ABSTRACT
Bacteriophage therapy is one potential strategy to treat antimicrobial resistant or persistent bacterial infections, and the year 2021 marked the centennial of Felix d'Hérelle's first publication on the clinical applications of phages. At the Center for Phage Biology & Therapy at Yale University, a preparatory modular approach has been established to offer safe and potent phages for single-patient investigational new drug applications while recognizing the time constraints imposed by infection(s). This study provides a practical walkthrough of the pipeline with an Autographiviridae phage targeting Pseudomonas aeruginosa (phage vB_PaeA_SB, abbreviated to ΦSB). Notably, a thorough phage characterization and the evolutionary selection pressure exerted on bacteria by phages, analogous to antibiotics, are incorporated into the pipeline.
Subject(s)
Bacteriophages , Phage Therapy , Pseudomonas Infections , Pseudomonas Phages , Humans , Pseudomonas aeruginosa , Universities , Pseudomonas Phages/genetics , Pseudomonas Infections/therapy , Pseudomonas Infections/microbiologyABSTRACT
Pseudomonas aeruginosa (PsA) is an opportunistic bacterial pathogen that causes life-threatening infections in individuals with compromised immune systems and exacerbates health concerns for those with cystic fibrosis (CF). PsA rapidly develops antibiotic resistance; thus, novel therapeutics are urgently needed to effectively combat this pathogen. Previously, we have shown that a novel cationic Zinc (II) porphyrin (ZnPor) has potent bactericidal activity against planktonic and biofilm-associated PsA cells, and disassembles the biofilm matrix via interactions with eDNA In the present study, we report that ZnPor caused a significant decrease in PsA populations in mouse lungs within an in vivo model of PsA pulmonary infection. Additionally, when combined with an obligately lytic phage PEV2, ZnPor at its minimum inhibitory concentration (MIC) displayed synergy against PsA in an established in vitro lung model resulting in greater protection of H441 lung cells versus either treatment alone. Concentrations above the minimum bactericidal concentration (MBC) of ZnPor were not toxic to H441 cells; however, no synergy was observed. This dose-dependent response is likely due to ZnPor's antiviral activity, reported herein. Together, these findings show the utility of ZnPor alone, and its synergy with PEV2, which could be a tunable combination used in the treatment of antibiotic-resistant infections.
ABSTRACT
The mechanisms by which excessive systemic activation of adaptive T lymphocytes, as in cytokine release syndrome (CRS), leads to innate immune cell-mediated acute lung injury (ALI) or acute respiratory distress syndrome, often in the absence of any infection, remains unknown. Here, we investigated the roles of IFN-γ and IL-17A, key T-cell cytokines significantly elevated in patients with CRS, in the immunopathogenesis of CRS-induced extrapulmonary ALI. CRS was induced in wild-type (WT), IL-17A- and IFN-γ knockout (KO) human leukocyte antigen-DR3 transgenic mice with 10 µg of the superantigen, staphylococcal enterotoxin B, given intraperitoneally. Several ALI parameters, including gene expression profiling in the lungs, were studied 4, 24, or 48 hours later. Systemic T-cell activation with staphylococcal enterotoxin B resulted in robust upregulation of several chemokines, S100A8/A9, matrix metalloproteases, and other molecules implicated in tissue damage, granulocyte as well as agranulocyte adhesion, and diapedesis in the lungs as early as 4 hours, which was accompanied by subsequent neutrophil/eosinophil lung infiltration and severe ALI in IFN-γ KO mice. These pathways were significantly underexpressed in IL-17A KO mice, which manifested mildest ALI and intermediate in WT mice. Neutralization of IFN-γ worsened ALI in WT and IL-17A KO mice, whereas neutralizing IL-17A did not mitigate lung injury in IFN-γ KO mice, suggesting a dominant protective role for IFN-γ in ALI and that IL-17A is dispensable. Ruxolitinib, a Janus kinase inhibitor, increased ALI severity in WT mice. Thus, our study identified novel mechanisms of ALI in CRS and its differential modulation by IFN-γ and IL-17A.
