IFN-γ receptor and STAT1 signaling in B cells are central to spontaneous germinal center formation and autoimmunity.

Spontaneously developed germinal centers (GCs [Spt-GCs]) harbor autoreactive B cells that generate somatically mutated and class-switched pathogenic autoantibodies (auto-Abs) to promote autoimmunity. However, the mechanisms that regulate Spt-GC development are not clear. In this study, we report that B cell-intrinsic IFN-γ receptor (IFN-γR) and STAT1 signaling are required for Spt-GC and follicular T helper cell (Tfh cell) development. We further demonstrate that IFN-γR and STAT1 signaling control Spt-GC and Tfh cell formation by driving T-bet expression and IFN-γ production by B cells. Global or B cell-specific IFN-γR deficiency in autoimmune B6.Sle1b mice leads to significantly reduced Spt-GC and Tfh cell responses, resulting in diminished antinuclear Ab reactivity and IgG2c and IgG2b auto-Ab titers compared with B6.Sle1b mice. Additionally, we observed that the proliferation and differentiation of DNA-reactive B cells into a GC B cell phenotype require B cell-intrinsic IFN-γR signaling, suggesting that IFN-γR signaling regulates GC B cell tolerance to nuclear self-antigens. The IFN-γR deficiency, however, does not affect GC, Tfh cell, or Ab responses against T cell-dependent foreign antigens, indicating that IFN-γR signaling regulates autoimmune, but not the foreign antigen-driven, GC and Tfh cell responses. Together, our data define a novel B cell-intrinsic IFN-γR signaling pathway specific to Spt-GC development and autoimmunity. This novel pathway can be targeted for future pharmacological intervention to treat systemic lupus erythematosus

Seasonal and Ontogenetic Changes in Movement Patterns of Sixgill Sharks

Understanding movement patterns is fundamental to population and conservation biology. The way an animal moves through its environment influences the dynamics of local populations and will determine how susceptible it is to natural or anthropogenic perturbations. It is of particular interest to understand the patterns of movement for species which are susceptible to human activities (e.g. fishing), or that exert a large influence on community structure, such as sharks.We monitored the patterns of movement of 34 sixgill sharks Hexanchus griseus using two large-scale acoustic arrays inside and outside Puget Sound, Washington, USA. Sixgill sharks were residents in Puget Sound for up to at least four years before making large movements out of the estuary. Within Puget Sound, sixgills inhabited sites for several weeks at a time and returned to the same sites annually. Across four years, sixgills had consistent seasonal movements in which they moved to the north from winter to spring and moved to the south from summer to fall. Just prior to leaving Puget Sound, sixgills altered their behavior and moved twice as fast among sites. Nineteen of the thirty-four sixgills were detected leaving Puget Sound for the outer coast. Three of these sharks returned to Puget Sound.For most large marine predators, we have a limited understanding of how they move through their environment, and this clouds our ability to successfully manage their populations and their communities. With detailed movement information, such as that being uncovered with acoustic monitoring, we can begin to quantify the spatial and temporal impacts of large predators within the framework of their ecosystems

Regulation of B Cell Responses in SLE by Three Classes of Interferons

There are three classes of interferons (type 1, 2, and 3) that can contribute to the development and maintenance of various autoimmune diseases, including systemic lupus erythematosus (SLE). Each class of interferons promotes the generation of autoreactive B cells and SLE-associated autoantibodies by distinct signaling mechanisms. SLE patients treated with various type 1 interferon-blocking biologics have diverse outcomes, suggesting that additional environmental and genetic factors may dictate how these cytokines contribute to the development of autoreactive B cells and SLE. Understanding how each class of interferons controls B cell responses in SLE is necessary for developing optimized B cell- and interferon-targeted therapeutics. In this review, we will discuss how each class of interferons differentially promotes the loss of peripheral B cell tolerance and leads to the development of autoreactive B cells, autoantibodies, and SLE

Spontaneous germinal centers and autoimmunity

Germinal centers (GCs) are dynamic microenvironments that form in the secondary lymphoid organs and generate somatically mutated high-affinity antibodies necessary to establish an effective humoral immune response. Tight regulation of GC responses is critical for maintaining self-tolerance. GCs can arise in the absence of purposeful immunization or overt infection (called spontaneous GCs, Spt-GCs). In autoimmune-prone mice and patients with autoimmune disease, aberrant regulation of Spt-GCs is thought to promote the development of somatically mutated pathogenic autoantibodies and the subsequent development of autoimmunity. The mechanisms that control the formation of Spt-GCs and promote systemic autoimmune diseases remain an open question and the focus of ongoing studies. Here, we discuss the most current studies on the role of Spt-GCs in autoimmunity

Decreased tumor progression and invasion by a novel anti-cell motility target for human colorectal carcinoma cells.

