FcRn-Targeted Mucosal Vaccination against Influenza Virus Infection.

The respiratory tract is constantly exposed to various airborne pathogens. Most vaccines against respiratory infections are designed for the parenteral routes of administration; consequently, they provide relatively minimal protection in the respiratory tract. A vaccination strategy that aims to induce the protective mucosal immune responses in the airway is urgently needed. The FcRn mediates IgG Ab transport across the epithelial cells lining the respiratory tract. By mimicking this natural IgG transfer, we tested whether FcRn delivers vaccine Ags to induce a protective immunity to respiratory infections. In this study, we designed a monomeric IgG Fc fused to influenza virus hemagglutinin (HA) Ag with a trimerization domain. The soluble trimeric HA-Fc were characterized by their binding with conformation-dependent HA Abs or FcRn. In wild-type, but not FcRn knockout, mice, intranasal immunization with HA-Fc plus CpG adjuvant conferred significant protection against lethal intranasal challenge with influenza A/PR/8/34 virus. Further, mice immunized with a mutant HA-Fc lacking FcRn binding sites or HA alone succumbed to lethal infection. Protection was attributed to high levels of neutralizing Abs, robust and long-lasting B and T cell responses, the presence of lung-resident memory T cells and bone marrow plasma cells, and a remarkable reduction of virus-induced lung inflammation. Our results demonstrate for the first time, to our knowledge, that FcRn can effectively deliver a trimeric viral vaccine Ag in the respiratory tract and elicit potent protection against respiratory infection. This study further supports a view that FcRn-mediated mucosal immunization is a platform for vaccine delivery against common respiratory pathogens.

TRPV4 is dispensable for the development of airway allergic asthma.

Allergic asthma is one of the most common immune-mediated disorders affecting the lungs. It is characterized clinically by airway hyperresponsiveness, eosinophilia, enhanced IL-4 and IL-13, peribronchial inflammation with mononuclear cell infiltration, and goblet cell hyperplasia associated with increased mucus production. However, chronic asthma with repeated exposures to inhaled allergens can result in subepithelial pulmonary fibrosis. The transient receptor potential cation channel subfamily V member 4 (TRPV4) protein can promote the generation of myofibroblasts and pulmonary fibrosis. Here, we investigated the possibility that TPRV4 facilitates the development of allergic asthma and subsequent pulmonary fibrosis in the lung. To test this, wild-type (WT) and TPRV4 gene knockout (KO) mice were repeatedly sensitized with chicken ovalbumin (OVA) and repeatedly subjected to aerosol challenge with 1% OVA. We found that there were no significant differences in the development of allergic asthma between the WT and TPRV4 KO mice. Both groups of mice exhibited similar levels of airway hyperresponsiveness, IL-13, IL-5, OVA-specific IgE, eosinophilia, mucus-secreting goblet cell hyperplasia, and deposition of collagen fiber, which is a hallmark of the pulmonary fibrosis. Thus, these data suggest that TPRV4 protein is dispensable in the initiation and development of airway asthma and subsequent fibrosis.

Regulatory Macrophages Inhibit Alternative Macrophage Activation and Attenuate Pathology Associated with Fibrosis.

Diversity and plasticity are the hallmarks of macrophages. The two most well-defined macrophage subsets are the classically activated macrophages (CAMϕs) and the IL-4-derived alternatively activated macrophages (AAMϕs). Through a series of studies, we previously identified and characterized a distinct population of macrophages with immunoregulatory functions, collectively termed regulatory macrophages (RMϕs). Although considerable advances have been made in understanding these various macrophage subsets, it is not known whether macrophages of one activation state can influence the other. In this study, we examined whether RMϕs capable of inhibiting inflammatory responses of CAMϕs could also inhibit AAMϕs and their profibrotic responses. Our results demonstrated that RMϕs significantly dampened the alternate activation phenotype of AAMϕs generated in vitro and intrinsically occurring AAMϕs from TACI-/- macrophages. Further, RMϕs inhibited AAMϕ-promoted arginase activity and fibroblast proliferation in vitro. This inhibition occurred regardless of the strength, duration, and mode of alternative activation and was only partially dependent on IL-10. In the chlorhexidine gluconate-induced peritoneal fibrosis model, AAMϕs worsened the fibrosis, but RMϕs rescued mice from AAMϕ-mediated pathological conditions. Taken together, our study demonstrates that RMϕs are a specialized subset of macrophages with a nonredundant role in limiting overt proregenerative functions of AAMϕs, a role distinct from their well-defined role of suppression of inflammatory responses by CAMϕs.

Human cytomegalovirus evades antibody-mediated immunity through endoplasmic reticulum-associated degradation of the FcRn receptor.