Subject(s)
Acute Lung Injury , Interleukin-17 , Humans , Mice , Animals , Cytokine Release Syndrome , Interferon-gamma , Cytokines , Lung/pathology , Acute Lung Injury/pathology , Mice, Knockout , Mice, Inbred C57BLABSTRACT
Aims: Studies have linked severe hyperoxia, or prolonged exposure to very high oxygen levels, with worse clinical outcomes. This study investigated the role of epidermal growth factor receptor (EGFR) in hyperoxia-induced lung injury at very high oxygen levels (>95%). Results: Effects of severe hyperoxia (100% oxygen) were studied in mice with genetically inhibited EGFR and wild-type littermates. Despite the established role of EGFR in lung repair, EGFR inhibition led to improved survival and reduced acute lung injury, which prompted an investigation into this protective mechanism. Endothelial EGFR genetic knockout did not confer protection. EGFR inhibition led to decreased levels of cleaved caspase-3 and poly (ADP-ribosyl) polymerase (PARP) and decreased terminal dUTP nick end labeling- (TUNEL-) positive staining in alveolar epithelial cells and reduced ERK activation, which suggested reduced apoptosis in vivo. EGFR inhibition decreased hyperoxia (95%)-induced apoptosis and ERK in murine alveolar epithelial cells in vitro, and CRISPR-mediated EGFR deletion reduced hyperoxia-induced apoptosis and ERK in human alveolar epithelial cells in vitro. Innovation. This work defines a protective role of EGFR inhibition to decrease apoptosis in lung injury induced by 100% oxygen. This further characterizes the complex role of EGFR in acute lung injury and outlines a novel hyperoxia-induced cell death pathway that warrants further study. Conclusion: In conditions of severe hyperoxia (>95% for >24 h), EGFR inhibition led to improved survival, decreased lung injury, and reduced cell death. These findings further elucidate the complex role of EGFR in acute lung injury.
Subject(s)
Acute Lung Injury , Hyperoxia , Lung Injury , Acute Lung Injury/metabolism , Adenosine Diphosphate/pharmacology , Animals , Apoptosis , Caspase 3/metabolism , ErbB Receptors/metabolism , Humans , Hyperoxia/complications , Hyperoxia/metabolism , Lung/metabolism , Lung Injury/etiology , Lung Injury/metabolism , Mice , Mice, Inbred C57BL , Oxygen/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/pharmacologyABSTRACT
Staphylococcal enterotoxin B (SEB), which is produced by the major human pathogen, Staphylococcus aureus, represents a powerful superantigenic toxin and is considered a bioweapon. However, the contribution of SEB to S. aureus pathogenesis has never been directly demonstrated with genetically defined mutants in clinically relevant strains. Many isolates of the predominant Asian community-associated methicillin-resistant S. aureus lineage sequence type (ST) 59 harbor seb, implying a significant role of SEB in the observed hypervirulence of this lineage. We created an isogenic seb mutant in a representative ST59 isolate and assessed its virulence potential in mouse infection models. We detected a significant contribution of seb to systemic ST59 infection that was associated with a cytokine storm. Our results directly demonstrate that seb contributes to S. aureus pathogenesis, suggesting the value of including SEB as a target in multipronged antistaphylococcal drug development strategies. Furthermore, they indicate that seb contributes to fatal exacerbation of community-associated methicillin-resistant S. aureus infection.
Subject(s)
Enterotoxins , Staphylococcal Infections , Animals , Methicillin-Resistant Staphylococcus aureus/pathogenicity , Mice , Staphylococcal Infections/pathology , VirulenceABSTRACT
Chimeric antigen receptor T cell (CART) therapy, administration of certain T cell-agonistic antibodies, immune check point inhibitors, coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) and Toxic shock syndrome (TSS) caused by streptococcal as well as staphylococcal superantigens share one common complication, that is T cell-driven cytokine release syndrome (CRS) accompanied by multiple organ dysfunction (MOD). It is not understood whether the failure of a particular organ contributes more significantly to the severity of CRS. Also not known is whether a specific cytokine or signaling pathway plays a more pathogenic role in precipitating MOD compared to others. As a result, there is no specific treatment available to date for CRS, and it is managed only symptomatically to support the deteriorating organ functions and maintain the blood pressure. Therefore, we used the superantigen-induced CRS model in HLA-DR3 transgenic mice, that closely mimics human CRS, to delineate the immunopathogenesis of CRS as well as to validate a novel treatment for CRS. Using this model, we demonstrate that (i) CRS is characterized by a rapid rise in systemic levels of several Th1/Th2/Th17/Th22 type cytokines within a few hours, followed by a quick decline. (ii) Even though multiple organs are affected, small intestinal immunopathology is the major contributor to mortality in CRS. (iii) IFN-γ deficiency significantly protected from lethal CRS by attenuating small bowel pathology, whereas IL-17A deficiency significantly increased mortality by augmenting small bowel pathology. (iv) RNA sequencing of small intestinal tissues indicated that IFN-γ-STAT1-driven inflammatory pathways combined with enhanced expression of pro-apoptotic molecules as well as extracellular matrix degradation contributed to small bowel pathology in CRS. These pathways were further enhanced by IL-17A deficiency and significantly down-regulated in mice lacking IFN-γ. (v) Ruxolitinib, a selective JAK-1/2 inhibitor, attenuated SAg-induced T cell activation, cytokine production, and small bowel pathology, thereby completely protecting from lethal CRS in both WT and IL-17A deficient HLA-DR3 mice. Overall, IFN-γ-JAK-STAT-driven pathways contribute to lethal small intestinal immunopathology in T cell-driven CRS.
