Structural and Biochemical Requirements for Secretory Component Interactions with Dimeric IgA.

Secretory (S) IgA is the predominant mucosal Ab that protects host epithelial barriers and promotes microbial homeostasis. SIgA production occurs when plasma cells assemble two copies of monomeric IgA and one joining chain (JC) to form dimeric (d) IgA, which is bound by the polymeric Ig receptor (pIgR) on the basolateral surface of epithelial cells and transcytosed to the apical surface. There, pIgR is proteolytically cleaved, releasing SIgA, a complex of the dIgA and the pIgR ectodomain, called the secretory component (SC). The pIgR's five Ig-like domains (D1-D5) undergo a conformational change upon binding dIgA, ultimately contacting four IgA H chains and the JC in SIgA. In this study, we report structure-based mutational analysis combined with surface plasmon resonance binding assays that identify key residues in mouse SC D1 and D3 that mediate SC binding to dIgA. Residues in D1 CDR3 are likely to initiate binding, whereas residues that stabilize the D1-D3 interface are likely to promote the conformational change and stabilize the final SIgA structure. Additionally, we find that the JC's three C-terminal residues play a limited role in dIgA assembly but a significant role in pIgR/SC binding to dIgA. Together, these results inform models for the intricate mechanisms underlying IgA transport across epithelia and functions in the mucosa.

BTB and CNC homology 1 deficiency disrupts intestinal IgA secretion through regulation of polymeric immunoglobulin receptor expression.

Immunoglobulin A (IgA)-mediated mucosal immunity is important for the host because it contributes to reducing infection risk and to establishing host-microbe symbiosis. BTB and CNC homology 1 (Bach1) is a transcriptional repressor with physiological and pathophysiological functions that are of particular interest for their relation to gastrointestinal diseases. However, Bach1 effects on IgA-mediated mucosal immunity remain unknown. For this study using Bach1-deficient (Bach1-/-) mice, we investigated the function of Bach1 in IgA-mediated mucosal immunity. Intestinal mucosa, feces, and plasma IgA were examined using immunosorbent assay. After cell suspensions were prepared from Peyer's patches and colonic lamina propria, they were examined using flow cytometry. The expression level of polymeric immunoglobulin receptor (pIgR), which plays an important role in the transepithelial transport of IgA, was evaluated using Western blotting, quantitative real-time PCR, and immunohistochemistry. Although no changes in the proportions of IgA-producing cells were observed, the amounts of IgA in the intestinal mucosa were increased in Bach1-/- mice. Furthermore, plasma IgA was increased in Bach1-/- mice, but fecal IgA was decreased, indicating that Bach1-/- mice have abnormal secretion of IgA into the intestinal lumen. In fact, Bach1 deficiency reduced pIgR expression in colonic mucosa at both the protein and mRNA levels. In the human intestinal epithelial cell line LS174T, suppression of Bach1 reduced pIgR mRNA stability. In contrast, the overexpression of Bach1 increased pIgR mRNA stability. These results demonstrate that Bach1 deficiency causes abnormal secretion of IgA into the intestinal lumen via suppression of pIgR expression.NEW & NOTEWORTHY The transcriptional repressor Bach1 has been implicated in diverse intestinal functions, but the effects of Bach1 on IgA-mediated mucosal immunity remain unclear. We demonstrate here that Bach1 deficiency causes abnormal secretion of IgA into the intestinal lumen, although the proportions of IgA-producing cells were not altered. Furthermore, Bach1 regulates the expression of pIgR, which plays an important role in the transepithelial transport of IgA, at the posttranscriptional level.

White spot syndrome virus directly activates mTORC1 signaling to facilitate its replication via polymeric immunoglobulin receptor-mediated infection in shrimp.

Previous studies have shown that the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway has antiviral functions or is beneficial for viral replication, however, the detail mechanisms by which mTORC1 enhances viral infection remain unclear. Here, we found that proliferation of white spot syndrome virus (WSSV) was decreased after knockdown of mTor (mechanistic target of rapamycin) or injection inhibitor of mTORC1, rapamycin, in Marsupenaeus japonicus, which suggests that mTORC1 is utilized by WSSV for its replication in shrimp. Mechanistically, WSSV infects shrimp by binding to its receptor, polymeric immunoglobulin receptor (pIgR), and induces the interaction of its intracellular domain with Calmodulin. Calmodulin then promotes the activation of protein kinase B (AKT) by interaction with the pleckstrin homology (PH) domain of AKT. Activated AKT phosphorylates mTOR and results in the activation of the mTORC1 signaling pathway to promote its downstream effectors, ribosomal protein S6 kinase (S6Ks), for viral protein translation. Moreover, mTORC1 also phosphorylates eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), which will result in the separation of 4EBP1 from eukaryotic translation initiation factor 4E (eIF4E) for the translation of viral proteins in shrimp. Our data revealed a novel pathway for WSSV proliferation in shrimp and indicated that mTORC1 may represent a potential clinical target for WSSV control in shrimp aquaculture.

