David, Chella S.

Mice with targeted disruption of the A beta gene of major histocompatibility complex class II molecules (Abo) were used to investigate the role of class II gene products in resistance or susceptibility to virus-induced chronic demyelination in the central nervous system (CNS). Class-II-deficient mice from the resistant H-2b [H-2b(Abo)] and nonmutant H-2b backgrounds were infected with Theiler's murine encephalomyelitis virus intracerebrally and examined for CNS virus persistence, demyelination, and neurologic clinical signs. Virus titers measured by plaque assays showed that 8 of 10 normally resistant nonmutant H-2b mice had cleared the virus within 21 days, whereas the other 2 mice had low titers. In contrast, all class II-deficient Abo mice had high virus titers for up to 90 days after infection (4.30 log10 PFU per g of CNS tissue). Virus antigens and RNA were localized to the brains (cortex, hippocampus, thalamus, and brain stem) and spinal cords of Abo mice. Colocalization identified persistent Theiler's murine encephalomyelitis virus in oligodendrocytes and astrocytes but not in macrophages. There was demyelination in 11 of 23 and 6 of 9 Abo mice 45 and 90 days after virus infection, respectively, whereas no demyelination was observed in infected nonmutant H-2b mice. Demyelinating lesions in Abo mice showed virus-specific CD8+ T cells and macrophages but no CD4+ T cells. Spasticity and paralysis were observed in chronically infected Abo mice but not in the nonmutant H-2b mice. These findings demonstrate that class II gene products are required for virus clearance from the CNS but not for demyelination and neurologic disease.

ABSTRACTCoxsackievirus B3 (CVB3) infection induces myocardial inflammation and myocyte necrosis in some, but not all, strains of mice. C57BL/6 mice, which inherently lack major histocompatibility complex (MHC) class II IE antigen, develop minimal cardiac lesions despite high levels of virus in the heart. The present experiments evaluate the relative roles of class II IA and IE expression on myocarditis susceptibility in four transgenic C57BL/6 mouse strains differing in MHC class II antigen expression. Animals lacking MHC class II IE antigen (C57BL/6 [IA+IE−] and ABo[IA−IE−]) developed minimal cardiac lesions subsequent to infection despite high concentrations of virus in the heart. In contrast, strains expressing IE (ABoEα [IA−IE+] and Bl.Tg.Eα [IA+IE+]) had substantial cardiac injury. Myocarditis susceptibility correlated to a Th1 (gamma interferon-positive) cell response in the spleen, while disease resistance correlated to a preferential Th2 (interleukin-4-positive) phenotype. Vγ/Vδ analysis indicates that distinct subpopulations of γδ+T cells are activated after CVB3 infection of C57BL/6 and Bl.Tg.Eα mice. Depletion of γδ+T cells abrogated myocarditis susceptibility in IE+animals and resulted in a Th1→Th2 phenotype shift. These studies indicate that the MHC class II antigen haplotype controls myocarditis susceptibility, that this control is most likely mediated through the type of γδ T cells activated during CVB3 infection, and finally that different subpopulations of γδ+T cells may either promote or inhibit Th1 cell responses.

ABSTRACTA newly identified intraerythrocyticBabesia-like organism, WA1, and its relatives were recently shown to be infectious for humans in the western United States. The purpose of the present study was to determine the susceptibilities of selected mouse genotypes to WA1 infection in an attempt to develop a murine model of the human disease. Several mouse strains were inoculated intraperitoneally with various passages of WA1-parasitized erythrocytes. Parasitemia was evaluated by blood smears and by PCR with blood samples collected at various intervals after inoculation. Hematologic parameters were monitored in blood samples at all intervals. C57BL/6 and C57BL/10 mice exhibited mortality rates of <10%. BALB/cJ, CBAJ, and 129/J mice had higher peak parasitemias than did C57BL mice, with mortality rates of 40, 50, and 50%, respectively. A/J, AKR/N, C3H, and DBA/1J mice also had higher peak parasitemia and mortality rates (>95%). An F1cross of C57BL/6 (resistant) and C3H.RKK (susceptible) mice had a mortality rate similar to that of the resistant parental strain. Histopathology of BALB/cJ and C3H mice at 9 and 14 days after inoculation revealed erythrophagocytosis and deposition of an iron-negative pigment in multiple organs. Morbidly ill C3H mice at 14 days had severe pulmonary edema, hemoglobinuria, and glomerulonephritis.

HLA‐DQ8 is associated with certain autoimmune diseases including celiac disease, juvenile diabetes mellitus, and rheumatoid arthritis. These diseases have significantly high morbidity and mortality rates and often require chronic, life‐long immunosuppressive therapy, especially in the case of rheumatoid arthritis. Recent studies have determined that commensal microorganisms from the gut flora can modulate the development of autoimmune diseases although the mechanism of action remains elusive. We examined the systemic immunological effects following oral exposure to live commensal bacteria within naive AEoDQ8 mice. Specifically, Prevotella species was isolated and cultured from the small intestine of celiac patients and administered by oral gavages every 48 hours for 2 weeks to AEo DQ8 mice. Significant decreases in serum IL‐17 expressed in pg/ml (2406 to 1662, □33%) and GM‐CSF (15.5 to 9.7, □40%) were detected in the sera of the group administered live bacteria as compared to the group administered sham gavages. The decrease in serum IL‐6, G‐CSF, MIP1a, and MIP1b levels did not reach statistical significance. Also, no difference in serum TGFbeta levels was observed between the two groups. However, FACS analysis revealed an increase in splenic CD4+CD25+FoxP3+ regulatory T‐cells in the group administered live bacteria as compared to the control group, 11.4% vs. 8.0%, respectively. These findings suggest that gram negative, commensal bacteria can suppress the expression of pro‐inflammatory IL‐17 at a systemic level.

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.

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.

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.