Claudia Veltkamp

Regulatory T cells are key cerebroprotective immunomodulators in acute experimental stroke

Systemic and local inflammatory processes have a key, mainly detrimental role in the pathophysiology of ischemic stroke. Currently, little is known about endogenous counterregulatory immune mechanisms. We examined the role of the key immunomodulators CD4+CD25+ forkhead box P3 (Foxp3)+ regulatory T lymphocytes (Treg cells), after experimental brain ischemia. Depletion of Treg cells profoundly increased delayed brain damage and deteriorated functional outcome. Absence of Treg cells augmented postischemic activation of resident and invading inflammatory cells including microglia and T cells, the main sources of deleterious cerebral tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), respectively. Early antagonization of TNF-α and delayed neutralization of IFN-γ prevented infarct growth in Treg cell–depleted mice. Intracerebral interleukin-10 (IL-10) substitution abrogated the cytokine overexpression after Treg cell depletion and prevented secondary infarct growth, whereas transfer of IL-10–deficient Treg cells in an adoptive transfer model was ineffective. In conclusion, Treg cells are major cerebroprotective modulators of postischemic inflammatory brain damage targeting multiple inflammatory pathways. IL-10 signaling is essential for their immunomodulatory effect.

IL-2-deficient mice raised under germfree conditions develop delayed mild focal intestinal inflammation

Interleukin-2 (IL-2) amplifies immune stimuli and influences B cell differentiation. IL-2-deficient mice spontaneously develop intestinal inflammation if raised under specific pathogen-free (SPF) conditions. We quantitatively determined the aggressiveness and kinetics of gastrointestinal and hepatic inflammation in the presence or absence of viable bacteria in IL-2-deficient mice. Breeding colonies were maintained under SPF and germfree (GF) conditions. Intestinal tissues, serum, and mesenteric lymph nodes were obtained from mice at different ages for blind histological scoring, immunoglobulin measurements, mucosal T cell infiltration, and cytokine secretion. GF IL-2 -/- mice developed mild, focal, and nonlethal intestinal inflammation with delayed onset, whereas the more aggressive inflammation in SPF IL-2 -/- mice led to their death between 28 and 32 wk. Periportal hepatic inflammation was equal in the presence or absence of bacterial colonization. Intestinal immunoglobulin secretion decreased significantly by 13 wk of age in IL-2 -/- mice in both GF and SPF environments. In contrast to other genetically engineered rodents, IL-2 -/- mice develop mild focal gastrointestinal and active portal tract inflammation in the absence of viable bacteria.Interleukin-2 (IL-2) amplifies immune stimuli and influences B cell differentiation. IL-2-deficient mice spontaneously develop intestinal inflammation if raised under specific pathogen-free (SPF) conditions. We quantitatively determined the aggressiveness and kinetics of gastrointestinal and hepatic inflammation in the presence or absence of viable bacteria in IL-2-deficient mice. Breeding colonies were maintained under SPF and germfree (GF) conditions. Intestinal tissues, serum, and mesenteric lymph nodes were obtained from mice at different ages for blind histological scoring, immunoglobulin measurements, mucosal T cell infiltration, and cytokine secretion. GF IL-2 -/- mice developed mild, focal, and nonlethal intestinal inflammation with delayed onset, whereas the more aggressive inflammation in SPF IL-2 -/- mice led to their death between 28 and 32 wk. Periportal hepatic inflammation was equal in the presence or absence of bacterial colonization. Intestinal immunoglobulin secretion decreased significantly by 13 wk of age in IL-2 -/- mice in both GF and SPF environments. In contrast to other genetically engineered rodents, IL-2 -/- mice develop mild focal gastrointestinal and active portal tract inflammation in the absence of viable bacteria.

Apoptosis of regulatory T lymphocytes is increased in chronic inflammatory bowel disease and reversed by anti-TNFα treatment

