Effects of short-chain fatty acid-butyrate supplementation on expression of circadian-clock genes, sleep quality, and inflammation in patients with active ulcerative colitis: a double-blind randomized controlled trial.

BACKGROUND: The regulation of the circadian clock genes, which coordinate the activity of the immune system, is disturbed in inflammatory bowel disease (IBD). Emerging evidence suggests that butyrate, a short-chain fatty acid produced by the gut microbiota is involved in the regulation of inflammatory responses as well as circadian-clock genes. This study was conducted to investigate the effects of sodium-butyrate supplementation on the expression of circadian-clock genes, inflammation, sleep and life quality in active ulcerative colitis (UC) patients. METHODS: In the current randomized placebo-controlled trial, 36 active UC patients were randomly divided to receive sodium-butyrate (600 mg/kg) or placebo for 12-weeks. In this study the expression of circadian clock genes (CRY1, CRY2, PER1, PER2, BMAl1 and CLOCK) were assessed by real time polymerase chain reaction (qPCR) in whole blood. Gene expression changes were presented as fold changes in expression (2^-ΔΔCT) relative to the baseline. The faecal calprotectin and serum level of high-sensitivity C-reactive protein (hs-CRP) were assessed by enzyme-linked immunosorbent assay method (ELIZA). Moreover, the sleep quality and IBD quality of life (QoL) were assessed by Pittsburgh sleep quality index (PSQI) and inflammatory bowel disease questionnaire-9 (IBDQ-9) respectively before and after the intervention. RESULTS: The results showed that sodium-butyrate supplementation in comparison with placebo significantly decreased the level of calprotectin (-133.82 ± 155.62 vs. 51.58 ± 95.57, P-value < 0.001) and hs-CRP (-0.36 (-1.57, -0.05) vs. 0.48 (-0.09-4.77), P-value < 0.001) and upregulated the fold change expression of CRY1 (2.22 ± 1.59 vs. 0.63 ± 0.49, P-value < 0.001), CRY2 (2.15 ± 1.26 vs. 0.93 ± 0.80, P-value = 0.001), PER1 (1.86 ± 1.77 vs. 0.65 ± 0.48, P-value = 0.005), BMAL1 (1.85 ± 0.97 vs. 0.86 ± 0.63, P-value = 0.003). Also, sodium-butyrate caused an improvement in the sleep quality (PSQI score: -2.94 ± 3.50 vs. 1.16 ± 3.61, P-value < 0.001) and QoL (IBDQ-9: 17.00 ± 11.36 vs. -3.50 ± 6.87, P-value < 0.001). CONCLUSION: Butyrate may be an effective adjunct treatment for active UC patients by reducing biomarkers of inflammation, upregulation of circadian-clock genes and improving sleep quality and QoL.

Post-translational modifications on the metal-sequestering protein calprotectin.

Human calprotectin (CP, S100A8/S100A9 oligomer) is an abundant neutrophil protein that contributes to innate immunity by sequestering nutrient metal ions in the extracellular space. This process starves invading microbial pathogens of essential metal nutrients, which can inhibit growth and colonization. Over the past decade, fundamental and clinical studies have revealed that the S100A8 and S100A9 subunits of CP exhibit a variety of post-translational modifications (PTMs). This review summarizes PTMs on the CP subunits that have been detected and highlights two recent studies that evaluated the structural and functional consequences of methionine and cysteine oxidation on CP. Collectively, these investigations indicate that the molecular speciation of extracellular CP is complex and composed of multiple proteoforms. Moreover, PTMs may impact biological function and the lifetime of the protein. It is therefore important that post-translationally modified CP species receive consideration and integration into the current working model for how CP functions in nutritional immunity.

Prostaglandins and calprotectin are genetically and functionally linked to the Inflammatory Bowel Diseases.