Subject(s)
Coronavirus Infections/pathology , Cytokine Release Syndrome/drug therapy , Interferon-gamma/genetics , Interleukin-17/genetics , Janus Kinase Inhibitors/therapeutic use , Pneumonia, Viral/pathology , Pyrazoles/therapeutic use , Animals , COVID-19 , Cells, Cultured , Coronavirus Infections/drug therapy , Cytokine Release Syndrome/pathology , Cytokine Release Syndrome/prevention & control , Cytokines/blood , Cytokines/immunology , HLA-DR3 Antigen/genetics , Intestine, Small/immunology , Intestine, Small/pathology , Lymphocyte Activation/drug effects , Mice , Mice, Knockout , Nitriles , Pandemics , Pneumonia, Viral/drug therapy , Pyrimidines , T-Lymphocytes, Helper-Inducer/immunologyABSTRACT
PURPOSE: Whole genome sequencing (WGS) analysis of Staphylococcus aureus is increasingly used in clinical practice. Although bioinformatics tools used in WGS analysis readily define the S. aureus virulome, the clinical value of this type of analysis is unclear. Here, virulence genes in S. aureus bacteremia (SAB) isolates were evaluated by WGS, with superantigens (SAgs) further evaluated by conventional PCR and functional assays, and results correlated with mortality. METHODS: 152 SAB isolates collected throughout 2015â¯at a large Minnesota medical center were studied and associated clinical data analyzed. Virulence genes were identified from previously-reported WGS data (https://doi.org/10.1371/journal.pone.0179003). SAg genes sea, seb, sec, sed, see, seg, seh, sei, sej, and tst were also assessed by individual PCR assays. Mitogenicity of SAgs was assessed using an in vitro proliferation assay with splenocytes from HLA-DR3 transgenic mice. RESULTS: Of the 152 SAB isolates studied, 106 (69%) were methicillin-susceptible S. aureus (MSSA). The number of deaths attributed and not attributed to SAB, and 30-day survivors were 24 (16%), 2 (1%), and 128 (83%), respectively. From WGS data, both MSSA and MRSA had high proportions of adhesion (>80%) and immune-evasion (>70%) genes. There was no difference in virulomes between survivor- and non-survivor-associated isolates. Although over 60% of SAB isolates produced functional SAgs, there were no differences in the distribution or prevalence of SAg genes between survivor- and non-survivor-associated isolates. CONCLUSION: In this study of one year of SAB isolates from a large medical center, the S. aureus virulome, as assessed by WGS, and also for SAgs using individual PCRs and phenotypic characterization, did not impact mortality.
Subject(s)
Bacteremia/microbiology , Bacteremia/mortality , Drug Resistance, Bacterial/genetics , Staphylococcal Infections/microbiology , Staphylococcal Infections/mortality , Staphylococcus aureus/genetics , Staphylococcus aureus/pathogenicity , Aged , Aged, 80 and over , Animals , Bacteremia/immunology , Bacterial Adhesion/genetics , Base Sequence , Cell Proliferation , Female , HLA-DR3 Antigen , Humans , Immune Evasion/genetics , Male , Methicillin-Resistant Staphylococcus aureus/genetics , Mice , Mice, Transgenic , Middle Aged , Minnesota , Polymerase Chain Reaction , Staphylococcal Infections/immunology , Superantigens/genetics , Superantigens/immunology , Virulence/genetics , Virulence Factors/geneticsABSTRACT
IgG4-related disease (IgG4-RD) is a chronic relapsing multi-organ fibro-inflammatory syndrome of presumed autoimmune etiology. It is characterized by increased serum levels of IgG4 and tissue infiltration by IgG4+ cells. Increased titers of autoantibodies against a spectrum of self-antigens and response to steroids have led to its characterization as an autoimmune disease. However, the pathognomonic antigens probably differ among manifestations, and different antigens or autoantibodies produce similar immune reactions in different organs. Little is known about the pathogenic effects, if any, of serum IgG4 or IgG4+ plasma cells in tissues. Despite several animal models of the disease, none truly recapitulates human IgG4-RD. Histologic analyses of tissues from patients with IgG4-RD reveal a dense lymphoplasmacytic infiltrate rich in IgG4+ plasma cells, storiform fibrosis, and obliterative phlebitis, although these features vary among organs. Typical presentation and imaging findings include mass-forming synchronous or metachronous lesions in almost any organ, but most commonly in the pancreas, bile duct, retroperitoneum, kidneys, lungs, salivary and lacrimal glands, orbit, and lymph nodes. In all organs, inflammation can be reduced by corticosteroids and drugs that deplete B cells, such as rituximab. Patients with IgG4-RD have relapses that respond to primary therapy. Intense fibrosis accompanies the inflammatory response, leading to permanent organ damage and insufficiency. Death from IgG4-RD is rare. IgG4-RD is a multi-organ disease with predominant pancreatico-biliary involvement. Despite its relapsing-remitting course, patients have an excellent prognosis.
