Eosinophilia and reduced STAT3 signaling affect neutrophil cell death in autosomal-dominant Hyper-IgE syndrome.
ABSTRACT: The autosomal-dominant hyper-IgE syndrome (HIES), caused by mutations in STAT3, is a rare primary immunodeficiency that predisposes to mucocutaneous candidiasis and staphylococcal skin and lung infections. This infection phenotype is suggestive of defects in neutrophils, but data on neutrophil functions in HIES are inconsistent. This study was undertaken to functionally characterize neutrophils in STAT3-deficient HIES patients and to analyze whether the patients` eosinophilia affects the neutrophil phenotype in S. aureus infection. Neutrophil functions and cell death kinetics were studied in eight STAT3-deficient patients. Moreover, the response of STAT3-deficient neutrophils to S. aureus and the impact of autologous eosinophils on pathogen-induced cell death were analyzed. No specific aberrations in neutrophil functions were detected within this cohort. However, the half-life of STAT3-deficient neutrophils ex vivo was reduced, which was partially attributable to the presence of eosinophils. Increased S. aureus-induced cell lysis, dependent on the staphylococcal virulence controlling accessory gene regulator (agr)-locus, was observed in STAT3-deficient neutrophils and upon addition of eosinophils. Accelerated neutrophil cell death kinetics may underlie the reported variability in neutrophil function testing in HIES. Increased S. aureus-induced lysis of STAT3-deficient neutrophils might affect pathogen control and contribute to tissue destruction during staphylococcal infections in HIES.
Project description:Autosomal dominant hyper IgE syndrome (AD-HIES) is a primary immunodeficiency caused by a loss-of-function mutation in the Signal Transducer and Activator of Transcription 3 (STAT3). This immune disorder is clinically characterized by increased susceptibility to cutaneous and sinopulmonary infections, in particular with Candida and Staphylococcus aureus. It has recently been recognized that the skin microbiome of patients with AD-HIES is altered with an overrepresentation of certain Gram-negative bacteria and Gram-positive staphylococci. However, these alterations have not been characterized at the species- and strain-level. Since S. aureus infections are influenced by strain-specific expression of virulence factors, information on colonizing strain characteristics may provide insights into host-pathogen interactions and help guide management strategies for treatment and prophylaxis. The aim of this study was to determine whether the immunodeficiency of AD-HIES selects for unique strains of colonizing S. aureus. Using multi-locus sequence typing (MLST), protein A (spa) typing, and PCR-based detection of toxin genes, we performed a detailed analysis of the S. aureus isolates (n = 13) found on the skin of twenty-one patients with AD-HIES. We found a low diversity of sequence types, and an abundance of strains that expressed methicillin resistance, Panton-Valentine leukocidin (PVL), and staphylococcal enterotoxins K and Q (SEK, SEQ). Our results indicate that patients with AD-HIES may often carry antibiotic-resistant strains that harbor key virulence factors.
Project description:Mechanisms underlying the enhanced virulence phenotype of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are incompletely defined, but presumably include evasion of killing by human polymorphonuclear leukocytes (PMNs or neutrophils). To better understand this phenomenon, we investigated the basis of rapid PMN lysis after phagocytosis of USA300, a prominent CA-MRSA strain. Survival of USA300 clinical isolates after phagocytosis ultimately resulted in neutrophil lysis. PMNs containing ingested USA300 underwent morphological changes consistent with apoptosis, but lysed rapidly thereafter (within 6 h), whereas cells undergoing FAS-mediated apoptosis or phagocytosis-induced cell death remained intact. Phagosome membranes remained intact until the point of PMN destruction, suggesting lysis was not caused by escape of S. aureus from phagosomes or the cytolytic action of pore-forming toxins. Microarray analysis of the PMN transcriptome after phagocytosis of representative community-associated S. aureus and healthcare-associated MRSA strains revealed changes unique to community-associated S. aureus strains, such as upregulation of transcripts involved in regulation of calcium homeostasis. Collectively, the data suggest that neutrophil destruction after phagocytosis of USA300 is in part a form of programmed necrosis rather than direct lysis by S. aureus pore-forming toxins. We propose that the ability of CA-MRSA strains to induce programmed necrosis of neutrophils is a component of enhanced virulence.