BACKGROUND: Genome wide association studies (GWAS) have identified and validated more than 200 genomic loci associated with the inflammatory bowel disease (IBD), although for most the causal gene remains unknown. Given the importance of myeloid cells in IBD pathogenesis, the current study aimed to uncover the role of genes within IBD genetic loci that are endogenously expressed in this cell lineage. METHODS: The open reading frames (ORF) of 42 genes from IBD-associated loci were expressed via lentiviral transfer in the THP-1 model of human monocytes and the impact of each of these on the cell's transcriptome was analyzed using a RNA sequencing-based approach. We used a combination of genetic and pharmacologic approaches to validate our findings in the THP-1 line with further validation in human induced pluripotent stem cell (hiPSC)-derived-monocytes. RESULTS: This functional genomics screen provided evidence that genes in four IBD GWAS loci (PTGIR, ZBTB40, SLC39A11 and NFKB1) are involved in controlling S100A8 and S100A9 gene expression, which encode the two subunits of calprotectin (CP). We demonstrated that increasing PTGIR expression and/or stimulating PTGIR signaling resulted in increased CP expression in THP-1. This was further validated in hiPSC-derived monocytes. Conversely, knocking-down PTGIR endogenous expression and/or inhibiting PTGIR signaling led to decreased CP expression. These analyses were extended to the known IBD gene PTGER4, whereby its specific agonist also led to increased CP expression. Furthermore, we demonstrated that the PTGIR and PTGER4 mediated control of CP expression was dependent on signaling via adenylate cyclase and STAT3. Finally, we demonstrated that LPS-mediated increases in CP expression could be potentiated by agonists of PTGIR and PTGER4, and diminished by their antagonists. CONCLUSION: Our results support a causal role for the PTGIR, PTGER4, ZBTB40, SLC39A11 and NFKB1 genes in IBD, with all five genes regulating the expression of CP in myeloid cells, as well as potential roles for the prostacyclin/prostaglandin biogenesis and signaling pathways in IBD susceptibility and pathogenesis.

Mn uptake system affects the virulence of Streptococcus suis by mediating oxidative stress.

Manganese (Mn) is an important micronutrient that is not readily available to pathogens during infection. Hosts resist the invasion of pathogens through nutritional immunity and oxidative stress. To overcome this nutrient restriction, bacteria utilize high affinity transporters to compete with nutrient-binding proteins (e.g., calprotectin). Little is known about the role of Mn in the pathophysiology of Streptococcus suis. Here, we revealed that the tolerance of S. suis to calprotectin and oxidative stress was associated with Mn. Inactivation of Mn uptake system, TroABCD, in S. suis decreased the tolerance to calprotectin and oxidative stress. Furthermore, Mn uptake system mutant strains reduced capacity for bacterial cellular survival, and attenuated virulence in a mouse model. To explore the regulatory mechanism, we determined the transcriptional start site of troABCD using capping rapid amplification of cDNA ends. Furthermore, we revealed that TroR was a transcriptional regulatory repressor of troABCD. In the absence of troR, transcription levels of troA, troB, troC, and troD were not inhibited by low or high Mn levels, and intracellular Mn contents of mutant strains were higher than that of the wild-type strain. Finally, we used electrophoretic mobility shift assay to demonstrate that TroR bound the promoter region of troABCD. Collectively, this study revealed that Mn acquisition was essential for pathogenesis of S. suis and Mn uptake systems should be targets for the development of new antimicrobials.

The Innate Immune Protein Calprotectin Interacts With and Encases Biofilm Communities of Pseudomonas aeruginosa and Staphylococcus aureus.

Calprotectin is a transition metal chelating protein of the innate immune response known to exert nutritional immunity upon microbial infection. It is abundantly released during inflammation and is therefore found at sites occupied by pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus. The metal limitation induced by this protein has previously been shown to mediate P. aeruginosa and S. aureus co-culture. In addition to the transition metal sequestration role of calprotectin, it has also been shown to have metal-independent antimicrobial activity via direct cell contact. Therefore, we sought to assess the impact of this protein on the biofilm architecture of P. aeruginosa and S. aureus in monomicrobial and polymicrobial culture. The experiments described in this report reveal novel aspects of calprotectin's interaction with biofilm communities of P. aeruginosa and S. aureus discovered using scanning electron microscopy and confocal laser scanning microscopy. Our results indicate that calprotectin can interact with microbial cells by stimulating encapsulation in mesh-like structures. This physical interaction leads to compositional changes in the biofilm extracellular polymeric substance (EPS) in both P. aeruginosa and S. aureus.

Effects of ivacaftor on systemic inflammation and the plasma proteome in people with CF and G551D.