Subject(s)
Autoimmune Diseases/immunology , Autoimmunity , Gastrointestinal Diseases/immunology , Gastrointestinal Tract/immunology , Immunoglobulin G/immunology , Animals , Anti-Inflammatory Agents/therapeutic use , Autoimmune Diseases/diagnosis , Autoimmune Diseases/drug therapy , Autoimmune Diseases/epidemiology , Autoimmunity/drug effects , Gastrointestinal Diseases/diagnosis , Gastrointestinal Diseases/drug therapy , Gastrointestinal Diseases/epidemiology , Gastrointestinal Tract/drug effects , Gastrointestinal Tract/pathology , Humans , Predictive Value of Tests , Prognosis , Risk FactorsABSTRACT
TNF-related apoptosis-inducing ligand (TRAIL) was initially described to induce apoptosis of tumor cells and/or virally infected cells, although sparing normal cells, and has been implicated in the pathogenesis of HIV disease. We previously identified TRAILshort, a TRAIL splice variant, in HIV-infected patients and characterized it as being a dominant negative ligand to subvert TRAIL-mediated killing. Herein, using single-cell genomics we demonstrate that TRAILshort is produced by HIV-infected cells, as well as by uninfected bystander cells, and that the dominant stimulus which induces TRAILshort production are type I IFNs and TLR7, TLR8, and TLR9 agonists. TRAILshort has a short t1/2 by virtue of containing a PEST domain, which targets the protein toward the ubiquitin proteasome pathway for degradation. Further we show that TRAILshort binds preferentially to TRAIL receptors 1 and 2 with significantly reduced interaction with the decoy TRAIL receptors 3 and 4. Recombinant TRAILshort is sufficient to protect cells against TRAIL-induced killing, whereas immunodepletion of TRAILshort with a specific Ab restores TRAIL sensitivity. Importantly we show that TRAILshort is shed in microvesicles into the cellular microenvironment and therefore confers TRAIL resistance not only on the cell which produces it, but also upon neighboring bystander cells. These results establish a novel paradigm for understanding and overcoming TRAIL resistance, in particular how HIV-infected cells escape immune elimination by the TRAIL:TRAILshort receptor axis.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Cellular Microenvironment/immunology , HIV Infections/immunology , Protein Isoforms/genetics , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , TNF-Related Apoptosis-Inducing Ligand/genetics , Alternative Splicing/genetics , Apoptosis , Bystander Effect/immunology , CD4-Positive T-Lymphocytes/virology , Cell Line, Tumor , Cell Membrane/immunology , HEK293 Cells , HIV Infections/pathology , HIV Infections/virology , HeLa Cells , Humans , Jurkat Cells , Protein Isoforms/biosynthesis , TNF-Related Apoptosis-Inducing Ligand/biosynthesisABSTRACT
Mature peripheral double negative T (DNT) cells expressing αß TCR but lacking CD4/CD8 coreceptors play protective as well as pathogenic roles. To better understand their development and functioning in vivo, we concomitantly inactivated CD4 and CD8 genes in mice with intact MHC class I and class II molecules with the hypothesis that this would enable the development of DNT cells. We also envisaged that these DNT cells could be activated by bacterial superantigens in vivo as activation of T cells by superantigens does not require CD4 and CD8 coreceptors. Because HLA class II molecules present superantigens more efficiently than murine MHC class II molecules, CD4 CD8 double knockout (DKO) mice transgenically expressing HLA-DR3 or HLA-DQ8 molecules were generated. Although thymic cellularity was comparable between wild type (WT) and DKO mice, CD3+ αß TCR+ thymocytes were significantly reduced in DKO mice, implying defects in thymic-positive selection. Splenic CD3+ αß TCR+ cells and Foxp3+ T regulatory cells were present in DKO mice but significantly reduced. However, the in vivo inflammatory responses and immunopathology elicited by acute challenge with the staphylococcal superantigen enterotoxin B were comparable between WT and DKO mice. Choric exposure to staphylococcal enterotoxin B precipitated a lupus-like inflammatory disease with characteristic lympho-monocytic infiltration in lungs, livers, and kidneys, along with production of anti-nuclear Abs in DKO mice as in WT mice. Overall, our results suggest that DNT cells can develop efficiently in vivo and chronic exposure to bacterial superantigens may precipitate a lupus-like autoimmune disease through activation of DNT cells.