Background and aims Inappropriate immune responses contribute to the continuous stimulation of the intestinal immune system in chronic inflammatory bowel disease (IBD). Among several pathogenic factors, a numerical deficiency of regulatory T (Treg) cells has been suggested to lead to an insufficient compensation of chronically activated T lymphocytes. This study was conducted to investigate whether increased apoptosis contributes to Treg cell deficiency in IBD and whether successful treatment with antitumour necrosis factor α (TNFα) is achieved by reducing of Treg cell apoptosis. Methods Apoptosis of CD4+Foxp3+ Treg cells in tissue sections of patients with active IBD was analysed by immunohistochemistry and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) staining. Apoptosis of peripheral blood CD4+CD25+Foxp3+ Treg cells was investigated by flow cytometry and annexin-V staining. In addition, caspase activity and apoptosis were measured in sera of patients with IBD treated with anti-TNFα by a luminometric caspase enzyme assay. Results It is demonstrated that patients with active IBD revealed increased apoptosis of local CD4+Foxp3+ Treg cells in the inflamed mucosa compared with non-inflamed control colon tissue. Moreover, in peripheral blood a reduced frequency and increased apoptosis of Treg cells were found and accompanied by elevated caspase activity in the serum. During anti-TNFα treatment, Treg cell apoptosis declined in close correlation with elevated peripheral Treg cell numbers and a decrease of caspase activation and disease activity. Conclusions These data suggest that increased apoptosis of Treg cells plays a potentially important role in the pathogenesis of IBD and can be reversed by anti-TNFα treatment. Measurement of Treg cell apoptosis and serum caspase activity might therefore represent promising tools for monitoring disease activity and treatment response in patients with IBD.

Hyperbaric oxygen induces rapid protection against focal cerebral ischemia

BACKGROUND AND PURPOSE The timing and mechanisms of protection by hyperbaric oxygen (HBO) in cerebral ischemia have only been partially elucidated. We monitored the early in vivo effects of HBO after 2 h transient focal ischemia using repetitive MRI. METHODS Wistar rats underwent filament occlusion of the middle cerebral artery (MCAO). 40 min after MCAO, rats were placed in a HBO chamber and breathed either 100% O(2) at 3.0 atmospheres absolute (ata; n = 24) or at 1.0 ata (control; n = 24) for 1 h. Diffusion, perfusion and T2-weighted MR-images were obtained after 15 min and 3, 6 and 24 h of reperfusion. In 6 axial MR slices, volume of abnormal diffusion and T2w signals were measured in the ischemic hemisphere. Furthermore, hemispheric mean apparent diffusion coefficient- (ADC) and T2 values were calculated for statistical analysis. RESULTS HBO significantly reduced volume of abnormal DWI signal beginning immediately after reperfusion (control: 92 +/- 28 mm(3); HBO: 64 +/- 17) and lesion size on T2w (control: 375 +/- 91 mm(3); HBO: 225 +/- 39) after 24 h. Correspondingly, mean ADC levels were lower and T2 values higher in the ischemic hemisphere in the control group. HBO reduced histological infarct size at 24 h. CONCLUSION High-dose intraischemic HBO therapy has an immediate protective on the brain which is superior to normobaric oxygen.

Oxygen therapy in permanent brain ischemia: Potential and limitations

BACKGROUND Both normobaric (NBO) and hyperbaric (HBO) oxygen therapy are protective in transient cerebral ischemia. In contrast, in permanent ischemia models, which reflect the majority of clinical strokes, the effectiveness of NBO is unknown, and the effectiveness of HBO is controversial. The goals of the present study were to compare both oxygen therapies in 2 models of permanent ischemia, to study the effect of time window, and to evaluate the combination of both oxygen therapies. METHODS Distal or proximal permanent occlusion of middle cerebral artery (MCAO) was induced by coagulation or filament, respectively. Mice received air, NBO, a single or repeated HBO (3 ata) treatments. Infarct sizes were quantified at 7 days (coagulation) and 24 h (filament), respectively. RESULTS Following MCA coagulation, infarct volume was 12.9+/-1.6 mm3 in mice breathing air. When started 45 min or 120 min after MCAO, NBO (10.8+/-2.2) and significantly more potently HBO (7.8+/-0.9) reduced infarct size. Repeated HBO treatments had no additional effect (8.3+/-2.3). HBO also significantly decreased TUNEL cell staining at 24 h. Combination of 60 min NBO plus 60 min HBO resulted in smaller cortical infarcts (8.7+/-1.5) than 120 min NBO alone (11.1+/-3.2). In contrast, infarct volumes in filament-induced permanent MCAO did not differ among rodents receiving air (50+/-24 mm3), NBO (48+/-16), or HBO (46+/-21). After filament-induced transient MCAO, however, HBO reduced infarct volume significantly. CONCLUSIONS NBO and more effectively HBO protect the brain against permanent cortical ischemia. In extensive focal ischemia, however, oxygen therapy is only effective in case of early recanalization.

Hyperbaric oxygen reduces basal lamina degradation after transient focal cerebral ischemia in rats.