Human cytomegalovirus (HCMV) can persistently infect humans, but how HCMV avoids humoral immunity is not clear. The neonatal Fc receptor (FcRn) controls IgG transport from the mother to the fetus and prolongs IgG half-life. Here we show that US11 inhibits the assembly of FcRn with ß2m and retains FcRn in the endoplasmic reticulum (ER), consequently blocking FcRn trafficking to the endosome. Furthermore, US11 recruits the ubiquitin enzymes Derlin-1, TMEM129 and UbE2J2 to engage FcRn, consequently initiating the dislocation of FcRn from the ER to the cytosol and facilitating its degradation. Importantly, US11 inhibits IgG-FcRn binding, resulting in a reduction of IgG transcytosis across intestinal or placental epithelial cells and IgG degradation in endothelial cells. Hence, these results identify the mechanism by which HCMV infection exploits an ER-associated degradation pathway through US11 to disable FcRn functions. These results have implications for vaccine development and immune surveillance.

Evaluation of humoral, mucosal, and cellular immune responses following co-immunization of HIV-1 Gag and Env proteins expressed by Newcastle disease virus.

The combination of multiple HIV antigens in a vaccine can broaden antiviral immune responses. In this study, we used NDV vaccine strain LaSota to generate rNDV (rLaSota/optGag) expressing human codon optimized p55 Gag protein of HIV-1. We examined the effect of co-immunization of rLaSota/optGag with rNDVs expressing different forms of Env protein gp160, gp120, gp140L [a version of gp140 that lacked cytoplasmic tail and contained complete membrane-proximal external region (MPER)] and gp140S (a version of gp140 that lacked cytoplasmic tail and distal half of MPER) on magnitude and breadth of humoral, mucosal and cellular immune responses in guinea pigs and mice. Our results showed that inclusion of rLaSota/optGag with rNDVs expressing different forms of Env HIV Gag did not affect the Env-specific humoral and mucosal immune responses in guinea pigs and that the potent immune responses generated against Env persisted for at least 13 weeks post immunization. The highest Env-specific humoral and mucosal immune responses were observed with gp140S+optGag group. The neutralizing antibody responses against HIV strains BaL.26 and MN.3 induced by gp140S+optGag and gp160+optGag were higher than those elicited by other groups. Inclusion of Gag with gp160, gp140S and gp140L enhanced the level of Env-specific IFN-γ-producing CD8(+) T cells in mice. Inclusion of Gag with gp160 and gp140L also resulted in increased Env-specific CD4(+) T cells. The level of Gag-specific CD8(+) and CD4(+) T cells was also enhanced in mice immunized with Gag along with gp140S and gp120. These results indicate lack of antigen interference in a vaccine containing rNDVs expressing Env and Gag proteins.

Actin-binding protein 1 links B-cell antigen receptors to negative signaling pathways.

Prolonged or uncontrolled B-cell receptor (BCR) signaling is associated with autoimmunity. We previously demonstrated a role for actin in BCR signal attenuation. This study reveals that actin-binding protein 1 (Abp1/HIP-55/SH3P7) is a negative regulator of BCR signaling and links actin to negative regulatory pathways of the BCR. In both Abp1(-/-) and bone marrow chimeric mice, in which only B cells lack Abp1 expression, the number of spontaneous germinal center and marginal zone B cells and the level of autoantibody are significantly increased. Serum levels of T-independent antibody responses and total antibody are elevated, whereas T-dependent antibody responses are markedly reduced and fail to undergo affinity maturation. Upon activation, surface BCR clustering is enhanced and B-cell contraction delayed in Abp1(-/-) B cells, concurrent with slow but persistent increases in F-actin at BCR signalosomes. Furthermore, BCR signaling is enhanced in Abp1(-/-) B cells compared with wild-type B cells, including Ca(2+) flux and phosphorylation of B-cell linker protein, the mitogen-activated protein kinase kinase MEK1/2, and ERK, coinciding with reductions in recruitment of the inhibitory signaling molecules hematopoietic progenitor kinase 1 and SH2-containing inositol 5-phosphatase to BCR signalosomes. Our results indicate that Abp1 negatively regulates BCR signaling by coupling actin remodeling to B-cell contraction and activation of inhibitory signaling molecules, which contributes to the regulation of peripheral B-cell development and antibody responses.

LuxS contributes to virulence in avian pathogenic Escherichia coli O78:K80:H9.

