstrated that circulating PMNs from infant mice expressed less constitutive CXCR2, and bacterial infection caused further reduction of CXCR2 on PMNs in infant mice compared with adult mice. As a result, infant PMNs exhibited defective in vitro chemotaxis toward the chemoattractant CXCL2. However, we found that the reduced CXCR2 and impaired chemotaxis characterized in infant PMNs was not due to the overexpression of GRK2, a serine-threonine kinase that causes downregulation of CXCR2 [30-32] as constitutive and bacteria-stimulated expression of GRK2 was identical between infant and adult PMNs. Thus, in response to bacterial challenge infant PMNs display impaired in vitro chemotaxis and in vivo migration, which is associated with a substantial reduction in their CXCR2 expression. These findings are consistent with previous reports of other PMN deficiencies in neonates and infants including reduced reactive oxygen species production and impaired neutrophil extracellular trap formation [22, 42].
Engulfment of the invaded microbial pathogens by the innate phagocytes and subsequent phagosome maturation are critical events in phagocyte-associated antimicrobial functions of the host innate immune system in response to bacterial infection [23, 24]. To further clarify the underlying mechanisms that might be responsible for the inability to clear bacteria observed in infant mice after septic challenges, we assessed phagocytic receptor expression, bacterial phagocytosis, and intracellular bacterial killing in macrophages from infant mice and compared them with adult macrophages. We observed significantly reduced constitutive and LPS- or BLP-stimulated expression of CR3 on infant macrophages. Both phagocytic receptors CR3 and FcγR contribute to the phagocyte-associated uptake, ingestion, and killing of the invaded bacteria [43, 44]. As a result, any defects in CR3 and/or FcγR may cause a downregulated antimicrobial response, whereas overexpression of these receptors leads to the enhanced bacterial clearance in a murine generalized peritonitis model . When exposed to either gram-positive or gram-negative bacteria however, bacterial phagocytosis by infant and adult macrophages was comparable, whereas intracellular bacterial killing by infant macrophages was significantly reduced compared with adult macrophages. With no obvious reduction in bacterial phagocytosis but a defective killing of the ingested bacteria, we propose that the impaired and/or interrupted processing of the ingested microbial pathogens by infant macrophages, such as a defect in phagosome formation and maturation may offer a plausible explanation for our observed results.
Phagosome maturation of the professional phagocytes after ingestion of microbial pathogens, characterized by phagosomal acidification and phagosome/lysosome fusion, is a critical step in the killing and degradation of the internalized pathogens and thus plays a key role in innate immunity against microbial infection [23-25]. We first measured phasosomal pH in infant macrophages and observed a substantially delayed and reduced phagosomal acidification in infant macrophages compared with adult macrophages after ingestion of either S. aureus or S. typhimurium. Consistent with the defective phagosomal acidification, infant macrophages also exhibited severely impaired phagolysosome fusion in response to both gram-positive and gram-negative bacterial challenges, as revealed by the impaired colocalization of either S. aureus-FITC or E. coli-FITC with LysoTraker red-labeled lysosomes in infant macrophages compared with adult macrophages. These data indicate that infant macrophages exhibit a defect in phagosome maturation into the late lysosomal stage.
Collectively, our results reveal the deficiency of infant mice in their innate phagocyte-associated antimicrobial functions in response to bacterial infection, which is characterized by diminished PMN in vitro chemotaxis and in vivo recruitment into the infections site, and impaired macrophage phagosome maturation and bactericidal activity. These defective innate immunity-mediated antimicrobial responses render infant mice more susceptible to microbial sepsis.
Materials and methods
Mice and bacterial infection
Two- and eight-week-old infant and adult C57BL/6 mice were purchased from Harlan (Oxon, U.K.) and maintained in the University Biological Services Unit, University College Cork / National University of Ireland. Mice were housed in barrier cages under controlled environmental conditions (12/12 h of light/dark cycle, 55% ± 5% humidity, 23°C) and had free access to standard laboratory chow and water. Animals were fasted 12 h before experiments and allowed water ad libitum. All animal procedures were carried out in the University Biological Services Unit under a license from the Department of Health (Republic of Ireland). All animal studies were conducted with ethical approval granted from the University College Cork Ethics Committee.
Gram-positive S. aureus and gram-negative S. typhimurium were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and the National University of Ireland Culture Collection, respectively. Bacteria were cultured at 37°C in trypticase soy broth (Merck, Darmstadt, Germany), harvested at the mid-logarithmic growth phase, washed twice, and resuspended in PBS for in vitro and in vivo use. The concentration of resuspended bacteria was determined and adjusted spectrophotometrically at 550 nm.
