Inhibition of PAD4 mediated neutrophil extracellular traps prevents fibrotic osseointegration failure in a tibial implant murine model : an animal study.

AIMS: Aseptic loosening is a leading cause of uncemented arthroplasty failure, often accompanied by fibrotic tissue at the bone-implant interface. A biological target, neutrophil extracellular traps (NETs), was investigated as a crucial connection between the innate immune system's response to injury, fibrotic tissue development, and proper bone healing. Prevalence of NETs in peri-implant fibrotic tissue from aseptic loosening patients was assessed. A murine model of osseointegration failure was used to test the hypothesis that inhibition (through Pad4-/- mice that display defects in peptidyl arginine deiminase 4 (PAD4), an essential protein required for NETs) or resolution (via DNase 1 treatment, an enzyme that degrades the cytotoxic DNA matrix) of NETs can prevent osseointegration failure and formation of peri-implant fibrotic tissue. METHODS: Patient peri-implant fibrotic tissue was analyzed for NETs biomarkers. To enhance osseointegration in loose implant conditions, an innate immune system pathway (NETs) was either inhibited (Pad4-/- mice) or resolved with a pharmacological agent (DNase 1) in a murine model of osseointegration failure. RESULTS: NETs biomarkers were identified in peri-implant fibrotic tissue collected from aseptic loosening patients and at the bone-implant interface in a murine model of osseointegration failure. Inhibition (Pad4-/- ) or resolution (DNase 1) of NETs improved osseointegration and reduced fibrotic tissue despite loose implant conditions in mice. CONCLUSION: This study identifies a biological target (NETs) for potential noninvasive treatments of aseptic loosening by discovering a novel connection between the innate immune system and post-injury bone remodelling caused by implant loosening. By inhibiting or resolving NETs in an osseointegration failure murine model, fibrotic tissue encapsulation around an implant is reduced and osseointegration is enhanced, despite loose implant conditions. Cite this article: Bone Joint J 2021;103-B(7 Supple B):135-144.

AAV-mediated gene transfer of DNase I in the liver of mice with colorectal cancer reduces liver metastasis and restores local innate and adaptive immune response.

Liver metastasis is the main cause of colorectal cancer (CRC)-related death. Neutrophil extracellular traps (NETs) play important roles in CRC progression. Deoxyribonuclease I (DNase I) has been shown to alter NET function by cleaving DNA strands comprising the NET backbone. Moreover, DNase I displays high antimetastatic activity in multiple tumor models. To circumvent long-term daily administrations of recombinant DNase I, we have developed an adeno-associated virus (AAV) gene therapy vector to specifically express DNase I in the liver. In this study, we demonstrate AAV-mediated DNase I liver gene transfer following a single intravenous injection suppresses the development of liver metastases in a mouse model of CRC liver metastasis. Increased levels of neutrophils and NET formation in tumors are associated with poor prognosis in many patients with advanced cancers. Neutrophil infiltration and NET formation were inhibited in tumor tissues with AAV-DNase I treatment. This approach restored local immune responses at the tumor site by increasing the percentage of CD8+ T cells while keeping CD4+ T cells similar between AAV-DNase I and AAV-null treatments. Our data suggest that AAV-mediated DNase I liver gene transfer is a safe and effective modality to inhibit metastasis and represents a novel therapeutic strategy for CRC.

Anti-DNase I Antibody: A New Serological Reactivity in Primary Sjögren Syndrome.

