Dissecting Integrin Expression and Function on Memory B Cells in Mice and Humans in Autoimmunity

Immunological memory ensures life-long protection against previously encountered pathogens, and in mice and humans the spleen is an important reservoir for long-lived memory B cells (MBCs). It is well-established that integrins play several crucial roles in lymphocyte survival and trafficking, but their involvement in the retention of MBCs in secondary lymphoid organs, and differences between B cell subsets in their adhesion capacity to ICAM-1 and/or VCAM-1 have not yet been confirmed. Here, we use an autoimmune mouse model, where MBCs are abundant, to show that the highest levels of LFA-1 and VLA-4 amongst B cells are found on MBCs. In vivo blockade of VLA-4 alone or in combination with LFA-1, but not LFA-1 alone, causes a release of MBCs from the spleen into the blood stream. In humans, we find that in peripheral blood, spleens, and tonsils from healthy donors the highest expression levels of the integrins LFA-1 and VLA-4 are also found on MBCs. Consistent with this, we found MBCs to have a higher capacity to adhere to ICAM-1 and VCAM-1 than naïve B cells. In patients with the autoimmune disease rheumatoid arthritis, it is the MBCs that have the highest levels of LFA-1 and VLA-4; moreover, compared with healthy donors, naïve B and MBCs of patients receiving anti-TNF medication have enhanced levels of the active form of LFA-1. Commensurate levels of the active αL subunit can be induced on B cells from healthy donors by exposure to the integrin ligands. Thus, our findings establish the selective use of the integrins LFA-1 and VLA-4 in the localization and adhesion of MBCs in both mice and humans

The role of IL-17A and IFNγ in vaccine-induced protection against Helicobacter pylori

It is estimated that half the world’s population is infected with Helicobacter pylori in the stomach. Chronic H. pylori infection can lead to peptic ulcer disease or gastric cancer, but only in a sub-population of infected individuals. Eradication of the bacteria with antibiotic treatment can be successful, but the emergence of antibiotic resistant strains of H. pylori is a problem in areas endemic with H. pylori infection. A mucosal vaccine would have the potential for boosting the immune response to H. pylori, preventing and thus reducing the prevalence of the infection. In spite of decades of intense research, no vaccine has yet been found to be effective against H. pylori infection in humans. The work in this thesis aimed to evaluate the impact of varying the adjuvant and route of mucosal vaccinations on the gastric immune response and protection against H. pylori infection in a mouse model. In particular, the role of cytokines induced by H. pylori infection was evaluated, with an overriding objective to separate the protective and pathogenic immune response in the stomach. In the first part of the thesis, the adjuvant effect of a detoxified mucosal adjuvant based on the E. coli heat labile toxin LT, double mutant heat-labile toxin R192G/L211A (dmLT) was evaluated. Furthermore, the thesis addressed the differences if any, in immune responses and protection against H. pylori infection after sublingual (SL; under the tongue) and intragastric (IG) route of vaccination with H. pylori antigens and the prototype mucosal adjuvant cholera toxin (CT). And finally, using gene knockout mice and neutralizing antibodies, the impact of cytokines IFNγ and IL-17A on bacterial load and immune responses was addressed. Sublingual vaccination with H. pylori antigens and dmLT as an adjuvant was efficient in reducing the bacterial load in the stomach of mice, similar to when using the potent adjuvant CT, which is highly toxic in humans. Compared to infected unvaccinated mice, sublingual vaccination with dmLT enhanced stomach IL-17A and IFNγ secretion and proliferative responses to H. pylori antigens in mesenteric lymph nodes and spleen. Furthermore, we could show that there was a tendency for SL route to be more efficient than the IG route of vaccination in reducing the bacterial load in the stomach. And that the sublingual route of vaccination enhanced both IFNγ and IL-17A responses in the draining lymph nodes compared to unvaccinated mice. Studies on the role of individual cytokines in vaccine-induced responses revealed that after sublingual vaccination, IFN knockout (IFNγ-/-) mice were protected against H. pylori infection and had elevated IL-17A production and lower inflammation scores in the stomach compared to vaccinated wild-type mice. Furthermore, neutralization of IL-17A in sublingually vaccinated IFNγ-/- mice abrogated protection against H. pylori infection. As IL-17A was found to be important for vaccine-induced protection, we next examined the mechanisms for induction and maintenance of IL-17A after sublingual vaccination by studying the role of cytokines IL-1β and IL-23. Our results show that after sublingual vaccination, IL-23, but not IL-1β, deficient mice were protected against H. pylori infection. Gastric IL-17A responses could not be induced after challenge in the absence of IL-1β, but could be maintained in the absence of IL-23. In summary, we report that dmLT can be considered as a strong candidate mucosal adjuvant for use in a H. pylori vaccine in humans particularly when administered via the sublingual route. Furthermore, we show that IL-17A might contribute to protective immune responses, while IFNγ may promote inflammation. The results presented in this thesis will facilitate the design and administration of a vaccine against H. pylori infection in humans