Hepatic pIgR-mediated secretion of IgA limits bacterial translocation and prevents ethanol-induced liver disease in mice.

OBJECTIVE: Alcohol-associated liver disease is accompanied by microbial dysbiosis, increased intestinal permeability and hepatic exposure to translocated microbial products that contribute to disease progression. A key strategy to generate immune protection against invading pathogens is the secretion of IgA in the gut. Intestinal IgA levels depend on the polymeric immunoglobulin receptor (pIgR), which transports IgA across the epithelial barrier into the intestinal lumen and hepatic canaliculi. Here, we aimed to address the function of pIgR during ethanol-induced liver disease. DESIGN: pIgR and IgA were assessed in livers from patients with alcohol-associated hepatitis and controls. Wild-type and pIgR-deficient (pIgR-/- ) littermates were subjected to the chronic-binge (NIAAA model) and Lieber-DeCarli feeding model for 8 weeks. Hepatic pIgR re-expression was established in pIgR-/- mice using adeno-associated virus serotype 8 (AAV8)-mediated pIgR expression in hepatocytes. RESULTS: Livers of patients with alcohol-associated hepatitis demonstrated an increased colocalisation of pIgR and IgA within canaliculi and apical poles of hepatocytes. pIgR-deficient mice developed increased liver injury, steatosis and inflammation after ethanol feeding compared with wild-type littermates. Furthermore, mice lacking pIgR demonstrated increased plasma lipopolysaccharide levels and more hepatic bacteria, indicating elevated bacterial translocation. Treatment with non-absorbable antibiotics prevented ethanol-induced liver disease in pIgR-/- mice. Injection of AAV8 expressing pIgR into pIgR-/- mice prior to ethanol feeding increased intestinal IgA levels and ameliorated ethanol-induced steatohepatitis compared with pIgR-/- mice injected with control-AAV8 by reducing bacterial translocation. CONCLUSION: Our results highlight that dysfunctional hepatic pIgR enhances alcohol-associated liver disease due to impaired antimicrobial defence by IgA in the gut.

Secretory Cells Are the Primary Source of pIgR in Small Airways.

Loss of secretory IgA (SIgA) is common in chronic obstructive pulmonary disease (COPD) small airways and likely contributes to disease progression. We hypothesized that loss of SIgA results from reduced expression of pIgR (polymeric immunoglobulin receptor), a chaperone protein needed for SIgA transcytosis, in the COPD small airway epithelium. pIgR-expressing cells were defined and quantified at single-cell resolution in human airways using RNA in situ hybridization, immunostaining, and single-cell RNA sequencing. Complementary studies in mice used immunostaining, primary murine tracheal epithelial cell culture, and transgenic mice with secretory or ciliated cell-specific knockout of pIgR. SIgA degradation by human neutrophil elastase or secreted bacterial proteases from nontypeable Haemophilus influenzae was evaluated in vitro. We found that secretory cells are the predominant cell type responsible for pIgR expression in human and murine airways. Loss of SIgA in small airways was not associated with a reduction in secretory cells but rather a reduction in pIgR protein expression despite intact PIGR mRNA expression. Neutrophil elastase and nontypeable H. influenzae-secreted proteases are both capable of degrading SIgA in vitro and may also contribute to a deficient SIgA immunobarrier in COPD. Loss of the SIgA immunobarrier in small airways of patients with severe COPD is complex and likely results from both pIgR-dependent defects in IgA transcytosis and SIgA degradation.

Patient pIgR-enriched extracellular vesicles drive cancer stemness, tumorigenesis and metastasis in hepatocellular carcinoma.

