Development of the expansive endoplasmic reticulum (ER) present in specialized secretory cell types requires X-box-binding protein-1 (Xbp-1). Enforced expression of XBP-1(S), a transcriptional activator generated by unfolded protein responsemediated splicing of Xbp-1 mRNA, is sufficient to induce proliferation of rough ER. We previously showed that XBP-1(S)-induced ER biogenesis in fibroblasts correlates with increased production of phosphatidylcholine (PtdCho), the primary phospholipid of the ER membrane, and enhanced activities of the choline cytidylyltransferase (CCT) and cholinephosphotransferase enzymes in the cytidine diphosphocholine (CDP-choline) pathway of PtdCho biosynthesis. Here, we report that the level and synthesis of CCT, the rate-limiting enzyme in the CDPcholine pathway, is elevated in fibroblasts overexpressing XBP-1(S). Furthermore, overexpression experiments demonstrated that raising the activity of CCT, but not cholinephosphotransferase, is sufficient to augment PtdCho biosynthesis in fibroblasts, indicating that XBP-1(S) increases the output of the CDP-choline pathway primarily via its effects on CCT. Finally, fibroblasts overexpressing CCT up-regulated PtdCho synthesis to a level similar to that in XBP-1(S)-transduced cells but exhibited only a small increase in rough ER and no induction of secretory pathway genes. The more robust XBP-1(S)-induced ER expansion was accompanied by induction of a wide array of genes encoding proteins that function either in the ER or at other steps in the secretory pathway. We propose that XBP-1(S) regulates ER abundance by coordinately increasing the supply of membrane phospholipids and ER proteins, the key ingredients for ER biogenesis. The endoplasmic reticulum (ER)3 is a multifunctional organelle responsible for the folding and assembly of all proteins targeted to the secretory pathway (1). As such, the ER can adapt to accommodate an increased load of nascent polypeptides. For example, when B-lymphocytes differentiate into antibody-secreting plasma cells, an elaborate network of rough ER develops to facilitate immunoglobulin production (2-4). Likewise, the rough ER is highly developed in other specialized secretory cell types such as pancreatic acinar cells that secrete copious amounts of digestive enzymes (5). In contrast, the ER is sparse in non-secretory cells, such as reticulocytes (6). ER abundance, therefore, is regulated according to the demands on the secretory pathway. However, the mechanisms that regulate ER biogenesis are incompletely defined (7).A key regulator of ER homeostasis is the unfolded protein response (UPR) pathway, a complex signaling system emanating from the ER membrane (8). When the protein folding capacity of the ER is challenged, the UPR relieves the resulting stress by repressing translation, increasing expression of ER chaperones and folding enzymes, and enhancing ER-associated degradation (8). In addition, recent studies have uncovered a connection between the UPR and ER abundance (9, 10). The UPR-regulated transcription fa...

Extended circulation of nanoparticles in blood is essential for most clinical applications. Nanoparticles are rapidly cleared by cells of the mononuclear phagocyte system (MPS). Approaches such as grafting polyethylene glycol onto particles (PEGylation) extend circulation times; however, these particles are still cleared, and the processes involved in this clearance remain poorly understood. Here, we present an intravital microscopybased assay for the quantification of nanoparticle clearance, allowing us to determine the effect of mouse strain and immune system function on particle clearance. We demonstrate that mouse strains that are prone to Th1 immune responses clear nanoparticles at a slower rate than Th2-prone mice. Using depletion strategies, we show that both granulocytes and macrophages participate in the enhanced clearance observed in Th2-prone mice. Macrophages isolated from Th1 strains took up fewer particles in vitro than macrophages from Th2 strains. Treating macrophages from Th1 strains with cytokines to differentiate them into M2 macrophages increased the amount of particle uptake. Conversely, treating macrophages from Th2 strains with cytokines to differentiate them into M1 macrophages decreased their particle uptake. Moreover, these results were confirmed in human monocyte-derived macrophages, suggesting that global immune regulation has a significant impact on nanoparticle clearance in humans. IntroductionThe potential clinical applications of nanoparticles and nanoformulations have been investigated for more than 30 years. Nanoparticle approaches have the potential to revolutionize drug delivery by allowing for the encapsulation of drugs with poor solubility or stability in a stable carrier particle. In addition, targeting nanoparticles to specific pathological sites may allow an increased effective dose of drug at the needed site, while decreasing systemic drug exposure, and therefore side effects. However, to date, only 2 nanoformulations for cancer treatment have been approved for clinical use (liposomal doxorubicin [Doxil] and protein-bound paclitaxel [Abraxane]) (1-3). One major obstacle for the use of nanoparticles in vivo is rapid clearance by the cells of the reticuloendothelial system (RES)/mononuclear phagocyte system (MPS) (4-7). In addition to rapid clearance, variable activity of the MPS among patients leads to widely variable pharmacokinetics of nanoformulations in the clinic, reducing the efficacy of both approved and future experimental nanoformulations (8).

