Identification of potent small-molecule inhibitors of STAT3 with anti-inflammatory properties in RAW 264.7 macrophages.

Signal transducer and activator of transcription 3 (STAT3) is a key mediator of the inflammatory response of macrophages and other immune cell types. The naturally occurring polyphenol resveratrol is associated with anti-proliferative and anti-inflammatory properties via mechanisms implicating inhibition of STAT3 signaling. Here, we report that the small-molecule analogs of resveratrol, RSVA314 and RSVA405, are potent inhibitors of STAT3. RSVA314 and RSVA405 inhibited both constitutive and stimulated STAT3 activity in HEK293 cells and lipopolysaccharide-stimulated RAW 264.7 macrophages, respectively. The small-molecule analogs inhibited STAT3 nearly 50 times more potently than did resveratrol (apparent IC(50) ~0.5 µM). We further show that RSVA405 interfered with the inflammatory response by RAW 264.7 cells upon lipopolysaccharide stimulation by inhibiting IκB kinase and IκBα phosphorylation and by decreasing the expression of several cytokines, including the NF-κB target genes tumor necrosis factor α and interleukin-6. Downstream activation of STAT1 upon lipopolysaccharide stimulation was also inhibited by RSVA405. Consequently, RSVA405 significantly interfered with the phagocytotic activity and proliferation of lipopolysaccharide-activated RAW 264.7 macrophages. Finally, we found that the effect of the two small-molecule analogs on STAT3 phosphorylation could be prevented by inhibitors of protein tyrosine phosphatases, indicating that the small molecules acted by promoting dephosphorylation of STAT3 by protein tyrosine phosphatases.

Selective regulation of autoreactive B cells by FcgammaRIIB.

FcgammaRIIB is an inhibitory receptor which plays a role in limiting B cell and DC activation. Since FcgammaRIIB is known to dampen the signaling strength of the BCR, we wished to determine the impact of FcgammaRIIB on the regulation of BCRs which differ in their affinity for DNA. For these studies, FcgammaRIIB deficient BALB/c mice were bred with mice expressing the transgene-encoded H chain of the R4A anti-DNA antibody which gives rise to BCRs which express high, low or no affinity for DNA. The deletion of FcgammaRIIB in R4A BALB/c mice led to an alteration in the B cell repertoire, allowing for the expansion and activation of high affinity DNA-reactive B cells. By 6-8 months of age, R4A x FcgammaRIIB-/- BALB/c mice spontaneously developed anti-DNA antibody titers. These mice also displayed an induction of IFN-inducible genes and an elevation in levels of the B cell survival factor, BAFF. These data demonstrate that FcgammaRIIB preferentially limits activation of high affinity autoreactive B cells and can influence the activation of DC through an immune complex-mediated mechanism.

Cutting Edge: Hormonal milieu, not antigenic specificity, determines the mature phenotype of autoreactive B cells.

Although both marginal zone and follicular B cells produce anti-DNA Abs in murine models of systemic lupus erythematosus, it has been unclear whether these distinct B cell subsets make identical or different Abs. Single-cell analysis demonstrates that the same DNA-reactive B cells can mature to either subset, depending on the hormonal environment. Anti-DNA B cells in estradiol-treated mice become marginal zone cells while identical cells from prolactin-treated mice become follicular cells. The B cell receptor signaling pathway is influenced by hormonal milieu. Thus, hormonal milieu and perhaps B cell receptor signaling, but not antigenic specificity, correlates with the differentiation pathway. These observations have implications for the pathogenesis and treatment of autoimmune disease.

Tamoxifen blocks estrogen-induced B cell maturation but not survival.

Estrogen treatment has been shown not only to exacerbate disease activity and accelerate death in spontaneous murine models of lupus but also to induce a lupus-like phenotype in non-spontaneously autoimmune mice. In mice transgenic for the H chain of an anti-DNA Ab, estrogen rescues naive autoreactive B cells that normally are deleted and causes them to mature to a marginal zone phenotype. Estrogen further leads to the activation of this population causing an elevation of serum anti-DNA Ab titers and renal disease. This study was designed to evaluate the therapeutic potential of tamoxifen, a selective estrogen receptor modulator, on estrogen-induced lupus. Mice treated with both estradiol and tamoxifen showed no elevation in anti-DNA Ab titers and consequently no glomerular IgG. The DNA-reactive B cell population that is rescued by estrogen was present in an anergic state in mice treated with both estradiol and tamoxifen. Estradiol enhances transitional B cell resistance to apoptosis and expands the population of marginal zone B cells; tamoxifen did not impede the enhanced resistance to apoptosis, but prevented the development of autoreactive cells as marginal zone B cells. Thus, estrogen-induced autoimmunity proceeds through two distinct molecular pathways, one affecting survival and the other maturation. Activation, but not survival, of autoreactive B cells can be abrogated by tamoxifen. Drugs that modulate even some of the effects of estrogen may be beneficial in patients with lupus. Eventually, understanding the pathways involved in survival and activation of autoreactive B cells will permit the development of therapeutics that target all relevant pathways.

Importance of uracil DNA glycosylase in Pseudomonas aeruginosa and Mycobacterium smegmatis, G+C-rich bacteria, in mutation prevention, tolerance to acidified nitrite, and endurance in mouse macrophages.

Uracil DNA glycosylase (Ung (or UDG)) initiates the excision repair of an unusual base, uracil, in DNA. Ung is a highly conserved protein found in all organisms. Paradoxically, loss of this evolutionarily conserved enzyme has not been seen to result in severe growth phenotypes in the cellular life forms. In this study, we chose G+C-rich genome containing bacteria (Pseudomonas aeruginosa and Mycobacterium smegmatis) as model organisms to investigate the biological significance of ung. Ung deficiency was created either by expression of a highly specific inhibitor protein, Ugi, and/or by targeted disruption of the ung gene. We show that abrogation of Ung activity in P. aeruginosa and M. smegmatis confers upon them an increased mutator phenotype and sensitivity to reactive nitrogen intermediates generated by acidified nitrite. Also, in a mouse macrophage infection model, P. aeruginosa (Ung-) shows a significant decrease in its survival. Infections of the macrophages with M. smegmatis show an initial increase in the bacterial counts that remain for up to 48 h before a decline. Interestingly, abrogation of Ung activity in M. smegmatis results in nearly a total abolition of their multiplication and a much-decreased residency in macrophages stimulated with interferon gamma. These observations suggest Ung as a useful target to control growth of G+C-rich bacteria.