Broad spectrum antibiotic enrofloxacin modulates contact sensitivity through gut microbiota in a murine model.

BACKGROUND: Medical advances in the field of infection therapy have led to an increasing use of antibiotics, which, apart from eliminating pathogens, also partially eliminate naturally existing commensal bacteria. It has become increasingly clear that less exposure to microbiota early in life may contribute to the observed rise in "immune-mediated" diseases, including autoimmunity and allergy. OBJECTIVE: We sought to test whether the change of gut microbiota with the broad spectrum antibiotic enrofloxacin will modulate contact sensitivity (CS) in mice. METHODS: Natural gut microbiota were modified by oral treatment with enrofloxacin prior to sensitization with trinitrophenyl chloride followed by CS testing. Finally, adoptive cell transfers were performed to characterize the regulatory cells that are induced by microbiota modification. RESULTS: Oral treatment with enrofloxacin suppresses CS and production of anti-trinitrophenyl chloride IgG1 antibodies. Adoptive transfer experiments show that antibiotic administration favors induction of regulatory cells that suppress CS. Flow cytometry and adoptive transfer of purified cells show that antibiotic-induced suppression of CS is mediated by TCR αß+CD4+CD25+FoxP3+ Treg, CD19+B220+CD5+ IL-10+, IL-10+ Tr1, and IL-10+ TCR γδ+ cells. Treatment with the antibiotic induces dysbiosis characterized by increased proportion of Clostridium coccoides (cluster XIVa), C coccoides-Eubacterium rectale (cluster XIVab), Bacteroidetes, and Bifidobacterium spp, but decreased segmented filamentous bacteria. Transfer of antibiotic-modified gut microbiota inhibits CS, but this response can be restored through oral transfer of control gut bacteria to antibiotic-treated animals. CONCLUSIONS: Oral treatment with a broad spectrum antibiotic modifies gut microbiota composition and promotes anti-inflammatory response, suggesting that manipulation of gut microbiota can be a powerful tool to modulate the course of CS.

Epicutaneous immunization with ovalbumin and CpG induces TH1/TH17 cytokines, which regulate IgE and IgG2a production.

BACKGROUND: Subcutaneous allergen-specific immunotherapy is a standard route for the immunotherapy of allergic diseases. It modulates the course of allergy and can generate long-term remission. However, subcutaneous allergen-specific immunotherapy can also induce anaphylaxis in some patients, and therefore additional routes of administration should be investigated to improve the safety and tolerability of immunotherapy. OBJECTIVE: We sought to determine whether epicutaneous treatment with antigen in the presence of a Toll-like receptor 9 agonist can suppress TH2-mediated responses in an antigen-specific manner. METHODS: Epicutaneous immunization was performed by applying a skin patch soaked with ovalbumin (OVA) plus CpG, and its suppressor activity was determined by using the mouse model of atopic dermatitis. Finally, adoptive cell transfers were implemented to characterize the regulatory cells that are induced by epicutaneous immunization. RESULTS: Epicutaneous immunization with OVA and CpG reduces the production of OVA-specific IgE and increases the synthesis of OVA-specific IgG2a antibodies in an antigen-specific manner. Moreover, eosinophil peroxidase activity in the skin and production of IL-4, IL-5, IL-10, and IL-13 are suppressed. The observed reduction of IgE synthesis is transferable with T-cell receptor (TCR) αß(+)CD4(+)CD25(-) cells, whereas IgG2a production is dependent on both TCRαß(+) and TCRγδ(+) T cells. Further experiments show that the described phenomenon is myeloid differentiation primary response 88, IFN-γ, and IL-17A dependent. Finally, the results suggest that epicutaneous immunization with OVA and CpG decreases the synthesis of OVA-specific IgE and skin eosinophil peroxidase activity in mice with ongoing skin allergy. CONCLUSION: Epicutaneous application of protein antigen in the presence of adjuvant could be an attractive needle-free and self-administered immunotherapy for allergic diseases.

Epicutaneous immunization with protein antigen in the presence of TLR4 ligand induces TCR alpha beta+CD4+ T contrasuppressor cells that reverse skin-induced suppression of Th1-mediated contact sensitivity.

Our previous work showed that epicutaneous (EC) immunization of mice with different protein Ags applied on the skin in the form of a patch induces a state of subsequent Ag-nonspecific unresponsiveness due to suppressor CD4+8+ T cells (Ts) that inhibit Th1-mediated contact sensitivity (CS) reactions via released TGF-beta. In the present work we show that EC immunization with Ag together with the TLR4 ligand LPS induced cells that could prevent suppression by the Ag-nonspecific Ts. These up-regulatory cells, called contrasuppressor T cells (Tcs), belong to a population of Ag-specific TCRalphabeta CD4+ lymphocytes and are different from Th1 CD4+ cells that mediate the CS reaction. Experiments using knockout mice showed that EC induced contrasuppression is MyD88, INF-gamma, and IL-12 dependent, whereas IL-6 is not involved in this phenomenon. Additional experiments with anti-IFN-gamma mAb showed that IFN-gamma is required for induction of Tcs cells but does not play a crucial role in the effector phase of contrasuppression. Additionally, treatment of CS effector cells with rIL-12 makes them resistant to EC induced suppression without affecting Ts cells, whereas IL-12 neutralization in vitro abrogates contrasuppression. These data show that IL-12 is indeed involved in the effector phase of EC induced contrasuppression and that this cytokine does not act directly on Ts cells. The mechanism of action of Tcs protects Th1 effector cells mediating CS from the nonspecific Ts, leaving suppression to other Ags intact. Ts and Tcs cells do not influence each other and can be induced simultaneously in the same animal.

