Fibrin- and collagen-based matrices attenuate inflammatory and procoagulant responses in human endothelial cell cultures exposed to Staphylococcus aureus.

Infective endocarditis (IE) remains a serious complication after heart valve replacement. Autologous valves constructed by matrix-based tissue engineering are under investigation to increase biocompatibility. The impact of the underlying matrix on the risk to develop IE is not known. The IE is characterized by bacterial adhesion and subsequent interactions of disseminating bacteria with endothelial cells (ECs) and monocytes, evoking endothelial proinflammatory and procoagulant activity, leading to heart valve destruction. In the current study, we, therefore, have seeded human ECs on a fibrin versus collagen gel matrix and, at confluence, infected them with Staphylococcus aureus, Streptococcus sanguis, and Staphylococcus epidermidis. Especially Sta. aureus infected ECs grown on fibrin (4.2% of the inoculum) and collagen (3.7%) matrices, more than on ECs grown on noncoated plates (1.2%; p<0.01). This was associated with higher monocyte adhesion (61%; p<0.01 on fibrin and 43%; p<0.05 on collagen) versus control cultures (30%), even at comparable EC surface expression of intercellular adhesion molecule-1 and vascular adhesion molecule-1. The collagen matrix attenuated the Sta. aureus-induced monocyte chemoattractant protein 1 expression 2.0-fold, compared with the noncoated control ECs. This reduction coincided with a 4.2-5.0-fold reduction of the procoagulant activity, triggered in ECs grown on noncoated wells, as a consequence of tissue factor (TF) expression by ECs, further stimulated by EC-bound monocytes. Overall, moderate responses were seen on infection with Str. sanguis and Sta. epidermidis for both gel matrices. Thus, even when fibrin and collagen gel matrices equally increase bacterial adhesion, and subsequent monocyte adhesion to infected ECs, these matrices modulate EC responses to these stimuli, thus resulting in attenuated cytokine production and attenuated adherent monocyte-dependent TF production by the ECs. Further investigations will need to confirm whether also in vivo, EC-matrix interactions can attenuate EC responses to bacteria and inflammatory cells to reduce IE at infected endovascular sites.

LL-37 directs macrophage differentiation toward macrophages with a proinflammatory signature.

The human cathelicidin LL-37 has broad-spectrum antimicrobial activity. It also participates at the interface of innate and adaptive immunity by chemoattracting immune effector cells, modulating the production of a variety of inflammatory mediators by different cell types, and regulating the differentiation of monocytes into dendritic cells. In this study, we investigated the effects of LL-37 on the differentiation of human monocytes into anti-inflammatory macrophages (MPhi-2; driven by M-CSF) versus proinflammatory macrophages (MPhi-1; driven by GM-CSF) as well as on fully differentiated MPhi-1 and MPhi-2. Results revealed that monocytes cultured with M-CSF in the presence of LL-37 resulted in macrophages displaying a proinflammatory signature, namely, low expression of CD163 and little IL-10 and profound IL-12p40 production on LPS stimulation. The effects of LL-37 on M-CSF-driven macrophage differentiation were dose- and time-dependent with maximal effects observed at 10 microg/ml when the peptide was present from the start of the cultures. The peptide enhanced the GM-CSF-driven macrophage differentiation. Exposure of fully differentiated MPhi-2 to LL-37 for 6 d resulted in macrophages that produced less IL-10 and more IL-12p40 on LPS stimulation than control MPhi-2. In contrast, LL-37 had no effect on fully differentiated MPhi-1. Peptide mapping using a set of 16 overlapping 22-mer peptides covering the complete LL-37 sequence revealed that the C-terminal portion of LL-37 is responsible for directing macrophage differentiation. Our results furthermore indicate that the effects of LL-37 on macrophage differentiation required internalization of the peptide. Together, we conclude that LL-37 directs macrophage differentiation toward macrophages with a proinflammatory signature.

Antimicrobial peptide hLF1-11 directs granulocyte-macrophage colony-stimulating factor-driven monocyte differentiation toward macrophages with enhanced recognition and clearance of pathogens.

The human lactoferrin-derived peptide hLF1-11 displays antimicrobial activities in vitro and is effective against infections with antibiotic-resistant bacteria and fluconazole-resistant Candida albicans in animals. However, the mechanisms underlying these activities remain largely unclear. Since hLF1-11 is ineffective in vitro at physiological salt concentrations, we suggested modulation of the immune system as an additional mechanism of action of the peptide. We investigated whether hLF1-11 affects human monocyte-macrophage differentiation and determined the antimicrobial activities of the resulting macrophages. Monocytes were cultured for 7 days with GM-CSF in the presence of hLF1-11, control peptide, or saline for various intervals. At day 6, the cells were stimulated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or heat-killed C. albicans for 24 h. Thereafter, the levels of cytokines in the culture supernatants, the expression of pathogen recognition receptors, and the antimicrobial activities of these macrophages were determined. The results showed that a short exposure of monocytes to hLF1-11 during GM-CSF-driven differentiation is sufficient to direct differentiation of monocytes toward a macrophage subset characterized by both pro- and anti-inflammatory cytokine production and increased responsiveness to microbial structures. Moreover, these macrophages are highly effective against C. albicans and Staphylococcus aureus. In conclusion, hLF1-11 directs GM-CSF-driven differentiation of monocytes toward macrophages with enhanced effector functions.