Avian pathogenic Escherichia coli (APEC) cause avian colibacillosis, a poultry disease characterized by multiple organ infections resulting in significant economic loss in the poultry industry. Several virulence factors are important for disease manifestation in APEC of which, role of quorum sensing has not been investigated. Quorum sensing is a population dependent cell-cell signaling system which modulates numerous physiological processes such as biofilm formation and virulence in multiple species. LuxS, a well-known controller in the QS, plays a role in regulating virulence in various bacterial species. Here we investigated the role of LuxS in regulating virulence in APEC O78:K80:H9. Mutation of luxS resulted in a significant reduction of virulence in APEC O78:K80:H9, evidenced by both in vivo and in vitro assays such as decreased invasion of internal organs in chicken embryo, reduced lethality in chicken embryo lethality assay, and altered lipopolysaccharide (LPS) profile. In addition, the abilities of the knockout strain to survive in chicken macrophage cell lines and to invade in chicken embryo fibroblast cells were significantly diminished. Further, structure and expression level of the LPS profile was significantly altered in the knockout strain, which may be one of the contributing factors for the persistence and virulence of APEC. Complementation of luxS gene in trans restored the virulence of the knockout strain to the level of wild-type bacteria. Taken together, these results show that LuxS contributes to the virulence in APEC O78:K80:H9 strain and partly explain the role played by LuxS in the pathogenesis of APEC strains.

Mutations in the cytoplasmic domain of the Newcastle disease virus fusion protein confer hyperfusogenic phenotypes modulating viral replication and pathogenicity.

The Newcastle disease virus (NDV) fusion protein (F) mediates fusion of viral and host cell membranes and is a major determinant of NDV pathogenicity. In the present study, we demonstrate the effects of functional properties of F cytoplasmic tail (CT) amino acids on virus replication and pathogenesis. Out of a series of C-terminal deletions in the CT, we were able to rescue mutant viruses lacking two or four residues (rΔ2 and rΔ4). We further rescued viral mutants with individual amino acid substitutions at each of these four terminal residues (rM553A, rK552A, rT551A, and rT550A). In addition, the NDV F CT has two conserved tyrosine residues (Y524 and Y527) and a dileucine motif (LL536-537). In other paramyxoviruses, these residues were shown to affect fusion activity and are central elements in basolateral targeting. The deletion of 2 and 4 CT amino acids and single tyrosine substitution resulted in hyperfusogenic phenotypes and increased viral replication and pathogenesis. We further found that in rY524A and rY527A viruses, disruption of the targeting signals did not reduce the expression on the apical or basolateral surface in polarized Madin-Darby canine kidney cells, whereas in double tyrosine mutant, it was reduced on both the apical and basolateral surfaces. Interestingly, in rL536A and rL537A mutants, the F protein expression was more on the apical than on the basolateral surface, and this effect was more pronounced in the rL537A mutant. We conclude that these wild-type residues in the NDV F CT have an effect on regulating F protein biological functions and thus modulating viral replication and pathogenesis.

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.

BarA-UvrY two-component system regulates virulence of uropathogenic E. coli CFT073.

Uropathogenic Escherichia coli (UPEC), a member of extraintestinal pathogenic E. coli, cause ∼80% of community-acquired urinary tract infections (UTI) in humans. UPEC initiates its colonization in epithelial cells lining the urinary tract with a complicated life cycle, replicating and persisting in intracellular and extracellular niches. Consequently, UPEC causes cystitis and more severe form of pyelonephritis. To further understand the virulence characteristics of UPEC, we investigated the roles of BarA-UvrY two-component system (TCS) in regulating UPEC virulence. Our results showed that mutation of BarA-UvrY TCS significantly decreased the virulence of UPEC CFT073, as assessed by mouse urinary tract infection, chicken embryo killing assay, and cytotoxicity assay on human kidney and uroepithelial cell lines. Furthermore, mutation of either barA or uvrY gene reduced the production of hemolysin, lipopolysaccharide (LPS), proinflammatory cytokines (TNF-α and IL-6) and chemokine (IL-8). The virulence phenotype was restored similar to that of wild-type by complementation of either barA or uvrY gene in trans. In addition, we discussed a possible link between the BarA-UvrY TCS and CsrA in positively and negatively controlling virulence in UPEC. Overall, this study provides the evidences for BarA-UvrY TCS regulates the virulence of UPEC CFT073 and may point to mechanisms by which virulence regulations are observed in different ways may control the long-term survival of UPEC in the urinary tract.

A neonatal Fc receptor-targeted mucosal vaccine strategy effectively induces HIV-1 antigen-specific immunity to genital infection.

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.

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.

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 (Ms) 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 Ms 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 Ms and DCs. The presentation of phagocytosed OVA-ICs to CD4(+) T cells was considerably enhanced on wild-type versus FcRn-deficient Ms, 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 Ms 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 Ms versus DCs.

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.