• Optimal isolation of functional Foxp3+ induced regulatory T cells using DEREG mice.

      Baru, Abdul Mannan; Untucht, Christopher; Ganesh, Venkateswaran; Hesse, Christina; Mayer, Christian T; Sparwasser, Tim (2012)
      Foxp3 reporter mice including DEREG (DEpletion of REGulatory T cells) mice have greatly helped in exploring the biology of Foxp3(+) Tregs. DEREG mice express a DTR-eGFP fusion protein under the control of a bacterial artificial chromosome (BAC)-encoded Foxp3 promoter, allowing the viable isolation and inducible depletion of Foxp3(+) Tregs. Adaptive Tregs differentiated in vitro to express Foxp3 (iTregs) are gaining high interest as potential therapeutics for inflammatory conditions such as autoimmunity, allergy and transplant rejection. However, selective isolation of Foxp3(+) iTregs with a stable phenotype still remains to be a problem, especially in the human setting. While screening for culture conditions to generate stable CD4(+)Foxp3(+) iTregs from DEREG mice, with maximum suppressive activity, we observed an unexpected dichotomy of eGFP and Foxp3 expression which is not seen in ex vivo isolated cells from DEREG mice. Further characterization of eGFP(+)Foxp3(-) cells revealed relatively lower CD25 expression and a lack of suppressive activity in vitro. Similarly, eGFP(-) cells isolated from the same cultures were not suppressive despite of a broad CD25 expression reflecting mere T cell activation. In contrast, eGFP(+)Foxp3(+) iTregs exhibited potent suppressive activity comparable to that of natural eGFP(+)Foxp3(+) Tregs, emphasizing the importance of isolating Foxp3 expressing iTregs. Interestingly, the use of plate-bound anti-CD3 and anti-CD28 or Flt3L-driven BMDC resulted in considerable resolution of the observed dichotomy. In summary, we defined culture conditions for efficient generation of eGFP(+)Foxp3(+) iTregs by use of DEREG mice. Isolation of functional Foxp3(+) iTregs using DEREG mice can also be achieved under sub-optimal conditions based on the magnitude of surface CD25 expression, in synergy with transgene encoded eGFP. Besides, the reported phenomenon may be of general interest for exploring Foxp3 gene regulation, given that Foxp3 and eGFP expression are driven from distinct Foxp3 loci and because this dichotomy preferentially occurs only under defined in vitro conditions.
    • Salmonella enterica serovar Typhimurium infection-induced CD11b+ Gr1+ cells ameliorate allergic airway inflammation.

      Ganesh, Venkateswaran; Baru, Abdul Mannan; Hesse, Christina; Friedrich, Christin; Glage, Silke; Gohmert, Melanie; Jänke, Christine; Sparwasser, Tim (2014-03)
      Allergies are mainly characterized as an unrestrained Th2-biased immune response. Epidemiological data associate protection from allergic diseases with the exposure to certain infectious agents during early stages of life. Modulation of the immune response by pathogens has been considered to be a major factor influencing this protection. Recent evidence indicates that immunoregulatory mechanisms induced upon infection ameliorate allergic disorders. A longitudinal study has demonstrated reduced frequency and incidence of asthma in children who reported a prior infection with Salmonella. Experimental studies involving Salmonella enterica serovar Typhimurium-infected murine models have confirmed protection from induced allergic airway inflammation; however, the underlying cause leading to this amelioration remains incompletely defined. In this study, we aimed to delineate the regulatory function of Salmonella Typhimurium infection in the amelioration of allergic airway inflammation in mice. We observed a significant increase in CD11b+ Gr1+ myeloid cell populations in mice after infection with S. Typhimurium. Using in vitro and in vivo studies, we confirmed that these myeloid cells reduce airway inflammation by influencing Th2 cells. Further characterization showed that the CD11b+ Gr1+ myeloid cells exhibited their inhibitory effect by altering GATA-3 expression and interleukin-4 (IL-4) production by Th2 cells. These results indicate that the expansion of myeloid cells upon S. Typhimurium infection could potentially play a significant role in curtailing allergic airway inflammation. These findings signify the contribution of myeloid cells in preventing Th2-mediated diseases and suggest their possible application as therapeutics.
    • Schistosomes induce regulatory features in human and mouse CD1d(hi) B cells: inhibition of allergic inflammation by IL-10 and regulatory T cells.

      van der Vlugt, Luciën E P M; Labuda, Lucja A; Ozir-Fazalalikhan, Arifa; Lievers, Ellen; Gloudemans, Anouk K; Liu, Kit-Yeng; Barr, Tom A; Sparwasser, Tim; Boon, Louis; Ngoa, Ulysse Ateba; et al. (2012)
      Chronic helminth infections, such as schistosomes, are negatively associated with allergic disorders. Here, using B cell IL-10-deficient mice, Schistosoma mansoni-mediated protection against experimental ovalbumin-induced allergic airway inflammation (AAI) was shown to be specifically dependent on IL-10-producing B cells. To study the organs involved, we transferred B cells from lungs, mesenteric lymph nodes or spleen of OVA-infected mice to recipient OVA-sensitized mice, and showed that both lung and splenic B cells reduced AAI, but only splenic B cells in an IL-10-dependent manner. Although splenic B cell protection was accompanied by elevated levels of pulmonary FoxP3(+) regulatory T cells, in vivo ablation of FoxP3(+) T cells only moderately restored AAI, indicating an important role for the direct suppressory effect of regulatory B cells. Splenic marginal zone CD1d(+) B cells proved to be the responsible splenic B cell subset as they produced high levels of IL-10 and induced FoxP3(+) T cells in vitro. Indeed, transfer of CD1d(+) MZ-depleted splenic B cells from infected mice restored AAI. Markedly, we found a similarly elevated population of CD1d(hi) B cells in peripheral blood of Schistosoma haematobium-infected Gabonese children compared to uninfected children and these cells produced elevated levels of IL-10. Importantly, the number of IL-10-producing CD1d(hi) B cells was reduced after anti-schistosome treatment. This study points out that in both mice and men schistosomes have the capacity to drive the development of IL-10-producing regulatory CD1d(hi) B cells and furthermore, these are instrumental in reducing experimental allergic inflammation in mice.