Carl-Fredrik Flach

B Lymphocytes Promote Expansion of Regulatory T Cells in Oral Tolerance: Powerful Induction by Antigen Coupled to Cholera Toxin B Subunit

Mucosal administration of Ag conjugated to cholera toxin B subunit (CTB) can efficiently induce peripheral immunologic tolerance, so-called oral tolerance, associated with development of Foxp3+CD25+CD4+ regulatory T (Treg) cells. Using an established sublingual tolerization regimen with Ag(OVA)/CTB conjugate, wherein CTB mediates Ag uptake and presentation by most B lymphocytes irrespective of their Ag specificity, we have assessed the importance of B cells for induction of Ag-specific Treg cells and oral tolerance. We found that Treg cells are reduced in μMT−/− B cell-deficient mice compared with wild-type (WT) mice. After sublingual Ag/CTB treatment, Treg cells increased much more in WT than in μMT−/− mice; however, adoptive transfer of B cells before treatment normalized Treg cell development and functional oral tolerance. B cells from OVA/CTB-treated mice expressed more IL-10 and less CD86 than control B cells. Adoptive transfer of these cells before parenteral immunization with OVA led to efficient suppression of proliferation and to induction of apoptotic depletion of Ag-specific CD25−CD4+ effector T cells associated with the expansion of Treg cells. However, also OVA/CTB-treated μMT−/− mice could suppress the immune response to parenteral immunization with OVA, which was associated with a strong increase in Foxp3−CD4+ T cells expressing LAP/TGF-β. Our results indicate that mucosal tolerance comprises at least two separate pathways: one being B cell dependent and associated with expansion of Treg cells and Treg-mediated suppression and depletion of effector T cells, and one being B cell independent and associated with development of Foxp3−LAP+TGF-β+ regulatory T cells.

Fluoroquinolones and qnr Genes in Sediment, Water, Soil, and Human Fecal Flora in an Environment Polluted by Manufacturing Discharges

There is increasing concern that environmental antibiotic pollution promotes transfer of resistance genes to the human microbiota. Here, fluoroquinolone-polluted river sediment, well water, irrigated farmland, and human fecal flora of local villagers within a pharmaceutical industrial region in India were analyzed for quinolone resistance (qnr) genes by quantitative PCR. Similar samples from Indian villages farther away from industrial areas, as well as fecal samples from Swedish study participants and river sediment from Sweden, were included for comparison. Fluoroquinolones were detected by MS/MS in well water and soil from all villages located within three km from industrially polluted waterways. Quinolone resistance genes were detected in 42% of well water, 7% of soil samples and in 100% and 18% of Indian and Swedish river sediments, respectively. High antibiotic concentrations in Indian sediment coincided with high abundances of qnr, whereas lower fluoroquinolone levels in well water and soil did not. We could not find support for an enrichment of qnr in fecal samples from people living in the fluoroquinolone-contaminated villages. However, as qnr was detected in 91% of all Indian fecal samples (24% of the Swedish) it suggests that the spread of qnr between people is currently a dominating transmission route.

Broad Up-Regulation of Innate Defense Factors during Acute Cholera

ABSTRACT We used a whole-genome microarray screening system (Affymetrix human GeneChips covering 47,000 different transcripts) to examine the gene expression in duodenal mucosa during acute cholera. Biopsies were taken from the duodenal mucosa of seven cholera patients 2 and 30 days after the onset of diarrhea, and the gene expression patterns in the acute- and convalescent-phase samples were compared pairwise. Of about 21,000 transcripts expressed in the intestinal epithelium, 29 were defined as transcripts that were up-regulated and 33 were defined as transcripts that were down-regulated during acute cholera. The majority of the up-regulated genes characterized were found to have an established or possible role in the innate defense against infections; these genes included the LPLUNC1, LF, VCC1, TCN1, CD55, SERPINA3, MMP1, MMP3, IL1B, LCN2, SOCS3, GDF15, SLPI, CXCL13, and MUC1 genes. The results of confirmative PCR correlated well with the microarray data. An immunohistochemical analysis revealed increased expression of lactoferrin in lamina propria cells and increased expression of CD55 in epithelial cells, whereas increased expression of the SERPINA3 protein (α1-antichymotrypsin) was detected in both lamina propria and epithelial cells during acute cholera. The expression pattern of CD55 and SERPINA3 in cholera toxin (CT)-stimulated Caco-2 cells was the same as the pattern found in the intestinal mucosa during acute cholera, indicating that the activation of the CD55 and SERPINA3 genes in intestinal epithelium was induced by CT. In conclusion, during acute cholera infection, innate defense mechanisms are switched on to an extent not described previously. Both direct effects of CT on the epithelial cells and changes in the lamina propria cells contribute to this up-regulation.

