Lymph node (LN) stromal cell populations expand during the inflammation that accompanies T cell activation. Interleukin 17 (IL-17)-producing T helper (TH17) cells promote inflammation through induction of cytokines and chemokines in peripheral tissues. We demonstrate a critical requirement for IL-17 in the proliferation of lymph node (LN) and spleen stromal cells, particularly fibroblastic reticular cells (FRCs), during experimental autoimmune encephalomyelitis and colitis. Without IL-17 receptor signaling, activated FRCs underwent cell cycle arrest and ultimately apoptosis, accompanied by signs of nutrient stress in vivo. IL-17 signaling in FRCs was not required for TH17 cell development, but failed FRC proliferation impaired germinal center formation and antigen-specific antibody production. IL-17 induction of the transcriptional coactivator IκBζ mediated increased glucose uptake and mitochondrial Cpt1a expression. Hence, IL-17 produced by locally differentiating TH17 cells is an important driver of inflamed LN stromal cell activation, through metabolic reprogramming required to support proliferation and survival.

The loading of cohesin onto chromatin requires the heterodimeric complex sister chromatid cohesion (Scc)2 and Scc4 (Scc2/4), which is highly conserved in all species. Here, we describe the purification of the human (h)-Scc2/4 and show that it interacts with hcohesin and the heterodimeric Smc1-Smc3 complex but not with the Smc1 or Smc3 subunit alone. We demonstrate that both h-Scc2/4 and h-cohesin are loaded onto dsDNA containing the prereplication complex (pre-RC) generated in vitro by Xenopus high-speed soluble extracts. The addition of geminin, which blocks pre-RC formation, prevents the loading of Scc2/4 and cohesin. Xenopus extracts depleted of endogenous Scc2/4 with specific antibodies, although able to form pre-RCs, did not support cohesin loading unless supplemented with purified h-Scc2/4. The results presented here indicate that the Xenopus or h-Scc2/4 complex supports the loading of Xenopus and/or h-cohesin onto pre-RCs formed by Xenopus high-speed extracts. We show that cohesin loaded onto pre-RCs either by h-Scc2/4 and/or the Xenopus complex was dissociated from chromatin by low salt extraction, similar to cohesin loaded onto chromatin in G 1 by HeLa cells in vivo. Replication of cohesin-loaded DNA, both in vitro and in vivo, markedly increased the stability of cohesin associated with DNA. Collectively, these in vitro findings partly recapitulate the in vivo pathway by which sister chromatids are linked together, leading to cohesion.cohesin stability | cell-cycle | cohesion establishment | sister chromatid cohesion N ewly replicated chromosomes are held together until their separation and equal distribution to daughter cells during cell division. Their association is mediated by cohesin, a foursubunit complex comprising (i) Rad21/sister chromatid cohesion (Scc)1/multiple chloroplast division (Mcd)1, (ii) structural maintenance of chromosomes (Smc)1, (iii) Smc3, and (iv) Scc3/ (stromal antigen) STAG/SA that stably links the sister chromatids together in S and G 2 (1). Smc1 and Smc3 are elongated coiled-coil proteins, each of which folds onto itself to form a globular ATPase head domain through the juxtaposition of the N and C terminus with a hinge domain at the other end. Smc1 and Smc3 interact through their hinge domains to form a heterodimer. The kleisin subunit, Scc1, associates with the head domains of Smc1 and Smc3 to stabilize their interaction and recruits the Scc3/SA subunit. The Smc1-Smc3-Scc1 complex forms a ring structure with an internal diameter of 40 nm, large enough to tether two chromatids (embrace model) (2). Although other models have been proposed to explain how the cohesin ring leads to the association of sister chromatids, substantial evidence supporting the "embrace model" has accumulated (3).Sister chromatid cohesion is a multistep process that includes cohesin loading onto chromatin and the establishment of cohesion during replication (3). Although cohesin loading required for cohesion occurs before the S phase, the timing of its chromosome association differs among species. In ...

Chromosome transmission fidelity 4 (Ctf4) is a conserved protein required for DNA replication. In this report, interactions between human Ctf4 (hCtf4) and the replicative helicase containing the cell division cycle 45 (Cdc45)/minichromosome maintenance 2-7 (Mcm2-7)/Go, Ichi, Nii, and San (GINS) (CMG) proteins [human CMG (hCMG) complex] were examined. The hCtf4-CMG complex was isolated following in vitro interaction of purified proteins (hCtf4 plus the hCMG complex), coinfection of Spodoptera frugiperda (Sf9) insect cells with viruses expressing the hCMG complex and hCtf4, and from HeLa cell chromatin after benzonase and immunoprecipitation steps. The stability of the hCtf4-CMG complex depends upon interactions between hCtf4 and multiple components of the hCMG complex. The hCtf4-CMG complex, like the hCMG complex, contains DNA helicase activity that is more salt-resistant than the helicase activity of the hCMG complex. We demonstrate that the hCtf4-CMG complex contains a homodimeric hCtf4 and a monomeric hCMG complex and suggest that the homodimeric hCtf4 acts as a platform linking polymerase α to the hCMG complex. The role of the hCMG complex as the core of the replisome is also discussed.replisome core structure | dimerization | replication initiation E ukaryotic DNA replication is a stepwise process by which protein complexes are assembled on chromatin. During the G1 phase of the cell cycle, the origin recognition complex (ORC) binds to replication origins and recruits cell division cycle 6 (Cdc6) (1). This complex leads to the association of cdc10 dependent transcript 1 (Cdt1)/Mcm2-7 with chromatin and the loading of the Mcm2-7 complex as a head-to-head dimer (2, 3). At the G1/S transition, this prereplication complex is altered further by a number of replication initiation factors whose actions are facilitated by the cyclin-dependent and cell division cycle 7 (Cdc7)-dumbbell former 4 (Dbf4) kinases (4). These replication initiation factors [which include synthetically lethal with dpb11-1 (Sld)2, Sld3, Sld7, DNA polymerase B possible subunit 11 (Dpb11), and DNA polymerase e (Pol e) in budding yeast] play critical roles resulting in the interaction of Cdc45 and GINS with the Mcm2-7 complex and the formation of the replicative DNA helicase complex containing Cdc45/Mcm2-7/GINS (CMG) (5). Other replication factors (including Mcm10 and Ctf4) contribute to the activation of the CMG helicase and association of proteins that recruit the replicative Pols to effect DNA replication (6, 7). Interactions between TopBP1-interacting, replication-stimulating protein (Treslin)

SUMMARY Inerleukin-23 (IL-23) is required for inflammatory Th17 cell function in experimental autoimmune encephalomyelitis (EAE), and IL-23 blockade reduces the number of effector Th17 cells in the CNS. We report that pro-inflammatory Th17 cells express high integrin β3 that is IL-23 dependent. Integrin β3 was not upregulated on all activated T cells; rather, integrin β3 was upregulated along with its functional partner integrin αv on effector Th17 cells and “ex-Th17” cells, and αvβ3hi RORγt+ cells expanded during EAE. Integrin αvβ3 inhibitors ameliorated clinical signs of EAE, and integrin β3 deficiency on CD4+ T cells alone was sufficient to block EAE induction. Furthermore, integrin-β3-deficient Th17 cells, but not Th1 cells, were impaired in their ability to induce EAE. Integrin β3−/− T cells induced smaller demyelinated lesions and showed reduced spread and accumulation within the CNS, corresponding with impaired extracellular-matrix-mediated migration. Hence, integrin β3 is required for Th17 cell-mediated autoimmune CNS inflammation.

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