Citations

Abstract Mature peripheral double negative T (DNT) cells expressing αβ 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+ αβ TCR+ thymocytes were significantly reduced in DKO mice, implying defects in thymic-positive selection. Splenic CD3+ αβ 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.

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 F io 2 (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.

AbstractToxic shock syndrome toxin‐1 (TSST‐1) is a potent superantigen produced by Staphylococcus aureus. In addition to menstrual and nonmenstrual toxic shock syndromes, TSST‐1 is also implicated in the immunopathogenesis of pneumonia, infective endocarditis, neonatal exanthematous disease, and atopic dermatitis among others. Superantigens first bind to major histocompatibility complex (MHC) class II molecules and then activate a large proportion of T cells by cross‐linking their T cell receptor. As binding to MHC class II molecules is a critical step in the robust activation of the immune system by TSST‐1 and other superantigens, polymorphic variations between different HLA‐DR alleles could potentially influence the magnitude of immune activation and immunopathology caused by TSST‐1. As TSST‐1 is highly toxic to humans and given that multiple variations of alleles of HLA‐DR and HLA‐DQ are expressed in each individual, it is difficult to determine how HLA‐DR polymorphisms quantitatively and qualitatively impact immune activation caused by TSST‐1 in humans. However, such investigations can be conducted on transgenic mice lacking all endogenous MHC class II molecules and expressing specific HLA class II alleles. Therefore, transgenic mice expressing different HLA‐DRB1 alleles (HLA‐DRB1*15:01, HLA‐DRB1*15:02, HLA‐DRB1*03:01, HLA‐DRB1*04:01), and sharing HLA‐A1*01:01 chain, were systemically challenged with purified TSST‐1 and multiple immune parameters were assessed. Among the HLA‐DR alleles, mice expressing HLA‐DRB1*15:01 allele elicited a significantly higher serum cytokine/chemokine response; greater splenic T cell expansion and most severe organ pathology. Our study highlights the potential utility of human leukocyte antigen (HLA) transgenic mice in understanding the impact of HLA polymorphisms on the outcomes of diseases caused by TSST‐1 and other superantigens.

AbstractAsthma is independently associated with HLA‐DR3 and increased risks of pneumococcal diseases. We aimed to determine whether HLA‐DR polymorphism (HLA‐DRB1*03), sensitization to house dust mite (HDM), or their interaction affects humoral immune responses to pneumococcal polysaccharide and protein antigens of intact pneumococci. Induction of serum titers of anti‐pneumococcal polysaccharide and anti‐surface protein IgM and IgG in response to immunization with intact pneumococci (Pn) serotype 14 was determined using humanized HLA‐DR3 and DR2 transgenic mice. Transgenic mice were sensitized by injecting HDM and challenged with intranasal HDM. Mice were subsequently immunized with heat‐killed Pn14 at day 24. Serum titers of anti‐phosphorylcholine (PC) IgM and IgG, anti‐pneumococcal polysaccharide, capsular type 14 (PPS14) IgM and IgG, and anti‐pneumococcal surface protein A (PspA) IgG were measured. We included a total of 44 mice (22 DR3 and 22 DR2 mice) and half of mice in each group were sensitized with HDM (i.e. 22 HDM‐sensitized and 22 control mice). HDM‐sensitized mice, irrespective of HLA‐DR polymorphism, had significantly lower humoral immune responses. HLA‐DR3 mice, irrespective of HDM sensitization, elicited a significantly lower anti‐PC IgG response. In contrast, the anti‐PspA IgG response was higher in DR3 relative to DR2 mice. The effect of HDM sensitization on lowering humoral immune responses to Pn14 was observed in DR3 mice regardless of the nature of the antigen, whereas such decreases were observed only for the anti‐PPS14 IgG and anti‐PC IgM responses in DR2 mice. HDM sensitization lowered humoral immune responses to intact pneumococcus and this effect was significantly modified by the HLA‐DR polymorphism.

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 Vβ8+CD4+ T cells was increased, while the proportion of Vβ8+CD8+ 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.

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 (DRβ1*0301) and devoid of endogenous class II (both I-A and I-E genes, AE0) on a lupus-prone NZM2328 background (NZM2328.DR3+AE0). Both NZM2328 and NZM2328.DR3+AE0 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+AE0 mice (7.2 ± 4.3 and 8.6 ± 5.7, respectively, p = 0.48), the onset of severe proteinuria occurred earlier in NZM2328.DR3+AE0 mice compared with NZM2328 mice (median, 5 and 9 mo respectively, p &lt; 0.001). Periarterial lymphoid aggregates, classic wire loop lesions, and occasional crescents were seen only in kidneys from NZM2328.DR3+AE0 mice. Interestingly, NZM2328.DR3+AE0 mice, but not NZM2328 mice, spontaneously developed anti-Smith (Sm) Abs. The anti-Sm Abs were seen in NZM2328.DR3+AE0 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.

Fab binding to CD3 couples with weak antigen recognition to enhance T cell responses and immunotherapy.

Abstract 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 AEo 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 Vβ8. Lungs, livers, and kidneys from these mice showed infiltration with CD4+ and CD11b+ cells. These findings were absent in B6 and AEo 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.