Thomas G. Blanchard

Bacterial Probiotic Modulation of Dendritic Cells

ABSTRACT Intestinal dendritic cells are continually exposed to ingested microorganisms and high concentrations of endogenous bacterial flora. These cells can be activated by infectious agents and other stimuli to induce T-cell responses and to produce chemokines which recruit other cells to the local environment. Bacterial probiotics are of increasing use against intestinal disorders such as inflammatory bowel disease. They act as nonpathogenic stimuli within the gut to regain immunologic quiescence. This study was designed to determine the ability of a bacterial probiotic cocktail VSL#3 to alter cell surface antigen expression and cytokine production in bone marrow-derived dendritic cell-enriched populations. Cell surface phenotype was monitored by monoclonal fluorescent antibody staining, and cytokine levels were quantitated by enzyme-linked immunosorbent assay. High-dose probiotic upregulated the expression of C80, CD86, CD40, and major histocompatibility complex class II I-Ad. Neither B7-DC or B7RP-1 was augmented after low-dose probiotic or Lactobacillus casei treatment, but B7RP-1 showed increased expression on dendritic cells stimulated with the gram-negative bacterium Escherichia coli. Functional studies showed that probiotic did not enhance the ability of dendritic cells to induce allogeneic T-cell proliferation, as was observed for E. coli. Substantial enhancement of interleukin-10 release was observed in dendritic cell-enriched culture supernatants after 3 days of probiotic stimulation. These results demonstrate that probiotics possess the ability to modulate dendritic cell surface phenotype and cytokine release in granulocyte-macrophage colony-stimulating factor-stimulated bone marrow-derived dendritic cells. Regulation of dendritic cell cytokines by probiotics may contribute to the benefit of these molecules in treatment of intestinal diseases.

A Mucosal IgA-Mediated Excretory Immune System In Vivo

The capacity of mucosal IgA Abs to serve as an excretory immune system in vivo was investigated. Mice expressing a transgenic TCR were immunized intragastrically with the cognate Ag to elicit a vigorous mucosal IgA Ab response. Soon after i.v. challenge, Ag was detected within the epithelial cells of the small intestinal crypts and to a lesser degree within the epithelial cells higher up the villi, paralleling the gradient in expression of the polymeric Ig receptor and the transport of its ligand, oligomeric IgA. Uptake of Ag into the epithelial cells occurred only from the basolateral aspect and only when Ag complexed to IgA Ab could be present in the lamina propria. The results support the concept that local IgA Abs can excrete Ags from the body by transporting them directly through mucosal epithelial cells, using the same mechanism that transports free IgA into the mucosal secretions.

Vaccination of mice against H pylori induces a strong Th-17 response and immunity that is neutrophil dependent.

BACKGROUND & AIMS Vaccine efficacy against gastric Helicobacter pylori infection has been shown in mice, but little is known about the mechanisms of bacterial clearance. Our aim was to investigate a possible T-cell/neutrophil pathway of vaccine-induced protection. METHODS Nonimmune and immunized mice were compared for their response to H pylori challenge. T-cell responses were assessed by recall assays. Interleukin (IL)-17-induced chemokine production was evaluated by cytokine enzyme-linked immunosorbent assay. In a kinetic study, biopsy specimens were collected at multiple time points postchallenge and assessed for bacterial load and inflammation. Relative levels of T cells, IL-17, interferon gamma, MIP-2, KC, and LIX were determined by quantitative polymerase chain reaction. The role of neutrophils was evaluated by antibody-mediated depletion of neutrophils following challenge. RESULTS Immunization induced strong interferon gamma- and IL-17-producing T-cell responses, and IL-17 was capable of inducing significant amounts of KC and MIP-2 from dendritic cells, macrophages, fibroblasts, and gastric epithelial cells. Challenge of immunized mice induced significantly greater gastritis than that of infected mice, preceding significantly lower bacterial loads by day 7. In immune mice, T-cell recruitment to the gastric mucosa correlated with a continuous rise in IL-17 and interferon gamma levels, followed by KC, MIP-2, and LIX production and the recruitment of significant numbers of neutrophils by day 5. Antibody-mediated depletion of neutrophils abrogated vaccine efficacy. CONCLUSIONS Vaccination of mice against H pylori results in a significant Th-17 cell recall response associated with increases in chemokines that attract neutrophils to the stomach, which are important for eradication of H pylori.

Protective Anti‐Helicobacter Immunity Is Induced with Aluminum Hydroxide or Complete Freund’s Adjuvant by Systemic Immunization

To determine whether systemic immunization against Helicobacter pylori could be achieved with an adjuvant approved for human use, the efficacy of vaccination with Helicobacter antigen in combination with aluminum hydroxide (AlOH) was evaluated in a murine model of Helicobacter infection. Immunization with antigen and AlOH induced interleukin-5-secreting, antigen-specific T cells, and immunization with antigen and complete Freunds adjuvant induced interferon-gamma-secreting, antigen-specific T cells, as determined by ELISPOT assay. Both immune responses conferred protection after challenge with either H. pylori or H. felis, as confirmed by the complete absence of any bacteria, as assessed by both histology and culture of gastric biopsy samples. Protection was antibody independent, as demonstrated with antibody-deficient muMT mice (immunoglobulin-gene knockout mice), and CD4(+) spleen T cells from immunized mice were sufficient to transfer protective immunity to otherwise immunodeficient rag1(-/-) recipients. These results suggest an alternative and potentially more expeditious strategy for development of a human-use H. pylori vaccine.