BACKGROUND & AIMS: Extracellular vesicles (EVs) play a pivotal role in connecting tumor cells with their local and distant microenvironments. Herein, we aimed to understand the role (on a molecular basis) patient-derived EVs play in modulating cancer stemness and tumorigenesis in the context of hepatocellular carcinoma (HCC). METHODS: EVs from patient sera were isolated, quantified and characterized. The EVs were vigorously tested, both in vitro and in vivo, through various functional assays. Proteomic analysis was performed to identify the functional components of EVs. The presence and level of polymeric immunoglobulin receptor (pIgR) in circulating EVs and tumor and non-tumorous tissues of patients with HCC were determined by ELISA, immunoblotting, immunohistochemistry and quantitative PCR. The functional role and underlying mechanism of EVs with enhanced pIgR expression were elucidated. Blockade of EV-pIgR with neutralizing antibody was performed in nude mice implanted with patient-derived tumor xenografts (PDTXs). RESULTS: Circulating EVs from patients with late-stage HCC (L-HCC) had significantly elevated pIgR expression compared to the EVs released by control individuals. The augmenting effect of L-HCC-EVs on cancer stemness and tumorigenesis was hindered by an anti-pIgR antibody. EVs enriched with pIgR consistently promoted cancer stemness and cancerous phenotypes in recipient cells. Mechanistically, EV-pIgR-induced cancer aggressiveness was abrogated by Akt and ß-catenin inhibitors, confirming that the role of EV-pIgR depends on the activation of the PDK1/Akt/GSK3ß/ß-catenin signaling axis. Furthermore, an anti-pIgR neutralizing antibody attenuated tumor growth in mice implanted with PDTXs. CONCLUSIONS: This study illustrates a previously unknown role of EV-pIgR in regulating cancer stemness and aggressiveness: EV-pIgR activates PDK1/Akt/GSK3ß/ß-catenin signaling cascades. The blockade of the intercellular communication mediated by EV-pIgR in the tumor microenvironment may provide a new therapeutic strategy for patients with cancer. LAY SUMMARY: The World Health Organization estimates that more than 1 million patients will die from liver cancer, mostly hepatocellular carcinoma (HCC), in 2030. Understanding the underlying mechanism by which HCC acquires aggressive attributes is crucial to improving the diagnosis and treatment of patients. Herein, we demonstrated that nanometer-sized extracellular vesicles released by tumors promote cancer stemness and tumorigenesis. Within these oncogenic vesicles, we identified a key component that functions as a potent modulator of cancer aggressiveness. By inhibiting this functional component of EVs using a neutralizing antibody, tumor growth was profoundly attenuated in mice. This hints at a potentially effective therapeutic alternative for patients with cancer.

Malondialdehyde-Acetaldehyde Adduct Formation Decreases Immunoglobulin A Transport across Airway Epithelium in Smokers Who Abuse Alcohol.

Alcohol misuse and smoking are risk factors for pneumonia, yet the impact of combined cigarette smoke and alcohol on pneumonia remains understudied. Smokers who misuse alcohol form lung malondialdehyde-acetaldehyde (MAA) protein adducts and have decreased levels of anti-MAA secretory IgA (sIgA). Transforming growth factor-ß (TGF-ß) down-regulates polymeric Ig receptor (pIgR) on mucosal epithelium, resulting in decreased sIgA transcytosis to the mucosa. It is hypothesized that MAA-adducted lung protein increases TGF-ß, preventing expression of epithelial cell pIgR and decreasing sIgA. Cigarette smoke and alcohol co-exposure on sIgA and TGF-ß in human bronchoalveolar lavage fluid and in mice instilled with MAA-adducted surfactant protein D (SPD-MAA) were studied herein. Human bronchial epithelial cells (HBECs) and mouse tracheal epithelial cells were treated with SPD-MAA and sIgA and TGF-ß was measured. Decreased sIgA and increased TGF-ß were observed in bronchoalveolar lavage from combined alcohol and smoking groups in humans and mice. CD204 (MAA receptor) knockout mice showed no changes in sIgA. SPD-MAA decreased pIgR in HBECs. Conversely, SPD-MAA stimulated TGF-ß release in both HBECs and mouse tracheal epithelial cells, but not in CD204 knockout mice. SPD-MAA stimulated TGF-ß in alveolar macrophage cells. These data show that MAA-adducted surfactant protein stimulates lung epithelial cell TGF-ß, down-regulates pIgR, and decreases sIgA transcytosis. These data provide a mechanism for the decreased levels of sIgA observed in smokers who misuse alcohol.

