Cytosolic PCNA interacts with p47phox and controls NADPH oxidase NOX2 activation in neutrophils.

Neutrophils produce high levels of reactive oxygen species (ROS) by NADPH oxidase that are crucial for host defense but can lead to tissue injury when produced in excess. We previously described that proliferating cell nuclear antigen (PCNA), a nuclear scaffolding protein pivotal in DNA synthesis, controls neutrophil survival through its cytosolic association with procaspases. We herein showed that PCNA associated with p47phox, a key subunit of NADPH oxidase, and that this association regulated ROS production. Surface plasmon resonance and crystallography techniques demonstrated that the interdomain-connecting loop of PCNA interacted directly with the phox homology (PX) domain of the p47phox. PCNA inhibition by competing peptides or by T2AA, a small-molecule PCNA inhibitor, decreased NADPH oxidase activation in vitro. Furthermore, T2AA provided a therapeutic benefit in mice during trinitro-benzene-sulfonic acid (TNBS)-induced colitis by decreasing oxidative stress, accelerating mucosal repair, and promoting the resolution of inflammation. Our data suggest that targeting PCNA in inflammatory neutrophils holds promise as a multifaceted antiinflammatory strategy.

Transgenic Mice Expressing Human Proteinase 3 Exhibit Sustained Neutrophil-Associated Peritonitis.

Proteinase 3 (PR3) is a myeloid serine protease expressed in neutrophils, monocytes, and macrophages. PR3 has a number of well-characterized proinflammatory functions, including cleaving and activating chemokines and controlling cell survival and proliferation. When presented on the surface of apoptotic neutrophils, PR3 can disrupt the normal anti-inflammatory reprogramming of macrophages following the phagocytosis of apoptotic cells. To better understand the function of PR3 in vivo, we generated a human PR3 transgenic mouse (hPR3Tg). During zymosan-induced peritonitis, hPR3Tg displayed an increased accumulation of neutrophils within the peritoneal cavity compared with wild-type control mice, with no difference in the recruitment of macrophages or B or T lymphocytes. Mice were also subjected to cecum ligation and puncture, a model used to induce peritoneal inflammation through infection. hPR3Tg displayed decreased survival rates in acute sepsis, associated with increased neutrophil extravasation. The decreased survival and increased neutrophil accumulation were associated with the cleavage of annexin A1, a powerful anti-inflammatory protein known to facilitate the resolution of inflammation. Additionally, neutrophils from hPR3Tg displayed enhanced survival during apoptosis compared with controls, and this may also contribute to the increased accumulation observed during the later stages of inflammation. Taken together, our data suggest that human PR3 plays a proinflammatory role during acute inflammatory responses by affecting neutrophil accumulation, survival, and the resolution of inflammation.

Cytoplasmic proliferating cell nuclear antigen connects glycolysis and cell survival in acute myeloid leukemia.

Cytosolic proliferating cell nuclear antigen (PCNA), a scaffolding protein involved in DNA replication, has been described as a key element in survival of mature neutrophil granulocytes, which are non-proliferating cells. Herein, we demonstrated an active export of PCNA involved in cell survival and chemotherapy resistance. Notably, daunorubicin-resistant HL-60 cells (HL-60R) have a prominent cytosolic PCNA localization due to increased nuclear export compared to daunorubicin-sensitive HL-60 cells (HL-60S). By interacting with nicotinamide phosphoribosyltransferase (NAMPT), a protein involved in NAD biosynthesis, PCNA coordinates glycolysis and survival, especially in HL-60R cells. These cells showed a dramatic increase in intracellular NAD+ concentration as well as glycolysis including increased expression and activity of hexokinase 1 and increased lactate production. Furthermore, this functional activity of cytoplasmic PCNA was also demonstrated in patients with acute myeloid leukemia (AML). Our data uncover a novel pathway of nuclear export of PCNA that drives cell survival by increasing metabolism flux.

Neutrophil-Expressed p21/waf1 Favors Inflammation Resolution in Pseudomonas aeruginosa Infection.