We have previously described a novel modulator of the actin cytoskeleton that also regulates Ras and mitogen-activated protein kinase activities in TGFβ-sensitive epithelial cells. Here we examined the functional role of this signaling regulatory protein (km23-1) in mediating the migration, invasion, and tumor growth of human colorectal carcinoma (CRC) cells. We show that small interfering RNA (siRNA) depletion of km23-1 in human CRC cells inhibited constitutive extracellular signal-regulated kinase (ERK) activation, as well as pro-invasive ERK effector functions that include phosphorylation of Elk-1, constitutive regulation of c-Fos-DNA binding, TGFβ1 promoter transactivation, and TGFβ1 secretion. In addition, knockdown of km23-1 reduced the paracrine effects of CRC cell-secreted factors in conditioned medium and in fibroblast co-cultures. Moreover, km23-1 depletion in human CRC cells reduced cell migration and invasion, as well as expression of the ERK-regulated, metastasis-associated scaffold protein Ezrin. Finally, km23-1 inhibition significantly suppressed tumor formation in vivo. Thus, our results implicate km23-1 as a novel anti-metastasis target for human colon carcinoma cells, capable of decreasing tumor growth and invasion via a mechanism involving suppression of various pro-migratory features of CRC. These include a reduction in ERK signaling, diminished TGFβ1 production, decreased expression of the plasma membrane-cytoskeletal linker Ezrin, as well as attenuation of the paracrine effects of colon carcinoma-secreted factors on fibroblast migration and mitogenesis. As such, km23-1 inhibitors may represent a viable therapeutic strategy for interfering with colon cancer progression and invasion

km23-1-siRNA blocks tumor growth of RKO cells <i>in vivo</i>.

<p><b>A:</b> NC-siRNA RKO and km23-1siRNA RKO clone #1 and #5 cells (5×10⁶) were inoculated subcutaneously behind the right anterior forelimb of nude mice (n = 5). Tumor volumes were calculated as (length×width²)/2. *p<0.05 compared to NC, ANOVA. <b>B:</b> At the termination of the study, ERK activation and km23-1 knockdown in RKO xenografts were confirmed by Western blotting using the indicated antibodies. DIC, loading control.</p

Depletion of km23-1 reduces Ezrin expression in human CRC cells.

<p><b>A:</b> The cell lysates used for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066439#pone-0066439-g001" target="_blank">Fig. 1D</a> were further subjected to Western blot analysis of Ezrin expression <b>(Top). Bottom</b>, DIC loading. <b>B:</b> HCT116 cells were infected with either pilenti-NC siRNA-GFP or the pilenti-km23-1 siRNA-GFP set. 24 h after infection, Western blotting was performed using the indicated antibodies. <b>Top</b>, confirms knockdown of endogenous km23-1. <b>Bottom</b>, DIC protein was assessed as a loading control. <b>C:</b> HCT116 stable pools that migrated through to the lower membrane in the Matrigel invasion assay were stained with an Ezrin Ab, followed by cy3-conjugated goat anti-rabbit IgG (red). DAPI staining permitted visualization of nuclei of individual cells (blue). GFP was used as a marker to designate cells transduced with siRNA (green). The cells were analyzed by an Olympus IX81 microscope at a magnification of 1000× with the appropriate filter sets.</p

Depletion of km23-1 inhibits cell migration and invasion of human CRC cells.

<p><b>A:</b> Transwell migration assays were performed as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066439#s2" target="_blank">Materials and methods</a>.” Briefly, HCT116 cells stably transduced with lentiviral vectors expressing either piLenti-NC siRNA-GFP or piLenti-km23-1 siRNA-GFP were seeded into the upper wells of the Costar Transwell System (8-µm pore size polycarbonate membrane, 6.5-mm diameter), and the cells on the lower surface of the well after 24 h were fixed in methanol and stained with DAPI. <b>Top</b>, representative images of the lower surface of the membrane are shown (100× magnification). <b>Bottom</b>, the number of migrating cells of both NC siRNA- and km23-1-siRNA-HCT116 stable transfectants were counted under a fluorescence microscope and statistically analyzed. *p<0.01 compared to NC siRNA. <b>B:</b> Matrigel invasion assays were performed with the indicated RKO stable cells clones (clones #1, 5) for 24 h using EGF (20 ng/ml) as the stimulus as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066439#s2" target="_blank">Materials and methods</a>.” Invaded cells were stained with 0.2% crystal violet. <b>Top</b>, representative images of the lower membrane surface from Matrigel are shown (100× magnification). <b>Bottom</b>, the number of invading cells for both NC siRNA and km23-1-siRNA HCT116 stable transfectants were counted under a light microscope and statistically analyzed. *p<0.01 compared to EV.</p

Depletion of km23-1 blocks constitutive ERK activation in human CRC cells.

<p><b>A.</b> EV, NC siRNA, and km23-1 siRNA stable transfectants (clones 1 and 5) were used to isolate RNA. RT-PCR was performed and products were analyzed as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066439#s2" target="_blank">Materials and Methods</a>.” The data plotted are the mean ± SE of three independent experiments. *p<0.01 compared to the NC siRNA. <b>B:</b> Western blotting of phospho- and total protein expression levels for ERK1/2 in RKO human CRC cells. <b>Bottom</b>, DIC protein was assessed as a loading control. Data are representative of three independent experiments. <b>C</b>: Western blotting of phospho- and total protein expression levels for ERK1/2 in HCT116 cells stably transduced with the lentiviral particles described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066439#s2" target="_blank">Materials and Methods</a>.” Top, confirms knockdown of endogenous km23-1. <b>Bottom</b>, GAPDH protein was assessed as a loading control. Data are representative of three independent experiments. <b>D:</b> CBS cells were infected with either pilenti-NC siRNA-GFP or pilenti-km23-1 siRNA-GFP pools. 24 h after infection, Western blotting was performed using the indicated antibodies. <b>Top</b>, confirms knockdown of endogenous km23-1. <b>Bottom</b>, DIC protein was assessed as a loading control. Three independent experiments were performed and representative figures are shown.</p