A complex balance of host pro- and anti-inflammatory responses to pathogens has been established (1). Sepsis is a syndrome of dysregulated host responses to systemic infection, independent of the organisms, resulting in organ dysfunction (2). Sepsis is the most common cause of death among patients in intensive care unit (3) and is a critically important worldwide health-care problem. Intestinal bacteria are the main sources of pathogen-associated molecular patterns (PAMPs) capable of immune activation, are essential for host immunity development, and gut-translocation of viable bacteria or bacterial molecules results in vigorous systemic inflammation (4). With this background, it is telling that gut permeability barrier failure is reported in sepsis (5, 6). While the significance of gut-translocation of bacterial molecules is appreciated (7), the impact of fungal molecules in bacterial sepsis is unknown.
(1→3)-β-D-glucan (BG), a key component of the cell wall in most fungi, has been used as a biomarker of fungal infection. Fungal BG, released from fungi during fungal growth and the tissue invasion process (8), activates immune responses through several receptors (9, 10). Interestingly, higher serum BG from gut-translocation in bacterial sepsis, without fungemia, is associated with greater sepsis severity (6). However, the importance of intestinal fungi in bacterial sepsis, in the absence of fungemia, has not been adequately explored. As C albicans is the predominant fungal species in human intestine but not in mouse (11), we investigated this using murine models in which C albicans was orally administered prior to sepsis induction by cecal ligation and puncture (CLP). The hypotheses investigated included that intestinal colonization with C albicans enhances bacterial sepsis severity through the gut translocation of BG in the absence of candidemia and that intestinal fungi suppression, by fluconazole, attenuates serum BG, mortality, and severity of inflammation as judged by serum IL-6.
Candida albicans preparation
Candida albicans ATCC 90028 (Fisher Scientific, Waltham, Mass), a fluconazole susceptible strain (minimal inhibitory concentration 0.25 μg/mL to 1 μg/mL) was used. C albicans was cultured overnight on Sabouraud dextrose broth (Thermo Scientific, Hampshire, UK) and counted in a hemocytometer (Bright-Line, Denver, Colo) before use. Heat-killed C albicans was prepared by immersion in a water bath at 60°C for 1 h.
For the in vitro experiments, C albicans lysate was prepared by vigorous sonication of heat-killed cells (Sonics Vibra Cell, VCX 750, Sonics and Materials Inc, Newtown, Conn) until a homogenous solution was formed. Both well-mixed complete homogenate and the supernatant after centrifugation of the preparation were used as heat-killed C albicans and lysate, respectively. BG titers >523 pg/mL were established for both the heat-killed C albicans and the cell lysate, demonstrating that administration of these preparations also included measurable BG.
Animals and animal models
The US National Institutes of Health animal care and use protocols (#85-23, revised 1985) were followed. Male, 8-week-old, ICR mice (National Laboratory Animal Center, Nakhornpathom, Thailand) were used. Only male mice were selected due to the gender difference in sepsis severity (12). The animal protocols were approved by the Institutional Animal Care and Use Committee of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
Cecal ligation and puncture at 3 h after oral administration of Candida albicans
The oral administration of high-dose Candida might induce candidemia after CLP surgery. To see the effect of intestinal Candida, in the absence of candidemia, upon bacterial sepsis, several oral dose levels of live C albicans, 1 × 104, 1 × 106, and 1 × 1010 colony forming units (CFU), were administered to each group of mice at 3 h prior to CLP surgery. CLP procedures were slightly modified from the previous publication (6). Briefly, cecum was ligated at 10 mm from cecal tip with silk 2-0 and then punctured twice with a 21-gauge needle through an abdominal incision under isoflurane anesthesia. Postoperative fentanyl, consisting of 0.03 mg/kg in 0.5 mL of normal saline solution (NSS), was administered subcutaneously for analgesia and fluid replacement immediately after CLP, and repeated 6 h later. Antibiotic [imipenem/cilastatin, 14 mg/kg in 0.3 mL of NSS] was administered subcutaneously at 6 h post-surgery. Subsequent to these experiments, live C albicans oral administration at 1 × 106 CFU was selected for further investigation due to the absence, at this dosage, of concomitant candidemia after CLP surgery (Fig. 1).