Neutrophil-associated inflammation during Pseudomonas aeruginosa lung infection is a determinant of morbidity in cystic fibrosis (CF). Neutrophil apoptosis is a key factor in inflammation resolution and is controlled by cytosolic proliferating cell nuclear antigen (PCNA). p21/Waf1, a cyclin-dependent kinase inhibitor, is a partner of PCNA, and its mRNA is up-regulated in human neutrophils during LPS challenge. We show here that, after 7 days of persistent infection with P. aeruginosa, neutrophilic inflammation was more prominent in p21(-/-) compared with wild-type (WT) mice. Notably, no intrinsic defect in the phagocytosis of apoptotic cells by macrophages was found in p21(-/-) compared with WT mice. Inflammatory cell analysis in peritoneal lavages after zymosan-induced peritonitis showed a significantly increased number of neutrophils at 48 hours in p21(-/-) compared with WT mice. In vitro analysis was consistent with delayed neutrophil apoptosis in p21(-/-) compared with WT mice. Ectopic expression of p21/waf1 in neutrophil-differentiated PLB985 cells potentiated apoptosis and reversed the prosurvival effect of PCNA. In human neutrophils, p21 messenger RNA was induced by TNF-α, granulocyte colony-stimulating factor, and LPS. Neutrophils isolated from patients with CF showed enhanced survival, which was reduced after treatment with a carboxy-peptide derived from the sequence of p21/waf1. Notably, p21/waf1 was detected by immunohistochemistry in neutrophils within lungs from patients with CF. Our data reveal a novel role for p21/waf1 in the resolution of inflammation via its ability to control neutrophil apoptosis. This mechanism may be relevant in the neutrophil-dominated inflammation observed in CF and other chronic inflammatory lung conditions.

Characterization of cytosolic proliferating cell nuclear antigen (PCNA) in neutrophils: antiapoptotic role of the monomer.

We have shown previously that PCNA, a nuclear factor involved in DNA replication and repair in proliferating cells, is localized exclusively in the cytoplasm of neutrophils, where it regulates their survival. Nuclear PCNA functions are tightly linked to its ring-shaped structure, which allows PCNA to bind to numerous partner proteins to orchestrate DNA-related processes. We have shown that only monomeric PCNA can expose its NES to be relocalized from nucleus to cytosol during granulocyte differentiation. This study tested the hypothesis that monomeric PCNA could have a biological role in neutrophils. With the use of a combination of cross-linking and gel-filtration experiments, trimeric and monomeric PCNAs were detected in neutrophil cytosol. The promyelocytic cell line PLB985 was next stably transfected to express the monomeric PCNAY114A mutant to examine its function compared with the WT trimeric PCNA. Monomeric PCNAY114A mutant potentiated DMF-induced differentiation, as evidenced by an increased percentage of CD11b- and gp91phox-positive PLB985PCNAY114A cells and by an increased, opsonized zymosan-triggered NADPH oxidase activity compared with PLB985PCNA or PLB985 cells overexpressing WT PCNA or the empty plasmid, respectively. Regarding antiapoptotic activity, DMF-differentiated PLB985 cells overexpressing WT or the monomeric PCNAY114A mutant displayed a similar antiapoptotic activity following treatment with gliotoxin or TRAIL compared with PLB985. The molecular basis through which cytoplasmic PCNA exerts its antiapoptotic activity in mature neutrophils may, at least in part, be independent of the trimeric conformation.

Nuclear-to-cytoplasmic relocalization of the proliferating cell nuclear antigen (PCNA) during differentiation involves a chromosome region maintenance 1 (CRM1)-dependent export and is a prerequisite for PCNA antiapoptotic activity in mature neutrophils.