Sublingual Immunization Protects against Helicobacter pylori Infection and Induces T and B Cell Responses in the Stomach

ABSTRACT Sublingual (SL) immunization has been described as an effective novel way to induce mucosal immune responses in the respiratory and genital tracts. We examined the potential of SL immunization against Helicobacter pylori to stimulate immune responses in the gastrointestinal mucosa and protect against H. pylori infection. Mice received two SL immunizations with H. pylori lysate antigens and cholera toxin as an adjuvant, and after challenge with live H. pylori bacteria, their immune responses and protection were evaluated, as were immune responses prior to challenge. SL immunization induced enhanced proliferative responses to H. pylori antigens in cervicomandibular lymph nodes and provided at least the same level of immune responses and protection as corresponding intragastric immunization. Protection in SL-immunized mice was associated with strong H. pylori-specific serum IgG and IgA antibody responses in the stomach and intestine, with strong proliferation and gamma interferon (IFN-γ) and interleukin-17 (IL-17) production by spleen and mesenteric lymph node T cells stimulated with H. pylori antigens in vitro, and with increased IFN-γ and IL-17 gene expression in the stomach compared to levels in infected unimmunized mice. Immunohistochemical studies showed enhanced infiltration of CD4+ T cells and CD19+ B cells into the H. pylori-infected stomach mucosa of SL-immunized but not unimmunized H. pylori-infected mice, which coincided with increased expression of the mucosal addressin cell adhesion molecule (MAdCAM-1) and T and B cell-attracting chemokines CXCL10 and CCL28. We conclude that, in mice, SL immunization can effectively induce protection against H. pylori infection in association with strong T and B cell infiltration into the stomach.

A Double Mutant Heat-Labile Toxin from Escherichia coli, LT(R192G/L211A), Is an Effective Mucosal Adjuvant for Vaccination against Helicobacter pylori Infection

ABSTRACT Helicobacter pylori infection in the stomach is a common cause of peptic ulcer disease and is a strong risk factor for the development of gastric adenocarcinoma, yet no effective vaccine against H. pylori infection is available to date. In mice, mucosal vaccination with H. pylori antigens when given together with cholera toxin (CT) adjuvant, but not without adjuvant, can induce protective immune responses against H. pylori infection. However, the toxicity of CT precludes its use as a mucosal adjuvant in humans. We evaluated a recently developed, essentially nontoxic double mutant Escherichia coli heat-labile toxin, LT(R192G/L211A) (dmLT), as a mucosal adjuvant in an experimental H. pylori vaccine and compared it to CT in promoting immune responses and protection against H. pylori infection in mice. Immunization via the sublingual or intragastric route with H. pylori lysate antigens and dmLT resulted in a significant decrease in bacterial load after challenge compared to that in unimmunized infection controls and to the same extent as when using CT as an adjuvant. Cellular immune responses in the sublingually immunized mice known to correlate with protection were also fully comparable when using dmLT and CT as adjuvants, resulting in enhanced in vitro proliferative and cytokine responses from spleen and mesenteric lymph node cells to H. pylori antigens. Our results suggest that dmLT is an attractive adjuvant for inclusion in a mucosal vaccine against H. pylori infection.

Proinflammatory Cytokine Gene Expression in the Stomach Correlates with Vaccine-Induced Protection against Helicobacter pylori Infection in Mice: an Important Role for Interleukin-17 during the Effector Phase

ABSTRACT CD4+ T cells have been shown to be essential for vaccine-induced protection against Helicobacter pylori infection in mice. Less is known about the relative contributions of individual cell subpopulations, such as Th1 and Th17 cells, and their associated cytokines. The aim of the present study was to find immune correlates to vaccine-induced protection and further study their role in protection against H. pylori infection. Immunized and unimmunized mice were challenged with H. pylori, and immune responses were compared. Vaccine-induced protection was assessed by measuring H. pylori colonization in the stomach. Gastric gene expression of Th1- and/or Th17-associated cytokines was analyzed by quantitative PCR, and contributions of individual cytokines to protection were evaluated by antibody-mediated in vivo neutralization. By analyzing immunized and unimmunized mice, a significant inverse correlation between the levels of interleukin-12p40 (IL-12p40), tumor necrosis factor alpha (TNF), gamma interferon (IFN-γ), and IL-17 gene expression and the number of H. pylori bacteria in the stomachs of individual animals after challenge could be demonstrated. In a kinetic study, upregulation of TNF, IFN-γ, and IL-17 coincided with vaccine-induced protection at 7 days after H. pylori challenge and was sustained for at least 21 days. In vivo neutralization of these cytokines during the effector phase of the immune response revealed a significant role for IL-17, but not for IFN-γ or TNF, in vaccine-induced protection. In conclusion, although both Th1- and Th17-associated gene expression in the stomach correlate with vaccine-induced protection against H. pylori infection, our study indicates that mainly Th17 effector mechanisms are of critical importance to protection.