BACKGROUND AND OBJECTIVE: Primary Sjögren syndrome (pSS) is a systemic autoimmune rheumatic disease that particularly affects exocrine glands. Dry eye is one of the most important features of this syndrome, and a recent study reported reduced deoxyribonuclease I (DNase I) activity in the tear of patients with dry eye. We therefore postulated that patients with pSS might have antibodies targeting DNAse I. METHODS: We have evaluated in a cross-sectional study 85 patients with pSS (2002 American-European Consensus Group Criteria), 50 rheumatoid arthritis (RA) patients (1987 American College of Rheumatology Criteria) without sicca symptoms, and 88 healthy volunteers. IgG anti-DNase I was detected by enzyme-linked immunosorbent assay using as antigen bovine pancreas enzyme and confirmed by immunoblotting. RESULTS: Age and sex were alike in the 3 groups (p > 0.05). Anti-DNase I was detected in 43.5% of the pSS patients. In contrast, this reactivity was absent in all RA patients (p = 0.0001). Additional comparison of pSS patients with (n = 37) or without (n = 48) anti-DNase I showed that the former group had higher IgG serum levels (2293.2 ± 666.2 vs 1483.9 ± 384.6 mg/dL, p = 0.0001) and greater rate of non-drug-induced leukopenia (43% vs 19%, p = 0.02). A multivariate logistic regression analysis identified that only IgG levels were independently associated with anti-DNase I. CONCLUSIONS: We describe a high frequency of anti-DNase I antibodies in pSS patients associated with higher serum IgG levels. The lack of this reactivity in RA patients without sicca symptoms suggests that this antibody may be helpful in the differential diagnosis of these diseases.

Human polyspecific immunoglobulin attenuates group A streptococcal virulence factor activity and reduces disease severity in a murine necrotizing fasciitis model.

OBJECTIVES: Streptococcus pyogenes causes life-threatening invasive infections including necrotizing fasciitis (NF). Current treatment guidelines recommend the use of a cell-wall-active antibiotic combined with a protein synthesis inhibitor and surgical debridement in NF patients. Adjunctive therapy with intravenous immunoglobulin (IVIG) has been proposed for superantigen-associated streptococcal toxic shock syndrome. So far, benefits of IVIG treatment remain unclear and prospective clinical studies are scarce. Thus, we aimed to assess the effects of IVIG on virulence factor activity in vitro, ex vivo in patients and in vivo in a NF mouse model. METHODS: We investigated the effect of IVIG on the activity of the virulence factors streptolysin O (SLO), streptodornase 1 (Sda1), S. pyogenes cell envelope protease and streptococcal pyrogenic exotoxin B in vitro and ex vivo in patient sera. Additionally, we assessed the influence of IVIG on the clinical outcome in a murine NF model. RESULTS: In vitro, IVIG inhibited various streptococcal virulence factors. Further, IVIG treatment of group A Streptococcus-infected mice led to a reduced skin lesion size (median (interquartile range) day 3 intraperitoneal administration: 12 mm2 (9-14.5) vs. 4 mm2 (0.8-10.5), subcutaneous: 10.3 mm2 (6.9-18.6) vs. 0.5 mm2 (0.1-6.8)) and lower SLO activity. After treatment with IVIG, patient sera showed an elevated titre of specific SLO (7/9) and Sda1 (5/9) antibodies, reducing SLO and Sda1 activity. CONCLUSIONS: The clear reduction in disease severity in IVIG-treated mice and inhibition of virulence factor activity in mouse and human sera suggest that IVIG may be beneficial in invasive group A Streptococcus infections such as NF in addition to streptococcal toxic shock syndrome.

Group A Streptococcal DNase Sda1 Impairs Plasmacytoid Dendritic Cells' Type 1 Interferon Response.

Group A Streptococcus causes severe invasive infections, including necrotizing fasciitis. The expression of an array of virulence factors targeting specific host immune functions impedes successful bacterial clearance. The virulence factor streptococcal DNase Sda1 was previously shown to interfere with the entrapment of bacteria through neutrophil extracellular traps and TLR9 signaling. In this study, we showed that plasmacytoid dendritic cells are recruited to the infected tissue during group A streptococcal necrotizing fasciitis. We found that the streptococcal DNase Sda1 impairs plasmacytoid dendritic cell recruitment by reducing IFN-1 levels at the site of infection. We found that streptococcal DNase Sda1 interferes with stabilization of the DNA by the host molecule HMGB1 protein, which may account for decreased IFN-1 levels at the site of infection.