BACKGROUND: Ivacaftor is a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator for people with CF and the G551D mutation. We aimed to investigate the biology of CFTR modulation and systemic effects of CFTR restoration by examining changes in circulating measurements of inflammation and growth and novel proteins with ivacaftor treatment. METHODS: Blood samples from 64 CF subjects with G551D-CFTR were analyzed for inflammatory and growth-related proteins at baseline, 1 and 6 months after ivacaftor initiation. In 30 subjects, plasma was assayed for 1,322 proteins using the SomaScan proteomic platform at baseline and 6 months post-ivacaftor. Correlations with clinical outcomes were assessed. MEASUREMENTS AND MAIN RESULTS: Significant reductions in high mobility group box-1 protein (HMGB-1), calprotectin, serum amyloid A, and granulocyte colony-stimulating factor (G-CSF), and an increase in insulin-like growth factor (IGF-1) occurred 1 month after ivacaftor. This treatment effect was sustained at 6 months for HMGB-1 and calprotectin. Correcting for multiple comparisons in the proteomic analysis, 9 proteins (albumin, afamin, leptin, trypsin, pancreatic stone protein [PSP], pituitary adenylate cyclase-activating polypeptide-38, repulsive guidance molecule A [RGMA], calreticulin, GTPase KRas) changed significantly with ivacaftor. Proteins changing with treatment are involved in lipid digestion and transport and extracellular matrix organization biological processes. Reductions in calprotectin and G-CSF and increases in calreticulin, and RGMA correlated with improved lung function, while increasing IGF-1, leptin and afamin and decreasing PSP correlated with increased weight. CONCLUSIONS: Ivacaftor led to changes in inflammatory, lipid digestion, and extracellular matrix proteins, lending insights into the extrapulmonary effects of CFTR modulation.

Formation of Calprotectin-Derived Peptides in the Airways of Children with Cystic Fibrosis.

Calprotectin is released by activated neutrophils along with myeloperoxidase (MPO) and proteases. It plays numerous roles in inflammation and infection, and is used as an inflammatory biomarker. However, calprotectin is readily oxidized by MPO-derived hypohalous acids to form covalent dimers of its S100A8 and S100A9 subunits. The dimers are susceptible to degradation by proteases. We show that detection of human calprotectin by ELISA declines markedly because of its oxidation by hypochlorous acid and subsequent degradation. Also, proteolysis liberates specific peptides from oxidized calprotectin that is present at inflammatory sites. We identified six calprotectin-derived peptides by mass spectrometry and detected them in the bronchoalveolar lavage fluid of children with cystic fibrosis (CF). We assessed the peptides as biomarkers of neutrophilic inflammation and infection. The content of the calprotectin peptide ILVI was related to calprotectin (r = 0.72, p = 0.01, n = 10). Four of the peptides were correlated with the concentration of MPO (r > 0.7, p ≤ 0.01, n = 21), while three were higher (p < 0.05) in neutrophil elastase-positive (n = 14) than -negative samples (n = 7). Also, five of the peptides were higher (p < 0.05) in the bronchoalveolar lavage fluid from children with CF with infections (n = 21) than from non-CF children without infections (n = 6). The specific peptides liberated from calprotectin will signal uncontrolled activity of proteases and MPO during inflammation. They may prove useful in tracking inflammation in respiratory diseases dominated by neutrophils, including coronavirus disease 2019.

Molecular Basis of Ca(II)-Induced Tetramerization and Transition-Metal Sequestration in Human Calprotectin.

