Host and microbiota interactions are critical for development of murine Crohn's-like ileitis.

Deregulation of host-microbiota interactions in the gut is a pivotal characteristic of Crohn's disease. It remains unclear, however, whether commensals and/or the dysbiotic microbiota associated with pathology in humans are causally involved in Crohn's pathogenesis. Here, we show that Crohn's-like ileitis in Tnf(ΔARE/+) mice is microbiota-dependent. Germ-free Tnf(ΔARE/+) mice are disease-free and the microbiota and its innate recognition through Myd88 are indispensable for tumor necrosis factor (TNF) overexpression and disease initiation in this model. The epithelium of diseased mice shows no major defects in mucus barrier and paracellular permeability. However, Tnf(ΔARE/+) ileitis associates with the reduction of lysozyme-expressing Paneth cells, mediated by adaptive immune effectors. Furthermore, we show that established but not early ileitis in Tnf(ΔARE/+) mice involves defective expression of antimicrobials and dysbiosis, characterized by Firmicutes expansion, including epithelial-attaching segmented filamentous bacteria, and decreased abundance of Bacteroidetes. Microbiota modulation by antibiotic treatment at an early disease stage rescues ileitis. Our results suggest that the indigenous microbiota is sufficient to drive TNF overexpression and Crohn's ileitis in the genetically susceptible Tnf(ΔARE/+) hosts, whereas dysbiosis in this model results from disease-associated alterations including loss of lysozyme-expressing Paneth cells.

TNFα-dependent development of lymphoid tissue in the absence of RORγt⁺ lymphoid tissue inducer cells.

Lymphoid tissue often forms within sites of chronic inflammation. Here we report that expression of the proinflammatory cytokine tumor necrosis factor α (TNFα) drives development of lymphoid tissue in the intestine. Formation of this ectopic lymphoid tissue was not dependent on the presence of canonical RORgt(+) lymphoid tissue-inducer (LTi) cells, because animals expressing increased levels of TNFα but lacking RORgt(+) LTi cells (TNF/Rorc(gt)(-/-) mice) developed lymphoid tissue in inflamed areas. Unexpectedly, such animals developed several lymph nodes (LNs) that were structurally and functionally similar to those of wild-type animals. TNFα production by F4/80(+) myeloid cells present within the anlagen was important for the activation of stromal cells during the late stages of embryogenesis and for the activation of an organogenic program that allowed the development of LNs. Our results show that lymphoid tissue organogenesis can occur in the absence of LTi cells and suggest that interactions between TNFα-expressing myeloid cells and stromal cells have an important role in secondary lymphoid organ formation.

Attenuation of TNF-driven murine ileitis by intestinal expression of the viral immunomodulator CrmD.

Tumor necrosis factor α (TNFα) is a key pathogenic factor in Crohn's disease and rheumatoid arthritis. TNF(ΔARE) mice express high levels of TNFα and present Crohn's-like ileitis and arthritis. Alterations in the chemokine network could underline the TNF-driven ileitis. The aim of this study was to evaluate the role of TNF and chemokines in ileitis using ectromelia virus cytokine response modifier D (CrmD), a protein that binds TNFα and a limited number of chemokines. We generated transgenic mice expressing CrmD in intestinal epithelial cells (vCrmD mice) and crossed them with the TNF(ΔARE) mice to test whether CrmD could affect TNF-driven inflammatory processes. During homeostasis, only the number of B cells in the lamina propria was reduced by CrmD expression. Interestingly, CrmD expression in the intestine markedly attenuated the inflammatory infiltrates in the ileum of TNF(ΔARE) mice, but did not affect development of arthritis. Our results suggest that CrmD affects development of ileitis by locally affecting both TNF and chemokine function in the ileum.

CXCL13 expression in the gut promotes accumulation of IL-22-producing lymphoid tissue-inducer cells, and formation of isolated lymphoid follicles.

The chemokine CXCL13 is overexpressed in the intestine during inflammation. To mimic this condition, we created transgenic mice-expressing CXCL13 in intestinal epithelial cells. CXCL13 expression promoted a marked increase in the number of B cells in the lamina propria and an increase in the size and number of lymphoid follicles in the small intestine. Surprisingly, these changes were associated with a marked increase in the numbers of RORgammat(+)NKp46(-)CD3(-)CD4(+) and RORgammat(+)NKp46(+) cells. The RORgammat(+)NKp46(-)CD3(-)CD4(+) cells expressed CXCR5, the receptor for CXCL13, and other markers of lymphoid tissue-inducer cells, such as LTalpha, LTbeta, and TNF-related activation-induced cytokine (TRANCE). RORgammat(+)NKp46(-)CD3(-)CD4(+) gut LTi cells produced IL-22, a cytokine implicated in epithelial repair; and expressed the IL-23 receptor, a key regulator of IL-22 production. These results suggest that overexpression of CXCL13 in the intestine during inflammatory conditions favors mobilization of B cells and of LTi and NK cells with immunomodulatory and reparative functions.

Hyper immunoglobulin E response in mice with monoclonal populations of B and T lymphocytes.

