Xiaoping Zhu

Transfer of IgG in the female genital tract by MHC class I-related neonatal Fc receptor (FcRn) confers protective immunity to vaginal infection.

IgG is a major Ig subclass in mucosal secretions of the human female genital tract, where it predominates over the IgA isotype. Despite the abundance of IgG, surprisingly little is known about where and how IgG enters the lumen of the genital tract and the exact role local IgG plays in preventing sexually transmitted diseases. We demonstrate here that the neonatal Fc receptor, FcRn, is expressed in female genital tract epithelial cells of humans and mice and binds IgG in a pH-dependent manner. In vitro we show that FcRn mediates bidirectional IgG transport across polarized human endometrial HEC-1-A monolayers and primary human genital epithelial cells. Furthermore, endosomal acidification appears to be a prerequisite for FcRn-mediated IgG transcytosis; IgG transcytosis was demonstrated in vivo by translocation of systemically administered IgG into the genital lumen in WT but not FcRn-KO mice. The biological relevance of FcRn-transported IgG was demonstrated by passive immunization using herpes simplex virus-2 (HSV-2)–specific polyclonal serum, which conferred significantly higher protection against intravaginal challenge infection by the HSV-2 186 strain in WT mice than in FcRn-KO mice. These studies demonstrate that FcRn-mediated transport is a mechanism by which IgG can act locally in the female genital tract in immune surveillance and in host defense against sexually transmitted diseases.

Efficient mucosal vaccination mediated by the neonatal Fc receptor

Almost all infectious diseases are initiated at mucosal surfaces, yet intramuscular or subcutaneous vaccination usually provides only minimal protection at sites of infection owing to suboptimal activation of the mucosal immune system. The neonatal Fc receptor (FcRn) mediates the transport of IgG across polarized epithelial cells lining mucosal surfaces. We mimicked this process by fusing a model antigen, herpes simplex virus type-2 (HSV-2) glycoprotein gD, to an IgG Fc fragment. Intranasal immunization, together with the adjuvant CpG, completely protected wild-type, but not FcRn knockout, mice after intravaginal challenge with virulent HSV-2 186. This immunization strategy induced efficient mucosal and systemic antibody, B- and T-cell immune responses, with stable protection for at least 6 months after vaccination in most of the immunized animals. The FcRn-IgG transcellular transport pathway may provide a general delivery route for subunit vaccines against many mucosal pathogens.

NF-κB Signaling Regulates Functional Expression of the MHC Class I-Related Neonatal Fc Receptor for IgG via Intronic Binding Sequences

The neonatal Fc receptor for IgG (FcRn) functions to transport maternal IgG to a fetus or newborn and to protect IgG from degradation. Although FcRn is expressed in a variety of tissues and cell types, the extent to which FcRn expression is regulated by immunological and inflammatory events remains unknown. Stimulation of intestinal epithelial cell lines, macrophage-like THP-1, and freshly isolated human monocytes with the cytokine TNF-α rapidly up-regulated FcRn gene expression. In addition, the TLR ligands LPS and CpG oligodeoxynucleotide enhanced the level of FcRn expression in THP-1 and monocytes. Treatment of TNF-stimulated THP-1 cells with the NF-κB-specific inhibitor or overexpression of a dominant negative mutant inhibitory NF-κB (IκBα; S32A/S36A) resulted in down-regulation of FcRn expression. By using chromatin immunoprecipitation we identified three NF-κB binding sequences within introns 2 and 4 of the human FcRn gene. An EMSA confirmed the p50/p50 and/or p65/p50 complex (s) bound to intron 2- or 4-derived oligonucleotides containing putative NF-κB binding sequences, respectively. The intronic NF-κB sequences in combination with the promoter or alone regulated the expression of a luciferase reporter gene in response to TNF-α stimulation or overexpression of NF-κB p65 and p50. DNA looping interactions potentially occurred after the stimulation between intronic NF-κB sequences and the FcRn promoter as shown by a chromosome conformation capture assay. Finally, TNF-α stimulations enhanced IgG transport across an intestinal Caco-2 epithelial monolayer. Together, these data provide the first evidence that NF-κB signaling via intronic sequences regulates FcRn expression and function.

The Neonatal FcR-Mediated Presentation of Immune-Complexed Antigen Is Associated with Endosomal and Phagosomal pH and Antigen Stability in Macrophages and Dendritic Cells

The FcγRs found on macrophages (Mϕs) and dendritic cells (DCs) efficiently facilitate the presentation or cross-presentation of immune-complexed Ags to T cells. We found that the MHC class I-related neonatal FcR for IgG (FcRn) in both Mϕs and DCs failed to have a strong effect on the cross-presentation of immune complex (IC) OVA Ag to CD8+ T cells. Interestingly, endosomal FcRn enhanced the presentation of the monomeric OVA-IC to CD4+ T cells robustly, whereas FcRn in phagosomes exerted distinctive effects on Ag presentation between Mϕs and DCs. The presentation of phagocytosed OVA-ICs to CD4+ T cells was considerably enhanced on wild-type versus FcRn-deficient Mϕs, but was not affected in FcRn-deficient DCs. This functional discrepancy was associated with the dependence of IgG–FcRn binding in an acidic pH. Following phagocytosis, the phagosomal pH dropped rapidly to <6.5 in Mϕs but remained in the neutral range in DCs. This disparity in pH determined the rate of degradation of phagocytosed ICs. Thus, our findings reveal that FcRn expression has a different effect on Ag processing and presentation of ICs to CD4+ T cells in the endosomal versus phagosomal compartments of Mϕs versus DCs.