Human calprotectin (CP, S100A8/S100A9 oligomer, MRP8/MRP14 oligomer) is an abundant innate immune protein that contributes to the host metal-withholding response. Its ability to sequester transition metal nutrients from microbial pathogens depends on a complex interplay of Ca(II) binding and self-association, which converts the αß heterodimeric apo protein into a Ca(II)-bound (αß)2 heterotetramer that displays enhanced transition metal affinities, antimicrobial activity, and protease stability. A paucity of structural data on the αß heterodimer has hampered molecular understanding of how Ca(II) binding enables CP to exert its metal-sequestering innate immune function. We report solution NMR data that reveal how Ca(II) binding affects the structure and dynamics of the CP αß heterodimer. These studies provide a structural model in which the apo αß heterodimer undergoes conformational exchange and switches between two states, a tetramerization-incompetent or "inactive" state and a tetramerization-competent or "active" state. Ca(II) binding to the EF-hands of the αß heterodimer causes the active state to predominate, resulting in self-association and formation of the (αß)2 heterotetramer. Moreover, Ca(II) binding causes local and allosteric ordering of the His3Asp and His6 metal-binding sites. Ca(II) binding to the noncanonical EF-hand of S100A9 positions (A9)D30 and organizes the His3Asp site. Remarkably, Ca(II) binding causes allosteric effects in the C-terminal region of helix αIV of S100A9, which stabilize the α-helicity at positions H91 and H95 and thereby organize the functionally versatile His6 site. Collectively, this study illuminates the molecular basis for how CP responds to high extracellular Ca(II) concentrations, which enables its metal-sequestering host-defense function.

Decrease in Mucosal IL17A, IFNγ and IL10 Expressions in Active Crohn's Disease Patients Treated with High-Dose Vitamin Alone or Combined with Infliximab.

BACKGROUND: Vitamin D treatment may reduce Crohn's disease (CD) activity by modulating the mucosal immune function. We investigated if high-dose vitamin D +/- infliximab modulated the mucosal cytokine expression in active CD. METHODS: Forty CD patients were randomized into: infliximab + vitamin D; infliximab + placebo-vitamin D; placebo-infliximab + vitamin D or placebo-infliximab + placebo-vitamin D. Infliximab (5 mg/kg) and placebo-infliximab were administered at weeks 0, 2 and 6. Oral vitamin D was administered as bolus 200,000 international units (IU) per week 0 followed by 20,000 IU/day for 7 weeks or placebo. Endoscopy with biopsies was performed at weeks 0 and 7 where endoscopic activity was measured and mucosal mRNA cytokine expression was examined. C-reactive protein (CRP), fecal calprotectin and Harvey-Bradshaw Index (HBI) were measured at weeks 0, 2 and 6. RESULTS: High-dose vitamin D treatment alone and combined with infliximab decreased the IL17A, IFNγ and IL10 expression. High-dose vitamin D alone did not significantly decrease the disease activity, CRP or calprotectin. Combined infliximab and vitamin D treatment was not clinically significantly superior to monotherapy with infliximab. CONCLUSIONS: High-dose vitamin D as monotherapy and combined with infliximab decreases IL17A, IFNγ and IL-10 expression in mucosa within treatment groups. This did not induce a statistically significant decreased disease activity. EudraCT no.2013-000971-34.

Identification of Zinc-Dependent Mechanisms Used by Group B Streptococcus To Overcome Calprotectin-Mediated Stress.

Nutritional immunity is an elegant host mechanism used to starve invading pathogens of necessary nutrient metals. Calprotectin, a metal-binding protein, is produced abundantly by neutrophils and is found in high concentrations within inflammatory sites during infection. Group B Streptococcus (GBS) colonizes the gastrointestinal and female reproductive tracts and is commonly associated with severe invasive infections in newborns such as pneumonia, sepsis, and meningitis. Although GBS infections induce robust neutrophil recruitment and inflammation, the dynamics of GBS and calprotectin interactions remain unknown. Here, we demonstrate that disease and colonizing isolate strains exhibit susceptibility to metal starvation by calprotectin. We constructed a mariner transposon (Krmit) mutant library in GBS and identified 258 genes that contribute to surviving calprotectin stress. Nearly 20% of all underrepresented mutants following treatment with calprotectin are predicted metal transporters, including known zinc systems. As calprotectin binds zinc with picomolar affinity, we investigated the contribution of GBS zinc uptake to overcoming calprotectin-imposed starvation. Quantitative reverse transcriptase PCR (qRT-PCR) revealed a significant upregulation of genes encoding zinc-binding proteins, adcA, adcAII, and lmb, following calprotectin exposure, while growth in calprotectin revealed a significant defect for a global zinc acquisition mutant (ΔadcAΔadcAIIΔlmb) compared to growth of the GBS wild-type (WT) strain. Furthermore, mice challenged with the ΔadcAΔadcAIIΔlmb mutant exhibited decreased mortality and significantly reduced bacterial burden in the brain compared to mice infected with WT GBS; this difference was abrogated in calprotectin knockout mice. Collectively, these data suggest that GBS zinc transport machinery is important for combatting zinc chelation by calprotectin and establishing invasive disease.IMPORTANCE Group B Streptococcus (GBS) asymptomatically colonizes the female reproductive tract but is a common causative agent of meningitis. GBS meningitis is characterized by extensive infiltration of neutrophils carrying high concentrations of calprotectin, a metal chelator. To persist within inflammatory sites and cause invasive disease, GBS must circumvent host starvation attempts. Here, we identified global requirements for GBS survival during calprotectin challenge, including known and putative systems involved in metal ion transport. We characterized the role of zinc import in tolerating calprotectin stress in vitro and in a mouse model of infection. We observed that a global zinc uptake mutant was less virulent than the parental GBS strain and found calprotectin knockout mice to be equally susceptible to infection by wild-type (WT) and mutant strains. These findings suggest that calprotectin production at the site of infection results in a zinc-limited environment and reveals the importance of GBS metal homeostasis to invasive disease.