Engineered nanoparticles have the potential to expand the breadth of pulmonary therapeutics, especially as respiratory vaccines. Notably, cationic nanoparticles have been demonstrated to produce superior local immune responses following pulmonary delivery; however, the cellular mechanisms of this increased response remains unknown. To this end, we systematically investigated the cellular response of lung APCS following pulmonary instillation of anionic and cationic charged nanoparticles. While nanoparticles of both surface charges were capable of trafficking to the draining lymph node and were readily internalized by alveolar macrophages, both CD11b and CD103 lung dendritic cell (DC) subtypes preferentially associated with cationic nanoparticles. Instillation of cationic nanoparticles resulted in the upregulation of Ccl2 and Cxc10, which likely contributes to the recruitment of CD11b DCs to the lung. In total, these cellular mechanisms explain the increased efficacy of cationic formulations as a pulmonary vaccine carrier and provide critical benchmarks in the design of pulmonary vaccine nanoparticles.

Pulmonary immunization enhances local humoral and cell-mediated mucosal protection, which are critical for vaccination against lung-specific pathogens such as influenza or tuberculosis. A variety of nanoparticle (NP) formulations have been tested preclinically for pulmonary vaccine development, yet the role of NP surface charge on downstream immune responses remains poorly understood. We used the Particle Replication in Non-Wetting Templates (PRINT) process to synthesize hydrogel NPs that varied only in surface charge and otherwise maintained constant size, shape, and antigen loading. Pulmonary immunization with ovalbumin (OVA)-conjugated cationic NPs led to enhanced systemic and lung antibody titers compared with anionic NPs. Increased antibody production correlated with robust germinal center B-cell expansion and increased activated CD4(+) T-cell populations in lung draining lymph nodes. Ex vivo treatment of dendritic cells (DCs) with OVA-conjugated cationic NPs induced robust antigen-specific T-cell proliferation with ∼ 100-fold more potency than soluble OVA alone. Enhanced T-cell expansion correlated with increased expression of surface MHCII, T-cell coactivating receptors, and key cytokines/chemokine expression by DCs treated with cationic NPs, which were not observed with anionic NPs or soluble OVA. Together, these studies highlight the importance of NP surface charge when designing pulmonary vaccines, and our findings support the notion that cationic NP platforms engender potent humoral and mucosal immune responses.

Background: Multiple functions have been ascribed to NLRC5 including MHC-I transcription and cytokine responses. Results: We generated Nlrc5 Ϫ/Ϫ mice and showed that Nlrc5 increases classical and nonclassical MHC-I and causes removal of

Summary Modern medical and hygienic practices have greatly improved human health and longevity; however, increased human lifespan occurs concomitantly with the emergence of metabolic and age-related diseases. Studies over the past decade have strongly linked host inflammatory responses to the etiology of several metabolic diseases including atherosclerosis, type 2 diabetes (T2D), obesity and gout. A common immunological factor to these diseases is the activation of the inflammasome and release of pro-inflammatory cytokines that promote disease progression. Here we review the molecular mechanism(s) of inflammasome activation in response to metabolic damage associated molecular patterns (DAMPs) and discuss potential targets for therapeutic intervention.

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Long-circulating nanoparticles are essential for increasing tumor accumulation to provide therapeutic efficacy. While it is known that tumor presence can alter the immune system, very few studies have explored this impact on nanoparticle circulation. In this report, we demonstrate how the presence of a tumor can change the local and global immune system, which dramatically increases particle clearance. We found that tumor presence significantly increased clearance of PRINT hydrogel nanoparticles from the circulation, resulting in increased accumulation in the liver and spleen, due to an increase in M2-like macrophages. Our findings highlight the need to better understand interactions between immune status and nanoparticle clearance, and suggest that further consideration of immune function is required for success in preclinical and clinical nanoparticle studies.