Contribution of (sub)domains of Staphylococcus aureus fibronectin-binding protein to the proinflammatory and procoagulant response of human vascular endothelial cells.

The Staphylococcus aureus fibronectin (Fn) -binding protein A (FnBPA) is involved in bacterium-endothelium interactions which is one of the crucial events leading to infective endocarditis (IE). We previously showed that the sole expression of S. aureus FnBPA was sufficient to confer to non-invasive Lactococcus lactis bacteria the capacity to invade human endothelial cells (ECs) and to launch the typical endothelial proinflammatory and procoagulant responses that characterize IE. In the present study we further questioned whether these bacterium-EC interactions could be reproduced by single or combined FnBPA sub-domains (A, B, C or D) using a large library of truncated FnBPA constructs expressed in L. lactis. Significant invasion of cultured ECs was found for L. lactis expressing the FnBPA subdomains CD (aa 604-877) or A4(+16) (aa 432-559). Moreover, this correlates with the capacity of these fragments to elicit in vitro a marked increase in EC surface expression of both ICAM-1 and VCAM-1 and secretion of the CXCL8 chemokine and finally to induce a tissue factor-dependent endothelial coagulation response. We thus conclude that (sub)domains of the staphylococcal FnBPA molecule that express Fn-binding modules, alone or in combination, are sufficient to evoke an endothelial proinflammatory as well as a procoagulant response and thus account for IE severity.

Staphylococcal peptidoglycan initiates an inflammatory response and procoagulant activity in human vascular endothelial cells: a comparison with highly purified lipoteichoic acid and TSST-1.

Staphylococcus aureus is one of the most significant pathogens in human sepsis and endocarditis. A hallmark of these endovascular S. aureus infections is that the coagulation system is triggered by a tissue factor (TF)-dependent pathway. This study demonstrates that highly purified S. aureus peptidoglycan, lipoteichoic acid (LTA) and TSST-1 increase TF mRNA and TF surface protein in human umbilical vein endothelial cells (ECs). Concomitantly, peptidoglycan- and LTA-activated ECs express significant TF-dependent procoagulant activity (TF PCA). In addition peptidoglycan, but not LTA or TSST-1, induced surface expression of the EC inflammation markers ICAM-1 and VCAM-1, which supported the adhesion of monocytes to these ECs. During the coculture of peptidoglycan-activated ECs and adherent monocytes, a marked additional increase of TF PCA was observed. Marginal increases in TF PCA were observed in cocultures of monocytes with LTA- or TSST-1-activated ECs. This study defines in particular staphylococcal peptidoglycan, previously known as a potent initiator of TF PCA in monocytes, as also being an activator of a coagulant response in human ECs that is further intensified by the presence of surface-bound monocytes.

Gamma interferon confers resistance to infection with Staphylococcus aureus in human vascular endothelial cells by cooperative proinflammatory and enhanced intrinsic antibacterial activities.

Vascular endothelium is an exposed target in systemic endovascular Staphylococcus aureus infections. We reported earlier that the proinflammatory and procoagulant activities of primary human umbilical vein endothelial cells (ECs) after binding and ingestion of S. aureus organisms provide the cells effective means for leukocyte-mediated bacterial elimination. Expanding on this, we now show that these ECs exhibit a modest intrinsic capacity for eliminating intracellular S. aureus that was influenced by cytokines relevant to S. aureus infections. Using various EC infection assays, we showed that gamma interferon (IFN-gamma), applied to cultures of ECs prior to or after infection with S. aureus, markedly reduced the level of infection, illustrated by lower percentages of S. aureus-infected ECs and less intracellular bacteria per infected cell. IFN-gamma-activated ECs had unaltered abilities to bind S. aureus and processed ingested bacteria by a seemingly conventional phagocytic pathway. IFN-gamma treatment rescued EC monolayers from severe injury by virulent clinical S. aureus strains or excessive bacterial numbers. Mechanistically, IFN-gamma controls S. aureus infection via IFN-gamma receptor, most likely through stimulation of intrinsic endothelial antibacterial mechanisms but independent of processes that deprive bacteria of intracellular L-tryptophan or iron. The antibacterial activity of IFN-gamma-stimulated ECs coincided with sustained or slightly elevated endothelial proinflammatory responses that supported monocyte recruitment. In conclusion, we identify IFN-gamma as a potent regulatory Th1 cytokine possessing exclusive abilities to augment intrinsic antistaphylococcal effector mechanisms in human ECs without ablating the S. aureus-induced proinflammatory EC responses and, as such, coordinating a protective efficacy of ECs against blood-borne S. aureus infection.

Fibronectin-binding proteins and clumping factor A in Staphylococcus aureus experimental endocarditis: FnBPA is sufficient to activate human endothelial cells.

