Krzysztof Guzik

A Potential New Pathway for Staphylococcus aureus Dissemination: The Silent Survival of S. aureus Phagocytosed by Human Monocyte-Derived Macrophages

Although considered to be an extracellular pathogen, Staphylococcus aureus is able to invade a variety of mammalian, non-professional phagocytes and can also survive engulfment by professional phagocytes such as neutrophils and monocytes. In both of these cell types S. aureus promptly escapes from the endosomes/phagosomes and proliferates within the cytoplasm, which quickly leads to host cell death. In this report we show that S. aureus interacted with human monocyte-derived macrophages in a very different way to those of other mammalian cells. Upon phagocytosis by macrophages, S. aureus persisted intracellularly in vacuoles for 3–4 days before escaping into the cytoplasm and causing host cell lysis. Until the point of host cell lysis the infected macrophages showed no signs of apoptosis or necrosis and were functional. They were able to eliminate intracellular staphylococci if prestimulated with interferon-γ at concentrations equivalent to human therapeutic doses. S. aureus survival was dependent on the alternative sigma factor B as well as the global regulator agr, but not SarA. Furthermore, isogenic mutants deficient in α-toxin, the metalloprotease aureolysin, protein A, and sortase A were efficiently killed by macrophages upon phagocytosis, although with different kinetics. In particular α-toxin was a key effector molecule that was essential for S. aureus intracellular survival in macrophages. Together, our data indicate that the ability of S. aureus to survive phagocytosis by macrophages is determined by multiple virulence factors in a way that differs considerably from its interactions with other cell types. S. aureus persists inside macrophages for several days without affecting the viability of these mobile cells which may serve as vehicles for the dissemination of infection.

Fas (CD95)-Fas Ligand Interactions Are Responsible for Monocyte Apoptosis Occurring as a Result of Phagocytosis and Killing of Staphylococcus aureus

ABSTRACT Human peripheral blood monocytes become apoptotic following phagocytosis of Staphylococcus aureus. In this study, we investigated the mechanisms involved in this phenomenon. Cells exposed to bacteria were examined for the surface expression of Fas and Fas ligand (FasL). The level of soluble form of FasL was also measured in the culture supernatants. As Fas-mediated apoptosis involves the activation of caspases, the activities of caspase-8 and caspase-3 were determined. Finally, the involvement of oxidative stress in apoptosis of infected monocytes was investigated. The data indicated that as a consequence of phagocytosis of S. aureus, FasL is released from the monocyte surface and induces apoptosis of phagocytic monocytes and to some extent the bystander cells. The importance of this mechanism was confirmed by demonstrating that blockage of CD95 preventsS. aureus-induced apoptosis of monocytes. Cell death occurring after phagocytosis of S. aureus involves the activation of caspase-3-like proteases, as the specific caspase-3 inhibitor suppressed apoptosis of infected cells. The generation of reactive oxygen intermediates by phagocytic monocytes by itself is not sufficient as a death signal but rather acts in up-regulating FasL shedding and possibly in modulating caspase activity.

Identification of interleukin-1 and interleukin-6-responsive genes in human monocyte-derived macrophages using microarrays

The transcriptome profile of human monocyte-derived macrophages stimulated in vitro by low doses of IL-1 or IL-6 was analyzed by microarrays (Affymetrix, HG-U133A) in 5 independent experiments. Out of 4886 probe sets consistently detected in all 5 array replicates we found approximately 300 genes (FDR<5%) modulated by IL-1 and/or IL-6, among which 34 may be regarded as novel cytokine-responsive macrophage genes of various function. Detailed analysis indicates that cytokine-responsive genes include 125 transcripts significantly up-regulated by IL-1 and only 39 transcripts up-regulated by IL-6, whereas the number of down-regulated transcripts is lower and almost equal for both cytokines. These data indicate that, in comparison to liver cells, IL-1 is more potent than IL-6 in modulating gene expression of human macrophages. Hierarchical clustering analysis of these transcripts yielded 7 separate gene clusters. The most abundant group contains genes strongly activated by IL-1 alone and coding for chemokines, cytokines and their receptors, the components of intracellular signaling as well as transcription factors from NF-kB family. In order to validate the results obtained by microarray analysis the expression of 5 genes from various clusters was determined by quantitative RT-PCR. Moreover, the putative promoter regions of all cytokine-responsive genes were subjected to the in silico identification of transcription factor binding sites (TFBS). We found that TFBS corresponding to RelA/NF-kB is the most strongly over-represented group and we demonstrated involvement of NF-kB in the expression of selected genes.

Increased IL-10 production during spontaneous apoptosis of monocytes.

