Formyl Peptide Receptor-1 Blockade Prevents Receptor Regulation by Mitochondrial Danger-Associated Molecular Patterns and Preserves Neutrophil Function After Trauma.

OBJECTIVES: Trauma predisposes to systemic sterile inflammation (systemic inflammatory response syndrome) as well as infection, but the mechanisms linking injury to infection are poorly understood. Mitochondrial debris contains formyl peptides. These bind formyl peptide receptor-1, trafficking neutrophils to wounds, initiating systemic inflammatory response syndrome, and wound healing. Bacterial formyl peptides, however, also attract neutrophils via formyl peptide receptor-1. Thus, mitochondrial formyl peptides might suppress neutrophils antimicrobial function. Also, formyl peptide receptor-1 blockade used to mitigate systemic inflammatory response syndrome might predispose to sepsis. We examined how mitochondrial formyl peptides impact neutrophils functions contributing to antimicrobial responses and how formyl peptide receptor-1 antagonists affect those functions. DESIGN: Prospective study of human and murine neutrophils and clinical cohort analysis. SETTING: University research laboratory and level 1 trauma center. PATIENTS: Trauma patients, volunteer controls. ANIMAL SUBJECTS: C57Bl/6, formyl peptide receptor-1, and formyl peptide receptor-2 knockout mice. INTERVENTIONS: Human and murine neutrophils functions were activated with autologous mitochondrial debris, mitochondrial formyl peptides, or bacterial formyl peptides followed by chemokines or leukotrienes. The experiments were repeated using formyl peptide receptor-1 antagonist cyclosporin H, "designer" human formyl peptide receptor-1 antagonists (POL7178 and POL7200), or anti-formyl peptide receptor-1 antibodies. Mouse injury/lung infection model was used to evaluate effect of formyl peptide receptor-1 inhibition. MEASUREMENTS AND MAIN RESULTS: Human neutrophils cytosolic calcium, chemotaxis, reactive oxygen species production, and phagocytosis were studied before and after exposure to mitochondrial debris, mitochondrial formyl peptides, and bacterial formyl peptides. Mitochondrial formyl peptide and bacterial formyl peptides had similar effects on neutrophils. Responses to chemokines and leukotrienes were suppressed by prior exposure to formyl peptides. POL7200 and POL7178 were specific antagonists of human formyl peptide receptor-1 and more effective than cyclosporin H or anti-formyl peptide receptor-1 antibodies. Formyl peptides inhibited mouse neutrophils responses to chemokines only if formyl peptide receptor-1 was present. Formyl peptide receptor-1 blockade did not inhibit neutrophils bacterial phagocytosis or reactive oxygen species production. Cyclosporin H increased bacterial clearance in lungs after injury. CONCLUSIONS: Formyl peptides both activate and desensitize neutrophils. Formyl peptide receptor-1 blockade prevents desensitization, potentially both diminishing systemic inflammatory response syndrome and protecting the host against secondary infection after tissue trauma or primary infection.

Conformational modulation of the farnesoid X receptor by prenylflavonoids: Insights from hydrogen deuterium exchange mass spectrometry (HDX-MS), fluorescence titration and molecular docking studies.

We report on the molecular interactions of the farnesoid X receptor (FXR) with prenylflavonoids, an emerging class of FXR modulators. FXR is an attractive therapeutic target for mitigating metabolic syndromes (MetS) because FXR activates the inhibitory nuclear receptor, small heterodimer partner (SHP), thereby inhibiting both gluconeogenesis and de novo lipogenesis. We and others have shown that xanthohumol (XN), the principal prenylflavonoid of the hop plant (Humulus lupulus L.), is a FXR agonist based on its ability to affect lipid and glucose metabolism in vivo and to induces FXR target genes in biliary carcinoma cells and HEK293 cells. However, studies are currently lacking to rationalize the molecular mechanisms of FXR modulation by prenylflavonoids. We addressed this deficiency and report the first systematic study of FXR prenylflavonoid interactions. We combined hydrogen deuterium exchange mass spectrometry (HDX-MS) with computational studies for dissecting molecular recognition and conformational impact of prenylflavonoid interactions on the ligand binding domain (LBD) of human FXR. Four prenylflavonoids were tested: xanthohumol, a prenylated chalcone, two prenylated flavonones, namely isoxanthohumol (IX) and 8-prenylnaringenin (8PN), and a semisynthetic prenylflavonoid derivative, tetrahydroxanthohumol (TX). Enhancement of the HDX protection profile data by in silico predicted models of FXR prenylflavonoid complexes resulted in mapping of the prenylflavonoid interactions within the canonical ligand binding pocket. Our findings provide a foundation for the exploration of the chemical scaffolds of prenylated chalcones and flavanones as leads for future structure activity studies of this important nuclear receptor with potential relevance for ameliorating lipid metabolic disorders associated with obesity and MetS.

