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A longstanding question in cell biology is how is the routing of intracellular organelles within cells regulated? Although data support the involvement of Rab4 and Rab11 GTPases in the recycling pathway, the function of Rab11 in particular is uncertain. Here we have analyzed the association of these two Rab GTPases with the Fc receptor, FcRn, during intracellular trafficking. This Fc receptor is both functionally and structurally distinct from the classical Fcγ receptors and transports immunoglobulin G (IgG) within cells. FcRn is therefore a recycling receptor that sorts bound IgG from unbound IgG in sorting endosomes. In the current study we have used dual color total internal reflection fluorescence microscopy (TIRFM) and wide-field imaging of live cells to analyze the events in human endothelial cells that are involved in the trafficking of FcRn positive (FcRn+) recycling compartments from sorting endosomes to exocytic sites at the plasma membrane. Our data are consistent with the following model for this pathway: FcRn leaves sorting endosomes in Rab4+Rab11+or Rab11+compartments. For Rab4+Rab11+compartments, Rab4 depletion occurs by segregation of the two Rab proteins into discrete domains that can separate. The Rab11+FcRn+vesicle or tubule subsequently fuses with the plasma membrane in an exocytic event. In contrast to Rab11, Rab4 is not involved in exocytosis.

IgG is the most abundant serum antibody and is an essential component of the humoral immune response. It is known that the 'neonatal' Fc receptor (FcRn) plays a role in maintaining constant serum IgG levels by acting as a protective receptor which binds and salvages IgG from degradation. However, the cellular mechanism that is involved in serum IgG homeostasis is poorly understood. In the current study we address this issue by analyzing the intracellular fate in human endothelial cells of IgG molecules which bind with different affinities to FcRn. The studies show that IgG which do not bind to FcRn accumulate in the lysosomal pathway, providing a cellular explanation for short serum persistence of such antibodies. We have also investigated the saturability of the homeostatic system and find that it has limited capacity. Our observations have direct relevance to the understanding and treatment of IgG deficiency, and to the effective application of therapeutic antibodies.

Myeloid lineage cells use TLRs to recognize and respond to diverse microbial ligands. Although unique transcription factors dictate the outcome of specific TLR signaling, whether lineage-specific differences exist to further modulate the quality of TLR-induced inflammation remains unclear. Comprehensive analysis of global gene transcription in human monocytes, monocyte-derived macrophages, and monocyte-derived dendritic cells stimulated with various TLR ligands identifies multiple lineage-specific, TLR-responsive gene programs. Monocytes are hyperresponsive to TLR7/8 stimulation that correlates with the higher expression of the receptors. While macrophages and monocytes express similar levels of TLR4, macrophages, but not monocytes, upregulate interferon-stimulated genes (ISGs) in response to TLR4 stimulation. We find that TLR4 signaling in macrophages uniquely engages transcription factor IRF1, which facilitates the opening of ISG loci for transcription. This study provides a critical mechanistic basis for lineage-specific TLR responses and uncovers IRF1 as a master regulator for the ISG transcriptional program in human macrophages.

Background: Systemic lupus erythematosus (SLE) is a clinically heterogeneous autoimmune disease characterized by the development of anti-nuclear antibodies. Susceptibility to SLE is multifactorial, with a combination of genetic and environmental risk factors contributing to disease development. Like other polygenic diseases, a significant proportion of estimated SLE heritability is not accounted for by common disease alleles analyzed by SNP array-based GWASs. Death-associated protein 1 (DAP1) was implicated as a candidate gene in a previous familial linkage study of SLE and rheumatoid arthritis, but the association has not been explored further. Results: We perform deep sequencing across the DAP1 genomic segment in 2032 SLE patients, and healthy controls, and discover a low-frequency functional haplotype strongly associated with SLE risk in multiple ethnicities. We find multiple cis-eQTLs embedded in a risk haplotype that progressively downregulates DAP1 transcription in immune cells. Decreased DAP1 transcription results in reduced DAP1 protein in peripheral blood mononuclear cells, monocytes, and lymphoblastoid cell lines, leading to enhanced autophagic flux in immune cells expressing the DAP1 risk haplotype. Patients with DAP1 risk allele exhibit significantly higher autoantibody titers and altered expression of the immune system, autophagy, and apoptosis pathway transcripts, indicating that the DAP1 risk allele mediates enhanced autophagy, leading to the survival of autoreactive lymphocytes and increased autoantibody. Conclusions: We demonstrate how targeted sequencing captures low-frequency functional risk alleles that are missed by SNP array-based studies. SLE patients with the DAP1 genotype have distinct autoantibody and transcription profiles, supporting the dissection of SLE heterogeneity by genetic analysis.

Introduction: The finding of antinuclear antibody (ANA) positivity in a healthy individual is usually of unknown significance and in most cases is benign. However, a subset of such individuals is at risk for development of autoimmune disease. We examined demographic and immunological features that are associated with ANA positivity in clinically healthy persons to develop insights into when this marker carries risk of progression to lupus. Methods: Biological samples from healthy individuals and patients with systemic lupus erythematosus (SLE) were obtained from the Dallas Regional Autoimmune Disease Registry (DRADR). Measurements carried out on serum samples included ANA, extractable nuclear antibodies (ENA) and autoantibody profiling using an array with more than 100 specificities. Whole blood RNA samples from a subset of individuals were used to analyze gene expression on the Illumina platform. Data were analyzed for associations of high ANA levels with demographic features, the presence of other autoantibodies and with gene expression profiles. Results: Overall, ANA levels are significantly higher in females than in males and this association holds in patients with the autoimmune diseases lupus and rheumatoid arthritis (RA) as well as in healthy controls (HC). Age was not significantly associated with ANA levels and the elevated ANA values could not be explained by higher IgG levels. Another autoantibody, anti- cyclic citrullinated peptide (CCP), did not show gender dimorphism in rheumatoid arthritis (RA) or healthy individuals. The autoantigen array showed significant elevations of other autoantibodies in high ANA HCs. Some of these autoantibodies were directed to antigens in skin and others were related to autoimmune conditions of kidney, thyroid or joints. Gene expression analyses showed a greater prevalence of significantly upregulated genes in HCs with negative ANA values than in those with significant ANA positivity. Genes upregulated in high ANA HCs included a celiac disease autoantigen and some components of the Type I interferon (IFN) gene signature. Conclusions: Risks for ANA positivity include female gender and organ-specific autoimmunity. Upregulation of skin-specific autoantibodies may indicate that early events in the break of tolerance take place in cutaneous structures. Some of these changes may be mediated by Type I IFN. Blood profiling for expressed autoantibodies and genes has the potential to identify individuals at risk for development of autoimmune diseases including lupus.