Monocytes/macrophages undergo apoptosis and are in contact with apoptotic cells both in vitro and in vivo. The data show that monocytes undergoing spontaneous apoptosis in vitro change their cytokine production profile. We demonstrate that the lipopolysaccharide (LPS)‐induced production of interleukin‐10 (IL‐10) is up‐regulated, while production of pro‐inflammatory cytokines such as tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) is either not affected or reduced. These differences seen both at the protein and mRNA level directly correlate with the appearance of apoptotic cells in the culture. Flow cytometry analysis using double staining, surface with annexin V and intracellular with anti‐IL‐10, suggested that annexin V‐negative monocytes are the predominant source of IL‐10. Analysis of sorted populations of monocytes indicated that the increase in IL‐10 synthesis appears to result from direct interactions between non‐apoptotic and apoptotic cells at the time of stimulation. Also non‐apoptotic, freshly isolated monocytes produced more IL‐10 upon stimulation with LPS, Staphylococcus aureus or zymosan when apoptotic neutrophils were added to the culture. In contrast, monocyte‐derived macrophages did not produce more IL‐10 in the presence of apoptotic neutrophils. Finally, we found that the presence of apoptotic monocytes in the culture may influence specific immune responses. The data show that in the presence of annexin V‐positive monocytes CD4‐positive memory T cells produce less IFN‐γ upon stimulation with purified protein derivative of tuberculin, which could be partially reversed by anti‐IL‐10 neutralizing antibodies. We conclude that these findings might illustrate the mechanisms operating within an inflammatory site and play an important immunoregulatory role during the resolution of inflammation and specific immune responses.

A New Pathway of Staphylococcal Pathogenesis: Apoptosis-Like Death Induced by Staphopain B in Human Neutrophils and Monocytes

Circulating neutrophils and monocytes form the first line of cellular defense against invading bacteria. Here, we describe a novel and specific mechanism of disabling and eliminating phagocytes by Staphylococcus aureus. Staphopain B (SspB) selectively cleaved CD11b on phagocytes, which rapidly acquired features of cell death. SspB-treated phagocytes expressed phosphatidylserine as well as annexin I and became permeable to propidium iodide, thus demonstrating distinctive features of both apoptosis and necrosis, respectively. The cell death observed was caspase and Syk tyrosine kinase independent, whilst cytochalasin D efficiently inhibited the staphopain-induced neutrophil killing. Neutrophil and monocyte cell death was not affected by integrin clustering ligands (ICAM-1 or fibrin) and was prevented, and even reversed, by IgG. This protective effect was dependent on the Fc fragment, collectively suggesting cooperation of the CD16 receptor and integrin Mac-1 (CD11b/CD18). We conclude that SspB, particularly in the presence of staphylococcal protein A, may reduce the number of functional phagocytes at infection sites, thus facilitating colonization and dissemination of S. aureus.

Staphylococcal cysteine protease staphopain B (SspB) induces rapid engulfment of human neutrophils and monocytes by macrophages

Abstract Circulating neutrophils and monocytes constitute the first line of antibacterial defence, which is responsible for the phagocytosis and killing of microorganisms. Previously, we have described that the staphylococcal cysteine proteinase staphopain B (SspB) cleaves CD11b on peripheral blood phagocytes, inducing the rapid development of features of atypical cell death in protease-treated cells. Here, we report that exposure of phagocytes to SspB critically impairs their antibacterial functions. Specifically, SspB blocks phagocytosis of Staphylococcus aureus by both neutrophils and monocytes, represses their chemotactic activity and induces extensive, nonphlogistic clearance of SspB-treated cells by macrophages. The proteinase also cleaves CD31, a major repulsion (‘do not-eat-me’) signal, on the surface of neutrophils. We suggest that both proteolytic degradation of repulsion signals and induction of ‘eat-me’ signals on the surface of leukocytes are responsible for the observed intensive phagocytosis of SspB-treated neutrophils by human monocyte-derived macrophages. Collectively, this may lead to the depletion of functional neutrophils at the site of infection, thus facilitating staphylococcal colonisation and spreading.

Heat shock protein and heat shock factor 1 expression and localization in vaccinia virus infected human monocyte derived macrophages

