Metal ions in macrophage antimicrobial pathways: emerging roles for zinc and copper

The immunomodulatory and antimicrobial properties of zinc and copper have long been appreciated. In addition, these metal ions are also essential for microbial growth and survival. This presents opportunities for the host to either harness their antimicrobial properties or limit their availability as defence strategies. Recent studies have shed some light on mechanisms by which copper and zinc regulation contribute to host defence, but there remain many unanswered questions at the cellular and molecular levels. Here we review the roles of these two metal ions in providing protection against infectious diseases in vivo, and in regulating innate immune responses. In particular, we focus on studies implicating zinc and copper in macrophage antimicrobial pathways, as well as the specific host genes encoding zinc transporters (SLC30A, SLC39A family members) and CTRs (copper transporters, ATP7 family members) that may contribute to pathogen control by these cells

Endogenous danger signals in infectious diseases

Bacterial infections present a significant burden on healthcare and cause considerable morbidity and mortality. Emerging resistance against antimicrobial therapy complicates the treatment of these infections. In addition, antibiotic therapy may not be sufficient to prevent mortality in severely ill patients. It is therefore important to expand our knowledge on host defense mechanisms that may help in the development of new treatment strategies. Invasive infection is associated with the release of endogenous molecules, also known as alarmins or damage associated molecular patterns. Alarmins activate innate immune cells and act as endogenous danger signals to initiate and perpetuate inflammation. Uncontrolled release of alarmins however, may lead to an overwhelming inflammatory response and collateral tissue damage. In this thesis we focused on the role of two well-characterized alarmins, High-mobility Group Box 1 and Myeloid-related Protein 8/14 (or calprotectin) in severe infections

Optimized bacterial DNA isolation method for microbiome analysis of human tissues

Recent advances in microbiome sequencing have rendered new insights into the role of the microbiome in human health with potential clinical implications. Unfortunately, the presence of host DNA in tissue isolates has hampered the analysis of host-associated bacteria. Here, we present a DNA isolation protocol for tissue, optimized on biopsies from resected human colons (~2-5 mm in size), which includes reduction of human DNA without distortion of relative bacterial abundance at the phylum level. We evaluated which concentrations of Triton and saponin lyse human cells and leave bacterial cells intact, in combination with DNAse treatment to deplete released human DNA. Saponin at a concentration of 0.0125% in PBS lysed host cells, resulting in a 4.5-fold enrichment of bacterial DNA while preserving the relative abundance of Firmicutes, Bacteroidetes, γ-Proteobacteria, and Actinobacteria assessed by qPCR. Our optimized protocol was validated in the setting of two large clinical studies on 521 in vivo acquired colon biopsies of 226 patients using shotgun metagenomics. The resulting bacterial profiles exhibited alpha and beta diversities that are similar to the diversities found by 16S rRNA amplicon sequencing. A direct comparison between shotgun metagenomics and 16S rRNA amplicon sequencing of 15 forceps tissue biopsies showed similar bacterial profiles and a similar Shannon diversity index between the sequencing methods. Hereby, we present the first protocol for enriching bacterial DNA from tissue biopsies that allows efficient isolation of all bacteria. Our protocol facilitates analysis of a wide spectrum of bacteria of clinical tissue samples improving their applicability for microbiome research

Myeloid-related protein-14 contributes to protective immunity in gram-negative pneumonia derived sepsis

Contains fulltext : 108788.pdf (publisher's version ) (Open Access)Klebsiella (K.) pneumoniae is a common cause of pneumonia-derived sepsis. Myeloid related protein 8 (MRP8, S100A8) and MRP14 (S100A9) are the most abundant cytoplasmic proteins in neutrophils. They can form MRP8/14 heterodimers that are released upon cell stress stimuli. MRP8/14 reportedly exerts antimicrobial activity, but in acute fulminant sepsis models MRP8/14 has been found to contribute to organ damage and death. We here determined the role of MRP8/14 in K. pneumoniae sepsis originating from the lungs, using an established model characterized by gradual growth of bacteria with subsequent dissemination. Infection resulted in gradually increasing MRP8/14 levels in lungs and plasma. Mrp14 deficient (mrp14(-/-)) mice, unable to form MRP8/14 heterodimers, showed enhanced bacterial dissemination accompanied by increased organ damage and a reduced survival. Mrp14(-/-) macrophages were reduced in their capacity to phagocytose Klebsiella. In addition, recombinant MRP8/14 heterodimers, but not MRP8 or MRP14 alone, prevented growth of Klebsiella in vitro through chelation of divalent cations. Neutrophil extracellular traps (NETs) prepared from wildtype but not from mrp14(-/-) neutrophils inhibited Klebsiella growth; in accordance, the capacity of human NETs to kill Klebsiella was strongly impaired by an anti-MRP14 antibody or the addition of zinc. These results identify MRP8/14 as key player in protective innate immunity during Klebsiella pneumonia

