Thomas W. Spahn

Mesenteric lymph nodes are critical for the induction of high-dose oral tolerance in the absence of Peyer's patches.

We have previously demonstrated the loss of oral tolerance (OT) in lymphotoxinα − / − (LTα − / −) and TNFα / lymphotoxinα deficient (TNFα / LTα − / −) mice which have defective Peyers patches (PP) and lymph node (LN) development. We have now studied OT in BALB / c mice with differential defects of the gut‐associated lymphoid tissue (GALT) caused by inhibition of LTβR signaling during fetal development. Treatment of pregnant mice with LTβR‐IgG (LTβRIgG) and TNFR I(55)‐IgG (TNFR55IgG) abrogates the formation of PP (LTβRIgG) or of PP and mesenteric LN (MLN) (LTβRIgG / TNFRIgG) without genetically deleting the respective cytokine pathways. OT was readily induced in mice without PP but retaining MLN (PP null / LN +). In contrast, OTcould not be induced in mice lacking both MLN and PP (PP null / MLN null) as shown by the inability of these mice to suppress IFN‐γ secretion or DTH reactions. We next assessed OT in 129 × B6 LTα − / − mice with and without MLN. Timed treatment of pregnant LTα − / − mice with an agonist anti‐LTβR mAb induces formation of MLN but not of PP in LTα − / − mice. LN + LTα −/ − mice developed OT while LN LTα − / − mice were resistant to OT induction. Taken collectively, the data show that in the presence of MLN PP are not required for OT induction and that the presence of MLN is sufficient for OT induction in the LTα − / − model.

Induction of oral tolerance to cellular immune responses in the absence of Peyer's patches

Systemic hyporesponsiveness occurs following oral administration of antigen (oral tolerance) and involves the uptake and processing of antigen by the gut‐associated lymphoid tissue (GALT), which includes Peyers patches (PP) lamina propria lymphocytes and mesenteric lymph nodes (MLN). Animals with targeted mutations of genes in the tumor necrosis factor (TNF) family have differential defects in the development of peripheral lymphoid organs including PP and MLN, and provide a unique opportunity to investigate the role of GALT structures in the induction of oral tolerance. Oral tolerance could not be induced in TNF/lymphotoxin (LT) α–/– mice, which are devoid of both PP and MLN, although these animals could be tolerized by intraperitoneal administration of antigen, demonstrating the requirement for GALT for oral tolerance induction. LTβ–/– mice and LTα/LTβ+/– animals do not have PP but could be orally tolerized, as measured by IFN‐γ production and delayed‐type hypersensitivity responses by administration of both low or high doses of ovalbumin. To further investigate the requirement for PP, we tested the progeny of LTβ‐receptor‐IgG‐fusion‐protein (LTβRigG)‐treated mice, which do not form PP but have an otherwise intact immune system. Although these animals had decreased fecal IgA production, they could be orally tolerized. Our results demonstrate that PP are not an absolute requirement for the induction of either high‐ or low‐dose oral tolerance, although oral tolerance could not be induced in animals devoid of both PP and MLN.

Oral tolerance induced by continuous feeding: enhanced up-regulation of transforming growth factor-β/interleukin-10 and suppression of experimental autoimmune encephalomyelitis

Oral administration of antigen leads to specific immune hyporesponsiveness termed as oral tolerance. Different doses and feeding regimens have been demonstrated to induce different types of tolerance and degrees of immune suppression. Herein, we compare distinct different regimens of feeding using equivalent final doses of antigen in order to investigate the role of frequency of antigen uptake in the induction of oral tolerance. We demonstrate that continuous feeding of antigen in the drinking water, as compared to a single feeding or feeding once per day over several days enhances suppression to both Th1 and Th2 type responses in B6D2F1 and BALB/c mice. Continuous feeding suppresses antibody responses in aged B6D2F1 mice, which are otherwise refractory to oral tolerance induction. Continuous feeding of ovalbumin (OVA) in high or low doses, as compared to control or single daily feeding over several days, up-regulates interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) production in both OVA TCR transgenic and BALB/c mice. In all regimens tested in wild type mice, low doses were more efficacious than high doses in inducing IL-10 and TGF-beta. Serial feeding (multiple low dose daily gavages) using OVA or myelin basic protein (MBP), also led to up-regulation of TGF-beta and IL-10 production in OVA TCR and MBP TCR transgenic mice, as well as enhanced inhibition of MBP-induced experimental autoimmune encephalomyelitis (EAE) in (PLxSJL) F1 mice. We did not find differences in the cytokine profile between serial (multiple low dose daily gavages) and continuous feeding regimens, suggesting that repetitive discrete delivery of oral antigen provides a sustained signal for the induction of oral tolerance. Thus, using different regimens of feeding that resemble natural feeding with equivalent final doses of antigen, we found enhancement of oral tolerance utilizing regimens that resemble natural feeding. Such feeding regimens may be advantageous in the application of oral tolerance for clinical purposes in the treatment of autoimmune and other inflammatory conditions.