Neutrophils are deprived of proliferative capacity and have a tightly controlled lifespan to avoid their persistence at the site of injury. We have recently described that the proliferating cell nuclear antigen (PCNA), a nuclear factor involved in DNA replication and repair of proliferating cells, is a key regulator of neutrophil survival. In neutrophils, PCNA was localized exclusively in the cytoplasm due to its nuclear-to-cytoplasmic relocalization during granulocytic differentiation. We showed here that leptomycin B, an inhibitor of the chromosome region maintenance 1 (CRM1) exportin, inhibited PCNA relocalization during granulocytic differentiation of HL-60 and NB4 promyelocytic cell lines and of human CD34(+) primary cells. Using enhanced green fluorescent protein fusion constructs, we have demonstrated that PCNA relocalization involved a nuclear export signal (NES) located from Ile-11 to Ile-23 in the PCNA sequence. However, this NES, located at the inner face of the PCNA trimer, was not functional in wild-type PCNA, but instead, was fully active and leptomycin B-sensitive in the monomeric PCNAY114A mutant. To test whether a defect in PCNA cytoplasmic relocalization would affect its antiapoptotic activity in mature neutrophils, a chimeric PCNA fused with the SV40 nuclear localization sequence (NLS) was generated to preclude its cytoplasmic localization. As expected, neutrophil-differentiated PLB985 cells expressing ectopic SV40NLS-PCNA had an increased nuclear PCNA as compared with cells expressing wild-type PCNA. Accordingly, the nuclear PCNA mutant did not show any antiapoptotic activity as compared with wild-type PCNA. Nuclear-to-cytoplasmic relocalization that occurred during myeloid differentiation is essential for PCNA antiapoptotic activity in mature neutrophils and is dependent on the newly identified monomerization-dependent PCNA NES.

Proliferating cell nuclear antigen acts as a cytoplasmic platform controlling human neutrophil survival.

Neutrophil apoptosis is a highly regulated process essential for inflammation resolution, the molecular mechanisms of which are only partially elucidated. In this study, we describe a survival pathway controlled by proliferating cell nuclear antigen (PCNA), a nuclear factor involved in DNA replication and repairing of proliferating cells. We show that mature neutrophils, despite their inability to proliferate, express high levels of PCNA exclusively in their cytosol and constitutively associated with procaspases, presumably to prevent their activation. Notably, cytosolic PCNA abundance decreased during apoptosis, and increased during in vitro and in vivo exposure to the survival factor granulocyte colony-stimulating factor (G-CSF). Peptides derived from the cyclin-dependent kinase inhibitor p21, which compete with procaspases to bind PCNA, triggered neutrophil apoptosis thus demonstrating that specific modification of PCNA protein interactions affects neutrophil survival. Furthermore, PCNA overexpression rendered neutrophil-differentiated PLB985 myeloid cells significantly more resistant to TNF-related apoptosis-inducing ligand- or gliotoxin-induced apoptosis. Conversely, a decrease in PCNA expression after PCNA small interfering RNA transfection sensitized these cells to apoptosis. Finally, a mutation in the PCNA interdomain-connecting loop, the binding site for many partners, significantly decreased the PCNA-mediated antiapoptotic effect. These results identify PCNA as a regulator of neutrophil lifespan, thereby highlighting a novel target to potentially modulate pathological inflammation.

Secretory IgA mediates retrotranscytosis of intact gliadin peptides via the transferrin receptor in celiac disease.

Celiac disease (CD) is an enteropathy resulting from an abnormal immune response to gluten-derived peptides in genetically susceptible individuals. This immune response is initiated by intestinal transport of intact peptide 31-49 (p31-49) and 33-mer gliadin peptides through an unknown mechanism. We show that the transferrin receptor CD71 is responsible for apical to basal retrotranscytosis of gliadin peptides, a process during which p31-49 and 33-mer peptides are protected from degradation. In patients with active CD, CD71 is overexpressed in the intestinal epithelium and colocalizes with immunoglobulin (Ig) A. Intestinal transport of intact p31-49 and 33-mer peptides was blocked by polymeric and secretory IgA (SIgA) and by soluble CD71 receptors, pointing to a role of SIgA-gliadin complexes in this abnormal intestinal transport. This retrotranscytosis of SIgA-gliadin complexes may promote the entry of harmful gliadin peptides into the intestinal mucosa, thereby triggering an immune response and perpetuating intestinal inflammation. Our findings strongly implicate CD71 in the pathogenesis of CD.

T84-intestinal epithelial exosomes bear MHC class II/peptide complexes potentiating antigen presentation by dendritic cells.