Both infant and adult mice received an intraperitoneal injection of live S. aureus (1.25 × 106 CFU/g body weight) or S. typhimurium (2.5 × 105 CFU/g body weight). Infant and adult mice were also subjected to polymicrobial infection induced by the cecal slurry method, as described previously . Briefly, cecal contents of adult C57BL/6 mice were suspended in 5% dextrose solution (Sigma-Aldrich, St. Louise, MO, USA) with a final concentration of 80 mg/mL. The cecal slurry was briefly vortexed before injection to create a homogenous suspension and was used within 2 h of preparation. Infant and adult mice received an intraperitoneal injection of the cecal content suspension (1.25 mg/g body weight). Survival was monitored for at least 14 days.
Serum cytokine measurement
Infant and adult mice were infected with live bacteria or underwent polymicrobial sepsis induced by the cecal slurry method. Blood samples were collected via intracardiac puncture at different time points post septic challenges. Serum TNF-α and IL-6 were assessed by cytometric bead array (BD Biosciences, San Jose, CA, USA).
Enumeration of bacteria in the blood and visceral organs
Bacterial counts were determined as described previously [45, 46]. Briefly, infant and adult mice were culled at 12, 24, and 48 h post septic challenges. Blood samples were obtained by intracardiac puncture, and the dissected liver, spleen, and lung were homogenized in sterile PBS. Serial 10-fold dilutions of heparinized blood and organ homogenates in sterile water containing 0.5% Triton X-100 (Sigma-Aldrich) were plated on trypticase soy agar (Merck) or brain heart infusion agar (BD Biosciences), and incubated for 24 h at 37°C for determination of bacterial CFU.
FACScan analysis of immunofluorescence
Heparinized blood and peritoneal lavage were collected from infant and adult mice before and after bacterial infection, and dual-stained with anti-Ly-6G (BD PharMingen, San Diego, CA, USA), anti-F4/80 (Serotec, Oxford, UK), anti-CR3 (BD PharMingen), anti-FcγR (BD PharMingen), and anti-CXCR2 (R&D Systems, Minneapolis, MN, USA) mAbs conjugated with PE or FITC. Erythrocytes were lysed using lysis buffer (BD Biosciences). FACScan analysis was performed from at least 10 000 events for detecting the surface expression of CR3, FcγR, and CXCR2 on macrophages (F4/80-positive cells) and PMNs (Ly-6G-positive cells), respectively, using CellQuest software (BD Biosciences). Intracellular GRK2 expression in PMNs was assessed by FACScan analysis after incubation with anti-GRK2 primary mAb (Abcam, Cambridge, MA, USA), followed by dual staining with FITC-conjugated secondary mAb (Abcam) and PE-conjugated anti-Ly-6G mAb (BD PharMingen).
Heparinized blood samples were collected from infant and adult mice before and after bacterial infection, and dual- or triple-stained with anti-Gr-1 (BD PharMingen), anti-CD11b (eBioscience, San Diego, CA, USA), anti-F4/80 (eBioscience), and anti-CD31 (BD PharMingen) mAbs conjugated with PerCp5.5, PE, allophycocyanin, or PECy7. FACScan analysis was performed for detection of circulating granulocytes (Gr-1+CD11b+ cells), circulating monocytes (F4/80+CD11b+ cells), and the monocytes (Gr-1+CD11b+F4/80+ cells) and immature cells (Gr-1+CD11b+CD31+ cells) in the circulating Gr-1+CD11b+ population.
PMN influx and chemotaxis
Peritoneal exudate cells collected from infant and adult mice before and after septic challenges were analyzed by FACScan analysis for PMN (Ly-6G-positive cells) and macrophage (F4/80-positive cells) subpopulations .
PMN chemotaxis was assessed as described previously [40, 47]. Briefly, PMNs were isolated from the BM of infant and adult mice. Isolated PMNs were incubated for 1 h with heat-killed S. aureus (1 × 106 CFU/mL), heat-killed S. typhimurium (1 × 106 CFU/mL), LPS (100 ng/mL), or BLP (100 ng/mL) in the presence or absence of a GRK2 inhibitor, methyl 5-(2-(5-nitro-2-furyl)vinyl)-2-furoate (150 μM) (Calbiochem, Billerica, MA, USA), plated onto 48-well chemotaxis plates (NeuroProbe, Gaithersburg, MD, USA), and allowed to migrate toward CXCL2 (30 ng/mL) (R&D Systems) or culture medium for 1 h.