Project description:Staphylococcus aureus is a commensal organism in approximately 30% of the human population and colonization is a significant risk factor for invasive infection. As a result of this, there is a great need to better understand how S. aureus overcomes human immunity. Neutrophils are essential during the innate immune response to S. aureus, yet this microorganism uses multiple evasion strategies to avoid killing by these immune cells, perhaps the most catastrophic of which is the rapid induction of neutrophil cell death. The aim of this study was to better understand the mechanisms underpinning S. aureus-induced neutrophil lysis, and how this contributes to pathogenesis in a whole organism model of infection. To do this we screened the genome-wide Nebraska Transposon Mutant Library (NTML) in the community acquired methicillin resistant S. aureus strain, USA300, for decreased ability to induce neutrophil cell lysis. Out of 1,920 S. aureus mutants, a number of known regulators of cell lysis (including the master regulators accessory gene regulator A, agrA and Staphylococcus exoprotein expression protein S, saeS) were identified in this blinded screen, providing validity to the experimental system. Three gene mutations not previously associated with cell death: purB, lspA, and clpP were found to be significantly attenuated in their ability to induce neutrophil lysis. These phenotypes were verified by genetic transductants and complemented strains. purB and clpP were subsequently found to be necessary for bacterial replication and pathogenesis in a zebrafish embryo infection model. The virulence of the clpP mutant was restored in a neutrophil-depleted zebrafish model, suggesting the importance of ClpP in mechanisms underpinning neutrophil immunity to S. aureus. In conclusion, our work identifies genetic components underpinning S. aureus pathogenesis, and may provide insight into how this commensal organism breaches innate immune barriers during infection.
Project description:The autosomal dominant hyper-IgE syndrome (HIES, 'Job's syndrome') is characterized by recurrent and often severe pulmonary infections, pneumatoceles, eczema, staphylococcal abscesses, mucocutaneous candidiasis, and abnormalities of bone and connective tissue. Mutations presumed to underlie HIES have recently been identified in stat3, the gene encoding STAT3 (signal transducer and activator of transcription 3) (refs 3, 4). Although impaired production of interferon-gamma and tumour-necrosis factor by T cells, diminished memory T-cell populations, decreased delayed-type-hypersensitivity responses and decreased in vitro lymphoproliferation in response to specific antigens have variably been described, specific immunological abnormalities that can explain the unique susceptibility to particular infections seen in HIES have not yet been defined. Here we show that interleukin (IL)-17 production by T cells is absent in HIES individuals. We observed that ex vivo T cells from subjects with HIES failed to produce IL-17, but not IL-2, tumour-necrosis factor or interferon-gamma, on mitogenic stimulation with staphylococcal enterotoxin B or on antigenic stimulation with Candida albicans or streptokinase. Purified naive T cells were unable to differentiate into IL-17-producing (T(H)17) T helper cells in vitro and had lower expression of retinoid-related orphan receptor (ROR)-gammat, which is consistent with a crucial role for STAT3 signalling in the generation of T(H)17 cells. T(H)17 cells have emerged as an important subset of helper T cells that are believed to be critical in the clearance of fungal and extracellular bacterial infections. Thus, our data suggest that the inability to produce T(H)17 cells is a mechanism underlying the susceptibility to the recurrent infections commonly seen in HIES.
Project description:BACKGROUND: Interest in phage therapy has grown over the past decade due to the rapid emergence of antibiotic resistance in bacterial pathogens. However, the use of bacteriophages for therapeutic purposes has raised concerns over the potential for immune response, rapid toxin release by the lytic action of phages, and difficulty in dose determination in clinical situations. A phage that kills the target cell but is incapable of host cell lysis would alleviate these concerns without compromising efficacy. RESULTS: We developed a recombinant lysis-deficient Staphylococcus aureus phage P954, in which the endolysin gene was rendered nonfunctional by insertional inactivation. P954, a temperate phage, was lysogenized in S. aureus strain RN4220. The native endolysin gene on the prophage was replaced with an endolysin gene disrupted by the chloramphenicol acetyl transferase (cat) gene through homologous recombination using a plasmid construct. Lysogens carrying the recombinant phage were detected by growth in presence of chloramphenicol. Induction of the recombinant prophage did not result in host cell lysis, and the phage progeny were released by cell lysis with glass beads. The recombinant phage retained the endolysin-deficient genotype and formed plaques only when endolysin was supplemented. The host range of the recombinant phage was the same as that of the parent phage. To test the in vivo efficacy of the recombinant endolysin-deficient phage, immunocompromised mice were challenged with pathogenic S. aureus at a dose that results in 80% mortality (LD80). Treatment with the endolysin-deficient phage rescued mice from the fatal S. aureus infection. CONCLUSIONS: A recombinant endolysin-deficient staphylococcal phage has been developed that is lethal to methicillin-resistant S. aureus without causing bacterial cell lysis. The phage was able to multiply in lytic mode utilizing a heterologous endolysin expressed from a plasmid in the propagation host. The recombinant phage effectively rescued mice from fatal S. aureus infection. To our knowledge this is the first report of a lysis-deficient staphylococcal phage.