Exogenous and Endogenous Triggers Differentially Stimulate Pigr Expression and Antibacterial Secretory Immunity in the Murine Respiratory Tract.

PURPOSE: Transport of secretory immunoglobulin A (SIgA) through the airway epithelial cell barrier into the mucosal lumen by the polymeric immunoglobulin receptor (pIgR) is an important mechanism of respiratory mucosal host defense. Identification of immunomodulating substances that regulate secretory immunity might have therapeutic implications with regard to an improved immune exclusion. Thus, we sought to analyze secretory immunity under homeostatic and immunomodulating conditions in different compartments of the murine upper and lower respiratory tract (URT&LRT). METHODS: Pigr gene expression in lung, trachea, and nasal-associated lymphoid tissue (NALT) of germ-free mice, specific pathogen-free mice, mice with an undefined microbiome, as well as LPS- and IFN-γ-treated mice was determined by quantitative real-time PCR. IgA levels in bronchoalveolar lavage (BAL), nasal lavage (NAL), and serum were determined by ELISA. LPS- and IFN-γ-treated mice were colonized with Streptococcus pneumoniae and bacterial CFUs were determined in URT and LRT. RESULTS: Respiratory Pigr expression and IgA levels were dependent on the degree of exposure to environmental microbial stimuli. While immunostimulation with LPS and IFN-γ differentially impacts respiratory Pigr expression and IgA in URT vs. LRT, only prophylactic IFN-γ treatment reduces nasal colonization with S. pneumoniae. CONCLUSION: Airway-associated secretory immunity can be partly modulated by exposure to microbial ligands and proinflammatory stimuli. Prophylactic IFN-γ-treatment modestly improves antibacterial immunity in the URT, but this does not appear to be mediated by SIgA or pIgR.

The polymeric immunoglobulin receptor-like protein from Marsupenaeus japonicus is a receptor for white spot syndrome virus infection.

Viral entry into the host cell is the first step towards successful infection. Viral entry starts with virion attachment, and binding to receptors. Receptor binding viruses either directly release their genome into the cell, or enter cells through endocytosis. For DNA viruses and a few RNA viruses, the endocytosed viruses will transport from cytoplasm into the nucleus followed by gene expression. Receptors on the cell membrane play a crucial role in viral infection. Although several attachment factors, or candidate receptors, for the infection of white spot syndrome virus (WSSV) were identified in shrimp, the authentic entry receptors for WSSV infection and the intracellular signaling triggering by interaction of WSSV with receptors remain unclear. In the present study, a receptor for WSSV infection in kuruma shrimp, Marsupenaeus japonicus, was identified. It is a member of the immunoglobulin superfamily (IgSF) with a transmembrane region, and is similar to the vertebrate polymeric immunoglobulin receptor (pIgR); therefore, it was designated as a pIgR-like protein (MjpIgR for short). MjpIgR was detected in all tissues tested, and its expression was significantly induced by WSSV infection at the mRNA and protein levels. Knockdown of MjpIgR, and blocking MjpIgR with its antibody inhibited WSSV infection in shrimp and overexpression of MjpIgR facilitated the invasion of WSSV. Further analyses indicated that MjpIgR could independently render non-permissive cells susceptible to WSSV infection. The extracellular domain of MjpIgR interacts with envelope protein VP24 of WSSV and the intracellular domain interacts with calmodulin (MjCaM). MjpIgR was oligomerized and internalized following WSSV infection and the internalization was associated with endocytosis of WSSV. The viral internalization facilitating ability of MjpIgR could be blocked using chlorpromazine, an inhibitor of clathrin dependent endocytosis. Knockdown of Mjclathrin and its adaptor protein AP-2 also inhibited WSSV internalization. All the results indicated that MjpIgR-mediated WSSV endocytosis was clathrin dependent. The results suggested that MjpIgR is a WSSV receptor, and that WSSV enters shrimp cells via the pIgR-CaM-Clathrin endocytosis pathway.

Role of Polymeric Immunoglobulin Receptor in IgA and IgM Transcytosis.