Using liquid smoke to control infestations of the ham mite, Tyrophagus putrescentiae, on dry-cured hams during aging.

Eight treatments of edible coatings and nets including liquid smoke (SP and 24P) and xanthan gum (XG) were used to evaluate their effectiveness at controlling mite growth on dry-cured hams. Mite growth was controlled (P < 0.05) in both coating and netting treatments of 1% SP + 1% XG. Increasing SP concentration from 1% to 2% in the SP only treatments without XG did not control mite growth (P > 0.05) in the coating but controlled mite growth (P < 0.05) when infused in the nets. Both coating and netting treatments with 2% 24P + 1% XG controlled mite growth (P < 0.05), and ham cubes with 1% and 2% 24P in infused nets had mite numbers of 4.6 and 9.4, respectively. SP did not impact the sensory attributes of the ham. Results indicate that liquid smoke can potentially be added in coatings or ham nets to control mites and used in an integrated pest management program for dry-cured hams.

Regulation of antimicrobial peptide gene expression by nutrients and by-products of microbial metabolism.

BACKGROUND: Antimicrobial peptides (AMPs) are synthesized and secreted by immune and epithelial cells that are constantly exposed to environmental microbes. AMPs are essential for barrier defense, and deficiencies lead to increased susceptibility to infection. In addition to their ability to disrupt the integrity of bacterial, viral and fungal membranes, AMPs bind lipopolysaccharides, act as chemoattractants for immune cells and bind to cellular receptors and modulate the expression of cytokines and chemokines. These additional biological activities may explain the role of AMPs in inflammatory diseases and cancer. Modulating the endogenous expression of AMPs offers potential therapeutic treatments for infection and disease. METHODS: The present review examines the published data from both in vitro and in vivo studies reporting the effects of nutrients and by-products of microbial metabolism on the expression of antimicrobial peptide genes in order to highlight an emerging appreciation for the role of dietary compounds in modulating the innate immune response. RESULTS: Vitamins A and D, dietary histone deacetylases and by-products of intestinal microbial metabolism (butyrate and secondary bile acids) have been found to regulate the expression of AMPs in humans. Vitamin D deficiency correlates with increased susceptibility to infection, and supplementation studies indicate an improvement in defense against infection. Animal and human clinical studies with butyrate indicate that increasing expression of AMPs in the colon protects against infection. CONCLUSION: These findings suggest that diet and/or consumption of nutritional supplements may be used to improve and/or modulate immune function. In addition, by-products of gut microbe metabolism could be important for communicating with intestinal epithelial and immune cells, thus affecting the expression of AMPs. This interaction may help establish a mucosal barrier to prevent invasion of the intestinal epithelium by either mutualistic or pathogenic microorganisms.

Cecal microbiota contribute to the development of woody breast myopathy.