Ascitic Calprotectin as an early predictor of hepatocellular carcinoma in patients with cirrhotic ascites.

BACKGROUND: Calprotectin is a heterodimer formed by S100A8 and S100A9 proteins which are enhanced during hepatic carcinogenesis and the increased expression of both proteins promotes malignant progression of hepatocellular carcinoma. The potential correlation between ascitic Calprotectin and HCC was not studied. METHODS: 100 patients were stratified into a case group which enrolled 50 patients with cirrhotic ascites and documented HCC and a control group consisted of 50 patients with cirrhotic ascites without HCC. They were evaluated by liver function tests, abdominal ultrasound and routine ascitic fluid examination including ascetic Calprotectin and results were validated in another group (n = 100). RESULTS: Calprotectin level was significantly higher in the HCC group with insignificant difference regarding total cell count, PNLs, ascitic albumin, LDH, CEA and SAAG. It correlated with serum creatinine (r = 0.245, p = 0.014) and number of focal hepatic lesions (r = 0.309, p = 0.002). In the validation group, 28 patients had elevated ascitic Calprotectin of which 21 patients had developed HCC (75%) after a mean period of 3.8 ± 1.54 months. A cut of value 126 ng/ml was accurate to predict HCC in liver cirrhosis with ascites with a sensitivity of 93.3% specificity 94%, AUC 0.950, Youden's J value = 0.873, p = 0.0001. CONCLUSION: Ascitic Calprotectin may offer an easy, affordable marker that can predict the early occurrence of HCC.

Group A Streptococcus AdcR Regulon Participates in Bacterial Defense against Host-Mediated Zinc Sequestration and Contributes to Virulence.

Colonization by pathogenic bacteria depends on their ability to overcome host nutritional defenses and acquire nutrients. The human pathogen group A streptococcus (GAS) encounters the host defense factor calprotectin (CP) during infection. CP inhibits GAS growth in vitro by imposing zinc (Zn) limitation. However, GAS counterstrategies to combat CP-mediated Zn limitation and the in vivo relevance of CP-GAS interactions to bacterial pathogenesis remain unknown. Here, we report that GAS upregulates the AdcR regulon in response to CP-mediated Zn limitation. The AdcR regulon includes genes encoding Zn import (adcABC), Zn sparing (rpsN.2), and Zn scavenging systems (adcAII, phtD, and phtY). Each gene in the AdcR regulon contributes to GAS Zn acquisition and CP resistance. The ΔadcC and ΔrpsN.2 mutant strains were the most susceptible to CP, whereas the ΔadcA, ΔadcAII, and ΔphtD mutant strains displayed less CP sensitivity during growth in vitro However, the ΔphtY mutant strain did not display an increased CP sensitivity. The varied sensitivity of the mutant strains to CP-mediated Zn limitation suggests distinct roles for individual AdcR regulon genes in GAS Zn acquisition. GAS upregulates the AdcR regulon during necrotizing fasciitis infection in WT mice but not in S100a9-/- mice lacking CP. This suggests that CP induces Zn deficiency in the host. Finally, consistent with the in vitro results, several of the AdcR regulon genes are critical for GAS virulence in WT mice, whereas they are dispensable for virulence in S100a9-/- mice, indicating the direct competition for Zn between CP and proteins encoded by the GAS AdcR regulon during infection.