Transcytosis of polymeric IgA and IgM from the basolateral surface to the apical side of the epithelium and subsequent secretion into mucosal fluids are mediated by the polymeric immunoglobulin receptor (pIgR). Secreted IgA and IgM have vital roles in mucosal immunity in response to pathogenic infections. Binding and recognition of polymeric IgA and IgM by pIgR require the joining chain (J chain), a small protein essential in the formation and stabilization of polymeric Ig structures. Recent studies have identified marginal zone B and B1 cell-specific protein (MZB1) as a novel regulator of polymeric IgA and IgM formation. MZB1 might facilitate IgA and IgM transcytosis by promoting the binding of J chain to Ig. In this review, we discuss the roles of pIgR in transcytosis of IgA and IgM, the roles of J chain in the formation of polymeric IgA and IgM and recognition by pIgR, and focus particularly on recent progress in understanding the roles of MZB1, a molecular chaperone protein.

Rab11-FIP1 and Rab11-FIP5 Regulate pIgR/pIgA Transcytosis through TRIM21-Mediated Polyubiquitination.

Polymeric immunoglobulin receptor (pIgR)-mediated polymeric immunoglobulin A (pIgA) transcytosis across mucosal epithelial cells plays an essential role in mucosal immunity. The general trafficking process has been well investigated, yet the elaborate regulatory mechanisms remain enigmatic. We identified a new pIgR interacting protein, the Rab11 effector Rab11-FIP1. Rab11-FIP1 and Rab11-FIP5 knockdown additively impaired pIgA transcytosis in both polarized and incompletely polarized cells. Moreover, Rab11-FIP1 and Rab11-FIP5 knockdown exhibited more significant inhibitory effects on pIgA transcytosis in incompletely polarized cells than in polarized cells. Interestingly, the trafficking process of pIgA in incompletely polarized cells is distinct from that in polarized cells. In incompletely polarized cells, the endocytic pIgR/pIgA was first transported from the basolateral plasma membrane to the vicinity of the centrosome where Rab11-FIP1 and Rab11-FIP5 bound to it, before the Rab11a-positive endosomes containing pIgR/pIgA, Rab11-FIP1 and Rab11-FIP5 were further transported to the apical plasma membrane via Golgi apparatus. During the trafficking process, TRIM21 mediated the K11-linked polyubiquitination of Rab11-FIP1 and the K6-linked polyubiquitination of Rab11-FIP5 to promote their activation and pIgA transcytosis. This study indicates that polyubiquitinated Rab11-FIP1 and Rab11-FIP5 mediated by TRIM21 cooperatively facilitate pIgA transcytosis and provides new insights into the intracellular trafficking process of pIgA in incompletely polarized cells.

Localization and Regulation of Polymeric Ig Receptor in Healthy and Diseased Human Kidney.

The polymeric Ig receptor (PIgR) constitutes an important part of the immune system by mediating transcytosis of dimeric IgA into mucosal fluids. Although well studied in organs such as the intestine, the regulation and localization of PIgR in human kidney are incompletely characterized. Herein, using immunohistochemistry, we show that in healthy human kidneys, PIgR is expressed by the progenitor-like tubular scattered cells of the proximal tubules and by parietal epithelial cells of glomeruli. We further show that proximal tubular expression of PIgR becomes widespread during kidney disease, correlating to elevated levels of urinary secretory IgA. Urinary secretory IgA levels also correlated to the degree of tubular fibrosis, plasma creatinine, and urea levels. In addition, primary tubular cells were cultured to study the function and regulation of PIgR in vitro. Cellular PIgR expression was induced by conditioned medium from activated human leukocytes, as well as by inflammatory cytokines, whereas transforming growth factor-ß1 caused decreased expression. Furthermore, interferon-γ increased the transcytosis of dimeric IgA in cultured tubular cells. Finally, a correlation study of mRNA data from the Genotype-Tissue Expression portal indicated that PIGR mRNA expression in kidney correlates to the expression of TNFSF13, a cytokine involved in plasma cell class switching to IgA. These results indicate that PIgR induction is an integral part of the injury phenotype of renal tubular cells.

Alterations in Intestinal Innate Mucosal Immunity of Weaned Pigs During Porcine Epidemic Diarrhea Virus Infection.

