Mohammed M. Nooh

HLA Transgenic Mice Provide Evidence for a Direct and Dominant Role of HLA Class II Variation in Modulating the Severity of Streptococcal Sepsis

Our epidemiologic studies on invasive Group A Streptococci (GAS) infections identified specific HLA class II haplotypes/alleles conferring high-risk or protection from streptococcal toxic shock syndrome with a strong protection conferred by the DRB1*15/DQB1*06 haplotype. We used HLA-transgenic mice to provide an in vitro and in vivo validation for the direct role of HLA class II allelic variation in streptococcal toxic shock syndrome. When splenocytes from mice expressing the protective HLA-DQB1*06 (DQ6) allele were stimulated with a mixture of streptococcal superantigens (SAgs), secreted by the prevalent M1T1 strain, both proliferative and cytokine responses were significantly lower than those of splenocytes from mice expressing the neutral DRB1*0402/DQB1*0302 (DR4/DQ8) alleles (p < 0.001). In crisscross experiments, the presentation of SAgs to pure T cells from either the DQ6 or the DR4/DQ8 mice resulted in significantly different levels of response depending on the HLA type expressed on the APCs. Presentation by HLA-DQ6 APCs elicited significantly lower responses than the presentation by HLA-DR4/DQ8 APCs. Our in vitro data were supported by in vivo findings, as the DQ6 mice showed significantly longer survival post-i.v. infection with live M1T1 GAS (p < 0.001) and lower inflammatory cytokine responses as compared with the DR4/DQ8 mice (p < 0.01). The data presented here provide evidence for a direct role of HLA class II molecules in modulating responses to GAS SAgs and underscore the dominant role of HLA class II allelic variation in potentiating the severity of GAS systemic infections.

An Unbiased Systems Genetics Approach to Mapping Genetic Loci Modulating Susceptibility to Severe Streptococcal Sepsis

Striking individual differences in severity of group A streptococcal (GAS) sepsis have been noted, even among patients infected with the same bacterial strain. We had provided evidence that HLA class II allelic variation contributes significantly to differences in systemic disease severity by modulating host responses to streptococcal superantigens. Inasmuch as the bacteria produce additional virulence factors that participate in the pathogenesis of this complex disease, we sought to identify additional gene networks modulating GAS sepsis. Accordingly, we applied a systems genetics approach using a panel of advanced recombinant inbred mice. By analyzing disease phenotypes in the context of mice genotypes we identified a highly significant quantitative trait locus (QTL) on Chromosome 2 between 22 and 34 Mb that strongly predicts disease severity, accounting for 25%–30% of variance. This QTL harbors several polymorphic genes known to regulate immune responses to bacterial infections. We evaluated candidate genes within this QTL using multiple parameters that included linkage, gene ontology, variation in gene expression, cocitation networks, and biological relevance, and identified interleukin1 alpha and prostaglandin E synthases pathways as key networks involved in modulating GAS sepsis severity. The association of GAS sepsis with multiple pathways underscores the complexity of traits modulating GAS sepsis and provides a powerful approach for analyzing interactive traits affecting outcomes of other infectious diseases.

Commercial peptidoglycan preparations are contaminated with superantigen-like activity that stimulates IL-17 production.

The immunomodulatory properties of peptidoglycan (PGN), a constituent of the bacterial cell wall, have been studied extensively but with contrasting results. Recent studies have demonstrated that the TLR2‐mediated inflammatory responses elicited by Gram‐positive PGN preparations are in fact a result of contaminating lipoproteins and lipoteichoic acid that can be removed only through sophisticated extraction procedures. Here, we report that commercial preparations of Staphylococcus aureus or Streptococcus pyogenes PGN are contaminated with bacterial superantigens (SAg). The T cell‐derived cytokines IL‐17A and IL‐17F were induced by PGN preparations but not by TLR agonists or nucleotide‐binding and oligomerization domain‐like receptor agonists in human PBMC. IL‐17 induction by PGN preparations was sensitive to protease digestion and required TCR signaling. Bacterial SAg could be detected by immunoblot in the PGN preparations, and purified recombinant SAg were powerful inducers of IL‐17. Finally, the PGN preparations stimulated proliferation and expansion of T cells bearing specific TCR Vβ elements. Our results suggest that a large body of literature that relied on commercial PGN preparations to study inflammatory diseases, such as arthritis, where IL‐17 also plays an important role, should be interpreted with caution and possibly revisited. Future studies aimed at characterizing the activities of PGN should use PGN preparations of proven purity.