Role of TLR ligands in epicutaneously induced contrasuppression.

Our previous work showed that epicutaneous (EC) immunization in mice with protein antigen (Ag) induced an Ag-independent unresponsiveness mediated by suppressor CD4(+)8(+) T cells (Ts), which inhibited contact hypersensitivity (CS). Simultaneous EC immunization with Ag and various Toll-like receptor (TLR) ligands reversed skin-induced suppression. Our present study shows that this process activates Ag-specific T contrasuppressor (Tcs) cells and leads to the protection of CS effector T cells from suppression. Epicutaneous immunization with Ag and the TLR4 ligand lipopolysaccharide (LPS) led to a significant increase in IFN-gamma production by lymph node and spleen cells. Ag and TLR ligands, like LPS, CpG or lipoteichoic acid did not need to be applied concomitantly to the skin. An identical contrasuppressive effect was observed when the Ag and TLR ligands were deposited on distant skin areas, suggesting that both the generation of Ts and Tcs are independent. To corroborate this finding, we used a model system that uses macrophages (Mf) as Ag-presenting cells. Mf labeled in vitro with Ag (Mf-Ag) induced, upon intravenous (iv) administration, an unresponsiveness reaction that was mediated by Ts cells. When treated simultaneously with LPS-treated Mf (Mf-Ag-LPS), a TLR-ligand could induce CS. Both the Ag and the LPS signal could be uncoupled i.e., Mf-Ag and Mf-LPS given at separate time points (with an 1 h interval between injections) induced immunity.We also found that LPS-treated Mf also produced significant amounts of IL-12, a cytokine that has well-known anti-tolerogenic properties. Our experiments suggest that reversal of EC-induced suppression by TLR-ligands may be a potential tool to increase the immunogenicity of weakly immunogenic antigens.

IgA-deficient humans exhibit gut microbiota dysbiosis despite secretion of compensatory IgM.

Immunoglobulin A is the dominant antibody isotype found in mucosal secretions and enforces host-microbiota symbiosis in mice, yet selective IgA-deficiency (sIgAd) in humans is often described as asymptomatic. Here, we determined the effects of IgA deficiency on human gut microbiota composition and evaluated the possibility that mucosal secretion of IgM can compensate for a lack of secretory IgA. We used 16S rRNA gene sequencing and bacterial cell sorting to evaluate gut microbiota composition and taxa-specific antibody coating of the gut microbiota in 15 sIgAd subjects and matched controls. Despite the secretion of compensatory IgM into the gut lumen, sIgAd subjects displayed an altered gut microbiota composition as compared to healthy controls. These alterations were characterized by a trend towards decreased overall microbial diversity as well as significant shifts in the relative abundances of specific microbial taxa. While secretory IgA in healthy controls targeted a defined subset of the microbiota via high-level coating, compensatory IgM in sIgAd subjects showed less specificity than IgA and bound a broader subset of the microbiota. We conclude that IgA plays a critical and non-redundant role in controlling gut microbiota composition in humans and that secretory IgA has evolved to maintain a diverse and stable gut microbial community.

What do physicians know about normal pressure hydrocephalus and when did they know it? A survey of 284 physicians.

Normal pressure hydrocephalus (NPH) is a relatively new neurologic disorder first described by Salamon Hakim of Bogotá, Colombia, in 1965. NPH is characterized by three symptoms - impaired gait, incontinence and dementia - and an anatomic abnormality, i.e., enlargement of the cerebral ventricles, which can be seen on computerized tomographic or magnetic resonance imaging. Surprisingly, the intracranial pressure is normal. The first author of this article, a Yale Medical School faculty member, developed NPH over the decade from 1992 to 2002, during which it was erroneously diagnosed as cerebral atrophy and/or Parkinson's disease. On recognizing the lack of awareness of NPH by physicians, he initiated a survey to explore this problem. He interviewed 166 practicing physicians who graduated from 50 American and 33 foreign medical schools, using a one-page, 10-point questionnaire (Part I). Almost one-third of the physicians had never heard of NPH. One-fifth had learned of NPH in medical school, and about half learned of it after medical school. Because there were insufficient physicians surveyed from 1986 to 2005, we recruited 118 additional physicians from the 20 Yale Medical School graduating classes from 1986 through 2005 (Part II). Two-thirds of them had learned of NPH in medical school, and one-fourth during residency and fellowship. Seven percent had never heard of NPH. The significance of these studies is discussed.