Surface molecules of Staphylococcus aureus are involved in the colonization of vascular endothelium which is a crucial primary event in the pathogenesis of infective endocarditis (IE). The ability of these molecules to also launch endothelial procoagulant and proinflammatory responses, which characterize IE, is not known. In the present study we investigated the individual capacities of three prominent S. aureus surface molecules; fibronectin-binding protein A (FnBPA) and B (FnBPB) and clumping factor A (ClfA), to promote bacterial adherence to cultured human endothelial cells (ECs) and to activate phenotypic and functional changes in these ECs. Non-invasive surrogate bacterium Lactococcus lactis, which, by gene transfer, expressed staphylococcal FnBPA, FnBPB or ClfA molecules were used. Infection of ECs increased 50- to 100-fold with FnBPA- or FnBPB-positive recombinant lactococci. This coincided with EC activation, interleukin-8 secretion and surface expression of ICAM-1 and VCAM-1 and concomitant monocyte adhesion. Infection with ClfA-positive lactococci did not activate EC. FnBPA-positive L. lactis also induced a prominent tissue factor-dependent endothelial coagulation response that was intensified by cell-bound monocytes. Thus S. aureus FnBPs, but not ClfA, confer invasiveness and pathogenicity to non-pathogenic L. lactis organisms indicating that bacterium-EC interactions mediated by these adhesins are sufficient to evoke inflammation as well as procoagulant activity at infected endovascular sites.

Adherence of Acinetobacter baumannii strains to human bronchial epithelial cells.

Acinetobacter baumannii is an important nosocomial pathogen, but the mechanisms contributing to its epidemicity and virulence are largely unknown. The organism is able to colonize skin and mucosal surfaces of the human host. Adherence of microorganisms to host cells is an important virulence factor as it is the initial step of the colonization process. In the present study, adherence of A. baumannii to human bronchial epithelial NCI-H(292) cells was examined by light and scanning electron microscopy. Thirty-seven strains were investigated including 18 from outbreaks, 16 not associated with outbreaks, and three for which an epidemic implication was unknown. Eight and 11 isolates belonged to European clone I and II, respectively. Two types of adherence were observed, dispersed adherence of bacteria to the cell, and adherence of clusters of bacteria at localized areas of the cells. Bacteria with dispersed adherence interacted with the epithelial cells through fimbriae, but were also entrapped by protrusions extending from the epithelial cells. Quantitative adherence varied considerably among strains but there was no significant correlation of the outbreak-associated strains with the percentage of infected cells. There was, however, a correlation between the clonal lineage and the percent of infected cells, with clone II being more adherent than clone I (P<0.05). Ten consecutive isolates from one outbreak were investigated to test whether adherence increased during passage among patients, but this appeared not to be the case. This study showed that A. baumannii adheres to human bronchial epithelial cells in vitro and that A. baumannii strains of clone II had a relatively high capacity for adhering to these cells.

Gamma irradiation or CD4+-T-cell depletion causes reactivation of latent Salmonella enterica serovar Typhimurium infection in C3H/HeN mice.

Upon infection with Salmonella, a host develops an immune response to limit bacterial growth and kill and eliminate the pathogen. Salmonella has evolved mechanisms to remain dormant within the body, only to reappear (reactivate) at a later time when the immune system is abated. We have developed an in vivo model for studying reactivation of Salmonella enterica serovar Typhimurium infection in mice. Upon subcutaneous infection, C3H/HeN (Ity(r)) mice showed an increase in bacterial numbers in livers and spleens, which reached a peak on day 19. After full recovery from the infection, these mice were irradiated or depleted of CD4(+) T cells. The mice displayed a secondary infection peak in livers and spleens with a course similar to that of the primary infection. We concluded that CD4(+) T cells are involved in active suppression of S. enterica serovar Typhimurium during latency. The role of CD4(+) T cells during primary infection with S. enterica serovar Typhimurium is well established. This is the first study to describe a role of CD4(+) T cells during the latent phase of S. enterica serovar Typhimurium infection.

Monocytes maintain tissue factor activity after cytolysis of bacteria-infected endothelial cells in an in vitro model of bacterial endocarditis.

Intravascular infection with Staphylococcus aureus, Staphylococcus epidermidis, or Streptococcus sanguis can initiate fibrin formation on endocardial tissue, causing bacterial endocarditis. The ability of these bacteria to injure intact endothelial cells (ECs) and to aggravate tissue factor (TF)-dependent coagulation in the presence of blood leukocytes was investigated. Cytolysis of ECs occurred after infection with S. aureus and, with membrane-bound monocytes or granulocytes present, also after infection with S. sanguis or S. epidermidis. Monocytes that subsequently bound to the resultant bacteria-infected subcellular EC matrix (ECM) elicited TF mRNA, TF antigen, and TF activity (TFA). This was most pronounced in ECM prepared after the cytolysis of ECs by infection with S. aureus or S. epidermidis. We demonstrate that monocytes continue and intensify fibrin formation after lysis of bacteria-infected ECs, which suggests that, during the course of intravascular infection, early fibrin formation shifts from being mediated by EC-derived TFA to being mediated by TFA of monocytes bound to bacteria-infected ECM.