Susceptibility to severe streptococcal sepsis: use of a large set of isogenic mouse lines to study genetic and environmental factors

Variation in responses to pathogens is influenced by exposure history, environment and the hosts genetic status. We recently demonstrated that human leukocyte antigen class II allelic differences are a major determinant of the severity of invasive group A streptococcal (GAS) sepsis in humans. While in-depth controlled molecular studies on populations of genetically well-characterized humans are not feasible, it is now possible to exploit genetically diverse panels of recombinant inbred BXD mice to define genetic and environmental risk factors. Our goal in this study was to standardize the model and identify genetic and nongenetic covariates influencing invasive infection outcomes. Despite having common ancestors, the various BXD strains (n strains=33, n individuals=445) showed marked differences in survival. Mice from all strains developed bacteremia but exhibited considerable differences in disease severity, bacterial dissemination and mortality rates. Bacteremia and survival showed the expected negative correlation. Among nongenetic factors, age – but not sex or weight – was a significant predictor of survival (P=0.0005). To minimize nongenetic variability, we limited further analyses to mice aged 40–120 days and calculated a corrected relative survival index that reflects the number of days an animal survived post-infection normalized to all significant covariates. Genetic background (strain) was the most significant factor determining susceptibility (P⩽0.0001), thus underscoring the strong effect of host genetic variation in determining susceptibility to severe GAS sepsis. This model offers powerful unbiased forward genetics to map specific quantitative trait loci and networks of pathways modulating the severity of GAS sepsis.

Streptococcal mitogenic exotoxin, SmeZ, is the most susceptible M1T1 streptococcal superantigen to degradation by the streptococcal cysteine protease, SpeB

Superantigens (SAgs) play an important role in the pathogenesis of severe invasive infections caused by Group A Streptococcus (GAS). We had shown earlier that the expression of streptococcal cysteine protease SpeB results in partial loss of the immune-stimulating activity of the native secreted GAS SAgs, namely the streptococcal pyrogenic exotoxins produced by the globally disseminated M1T1 GAS strain, associated with invasive infections worldwide. In this study, we examined the susceptibility of each of the M1T1 recombinant SAgs to degradation by rSpeB. Whereas SmeZ was degraded completely within 30 min of incubation with rSpeB, SpeG, and SpeA were more resistant and SpeJ was completely unaffected by the proteolytic effects of this protease. Proteomic analyses demonstrated that the order of susceptibility of the M1T1 SAgs to SpeB proteolysis is unaltered when they are present in a mixture that reflects their native physiological status. As expected, the degradation of SmeZ abolished its immune stimulatory activity. In silico sequence disorder and structural analyses revealed that SmeZ, unlike the three other structurally related SAgs, possesses a putative SpeB cleavage site within an area of the protein likely to be exposed to the surface. The study provides evidence for the effect of subtle structural differences between highly similar SAgs on their biological activity.

Molecular Requirements for MHC Class II α-Chain Engagement and Allelic Discrimination by the Bacterial Superantigen Streptococcal Pyrogenic Exotoxin C

Superantigens (SAgs) are microbial toxins that bind to both TCR β-chain variable domains (Vβs) and MHC class II molecules, resulting in the activation of T cells in a Vβ-specific manner. It is now well established that different isoforms of MHC II molecules can play a significant role in the immune response to bacterial SAgs. In this work, using directed mutational studies in conjunction with functional analyses, we provide a complete functional map of the low-affinity MHC II α-chain binding interface of the SAg streptococcal pyrogenic exotoxin C (SpeC) and identify a functional epitope in the β-barrel domain that is required for the activation of T cells. Using cell lines that exclusively express individual MHC II isoforms, our studies provide a molecular basis for the selectivity of SpeC-MHC II recognition, and provide one mechanism by how SAgs are capable of distinguishing between different MHC II alleles.