Vaccinating against Helicobacter pylori infection

Helicobacter pylori infection of the gastric mucosa remains a cause of significant morbidity and mortality almost 30 years after its discovery. H. pylori infection can lead to several gastric maladies, including gastric cancer, and although antimicrobial therapies for the infection exist, the cost of treatment for gastric cancer and the prognosis of individuals who present with this disease make vaccine development a cost effective alternative to bacterial eradication. Experimental mucosal and systemic H. pylori vaccines in mice significantly reduce bacterial load and sometimes provide sterilizing immunity. Clinical trials of oral vaccines consisting of H. pylori proteins with bacterial exotoxin adjuvants or live attenuated bacterial vectors expressing H. pylori proteins induce adaptive immune mechanisms but fail to consistently reduce bacterial load. Clinical trials and murine studies demonstrate that where H. pylori is killed, either spontaneously or following vaccination, the host demonstrated cellular immunity. Improved efficacy of vaccines may be achieved in new trials of vaccine formulations that include multiple antigens and use methods to optimize cellular immunity. Unfortunately, the industrial sponsors that served as the primary engine for much of the previous animal and human research have withdrawn their support. A renewed or expanded commitment from the biotechnology or pharmaceutical industry that could exploit recent advances in our understanding of the host immune response to H. pylori is necessary for the advancement of an H. pylori vaccine.

Induction of CTLA-4-Mediated Anergy Contributes to Persistent Colonization in the Murine Model of Gastric Helicobacter pylori Infection

Helicobacter pylori infection induces gastric inflammation but the host fails to generate protective immunity. Therefore, we evaluated the immunologic mechanisms that contribute to the failure of the T cells to promote active immunity to H. pylori in the mouse model of H. pylori infection. Spleen cells from infected C57BL/6 mice underwent significantly less proliferation and cytokine production than cells from immune mice upon in vitro stimulation with H. pylori lysate. Similar results were observed when stimulating with Ag-pulsed macrophages demonstrating that hyporesponsiveness was not due to a direct effect of H. pylori virulence factors on the T cells. Ag-specific hyporesponsiveness could be reversed by the addition of high-dose IL-2 but not by removal of CD4+CD25+ T cells, indicating that hyporesponsiveness was due to anergy and not due to active suppression. Cells from infected mice lacked significant suppressor activity as shown by the failure to reduce the recall response of cells from immune mice in coculture at physiologic ratios. Direct blockade of CTLA-4 using anti-CTLA-4 Fabs or indirect blockade using CTLA-4 Ig plus anti-CD28 Ab resulted in significantly increased T cell activation in vitro. The importance of CTLA-4 in establishing anergy was confirmed in an in vivo model of H. pylori infection in which mice that received anti-CTLA-4 Fabs responded to H. pylori challenge with significantly greater inflammation and significantly reduced bacterial load. These results suggest that CTLA-4 engagement induces and maintains functional inactivation of H. pylori-specific T cells during H. pylori infection resulting in a reduced immune response.

Muc1 Cell Surface Mucin Attenuates Epithelial Inflammation in Response to a Common Mucosal Pathogen

Helicobacter pylori infection of the gastric mucosa causes an active-chronic inflammation that is strongly linked to the development of duodenal and gastric ulcers and stomach cancer. However, greater than 80% of individuals infected with H. pylori are asymptomatic beyond histologic inflammation, and it is unknown what factors influence the incidence and character of bacterial-associated gastritis and related disorders. Because previous studies demonstrated that the Muc1 epithelial glycoprotein inhibited inflammation during acute lung infection by Pseudomonas aeruginosa, we asked whether Muc1 might also counter-regulate gastric inflammation in response to H. pylori infection. Muc1−/− mice displayed increased bacterial colonization of the stomach and greater TNF-α and keratinocyte chemoattractant transcript levels compared with Muc1+/+ mice after experimental H. pylori infection. Knockdown of Muc1 expression in AGS human gastric epithelial cells by RNA interference was associated with increased phosphorylation of IκBα, augmented activation and nuclear translocation of NF-κB, and enhanced production of interleulin-8 compared with Muc1-expressing cells. Conversely, Muc1 overexpression was correlated with decreased NF-κB activation, reduced interleulin-8 production, and diminished IκB kinase β (IKKβ)/IKKγ coimmunoprecipitation compared with cells expressing Muc1 endogenously. Cotransfection of AGS cells with Muc1 plus IKKβ, but not a catalytically inactive IKKβ mutant, reversed the Muc1 inhibitory effect. Finally, Muc1 formed a coimmunoprecipitation complex with IKKγ but not with IKKβ. These results are consistent with the hypothesis that Muc1 binds to IKKγ, thereby inhibiting formation of the catalytically active IKK complex and blocking the ability of H. pylori to stimulate IκBα phosphorylation, NF-κB activation, and downstream inflammatory responses.