The objective of this study was to characterize the bacterial diversity of cecal microbiota in broilers related to breast phenotype, diet, and genetic strain. Broilers from 2 genetic strains (120 birds/strain) were fed a control diet (15 birds/pen) and an amino acid reduced diet (15 birds/pen, digestible lysine, total sulfur amino acids, and threonine reduced by 20% compared to the control diet). At 8 wk of age, 4 male broilers with normal breast (NB, 1 chick per pen) and 4 male broilers with woody breast (WB, 1 chick per pen) were selected for each treatment (strain × diet). The DNA of cecal samples was extracted and the 16S rRNA genes were sequenced and analyzed. There were no differences (P > 0.05) in the alpha diversity of gut microbiota between 2 phenotypes (NB vs. WB), 2 strains, or 2 diets (control vs. reduced). However, principal coordinate analysis plots (beta diversity) revealed that there were composition differences in samples between the 2 phenotypes (P = 0.001) and the 2 diets (P = 0.024). The most abundant phyla in all samples were Firmicutes, followed by Bacteroidetes and Proteobacteria. There were differences (false discovery rate, FDR < 0.05) in bacterial relative abundance between phenotypes and between diet treatments, but not (FDR > 0.05) between the 2 genetic strains. Selenomonas bovis (12.6%) and Bacteroides plebeius (12.3%) were the top 2 predominant bacteria in the ceca of WB birds; however, the relative abundances of these 2 bacteria were only 5.1% and 1.2% in NB birds, respectively. Function analysis predicted that the metabolic activities differed (q < 0.05) only between phenotypes. The microbiota of WB birds was characterized as reduced glycolysis and urea cycle but increased tricarboxylic acid (TCA) cycles, sugar degradation, and purine and pyrimidine nucleotides biosynthesis. Further studies are needed to investigate if WB incidence could be reduced by regulating gut microbiota and the potential mechanism that leads to decreased WB incidence.

Broiler genetics influences proteome profiles of normal and woody breast muscle.

Wooden or woody breast (WB) is a myopathy of the pectoralis major in fast-growing broilers that influences the quality of breast meat and causes an economic loss in the poultry industry. The objective of this study was to evaluate growth and proteome differences between 5 genetic strains of broilers that yield WB and normal breast (NB) meat. Eight-week-old broilers were evaluated for the WB myopathy and divided into NB and WB groups. Differential expression of proteins was analyzed using 2-dimensional gel electrophoresis and LC-MS/MS to elucidate the mechanism behind the breast myopathy because of the genetic backgrounds of the birds. The percentages of birds with WB were 61.3, 68.8, 46.9, 45.2, and 87.5% for strains 1-5, respectively, indicating variability in WB myopathy among broiler strains. Birds from strains 1, 3, and 5 in the WB group were heavier than those in the NB group (P < 0.05). Woody breast meat from all strains were heavier than NB meat (P < 0.05). Within WB, strain 5 had a greater breast yield than strains 1, 3, and 4 (P < 0.0001). Woody breast from strains 2, 3, 4, and 5 had a greater breast yield than NB (P < 0.05). Six proteins were more abundant in NB of strain 5 than those of strains 2, 3, and 4, and these proteins were related to muscle growth, regeneration, contraction, apoptosis, and oxidative stress. Within WB, 14 proteins were differentially expressed between strain 5 and other strains, suggesting high protein synthesis, weak structural integrity, intense contraction, and oxidative stress in strain 5 birds. The differences between WB from strain 3 and strains 1, 2, and 4 were mainly glycolytic. In conclusion, protein profiles of broiler breast differed because of both broiler genetics and the presence of WB myopathy.

A mouse model for vitamin D-induced human cathelicidin antimicrobial peptide gene expression.

In humans and other primates, 1,25(OH)2vitamin D3 regulates the expression of the cathelicidin antimicrobial peptide (CAMP) gene via toll-like receptor (TLR) signaling that activates the vitamin D pathway. Mice and other mammals lack the vitamin D response element (VDRE) in their CAMP promoters. To elucidate the biological importance of this pathway, we generated transgenic mice that carry a genomic DNA fragment encompassing the entire human CAMP gene and crossed them with Camp knockout (KO) mice. We observed expression of the human transgene in various tissues and innate immune cells. However, in mouse CAMP transgenic macrophages, TLR activation in the presence of 25(OH)D3 did not induce expression of either CAMP or CYP27B1 as would normally occur in human macrophages, reinforcing important species differences in the actions of vitamin D. Transgenic mice did show increased resistance to colonization by Salmonella typhimurium in the gut. Furthermore, the human CAMP gene restored wound healing in the skin of Camp KO mice. Topical application of 1,25(OH)2vitamin D3 to the skin of CAMP transgenic mice induced CAMP expression and increased killing of Staphylococcus aureus in a wound infection model. Our model can help elucidate the biological importance of the vitamin D-cathelicidin pathway in both pathogenic and non-pathogenic states.

Technological characteristics of pre- and post-rigor deboned beef mixtures from Holstein steers and quality attributes of cooked beef sausage.