BACKGROUND & AIMS: Intestinal epithelial cells release antigen-presenting vesicles (exosomes) bearing major histocompatibility complex class II/peptide complexes stimulating specific immune responses in vivo. To characterize further the role of human epithelial exosomes in antigen presentation, their capacity to load antigenic peptides, bind immune target cells, and induce T-cell activation was analyzed in vitro. METHODS: The capacity of exosomes derived from the HLA-DR4-expressing, intestinal epithelial cell line T84 to load the HLA-DR4-specific peptide (3)H-HSA 64-76 and to activate a HLA-DR4-restricted T-cell hybridoma was tested in the presence or absence of human monocyte-derived dendritic cells (DCs). Interaction of fluorescein isothiocyanate-labeled exosomes with T cells and DCs was analyzed by flow cytometry and confocal microscopy. RESULTS: T84-derived exosomes, enriched in CD9, CD81, CD82, and A33 antigen, were capable of binding specifically human serum albumin (HSA) 64-76 peptide on HLA-DR4 molecules and of interacting preferentially with DCs. HSA-loaded exosomes were unable to activate the T-cell hybridoma directly but induced a productive T-cell activation through DCs. When HSA peptide was bound to exosomal HLA-DR4 molecules instead of in a soluble form, the threshold of peptide presentation by DCs was markedly decreased (x10(-3)). CONCLUSIONS: Exosomes released by intestinal epithelial cells bear exogenous peptides complexed to major histocompatibility complex class II molecules and interact preferentially with DCs, strongly potentiating peptide presentation to T cells. Epithelial exosomes constitute a powerful link between luminal antigens and local immune cells by mediating the transfer of tiny amounts of luminal antigenic information and facilitating immune surveillance at mucosal surfaces.

Effect of rebamipide on the colonic barrier in interleukin-10-deficient mice.

Our aim was to study the effect of a mucosal protective agent, rebamipide, on the colonic barrier and the immune response in colitis-prone interleukin-10-deficient (IL-10-/-) C57BL/6 mice infected with Helicobacter hepaticus. After sacrifice, in all mice, control, or previously infected with H. hepaticus, or previously infected and treated with rebamipide enema, a histological examination of colonic samples was performed, intestinal permeability was studied in Ussing chamber, and mesenteric lymph node proliferation and cytokine secretion were measured. Mice treated with rebamipide presented a reinforcement of the distal colonic epithelial barrier, an increase of mesenteric lymph node cells proliferation, and of IFNgamma and IL-12 secretion. These results indicate that in IL-10-/- mice with mild colitis, rectally administered rebamipide reinforces the distal colonic barrier and has a slight Th1 immuno-stimulatory effect on mesenteric lymph node cells. These properties could be helpful in the management of some inflammatory bowel diseases.

Stimulation of immunity without alteration of oral tolerance in mice fed with heat-treated fermented infant formula.

OBJECTIVES: Little information is available on the properties of fermented milk formula intended to healthy infants. This study analyzes the effect of long-term ingestion of a heat-treated, fermented milk formula on the development of oral tolerance or systemic immune response to soluble antigens in mice. MATERIALS AND METHODS: The C3H/HeN mice, fed with a heat-treated fermented (Bifidobacterium breve C50 and Streptococcus thermophilus 065) infant formula (htFF) or a matched control diet (control), were immunized with ovalbumin (OVA) with or without gavage of 20 mg OVA to induce tolerance or immunity, respectively. Systemic and local anti-OVA immune responses and intestinal barrier function were measured after 5 to 6 weeks. RESULTS: Oral tolerance to OVA developed similarly in htFF- and control-fed mice, attested to by the downregulation of OVA-specific immunoglobulin (Ig) G and IgE after oral OVA administration. In contrast, immunization with OVA led to significantly higher titers in htFF-fed mice than in control-fed mice (log2 IgG titers, 16.45 +/- 1.24 and 15.46 +/- 0.79, respectively; P = 0.012). Jejunal interferon gamma, interleukin 12p40 and interleukin 10 expressions were significantly higher in tolerized mice fed with htFF compared with those fed with the control diet. Mucosal to serosal intact horseradish peroxidase fluxes were lower in htFF-fed mice than in control-fed mice (39 +/- 8 and 118 +/- 38 ng/h x cm2, respectively; P < 0.0001), indicating that the htFF diet reinforces intestinal barrier capacity to macromolecules. CONCLUSIONS: In mice, htFF strengthens intestinal barrier and enhances systemic immune responses to antigens without interfering with the development of oral tolerance, suggesting a potential beneficial effect in host defence and vaccination.

