Margareta Verdrengh

Intra-articularly localized bacterial DNA containing CpG motifs inducesarthritis

Unmethylated CpG motifs are often found in bacterial DNA, and exert immunostimulatory effects on hematopoetic cells. Bacteria produce severe joint inflammation in septic and reactive arthritides; bacterial DNA may be involved in this process. We injected bacterial DNA originating from Escherichia coli and Staphylococcus aureus and oligonucleotides containing CpG directly into the knee joints of mice of different strains. Arthritis was seen by histopathology within 2 hours and lasted for at least 14 days. Unmethylated CpG motifs were responsible for this induction of arthritis, as oligonucleotides containing these motifs produced the arthritis. The arthritis was characterized by an influx of monocytic, Mac-1+ cells and by a lack of T lymphocytes. Depletion of monocytes resulted in abrogation of the synovial inflammation. Tumor necrosis factor (TNF)-α, a cytokine produced by cells of the monocyte/macrophage lineage, is an important mediator of this disease, as expression of mRNA for TNF-α was evident in the inflamed joints, and the CpG-mediated inflammation was abrogated in mice genetically unable to produce this cytokine. These findings demonstrate that bacterial DNA containing unmethylated CpG motifs induces arthritis, and indicate an important pathogenic role for bacterial DNA in septic arthritis.

Genistein as an anti-inflammatory agent

AbstractObjective and design: The aim of this study was to investigate the impact of the isoflavone genistein on in vivo cell-mediated responses. In addition, we wanted to study the influence of genistein on collagen induced arthritis (CIA) in mice. Methods: Delayed type hypersensitivity reaction (DTH) to oxazolone and the inflammatory response to olive oil were measured in mice treated with genistein. In addition, the impact of genistein treatment on disease progression and outcome of collagen induced arthritis (CIA) was examined. Results: The DTH reaction to oxazolone and the granulocyte-mediated response were significantly suppressed in genistein-treated as compared to control mice. Also, genistein treatment led to decreased levels of oxazolone-specific antibodies. Histologically, mice exposed to genistein and immunized with collagen II displayed somewhat lower degree of inflammation and joint destruction. In addition, serum levels of autoantibodies to collagen II were significantly lower following genistein-treatment in immunized mice. Conclusion: We conclude that genistein exerts evident anti-inflammatory properties affecting granulocytes, monocytes, and lymphocytes.

Role of Neutrophil Leukocytes in Cutaneous Infection Caused by Staphylococcus aureus

ABSTRACT Despite the high prevalence of cutaneous infections, little is known about the role of host immune responsiveness duringStaphylococcus aureus dermatitis. We have recently described a murine model of infectious dermatitis induced by superantigen-producing S. aureus. To assess the role of neutrophils in staphylococcal dermatitis, mice were given granulocyte-depleting monoclonal antibody prior to and on several occasions following intracutaneous inoculation with staphylococci. The granulocyte-depleted mice that had been intradermally inoculated withS. aureus developed crusted ulcerations which tended not to heal, whereas animals injected with control monoclonal antibody displayed only minor and transient skin lesions. The finding of severe ulcerations in neutropenic mice correlated with a significantly higher burden of bacteria in the blood and skin during the early phase of the infection. Importantly, while mice with an intact granulocyte population showed only limited skin infection, bacteremia occurred in the great majority of the neutrophil-depleted animals. As a consequence, the latter individuals exhibited significantly increased levels of the proinflammatory cytokine interleukin-6 and specific antibodies to staphylococcal cell wall components and toxic shock syndrome toxin-1 in the serum. Our data point to a crucial protective role of granulocytes in S. aureus dermatitis.