A Neonatal Fc Receptor-Targeted Mucosal Vaccine Strategy Effectively Induces HIV-1 Antigen-Specific Immunity to Genital Infection

ABSTRACT Strategies to prevent the sexual transmission of HIV include vaccines that elicit durable, protective mucosal immune responses. A key to effective mucosal immunity is the capacity for antigens administered locally to cross epithelial barriers. Given the role of neonatal Fc receptor (FcRn) in transferring IgG across polarized epithelial cells which line mucosal surfaces, FcRn might be useful for delivering HIV vaccine antigens across mucosal epithelial barriers to the underlying antigen-presenting cells. Chimeric proteins composed of HIV Gag (p24) fused to the Fc region of IgG (Gag-Fc) bind efficiently to airway mucosa and are transported across this epithelial surface. Mice immunized intranasally with Gag-Fc plus CpG adjuvant developed local and systemic immunity, including durable B and T cell memory. Gag-specific immunity was sufficiently potent to protect against an intravaginal challenge with recombinant vaccinia virus expressing the HIV Gag protein. Intranasal administration of a Gag-Fc/CpG vaccine protected at a distal mucosal site. Our data suggest that targeting of FcRn with chimeric immunogens may be an important strategy for mucosal immunization and should be considered a new approach for preventive HIV vaccines.

Intracellular neutralization of viral infection in polarized epithelial cells by neonatal Fc receptor (FcRn)-mediated IgG transport.

IgG was traditionally thought to neutralize virions by blocking their attachment to or penetration into mucosal epithelial cells, a common site of exposure to viruses. However, we describe an intracellular neutralizing action for an influenza hemagglutinin-specific monoclonal antibody, Y8-10C2 (Y8), which has neutralizing activity only at an acidic pH. When Y8 was applied to the basolateral surface of Madin–Darby canine kidney cells expressing the rat neonatal Fc receptor for IgG (FcRn), it significantly reduced viral replication following apical exposure of the cell monolayer to influenza virus. Virus neutralization by Y8 mAb was dependent on FcRn expression and its transport of IgG. As both FcRn and Y8 mAb bind their partners only at acidic pH, the Y8 mAb is proposed to carry out its antiviral activity intracellularly. Furthermore, the virus, Y8 mAb, and FcRn colocalized within endosomes, possibly inhibiting the fusion of viral envelopes with endosomal membranes during primary uncoating, and preventing the accumulation of the neutralized viral nucleoprotein antigen in the nucleus. Prophylactic administration of Y8 mAb before viral challenge in WT mice, but not FcRn-KO mice, conferred protection from lethality, prevented weight loss, resulted in a significant reduction in pulmonary virus titers, and largely reduced virus-induced lung pathology. Thus, this study reveals an intracellular mechanism for viral neutralization in polarized epithelial cells that is dependent on FcRn-mediated transport of neutralizing IgG.

Activation of the JAK/STAT-1 signaling pathway by IFN-γ can down-regulate functional expression of the MHC class I-related neonatal Fc receptor for IgG

Expression of many MHC genes is enhanced at the transcriptional or posttranscriptional level following exposure to the cytokine IFN-γ. However, in this study we found that IFN-γ down-regulated the constitutive expression of the neonatal Fc receptor (FcRn), an MHC class I-related molecule that functions to transport maternal IgG and protect IgG and albumin from degradation. Epithelial cell, macrophage-like THP-1 cell, and freshly isolated human PBMC exposure to IFN-γ resulted in a significant decrease of FcRn expression as assessed by real-time RT-PCR and Western blotting. The down-regulation of FcRn was not caused by apoptosis or the instability of FcRn mRNA. Chromatin immunoprecipitation and gel mobility shift assays showed that STAT-1 bound to an IFN-γ activation site in the human FcRn promoter region. Luciferase expression from an FcRn promoter-luciferase reporter gene construct was not altered in JAK1- and STAT-1-deficient cells following exposure to IFN-γ, whereas expression of JAK1 or STAT-1 protein restored the IFN-γ inhibitory effect on luciferase activity. The repressive effect of IFN-γ on the FcRn promoter was selectively reversed or blocked by mutations of the core nucleotides in the IFN-γ activation site sequence and by overexpression of the STAT-1 inhibitor PIAS1 or the dominant negative phospho-STAT-1 mutations at Tyr-701 and/or Ser-727 residues. Furthermore, STAT-1 might down-regulate FcRn transcription through sequestering the transcriptional coactivator CREB binding protein/p300. Functionally, IFN-γ stimulation dampened bidirectional transport of IgG across a polarized Calu-3 lung epithelial monolayer. Taken together, our results indicate that the JAK/STAT-1 signaling pathway was necessary and sufficient to mediate the down-regulation of FcRn gene expression by IFN-γ.