Subject(s)
CD4 Antigens/genetics , CD4 Antigens/immunology , CD8 Antigens/genetics , CD8 Antigens/immunology , Enterotoxins/immunology , Superantigens/immunology , T-Lymphocyte Subsets/immunology , Animals , HLA-DQ Antigens/genetics , HLA-DQ Antigens/immunology , HLA-DR3 Antigen/genetics , HLA-DR3 Antigen/immunology , Histocompatibility Antigens Class II/immunology , Mice , Mice, Knockout , Mice, Transgenic , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/immunology , Spleen/cytology , Spleen/immunology , Thymus Gland/cytology , Thymus Gland/immunologyABSTRACT
OBJECTIVES: Effective pharmacologic treatments directly targeting lung injury in patients with the acute respiratory distress syndrome are lacking. Early treatment with inhaled corticosteroids and beta agonists may reduce progression to acute respiratory distress syndrome by reducing lung inflammation and enhancing alveolar fluid clearance. DESIGN: Double-blind, randomized clinical trial (ClinicalTrials.gov: NCT01783821). The primary outcome was longitudinal change in oxygen saturation divided by the FIO2 (S/F) through day 5. We also analyzed categorical change in S/F by greater than 20%. Other outcomes included need for mechanical ventilation and development of acute respiratory distress syndrome. SETTING: Five academic centers in the United States. PATIENTS: Adult patients admitted through the emergency department at risk for acute respiratory distress syndrome. INTERVENTIONS: Aerosolized budesonide/formoterol versus placebo bid for up to 5 days. MEASUREMENTS AND MAIN RESULTS: Sixty-one patients were enrolled from September 3, 2013, to June 9, 2015. Median time from presentation to first study drug was less than 9 hours. More patients in the control group had shock at enrollment (14 vs 3 patients). The longitudinal increase in S/F was greater in the treatment group (p = 0.02) and independent of shock (p = 0.04). Categorical change in S/F improved (p = 0.01) but not after adjustment for shock (p = 0.15). More patients in the placebo group developed acute respiratory distress syndrome (7 vs 0) and required mechanical ventilation (53% vs 21%). CONCLUSIONS: Early treatment with inhaled budesonide/formoterol in patients at risk for acute respiratory distress syndrome is feasible and improved oxygenation as assessed by S/F. These results support further study to test the efficacy of inhaled corticosteroids and beta agonists for prevention of acute respiratory distress syndrome.
Subject(s)
Adrenal Cortex Hormones/administration & dosage , Adrenergic beta-Agonists/administration & dosage , Budesonide, Formoterol Fumarate Drug Combination/administration & dosage , Hypoxia/drug therapy , Respiratory Distress Syndrome/prevention & control , Academic Medical Centers , Administration, Inhalation , Aged , Aged, 80 and over , Biomarkers , Double-Blind Method , Drug Therapy, Combination , Female , Humans , Hypoxia/complications , Male , Middle Aged , Oxygen/blood , Patient Acuity , Respiration, Artificial , Respiratory Distress Syndrome/etiology , Risk Factors , United StatesABSTRACT
Drugs such as linezolid that inhibit bacterial protein synthesis may be beneficial in treating infections caused by toxigenic Staphylococcus aureus. As protein synthesis inhibitors have no effect on preformed toxins, neutralization of pathogenic exotoxins with anti-toxin antibodies may be beneficial in conjunction with antibacterial therapy. Herein, we evaluated the efficacy of human-mouse chimeric high-affinity neutralizing anti-staphylococcal enterotoxin B (SEB) antibodies in the treatment of experimental pneumonia caused by SEB-producing S. aureus. Since HLA class II transgenic mice mount a stronger systemic immune response following challenge with SEB and are more susceptible to SEB-induced lethal toxic shock than conventional mice strains, HLA-DR3 transgenic mice were used. Lethal pneumonia caused by SEB-producing S. aureus in HLA-DR3 transgenic mice was characterized by robust T cell activation and elevated systemic levels of several pro-inflammatory cytokines and chemokines. Prophylactic administration of a single dose of linezolid 30 min prior to the onset of infection attenuated the systemic inflammatory response and protected from mortality whereas linezolid administered 60 min after the onset of infection failed to confer significant protection. Human-mouse chimeric high-affinity neutralizing anti-SEB antibodies alone, but not polyclonal human IgG, mitigated this response and protected from death when administered immediately after initiation of infection. Further, anti-SEB antibodies as well as intact polyclonal human IgG, but not its Fab or Fc fragments, protected from lethal pneumonia when followed with linezolid therapy 60 min later. In conclusion, neutralization of superantigens with high-affinity antibodies may have beneficial effects in pneumonia.