On the Role of Staphylococcus aureus Sortase and Sortase-Catalyzed Surface Protein Anchoring in Murine Septic Arthritis

Anchoring of Staphylococcus aureus surface protein to the cell wall is catalyzed by sortase, a transpeptidase. The contribution of staphylococcal surface proteins to establishment of infection was examined using a murine septic arthritis model. Intravenous inoculation of mice with the sortase-deficient mutant S. aureus strain SMK3 did not result in weight loss or severe septic arthritis, in contrast to the parent strain, S. aureus Newman. Direct inoculation of the sortase mutant into joint cavities also failed to cause severe synovitis or erosive arthritis. Furthermore, intravenous inoculation with staphylococci resulted in the rapid clearing of the sortase mutant from the bloodstream. This phenomenon demonstrates the involvement of host neutrophils; when these cells were depleted, sortase mutant staphylococci caused severe systemic infection, although not septic arthritis. These results suggest that sortase mutant staphylococci are significantly less virulent than the parent strain, Newman: the sortase mutant has decreased ability to reach target organs and, once there, to induce an inflammatory response.

Protection against Staphylococcus auveus Sepsis by Vaccination with Recombinant Staphylococcal Enterotoxin A Devoid of Superantigenicity

Staphylococcal exotoxins are virulence determinants in Staphylococcus aureus arthritis and septicemia. To assess the utility of enterotoxins as vaccine candidates for these diseases, a genetically modified staphylococcal enterotoxin A (SEA) that lacks superantigenic properties was used. Mice immunized with recombinant (r) SEA had significantly longer survival than control immunized mice and lost significantly less weight than the controls. Transfer of SEA-specific antibodies to naive mice resulted in good protection against death in staphylococcal sepsis. In vitro proliferative responses to SEA by naive lymphocytes were almost totally abolished on incubation with serum from rSEA but not with control antigen-immunized mice. These results suggest that immunization with rSEA devoid of superantigenic properties provides good protection against S. aureus sepsis. In addition, the data indicate that the protection is at least in part mediated by SEA neutralizing antibodies.

Model systems: modeling human staphylococcal arthritis and sepsis in the mouse.

The staphylococci have been recognized as serious pathogens for over a century and are the etiological agent of a variety of diseases ranging from mild cutaneous infections to often fatal forms of septic arthritis, endocarditis, toxic shock syndrome and sepsis. Despite intensive efforts to halt their spread, they remain the most common cause of community- and nosocomially acquired bacteremia. Murine models of Staphylocococus aureus-mediated arthritis and sepsis exist and are being used to gain a better understanding of the host-bacterium relationship as well to develop better methods of prevention and treatment.

Ethanol prevents development of destructive arthritis

Environmental factors are thought to play a major role in the development of rheumatoid arthritis. Because the use of ethanol is widespread, we assessed the role of ethanol intake on the propensity to develop chronic arthritis. Collagen type II-immunized mice were given water or water containing 10% (vol/vol) ethanol or its metabolite acetaldehyde. Their development of arthritis was assessed, as well as the impact of ethanol on leukocyte migration and activation of intracellular transcription factors. Mice exposed daily to this dose of ethanol did not display any liver toxicity, and the development of erosive arthritis was almost totally abrogated. In contrast, the antibody-mediated effector phase of collagen-induced arthritis was not influenced by ethanol exposure. Also, the major ethanol metabolite, acetaldehyde, prevented the development of arthritis. This antiinflammatory and antidestructive property of ethanol was mediated by (i) down-regulation of leukocyte migration and (ii) up-regulation of testosterone secretion, with the latter leading to decreased NF-κB activation. We conclude that low but persistent ethanol consumption delays the onset and halts the progression of collagen-induced arthritis by interaction with innate immune responsiveness.

Role of macrophages in Staphylococcus aureus-induced arthritis and sepsis.