Decreased severity of myelin oligodendrocyte glycoprotein peptide 33 – 35‐induced experimental autoimmune encephalomyelitis in mice with a disrupted TCR δ chain gene

Immunization of C57BL / 6 mice with myelin oligodendrocyte glycoprotein (MOG) peptide (p) 35 – 55 induces chronic experimental autoimmune encephalomyelitis (EAE). The role of γ δ T cells in the regulation of EAE is unclear. We investigated γ δ T cells in C57BL / 6 wild‐type mice and C57BL / mice with a disrupted TCRδ chain gene (δ– / – mice) using MOG p35 – 55. We found significantly less disease in δ– / – mice immunized with MOG / complete Freunds adjuvant (mean maximal EAE score 4.3 ± 0.8 in wild‐type vs. 2.3 ± 0.5 in δ– / – mice). Transfer of wild‐type spleen cells restored the ability of δ– / – mice to develop equally severe EAE as wild‐type mice. In addition to IFN‐γ, IL‐2, IL‐5 and IL‐10 was decreased in δ– / – mice. Decreased immune responses were also seen in δ– / – animals immunized with OVA peptide or protein and in concanavalin A‐stimulated splenocytes from δ– / – mice. Enriched dendritic cells from δ– / – mice secreted significantly less TNF‐α in response to lipopolysaccharide stimulation. Furthermore, when EAE was induced by adoptive transfer of an anti‐MOG p35 – 55 α β T cell line, there was a striking reduction of disease incidence (0 %) and severity in δ– / – as compared to wild‐type mice (83 % incidence). δ– / – mice showed no cellular infiltration in the spinal cord whereas wild‐type animals had infiltration of macrophages, B cells, α β‐ and γ δ T cells. In adoptive transfer EAE, there was reduced IL‐2 and IFN‐γ secretion in δ– / – mice. These results demonstrate an impaired immune response in the δ– / – mouse that is associated with a defect in developing both actively induced and adoptively transferred EAE.

Human Intestinal Microvascular Endothelial Cells Express Toll-Like Receptor 5: A Binding Partner for Bacterial Flagellin

Bacterial flagellin has recently been identified as a ligand for Toll-like receptor 5 (TLR5). Human sites known to specifically express TLR5 include macrophages and gastric and intestinal epithelium. Because infection of intestinal epithelial cells with Salmonella leads to an active transport of flagellin to the subepithelial compartment in proximity to microvessels, we hypothesized that human intestinal endothelial cells functionally express TLR5, thus enabling an active inflammatory response upon binding of translocated flagellin. Endothelial expression of TLR5 in human macro- and microvascular endothelial cells was examined by RT-PCR, immunoblot analysis, and immunofluorescence. Endothelial expression of TLR5 in vivo was verified by immunohistochemistry. Endothelial modulation of ICAM-1 expression was quantitated using flow cytometry, and leukocyte transmigration in vitro was assessed by an endothelial transmigration assay. Epithelial-endothelial cellular interactions upon infection with viable Salmonella were investigated using a coculture system in vitro. We found that Salmonella-infected intestinal epithelial cells induce endothelial ICAM-1 expression in cocultured human endothelial cells. Both macro- (HUVEC) and microvascular endothelial cells derived from human skin (human dermal microvascular endothelial cell 1) and human colon (human intestinal microvascular endothelial cells) were found to express high constitutive amounts of TLR5 mRNA and protein. These findings were paralleled by strong immunoreactivity for TLR5 of normal human colonic microvessels in vivo. Furthermore, incubation of human dermal microvascular endothelial cells with flagellin from clinical isolates of Escherichia and Salmonella strains led to a marked up-regulation of ICAM-1, as well as to an enhanced leukocyte transendothelial cell migration. These results suggest that endothelially expressed TLR5 might play a previously unrecognized role in the innate immune response toward bacterial Ags.