Defining the Roles of IFN-γ and IL-17A in Inflammation and Protection against <i>Helicobacter pylori</i> Infection

<div><p>CD4⁺ T cells have been shown to be essential for vaccine-induced protection against <i>Helicobacter pylori</i> infection. However, the effector mechanisms leading to reductions in the gastric bacterial loads of vaccinated mice remain unclear. We have investigated the function of IFN-γ and IL-17A for vaccine-induced protection and inflammation (gastritis) using IFN-γ-gene-knockout (IFN-γ^-/-) mice, after sublingual or intragastric immunization with <i>H</i>. <i>pylori</i> lysate antigens and cholera toxin. Bacteria were enumerated in the stomachs of mice and related to the gastritis score and cellular immune responses. We report that sublingually and intragastrically immunized IFN-γ^-/- mice had significantly reduced bacterial loads similar to immunized wild-type mice compared to respective unimmunized infection controls. The reduction in bacterial loads in sublingually and intragastrically immunized IFN-γ^-/- mice was associated with significantly higher levels of IL-17A in stomach extracts and lower gastritis scores compared with immunized wild-type mice. To study the role of IL-17A for vaccine-induced protection in sublingually immunized IFN-γ^-/- mice, IL-17A was neutralized <i>in vivo</i> at the time of infection. Remarkably, the neutralization of IL-17A in sublingually immunized IFN-γ^-/- mice completely abolished protection against <i>H</i>. <i>pylori</i> infection and the mild gastritis. In summary, our results suggest that IFN-γ responses in the stomach of sublingually immunized mice promote vaccine-induced gastritis, after infection with <i>H</i>. <i>pylori</i> but that IL-17A primarily functions to reduce the bacterial load.</p></div

Relative Gene expression in stomach tissue after SL vaccination and IL-17A neutralization compared to non-immunized controls.

<p>§ RT-PCR array on stomach tissue. The difference between housekeeping gene β-actin and the target gene (∆<i>CT</i>) was determined, and the relative expression was calculated using the formula 2^∆<i>CT</i>. Values are then expressed as fold change in gene expression in vaccinated mice compared to stomach tissue taken from unvaccinated infected mice.</p><p>Relative Gene expression in stomach tissue after SL vaccination and IL-17A neutralization compared to non-immunized controls.</p

Neutralization of IL-17A abrogates protection, and reduces gastric inflammation and proliferation of MLN cells in sublingually immunized IFN-γ^-/- mice.

<p>IFN-γ^<b>-/-</b> mice were sublingually immunized with <i>H</i>. <i>pylori</i> lysate antigens and CT (SL) or left unimmunized (Inf) and infected with live <i>H</i>. <i>pylori</i> bacteria. Mice were injected intraperitoneally neutralizing IL-17A antibody (αIL-17A) or control IgG antibody (IgG). Two weeks post infection mice were sacrificed. <b>A.</b> Stomach tissue was analyzed for <i>H</i>. <i>pylori</i> colonization by quantitative culture and expressed as mean log₁₀ cfu per stomach, and SEM. <b>B.</b> analysis of IL-17A secretion in stomach tissue extracts and <b>C.</b> Atrophy and <b>D.</b> Infiltration in stomach tissue was scored. n = 6–11 mice/group, pool of two experiments. Bars represent mean. Statistically significant difference between sublingually immunized IFN-γ^<b>-/-</b> mice injected neutralizing IL-17A antibody compared to immunized mice injected isotype control antibody was calculated by an unpaired two-tailed t-test with Welch correction and denoted by * (p<0.05), ** (p<0.01), *** (p<0.001). <b>F.</b> single cell suspensions of MLN were prepared and cultured <i>in vitro</i> with <i>H</i>. <i>pylori</i> lysate antigens. Counts per minute (cpm) of incorporated radioactive thymidine was used as a measure of proliferation of the cells. Bars represent mean value and standard deviation (SD) counts in 6 individual wells in pooled mice (n = 5–7 mice/group) <b>G.</b> Supernatants were collected from <i>in vitro</i> cultured MLN (from D) and assessed for IL-17A shown in pg/ml, of six pooled wells. Data pool of two independent experiments. <b>E.</b> Stomach tissue was analyzed for gene expression of Lcn (Lipocalin-2) and expressed as relative gene expression where unimmunized infection control was set to 1. Statistically significant difference between sublingually immunized IFN-γ^<b>-/-</b> mice injected neutralizing IL-17A antibody compared to immunized mice injected isotype control antibody was calculated by an unpaired two-tailed t-test with Welch’s correction and denoted by *** (p<0.001).</p

