Regulatory T cells may participate in Helicobacter pylori persistence in gastric MALT lymphoma: lessons from an animal model

Abstract

It has been postulated that the emergence of autoimmune gastritis in neonatal thymectomised (d3Tx) BALB/c mice may be a consequence of post-surgery deficit in Tregs. In this study, previously obtained samples from d3Tx mice were used in order to determine whether thymectomy creates a deficit in this T cell subset thereby allowing the emergence of autoimmune phenomena as a prerequisite for GML. The splenic Treg reserve and the local recruitment of these cells in the gastric mucosa were investigated using complementary molecular and immunohistochemistry approaches. Higher Foxp3/CD3 ratios were found in the spleen of non-infected d3Tx mice compared to non-thymectomised (NTx) controls. These results indicate a relative enrichment of Tregs following thymectomy in adult mice. The absence of Treg depletion in d3Tx mice is in line with the absence of auto-immune gastritis in non-infected d3Tx mice. Higher levels of T cell and Treg infiltration were also found in the stomach of GML-developing d3Tx mice versus NTx mice. Surprisingly, inflammatory scores inversely correlated with the bacterial inoculum. The presence of a small Treg containing compartment among gastric biopsies of GML developing d3Tx mice may play a role in perseverance of a minimal bacterial numbers thereby maintaining an antigen-dependent stimulation and proliferation.

Keywords: Helicobacter pylori; MALT lymphoma; animal model; regulatory T cell.

Figure 1. Evaluation of total T cells (CD3ε+) and Tregs (Foxp3+) reserve in non-infected (NI) mice spleens

A. Relative expression levels of Foxp3, CD3ε and Foxp3/CD3ε ratio quantified by qRT-PCR in NI non-thymectomised (NTx) (n = 10) and NI thymectomised (d3Tx) (n = 10) mice spleens. B. CD3ε and Foxp3 IHC stainings in one representative spleen from a NI d3Tx mice at 12 months post-infection. The quantification of these stainings, performed as described in the material and methods, on NI NTx (n = 10) and NI d3Tx (n = 9) mice spleens are represented as a Foxp3/CD3 ratio. Graphic representations are box plots, with the box representing 50% of the values around the median (horizontal line) and the whiskers representing the minimum and maximum of all the data, *p < 0.05, ns = non significant.

Figure 2. Evaluation of the lymphocytic infiltration in infected d3Tx mice stomachs

A. Relative expression levels of Foxp3 and CD3ε quantified by qRT-PCR in NI d3Tx (n = 10) or NI NTx (n = 8) as well as infected d3Tx (n = 29) or infected NTx (n = 39) mice stomachs. B. Evolution of relative expression levels of Foxp3 and CD3ε in d3Tx mice stomachs in comparison with inflammatory scores (NI, n = 10) (infected, n = 8, 10, 8 and 3 for scores 1, 2, 3 and 4, respectively). In A and B: Data are plotted as bar graphs displaying the mean ± standard deviation. ns: non-significant; *p < 0.05 versus NI mice for each group of mice (NTx or d3Tx); #p < 0.05 d3Tx compared to NTx. C. Example of B cells (CD45R+), T cells (CD3ε+) and Tregs (Foxp3+) after IHC staining on sections of a H. pylori-infected d3Tx mouse stomach (strain B47). Scale bars are indicated in μm. A higher magnification of Foxp3 IHC is shown (bar = 50 μm). D. Semi-quantitative evaluation of CD45R and CD3ε stainings in leucocyte infiltrates in d3Tx infected mice (n = 11). E. Semi-quantitative evaluation of Foxp3 and CD3ε stainings in leucocyte infiltrates in d3Tx infected mice (n = 11). In D and E, the results are expressed as percentage calculated with the ratio of surface of positive cells/the total surface of selected area for each marker, analyzed with the software Mercator. Graphic representations are box plots, with the box representing 50% of values around the median (horizontal line) and the whiskers representing the minimum and maximum of all the data.

Figure 3. Quantification of the bacterial load in gastric biopsies from infected mice

Results obtained by quantitative PCR. A. Bacteria/murine cell ratio in NTx (n = 40) versus d3Tx (n = 32) mice stomachs. B. Bacteria/murine cell ratio in NTx (n = 10 for each bacterial strain) versus d3Tx (n = 8 for each bacterial strain) mice stomachs classified according to the infecting bacterial strain: B38, B47, SS1 and TN2GF4. C. Bacteria/murine cell ratio in B47- and SS1-infected d3Tx mice stomachs only, classified according to inflammation scores obtained for each mouse (n = 3 and 11 for scores of 1 and 2–4, respectively). Data are presented as bar graphs displaying the mean ± standard deviation for each group, *p < 0.05.