EXPRESSION AND FUNCTION OF MYELOID-RELATED PROTEIN (MRP)-8/14 IN TYPHOID FEVER

Immunogenetics and cellular immunology of bacterial infectious disease

Toll-like receptor 9 enhances bacterial clearance and limits lung consolidation in murine pneumonia caused by methicillin resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen in pneumonia, associated with severe lung damage. Tissue injury causes release of Damage Associated Molecular Patterns (DAMPs), which may perpetuate inflammation. DNA has been implicated as a DAMP that activates inflammation through Toll-like receptor (TLR)9. The aim of this study was to evaluate the role of TLR9 in MRSA pneumonia. Wild-type (Wt) and TLR9 knockout (tlr9(-/-)) mice were infected intranasally with MRSA USA300 (BK 11540) (5(E7)CFU) and euthanized at 6,24,48 or 72 hours for analyses. MRSA pneumonia was associated with profound release of cell-free host DNA in the airways, as reflected by increases in nucleosome and DNA levels in bronchoalveolar lavage fluid (BALF), accompanied by transient detection of pathogen DNA in MRSA-free BALF supernatants. In BALF, as compared to Wt -mice tlr9(-/-) mice showed reduced TNFα and IL-6 levels at 6 hours and reduced bacterial clearance at 6 and 24 hours post infection. Furthermore, tlr9(-/-) mice exhibited a greater influx of neutrophils in BALF and increased lung consolidation at 24 and 48 hours. This study demonstrates the release of host- and pathogen-derived TLR9 ligands (DNA) into the alveolar space after infection with MRSA via the airways and suggests that TLR9 has pro-inflammatory effects during MRSA pneumonia associated with enhanced bacterial clearance and limitation of lung consolidatio

S100A12 and soluble receptor for advanced glycation end products levels during human severe sepsis

S100A12 is highly expressed, and serum levels correlate with individual disease activity in patients with inflammatory diseases. We here sought to determine the extent of S100A12 release and its soluble high-affinity receptor for advanced glycation end products (sRAGE) in patients with severe sepsis stratified to the three most common infectious sources (lungs, abdomen, and urinary tract) and to determine S100A12 and sRAGE concentrations at the site of infection during peritonitis. Two patient populations were studied: (a) 51 patients with sepsis due to (i) peritonitis (n = 12), (ii) pneumonia (n = 29), or (iii) urinary tract infection (n = 10); and (b) 17 patients with peritonitis. In addition, eight healthy humans were studied after intravenous injection of lipopolysaccharide (4 ng/kg). Compared with healthy volunteers, patients with severe sepsis displayed increased circulating S100A12 concentrations at day 0 (591.2 ± 101.0 vs. 106.2 ± 15.6 ng/mL [control subjects], P < 0.0001) and at day 3 (637.2 ± 111.2 vs. 106.2 ± 15.6 ng/mL [control subjects], P < 0.0001). All three severe sepsis subgroups had elevated serum S100A12 concentrations at both time points (sepsis due to [i] peritonitis [393.5 ± 89.9 at day 0 and 337.9 ± 97.2 at day 3 vs. 106.2 ± 15.6 ng/mL, control subjects, P < 0.005 and P < 0.05, respectively]; [ii] pneumonia [716.9 ± 167.0 at day 0 and 787.5 ± 164.7 at day 3 vs. 106.2 ± 15.6 ng/mL, control subjects, both P < 0.0001]; and [iii] urinary tract infection [464.2 ± 115.6 at day 0 and 545.6 ± 254.9 at day 3 vs. 106.2 ± 15.6 ng/mL, control subjects, P < 0.0001 and P < 0.05, respectively]). Remarkably, patients with sepsis due to pneumonia had the highest S100A12 levels (716.9 ± 167.0 and 787.5 ± 164.7 ng/mL at days 0 and 3, respectively). S100A12 levels were not correlated to either Acute Physiology and Chronic Health Evaluation II scores (r = -0.185, P = 0.19) or Sepsis-Related Organ Failure Assessment scores (r = -0.194, P = 0.17). Intravenous lipopolysaccharide injection in healthy humans elevated systemic S100A12 levels (peak levels at 3 h of 59.6 ± 22.0 vs. 12.4 ± 3.6 ng/mL; t = 0 h, P < 0.005). In contrast to S100A12, sRAGE concentrations did not change during severe sepsis or human endotoxemia. During peritonitis, S100A12 concentrations in abdominal fluid (12945.8 ± 4142.1 ng/mL) were more than 100-fold higher than in concurrently obtained plasma (121.2 ± 80.4 ng/mL, P < 0.0005), whereas sRAGE levels in abdominal fluid (148.8 ± 36.0 pg/mL) were lower than those in plasma (648.7 ± 145.6 pg/mL, P < 0.005) and did not increase. In conclusion, in severe sepsis, S100A12 is released systemically irrespective of the primary source of infection. During abdominal sepsis, S100A12 release likely predominantly occurs at the site of infection. Concentrations of its high-affinity sRAGE do not change during infection or human endotoxemia