Anti-dsDNA antibodies and resident renal cells - Their putative roles in pathogenesis of renal lesions in lupus nephritis.

Lupus nephritis affects up to 70% of patients with systemic lupus erythematosus and is an important treatable cause of kidney failure. Cardinal features of lupus nephritis include loss of self-tolerance, production of autoantibodies, immune complex deposition and immune-mediated injury to the kidney, resulting in increased cell proliferation, apoptosis, and induction of inflammatory and fibrotic processes that destroy normal nephrons. The production anti-dsDNA antibodies is a cardinal feature in lupus and their level correlates with disease activity. In addition to the formation of immune complexes thereby triggering complement activation, how anti-dsDNA antibodies home to the kidney and induce pathological processes in the renal parenchyma remain to be fully elucidated. Data from our laboratory and other investigators show that the properties of anti-dsDNA antibodies vary between patients and change over time, and that anti-dsDNA antibodies could bind directly to integral cell surface molecules such as annexin II or α-actinin, or indirectly through chromatin material deposited on the cell surface. The binding of anti-dsDNA antibodies to mesangial cells and proximal renal tubular epithelial cells triggers downstream inflammatory and fibrotic pathways, which include the activation of the PKC and MAPK signaling pathways, increased secretion of pro-inflammatory cytokines and matrix protein deposition that contribute to pathological processes in the renal parenchyma.

Distal regulation of c-myb expression during IL-6-induced differentiation in murine myeloid progenitor M1 cells.

The c-Myb transcription factor is a major regulator that controls differentiation and proliferation of hematopoietic progenitor cells, which is frequently deregulated in hematological diseases, such as lymphoma and leukemia. Understanding of the mechanisms regulating the transcription of c-myb gene is challenging as it lacks a typical promoter and multiple factors are involved. Our previous studies identified some distal regulatory elements in the upstream regions of c-myb gene in murine myeloid progenitor M1 cells, but the detailed mechanisms still remain unclear. In the present study, we found that a cell differentiation-related DNase1 hypersensitive site is located at a -28k region upstream of c-myb gene and that transcription factors Hoxa9, Meis1 and PU.1 bind to the -28k region. Circular chromosome conformation capture (4C) assay confirmed the interaction between the -28k region and the c-myb promoter, which is supported by the enrichment of CTCF and Cohesin. Our analysis also points to a critical role for Hoxa9 and PU.1 in distal regulation of c-myb expression in murine myeloid cells and cell differentiation. Overexpression of Hoxa9 disrupted the IL-6-induced differentiation of M1 cells and upregulated c-myb expression through binding of the -28k region. Taken together, our results provide an evidence for critical role of the -28k region in distal regulatory mechanism for c-myb gene expression during differentiation of myeloid progenitor M1 cells.

Anti-DNase I antibodies in systemic lupus erythematosus: diagnostic value and share in the enzyme inhibition.

Diagnostic accuracy of anti-DNase I antibodies measurement in a differentiation between SLE and other autoimmune rheumatic diseases was evaluated. The share of anti-DNase I and actin in the DNase I activity decrease in SLE was established. Serum samples were obtained from 54 patients with verified SLE, 52 control patients with other autoimmune rheumatic diseases, and 44 healthy persons. Anti-DNase I concentrations were measured by ELISA. Free and actin inhibited DNase I activities were evaluated in the fresh serum samples. The appraisal of antibodies and actin effects on DNase I activity was made using multiple regression. Anti-DNase I antibodies were positive in 35 SLE and 8 control patients, without significant difference between the mean antibody concentrations. Sensitivity of this test was 64.81 %, and specificity-84.62 %. Mean free DNase I activity in SLE was somewhat lower than in the control group as a result of augmented frequency of extremely low enzyme activities. On the contrary, after the exclusion of the latter cases we have revealed elevated mean free DNase I activity in the other SLE patients comparing to the similar control subgroup. Unlike the controls, low serum DNase I activity in SLE arose not only from actin and antibody action, but also, in half of the cases, from unidentified factor, related to active SLE. The accuracy of the anti-DNase I antibodies measurement is approximate to the present reference standard of SLE diagnostics. We first demonstrated that neither antibodies nor actin caused DNase I activity decrease in SLE.