Phagocytosis and intracellular killing of bacteria
Phagocytosis and intracellular killing of S. aureus or S. typhimurium by macrophages were determined, as described previously [45, 48]. Briefly, S. aureus and S. typhimurium were heat-killed at 95°C for 20 min and labeled with 0.1% FITC (Sigma-Aldrich). Peritoneal macrophages isolated from infant and adult mice were incubated with heat-killed, FITC-labeled S. aureus or S. typhimurium (macrophage/bacteria = 1:20) at 37°C for different time periods. Bacterial phagocytosis by macrophages was assessed by FACScan analysis after the external fluorescence of the bound, but noningested, bacteria was quenched with 0.025% crystal violet (Sigma-Aldrich). Intracellular bacterial killing was determined by incubation of macrophages with live S. aureus or S. typhimurium (macrophage/bacteria = 1:20) at 37°C for 60 min in the presence or absence of cytochalasin B (5 μg/mL) (Sigma-Aldrich). After macrophages were lysed, total and extracellular bacterial killing were determined by incubation of serial 10-fold dilutions of the lysates on tryptone soy agar (Merck) plates at 37°C for 24 h. Intracellular bacterial killing was calculated according to the total and extracellular bacterial killing.
Measurement of phagosomal pH
Phagosome luminal pH was assessed, as described previously [46, 49, 50]. Briefly, heat-killed S. aureus and S. typhimurium were doubly labeled with 5 μg/mL carboxyfluorescein-SE (a pH-sensitive fluorescent probe) (Molecular Probes, Eugene, OR, USA) and 10 μg/mL carboxytetramethylrhodamine-SE (a pH-insensitive fluorescent probe) (Molecular Probes). Isolated peritoneal macrophages were pulsed with the labeled bacteria (macrophage/bacteria = 1:20) for 20 min and then chased at 37°C for the indicated time periods. Macrophage-based MFI of fluorescein on FL1 and rhodamine on FL2 were simultaneously analyzed by an FACScan flow cytometer (BD Bioscience). Phagosomal pH was calculated according to the ratio of fluorescein/rhodamine fluorescence using a calibration curve.
Phagosome/lysosome fusion assay
Phagolysosome fusion was determined, as described previously . Briefly, peritoneal macrophages were harvested and plated into eight-well chamber slides (Lab-Tek™, Nunc, Rochester, NY, USA) at 1 × 105 cells/well. After resting in RPMI1640 containing 1% FCS for 6 h, cells were loaded with 50 nM LysoTracker red (Molecular Probes) at 37°C for 30 min and further incubated with FITC-conjugated bacteria (Molecular Probes) of either S. aureus or E. coli (macrophage/bacteria = 1:20) for various time periods. LysoTracker red was replenished every hour of incubation. After each time point, slides were vigorously washed five times in cold PBS and fixed in 2% paraformaldehyde (Sigma-Aldrich). Cell nuclei were stained with DAPI (Molecular Probes). Slides were mounted with coverslips and examined under a fluorescent Olympus BX61-TRF microscope (Olympus, Tokyo, Japan). Fluorescent images were acquired using the cell imaging software for life sciences microscopy (Olympus Soft Imaging Solutions, Munster, Germany). Unfused phagosomes containing FITC-bacteria and lysosomes labeled with LysoTracker red were stained in green and red, respectively, whereas phagosomes containing FITC-bacteria after being fused with LysoTracker red-labeled lysosomes were stained in yellow due to the coexistence of the two fluorochromes.
All data are expressed as the mean ± SD. Statistical analysis was performed using the log rank test for survival and the Mann-Whitney U test for all others, with GraphPad software, version 5.01 (Prism, La Jolla, CA, USA). A p-value <0.05 was judged statistically significant.
This work was supported by the National Natural Science Foundation of China (Grant 81272143), the Natural Science Foundation of Jiangsu Province (Grant K200509), Jiangsu Innovation Team (Grant LJ201141), Jiangsu Province Program of Innovative and Entrepreneurial Talents (2011–2014), and in part by the Science Foundation Ireland Research Frontiers Programme (Grant SFI/08/RFP/BIC1734).
Conflict of interest
The authors declare no financial or commercial conflict of interest.