In Vitro Inhibitory Effect of Recombinant Human Calprotectin on Nalm6 Leukemia Cell Line.

BACKGROUND & PURPOSE: In evaluating new drugs for the treatment of various types of cancer, investigations have been made to discover a variety of anti-tumor compounds with less side effects on normal cells. Investigations have shown that the heterodimers S100A8 and S100A9 inhibit the enzyme casein kinase 2 and then prevent the activation of the E7 oncoprotein. Therefore, the aim of this study was to evaluate the effect of calprotectin as an antitumor compound on the Nalm6 (B cell precursor leukemia cell line). MATERIALS & METHODS: Transformation of genes encoding S100A8 and S100A9 human, designed in the pQE32 plasmid, was performed by the thermal shock method into E. coli M15 bacteria. After bacterial growth in LB medium, the expression of two S100A8 and S100A9 subunits, the solubility of the protein by SDS-PAGE method was determined. Finally, the S100A8 / A9 complex was equally placed in the microtube. In the next step, the cytotoxic effects of calprotectin produced on the Nalm6 cell line were evaluated using the wst1 test. Then, the apoptosis in these cells was measured using flow cytometry methods with Annexin-V coloration. RESULTS: In the current study, the results showed that the cytotoxic effects of Calprotectin are time and concentration- dependent. Therefore, it can reduce the tumor expression and had a beneficial effect by induced apoptosis in Nalm6 cell line. CONCLUSION: Calprotectin has an anti-tumor effect on the Nalm6 cell line by increasing apoptosis.

The effects of a multispecies synbiotic on microbiome-related side effects of long-term proton pump inhibitor use: A pilot study.

Side effects of proton pump inhibitors (PPI) can be linked to the changes in the intestinal microbiome that occur during therapy, especially in long-term users. Therefore, the microbiome might also be a key player in the reduction of PPI side effects. We tested the effects of a three-month intervention with a multispecies synbiotic on intestinal inflammation, gut barrier function, microbiome composition, routine laboratory parameters and quality of life in patients with long-term PPI therapy. Thirty-six patients received a daily dose of a multispecies synbiotic for three months and were clinically observed without intervention for another three months. After intervention 17% of patients reached normal calprotectin levels; the overall reduction did not reach statistical significance (-18.8 ng/mg; 95%CI: -50.5; 12.9, p = 0.2). Elevated zonulin levels could be significantly reduced (-46.3 ng/mg; 95%CI: -71.4; -21.2; p < 0.001). The abundance of Stomatobaculum in the microbiome was reduced and Bacillus increased during the intervention. Furthermore, albumin, alkaline phosphatase and thrombocyte count were significantly increased and aspartate transaminase was significantly decreased during intervention. Gastrointestinal quality of life showed significant improvements. In conclusion, microbiome-related side effects of long-term PPI use can be substantially reduced by synbiotic intervention. Further studies are warranted to optimize dosage and duration of the intervention.

Proinflammatory Effects of Calprotectin in Graves' Orbitopathy.

Purpose: Early detection and control of inflammation are important to manage Graves' orbitopathy (GO). We investigated the effects of calprotectin (S100A8/A9) on orbital fibroblast inflammation and GO pathogenesis.Methods: We measured serum calprotectin, S100A8 and S100A9 mRNA expression in orbital fat/connective tissue from GO patients and healthy controls, and proinflammatory cytokines in primary cultured orbital fibroblasts.Results: The serum levels of S100A8/A9 and the expression of S100A8/A9 mRNA in orbital tissue were higher in the GO patients than in the healthy controls. The serum calprotectin levels positively correlated with the clinical activity score and serum thyroid-stimulating immunoglobulin levels. In cultured GO orbital fibroblasts, S100A8/A9 increased the expression of interleukin (IL)-6, IL-8, and monocyte chemotactic protein-1, as well as the phosphorylation of extracellular signal-regulated kinase and nuclear factor-κB.Conclusion: We demonstrated the potential of calprotectin as a biomarker of GO severity and proinflammatory responses to S100A8/A9 in GO orbital fibroblasts.

The Immune Protein Calprotectin Impacts Clostridioides difficile Metabolism through Zinc Limitation.