Subject(s)
Antibodies, Bacterial/administration & dosage , Antibodies, Monoclonal, Humanized/administration & dosage , Enterotoxins/immunology , Immunization, Passive , Pneumonia/therapy , Staphylococcal Infections/therapy , Staphylococcus aureus/drug effects , Animals , Cytokines/genetics , Cytokines/immunology , Enterotoxins/genetics , HLA-DR3 Antigen/genetics , HLA-DR3 Antigen/immunology , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Pneumonia/genetics , Pneumonia/immunology , Pneumonia/microbiology , Staphylococcal Infections/genetics , Staphylococcal Infections/immunology , Staphylococcal Infections/microbiology , Staphylococcus aureus/genetics , Staphylococcus aureus/immunology , T-Lymphocytes/immunologyABSTRACT
Staphylococcus aureus can exist as a colonizer or can cause a spectrum of diseases. S. aureus elaborates several exotoxins and the superantigens are one among them. Staphylococcal superantigens (SSAg) cause robust activation of the immune system and acute exposure to significant amounts of SSAg can be potentially lethal. However, chronic exposure to SSAg is also possible. Administering SSAg using mini-osmotic pumps may mimic chronic recurrent exposure to SSAg. This is a relatively simple and safe way to administer purified SSAg or any other toxin/agent. In this chapter, we describe the mini-osmotic pump-mediated delivery of SSAg.
Subject(s)
Disease Models, Animal , Staphylococcal Infections/immunology , Staphylococcus/immunology , Superantigens/administration & dosage , Superantigens/immunology , Animals , Infusion Pumps, Implantable , MiceABSTRACT
Staphylococcus aureus, the most common cause of wound infection, produces several exotoxins, including superantigens (SAgs). SAgs are the potent activators of the immune system. Given this unique property, we hypothesized that SAgs produced by S. aureus in wounds would have local, as well as systemic immunologic effects. We tested our hypothesis using a novel staphylococcal skin wound infection model in transgenic mice expressing HLA-DR3. Skin wounds were left uninfected or colonized with S. aureus strains producing SAgs or an isogenic strain not producing any SAg. Animals with wounds challenged with SAg-producing S. aureus had increased morbidity and lower serum IL-17 levels compared to those challenged with the SAg non-producing S. aureus (p = 0.027 and p = 0.032, respectively). At Day 8 following microbial challenge, compared to mice with uninfected wounds, the proportion of Vß8âºCD4⺠T cells was increased, while the proportion of Vß8âºCD8⺠T cells was decreased only in the spleens of mice challenged with SAg-producing S. aureus (p < 0.001). No such changes were measured in mice challenged with SAg non-producing S. aureus. Lungs, livers and kidneys from mice challenged with SAg-producing, but not SAg non-producing, S. aureus showed inflammatory changes. Overall, SAg-mediated systemic immune activation in wounds harboring S. aureus may have clinical implications.
Subject(s)
Antigens, Bacterial/immunology , Skin Diseases/immunology , Staphylococcal Infections/immunology , Staphylococcus aureus/immunology , Superantigens/immunology , Wound Healing/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Interleukin-17/blood , Kidney/pathology , Liver/pathology , Lung/pathology , Mice, Transgenic , Skin/immunology , Skin/injuries , Skin Diseases/blood , Staphylococcal Infections/bloodABSTRACT
Adaptive immunity is mediated by antigen receptors that can induce weak or strong immune responses depending on the nature of the antigen that is bound. In T lymphocytes, antigen recognition triggers signal transduction by clustering T cell receptor (TCR)/CD3 multiprotein complexes. In addition, it hypothesized that biophysical changes induced in TCR/CD3 that accompany receptor engagement may contribute to signal intensity. Nonclustering monovalent TCR/CD3 engagement is functionally inert despite the fact that it may induce changes in conformational arrangement or in the flexibility of receptor subunits. We report that the intrinsically inert monovalent engagement of TCR/CD3 can specifically enhance physiologic T cell responses to weak antigens in vitro and in vivo without stimulating antigen-unengaged T cells and without interrupting T cell responses to strong antigens, an effect that we term as "co-potentiation." We identified Mono-7D6-Fab, which biophysically altered TCR/CD3 when bound and functionally enhanced immune reactivity to several weak antigens in vitro, including a gp100-derived peptide associated with melanoma. In vivo, Mono-7D6-Fab induced T cell antigen-dependent therapeutic responses against melanoma lung metastases, an effect that synergized with other anti-melanoma immunotherapies to significantly improve outcome and survival. We conclude that Mono-7D6-Fab directly co-potentiated TCR/CD3 engagement by weak antigens and that such concept can be translated into an immunotherapeutic design. The co-potentiation principle may be applicable to other receptors that could be regulated by otherwise inert compounds whose latent potency is only invoked in concert with specific physiologic ligands.