A truncated form of HpaA is a promising antigen for use in a vaccine against Helicobacter pylori.

HpaA is a Helicobacter pylori-specific lipoprotein that has been shown to be an effective protective antigen for mucosal vaccination against H. pylori infection in mice. However, detergents are needed for the purification of full-length HpaA (HpaA(full)), which might confer toxicity, thus making HpaA(full) unsuitable for use in a human vaccine. We here describe a recombinantly produced truncated version of HpaA (HpaA(trunc)), which is easily purified without the use of detergents. Evaluation in the murine H. pylori infection model showed that sublingual immunization with HpaA(trunc) was equally immunogenic and protective as immunization with HpaA(full). Immunization with a combination of HpaA(trunc) and recombinant UreB protein induced strong immune responses to both antigens and importantly had a strong synergistic effect on protection, associated with synergistically increased expression of IL-17 in the stomach. Notably, sublingual immunization with HpaA(trunc) and UreB was superior to corresponding intragastric immunization with regard to the level of protection induced. In conclusion, HpaA(trunc) is a promising, readily produced, non-toxic recombinant antigen for inclusion in a mucosal vaccine against H. pylori infection, which may preferably be given sublingually together with UreB.

An assay for determining minimal concentrations of antibiotics that drive horizontal transfer of resistance.

Ability to understand the factors driving horizontal transfer of antibiotic resistance from unknown, harmless bacteria to pathogens is crucial in order to tackle the growing resistance problem. However, current methods to measure effects of stressors on horizontal gene transfer have limitations and often fall short, as the estimated endpoints can be a mix of both the number of transfer events and clonal growth of transconjugants. Our aim was therefore to achieve a proper strategy for assessing the minimal concentration of a stressor (exemplified by tetracycline) that drives horizontal transfer of antibiotic resistance from a complex community to a model pathogen. Conditions were optimized to improve a culture-based approach using the bacterial community of treated sewage effluent as donor, and fluorescent, traceable Escherichia coli as recipient. Reduced level of background resistance, differentiation of isolates as well as decreased risk for measuring effects of selection were achieved through the use of chromogenic medium, optimization of conjugation time as well as applying a different antibiotic for isolation of transconjugants than the one tested for its ability to drive transfer. Using this assay, we showed that a very low concentration of tetracycline, 10μg/L i.e. 150 times below the minimal inhibitory concentration of the recipient, promoted horizontal transfer of multiple antibiotic-resistance determinants. Higher concentrations favoured selection of a tetracycline-resistance phenotype along with a decline in the number of detectable transfer events. The described method can be used to evaluate different environmental conditions and factors that trigger horizontal dissemination of mobile resistance elements, eventually resulting in the formation of drug-resistant pathogens.

Minimal selective concentrations of tetracycline in complex aquatic bacterial biofilms

Selection pressure generated by antibiotics released into the environment could enrich for antibiotic resistance genes and antibiotic resistant bacteria, thereby increasing the risk for transmission to humans and animals. Tetracyclines comprise an antibiotic class of great importance to both human and animal health. Accordingly, residues of tetracycline are commonly detected in aquatic environments. To assess if tetracycline pollution in aquatic environments promotes development of resistance, we determined minimal selective concentrations (MSCs) in biofilms of complex aquatic bacterial communities using both phenotypic and genotypic assays. Tetracycline significantly increased the relative abundance of resistant bacteria at 10 μg/L, while specific tet genes (tetA and tetG) increased significantly at the lowest concentration tested (1 μg/L). Taxonomic composition of the biofilm communities was altered with increasing tetracycline concentrations. Metagenomic analysis revealed a concurrent increase of several tet genes and a range of other genes providing resistance to different classes of antibiotics (e.g. cmlA, floR, sul1, and mphA), indicating potential for co-selection. Consequently, MSCs for the tet genes of ≤ 1 μg/L suggests that current exposure levels in e.g. sewage treatment plants could be sufficient to promote resistance. The methodology used here to assess MSCs could be applied in risk assessment of other antibiotics as well.

Cholera toxin induces expression of ion channels and carriers in rat small intestinal mucosa

Cholera toxin causes cyclic adenosine monophosphate (cAMP)‐induced electrolyte and water secretion in the small intestine. The toxin‐induced change in gene expression in rat small intestine was evaluated with microarray technique and the results were confirmed by semiquantitative polymerase chain reaction (PCR). The transporter CNT2 for nucleosides was upregulated between 6 and 18 h after challenge, whereas the level of GLUT1 transporter for glucose became elevated at 6 h. Both changes probably facilitate uptake of these nutrients in the gut. At 18 h, the major chloride channel in the villus, ClC2, was upregulated. Aquaporin 8 was downregulated at 6 h and two mucin‐producing genes were upregulated 18 h after toxin challenge. The expression was back to normal after 72 h, which is the turnover time for intestinal epithelial cells.