Cecal ligation and puncture at 5 days postoral administration of Candida albicans (CLP with 5 day Candida colonization model)
All patients with sepsis receive antibiotics, an intervention that may enhance intestinal C albicans colonization (13). Oral administration of Candida, with antibiotics, but not antibiotic administration alone, has been shown to lead to significant intestinal Candida overgrowth in mice (14). Hence, an animal model of Candida colonization with antibiotic-administration should more closely resemble the septic patient. We administered a single dose of C albicans at 1 × 106, 1 × 104, or 1 × 102 CFU together with daily antibiotic-cocktails containing gentamicin (3.5 mg/kg), colistin (4.2 mg/kg), metronidazole (21.5 mg/kg), and vancomycin (4.5 mg/kg) (separately purchased from Sigma-Aldrich, St Louis, Mo), by twice-daily gavage, for 5 days prior to CLP surgery. To attempt to influence serum (1→3)-β-D-glucan (BG) burdens, due to growth of Candida, fluconazole, (Sigma-Aldrich, St Louis, Mo) at 10 mg/kg in 0.5 mL of NSS or NSS alone was administered orally at 3 and 0.5 h preoperation and at 6 h post-CLP. Of note, fluconazole at lower doses and exposure frequency did not reduce fecal Candida in our pilot experiments (data not shown). A high dose of fluconazole was shown to be necessary for the reduction of fecal fungi in a previously described Candida-administered mouse model (15).
Mouse blood sample analysis
Blood (50 μL) was collected through tail vein nicking at the indicated time-point and with cardiac puncture under isoflurane anesthesia at sacrifice. For quantitative bacterial analysis of blood, 25 μL of blood was spread directly onto blood agar plates (Oxoid, Hampshire, UK), kept at 37°C under aerobic conditions, and bacterial colonies were enumerated at 48 h. The colonies were speciated by standard biochemical tests. The rest of the blood was centrifuged to separate serum and kept at −80°C until analysis. Serum creatinine (Scr) and alanine transaminase (ALT) was measured with QuantiChrom Creatinine Assay kit (DICT-500, Bioassay, Hayward, Calif) and EnzyChrom Alanine Transaminase Assay kit (EALT-100, BioAssay, Hayward, Calif), respectively. Serum cytokines (TNF-α, IL-6, and IL-10) were measured with ELISA (ReproTech, NJ). BG was analyzed with Fungitell (Associates of Cape Cod Inc, East Falmouth, Mass). BG values at <7.8 pg/mL and >523.4 pg/mL (beyond the lower and upper ranges of the standard curve) were recorded as 0 pg/mL and 523 pg/mL, respectively. All assays were performed according to the manufacturer's protocol.
Culture of fecal fungi
Feces were collected by placing an individual mouse in an empty cage for 0.5 to 1 h before CLP for the 0 h time-point. At 6 and 18 h post CLP, mice were sacrificed and feces from descending colon and/or rectum were collected. Feces were well-mixed with phosphate buffer solution (PBS) in a ratio of 1 μg/ 1 μL before plating directly in sabouraud dextrose agar with 0.1% chloramphenicol (Thermo Scientific) kept at 35°C for 72 h for fungal colony enumeration.
