Iron Sequestration by Murine Calprotectin Induces Starvation Responses in Pseudomonas aeruginosa.

Pathogen sensing by the mammalian host induces a pro-inflammatory response that involves release of the antimicrobial metal-sequestering protein calprotectin (CP, S100A8/S100A9 heterooligomer, MRP8/MRP14 heterooligomer) from neutrophils. Biochemical investigations on human CP (hCP) have informed the molecular basis of how this protein sequesters metal ions. Murine models of infection have provided invaluable insights into the ability of murine CP (mCP) to compete with bacterial pathogens for essential metal nutrients. Despite this extensive work, our knowledge of how mCP sequesters metals from bacterial pathogens and its impacts on bacterial physiology is limited. Moreover, whether mCP sequesters iron and induces iron-starvation responses in bacterial pathogens has not been evaluated. Here, we examine the ability of mCP to withhold iron from Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen that causes severe infections in immunocompromised individuals and cystic fibrosis patients. We demonstrate that mCP prevents iron uptake and induces iron-starvation responses in P. aeruginosa laboratory strains PA14 and PAO1 and the JSRI-1 clinical isolate from a cystic fibrosis patient. We also show that mCP prevents iron uptake and induces an iron-starvation response in the Gram-positive bacterial pathogen Staphylococcus aureus. The His6 site of mCP is the iron-sequestering site; it exhibits Ca(II)-dependent Fe(II) affinity and binds Fe(II) with subpicomolar affinity in the presence of excess Ca(II) ions. This work is important for understanding the structure, function, and physiological consequences of mCP and how the mammalian host and bacterial pathogens compete for essential metal nutrients.

The effects of torula yeast as a protein source on apparent total tract digestibility, inflammatory markers, and fecal microbiota dysbiosis index in Labrador Retrievers with chronically poor stool quality.

This study examined the effects of varying protein sources on apparent total tract digestibility, inflammatory markers, and fecal microbiota in Labrador Retrievers with historically poor stool quality. Thirty dogs (15 male, 15 female; aged 0.93 to 11.7 yr) with stool quality scores ≤2.5 on a 5-point scale (1 representing liquid stool and 5 representing firm stool) were randomly assigned to 1 of 3 nutritionally complete diets with differing protein sources and similar macronutrient profiles: 1) chicken meal (n = 10); 2) 10% brewer's yeast (n = 10); or 3) 10% torula yeast (n = 10). Another 10 dogs (five male, five female) with normal stool quality (scores ranging from 3 to 4) received diet 1 and served as negative control (NC). All dogs were fed diet 1 for 7 days, then provided their assigned treatment diets from days 7 to 37. Daily stool scores and weekly body weights were recorded. On days 7, 21, and 36, blood serum was analyzed for c-reactive protein (CRP), and feces for calgranulin C (S100A12), α1-proteinase inhibitor (α1-PI), calprotectin, and microbiota dysbiosis index. Apparent total tract digestibility was assessed using the indicator method with 2 g titanium dioxide administered via oral capsules. Stool scores were greater in NC (P < 0.01) as designed but not affected by treatment × time interaction (P = 0.64). Body weight was greater (P = 0.01) and CRP lower (P < 0.01) in NC dogs. Dry matter and nitrogen-free extract digestibility did not differ among groups (P ≥ 0.14). Negative controls had greater fat digestibility compared to BY (94.64 ±â€…1.33% vs. 91.65 ±â€…1.25%; P = 0.02). The overall effect of treatment was significant for protein digestibility (P = 0.03), but there were no differences in individual post hoc comparisons (P ≥ 0.07). Treatment did not affect S100A12 or α1-PI (P ≥ 0.44). Calprotectin decreased at a greater rate over time in TY (P < 0.01). The dysbiosis index score for BY and TY fluctuated less over time (P = 0.01). Blautia (P = 0.03) and Clostridium hiranonis (P = 0.05) abundances were reduced in BY and TY. Dogs with chronically poor stool quality experienced reduced body weights and increased serum CRP, but TY numerically increased protein digestibility, altered the microbiome, and reduced fecal calprotectin. Torula yeast is a suitable alternative protein source in extruded canine diets, but further research is needed to understand the long-term potential for improving the plane of nutrition and modulating gut health.

Pet and human populations continue to grow and compete for nutritious, sustainable protein sources. The incorporation of alternative proteins like torula yeast can provide a solution to this problem. Torula yeast also may have additional health benefits like reducing gut inflammation. To test its effects in dogs, we fed Labrador Retrievers with chronically poor stool quality either a control diet with chicken meal, a diet with 10% brewer's yeast, or a diet with 10% torula yeast. We compared their responses to dogs with normal stool quality fed the control diet. Dogs with chronically poor stool quality had lower body weights and increased systemic inflammation compared to those with good stool quality. Calprotectin, a marker of gut inflammation, was reduced more in dogs fed torula yeast than in dogs fed chicken meal. Torula and brewer's yeast also changed the abundance of certain gut bacteria. Torula yeast may be added to dog diets with no negative effects and can alter the gut environment in Labrador Retrievers with chronically poor stool quality.