Homogeneous Immunochemical Assay on the Lateral Flow Strip for Measurement of DNase I Activity.

Deoxyribonuclease I (DNase I) is an important enzyme that cleaves both double-stranded and single-stranded DNA at their phosphate backbone. DNase I is a useful biomarker. Previous studies have shown that patients with prostate cancer and systemic lupus erythematosus exhibit reduced DNase I activity, and patients with myocardial infarction exhibit increased DNase I activity. Current methods of measuring DNase I relies either on an immunochemical assay, which requires multiple washing steps, or on a single radial enzyme diffusion assay, which requires a long digestion time and an expensive fluorescence detection system. We have developed a lateral flow immunochemical assay for the measurement of DNase I activity on the test strip. The assay utilized a dually labeled double-stranded DNA as the reporter probe. The biotin-labeled terminal of the probe bound to the streptavidin immobilized on the lateral flow test strip, and the fluorescein-labeled terminal bound to the antibody-conjugated gold nanoparticles, resulting in a visible test line. The presence of DNase I would cleave the reporter probe and lead to reduced test line intensity. Using the DNase I test strip, we have successfully measured the DNase I activity and determined the factors that influence the sensitivity and linear dynamic range of the assay. We have also investigated the conditions that inhibited the DNase I activity. The combined advantage of a wash-free assay format and colorimetric readout would make the lateral flow DNase I test strip a suitable platform for point-of-care diagnostics.

The Mga Regulon but Not Deoxyribonuclease Sda1 of Invasive M1T1 Group A Streptococcus Contributes to In Vivo Selection of CovRS Mutations and Resistance to Innate Immune Killing Mechanisms.

Invasive M1T1 group A Streptococcus (GAS) can have a mutation in the regulatory system CovRS, and this mutation can render strains hypervirulent. Interestingly, via mechanisms that are not well understood, the host innate immune system's neutrophils select spontaneous M1T1 GAS CovRS hypervirulent mutants, thereby enhancing the pathogen's ability to evade immune killing. It has been reported that the DNase Sda1 is critical for the resistance of M1T1 strain 5448 to killing in human blood and provides pressure for in vivo selection of CovRS mutations. We reexamined the role of Sda1 in the selection of CovRS mutations and in GAS innate immune evasion. Deletion of sda1 or all DNase genes in M1T1 strain MGAS2221 did not alter emergence of CovRS mutants during murine infection. Deletion of sda1 in strain 5448 resulted in Δsda1 mutants with (5448 Δsda1(M+) strain) and without (5448 Δsda1(M-) strain) M protein production. The 5448 Δsda1(M+) strain accumulated CovRS mutations in vivo and resisted killing in the bloodstream, whereas the 5448 Δsda1(M-) strain lost in vivo selection of CovRS mutations and was sensitive to killing. The deletion of emm and a spontaneous Mga mutation in MGAS2221 reduced and prevented in vivo selection for CovRS mutants, respectively. Thus, in contrast to previous reports, Sda1 is not critical for in vivo selection of invasive M1T1 CovRS mutants and GAS resistance to innate immune killing mechanisms. In contrast, M protein and other Mga-regulated proteins contribute to the in vivo selection of M1T1 GAS CovRS mutants. These findings advance the understanding of the progression of invasive M1T1 GAS infections.

A biomarker based detection and characterization of carcinomas exploiting two fundamental biophysical mechanisms in mammalian cells.