Absence of CCR6 Inhibits CD4+ Regulatory T-Cell Development and M-Cell Formation inside Peyer's Patches

The chemokine Mip3alpha is specifically expressed by the follicle-associated epithelia (FAE) covering intestinal Peyers patches (PPs) and is the only known chemokine ligand for the chemokine receptor CCR6. Although CCR6-deficient mice are known to have a perturbed intestinal immune system, little is known about the specific impact of this interaction for Peyers patch formation. To elucidate the effect of Mip3alpha on PP lymphocyte development, we used a CCR6/enhanced green fluorescent protein (EGFP) knock-in mouse model and analyzed lymphocyte development by immunohistochemistry and flow cytometry. PPs of CCR6-/- mice were significantly size-reduced with a proportional loss of B cells and T cells, whereas T-cell subsets were disturbed with a decreased CD4/CD8 ratio paralleled with a loss of regulatory CD4+ CD45Rb(low) T cells. The analysis of cytokine production by CCR6-expressing cells could demonstrate that CCR6 is involved in the regulation of cytokine secretion such as interleukin-12 by dendritic cells. Quantification of UEA-1+ cells inside the FAE showed reduced M-cell numbers in CCR6-deficient mice. These results suggest that the interaction of CCR6 with its ligand Mip3alpha is important for immune responses generated inside the PPs, particularly for the generation of regulatory CD4+ T cells residing inside PPs and for the formation of M cells.

Induction of Colitis in Mice Deficient of Peyer’s Patches and Mesenteric Lymph Nodes Is Associated with Increased Disease Severity and Formation of Colonic Lymphoid Patches

Inflammatory bowel disease is associated with immune activation in Peyers patches and mucosal lymph nodes. The role of these organs in dextran sodium sulfate (DSS)-induced colitis was investigated. We used mice lacking Peyers patches and/or lymph nodes because of lymphotoxin-alpha gene deficiency or treatment in utero with lymphotoxin-beta-receptor IgG and tumor necrosis factor-receptor-I (55)-IgG fusion proteins. Mice lacking Peyers patches and lymph nodes because of lymphotoxin-alpha deficiency or in utero fusion protein treatment developed more severe colitis than control mice as indicated by more severe intestinal shrinking, longer colonic ulcers, and higher histological disease scores. Oral DSS triggered the formation of colonic submucosal lymphoid patches in these mice and caused an increase in the number of submucosal lymphoid patches in mice treated in utero with the fusion proteins. Mice lacking Peyers patches only showed more submucosal lymphoid patches whereas intestinal length and histological disease score were similar to control mice. In conclusion, more severe DSS-induced colitis correlates with the loss of the mesenteric lymph nodes. However, neither the absence of Peyers patches nor the presence of colonic lymphoid patches were correlated with increased disease severity.

Resistance of Chemokine Receptor 6-Deficient Mice to Yersinia Enterocolitica Infection : Evidence of Defective M-Cell Formation in Vivo

M cells, specialized cells within Peyers patches (PPs), are reduced in number in chemokine receptor 6 (CCR6)-deficient mice. The pathogenic microorganism Yersinia enterocolitica exploits M cells for the purpose of mucosal tissue invasion exclusively through PPs. The aim of this study was to evaluate the course of yersiniosis in CCR6-deficient mice and to investigate whether these mice might be used as an in vivo model to determine M-cell function. After oral challenge with Y. enterocolitica, control mice suffered from lethal septic infection whereas CCR6-deficient mice showed very limited symptoms of infection. Immunohistochemical analysis demonstrated PP invasion by Y. enterocolitica in control mice whereas no bacteria could be found in CCR6-deficient mice. In addition, a significant induction of proinflammatory cytokines could be found in control mice whereas proinflammatory cytokine levels in CCR6-deficient mice remained unchanged. In contrast, intraperitoneal infection resulted in severe systemic yersiniosis in both mouse groups. Abrogated oral Y. enterocolitica infection in CCR6-deficient mice demonstrates the importance of CCR6 expression in the physiological and pathological immune responses generated within PPs by influencing M-cell differentiation, underscoring the important role of M cells in the process of microbial uptake. CCR6-deficient mice may therefore represent a suitable model for the study of M-cell function in vivo.