In the small intestine, localized innate mucosal immunity is critical for intestinal homeostasis. Porcine epidemic diarrhea virus (PEDV) infection induces villus injury and impairs digestive function. Moreover, the infection might comprise localized innate mucosal immunity. This study investigated specific enterocyte subtypes and innate immune components of weaned pigs during PEDV infection. Four-week-old pigs were orally inoculated with PEDV IN19338 strain (n = 40) or sham-inoculated (n = 24). At day post inoculation (DPI) 2, 4, and 6, lysozyme expression in Paneth cells, cellular density of villous and Peyer's patch microfold (M) cells, and the expression of polymeric immunoglobulin receptor (pIgR) were assessed in the jejunum and ileum by immunohistochemistry, and interleukin (IL)-1ß and tumor necrosis factor (TNF)-α were measured in the jejunum by ELISA. PEDV infection led to a decrease in the ratios of villus height to crypt depth (VH-CD) in jejunum at DPI 2, 4, and 6 and in ileum at DPI 4. The number of villous M cells was reduced in jejunum at DPI 4 and 6 and in ileum at DPI 6, while the number of Peyer's patch M cells in ileum increased at DPI 2 and then decreased at DPI 6. PEDV-infected pigs also had reduced lysozyme expression in ileal Paneth cells at DPI 2 and increased ileal pIgR expression at DPI 4. There were no significant changes in IL-1ß and TNF-α expression in PEDV-infected pigs compared to controls. In conclusion, PEDV infection affected innate mucosal immunity of weaned pigs through alterations in Paneth cells, villous and Peyer's patch M cells, and pIgR expression.

Polymeric immunoglobulin receptor promotes tumor growth in hepatocellular carcinoma.

Deregulation of the immune system is believed to contribute to cancer malignancy, which has led to recent therapeutic breakthroughs facilitating antitumor immunity. In a malignant setting, immunoglobulin receptors, which are fundamental components of the human immune system, fulfill paradoxical roles in cancer pathogenesis. This study describes a previously unrecognized pro-oncogenic function of polymeric immunoglobulin receptor (pIgR) in the promotion of cell transformation and proliferation. Mechanistically, pIgR overexpression is associated with YES proto-oncogene 1, Src family tyrosine kinase (Yes) activation, which is required for pIgR-induced oncogenic growth. Specifically, pIgR activates the Yes-DNAX-activating protein of 12 kDa-spleen tyrosine kinase-Rac1/CDC42-MEK (extracellular signal-regulated kinase kinase)/ERK (extracellular signal-regulated kinase) cascade in an immunoreceptor tyrosine-based activating motif (ITAM)-dependent manner to promote cell transformation and tumor growth, although pIgR itself does not contain an ITAM sequence. Additionally, the combination of pIgR and phosphorylated Yes (p-Yes) levels serves as a prognostic biomarker for hepatitis B surface antigen-positive and early-stage hepatocellular carcinoma (HCC) patients. Moreover, pharmacological targeting of MEK/ERK or Yes represents a therapeutic option for the subgroup of patients with pIgR/p-Yes-positive HCC based on our results with both cancer cell-line-based xenografts and primary patient-derived xenografts. CONCLUSION: Our findings reveal the molecular mechanism by which pIgR promotes cancer malignancy, suggest the clinical potential of targeting this pathway in HCC, and provide new insight into the oncogenic role of immunoglobulin receptors. (Hepatology 2017;65:1948-1962).

The Appearance and Diversification of Receptors for IgM During Vertebrate Evolution.

Three different receptors that interact with the constant domains of IgM have been identified: the polymeric immunoglobulin (Ig) receptor (PIGR), the dual receptor for IgA/IgM (FcαµR) and the IgM receptor (FcµR). All of them are related in structure and located in the same chromosomal region in mammals. The functions of the PIGRs are to transport IgM and IgA into the intestinal lumen and to saliva and tears, whereas the FcαµRs enhance uptake of immune complexes and antibody coated bacteria and viruses by B220+ B cells and phagocytes, as well as dampening the Ig response to thymus-independent antigens. The FcµRs have broad-spectrum effects on B-cell development including effects on IgM homeostasis, B-cell survival, humoral immune responses and also in autoantibody formation. The PIGR is the first of these receptors to appear during vertebrate evolution and is found in bony fish and all tetrapods but not in cartilaginous fish. The FcµR is present in all extant mammalian lineages and also in the Chinese and American alligators, suggesting its appearance with early reptiles. Currently the FcαµR has only been found in mammals and is most likely the evolutionary youngest of the three receptors. In bony fish, the PIGR has either 2, 3, 4, 5 or 6 extracellular Ig-like domains, whereas in amphibians, reptiles and birds it has 4 domains, and 5 in all mammals. The increase in domain number from 4 to 5 in mammals has been proposed to enhance the interaction with IgA. Both the FcαµRs and the FcµRs contain only one Ig domain; the domain that confers Ig binding. In both of these receptors this domain shows the highest degree of sequence similarity to domain 1 of the PIGR. All Ig domains of these three receptors are V type domains, indicating they all have the same origin although they have diversified extensively in function during vertebrate evolution by changing expression patterns and cytoplasmic signaling motifs.