BACKGROUND: Biomarkers allowing the characterization of malignancy and therapy response of oral squamous cell carcinomas (OSCC) or other types of carcinomas are still outstanding. The biochemical suicide molecule endonuclease DNaseX (DNaseI-like 1) has been used to identify the Apo10 protein epitope that marks tumor cells with abnormal apoptosis and proliferation. The transketolase-like protein 1 (TKTL1) represents the enzymatic basis for an anaerobic glucose metabolism even in the presence of oxygen (aerobic glycolysis/Warburg effect), which is concomitant with a more malignant phenotype due to invasive growth/metastasis and resistance to radical and apoptosis inducing therapies. METHODS: Expression of Apo10 and TKTL1 was analysed retrospectively in OSCC specimen (n = 161) by immunohistochemistry. Both markers represent independent markers for poor survival. Furthermore Apo10 and TKTL1 have been used prospectively for epitope detection in monocytes (EDIM)-blood test in patients with OSCC (n = 50), breast cancer (n = 48), prostate cancer (n = 115), and blood donors/controls (n = 74). RESULTS: Positive Apo10 and TKTL1 expression were associated with recurrence of the tumor. Multivariate analysis demonstrated Apo10 and TKTL1 expression as an independent prognostic factor for reduced tumor-specific survival. Apo10+/TKTL1+ subgroup showed the worst disease-free survival rate in OSCC.EDIM-Apo10 and EDIM-TKTL1 blood tests allowed a sensitive and specific detection of patients with OSCC, breast cancer and prostate cancer before surgery and in after care. A combined score of Apo10+/TKTL1+ led to a sensitivity of 95.8% and a specificity of 97.3% for the detection of carcinomas independent of the tumor entity. CONCLUSIONS: The combined detection of two independent fundamental biophysical processes by the two biomarkers Apo10 and TKTL1 allows a sensitive and specific detection of neoplasia in a noninvasive and cost-effective way. Further prospective trials are warranted to validate this new concept for the diagnosis of neoplasia and tumor recurrence.

Macrophage clearance of neutrophil extracellular traps is a silent process.

Neutrophil extracellular traps (NETs) facilitate the extracellular killing of pathogens. However, in recent years, excessive NET formation has been implicated in several pathological conditions. Indeed, NETs that are not removed from tissues or from the circulation might serve to trigger autoimmune responses. We observed that physiological amounts of DNase I do not suffice to completely degrade NETs in vitro, suggesting that additional mechanisms are required for the removal of these extracellular structures. We show in this article that human monocyte-derived macrophages are able to engulf NETs in a cytochalasin D-dependent manner, indicating that this is an active, endocytic process. Furthermore, preprocessing of NETs by DNase I facilitated their clearance by macrophages. In addition, both recombinant C1q and endogenous C1q derived from human serum were found to opsonize NETs, and this facilitated NET clearance. Upon internalization, NETs were apparently degraded in lysosomes, as treatment with chloroquine led to accumulation of extranuclear DNA in human monocyte-derived macrophages. Finally, uptake of NETs alone did not induce proinflammatory cytokine secretion, whereas LPS-induced production of IL-1ß, IL-6, and TNF-α was promoted by the uptake of NETs. In summary, we show that macrophages are capable of clearance of NETs and that this occurs in an immunologically silent manner.

DNase Sda1 allows invasive M1T1 Group A Streptococcus to prevent TLR9-dependent recognition.

Group A Streptococcus (GAS) has developed a broad arsenal of virulence factors that serve to circumvent host defense mechanisms. The virulence factor DNase Sda1 of the hyperinvasive M1T1 GAS clone degrades DNA-based neutrophil extracellular traps allowing GAS to escape extracellular killing. TLR9 is activated by unmethylated CpG-rich bacterial DNA and enhances innate immune resistance. We hypothesized that Sda1 degradation of bacterial DNA could alter TLR9-mediated recognition of GAS by host innate immune cells. We tested this hypothesis using a dual approach: loss and gain of function of DNase in isogenic GAS strains and presence and absence of TLR9 in the host. Either DNA degradation by Sda1 or host deficiency of TLR9 prevented GAS induced IFN-α and TNF-α secretion from murine macrophages and contributed to bacterial survival. Similarly, in a murine necrotizing fasciitis model, IFN-α and TNF-α levels were significantly decreased in wild type mice infected with GAS expressing Sda1, whereas no such Sda1-dependent effect was seen in a TLR9-deficient background. Thus GAS Sda1 suppressed both the TLR9-mediated innate immune response and macrophage bactericidal activity. Our results demonstrate a novel mechanism of bacterial innate immune evasion based on autodegradation of CpG-rich DNA by a bacterial DNase.