Project description:Hyper-immunoglobulin E syndrome (HIES) is a primary immune deficiency characterized by abnormal and devastating susceptibility to a narrow spectrum of infections, most commonly Staphylococcus aureus and Candida albicans. Recent investigations have identified mutations in STAT3 in the majority of HIES patients studied. Despite the identification of the genetic cause of HIES, the mechanisms underlying the pathological features of this disease remain to be elucidated. Here, we demonstrate a failure of CD4+ T cells harboring heterozygous STAT3 mutations to generate interleukin 17-secreting (i.e., T helper [Th]17) cells in vivo and in vitro due to a failure to express sufficient levels of the Th17-specific transcriptional regulator retinoid-related orphan receptor t. Because Th17 cells are enriched for cells with specificities against fungal antigens, our results may explain the pattern of infection susceptibility characteristic of patients with HIES. Furthermore, they underscore the importance of Th17 responses in normal host defense against the common pathogens S. aureus and C. albicans.
Project description:Autosomal dominant hyper IgE syndrome (AD-HIES), or Job's syndrome, is a primary immune deficiency caused by dominant-negative mutations in STAT3. Recurrent Staphylococcus aureus skin abscesses are a defining feature of this syndrome. A widely held hypothesis that defects in peripheral Th17 differentiation confer this susceptibility has never been directly evaluated. To assess the cutaneous immune response in AD-HIES, we induced suction blisters in healthy volunteers (HVs) and patients with AD-HIES and then challenged the wound with lethally irradiated bacteria. We show that cutaneous production of IL-17A and IL-17F was normal in patients with AD-HIES. Overproduction of TNF-? differentiated the responses in AD-HIES from HVs. This was associated with reduced IL-10 family signaling in blister-infiltrating cells and defective epithelial cell function. Mouse models of AD-HIES recapitulated these aberrant epithelial responses to S. aureus and involved defective epithelial-to-mesenchymal transition (EMT) rather than a failure of bacterial killing. Defective responses in mouse models of AD-HIES and primary keratinocyte cultures from patients with AD-HIES could be reversed by TNF-? blockade and by drugs with reported modulatory effects on EMT. Our results identify these as potential therapeutic approaches in patients with AD-HIES suffering S. aureus infections.
Project description:The roles of eosinophils in antimicrobial defense remain incompletely understood. In ovalbumin-sensitized mice, eosinophils are selectively recruited to the peritoneal cavity by antigen, eotaxin, or leukotriene(LT)B4, a 5-lipoxygenase (5-LO) metabolite. 5-LO blockade prevents responses to both antigen and eotaxin. We examined responses to eotaxin in the absence of sensitization and their dependence on 5-LO. BALB/c or PAS mice and their mutants (5-LO-deficient ALOX; eosinophil-deficient GATA-1) were injected i.p. with eotaxin, eosinophils, or both, and leukocyte accumulation was quantified up to 24?h. Significant recruitment of eosinophils by eotaxin in BALB/c, up to 24?h, was accompanied by much larger numbers of recruited neutrophils and monocytes/macrophages. These effects were abolished by eotaxin neutralization and 5-LO-activating protein inhibitor MK886. In ALOX (but not PAS) mice, eotaxin recruitment was abolished for eosinophils and halved for neutrophils. In GATA-1 mutants, eotaxin recruited neither neutrophils nor macrophages. Transfer of eosinophils cultured from bone-marrow of BALB/c donors, or from ALOX donors, into GATA-1 mutant recipients, i.p., restored eotaxin recruitment of neutrophils and showed that the critical step dependent on 5-LO is the initial recruitment of eosinophils by eotaxin, not the secondary neutrophil accumulation. Eosinophil-dependent recruitment of neutrophils in naive BALB/c mice was associated with increased binding of bacteria.
Project description:The hyper-IgE syndromes (HIES; originally named Job's syndrome) are a collection of primary immunodeficiency syndromes resulting in elevated serum IgE levels and typified by recurrent staphylococcal skin abscesses, eczema and pulmonary infections. The disorder has autosomal dominant and recessive forms. Autosomal dominant HIES has been shown to be mainly due to STAT3 mutations and additionally results in connective tissue, skeletal, vascular and dental abnormalities. Autosomal recessive HIES has been shown to be mainly due to mutations in DOCK8; these patients are more prone to viral skin infections instead. This review article discusses the common clinical features of the syndrome, the genetic mutations responsible and the pathogenesis of the disease, as well as treatments currently used.
Project description:Calprotectin, the most abundant cytoplasmic protein in neutrophils, suppresses the growth of Staphylococcus aureus by sequestering the nutrient metal ions Zn and Mn. Here we show that calprotectin can also enhance the activity of the SaeRS two component system (TCS), a signaling system essential for production of over 20 virulence factors in S. aureus. The activity of the SaeRS TCS is repressed by certain divalent ions found in blood or neutrophil granules; however, the Zn bound-form of calprotectin relieves this repression. During staphylococcal encounter with murine neutrophils or staphylococcal infection of the murine peritoneal cavity, calprotectin increases the activity of the SaeRS TCS as well as the production of proinflammatory cytokines such as IL-1? and TNF-?, resulting in higher murine mortality. These results suggest that, under certain conditions, calprotectin can be exploited by S. aureus to increase bacterial virulence and host mortality.