Intragastrically or sublingually immunized IFN-γ^-/- and wild-type mice have higher Inflammation and atrophy scores.

<p>Groups of IFN-γ^<b>-/-</b> and wild-type (WT) were immunized via the SL or IG route with <i>H</i>. <i>pylori</i> lysate antigens and CT (SL or IG) and infected with live <i>H</i>. <i>pylori</i> bacteria. Unimmunized (Naïve) mice infected at the same time point served as infected controls (Inf). At 2 weeks post-infection the mice were sacrificed. Stomach tissue was sampled to determine the score the inflammation. <b>A.</b> Atrophy and <b>B.</b> Infiltration scores of formalin fixed stomach tissue stained with hematoxylin and eosin. Data represent pool of two independent experiments of three with similar results, bars represent mean values of n = 6–11 mice/group. Gastritis scores from immunized mice were compared to their respective infected controls and was assessed with the unpaired two-tailed t-test with Welch’s correction and denoted by * (p<0.05), ** (p<0.01), *** (p<0.001). <b>C.</b> Microscopy images (100x) of hematoxylin and eosin stained sections of a representative corpus area of stomach from unimmunized infected (Inf), SL and IG immunized IFNγ^<b>-/-</b> mice (top row) and wild-type mice (bottom row).</p

Splenocytes from both intragastrically and sublingually immunized IFN-γ^-/- mice and wild-type mice have increased <i>in vitro</i> proliferative responses, and cytokine secretion.

<p>Groups of IFNγ^<b>-/-</b> and wild-type (WT) mice were sublingually (SL) or intragastrically (IG) immunized with <i>H</i>. <i>pylori</i> lysate antigens and CT or left unimmunized (Inf) and then challenged with live <i>H</i>. <i>pylori</i> bacteria. Mice were sacrificed and spleen cells were isolated <b>A.</b> Single cell suspensions were prepared and cultured in 6 replicate wells together with <i>H</i>. <i>pylori</i> lysate antigens. Counts per minute (cpm) of incorporated radioactive thymidine were used as a measure of proliferation of the cells. Each dot represents an individual mouse and the bars the mean of the group. Representative data of one of three independent experiments. Supernatants were collected from <i>in vitro</i> cultured spleen cells (from A) and assessed for cytokines <b>B.</b> IL-17A and <b>C.</b> IFN-γ. Values are shown in pg/ml. Levels in immunized mice were compared to their respective infection controls and was assessed with the unpaired two-tailed t-test with Welch’s correction and denoted by * (p<0.05), ** (p<0.01), *** (p<0.001). ND: Not Detected, ns: not significant</p

IL-17A and IFN-γ production from Spleen and MLN cells of intragastrically or sublingually immunized IFN-γ^-/- and wild-type mice.

<p>Groups of IFN-γ^<b>-/-</b> and wild-type (WT) were immunized via the SL or IG route with <i>H</i>. <i>pylori</i> lysate antigens and CT (SL or IG) or left unimmunized (Inf) and then challenged with live <i>H</i>. <i>pylori</i> bacteria. At three weeks post challenge mice were sacrificed and spleen and MLN was taken for flow cytometric analysis. Spleen and MLN was taken from individual mice (n = 4) and single cell single cell suspensions were prepared and stimulated <i>in vitro</i> with PMA and Ionomycin and then stained for further flow cytometric analysis. <b>A</b> Gating strategy showing analysis of IL-17A and IFN-γ secreting cells among live CD3+ T cells. <b>B</b> Bar graph shows frequency mean + SD of IL-17A^<b>+</b> cells in spleen and MLN. Data represent one of two independent experiments.</p