Bifidobacterium breve and Streptococcus thermophilus secretion products enhance T helper 1 immune response and intestinal barrier in mice.

Lactic acid bacteria or their secretion products can modulate immune responses differently in normal and inflammatory conditions. This comparative study analyzes the effect of oral administration of living lactic acid bacteria, or their conditioned media, on the epithelial and immune functions of colitis-prone C57BL/6 IL-10-deficient mice. Mice were untreated (control) or infected with Helicobacter hepaticus with or without oral treatment with living bacteria, Bifidobacterium breve C50 and Streptococcus thermophilus 065 (LB), or their culture-conditioned media (CM). Histology, cytokine mRNA, electrical resistance, and barrier capacity of colonic samples as well as cytokine secretion by mesenteric lymph node (MLN) cells were studied. Helicobacter hepaticus mice developed only mild colitis, which was not modified in LB or CM groups. In the CM (but not the LB) group, the colonic barrier was reinforced as compared to the other groups, as evidenced by decreased horseradish peroxidase (HRP) transcytosis and mannitol fluxes and increased electrical resistance. In MLN, the percentage of CD4+ and CD8+ T cells secreting IFNgamma was significantly higher in CM (2.06% and 1.98%, respectively) mice than in H. hepaticus (1.1% and 0.47%, P < 0.05) or control mice. In addition, the nonspecific stimulation of IFNgamma, TNFalpha, and IL-12 secretion by MLN cells was significantly higher in the CM group as compared to the other groups. In the absence of severe colitis, Bifidobacterium breve C50- and Streptococcus thermophilus 065-conditioned media can reinforce intestinal barrier capacity and stimulate Th1 immune response, highlighting the involvement of lactic acid bacteria-derived components in host defense.

Limited efficiency of prolyl-endopeptidase in the detoxification of gliadin peptides in celiac disease.

BACKGROUND & AIMS: The resistance of prolamines to digestive enzymes is thought to be a key contributor to the pathogenesis of celiac disease by promoting the intestinal entrance of peptides able to trigger inflammation in at-risk individuals. Oral administration of a bacterial prolyl-endopeptidase (PEP) therefore was proposed as a treatment for celiac disease. To delineate the feasibility of this treatment, the effect of PEP on gliadin peptides was assessed in vitro, and ex vivo during their transport across intestinal biopsy specimens of active celiac disease patients. METHODS: In vitro degradation by PEP of 3H-labeled gliadin peptides 56-88 (33-mer) and 31-49, was analyzed by radio-reverse-phase high-performance liquid chromatography and mass spectrometry. For ex vivo studies, PEP and 3H-peptides were added together onto the mucosal side of duodenal biopsy specimens mounted in Ussing chambers, and peptide transport and digestion were assessed by radio-reverse-phase high-performance liquid chromatography. RESULTS: Gliadin peptides were degraded partly by 20 mU/mL PEP both in vitro and ex vivo. This concentration of PEP decreased the amount of intact peptides 31-49 and 56-88 crossing the intestinal biopsy specimens of celiac disease patients, but could not prevent the intestinal passage of toxic or immunostimulatory metabolites. PEP concentrations of at least 500 mU/mL for 3 hours were required to achieve full detoxification of peptides and to prevent intestinal transport of active fragments. CONCLUSIONS: After prolonged exposure to high concentrations of PEP, the amount of immunostimulatory gliadin peptides reaching the local immune system in celiac patients is decreased. These results provide a basis to establish whether such conditions are achievable in vivo.

Food allergens are protected from degradation during CD23-mediated transepithelial transport.