Role of Lymphotoxin in Experimental Models of Infectious Diseases: Potential Benefits and Risks of a Therapeutic Inhibition of the Lymphotoxin-β Receptor Pathway

The immune system provides different mechanisms to protect organisms against pathogens, most of which are infectious agents. Simultaneously, immune activation secondary to genetic factors and/or environmental signals can induce detrimental autoimmunity. The effector pathways in host defense and autoimmunity use similar cytokines and chemokines. Accordingly, tumor necrosis factor alpha (TNF-α), for instance, is similarly important in the control of various infections and in the induction of autoimmunity. Hallmarks of the adaptive immune system are antigen-specific cellular and humoral immune responses. Secondary lymphoid organs serve as sites of contact between antigen-presenting cells (APCs) and immune effector T and B lymphocytes. Chemokines and cytokines serve as messengers determining the type of immune response to a given antigen. The TNF family cytokine lymphotoxin (LT) plays a pivotal role in the development of secondary lymphoid organs. The chronic and relapsing course of many autoimmune diseases calls for new biological agents capable of suppressing the underlying inflammatory disorders. Recent studies indicate that inhibition of LTβ receptor (LTβR)-mediated signaling in adult animals suppresses autoimmunity by modulating the cellular structure of secondary lymphoid organs (reviewed in reference 22). Because of the wide range of autoimmune diseases positively influenced by this treatment, blockade of the LTβR might serve as a new treatment principle for human autoimmune diseases. However, immune responses to infectious pathogens are also altered in mice with disrupted LTβR signaling. While the course of virus- and lipopolysaccharide (LPS)-induced shock, experimental Trypanosoma brucei infection, cerebral malaria, and experimental prion disease are less severe, inhibition of the LTβR is also associated with exacerbation of mycobacterial infection and infectious colitis. This review summarizes the findings of studies using mice with disrupted LTβR signaling in models of infectious diseases and discusses the relevance of these observations in considering LTβR blockade as a potential treatment for human autoimmune diseases.

The Absence of Cutaneous Lymph Nodes Results in a Th2 Response and Increased Susceptibility to Leishmania major Infection in Mice

ABSTRACT Lymph nodes (LNs) are important sentinel organs where antigen-presenting cells interact with T cells to induce adaptive immune responses. In cutaneous infection of mice with Leishmania major, resistance depends on the induction of a T-helper-cell-1 (Th1)-mediated cellular immune response in draining, peripheral LNs. We investigated whether draining, peripheral LNs are absolutely required for resistance against L. major infection. We investigated the course of experimental leishmaniasis in wild-type (wt) mice lacking peripheral LNs (pLNs), which we generated by in utero blockade of membrane-bound lymphotoxin, and in mice lacking pLNs or all LNs due to genetic deletion of lymphotoxin ligands or receptors. wt mice of the resistant C57BL/6 strain without local skin-draining LNs were still able to generate specific T-cell responses, but this yielded Th2 cells. This switch to a Th2 response resulted in severe systemic infection. We also confirmed these results with mice lacking pLNs due to genetic depletion of lymphotoxin-β. The complete absence of LNs due to a genetic depletion of the lymphotoxin-β receptor also resulted in a marked deterioration of disease and a Th2 response. Thus, in the absence of pLNs, an L. major-specific Th2 response is induced in the remaining secondary lymphoid organs, such as the spleen and non-skin-draining LNs. This indicates a critical requirement for pLNs to induce protective Th1 immunity and suggests that whether Th1 or Th2 priming to the same antigen occurs depends on the site of the primary antigen recognition.