Effects of a milk oligosaccharide biosimilar on fecal characteristics, microbiota, and bile acid, calprotectin, and immunoglobulin concentrations of healthy adult dogs treated with metronidazole.

In recent dog and cat experiments, a novel milk oligosaccharide biosimilar (GNU100) positively modulated fecal microbiota and metabolite profiles, suggesting benefits to gastrointestinal health. The objective of this study was to investigate the effects of GNU100 on the fecal characteristics, microbiota, and bile acid (BA) concentrations of healthy adult dogs treated with antibiotics. Twelve healthy adult female dogs (mean age: 3.74 ± 2.4 yr) were used in an 8-wk crossover design study (dogs underwent both treatments). All dogs were fed a control diet during a 2-wk baseline, then randomly allotted to 1 of 2 treatments (diet only or diet + 1% GNU100) for another 6 wk. From weeks 2 to 4, dogs were orally administered metronidazole (20 mg/kg BW) twice daily. Fecal scores were recorded daily and fresh fecal samples were collected at weeks 2, 4, 5, 6, and 8 for measurement of pH, dry matter, microbiota populations, and BA, immunoglobulin A, and calprotectin concentrations. On weeks 0, 4, and 8, blood samples were collected for serum chemistry and hematology analysis. All data were analyzed as repeated measures using the Mixed Models procedure of SAS version 9.4, with significance considered P < 0.05. Metronidazole increased (P < 0.0001) fecal scores (looser stools) and modified (P < 0.05) fecal microbiota and BA profiles. Using qPCR, metronidazole reduced fecal Blautia, Fusobacterium, Turicibacter, Clostridium hiranonis, and Faecalibacterium abundances, and increased fecal Streptococcus and Escherichia coli abundances. DNA sequencing analysis demonstrated that metronidazole reduced microbial alpha diversity and influenced the relative abundance of 20 bacterial genera and families. Metronidazole also increased primary BA and reduced secondary BA concentrations. Most antibiotic-induced changes returned to baseline by week 8. Fecal scores were more stable (P = 0.01) in GNU100-fed dogs than controls after antibiotic administration. GNU100 also influenced fecal microbiota and BA profiles, reducing (P < 0.05) the influence of metronidazole on microbial alpha diversity and returning some fecal microbiota and secondary BA to baseline levels at a quicker (P < 0.05) rate than controls. In conclusion, our results suggest that GNU100 supplementation provides benefits to dogs treated with antibiotics, providing more stable fecal scores, maintaining microbial diversity, and allowing for quicker recovery of microbiota and secondary BA profiles which play an essential role in gut health.

Our objective was to test the effects of a novel milk oligosaccharide biosimilar (GNU100) on the fecal characteristics, microbiota, and bile acid (BA) concentrations of healthy adult dogs treated with antibiotics. Dogs were fed a control diet during a 2-wk baseline, then randomly allotted to 1 of 2 treatments (diet only or diet + 1% GNU100) for another 6 wk. From weeks 2 to 4, dogs were given an oral antibiotic. Fecal scores were recorded and fresh fecal samples were collected over time to assess fecal characteristics, microbiota populations, and BA concentrations. The antibiotic was shown to increase fecal scores (looser stools) and modify fecal microbiota populations (altered diversity and ~20 bacterial genera and families) and BA profiles (increased primary and reduced secondary BA). Most antibiotic-induced changes returned to baseline by week 8. In dogs fed GNU100, fecal scores were more stable and changes to microbial diversity were lower than controls after antibiotic administration. Fecal microbiota and secondary BA of GNU100-fed dogs also returned to baseline levels at a quicker rate than controls. These results suggest that GNU100 provides benefits to dogs given antibiotics, providing more stable fecal scores, maintaining microbial diversity, and allowing for quicker recovery of microbiota and BA profiles.

Zinc-binding metallophores protect Pseudomonas aeruginosa from calprotectin-mediated metal starvation.

Pseudomonas aeruginosa is known to exhibit considerable resistance to the antimicrobial activity of the metal-sequestering protein calprotectin (CP). In this study, we demonstrate that although CP induces zinc deficiency in P. aeruginosa, a strain unable to import zinc through the two most important metal acquisition systems, namely ZnuABC and ZrmABCD, maintains significant growth capacity in the presence of high concentrations of CP. Furthermore, we have shown that nicotianamine, a molecule structurally similar to the metallophore pseudopaline, can favor the acquisition of the metal even in the presence of CP. To gain insights into the mechanisms through which metallophores can promote zinc acquisition, we analyzed the effect of nicotianamine on the activity of the metallo-ß-lactamase VIM-1. Our data suggest that metallophores released by bacteria in response to zinc deficiency can extract the protein-bound metal. The ability to interfere with the binding of metals to proteins, as well as favoring the acquisition of zinc, may contribute to increasing the resistance of P. aeruginosa to the antimicrobial action of CP.