A key event in the pathogenesis of allergies is the production of antibodies of the immunoglobulin (Ig)E class. In normal individuals the levels of IgE are tightly regulated, as illustrated by the low serum IgE concentration. In addition, multiple immunizations are usually required to generate detectable IgE responses in normal experimental animals. To define the parameters that regulate IgE production in vivo, we generated mice bearing monoclonal populations of B and T lymphocytes specific for influenza virus hemagglutinin (HA) and chicken ovalbumin (OVA), respectively. A single immunization of the monoclonal mice with the cross-linked OVA-HA antigen led to serum IgE levels that reached 30-200 microg/ml. This unusually high IgE response was prevented by the infusion of regulatory alpha/beta CD4(+) T cells belonging to both CD25(+) and CD25(-) subpopulations. The regulation by the infused T cells impeded the development of fully competent OVA-specific effector/memory Th2 lymphocytes without inhibiting the initial proliferative response of T cells or promoting activation-induced cell death. Our results indicate that hyper IgE responses do not occur in normal individuals due to the presence of regulatory T cells, and imply that the induction of regulatory CD4(+) T cells could be used for the prevention of atopy.

Regulatory T cells in spontaneous autoimmune encephalomyelitis.

Spontaneous experimental autoimmune encephalomyelitis (EAE) develops in 100% of mice harboring a monoclonal myelin basic protein (MBP)-specific CD4+ alphabeta T-cell repertoire. Monoclonality of the alphabeta T-cell repertoire can be achieved by crossing MBP-specific T-cell receptor (TCR) transgenic mice with either RAG-/- mice or TCR alpha-/-/TCR beta-/- double knockout mice. Spontaneous EAE can be prevented by a single administration of purified CD4+ splenocytes or thymocytes obtained from wild-type syngeneic mice. The regulatory T cells (T-reg) that protect from spontaneous EAE need not express the CD25 marker, as effective protection can be attained with populations depleted of CD25+ T cells. Although the specificity of the regulatory T cells is important for their generation or regulatory function, T cells that protect from spontaneous EAE can have a diverse TCR alpha and beta chain composition. T-reg cells expand poorly in vivo, and appear to be long lived. Finally, precursors for T-reg are present in fetal liver as well as in the bone marrow of aging mice. We propose that protection of healthy individuals from autoimmune diseases involves several layers of regulation, which consist of CD4+CD25+ regulatory T cells, CD4+CD25- T-reg cells, and anti-TCR T cells, with each layer potentially operating at different stages of T-helper cell-mediated immune responses.

Role of membrane-bound IgM in Trypanosoma cruzi evasion from immune clearance.

We have recently described that Trypanosoma cruzi parasites of the reticulotropic Y strain increase their resistance to antibody-induced clearance during their interaction with the vertebrate host immune system. In the present study, we observed that trypomastigotes of the myotropic CL strain isolated from normal host also display an increased resistance to immune clearance when compared to parasites obtained from immunosuppressed donors. Through fluorescence-activated cell sorting analysis, we have observed that the high expression of membrane-bound IgM antibodies on Y and CL trypomastigotes correlates with their enhanced resistance to Ig-induced clearance. Trypomastigotes from normal mice were essentially refractory to the in vitro binding of immunoglobulins, showing that their membrane structures were completely covered by IgM antibodies. These findings suggest that this isotype does not efficiently mediate immune clearance. Moreover, membrane-bound IgM antibodies limited the amount of IgG attached to the parasite and, as a consequence, impaired efficient immune clearance. Through this mechanism, trypomastigotes of T. cruzi could increase their persistence in the bloodstream thus favoring parasite transmission to its hematophagous host vector in the early acute phase of the disease.

Ig-isotype patterns of primary and secondary B cell responses to Plasmodium chabaudi chabaudi correlate with IFN-gamma and IL-4 cytokine production with CD45RB expression by CD4+ spleen cells.

In this work, the authors analysed T and B lymphocyte subsets and cytokine production in the spleen of BALB/c mice during polyclonal lymphocyte activation (primary infection) and parasite-specific response to Plasmodium chabaudi chabaudi (secondary infection). The secondary response was evaluated in fully immunoprotected animals, 60 days after a chloroquine-cured infection. The authors observed that in polyclonal lymphocyte activation antibody-secreting cells of all isotypes increased, with predominance of IgG2a and IgG3 classes. At that time, IFN-gamma was largely produced, but IL-4/IL-5 were just slightly enhanced. In mice re-infected after 60 days, the Ig-isotype pattern was restricted to IgG1 and only IL-4/IL-5 were produced. In both responses, however, the levels of IL-2 were greatly reduced, while those of IL-10 were enhanced to similar levels. The different involvement of Th1 and Th2 cells in both responses was confirmed through analysis of CD45RB expression by CD4+ cells. The authors observed that CD45RBhigh cells were the major CD4+ subpopulation in primary infected mice, while CD45RBlow cells predominated in 60 days re-infected animals. Moreover, the great majority of activated (large) CD4+ cells in the primary infection belonged to the CD45RBhigh subset, while after reinfection most of the CD4+ large had a CD45RBlow phenotype.

