Context-dependent induction of autoimmunity by TNF signaling deficiency.

TNF inhibitors are widely used to treat inflammatory diseases; however, 30%-50% of treated patients develop new autoantibodies, and 0.5%-1% develop secondary autoimmune diseases, including lupus. TNF is required for formation of germinal centers (GCs), the site where high-affinity autoantibodies are often made. We found that TNF deficiency in Sle1 mice induced TH17 T cells and enhanced the production of germline encoded, T-dependent IgG anti-cardiolipin antibodies but did not induce GC formation or precipitate clinical disease. We then asked whether a second hit could restore GC formation or induce pathogenic autoimmunity in TNF-deficient mice. By using a range of immune stimuli, we found that somatically mutated autoantibodies and clinical disease can arise in the setting of TNF deficiency via extrafollicular pathways or via atypical GC-like pathways. This breach of tolerance may be due to defects in regulatory signals that modulate the negative selection of pathogenic autoreactive B cells.

The Yaa locus and IFN-α fine-tune germinal center B cell selection in murine systemic lupus erythematosus.

Male NZW/BXSB.Yaa (W/B) mice express two copies of TLR7 and develop pathogenic autoantibodies, whereas females with only one copy of TLR7 have attenuated disease. Our goal was to analyze the regulation of the autoantibody response in male and female W/B mice bearing the autoreactive site-directed H chain transgene 3H9. Serum anti-dsDNA Abs appeared in males at 12 wk, and most had high-titer IgG anti-dsDNA and anti-cardiolipin Abs and developed >300 mg/dl proteinuria by 8 mo. Females had only low-titer IgG anti-cardiolipin Abs, and none developed proteinuria by 1 y. Males had a smaller marginal zone than females with a repertoire that was distinct from the follicular repertoire, indicating that the loss of marginal zone B cells was not due to diversion to the follicular compartment. Vk5-43 and Vk5-48, which were rare in the naive repertoire, were markedly overrepresented in the germinal center repertoire of both males and females, but the VJ junctions differed between males and females with higher-affinity autoreactive B cells being selected into the germinal centers of males. Administration of IFN-α to females induced anti-cardiolipin and anti-DNA autoantibodies and proteinuria and was associated with a male pattern of junctional diversity in Vk5-43 and Vk5-48. Our studies are consistent with the hypothesis that presence of the Yaa locus, which includes an extra copy of Tlr7, or administration of exogenous IFN-α relaxes the stringency for selection in the germinal centers resulting in increased autoreactivity of the Ag-driven B cell repertoire.

The effects of glutathione, insulin and oxidative stress on cultured spermatogenic cysts.

Spermatogenesis is a series of cellular processes that leads to the development of motile, elongate sperm cells. Mitotic expansion of spermatogenic stem cells is followed by two meiotic cell divisions that yield haploid round spermatids which then transform from a spherical form into an elongate, highly polarized form. In Drosophila, spermatogenesis takes place within encapsulating cysts that contain spermatogenic cells. Spermatogenic cysts were isolated and grown in culture over the course of 96 hours. Cultures were treated with buthionine sulfoximine (BSO), glutathione (GSH), insulin and GSH+insulin in order to test the effects of these agents on cyst viability. The addition of glutathione and exogenous insulin to cultured spermatogenic cysts each appeared to have a positive effect on early spermatogenic cyst survival in vitro at some timepoints. The addition of GSH+insulin together had no significant effect on early spermatogenic cyst survival in vitro. Oxidative stress induced by BSO resulted in a significant decrease and/or complete loss of specific early spermatogenic cyst types and the abnormal development of elongating cysts in culture. This culture system offers the opportunity for high-resolution analysis of spermatogenic processes not previously possible.

Spermatogenic cyst and organ culture in Drosophila pseudoobscura.

Spermatogenesis is a complex series of processes that involves (1) the maintenance of a renewable pool of diploid stem cells within a niche, (2) the mitotic expansion of a subpopulation of stem cells committed to the spermatogenic pathway, and (3) the differentiation of diploid cells into highly specialized, haploid spermatozoa through meiotic and post-meiotic cellular transformations. Drosophila melanogaster is a desirable model for studying spermatogenesis, as similarities exist between mammalian and fly spermatogenesis. Like mammals, flies maintain a spermatogenic stem cell niche; the steps involved in mammalian spermatogenesis are mimicked in flies, with the main difference being that fly sperm develop within cysts rather than an epithelial cell layer. Here, we report a reliable robust system for culturing whole testes and individual spermatogenic cysts obtained from mid- to late-pupal stages of Drosophila pseudoobscura. D. pseudoobscura testes can be easily distinguished in later pupal stages because of their intense red pigmentation and are easily handled because of their simple ellipsoidal morphology. Cultured cysts are comparable in length to cysts obtained from adult flies, and motility is consistently achieved in vitro. This system not only offers a method for dissecting the mechanisms involved in meiotic and post-meiotic cellular transformations, but also can be used for the study of signaling during spermatogenesis.