Ulcerative Colitis in Response to Fecal Microbiota Transplantation via Modulation of Gut Microbiota and Th17/Treg Cell Balance.

Background: Fecal microbiota transplantation (FMT) may contribute to disease remission in ulcerative colitis (UC). We studied the microbiota change and its regulation on T cells after FMT. Methods: Patients with mild to moderately active UC were included to receive FMT. The intestinal histopathological changes and barrier function were evaluated. The fecal samples of donors and patients were analyzed by 16S rRNA gene-based microbiota analysis, and the colon Th17 and Treg cells were assessed. Results: Fifteen patients completed the 8-week-follow-up. A total of 10 patients (66.7%) were in the responders (RE) group and five in the non-responders (NR) group. The Nancy histological index and fecal calprotectin decreased (p < 0.001, p = 0.06, respectively) and Occludin and Claudin1 increased in the RE group. The abundance of Faecalibaterium increased significantly by 2.3-fold in the RE group at week 8 (p = 0.043), but it was suppressed in the NR group. Fecal calprotectin (r = −0.382, p = 0.003) and Nancy index (r = −0.497, p = 0.006) were correlated inversely with the abundance of Faecalibacterium, respectively. In the RE group the relative mRNA expression of RORγt decreased and Foxp3 increased. Significantly decreased CD4+ RORγt+ Th17 and increased CD4+ Foxp3+ Treg were also observed in the RE group. The relative abundance of Faecalibacterium correlated with CD4+ RORγt+ Th17 (r = −0.430, p = 0.018) and CD4+ Foxp3+ Treg (r = 0.571, p = 0.001). Conclusions: The long-term Faecalibaterium colonization following FMT plays a crucial role in UC remission by alleviating intestinal inflammation. This anti-inflammatory effect of Faecalibacterium may be achieved by regulating the imbalance of Th17/Treg levels in UC.

Elucidating the role of the gut microbiota in the physiological effects of dietary fiber.

BACKGROUND: Dietary fiber is an integral part of a healthy diet, but questions remain about the mechanisms that underlie effects and the causal contributions of the gut microbiota. Here, we performed a 6-week exploratory trial in adults with excess weight (BMI: 25-35 kg/m2) to compare the effects of a high-dose (females: 25 g/day; males: 35 g/day) supplement of fermentable corn bran arabinoxylan (AX; n = 15) with that of microbiota-non-accessible microcrystalline cellulose (MCC; n = 16). Obesity-related surrogate endpoints and biomarkers of host-microbiome interactions implicated in the pathophysiology of obesity (trimethylamine N-oxide, gut hormones, cytokines, and measures of intestinal barrier integrity) were assessed. We then determined whether clinical outcomes could be predicted by fecal microbiota features or mechanistic biomarkers. RESULTS: AX enhanced satiety after a meal and decreased homeostatic model assessment of insulin resistance (HOMA-IR), while MCC reduced tumor necrosis factor-α and fecal calprotectin. Machine learning models determined that effects on satiety could be predicted by fecal bacterial taxa that utilized AX, as identified by bioorthogonal non-canonical amino acid tagging. Reductions in HOMA-IR and calprotectin were associated with shifts in fecal bile acids, but correlations were negative, suggesting that the benefits of fiber may not be mediated by their effects on bile acid pools. Biomarkers of host-microbiome interactions often linked to bacterial metabolites derived from fiber fermentation (short-chain fatty acids) were not affected by AX supplementation when compared to non-accessible MCC. CONCLUSION: This study demonstrates the efficacy of purified dietary fibers when used as supplements and suggests that satietogenic effects of AX may be linked to bacterial taxa that ferment the fiber or utilize breakdown products. Other effects are likely microbiome independent. The findings provide a basis for fiber-type specific therapeutic applications and their personalization. TRIAL REGISTRATION: Clinicaltrials.gov, NCT02322112 , registered on July 3, 2015. Video Abstract.

Lycopodium Mitigates Oxidative Stress and Inflammation in the Colonic Mucosa of Acetic Acid-Induced Colitis in Rats.