The microbiota analysis protocol was performed as previously reported (16). In short, feces from individual mice were used for metagenomic DNA extractions, with three independent extractions of 0.25 g performed per sample. The DNA Isolation Kit (MoBio, Carlsbad, Calif) was used to extract total nucleic acid. Metagenomic DNA quality was assessed by agarose gel electrophoresis and nanodrop spectrophotometry. Universal prokaryotic 515F (forward; (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (reverse; 5′-GGACTACHVGGGTWTCTAAT-3′), with appended 5′ Illumina adapter and 3′ Golay barcode sequences, were used for 16S rRNA gene V4 library construction (16). Independent triplicate PCR experiments were performed and pooled to prevent stochastic PCR bias. The 16S rDNA amplicons of 381 base pairs (bp) were purified by the GenepHlow Gel Extraction Kit (Geneaid Biotech Ltd, New Taipei City, Taiwan), and quantified with Picogreen (Invitrogen, Eugene, Ore). Samples, at 240 ng, were pooled for sequencing by Miseq300 platform (Illumina, San Diego, Calif) (17). Raw sequences were quality screened by Mothur's MiSeq platform procedures (18). Quality screening steps including the removal of reads that have ambiguous bases, >1 mismatch in the reverse primer sequence, >10 homopolymers, a minimum quality score of <35 over a 50-bp window, a read length of <350 bases, and chimeric sequence. Quality sequences were aligned and assigned taxon (operational taxonomic unit [OTU]) based on a default parameter, as previously published (18). Samples were normalized to an equal sampling depth (N=118121 reads per sample) (18). Good's Coverage was used to estimate the OTU coverage by sequencing (all samples showed 99.7%–100% coverage). Non-metric multidimensional scaling (NMDS) was used for the data visualization.
Bone marrow-derived macrophage preparation
Macrophages were derived from bone marrow (BM) following previous published methods (19). Briefly, femur BM cells were obtained and incubated for 7 days in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid with sodium pyruvate, and 20% conditioned medium of the L929 cell line (containing macrophage-colony stimulating factor (M-CSF)) in a humidified 5% CO2 incubator at 37°C. Cells were harvested with cold PBS and the macrophage phenotype was confirmed with anti-F4/80 and anti-CD11c antibody staining (BioLegend, San Diego, Calif) by flow cytometry.
Macrophage killing activity protocol
The protocol followed a previously published method (20). In brief, 1 × 105 cells of BM-derived macrophages were plated in a 96-well plate. Macrophages were treated for 24 h with lipopolysaccharide (LPS) of Escherichia coli 026:B6 (Sigma-Aldrich) at the concentration 10 ng/mL or 1,000 ng/mL with a crude preparation of 5 × 105 cells of heat-killed C albicans or the lysate (described previously) or an equivalent volume of PBS. E coli at 1 × 107 CFU was added to each well in 25 μL of normal mouse serum (added as an opsonin) and incubated with the macrophages for 15 min followed by washing to remove non-phagocytized microorganisms. Subsequently, gentamicin 100 μg/mL at 100 μL was added for 1 h to eradicate the viable extracellular E coli and then the plate was washed, gently scraped, lysis induced with 200 μL of distilled water. The serial dilutions of lysate were streaked on Tryptic soy agar (Thermo Scientific) and then incubated at 37°C overnight before bacterial colony enumeration. The number of bacteria from the macrophage cell lysate was inversely associated with the intracellular killing activity.
Induction of macrophage cytokine production protocol
E coli 026:B6 (Sigma-Aldrich) LPS at a concentration of 1,000 ng/mL, in combination with heat-killed C albicans at a dose of 5 × 105 yeast cells or lysate or PBS, was incubated with macrophages (1×105 cells/well) in culture plates for 24 h. Then the supernatant was collected and measured for cytokines using ELISA (ReproTech) procedures.
Data was analyzed as mean ± standard error and the differences between groups were examined for statistical significance by one-way analysis of variance (ANOVA) followed by Bonferroni analysis for the experiments with multiple groups comparison. The survival analysis was determined by log-rank test. In addition, the two-way ANOVA was used for the analysis of the time-course experiments. All statistical analyses were performed with SPSS 11.5 software (SPSS, Ill). A P value < 0.05 was considered to be statistically significant.
Oral administration of C albicans was performed at different doses 3 h before CLP to observe the influence of intestinal organisms on sepsis severity in the CLP model. Interestingly, mortality was somewhat dose-dependent with the administration of Candida. All CLP mice dosed with C albicans at 1 × 106 and 1 × 1010 CFU died within 36 and 24 h, respectively. Candidemia was detectable in 6/7 CLP mice (86%) at 18 h only, at the 1 × 1010 CFU dose (Fig. 1). Hence, for the determination of the influence of intestinal Candida in bacterial sepsis in the absence of candidemia, oral administration of C albicans at 1 × 106 CFU was selected for further experimentation.