BACKGROUND: CD23 (FcepsilonRII) is expressed by intestinal epithelial cells (IEC) following allergic stimulation and increases the uptake of IgE/allergen complexes. The aim of this study was to further analyze the role of CD23 in the intraepithelial processing of food allergens during transepithelial transport. METHODS: Balb-C mice were sensitized intraperitoneally with horseradish peroxidase (HRP) or beta-lactoglobulin (beta-LG) in the presence of pertussis toxin. In control and sensitized mice, 3H-HRP, intact HRP, or 14C-beta-LG fluxes were measured across jejunal segments mounted in Ussing chambers, in the presence or absence of mucosal anti-CD23 antibodies. HPLC analysis of serosal buffer was performed to detect HRP- or beta-LG-derived radiolabelled metabolites generated during transepithelial transport. RESULTS: In HRP-sensitized mice, 3H-HRP fluxes and intact HRP fluxes (3,836 +/- 476 and 290 +/- 86 ng/h x cm2, respectively) were significantly increased compared to control mice (1,677 +/- 297 ng/h x cm2, p < 0.01, and 106 +/- 23 ng/h x cm2, p < 0.02, respectively). HPLC analysis indicated the presence of intact HRP in the serosal compartment already 10 min after addition of HRP to the mucosal compartment, a result not observed in the control mice. In the presence of anti-CD23 antibodies, intact HRP fluxes were significantly decreased (131 +/- 27 ng/h x cm2) compared to control values in sensitized mice (290 +/- 86 ng/h x cm2, p < 0.02), suggesting that CD23 is involved is this 'protected' transport pathway. A similar protection during intestinal transport was observed for beta-LG in beta-LG sensitized mice. CONCLUSIONS: These results confirm that CD23 is involved in the rapid transepithelial transport of intact allergens in sensitized animals, and indicate that CD23 opens a 'protected' transport pathway in IECs.

Alterations of the intestinal transport and processing of gliadin peptides in celiac disease.

BACKGROUND & AIMS: The hypothesis of a defect in the intestinal transport and processing of toxic (31-49) or immunostimulant (57-68 and the 33-mer 56-89) gliadin peptides was tested in patients with active celiac disease (ACD), patients with treated celiac disease (TCD), and controls. METHODS: Using duodenal biopsy specimens mounted in Ussing chambers, we measured electrical resistance, mucosal-to-serosal radiolabeled-peptide fluxes, and peptide processing during transport using radio-reverse-phase high-performance liquid chromatography. RESULTS: Peptide 31-49 fluxes (24.7 microg x 3 h(-1). cm(-2)) were increased in patients with ACD compared with controls and patients with TCD (12.7 and 12.3 microg x 3 h(-1). cm(-2); P < 0.01). In contrast, no increase was observed for peptide 57-68 or 56-89 (33-mer). Electrical resistance was decreased in patients with ACD versus controls (15.3 vs. 23.9 ohms. cm(2); P < 0.001). Peptide 57-68 was partially degraded by brush-border peptidases in controls but not in patients with celiac disease. However, it was totally degraded after intestinal transport both in controls and patients with celiac disease. Peptides 31-49 and 56-89 were resistant to brush-border peptidases in all groups of patients but were totally degraded during intestinal transport in controls and patients with TCD. In patients with ACD, however, 50% of peptide 31-49 was delivered intact into the serosal compartment and only partial degradation of the 33-mer was observed. These abnormalities were not related to a nonspecific paracellular leakage. CONCLUSIONS: Our data indicate that gliadin peptides, although poorly or not digested by intraluminal enzymes, can be fully digested by enterocytes in controls and patients with TCD. In patients with ACD, incomplete degradation of the 33-mer and protected transport of the peptide 31-49 might favor their respective immunostimulatory and toxic effects.

In vitro transfer of clarithromycin and amoxicillin across the epithelial barrier: effect of Helicobacter pylori.

The mode of absorption of amoxicillin and clarithromycin, two antibiotics used in the treatment of Helicobacter pylori infection, has not been completely elucidated. The aims of this study were to investigate the passage of these antibiotics across normal and infected epithelium and to measure their accumulation in HT29-19A or Caco2 epithelial cell monolayers. In non-infected cultures, basal-to-apical fluxes were significantly higher than apical-to-basal fluxes for both antibiotics, but this difference disappeared in monolayers infected with H. pylori. In 24 h studies, clarithromycin, but not amoxicillin, showed rapid intracellular accumulation. No difference was found between the transepithelial passage of amoxicillin across the HT29-19A and Caco2 monolayers.