Inflammatory bowel diseases (IBDs) such as ulcerative colitis (UC) and Crohn's disease (CD) are diseases of the gastrointestinal system involving genetic and environmental factors attributed to oxidative stress and inflammation. Targeting oxidative stress and inflammation by novel dietary compounds of natural origin convincingly appears to be one of the important therapeutic strategies to keep the disease in remission. As there is no permanent cure for IBD except for chronic long-term treatment or surgery, it is therefore imperative to investigate plant-based agents that are receiving attention for their therapeutic benefits to overcome the debilitating clinical conditions of IBD. Lycopodium (LYCO), a plant of tropical and subtropical origin and known by numerous names such as ground pine, club moss, or devil's claw, has been popularly used for centuries in traditional medicine including Chinese and Indian medicines. In the present study, the effect of LYCO has been investigated in an acetic acid (AA)-induced colitis model in Wistar rats. LYCO was orally administered at the dose of 50 mg/kg/day either 3 days before or 30 min after the induction of IBD and continued for 7 days by intrarectal administration of AA. The changes in body weight and macroscopic and microscopic analysis of the colon of rats of different experimental groups were observed on days 0, 2, 4, and 7. The levels of myeloperoxidase (MPO), reduced glutathione (GSH), and malondialdehyde (MDA) were measured. AA caused a significant reduction in body weight and increased macroscopic and microscopic ulcer scores along with a significant decline in antioxidant enzymes, superoxide dismutase (SOD), and catalase and antioxidant substrate, glutathione (GSH). There was a concomitant increased formation of malondialdehyde (MDA), a marker of lipid peroxidation, and raised myeloperoxidase (MPO) activity, a marker of neutrophil activation. Treatment with LYCO significantly improved IBD-induced reduction in body weight, improved histology, inhibited MDA formation, and restored antioxidants along with reduced MPO activity. AA also caused the release of proinflammatory cytokines such as interleukin-1ß (IL-1ß) and interleukin-23 (IL-23). Furthermore, AA also increased the levels of calprotectin, a protein released by neutrophils under inflammatory conditions of the gastrointestinal tract. LYCO treatment significantly reduced the release of calprotectin and proinflammatory cytokines. The results demonstrate that LYCO treatment has the potential to improve disease activity by inhibiting oxidative stress, lipid peroxidation, and inflammation along with histological preservation of colonic tissues.

[Unintended Observations Leading to Macrophage Growth and Neutrophil Factor Research].

My research area in the pharmaceutical industry is innate immunity, especially in phagocytic cells. First, I studied the heat-stable growth factor of peripheral macrophages in tumorous ascitic fluid and found that lipoproteins are an influencing factor. Later, my colleagues and I found that lipid-containing substances, namely, oxidized low-density lipoprotein, dead neutrophils, or purified lipids that could be scavenged by macrophages, induce their growth. From the series of this study, I concluded that phagocytic substances induce macrophage growth by autocrine stimulation of granulocyte-macrophage colony-stimulating factor (GM-CSF). During the study, we found that neutrophils have growth-inhibitory effects against a variety of cells. Then, I elucidated that the primary factor is a zinc-binding protein, calprotectin, an abundant protein complex in the neutrophil cytosol. I found that calprotectin induces apoptosis in many cell types, including tumor cells and normal fibroblasts, and that the zinc-binding capacity is essential for its activity. Microscopic observations revealed that neutrophil extract contains factor-inducing three-dimensional cell aggregation of human mammary carcinoma, MCF-7. I elucidated that cathepsin G is responsible for this activity and that its effect is dependent on the activation of insulin-like growth factor-1. I believe that this modest, albeit novel, observation was crucial to my thirty-nine-year-long career researching phagocytic cells.

Effects of Resistant Starch on Symptoms, Fecal Markers, and Gut Microbiota in Parkinson's Disease - The RESISTA-PD Trial.

The composition of the gut microbiota is linked to multiple diseases, including Parkinson's disease (PD). Abundance of bacteria producing short-chain fatty acids (SCFAs) and fecal SCFA concentrations are reduced in PD. SCFAs exert various beneficial functions in humans. In the interventional, monocentric, open-label clinical trial "Effects of Resistant Starch on Bowel Habits, Short Chain Fatty Acids and Gut Microbiota in Parkinson'sDisease" (RESISTA-PD; ID: NCT02784145), we aimed at altering fecal SCFAs by an 8-week prebiotic intervention with resistant starch (RS). We enrolled 87 subjects in three study-arms: 32 PD patients received RS (PD + RS), 30 control subjects received RS, and 25 PD patients received solely dietary instructions. We performed paired-end 100 bp length metagenomic sequencing of fecal samples using the BGISEQ platform at an average of 9.9 GB. RS was well-tolerated. In the PD + RS group, fecal butyrate concentrations increased significantly, and fecal calprotectin concentrations dropped significantly after 8 weeks of RS intervention. Clinically, we observed a reduction in non-motor symptom load in the PD + RS group. The reference-based analysis of metagenomes highlighted stable alpha-diversity and beta-diversity across the three groups, including bacteria producing SCFAs. Reference-free analysis suggested punctual, yet pronounced differences in the metagenomic signature in the PD + RS group. RESISTA-PD highlights that a prebiotic treatment with RS is safe and well-tolerated in PD. The stable alpha-diversity and beta-diversity alongside altered fecal butyrate and calprotectin concentrations call for long-term studies, also investigating whether RS is able to modify the clinical course of PD.

The Sensor Histidine Kinase ArlS Is Necessary for Staphylococcus aureus To Activate ArlR in Response to Nutrient Availability.