Five-day colonization with Candida albicans before cecal ligation and puncture increased serum (1→3)-β-D-glucan and worsened sepsis severity
A single-oral administration of C albicans (1 × 106 CFU) 5 days prior to CLP with/without daily antibiotic-cocktail administration was used as a representative model for patients receiving broad-spectrum antibiotics. Oral-antibiotics plus Candida administration worsened CLP mortality (Fig. 2A). All mice fed Candida died regardless of oral antibiotic administration. The survival rate was 30% and 11% in the CLP-NSS control and CLP-antibiotic-alone groups, respectively (Fig. 2A). In addition, CLP with Candida administration worsened sepsis as determined by ALT, TNF-α, and IL-6, but not Scr, regardless of oral-antibiotics administration (Fig. 2B–E).
Fecal fungi were undetectable by the culture method in control and CLP mice (data not shown) as previously observed (11, 13, 21). Oral live-Candida administration converted fecal Candida culture after CLP from negative to positive in a time-dependent manner, in the absence of candidemia. Fecal Candida was culturable only in mice with Candida administration, but not from mice receiving antibiotics alone, as observed previously (22, 23). Fecal fungal abundance was also higher in mice with Candida plus oral antibiotics (Candida-ATB) than Candida without ATB (Candida-NSS) (Fig. 3A). Fecal Candida colony counts at 0, 6, and 18 h CLP without—versus with—ATB were 29 ± 7 CFU/g, 60 ± 25 CFU/g, and 117 ± 44 CFU/g versus 149 ± 37 CFU/g, 138 ± 35 CFU/g, and 324 ± 92 CFU/g, respectively (Fig. 3A), in the absence of candidemia.
On the other hand, fecal microbiota analysis showed increased pathogenic intestinal bacteria in models with oral ATB and this was more severe with Candida-ATB (Figs. 4 and 5). Candida administration alone did not significantly change the gut microbiota composition. Oral antibiotic administration alone induced a mild increase in Bacillus spp. (potentially pathogenic bacteria) and a large increase in Lactobacillus spp. (potentially probiotic bacteria) (Fig. 4). In conjunction with oral ATB, the addition of Candida reduced Lactobacillus spp. and increased the pathogenic bacteria, Shigella and Enterobacteriaceae. Interestingly, intestinal abundance of both Candida spp. (Fig. 3A) and pathogenic bacteria (Fig. 4) in Candida-ATB was more prominent than in ATB alone and occurred with a higher CLP mortality rate (Fig. 2A). In parallel, serum BG and mortality, but not bacteremia, were higher in the CLP-Candida-ATB-treated mice than those with CLP-ATB alone (Figs. 2A, 3B and C). This implied a greater association of serum BG titer with sepsis severity.
Despite negative fecal fungal culture in CLP-ATB-treated mice, serum BG at 18 h was higher than CLP-control (CLP-NSS); 223 ± 18 pg/mL versus 114 ± 26 pg/mL (Fig. 3B). CLP-Candida-ATB resulted in higher serum BG compared with CLP-ATB at 18 h; 495 ± 28 pg/mL versus 283 ± 13 pg/mL (Fig. 3B). In parallel, at 18 h after CLP, bacterial burdens in blood, mesenteric lymph node, and liver, but not in peritoneal fluid, of CLP-Candida-ATB and CLP-ATB were higher than CLP-NSS and CLP-Candida (Fig. 3, C–E).
Although polymicrobial bacteremia presented in all CLP mice, E coli bacteremia was predominant in mice treated with antibiotics (Fig. 3C), perhaps due to an increase of pathogenic bacteria in the gut (Fig. 4). Of note, fungal culture was negative in samples of blood, peritoneal fluid, mesenteric lymph node, and internal organs (data not shown).