Bioinformatics analysis of immune response to group A streptococcal sepsis integrating quantitative trait loci mapping with genome-wide expression studies

Individuals infected with genetically identical group Astreptococcal (GAS) strains develop starkly different dis-ease progression and outcome [1]. We reported that HLAclass II allelic variation contributes to differences in sys-temic disease severity by modulating host responses tostreptococcal superantigens [2]. Inasmuch as the bacteriaproduce additional virulence factors, we sought to iden-tify additional host gene networks modulating GAS sep-sis. Accordingly, we used two parallel approaches todefine these gene networks, quantitative trait loci (QTL)mapping and genome-wide transcriptome analyses. Tomap QTLs modulating response to severe GAS sepsis, weused advanced recombinant inbred (ARI) strains, whichare genetically diverse strains that have common ancestralparents [3]. We chose to use BXD strains of ARI mice, asparental strains C57Bl/6J (B6) and DBA/2J (D2) show dif-ferential response to GAS sepsis and BXD strains are heav-ily genotyped at 13377 SNPs and microsatellite markers.BXD strains, derived from B6 and D2 parental strains, arehomozygous inbred lines, each of which is genetically dis-tinct. Using 30 different BXD strains (n = 5–26 mice perstrain), we identified significant QTLs on chromosome 2that strongly modulate disease severity [4]. To narrowdown these mapped QTLs, we applied bioinformaticstools including: linkage, interval specific haplotype analy-ses, and gene ontology and we identified multiple candi-date gene networks modulating immune response tosepsis.As a parallel approach, we performed genome-wide tran-scriptome analyses comparing resistant and susceptiblestrains. This comparison revealed 93 genes that were dif-ferentially regulated in mice spleens 36 h post-infection.These genes belonged to gene networks involvingimmune response to sepsis; particularly notable exampleswere prostaglandin (Ptges) and interleukin1 (IL-1) familypathways. Quantitative expression analyses, using realtime PCR, of prostaglandin E synthase (

Resveratrol and Montelukast Alleviate Paraquat-Induced Hepatic Injury in Mice: Modulation of Oxidative Stress, Inflammation, and Apoptosis

Paraquat (PQ) is one of the most used herbicide worldwide. Its cytotoxicity is attributed to reactive radical generation. Resveratrol (Res) and montelukast (MK) have anti-inflammatory and antioxidant properties. The protective effects of Res, MK, or their combination against PQ-induced acute liver injury have not been investigated before. Therefore, we explored the protective potential of Res and/or MK against PQ hepatic toxicity in a mouse model. Mice were randomly assigned to five groups: group I served as the normal control and group II received a single dose of PQ (50u2009mg/kg, i.p.). Groups III, IV, and V received PQ plus oral Res (5u2009mg/kg/day), MK (10u2009mg/kg/day), and Res/MK combination, respectively. Res and/or MK reduced PQ-induced liver injury, evidenced by normalization of serum total protein, ALT, and AST. Res and/or MK significantly reversed PQ-induced oxidative stress markers glutathione and malondialdehyde. Res and/or MK significantly reduced PQ-induced inflammation reflected in TNF-α levels. Furthermore, Res and/or MK reversed PQ-induced apoptosis assessed by differential expression of p53, Bax, and Bcl-2. Histopathologic examination supported the biochemical findings. Although Res and MK displayed antioxidative, anti-inflammatory, and antiapoptotic activities, their combination was not always synergistic.

Beta-adrenergic receptor signaling by the isomers of isoproterenol and like drugs in retinal endothelial cells and Müller cells

Diabetic retinopathy is the leading cause of blindness to working-age adults. Complications of diabetic retinopathy include pericyte loss, basement membrane thickening of capillaries, microaneurysm formation, and an increase in inflammatory marker levels. We have shown that stimulation of β-adrenergic receptors by β-adrenergic receptor agonists significantly reduces key inflammatory markers in retinal endothelial cells (REC) or Muller cells cultured in high glucose, indicating that restoration of β-adrenergic receptor signaling may be protective to the retina. REC and Muller cells responded to increased glucose concentrations with an increase in inflammatory markers and apoptosis, which is reduced following treatment with β-adrenergic receptor agonists. In our initial studies, we evaluated the ability of optically active R-(−)-isoproterenol (R-1R-1), S-(+)-isoproterenol (S-1S-1S-1), and racemic mixture of (±)-isoproterenol (1), a non-selective β-adrenergic receptor agonist, to reduce the cleavage of caspase 3 and TNFα levels. We observed R-(−)-isoproterenol is more effective than S-(+)-isoproterenol, thus we developed novel optically active analogs of R-(−)-isoproterenol. Of the analogs, we found that compound 12 significantly reduced both caspase 3 and TNFα levels in REC, but did not reduce both markers in Muller cells. Binding data of compound 12 suggest that it is not a β-adrenergic receptor true agonist, but appears to decrease inflammatory levels and apoptosis in REC through an alternate mechanism.