Staphylococcus aureus is a versatile opportunistic pathogen whose success is driven by its ability to adapt to diverse environments and host-imposed stresses. Two-component signal transduction systems, such as ArlRS, often mediate these adaptations. Loss of ArlRS or the response regulator ArlR alone impairs the ability of S. aureus to respond to host-imposed manganese starvation and glucose limitation. As sensor histidine kinases and response regulators frequently work as pairs, it has been assumed that ArlS senses and activates ArlR in response to these stimuli. However, recent work suggests that the sensor histidine kinase GraS can also activate ArlR, calling the contribution of ArlS in responding to manganese and glucose availability into question. The results of current studies reveal that ArlS is necessary to activate ArlR in response to manganese sequestration by the host immune effector calprotectin and glucose limitation. Although the loss of ArlS does not completely eliminate ArlR activity, this response regulator is no longer responsive to manganese or glucose availability in the absence of its cognate histidine kinase. Despite the residual activity of ArlR in the absence of ArlS, ArlR phosphorylation by ArlS is required for S. aureus to resist calprotectin-imposed metal starvation. Cumulatively, these findings contribute to the understanding of S. aureus signal transduction in response to nutritional immunity and support the previous observation indicating that ArlRS is activated by a common signal derived from host-imposed manganese and glucose limitation. IMPORTANCE The ability of pathogens, including Staphylococcus aureus, to sense and adapt to diverse environments partially relies on two-component systems, such as ArlRS. Recent work revealed that the response regulator ArlR can be cross-activated by the sensor histidine kinase GraS, rendering the role of its cognate partner, ArlS, in response to manganese and glucose limitation uncertain. The results of this study reveal that ArlS is necessary for the activation of ArlR in response to calprotectin and glucose limitation. Although a low level of ArlR activity remains in the absence of ArlS, ArlS phosphotransfer to ArlR is required for S. aureus to overcome calprotectin-induced nutritional stress. Collectively, this study provides fundamental information to understand how ArlRS mediates staphylococcal adaptation during infection.

The Human Innate Immune Protein Calprotectin Elicits a Multimetal Starvation Response in Pseudomonas aeruginosa.

To combat infections, the mammalian host limits availability of essential transition metals such as iron (Fe), zinc (Zn), and manganese (Mn) in a strategy termed "nutritional immunity." The innate immune protein calprotectin (CP) contributes to nutritional immunity by sequestering these metals to exert antimicrobial activity against a broad range of microbial pathogens. One such pathogen is Pseudomonas aeruginosa, which causes opportunistic infections in vulnerable populations, including individuals with cystic fibrosis. CP was previously shown to withhold Fe(II) and Zn(II) from P. aeruginosa and induce Fe and Zn starvation responses in this pathogen. In this work, we performed quantitative, label-free proteomics to further elucidate how CP impacts metal homeostasis pathways in P. aeruginosa. We report that CP induces an incomplete Fe starvation response, as many Fe-containing proteins that are repressed by Fe limitation are not affected by CP treatment. The Zn starvation response elicited by CP seems to be more complete than the Fe starvation response and includes increases in Zn transporters and Zn-independent proteins. CP also induces the expression of membrane-modifying proteins, and metal depletion studies indicate this response results from the sequestration of multiple metals. Moreover, the increased expression of membrane-modifying enzymes upon CP treatment correlates with increased tolerance to polymyxin B. Thus, the response of P. aeruginosa to CP treatment includes both single- and multimetal starvation responses and includes many factors related to virulence potential, broadening our understanding of this pathogen's interaction with the host. IMPORTANCE Transition metal nutrients are critical for growth and infection by all pathogens, and the innate immune system withholds these metals from pathogens to limit their growth in a strategy termed "nutritional immunity." While multimetal depletion by the host is appreciated, the majority of studies have focused on individual metals. Here, we use the innate immune protein calprotectin (CP), which complexes with several metals, including iron (Fe), zinc (Zn), and manganese (Mn), and the opportunistic pathogen Pseudomonas aeruginosa to investigate multimetal starvation. Using an unbiased label-free proteomics approach, we demonstrate that multimetal withholding by CP induces a regulatory response that is not merely additive of individual metal starvation responses, including the induction of lipid A modification proteins.

Heme protects Pseudomonas aeruginosa and Staphylococcus aureus from calprotectin-induced iron starvation.

Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic bacterial pathogens that cause severe infections in immunocompromised individuals and patients with cystic fibrosis. Both P. aeruginosa and S. aureus require iron to infect the mammalian host. To obtain iron, these pathogens may rely on siderophore-mediated ferric iron uptake, ferrous iron uptake, or heme uptake at different points during infection. The preferred iron source depends on environmental conditions, including the presence of iron-sequestering host-defense proteins. Here, we investigate how the presence of heme, a highly relevant iron source during infection, affects bacterial responses to iron withholding by the innate immune protein calprotectin (CP). Prior work has shown that P. aeruginosa is starved of iron in the presence of CP. We report that P. aeruginosa upregulates expression of heme uptake machinery in response to CP. Furthermore, we show that heme protects P. aeruginosa from CP-mediated inhibition of iron uptake and iron-starvation responses. We extend our study to a second bacterial pathogen, S. aureus, and demonstrate that CP also inhibits iron uptake and induces iron-starvation responses by this pathogen. Similarly to P. aeruginosa, we show that heme protects S. aureus from CP-mediated inhibition of iron uptake and iron-starvation responses. These findings expand our understanding of microbial responses to iron sequestration by CP and highlight the importance of heme utilization for bacterial adaptation to host iron-withholding strategies.