Antibiotics and autoimmune and allergy diseases: Causative factor or treatment?

The newborn infant emerges from an almost sterile environment into a world of bacteria. Bacteria colonize the infant's skin, lungs, and, of most importance, the gut. The process of bacterial colonization is coordinated, and each body niche acquires a unique composition of bacteria. In the gut, most bacteria belong to the Firmicutes and Bacteroidetes phyla, while Actinobacteria and Proteobacteria are far less abundant. Some of these bacteria possess strong immunoregulatory properties. Bacterial colonization is essential to skew the newborn's immune response away from the allergy-favoring Type-2 response towards a Type-1 immune response, which is essential for pathogen elimination. Imbalance between Type 1 and Type 2 responses, however, can promote autoimmunity. In addition, the microbiota shapes immune responses in adults. Autoimmune and allergic diseases are commonly associated with an altered composition of resident bacteria, which is known as dysbiosis. Perhaps the most common cause of disruption and alteration of the bacterial colonization of newborns is the use of antibiotics. It is not known whether the dysbiosis precedes or is the consequence of allergic and autoimmune disorders, and whether antibiotics can be a trigger for these disorders, depending on the type of antibiotic used and the maturity of immune system. In this review, we discuss the development of the microbiota in different body niches and their immunomodulatory potential. We evaluate the impact of antibiotics, both in mice and in humans, on microbial communities and how that may impact the development and manifestation of diseases through all stages of life: the prenatal period, childhood, and adulthood.

Toll-like receptor ligands reverse suppression of contact hypersensitivity reactions induced by epicutaneous immunization with protein antigen.

BACKGROUND: Epicutaneous (EC) immunization with protein antigens has been shown to induce antigen nonspecific suppression of subsequent T cell-dependent contact hypersensitivity (CS) reactions after active immunization. The aim of this work was to test if EC application of Toll-like receptor (TLR) ligands together with protein antigen could reverse suppression of CS. METHODS: Mice were EC immunized by applying gauze patches soaked with a solution of protein antigen alone or in the presence of crude bacterial material (bacterial lysates or heat-killed bacteria) or purified TLR ligands and then tested for CS response. To test if reversal of EC-induced suppression is antigen-specific, mice were patched with TNP- or OX-substituted mouse Ig alone or together with LPS and then tested for CS with corresponding or non-cross-reacting hapten. Influence of EC immunization on cytokine production by lymph node cells was measured by ELISA. RESULTS: EC immunization with protein antigen induces antigen nonspecific suppression that can be reversed by crude bacterial material as well as purified TLR-2, TLR-3, TLR-4, and TLR-9 ligands. The effect of TLR-4 ligand LPS was not observed in the Tlr-4 mutant C3H/HeJ mouse, indicating that this effect was dependent upon intact TLR-4 signaling. Unlike the antigen nonspecific suppression of CS by EC immunization with antigen alone, the reversal of suppression by TLR ligands was specific for the protein antigen applied in the EC protocol. CONCLUSIONS: Our results strongly suggest that EC immunization with protein antigen together with TLR ligands induces a particular antigen-specific cell population, akin to previously described contrasuppressor cells, which protects immune cells against the action of suppressor cells but have no direct influence on antigen nonspecific suppressor cells induced by antigen alone.

CD40 ligand-deficient T cells from X-linked hyper-IgM syndrome carriers have intrinsic priming capability.

Deficiency in CD40 ligand (CD40L) expression is associated with impaired T cell immunity in mouse models and in humans. Previous studies have indicated that this is due to the failure of induction of extrinsic costimulatory molecules. However, other studies have suggested that CD40L is an intrinsic costimulatory molecule. The X-linked hyper-IgM syndrome (XHIM) is a primary immunodeficiency caused by mutations in CD40L, resulting in impaired Ab production and T cell immunity. CD4+ T cells from female carriers of XHIM express a variable degree of normal CD40L based on random X chromosome inactivation. We have examined T cells from XHIM carriers to investigate whether CD40L supports T cell function by acting as an intrinsic costimulator or by induction of other costimulatory molecules by examining coexpression of CD40L and markers of T lymphocyte priming. These carriers provide a unique model for comparison of CD40L-expressing and -nonexpressing lymphocytes in that all factors, including immunological experience, are equivalent between the two populations. Our results show that compared with CD40L-deficient T cells, T cells that express CD40L normally have a minimal advantage in becoming primed, as defined by CD45 RO isoform expression and production of IFN-gamma and TNF-alpha. Conversely, CD40L-deficient T lymphocytes clearly were capable of becoming primed as defined by the same parameters. These findings imply that the intrinsic costimulatory activity of CD40L is not required for attaining primed status, and that CD40L primarily supports T cell function by inducing extrinsic factors that can be shared by CD40L-deficient cells.