Immune status, antibiotic medication and pH are associated with changes in the stomach fluid microbiota.

The stomach acts as a barrier to ingested microbes, thereby influencing the microbial ecology of the entire gastrointestinal (GI) tract. The stomach microbiota and the role of human host and environmental factors, such as health status or medications, in shaping its composition remain largely unknown. We sought to characterize the bacterial and fungal microbiota in the stomach fluid in order to gain insights into the role of the stomach in GI homeostasis. Gastric fluid was collected from 25 patients undergoing clinically indicated upper endoscopy. DNA isolates were used for PCR amplification of bacterial 16S ribosomal RNA (rRNA) genes and fungal internal transcribed spacers (ITS). RNA isolates were used for 16S rRNA cDNA generation and subsequent PCR amplification. While all stomach fluid samples are dominated by the phyla Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria and Fusobacteria (>99% of sequence reads), the transcriptionally active microbiota shows significant reduction in Actinobacteria (34%) and increase in Campylobacter (444%) (P<0.003), specifically the oral commensal and suspected intestinal pathogen Campylobacter concisus. Bacterial but not fungal diversity is reduced by antibiotic treatment (28%; P<0.02), immunosuppression in transplant recipients and HIV/AIDS patients (42%; P<0.001) and gastric fluid pH >4 (70%; P<0.05). Immunosuppression correlates with decreased abundance of Prevotella (24%), Fusobacterium (2%) and Leptotrichia (6%) and increased abundance of Lactobacillus (3844%) (P<0.003). We have generated the first in-depth characterization of the human gastric fluid microbiota, using bacterial 16S rRNA gene and transcript, and fungal ITS amplicon sequencing and provide evidence for a significant impact of the host immune status on its composition with likely consequences for human health.

Severe Inflammation and Reduced Bacteria Load in Murine Helicobacter Infection Caused by Lack of Phagocyte Oxidase Activity

The vaccine-induced immune mechanisms that protect against Helicobacter pylori infection in the mouse model have not been identified. This study investigated the contribution of reactive oxygen and nitrogen intermediates to Helicobacter pathogenesis and immunity. Mice deficient in nicotinamide-adenine dinucleotide phosphate oxidase activity (gp91(phox-/-)), nitric oxide synthase activity (NOS2(-/-)), or both (gp91(phox-/-)/NOS2(-/-)) were infected with Helicobacter organisms and evaluated for inflammation and bacteria load. Infection of all 3 transgenic strains resulted in significantly more inflammation than found in infected C57BL/6 wild-type mice. However, only gp91(phox-/-) and gp91(phox-/-)/NOS2(-/-) mice had significantly reduced numbers of infected gastric glands. Intranasal immunization of NOS2(-/-) or gp91(phox-/-)/NOS2(-/-) mice against H. pylori resulted in protective immunity comparable to that seen in C57BL/6 control mice. Therefore, reactive oxygen species may play a role in limiting the inflammatory response associated with H. pylori infection of the gastric mucosa but may also limit the hosts ability to eradicate Helicobacter organisms.

RECOMBINANT CHOLERA TOXIN B SUBUNIT IS NOT AN EFFECTIVE MUCOSAL ADJUVANT FOR ORAL IMMUNIZATION OF MICE AGAINST HELICOBACTER FELIS

Cholera toxin is a potent oral mucosal adjuvant for enteric imm_unization. Several studies suggest that commercial cholera toxin B subunit (cCTB; purified from holotoxin) may be an effective non‐toxic alternative for oral imm_unization. The present study was performed, using an infectious disease model, to determine if the oral mucosal adjuvanticity of CTB is dependent on contaminating holotoxin. Mice were orally imm_unized with Helicobacter felis sonicate and either cholera holotoxin, cCTB or recombinant cholera toxin B subunit (rCTB). Serum imm_unoglobulin G (IgG) and intestinal imm_unoglobulin A (IgA) antibody responses were determined and the mice were challenged with live H. felis to determine the degree of protective imm_unity induced. All orally imm_unized mice responded with serum IgG antibody titres regardless of the adjuvant used. However, only mice imm_unized with either holotoxin or the cCTB responded with an intestinal mucosal IgA response. Consistent with the production of mucosal antibodies, mice imm_unized with either holotoxin or cCTB as adjuvants were protected from challenge while mice receiving H. felis sonicate and rCTB all became infected. cCTB induced the accumulation of cAMP in mouse thymocytes at a level equal to 0.1% of that induced by holotoxin, whereas rCTB was devoid of any activity. These results indicate that CTB possesses no intrinsic mucosal adjuvant activity when administered orally. Therefore, when used as an oral adjuvant, CTB should also include small, non‐toxic doses of cholera toxin.