Metal Sequestration and Antimicrobial Activity of Human Calprotectin Are pH-Dependent.

Human calprotectin (CP, S100A8/S100A9 oligomer) is an abundant innate immune protein that sequesters transition metal ions in the extracellular space to limit nutrient availability and the growth of invading microbial pathogens. Our current understanding of the metal-sequestering ability of CP is based on biochemical and functional studies performed at neutral or near-neutral pH. Nevertheless, CP can be present throughout the human body and is expressed at infection and inflammation sites that tend to be acidic. Here, we evaluate the metal binding and antimicrobial properties of CP in the pH range of 5.0-7.0. We show that Ca(II)-induced tetramerization, an important process for the extracellular functions of CP, is perturbed by acidic conditions. Moreover, a low pH impairs the antimicrobial activity of CP against some bacterial pathogens, including Staphylococcus aureus and Salmonella enterica serovar Typhimurium. At a mildly acidic pH, CP loses the ability to deplete Mn from microbial growth medium, indicating that Mn(II) sequestration is attenuated under acidic conditions. Evaluation of the Mn(II) binding properties of CP at pH 5.0-7.0 indicates that mildly acidic conditions decrease the Mn(II) binding affinity of the His6 site. Lastly, CP is less effective at preventing capture of Mn(II) by the bacterial solute-binding proteins MntC and PsaA at low pH. These results indicate that acidic conditions compromise the ability of CP to sequester Mn(II) and starve microbial pathogens of this nutrient. This work highlights the importance of considering the local pH of biological sites when describing the interplay between CP and microbes in host-pathogen interactions.

Myeloid-Related Protein 8/14 Participates in the Progression of Experimental Pneumococcal Meningitis by Augmentation of Inflammation.

It has been reported that myeloid-related protein 8/14 (MRP8/14) participates in the progression of inflammation after release from neutrophils and monocytes. This study aimed to clarify the mechanism(s) of the MRP8/14-augmented inflammatory response in mice with pneumococcal meningitis. Streptococcus pneumoniae (SP) meningitis was established by intracerebral injection of SP suspension. Balb/c mice were randomly divided into four groups and received the following injections: phosphate-buffer saline (PBS), MRP8/14 alone, SP alone, and SP plus MRP8/14. At 6 h, 24 h and 48 h postinfection, the clinical disease status was measured by the modified neurological severity score test, body weight loss and degree of cerebral edema; mice were anaesthetized, blood samples and brain samples were collected and brain inflammation was detected by haematoxylin and eosin (HE) staining; tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), C-reactive protein (CRP) and monocyte chemoattractant protein-1 (MCP-1) levels in serum and brain homogenates were assessed by an enzyme-linked immunosorbent assay (ELISA), and the mRNA levels of the above cytokines in brain homogenates were measured by polymerase chain reaction (PCR); and the expression of nuclear factor-kappa B (NF-κB) p65 in brain tissues was determined by immunohistochemical assay. In this study, we identified that MRP8/14 substantially augmented the SP-stimulated inflammatory response, aggravated clinical disease status and exacerbated SP-induced brain edema in a murine model of pneumococcal meningitis. Exogenous administration of MRP8/14 significantly enhanced mRNA and protein expression of the proinflammatory cytokines and chemokines TNF-α, CRP, IL-6 and MCP-1 in brain homogenates and serum from mice with pneumococcal meningitis, which may be related to the NF-κB signalling pathway. We further found that MRP8/14 strongly augmented SP-induced phosphorylation of NF-κB p65 in brain tissue slices from the same model. In conclusion, our results indicated that MRP8/14 augmented the inflammatory response in mice with pneumococcal meningitis and contributed to the development of disease, which was probably through NF-κB signalling pathway activation.

Calprotectin in pregnancy and pregnancy-associated diseases: a systematic review and prospective cohort study.