Gastrointestinal fungal abundance associated with the level of serum (1→3)-β-D-glucan and sepsis severity
The possibility that the abundance of intestinal fungi influenced serum BG burden and sepsis severity was investigated. Candida was administered, at different doses, in the 5-day-colonization model. Survival of CLP mice with Candida at 106 CFU was lower than CLP with 102 CFU, P = 0.042, by Log-rank test (Fig. 6). As hypothesized, fecal-Candida abundance was Candida dose-dependent (Fig. 7A). Fecal Candida levels at 0, 6, and 18 h post-CLP, with the administration of 102 CFU, were 7 ± 4 CFU/g, 47 ± 12 CFU/g, and 21 ± 14 CFU/g; at 104 CFU they were 66 ± 17 CFU/g, 85 ± 24 CFU/g, and 149 ± 23 CFU/g, and at 106 CFU were 135 ± 25 CFU/g, 140 ± 22 CFU/g, and 288 ± 63 CFU/g (Fig. 7A). Despite the absence of candidemia at all Candida doses, serum BG was detectable in an oral dose-dependent manner at 18 h post-CLP (Fig. 7B). Serum BG at 18 h after CLP with Candida (CLP-Candida) at 106 CFU, 104 CFU, and 102 CFU was 451 ± 31 pg/mL, 301 ± 74 pg/mL, and 239 ± 31 pg/mL, respectively (Fig. 7B). Additionally, serum IL-6 was higher in CLP-Candida at 106 CFU as early as 3 h after CLP in compared with CLP-Candida at 102 CFU (Fig. 7C).
Importantly, fluconazole administration attenuated sepsis severity, and serum BG at 18 h post-CLP in the 104 and 102Candida CFU, but not 106Candida CFU groups (Fig. 7, D–F). The survival rates of CLP-Candida mice at 104 and 102 CFU with fluconazole were 9% and 27%, respectively (Fig. 6, B and C). In parallel, with fluconazole, serum BG was reduced from 311 ± 75 pg/mL to 157 ± 45 pg/mL and from 239 ± 31 pg/mL to 74 ± 13 pg/mL in CLP-Candida at 104 and 102 CFU, respectively. However, fluconazole did not attenuate serum BG or mortality in the CLP-Candida mice at 106 CFU (Figs. 6A and 7D). In contrast to negative fecal fungal culture in fluconazole-treated CLP-Candida mice at 104 CFU and 102 CFU, fecal fungal culture was positive in fluconazole-treated CLP-Candida at 106 CFU. Fecal fungal burdens at 18 h-post CLP in 106 CFU Candida-treated mice, with and without fluconazole treatment, were 65 ± 35 CFU/g and 132 ± 57 CFU/g of feces, respectively, which was not statistically difference by the Student t test.
Heat-killed Candida or Candida cell lysate reduced macrophage killing activity and increased macrophage cytokine production
Macrophages are important in sepsis pathogenesis (24). Macrophages have also been shown to be subject to immunoregulatory influences by BG exposure (25, 26). As BG properties are dependent upon sources and isolation methods (27), macrophages were treated with a BG-containing crude extract of Candida, but not highly purified BG, to mimic exposure to intestinal Candida. In an in vitro challenge model, with macrophages alone (control) or LPS alone, macrophages eliminated all E coli (Fig. 8 A and B). However, macrophage killing activity was decreased with exposure to heat-killed Candida (Fig. 8A). Conversely, addition of LPS restored macrophage killing of E coli in a dose-related manner (Fig. 8A). In parallel, lysate of heat-killed Candida also inhibited macrophage-killing-activity but with much less potency and this was not restored by LPS addition (Fig. 8B). Additionally, LPS (1,000 ng), heat-killed Candida, or the lysate alone induced TNF-α and IL-6 production but with differing potency (Fig. 8, C and D). Interestingly, the cytokine induction potency was significantly enhanced by combining LPS and heat-killed Candida or Candida lysate (Fig. 8, C and D).
The possibility that gut translocation of PAMPs from commensal fungi (such as BG) may enhance sepsis severity through macrophage function interference and synergistic stimulation of pro-inflammatory cytokines was explored. Commensal organisms are crucial for immune system maturation and intestinal barrier function (28) and they activate inflammatory immune responses through PAMPs (29). C albicans is the predominant commensal fungal species of the human intestine (30). As such, the “Candida colonization index” is used for candidiasis prediction in sepsis but not for sepsis severity determination (31). In contrast, C albicans is not often present in mouse intestine. To study the impact of commensal Candida in a murine sepsis model, artificial introduction of Candida was required. Additionally, appropriate dose selection of oral C albicans was required to avoid candidemia after CLP surgery.