Hyperbaric oxygen (HBO) has been shown to preserve the integrity of the blood-brain barrier after cerebral ischemia. However, the underlying molecular mechanisms are currently unknown. We examined the effect of HBO on postischemic expression of the basal laminar component laminin-5 and on plasma matrix metalloproteinase-9 (MMP) levels. Wistar rats underwent occlusion of the middle cerebral artery (MCAO) for 2 h. With a delay of 45 min after filament introduction, animals breathed either 100% O2 at 1.0 atmosphere absolute (ata; NBO) or at 3.0 ata (HBO) for 1 h in an HBO chamber. Laminin-5 expression was quantified on immunohistochemical sections after 24 h of reperfusion. Plasma MMP-9 levels were measured using gelatin zymography before MCAO as well as 0, 6 and 24 h after reperfusion. Immunohistochemistry 24 h after ischemia revealed a decrease of vascular laminin-5 staining in the ischemic striatum to 43 +/- 26% of the contralateral hemisphere in the NBO group which was significantly attenuated to 73 +/- 31% in the HBO group. Densitometric analysis of zymography bands yielded significantly larger plasma MMP-9 levels in the NBO group compared to the HBO group 24 h after ischemia. In conclusion, HBO therapy attenuates ischemic degradation of cerebral microvascular laminin-5 and blocks postischemic plasma MMP-9 upregulation.

CD4+CD25+ cell depletion from the normal CD4+ T cell pool prevents tolerance toward the intestinal flora and leads to chronic colitis in immunodeficient mice

Background: CD4+CD25+ regulatory T cells have been shown to prevent immune‐mediated colitis in mice; however, it is unclear whether the absence of CD4+CD25+ in the normal CD4+ T cell pool is responsible for the development of chronic colitis. Using the T cell‐deficient Tg∈26 mouse model, we show that CD4+CD25− cells but not CD4+CD25+ cells induce a severe intestinal inflammation. Transfer of CD4+CD25+ cells, together with CD4+CD25− cells, ameliorated intestinal inflammation, and reconstitution with the whole mesenteric lymph node cell pool did not induce colitis in recipients. Transferred CD4+CD25− cells were found mainly in the mesenteric lymph nodes, where they showed an activated TH1‐like phenotype. In the absence of regulatory CD4+CD25+ T cells, recipient CD4+ cells secreted IFN‐&ggr; in response to stimulation with intestinal bacterial antigen that was prevented in vivo and in vitro by regulatory CD4+CD25+ cells. These studies suggest that CD4+CD25− cells have a strong colitogenic effect in the Tg∈26 colitis model and that CD4+CD25+ cells may be the main regulators that prevent or downregulate the proinflammatory effect of colitogenic T cells in the Tg∈26 mouse model.

Regulatory CD4+CD25+ cells reverse imbalances in the T cell pool of bone marrow transplanted TGε26 mice leading to the prevention of colitis

Background and aims: Erroneous thymic selection of developing T lymphocytes may be responsible for the expansion of self reactive T cells or may contribute to the absence of regulatory T cells important in controlling peripheral inflammatory processes. Colitis in bone marrow (BM) transplanted Tgε26 mice is induced by abnormally activated T cells developing in an aberrant thymic microenvironment. We investigated the protective role of regulatory CD4+CD25+ T cells in this model. Methods: BM from (C57BL/6×CBA/J) F1 mice was transplanted into specific pathogen free Tgε26 mice (BM⇒Tgε26). Transplanted mice received no cells (control), sorted CD4+CD25+, or CD4+CD25− cells from mesenteric lymph nodes (MLN) of normal mice. MLN cell subsets were analysed using membrane markers. Cytokine secretion of MLN cells was measured using intracellular cytokine staining and cytokine secretion in anti-CD3 stimulated cell cultures. Colitis was measured by histological scores. Results: CD4+CD25+ cells were reduced in the MLNs of BM⇒Tgε26 mice. Transfer of regulatory CD4+CD25+ but not of CD4+CD25− cells reduced the number of MLN CD4+ T cells in BM⇒Tgε26 recipients and increased the number of MLN CD8+ cells, thereby normalising the CD4+/CD8+ ratio. CD4+CD25+ but not CD4+CD25− cell transfer into BM⇒Tgε26 mice reduced the number of tumour necrosis factor α+ CD4+ cells and increased the secretion of transforming growth factor β by MLN cells. Transfer of 3×105 CD4+CD25+ cells after BM transplantation into Tgε26 mice prevented colitis whereas CD4+CD25− cells had no protective effect. Conclusions: These results suggest that defective selection or induction of regulatory T cells in the abnormal thymus is responsible for the development of colitis in BM⇒Tgε26 mice. Transfer of CD4+CD25+ cells can control intestinal inflammation in BM⇒Tgε26 mice by normalising the number and function of the MLN T cell pool.