The intestines house a diverse microbiota that must compete for nutrients to survive, but the specific limiting nutrients that control pathogen colonization are not clearly defined. Clostridioides difficile colonization typically requires prior disruption of the microbiota, suggesting that outcompeting commensals for resources is critical to establishing C. difficile infection (CDI). The immune protein calprotectin (CP) is released into the gut lumen during CDI to chelate zinc (Zn) and other essential nutrient metals. Yet, the impact of Zn limitation on C. difficile colonization is unknown. To define C. difficile responses to Zn limitation, we performed RNA sequencing on C. difficile exposed to CP. In medium containing CP, C. difficile upregulated genes involved in metal homeostasis and amino acid metabolism. To identify CP-responsive genes important during infection, we measured the abundance of select C. difficile transcripts in a mouse CDI model relative to expression in vitro Gene transcripts involved in selenium (Se)-dependent proline fermentation increased during infection and in response to CP. Increased proline fermentation gene transcription was dependent on CP Zn binding and proline availability, yet proline fermentation was only enhanced when Se was supplemented. CP-deficient mice could not restrain C. difficile proline fermentation-dependent growth, suggesting that CP-mediated Zn sequestration along with limited Se restricts C. difficile proline fermentation. Overall, these results highlight how C. difficile colonization depends on the availability of multiple nutrients whose abundances are dynamically influenced by the host response.IMPORTANCEClostridioides difficile infection (CDI) is the leading cause of postantibiotic nosocomial infection. Antibiotic therapy can be successful, yet up to one-third of individuals suffer from recurrent infections. Understanding the mechanisms controlling C. difficile colonization is paramount in designing novel treatments for primary and recurrent CDI. Here, we found that limiting nutrients control C. difficile metabolism during CDI and influence overall pathogen fitness. Specifically, the immune protein CP limits Zn availability and increases transcription of C. difficile genes necessary for proline fermentation. Paradoxically, this leads to reduced C. difficile proline fermentation. This reduced fermentation is due to limited availability of another nutrient required for proline fermentation, Se. Therefore, CP-mediated Zn limitation combined with low Se levels overall reduce C. difficile fitness in the intestines. These results emphasize the complexities of how nutrient availability influences C. difficile colonization and provide insight into critical metabolic processes that drive the pathogen's growth.

Intracellular calprotectin (S100A8/A9) controls epithelial differentiation and caspase-mediated cleavage of EGFR in head and neck squamous cell carcinoma.

OBJECTIVES: Calprotectin (S100A8/A9) appears to function as a tumor suppressor in head and neck squamous cell carcinoma (HNSCC) and expression in the carcinoma cells and patient survival rates are directly related. We seek to characterize the suppressive role of calprotectin in HNSCC. AIMS: (1) Investigate changes in S100A8/A9 expression as oral carcinogenesis progresses and (2) determine whether intracellular calprotectin can regulate epidermal growth factor receptor (EGFR), a negative prognostic factor, in HNSCC. MATERIALS AND METHODS: Using immunohistochemistry (IHC), S100A8/A9 was analyzed in HNSCC specimens (N = 46), including well-differentiated (WD, N = 19), moderately-differentiated (MD, N = 14), poorly-differentiated (PD, N = 5) and non-keratinizing/basaloid (NK/BAS, N = 8), and premalignant epithelial dysplasias (PED, N = 16). Similarly, EGFR was analyzed in HNSCCs (N = 21). To determine whether calprotectin and EGFR expression are mechanistically linked, TR146 HNSCC cells that are S100A8/A9-expressing or silenced (shRNA) were compared for EGFR levels and caspase-3/7 activity using western blotting and immunofluorescence microscopy. RESULTS: In normal oral mucosal epithelium, S100A8/A9 stained strongly in the cytoplasm and nucleus of suprabasal cells; basal cells were consistently S100A8/A9 negative. In PED and HNSCC, S100A8/A9 expression was lower than in adjacent normal epithelial tissues (NAT) and declined progressively in WD, MD, PD and NK/BAS HNSCCs. S100A8/A9 and EGFR levels appeared inversely related, which was simulated in vitro when S100A8/A9 was silenced in TR146 cells. Silencing S100A8/A9 significantly reduced caspase-3/7 activity, whereas EGFR levels increased. CONCLUSIONS: In HNSCC, S100A8/A9 is directly associated with cellular differentiation and appears to promote caspase-3/7-mediated cleavage of EGFR, which could explain why patients with S100A8/A9-high tumors survive longer.