ABSTRACT
MHC, especially HLA-DR3 and HLA-DR2, is one of the most important genetic susceptibility regions for systemic lupus erythematosus. Human studies to understand the role of specific HLA alleles in disease pathogenesis have been hampered by the presence of strong linkage disequilibrium in this region. To overcome this, we produced transgenic mice expressing HLA-DR3 (DRß1*0301) and devoid of endogenous class II (both I-A and I-E genes, AE(0)) on a lupus-prone NZM2328 background (NZM2328.DR3(+)AE(0)). Both NZM2328 and NZM2328.DR3(+)AE(0) mice developed anti-dsDNA and glomerulonephritis, but anti-dsDNA titers were higher in the latter. Although kidney histological scores were similar in NZM2328 and NZM2328.DR3(+)AE(0) mice (7.2 ± 4.3 and 8.6 ± 5.7, respectively, p = 0.48), the onset of severe proteinuria occurred earlier in NZM2328.DR3(+)AE(0) mice compared with NZM2328 mice (median, 5 and 9 mo respectively, p < 0.001). Periarterial lymphoid aggregates, classic wire loop lesions, and occasional crescents were seen only in kidneys from NZM2328.DR3(+)AE(0) mice. Interestingly, NZM2328.DR3(+)AE(0) mice, but not NZM2328 mice, spontaneously developed anti-Smith (Sm) Abs. The anti-Sm Abs were seen in NZM2328.DR3(+)AE(0) mice that were completely devoid of endogenous class II (AE(-/) (-)) but not in mice homozygous (AE(+/+)) or heterozygous (AE(+/-)) for endogenous MHC class II. It appears that only HLA-DR3 molecules can preferentially select SmD-reactive CD4(+) T cells for generation of the spontaneous anti-Sm immune response. Thus, our mouse model unravels a critical role for HLA-DR3 in generating an autoimmune response to SmD and lupus nephritis in the NZM2328 background.
Subject(s)
Antibodies, Antinuclear/immunology , Glomerulonephritis/immunology , HLA-DR3 Antigen/immunology , Lupus Nephritis/immunology , snRNP Core Proteins/immunology , Animals , Antibodies, Antinuclear/genetics , CD4-Positive T-Lymphocytes/immunology , DNA/immunology , Disease Models, Animal , Genetic Predisposition to Disease , Glomerulonephritis/genetics , HLA-DR2 Antigen/immunology , HLA-DR3 Antigen/genetics , HLA-DRB1 Chains/genetics , HLA-DRB1 Chains/immunology , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/immunology , Lupus Nephritis/genetics , Mice , Mice, KnockoutABSTRACT
SAgs, produced by Staphylococcus aureus, play a major role in the pathogenesis of invasive staphylococcal diseases by inducing potent activation of the immune system. However, the role of SAgs, produced by S. aureus, associated with indwelling devices or tissues, are not known. Given the prevalence of device-associated infection with toxigenic S. aureus in clinical settings and the potency of SAgs, we hypothesized that continuous exposure to SAgs produced by catheter-associated S. aureus could have systemic consequences. To investigate these effects, we established a murine in vivo catheter colonization model. One centimeter long intravenous catheters were colonized with a clinical S. aureus isolate producing SAgs or isogenic S. aureus strains, capable or incapable of producing SAg. Catheters were subcutaneously implanted in age-matched HLA-DR3, B6, and AE(o) mice lacking MHC class II molecules and euthanized 7 d later. There was no evidence of systemic infection. However, in HLA-DR3 transgenic mice, which respond robustly to SSAgs, the SSAg-producing, but not the nonproducing strains, caused a transient increase in serum cytokine levels and a protracted expansion of splenic CD4(+) T cells expressing SSAg-reactive TCR Vß8. Lungs, livers, and kidneys from these mice showed infiltration with CD4(+) and CD11b(+) cells. These findings were absent in B6 and AE(o) mice, which are known to respond poorly to SSAgs. Overall, our novel findings suggest that systemic immune activation elicited by SAgs, produced by S. aureus colonizing foreign bodies, could have clinical consequences in humans.