Laminin on Toxoplasma gondii mediates parasite binding to the beta 1 integrin receptor alpha 6 beta 1 on human foreskin fibroblasts and Chinese hamster ovary cells.

We investigated the role of parasite-bound laminin and the host cell beta 1 integrin receptors for this extracellular matrix protein in Toxoplasma gondii binding to fibroblasts. Laminin but not fibronectin was detected on extracellular tachyzoites by immunofluorescence and immunoblotting. Binding of parasites to CHO cells was inhibited by polyclonal antibodies to laminin and by a monoclonal antibody directed against the globular carboxyl-terminal portion of the long arm of laminin (at or near the suggested ligand-binding sites for alpha 3 beta 1 and alpha 6 beta 1), but not by a monoclonal antibody directed against the lateral short arms of laminin near the cross region of the molecule. Antibodies to the alpha 6 but not the alpha 2, alpha 3, or alpha 5 chains of the beta 1 family of integrins blocked parasite attachment to human foreskin fibroblasts and CHO cells. Attachment of T. gondii to cells via laminin on the parasite surface and laminin receptors on the mammalian cell is consistent with the capacity of the parasite to invade almost all nucleated cells.

Laminin enhances binding of Toxoplasma gondii tachyzoites to J774 murine macrophage cells.

We investigated the effects of the extracellular matrix proteins laminin and fibronectin on the attachment of tachyzoites of Toxoplasma gondii to the murine macrophage cell line J774. Laminin but not fibronectin increased parasite attachment to J774 cells in a dose-dependent fashion. Cyclic YIGSR, a laminin-derived peptide which inhibits laminin binding to the 32/67-kDa laminin-binding protein on host cells, blocked the laminin-mediated enhancement of parasite attachment. An antiserum to the 32/67-kDa laminin-binding protein also inhibited binding of parasites to J774 cells. These results, in conjunction with our previous observations (G. C. Furtado, F. L. Collins, and K. A. Joiner, submitted for publication), demonstrate that tachyzoites bearing surface laminin bind to multiple laminin receptors in attaching to different target cells.

Characterization of host cell-derived membrane proteins of the vacuole surrounding different intracellular forms of Trypanosoma cruzi in J774 cells. Evidence for phagocyte receptor sorting during the early stages of parasite entry.

Trypanosoma cruzi, an obligate intracellular protozoan parasite, exhibits developmental regulation of virulence. Although both noninfective epimastigote and infective trypomastigote stages of T. cruzi enter phagocytic cells via the formation of a parasitophorous vacuole (PV), only the latter developmental stages survive ingestion and perpetuate the infection. To determine whether the membrane composition of PV surrounding these different stages might contribute to differences in the outcome of infection, we identified selected membrane constituents by immunofluorescence and intracellular radioiodination, and studied their incorporation into PV. Complement receptors (CR3) are incorporated preferentially into the PV membrane surrounding serum-opsonized epimastigotes but not culture-derived metacyclic trypomastigotes. FcR are not preferentially incorporated into PV membranes unless epimastigotes or culture-derived metacyclic trypomastigotes are opsonized with anti-T. cruzi antibody. PV surrounding either parasite stage contain beta 1 integrins and lysosomal membrane glycoproteins (lgp). These results indicate that the plasma membrane glycoproteins incorporated into the surrounding PV membrane differ depending upon the stage of parasite being internalized, and that these differences reflect, at least in part, selective ligation of cell surface receptors mediating uptake. Furthermore, they imply that although virulent trypomastigote stages may avoid host cell uptake by conventional phagocytic receptors, i.e., CR3 or FcR, they do not escape fusion with an lgp-containing vacuole where they could still be exposed to lysosomal antimicrobial mechanisms.

Purification of F(ab')2 anti-snake venom by caprylic acid: a fast method for obtaining IgG fragments with high neutralization activity, purity and yield.

Pooled horse plasma containing antibodies against Crotalus durissus terrificus whole venom were digested with pepsin at an enzyme-substrate ratio of 8:1, pH 3.1, for 40 min and the F(ab')2M fragments purified by adding 8.7% caprylic acid (pH 5.0). For comparison, F(ab')2B purified by precipitation with ammonium sulphate and uncleaved IgG purified with caprylic acid were also prepared. Fab' fragments were obtained by reduction and alkylation of F(ab')2B. The anti-whole C.d. terrificus venom titers, determined by Dot-Blot were 12,800 (IgG), 6400 [F(ab')2B], 4800 [F(ab')2M] and 3200 (Fab'B). Immunochemical analysis of these fragments by SDS gel electrophoresis, Western blot and by double immunodiffusion revealed that the solution containing F(ab')2M was free of IgG and of other plasma proteins, whereas that containing F(ab')2B was not. One milligram of either F(ab')2B, F(ab')2M or Fab'B was able to neutralize respectively 20.7 micrograms, 20.2 micrograms and 13.8 micrograms of C.d. terrificus venom.