The MHC Class II-Associated Invariant Chain Interacts with the Neonatal Fcγ Receptor and Modulates Its Trafficking to Endosomal/Lysosomal Compartments

The neonatal Fc receptor for IgG (FcRn) transfers maternal IgG to the offspring and protects IgG from degradation. The FcRn resides in an acidic intracellular compartment, allowing it to bind IgG. In this study, we found the association of FcRn and invariant chain (Ii). The interaction was initiated within the endoplasmic reticulum by Ii binding to either the FcRn H chain alone or FcRn H chain-β2-microglobulin complex and appeared to be maintained throughout the endocytic pathway. The CLIP in Ii was not required for FcRn-Ii association. The interaction was also detected in IFN-γ-treated THP-1, epithelial and endothelial cells, and immature mouse DCs. A truncated FcRn without the cytoplasmic tail was unable to traffic to early endosomes; however, its location in early endosomes was restored by Ii expression. FcRn was also detected in the late endosome/lysosome only in the presence of Ii or on exposure to IFN-γ. In immature human or mouse DCs, FcRn was barely detected in the late endosome/lysosome in the absence of Ii. Furthermore, the cytoplasmic tail of Ii conferred tailless FcRn to route to both the early endosome and late endosome/lysosome in a hybrid molecule. Because the FcRn is expressed in macrophages and DCs or epithelial and endothelial cells where Ii is induced under inflammation and infection, these results reveal the complexity of FcRn trafficking in which Ii is capable of expanding the boundary of FcRn trafficking. Taken together, the intracellular trafficking of FcRn is regulated by its intrinsic sorting information and/or an interaction with Ii chain.

CD23-Dependent Transcytosis of IgE and Immune Complex across the Polarized Human Respiratory Epithelial Cells

IgE-mediated allergic inflammation occurs when allergens cross-link IgE on the surface of immune cells, thereby triggering the release of inflammatory mediators as well as enhancing Ag presentations. IgE is frequently present in airway secretions, and its level can be enhanced in human patients with allergic rhinitis and bronchial asthma. However, it remains completely unknown how IgE appears in the airway secretions. In this study, we show that CD23 (FcεRII) is constitutively expressed in established or primary human airway epithelial cells, and its expression is significantly upregulated when airway epithelial cells were subjected to IL-4 stimulation. In a transcytosis assay, human IgE or IgE-derived immune complex (IC) was transported across a polarized Calu-3 monolayer. Exposure of the Calu-3 monolayer to IL-4 stimulation also enhanced the transcytosis of either human IgE or the IC. A CD23-specific Ab or soluble CD23 significantly reduced the efficiency of IgE or IC transcytosis, suggesting a specific receptor-mediated transport by CD23. Transcytosis of both IgE and the IC was further verified in primary human airway epithelial cell monolayers. Furthermore, the transcytosed Ag–IgE complexes were competent in inducing degranulation of the cultured human mast cells. Because airway epithelial cells are the first cell layer to come into contact with inhaled allergens, our study implies CD23-mediated IgE transcytosis in human airway epithelial cells may play a critical role in initiating and contributing to the perpetuation of airway allergic inflammation.

Pleiotropic roles of uvrY on biofilm formation, motility and virulence in uropathogenic Escherichia coli CFT073.

Urinary tract infections primarily caused by uropathogenic strains of Escherichia coli (E. coli) remain a significant public health problem in both developed and developing countries. An important virulence determinant in uropathogenesis is biofilm formation which requires expression of fimbriae, flagella, and other surface components such as lipopolysaccharides. In this study, we explored the regulation of uvrY and csrA genes in biofilm formation, motility and virulence determinants in uropathogenic E. coli. We found that mutation in uvrY suppressed biofilm formation on abiotic surfaces such as polyvinyl chloride, polystyrene and glass, and complementation of uvrY in the mutant restored the biofilm phenotype. We further evaluated the role of uvrY gene in expression of type 1 fimbriae, an important adhesin that facilitates adhesion to various abiotic surfaces. We found that phase variation of type 1 fimbriae between fimbriated and afimbriated mode was modulated by uvrY at its transcriptional level. Deletion mutant of uvrY lowered expression of fimbrial recombinase genes, such as fimB, fimE, and fimA, a gene encoding major fimbrial subunit. Furthermore, transcription of virulence specific genes such as papA, hlyB and galU was also reduced in the deletion mutant. Swarming motility and expression of flhD and flhC was also diminished in the mutant. Taken together, our findings unravel a possible mechanism in which uvrY facilitates biofilm formation, persistence and virulence of uropathogenic E. coli.