Fructo-oligosaccharide-Induced Transient Increases in Cecal Immunoglobulin A Concentrations in Rats Are Associated with Mucosal Inflammation in Response to Increased Gut Permeability.

Background: The mechanism underlying transient increases in immunoglobulin (Ig) A concentrations in the cecal contents of rats fed fructo-oligosaccharide (FOS) is unclear.Objective: This study was designed to test whether increased IgA concentrations represent one aspect of the inflammatory response to increased permeability induced by FOS in the cecum.Methods: Seven-week-old male Wistar rats were fed a fiber-free semipurified diet (FFP) with or without supplemental FOS (60 g/kg diet) for 9 or 58 d [experiment (expt.) 1], 7 d (expt. 2), or 7 or 56 d (expt. 3). In addition to measuring IgA concentrations in cecal content, we assessed gut permeability, inflammatory responses (expt. 1), the number of IgA plasma cells in the cecal lamina propria, polymeric Ig receptor (pIgR) expression in the cecal mucosa (expt. 2), and the condition of the cecal mucus layer (expt. 3).Results: The cecal IgA concentration in the FOS-fed rats was 15-fold higher than that of the rats fed FFP for 9 d (P < 0.05). Gut permeability estimated by urinary chromium-EDTA excretion, bacterial translocation to mesenteric lymph nodes, myeloperoxidase activity, and expression of inflammatory cytokine genes in the cecal mucosa was greater in the FOS-fed rats than in the rats fed FFP for 9 d. These effects were not observed in the rats fed FOS for 58 d (expt. 1). Accompanying the higher cecal IgA concentration, pIgR protein and the number of IgA plasma cells in the cecal mucosa were higher in the FOS-fed rats than in the rats fed FFP for 7 d (expt. 2). Destruction of the mucus layer on the epithelial surface, as evidenced by Alcian blue staining in the cecal sections, was evident in the rats fed FOS for 7 d, but the mucus layer appeared normal in the rats fed FOS for 56 d (expt. 3).Conclusions: These findings suggest that transient increases in cecal IgA concentrations induced by FOS in rats are associated with mucosal inflammation in response to increased gut permeability; these are presumably evoked by disruption of the cecal mucus barrier. The observed responses could contribute to the maturation of the gut immune system.

Expression of polymeric immunoglobulin receptor and stromal activity in pancreatic ductal adenocarcinoma.

BACKGROUND/OBJECTIVES: Polymeric immunoglobulin receptor (pIgR) traffics Immunoglobulins (IgA and IgM) through epithelial cells in normal mucosae but neither are expressed in the normal pancreas. Recent work from our laboratory suggested pIgR may be upregulated in pancreatic ductal adenocarcinoma (PDAC). Our aim was to assess the role of pIgR in human PDAC. METHODS: pIgR expression was manipulated (siRNA and shRNA) in cell lines to evaluate its subsequent effect on cell behaviour in 2D assays as well as 3D organotypics models. Tissue Microarrays of 88 patients with PDAC were analysed after pIgR, αSMA, E-Cadherin and Picrosirius Red staining to assess their role as a combined bio-marker panel. RESULTS: Cytokines such as interleukin 4 (IL4) and Tumour Necrosis Factor (TNFα) could not modulate pIgR expression in PDAC cell lines despite this effect being seen in other studies. Down-regulation in pIgR expression in Capan1 cancer cell line resulted in reduction of cellular proliferation, adhesion and migration in 2D assays. In 3D physiomimetic organotypic models, pIgR downregulation resulted in reduced cancer cell invasion, alteration of apico-basal polarity and diminished stromal activity. In human PDAC, decreased E-cadherin expression correlates with increased pIgR expression through pancreatic intra-epithelial neoplasia (PanIN) progression. In combination with enhanced stromal indices (α-smooth muscle action (SMA) and Picrosirius red), low pIgR scores had a trend towards better survival. CONCLUSION: pIgR may be involved in PDAC progression and may be linked stromal activity. Further work on its precise role is mandated in in vivo models, to understand its influence on cancer progression.

Identification of sea bass pIgR shows its interaction with vitellogenin inducing antibody-like activities in HEK 293T cells.