The objective of this study was to determine the effects of deboning time (pre- and post-rigor), processing steps (grinding - GB; salting - SB; batter formulation - BB), and storage time on the quality of raw beef mixtures and vacuum-packaged cooked sausage, produced using a commercial formulation with 0.25% phosphate. The pH was greater in pre-rigor GB and SB than in post-rigor GB and SB (P < .001). However, deboning time had no effect on metmyoglobin reducing activity, cooking loss, and color of raw beef mixtures. Protein solubility of pre-rigor beef mixtures (124.26 mg/kg) was greater than that of post-rigor beef (113.93 mg/kg; P = .071). TBARS were increased in BB but decreased during vacuum storage of cooked sausage (P ≤ .018). Except for chewiness and saltiness being 52.9 N-mm and 0.3 points greater in post-rigor sausage (P = .040 and 0.054, respectively), texture profile analysis and trained panelists detected no difference in texture between pre- and post-rigor sausage.

Effect of deboning time on the growth of Salmonella, E. coli, aerobic, and lactic acid bacteria during beef sausage processing and storage.

The objective of the current study was to determine the effects of deboning time, three steps of sausage processing (grinding, salting, and batter formulation), and storage time (of raw materials and cooked sausage) on the growth (log CFU/g) of aerobic bacteria, lactic acid bacteria, and inoculated Salmonella and E. coli. Beef deboning time did not influence bacterial counts (P≥0.138). However, salting of raw ground beef resulted in a 0.4-log reduction in both aerobic plate count (APC) and Salmonella (P≤0.001). Lactic acid bacteria were increased from non-detectable concentration (0.54 log) on d 0 to 3.8 log on d 120 of vacuum storage (P≤0.019). Salmonella counts were increased (P<0.001) over storage time (3.2 to 3.3 log CFU/g from d 0 to 10). Results indicated that salting and batter formulation had a greater impact on bacterial counts than rigor state of raw beef.

A subset of five human mitochondrial formyl peptides mimics bacterial peptides and functionally deactivates human neutrophils.

BACKGROUND: Trauma causes inflammation by releasing mitochondria that act as Danger-Associated Molecular Patterns (DAMPs). Trauma also increases susceptibility to infection. Human mitochondria contain 13 N-formyl peptides (mtFPs). We studied whether mtFPs released into plasma by clinical injury induce neutrophil (PMN) inflammatory responses, whether their potency reflects their similarity to bacterial FPs and how their presence at clinically relevant concentration affects PMN function. METHODS: N-terminal sequences of the 13 mtFPs were synthesized. Changes in human PMN cytosolic Ca concentration ([Ca]i) and chemotactic responses to mtFPs were studied. Sequence similarity of mtFPs to the canonical bacterial peptide f-Met-Leu-Phe (fMLF/fMLP) was studied using the BLOcks SUbstitution Matrix 62 (BLOSUM 62) system. The presence of mtFPs in plasma of trauma patients was assayed by Enzyme-linked immunosorbent assay (ELISA). The effects of the most potent mtFP (ND6) on PMN signaling and function were then studied at ambient clinical concentrations by serial exposure of native PMN to ND6, chemokines and leukotrienes. RESULTS: Five mtFPs (ND6, ND3, ND4, ND5, and Cox 1) induced [Ca]i flux and chemotaxis in descending order of potency. Evolutionary similarity to fMLF predicted [Ca]i flux and chemotactic potency linearly (R = 0.97, R = 0.95). Chemoattractant potency was also linearly related to [Ca]i flux induction (R = 0.92). Active mtFPs appear to circulate in significant amounts immediately after trauma and persist through the first week. The most active mtFP, ND6, suppresses responses to physiologic alveolar chemoattractants (CXCL-1, leukotriene B4) as well as to fMLF where CXCL-1 and leukotriene B4 do not suppress N-formyl peptide receptor (FPR)-1 responses to mtFPs. Prior FPR-1 inhibition rescues PMN from heterologous suppression of CXCR-1 and BLT-1 by mtFPs. CONCLUSION: The data suggest mtFPs released by injured tissue may attract PMN to trauma sites while suppressing PMN responses to other chemoattractants. Inhibition of mtFP-FPR1 interactions might increase PMN recruitment to lung bacterial inoculation after trauma. These findings suggest new paradigms for preventing infections after trauma. LEVEL OF EVIDENCE: Therapeutic, Level IV.