PURPOSE: Calprotectin, a marker of acute and chronic inflammation, may play a role in pregnancy-associated disorders. We aimed to summarize available clinical data on calprotectin in pregnancy and to establish normal values of calprotectin during the course of pregnancy. METHODS: We performed a systematic review of the databases PubMed and Cochrane Central Register of Controlled Trials to identify experimental and clinical evidence assessing the role of calprotectin in pregnancy. In addition, we performed a prospective cohort study assessing serum and urine calprotectin throughout pregnancy. RESULTS: We identified 17 studies investigating 1638 pregnant women, 151 newborns, and 99 non-pregnant controls, measuring calprotectin in different compartments. Calprotectin was present in meconium and elevated in fecal samples of pregnant women with active inflammatory bowel disease. In women with pregnancy-induced hypertension, mild and severe preeclampsia (PE), calprotectin was significantly elevated in maternal plasma and serum, but not in fetal serum, amniotic fluid, and umbilical cord blood. For the cohort study, we recruited 196 pregnant women. PE and concomitant renal disease were present in 6/196 (3%) and 11/196 (5.6%) of women, respectively. Throughout pregnancy, median serum and urine levels of calprotectin largely exceed reported concentrations of the healthy non-pregnant population, but showed no significant variations between trimesters 1-3 and post-partum. Calprotectin in serum was correlated with systolic blood pressure and in urine with leukocytes and total protein. No significant differences were found in subgroup analyses of smokers vs. non-smokers, PE vs. none, and renal disease (kidney stones, reflux) vs. none. CONCLUSION: Calprotectin concentrations in amnion fluid and stools serve as potential indicators of inflammatory states during pregnancy. Urinary calprotectin concentrations are continuously high during pregnancy and show no significant variations between trimesters 1-3 and post-partum.

Multi-metal Restriction by Calprotectin Impacts De Novo Flavin Biosynthesis in Acinetobacter baumannii.

Calprotectin (CP) inhibits bacterial viability through extracellular chelation of transition metals. However, how CP influences general metabolism remains largely unexplored. We show here that CP restricts bioavailable Zn and Fe to the pathogen Acinetobacter baumannii, inducing an extensive multi-metal perturbation of cellular physiology. Proteomics reveals severe metal starvation, and a strain lacking the candidate ZnII metallochaperone ZigA possesses altered cellular abundance of multiple essential Zn-dependent enzymes and enzymes in de novo flavin biosynthesis. The ΔzigA strain exhibits decreased cellular flavin levels during metal starvation. Flavin mononucleotide provides regulation of this biosynthesis pathway, via a 3,4-dihydroxy-2-butanone 4-phosphate synthase (RibB) fusion protein, RibBX, and authentic RibB. We propose that RibBX ensures flavin sufficiency under CP-induced Fe limitation, allowing flavodoxins to substitute for Fe-ferredoxins as cell reductants. These studies elucidate adaptation to nutritional immunity and define an intersection between metallostasis and cellular metabolism in A. baumannii.

The human innate immune protein calprotectin induces iron starvation responses in Pseudomonas aeruginosa.

Most microbial pathogens have a metabolic iron requirement, necessitating the acquisition of this nutrient in the host. In response to pathogen invasion, the human host limits iron availability. Although canonical examples of nutritional immunity are host strategies that limit pathogen access to Fe(III), little is known about how the host restricts access to another biologically relevant oxidation state of this metal, Fe(II). This redox species is prevalent at certain infection sites and is utilized by bacteria during chronic infection, suggesting that Fe(II) withholding by the host may be an effective but unrecognized form of nutritional immunity. Here, we report that human calprotectin (CP; S100A8/S100A9 or MRP8/MRP14 heterooligomer) inhibits iron uptake and induces an iron starvation response in Pseudomonas aeruginosa cells by sequestering Fe(II) at its unusual His6 site. Moreover, under aerobic conditions in which the Fe(III) oxidation state is favored, Fe(II) withholding by CP was enabled by (i) its ability to stabilize this redox state in solution and (ii) the production and secretion of redox-active, P. aeruginosa-produced phenazines, which reduce Fe(III) to Fe(II). Analyses of the interplay between P. aeruginosa secondary metabolites and CP indicated that Fe(II) withholding alters P. aeruginosa physiology and expression of virulence traits. Lastly, examination of the effect of CP on cell-associated metal levels in diverse human pathogens revealed that CP inhibits iron uptake by several bacterial species under aerobic conditions. This work implicates CP-mediated Fe(II) sequestration as a component of nutritional immunity in both aerobic and anaerobic milieus during P. aeruginosa infection.

High Glucose-Mediated Cytokine Regulation in Gingival Fibroblasts and THP-1 Macrophage: a Possible Mechanism of Severe Periodontitis with Diabetes.