Fungal colonization augmented with oral-antibiotic administration (ATB), a model close to the patients with broad-spectrum ATB administration, produced more elevated serum BG, high prevalence of E coli bacteremia, and more severe sepsis than CLP with ATB without Candida. Although serum BG was detectable in sepsis alone without Candida gavage, the level was higher in sepsis with Candida. Interestingly, serum BG increased in CLP without Candida administration, despite the nonobvious fungal presence in feces as determined by undetectable fungi in fecal culture. The time-dependent increase of serum BG in mice without Candida gavage might be due to the progression of sepsis-induced gut leakage (6). With Candida-administration, fecal fungi are detectable by culture, along with higher serum BG levels, after CLP. With CLP and no Candida gavage, fecal fungi are undetectable by culture. Fungal culture of the mesenteric lymph node was also negative despite high serum BG, implying gut-translocation of fungal molecules but not viable Candida cells. In contrast, bacteria were detectable in mesenteric lymph node in all models with positive bacteremia, implying gut-translocation of viable bacteria.
Candida administration in the absence of antibiotics enhanced serum BG and sepsis mortality, but not blood bacteria burdens or intestinal pathogenic bacteria, in comparison with CLP without Candida gavage. This suggests potential influence of serum BG in enhancing sepsis severity. On the other hand, CLP with Candida plus ATB increased sepsis mortality along with serum BG and intestinal pathogenic bacteria, but not bacteria in mesenteric lymph node or in blood, in comparison with CLP-ATB without Candida. More severe sepsis in the CLP-Candida-ATB model might be due to bacterial factors (gut bacterial burdens and E coli bacteremia) or fungal factors (BG) or both. However, the data that sepsis severity increased dose-dependently with Candida administration and antifungal administration (fluconazole), without additional antibacterial drugs, attenuated sepsis severity in the CLP-Candida group implied an important role for fungal factors over bacterial factors in sepsis severity, in our models.
Of note, the alteration of gut bacteria in our models is interesting. The predominant bacteria in mice with Candida without ATB and the control groups were Bacteroides. With antibiotics, Fermicutes, and Proteobacteria became more prominent in the mice with ATB alone and ATB with Candida, respectively. This suggests that the presence of Candida in the murine gastrointestinal tract influences its microbiota.
Importantly, with the different levels of oral Candida administration in the 5 day-fungal colonization model, sepsis severity increased in a dose-dependent manner. Although the influence upon mortality is difficult to discern given the 100% mortality, serum cytokines and organ injuries indicated more severe sepsis. Fluconazole-related fungal suppression may have reduced gut levels of fungal material for translocation, resulting in less immune response enhancement.
Reduced-macrophage killing activity and enhanced-cytokine production by heat-killed Candida or its lysate were demonstrated in vitro. These data support the alteration of macrophage functions by the combination of bacterial and fungal molecules (32, 33), possibly due to complex responses from the combined activation of several pattern recognition receptors (25, 26, 34). The pro-inflammatory synergy of BG with different Toll-Like receptor ligands has also been demonstrated in a human whole blood model (35). Further investigations of the modulation of macrophage bactericidal activity by fungal factors, including specific tissue-resident macrophages, are needed. Translationally, an evaluation of fungal abundance in patient stool might discern associations with sepsis severity and, if so, the reduction of intestinal fungi and/or BG might attenuate bacterial sepsis. As serum BG associated with intestinal fungal abundance in sepsis (the present data), correlated with impaired gut-permeability (6) and as stool collection and culture in critical care conditions are too labor intensive, we propose the evaluation of serum BG, but not stool fungi, as an additional potential biomarker for sepsis severity.
In conclusion, increased intestinal burdens of fungal material were shown to cause increased sepsis severity. This was associated, at least in part, with the translocation of fungal material from the gut lumen, reduced macrophage killing activity, and enhanced cytokine production resulting in more profound inflammation.
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