BackgroundViruses remain one of the inducers of the stress response in the infected cells. Heat shock response induced by vaccinia virus (VV) infection was studied in vitro in human blood monocyte derived macrophages (MDMs) as blood cells usually constitute the primary site of the infection.MethodsHuman blood monocytes were cultured for 12 – 14 days. The transcripts of heat shock factor 1 (HSF1), heat shock protein 70 (HSP70), heat shock protein 90 (HSP90) and two viral genes (E3L and F17R) were assayed by reverse transcriptase-polymerase chain reaction (RT-PCR), and the corresponding proteins measured by Western blot. Heat shock factor 1 DNA binding activities were estimated by electrophoretic mobility shift assay (EMSA) and its subcellular localization analyzed by immunocytofluorescence.ResultsIt appeared that infection with vaccinia virus leads to activation of the heat shock factor 1. Activation of HSF1 causes increased synthesis of an inducible form of the HSP70 both at the mRNA and the protein level. Although HSP90 mRNA was enhanced in vaccinia virus infected cells, the HSP90 protein content remained unchanged. At the time of maximum vaccinia virus gene expression, an inhibitory effect of the infection on the heat shock protein and the heat shock factor 1 was most pronounced. Moreover, at the early phase of the infection translocation of HSP70 and HSP90 from the cytoplasm to the nucleus of the infected cells was observed.ConclusionPreferential nuclear accumulation of HSP70, the major stress-inducible chaperone protein, suggests that VV employs this particular mechanism of cytoprotection to protect the infected cell rather than to help viral replication. The results taken together with our previuos data on monocytes or MDMs infected with VV or S. aureus strongly argue that VV employs multiple cellular antiapoptotic/cytoprotective mechanisms to prolong viability and proinflammatory activity of the cells of monocytic-macrophage lineage.

Oxidized LDLs Inhibit TLR-induced IL-10 Production by Monocytes: A New Aspect of Pathogen-Accelerated Atherosclerosis

It is widely accepted that oxidized low-density lipoproteins and local infections or endotoxins in circulation contribute to chronic inflammatory process at all stages of atherosclerosis. The hallmark cells of atherosclerotic lesions—monocytes and macrophages—are able to detect and integrate complex signals derived from lipoproteins and pathogens, and respond with a spectrum of immunoregulatory cytokines. In this study, we show strong inhibitory effect of oxLDLs on anti-inflammatory interleukin-10 production by monocytes responding to TLR2 and TLR4 ligands. In contrast, pro-inflammatory tumor necrosis factor secretion was even slightly increased, when stimulated with lipopolysaccharide from Porphyromonas gingivalis—an oral pathogen associated with atherosclerosis. The oxLDLs modulatory activity may be explained by altered recognition of pathogen-associated molecular patterns, which involves serum proteins, particularly vitronectin. We also suggest an interaction between vitronectin receptor, CD11b, and TLR2. The presented data support a novel pathway for pathogen-accelerated atherosclerosis, which relies on oxidized low-density lipoprotein-mediated modulation of anti-inflammatory response to TLR ligands.

Staphylococcus aureus Proteases Degrade Lung Surfactant Protein A Potentially Impairing Innate Immunity of the Lung

The pulmonary surfactant is a complex mixture of lipids and proteins that is important for respiratory lung functions, which also provides the first line of innate immune defense. Pulmonary surfactant protein-A (SP-A) is a major surfactant component with immune functions with importance during Staphylococcus aureus infections that has been demonstrated in numerous studies. The current study showed that S. aureus can efficiently cleave the SP-A protein using its arsenal of proteolytic enzymes. This degradation appears to be mediated by cysteine proteases, in particular staphopain A (ScpA). The staphopain-mediated proteolysis of SP-A resulted in a decrease or complete abolishment of SP-A biological activity, including the promotion of S. aureus phagocytosis by neutrophils, aggregation of Gram-negative bacteria and bacterial cell adherence to epithelium. Significantly, ScpA has also efficiently degraded SP-A in complete bronchi-alveolar lavage fluid from human lungs. This indicates that staphopain activity in the lungs is resistant to protease inhibitors, thus suggesting that SP-A can be cleaved in vivo. Collectively, this study showed that the S. aureus protease ScpA is an important virulence factor that may impair innate immunity of the lungs.

Rapid Decrease of CD16 (FcγRIII) Expression on Heat-Shocked Neutrophils and Their Recognition by Macrophages

Accumulation of neutrophils in the site of inflammation is a typical mechanism of innate immunity. The accumulated neutrophils are exposed to stressogenic factors usually associated with inflammation. Here, we studied response of human peripheral blood neutrophils subjected to short, febrile-range heat stress. We show that 90 min heat stress slowed down the spontaneous apoptosis of neutrophils. In the absence of typical markers of apoptosis the heat-shocked neutrophils induced antiinflammatory effect in human monocyte-derived macrophages (hMDMs), yet without being engulfed. Importantly, the expression of FcγRIII (CD16) was sharply reduced. Surprisingly, concentration of the soluble CD16 did not change in heat-shocked neutrophil supernates indicating that the reduction of the cell surface CD16 was achieved mainly by inhibition of fresh CD16 delivery. Inhibitors of 90 kDa heat shock protein (HSP90), a molecular chaperone found in membrane platforms together with CD16 and CD11b, significantly increased the observed effects caused by heat shock. The presented data suggest a novel systemic aspect of increased temperature which relies on immediate modification by heat of a neutrophil molecular pattern. This effect precedes cell death and may be beneficial in the initial phase of inflammation providing a nonphlogistic signal to macrophages before it comes from apoptotic cells.