OBJECTIVE A model of hematogenously induced Staphylococcus aureus arthritis was used to analyze the role of macrophages in this highly destructive condition. In this model, the majority of cells in the cartilage-synovial junction that participate in the destructive process are macrophages. METHODS To assess the role of monocytes/macrophages in staphylococcal arthritis, mice were inoculated with S aureus or given phosphate buffered saline as control. Mice were rendered monocytopenic by administration of etoposide, a drug that selectively depletes the monocyte/macrophage population. RESULTS Throughout the course of infection, the etoposide-treated mice exhibited a significantly less severe arthritis than the control animals. These data were confirmed by histopathologic analysis of the joints. The down-regulation of development of arthritis was accompanied by decreased serum levels of the proinflammatory cytokines tumor necrosis factor alpha and interleukin-6. In contrast, infection-triggered mortality was increased in the etoposide-treated mice as compared with the control animals. Notably, the monocytopenic mice exhibited elevated bacterial burden in the blood and kidneys on days 3 and 7 after inoculation with staphylococci. CONCLUSION This study indicates a dual role of mononuclear phagocytes in the pathogenesis of S aureus-induced infection. On the one hand, absence of macrophages leads to a favorable outcome concerning the severity of arthritic lesions, but on the other hand, the clearance of bacteria by monocytes/macrophages is decreased, resulting in poor survival.

The major role of macrophages and their product tumor necrosis factor α in the induction of arthritis triggered by bacterial DNA containing CpG motifs

OBJECTIVE To understand the mechanisms of arthritis triggered by CpG-containing oligonucleotides (ODN). METHODS Following the induction of CpG ODN-triggered arthritis in mice, we analyzed the impact of depletion of immune cells, including neutrophils, natural killer (NK) cells, and monocyte/macrophages, on the arthritis, as well as the impact in SCID mice lacking T and B cells. In addition, tumor necrosis factor alpha (TNFalpha) knockout mice were studied, and intraarticular administration of p65 antisense to nuclear factor kappaB (NF-kappaB) was used to examine effects in CpG ODN-triggered arthritis. Cytokine messenger RNA expression in synovial tissue was evaluated by in situ hybridization. RESULTS The presence of macrophages was mandatory for the mediation of arthritis triggered by CpG ODN, whereas the absence of neutrophils, NK cells, T cells, and B cells was of minor importance in this regard. The proinflammatory cytokines TNFalpha, interleukin-1beta, and interleukin-12, which originate from macrophages, were frequently found in the inflamed joints, and TNFalpha was confirmed to be an important mediator in the development of arthritis, since the incidence and severity of joint inflammation were markedly reduced in TNFalpha knockout mice. NF-kappaB exerted an important regulatory role in the development of CpG ODN-mediated arthritis, since local administration of antisense to the p65 subunit of NF-kappaB diminished the incidence of inflammation by 50%. CONCLUSION Macrophages and their products play an important role in the development of arthritis triggered by bacterial DNA containing CpG motifs.

Reactive Oxygen Species Produced by the NADPH Oxidase 2 Complex in Monocytes Protect Mice from Bacterial Infections

Chronic granulomatous disease (CGD) is an inherited disorder characterized by recurrent life-threatening bacterial and fungal infections. CGD results from defective production of reactive oxygen species by phagocytes caused by mutations in genes encoding the NADPH oxidase 2 (NOX2) complex subunits. Mice with a spontaneous mutation in Ncf1, which encodes the NCF1 (p47phox) subunit of NOX2, have defective phagocyte NOX2 activity. These mice occasionally develop local spontaneous infections by Staphylococcus xylosus or by the common CGD pathogen Staphylococcus aureus. Ncf1 mutant mice were more susceptible to systemic challenge with these bacteria than were wild-type mice. Transgenic Ncf1 mutant mice harboring the wild-type Ncf1 gene under the human CD68 promoter (MN+ mice) gained the expression of NCF1 and functional NOX2 activity specifically in monocytes/macrophages, although minimal NOX2 activity was also detected in some CD11b+Ly6G+ cells defined as neutrophils. MN+ mice did not develop spontaneous infection and were more resistant to administered staphylococcal infections compared with MN− mice. Most strikingly, MN+ mice survived after being administered Burkholderia cepacia, an opportunistic pathogen in CGD patients, whereas MN− mice died. Thus, monocyte/macrophage expression of functional NCF1 protected against spontaneous and administered bacterial infections.