Fish vitellogenin (Vg) has been shown to mediate the phagocytosis via interaction with a Fcγ-like phagocytic receptor on macrophages, but identification of such a receptor and its functional characterization remains lacking. In this study, we isolated a cDNA of polymeric immunoglobulin receptor (pIgR) from sea bass, which encoded a single-spanning transmembrane protein of 326 amino acids including a 21-amino acid signal peptide, an extracellular region, a transmembrane region and a 36-amino acid intracellular region included two Ig-like domains (ILDs), and was expressed in multiple lymphoid organs. We then showed that recombinant extracellular domain of sea bass pIgR was capable of binding to Vg as well as IgG and IgM. We also showed that Vg as well as IgG and IgM interacted with pIgR-expressing HEK 293T cells. Importantly, we demonstrated that Vg as well as IgG and IgM were all capable of enhancing phagocytosis by HEK 293T cells and inducing expression of tnf-α and il-1ß, via interacting with pIgR. Collectively, these results suggest that fish Vg, analogous to IgG and IgM, can interact with pIgR and result in similar down-stream immune responses, providing an additional evidence that Vg plays an antibody-like role.

Role of Netrin-1 Signaling in Nerve Regeneration.

Netrin-1 was the first axon guidance molecule to be discovered in vertebrates and has a strong chemotropic function for axonal guidance, cell migration, morphogenesis and angiogenesis. It is a secreted axon guidance cue that can trigger attraction by binding to its canonical receptors Deleted in Colorectal Cancer (DCC) and Neogenin or repulsion through binding the DCC/Uncoordinated (Unc5) A-D receptor complex. The crystal structures of Netrin-1/receptor complexes have recently been revealed. These studies have provided a structure based explanation of Netrin-1 bi-functionality. Netrin-1 and its receptor are continuously expressed in the adult nervous system and are differentially regulated after nerve injury. In the adult spinal cord and optic nerve, Netrin-1 has been considered as an inhibitor that contributes to axon regeneration failure after injury. In the peripheral nervous system, Netrin-1 receptors are expressed in Schwann cells, the cell bodies of sensory neurons and the axons of both motor and sensory neurons. Netrin-1 is expressed in Schwann cells and its expression is up-regulated after peripheral nerve transection injury. Recent studies indicated that Netrin-1 plays a positive role in promoting peripheral nerve regeneration, Schwann cell proliferation and migration. Targeting of the Netrin-1 signaling pathway could develop novel therapeutic strategies to promote peripheral nerve regeneration and functional recovery.

Effect of Bovine Lactoferrin Treatment Followed by Acute Stress on the IgA-Response in Small Intestine of BALB/c Mice.

Secretory IgA (SIgA) has a pivotal role in gut homeostasis, which can be disturbed by stress. SIgA is formed by IgA-dimers (associated by the J-chain) and the secretory component, a protein derivative of polymeric immunoglobulin receptor (pIgR). Given the gut immuno-modulatory properties of bovine lactoferrin (bLf), the aim of this study was to compare, after bLf treatment followed by acute stress, the IgA response and IgA-associated parameters in proximal versus distal small intestine. Male BALB/c mice (n = 6) were orally treated with bLf (50, 500, and 5000 µg) for 7 days, then stressed by immobilization for 1 h, and sacrificed. In proximal and distal segments, levels were determined of IgA in gut secretions (enzyme-linked immunosorbent assay [ELISA]), the α-/J-chain and pIgR proteins in epithelial cells (Western-blot), and mRNA expression of the α-/J-chain, pIgR, and interleukins (ILs) in mucosa (RT-PCR). Data were compared by two-way analysis of variance (ANOVA) (significance at P < 0.05). Under acute stress, bLf triggered higher levels of IgA, SIgA, and anti-bLf-IgA as well as greater mRNA expression of pIgR, IL-4, and IL-6 (500 µg) in proximal intestine, while inducing higher levels of the total IgA, α-/J-chain, and pIgR proteins as well as greater mRNA expression of the α-chain and IL-4 (5000 µg) in distal intestine. Compared to unstressed/bLf-untreated mice, plasma corticosterone (a stress biomarker, measured by ELISA) increased in stressed/bLf-treated (0, 50 and 500 µg) and unstressed/bLf-treated (5000 µg) mice. The interplay of corticosterone with gut neuroendocrine factors may have elicited signals creating anti-inflammatory conditions for an IgA-response profile in each intestinal region, according to the bLf concentration administered.