Myeloid related protein 8/14 is a new candidate biomarker and therapeutic target for abdominal aortic aneurysm.

Open surgical or percutaneous endovascular (EVAR) intervention is the only approved treatment for abdominal aortic aneurysm (AAA). Novel targeted therapy and companion diagnostic are needed. Our study aimed to investigate the expression of myeloid related protein 8/14 (MRP8/14) in AAA and explore whether MRP8/14 can be a biomarker and therapeutic target for AAA. The serum levels of MRP8/14 and inflammatory factors including TNF-α, IL-1ß, and IL-6 were increased in 20 human AAA patients compared to healthy people by ELISA assay. The mRNA and protein expressions of MRPs in AAA tissues of AAA patient were significantly elevated when compared with the levels in adjacent non-aneurysmal aortic segments in AAA patients. through PCR and western blot analysis. Correlation analysis and receiver operating characteristic (ROC) curve analysis confirmed that MRP8/14 was secreted into the serum and possessed the potential diagnostic value. Rat AAA models established by perfusion porcine pancreatic elastase and murine macrophage RAW264.7 cells were used to evaluate the mechanisms of MRP8/14 after treatment with antibodies. Similarly, MRP8, MRP14, and MRP8/14 were highly expressed in rat AAA model, while the administrations of antibodies of MRPs significantly reversed the improvement expressions of MRP8 and MRP14. In RAW264.7 cells, MRPs especially for MRP8/14 obviously increased the levels of MMP-2 and MMP-9. Under MRP8/14 stimulation, the antibodies of MRPs recovered the expressions of MMP-2 and MMP-9. Anti MRP8/14 antibody exhibited the strongest effect in the experiments. Our results indicated that MRP8/14 was associated with AAA presence and progression and could be considered as a biomarker for AAA diagnose. Anti MRP8/14 can be a potential therapeutic target for treatment of AAA.

Preparation and Iron Redox Speciation Study of the Fe(II)-Binding Antimicrobial Protein Calprotectin.

Calprotectin (CP, S100A8/S100A9 heterooligomer) is an abundant metal-sequestering host-defense protein expressed by neutrophils, other white blood cells, and epithelial cells. The apoprotein is a S100A8/S100A9 heterodimer that contains two sites for transition metal binding at the S100A8/S100A9 interface: a His3Asp motif (site 1) and a His6 motif (site 2). In this chapter, we provide a step-by-step protocol for the overexpression and purification of the human and murine orthologues of CP that affords each apo heterodimer in high yield and purity. In these procedures, the S100A8 and S100A9 subunits are overexpressed in Escherichia coli BL21(DE3), and each apo heterodimer is obtained following cell lysis, folding, column chromatography, and dialysis against Chelex resin to reduce metal contamination. Recent studies demonstrated that human CP coordinates Fe(II) and that the protein affects the redox speciation of Fe in solution. An Fe redox speciation assay employing ferrozine is described that demonstrates the ability of both the human and murine orthologues of CP to shift the redox speciation of Fe from the ferric to the ferrous oxidation state over time.

Transition Metal Sequestration by the Host-Defense Protein Calprotectin.

In response to microbial infection, the human host deploys metal-sequestering host-defense proteins, which reduce nutrient availability and thereby inhibit microbial growth and virulence. Calprotectin (CP) is an abundant antimicrobial protein released from neutrophils and epithelial cells at sites of infection. CP sequesters divalent first-row transition metal ions to limit the availability of essential metal nutrients in the extracellular space. While functional and clinical studies of CP have been pursued for decades, advances in our understanding of its biological coordination chemistry, which is central to its role in the host-microbe interaction, have been made in more recent years. In this review, we focus on the coordination chemistry of CP and highlight studies of its metal-binding properties and contributions to the metal-withholding innate immune response. Taken together, these recent studies inform our current model of how CP participates in metal homeostasis and immunity, and they provide a foundation for further investigations of a remarkable metal-chelating protein at the host-microbe interface and beyond.