[Recurrent toxin-mediated perineal erythema in an 11-year-old child]. / Érythème périnéal récidivant toxinique chez un enfant de 11 ans.

INTRODUCTION: Recurrent perineal erythema is a rare toxin-mediated disease. We report the case of recurrent toxin-mediated perineal erythema in a child. CASE REPORT: An 11-year-old boy was hospitalized for erythematous pustular eruption involving the perineum and the axillary area. This erythema started a few days after the onset of pharyngotonsillitis and the patient's personal history involved another episode of pharyngotonsillitis which was followed by an identical cutaneous eruption. Laboratory analysis confirmed the diagnosis of recurrent toxin-mediated perineal erythema. The skin disorder quickly improved and antistreptococcal antibiotic treatment was initiated to eradicate bacteria. DISCUSSION: Recurrent toxin-mediated perineal erythema is a cutaneous disease mediated by superantigens which are toxins produced by staphylococci and streptococci. It is characterized by recurrent macular erythema involving the perineum. Streptococcus pyogenes is the most common cause of recurrent toxin-mediated perineal erythema, with Staphylococcus aureus being isolated most rarely. This observation emphasizes the possibility of atypical clinical presentation with pustular lesions, and dermatologists must be mindful of this aetiology in order to isolate bacterial toxins and to initiate appropriate antibiotics.

DNase, RNase, and phosphatase activities of antibodies formed upon immunization by DNA, DNase I, and DNase II.

Relative DNase, RNase (efficiency of hydrolysis of ribo- and deoxyribooligonucleotides (ON)), and phosphatase (removal of the ON 5' terminal phosphate) catalytic activities of antibodies (AB) obtained after rabbit immunization by DNA, DNase I, and DNase II were compared. It is shown that electrophoretically homogeneous preparations of polyclonal AB from non-immunized rabbits did not exhibit such activities. Immunization of rabbits by DNA, DNase I, and DNase II results in generation of IgG abzymes that exhibit high activity in the ON hydrolysis reaction and even higher activity in cleavage of 5' terminal phosphate of ON. In this case K(m) values for supercoiled plasmid DNA and ON found in reactions of their AB-dependent nuclease hydrolysis and phosphatase cleavage of 5' terminal phosphate differ by 2-4 orders of magnitude. This shows that nuclease and phosphatase activities belong to different abzyme fractions within polyclonal AB. Thus, in this work data indicative of the possibility of a formation of antibodies exhibiting phosphatase activity after immunization of animals with DNA, DNase I, and DNase II, were obtained for the first time. Possible reasons for production of AB with phosphatase activity after immunization of rabbits with these immunogens are discussed.

Treatment with DNAse I fosters binding to nec PBMC of CRP.

CRP is an important inflammatory marker, however, CRP levels are relatively low in patients with SLE. In addition patients with SLE often display low activities and serum levels for DNase I and complement, respectively. Here we show that DNase I treatment of nec PBMC increased their binding of CRP. Consequently, reduced DNase I activity in patients with SLE may contribute to the impaired opsonisation by CRP of dead cells, exacerbating the clearance defect in SLE of apo and nec cells.

Development of a sensitive enzyme-linked immunosorbent assay for measurement of DNase I in human serum.