BACKGROUND/AIMS: Diabetic patients are susceptible to severe periodontitis, but the precise mechanism is not fully understood. Aim of this study was to explore the biological pathogenesis of severe periodontitis in diabetic patients focusing on the crosstalk of human gingival fibroblasts (HGFs) and macrophages. METHODS: A total of 70 periodontitis patients with or without diabetes mellitus (DM) were enrolled, and the statistical relationships of diabetic conditions to the periodontal inflammatory parameters were examined by cross-sectional study. In in vitro study, HGFs cell line CRL-2014® (ATCC) and differentiated THP-1 macrophages were cultured with normal glucose (NG: 5.5 mM) or high glucose (HG: 25 mM) condition, and treated with indicated inflammatory factors such as calprotectin (CPT), interleukin (IL)-1ß and IL-6. To examine the effects of HG on soluble IL-6 receptor (sIL-6R) production in THP-1 macrophages, the supernatants were collected and the sIL-6R levels were measured by ELISA. To examine the effects of HG on IL-1ß or IL-6-induced matrix metalloproteinase (MMPs) production in HGFs, the supernatants were collected. Levels of MMP-1 and tissue inhibitor of MMP-1 (TIMP-1) were measured by ELISA. Finally, after conditioned medium (CM) from THP-1 macrophages cultured with NG or HG conditions was collected, HGFs were treated with the CM. The supernatants were collected 24 hours later and the levels of MMP-1 and TIMP-1 were measured. To examine the specific effects of IL-1ß contained in CM on MMP-1 and TIMP-1 production in HGFs, IL-1 receptor antagonist (IL-1ra) was used. RESULTS: There were statistical correlation between IL-1ß and sIL-6R levels in gingival crevicular fluid (GCF) and HbA1c in periodontitis patients with DM (IL-1ß: P=0.035, sIL-6R: P=0.040). HG and CPT significantly induced sIL-6R production in THP-1 macrophages. HG significantly enhanced IL-1ß or IL-6/sIL-6R-induced MMP-1 production in HGFs. The increase of MMP-1 by both IL-1ß and IL-6/sIL-6R was significantly inhibited by specific ERK or IκB inhibitors. Corresponding to the regulation of MMP-1 production, HG condition increased the phosphorylation of p44/42 MAPK and IκBα in HGFs treated with IL-1ß or IL-6/sIL-6R. Finally, MMP-1 production in HGFs cultured with HG increased significantly by CM from THP-1 macrophages cultured with HG. The induction of MMP-1 by the CM from THP-1 macrophages cultured with HG was significantly inhibited by dose dependent of IL-1ra in HGFs cultured with HG. CONCLUSION: Diabetic conditions such as HG induce IL-1ß and sIL-6R production from macrophages in inflammatory periodontal tissues and may exacerbate the periodontitis synergistically via MMP-1 production from HGFs.

Antimicrobial action of calprotectin that does not involve metal withholding.

Calprotectin is a potent antimicrobial that inhibits the growth of pathogens by tightly binding transition metals such as Mn and Zn, thereby preventing their uptake and utilization by invading microbes. At sites of infection, calprotectin is abundantly released from neutrophils, but calprotectin is also present in non-neutrophil cell types that may be relevant to infections. We show here that in patients infected with the Lyme disease pathogen Borreliella (Borrelia) burgdorferi, calprotectin is produced in neutrophil-free regions of the skin, in both epidermal keratinocytes and in immune cells infiltrating the dermis, including CD68 positive macrophages. In culture, B. burgdorferi's growth is inhibited by calprotectin, but surprisingly, the mechanism does not involve the classical withholding of metal nutrients. B. burgdorferi cells exposed to calprotectin cease growth with no reduction in intracellular Mn and no loss in activity of Mn enzymes including the SodA superoxide dismutase. Additionally, there is no obvious loss in intracellular Zn. Rather than metal depletion, we find that calprotectin inhibits B. burgdorferi growth through a mechanism that requires physical association of calprotectin with the bacteria. By comparison, calprotectin inhibited E. coli growth without physically interacting with the microbe, and calprotectin effectively depleted E. coli of intracellular Mn and Zn. Our studies with B. burgdorferi demonstrate that the antimicrobial capacity of calprotectin is complex and extends well beyond simple withholding of metal micronutrients.

miR­185­5p inhibits F­actin polymerization and reverses epithelial mesenchymal transition of human breast cancer cells by modulating RAGE.

In our previous study, advanced glycosylation end­product specific receptor (RAGE) was observed to bind to S100A8/A9 and cause epithelial mesenchymal transition (EMT). The results from target gene prediction revealed that microRNA (miR)­185­5p had a RAGE binding site. However, the function of miR­185­5p in the invasion and migration of breast cancer remains ambiguous. In the present study, the expression of miR­185­5p was examined in breast cancer tissues and cells. Clinical features revealed a negative correlation between miR­185­5p and tumor size, as well as in tumor differentiation and lymph node metastasis in breast cancer. In addition, miR­185­5p was negatively associated with RAGE, and this miRNA reversed the EMT of breast cancer by modulating RAGE in vitro. In addition, miR­185­5p inhibited the S100A8/A9­induced EMT of breast cancer cells by the nuclear factor­κB/Snail signaling pathway. Notably, miR­185­5p upregulation inhibited the F­actin polymerization induced by S100A8/A9 in breast cancer. Furthermore, overexpression of miR­185­5p and reduction of RAGE inhibited lung metastasis node in vivo. Thus, miR­185­5p represents a potential therapeutic target in breast cancer by modulating RAGE.