Subject(s)
Catheter-Related Infections/immunology , Enterotoxins/biosynthesis , Staphylococcal Infections/immunology , Staphylococcus aureus/pathogenicity , Superantigens/biosynthesis , Animals , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/microbiology , CD4-Positive T-Lymphocytes/pathology , Catheter-Related Infections/genetics , Catheter-Related Infections/microbiology , Catheter-Related Infections/pathology , Catheters, Indwelling , Enterotoxins/immunology , Gene Deletion , Histocompatibility Antigens/genetics , Histocompatibility Antigens/immunology , Humans , Kidney/immunology , Kidney/microbiology , Kidney/pathology , Liver/immunology , Liver/microbiology , Liver/pathology , Lung/immunology , Lung/microbiology , Lung/pathology , Lymphocyte Activation , Mice , Mice, Transgenic , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/immunology , Spleen/immunology , Spleen/microbiology , Spleen/pathology , Staphylococcal Infections/genetics , Staphylococcal Infections/microbiology , Staphylococcal Infections/pathology , Staphylococcus aureus/immunology , Superantigens/immunologyABSTRACT
Staphylococcus aureus is a common cause of prosthetic joint infection (PJI). The prevalence of superantigens (SAgs) among PJI-associated S. aureus is unknown. Eighty-four S. aureus isolates associated with PJI isolated between 1999 and 2006 were studied. SAg genes, sea, seb, sec, sed, see, seg, seh, sei, and tst, were assayed by PCR. Seventy-eight (92.9%) isolates carried at least 1 SAg gene studied, with 61 (72.6%) harboring more than 1. seg was most commonly (70.2%), and seh was least frequently (4.8%) detected. tst-positive isolates were associated with early infection and increased erythrocyte sedimentation rate at diagnosis (P=0.006 and P=0.021, respectively). seg and sei were associated with methicillin resistance (P=0.008 and P=0.002, respectively). A majority of PJI-associated isolates studied produced biologically active SAgs in both planktonic and biofilm growth modes. SAg genes are prevalent in S. aureus causing PJI.
Subject(s)
Arthritis/microbiology , Prosthesis-Related Infections/microbiology , Staphylococcal Infections/microbiology , Staphylococcus aureus/genetics , Superantigens/genetics , Adult , Aged , Aged, 80 and over , Animals , Cell Proliferation/drug effects , Cells, Cultured , Female , Genotype , Humans , Leukocytes, Mononuclear/drug effects , Male , Mice, Transgenic , Middle Aged , Polymerase Chain Reaction , Staphylococcus aureus/isolation & purification , Superantigens/analysisABSTRACT
Life-threatening infections caused by Staphylococcus aureus, particularly the community-acquired methicillin-resistant strains of S. aureus, continue to pose serious problems. Greater virulence and increased pathogenicity of certain S. aureus strains are attributed to higher prevalence of exotoxins. Of these exotoxins, the superantigens (SAg) are likely most pathogenic because of their ability to rapidly and robustly activate the T cells even in extremely small quantities. Therefore, countering SAg-mediated T cell activation using T regulatory cells (Tregs) might be beneficial in diseases such as toxic shock syndrome (TSS). As the normal numbers of endogenous Tregs in a typical host are insufficient, we hypothesized that increasing the Treg numbers by administration of IL-2/anti-IL-2 Ab immune complexes (IL2C) or by adoptive transfer of ex vivo expanded Tregs might be more effective in countering SAg-mediated immune activation. HLA-DR3 transgenic mice that closely recapitulate human TSS were treated with IL2C to increase endogenous Tregs or received ex vivo expanded Tregs. Subsequently, they were challenged with SAg to induce TSS. Analyses of various parameters reflective of TSS (serum cytokine/chemokine levels, multiple organ pathology, and SAg-induced peripheral T cell expansion) indicated that increasing the Tregs failed to mitigate TSS. On the contrary, serum IFN-γ levels were increased in IL2C-treated mice. Exploration into the reasons behind the lack of protective effect of Tregs revealed IL-17 and IFN-γ-dependent loss of Tregs during TSS. In addition, significant upregulation of glucocorticoid-induced TNFR family-related receptor on conventional T cells during TSS could render them resistant to Treg-mediated suppression, contributing to failure of Treg-mediated immune regulation.