BACKGROUND: Serum deoxyribonuclease I (DNase I) activity was reported to increase in the early phase after onset of acute myocardial infarction (AMI). Up to now, DNase I activity has been quantified by the single radial enzyme diffusion (SRED) method, which unfortunately requires a long incubation time. Therefore it is necessary to develop another assay suitable for measurement of serum DNase I concentrations in a clinical setting. METHODS: A sandwich ELISA was established for measurement of DNase I protein using a polyclonal antibody directed against DNase I protein and a biotinylated monoclonal for subsequent detection. Concentrations of serum DNase I protein were measured in healthy individuals and patients with AMI. RESULTS: This method was as precise as SRED, and took less time than SRED. A significant correlation was observed between DNase I concentration and enzyme activity (r=0.839; P<0.001). The average of serum DNase I in AMI patients within 0-12 h of chest pain was significantly higher than that in healthy individuals (P<0.001), and decreased with time. CONCLUSIONS: We have developed a sensitive ELISA capable of measuring DNase I protein concentrations. This method may be a useful alternative to SRED as an aid to diagnosis of AMI based on the serum DNase I level.

Serum DNase I, soluble Fas/FasL levels and cell surface Fas expression in patients with SLE: a possible explanation for the lack of efficacy of hrDNase I treatment.

The objectives of the study are to evaluate DNase I serum levels and their correlation with soluble Fas (sFas) and soluble Fas ligand (sFasL) and with cell surface Fas expression in patients with systemic lupus erythematosus (SLE), thus contributing to the dysregulated apoptosis typical of the disease. The methods include the following: Serum DNase I levels in patients and in controls were detected using the dot blot method and quantified by densitometry; sFas and sFasL were quantified using an ELISA system. Cell surface Fas expression was evaluated by FACS analysis. Apoptosis was studied by means of internucleosomal DNA degradation using a commercially available kit. The results are as follows: We found a significant difference in DNase I, sFas and sFasL serum levels between patients and controls. Levels of DNase I <7.79 ng ml(-1) are more represented in patients with SLE. Active SLE is strongly associated with high sFas levels and detectable sFasL. DNase I does not correlate with sFas or sFasL, whereas it correlates with T cell surface Fas expression that is higher in patients with active SLE than in healthy controls. Finally, administration of exogenous human recombinant DNase (hrDNase) I to freshly isolated T cells up-regulates cell surface Fas expression and induces increased susceptibility to Fas-mediated apoptosis. In conclusion, our findings confirm that DNase I is low in SLE and suggest that it may play a role in apoptosis in SLE by regulating the surface expression of the cell death molecule Fas. This role may contribute to explain the inefficacy of hrDNase I in SLE, a treatment proposed for the ability of DNase I to remove DNA from auto-antigenic nucleoprotein complexes.

DNase I inhibits a late phase of reactive oxygen species production in neutrophils.

Neutrophils kill bacteria on extracellular complexes of DNA fibers and bactericidal proteins known as neutrophil extracellular traps (NETs). The NET composition and the bactericidal mechanisms they use are not fully understood. Here, we show that treatment with deoxyribonuclease (DNase I) impairs a late oxidative response elicited by Gram-positive and Gram-negative bacteria and also by phorbol ester. Isoluminol-dependent chemiluminescence elicited by opsonized Listeria monocytogenes-stimulated neutrophils was inhibited by DNase I, and the DNase inhibitory effect was also evident when phagocytosis was blocked, suggesting that DNase inhibits an extracellular mechanism of reactive oxygen species (ROS) generation. The DNase inhibitory effect was independent of actin polymerization. Phagocytosis and cell viability were not impaired by DNase I. Immunofluorescence analysis shows that myeloperoxidase is present on NETs. Furthermore, granular proteins were detected in NETs from Rab27a-deficient neutrophils which have deficient exocytosis, suggesting that exocytosis and granular protein distribution on NETs proceed by independent mechanisms. NADPH oxidase subunits were also detected on NETs, and the detection of extracellular trap-associated NADPH oxidase subunits was abolished by treatment with DNase I and dependent on cell stimulation. In vitro analyses demonstrate that MPO and NADPH oxidase activity are not directly inhibited by DNase I, suggesting that its effect on ROS production depends on NET disassembly. Altogether, our data suggest that inhibition of ROS production by microorganism-derived DNase would contribute to their ability to evade killing.