Examining the Role of Microbiota in Emotional Behavior: Antibiotic Treatment Exacerbates Anxiety in High Anxiety-Prone Male Rats.

Intestinal microbiota are essential for healthy gastrointestinal function and also broadly influence brain function and behavior, in part, through changes in immune function. Gastrointestinal disorders are highly comorbid with psychiatric disorders, although biological mechanisms linking these disorders are poorly understood. The present study utilized rats bred for distinct emotional behavior phenotypes to examine relationships between emotionality, the microbiome, and immune markers. Prior work showed that Low Novelty Responder (LR) rats exhibit high levels of anxiety- and depression-related behaviors as well as myriad neurobiological differences compared to High Novelty Responders (HRs). Here, we hypothesized that the divergent HR/LR phenotypes are accompanied by changes in fecal microbiome composition. We used next-generation sequencing to assess the HR/LR microbiomes and then treated adult HR/LR males with an antibiotic cocktail to test whether it altered behavior. Given known connections between the microbiome and immune system, we also analyzed circulating cytokines and metabolic factors to determine relationships between peripheral immune markers, gut microbiome components, and behavioral measures. There were no baseline HR/LR microbiome differences, and antibiotic treatment disrupted the microbiome in both HR and LR rats. Antibiotic treatment exacerbated aspects of HR/LR behavior, increasing LRs' already high levels of anxiety-like behavior while reducing passive stress coping in both strains. Our results highlight the importance of an individual's phenotype to their response to antibiotics, contributing to the understanding of the complex interplay between gut microbes, immune function, and an individual's emotional phenotype.

Citrobacter rodentium: a model enteropathogen for understanding the interplay of innate and adaptive components of type 3 immunity.

Citrobacter rodentium is a natural murine intestinal pathogen that shares a core set of virulence factors with the related human pathogens enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC). C. rodentium is now the most widely used small animal model for studying the molecular underpinnings of EPEC and EHEC infections in vivo, including: enterocyte attachment; virulence; colonization resistance; and mucosal immunity. In this review, we discuss type 3 immunity in the context of C. rodentium infection and discuss recent publications that use this model to understand how the innate and adaptive components of immunity intersect to mediate host protection against enteric pathogens and maintain homeostasis with the microbiota.

Free radical-producing myeloid-derived regulatory cells: potent activators and suppressors of lung inflammation and airway hyperresponsiveness.

Levels of reactive free radicals are elevated in the airway during asthmatic exacerbations, but their roles in the pathophysiology of asthma remain unclear. We have identified subsets of myeloid-derived suppressor-like cells as key sources of nitric oxide and superoxide in the lungs of mice with evolving experimental allergic airway inflammation and established these cells as master regulators of the airway inflammatory response. The profiles of free radicals they produced depended on expression of inducible nitric oxide synthase (iNOS), arginase, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. These radicals controlled the pro- and anti-inflammatory potential of these cells, and also regulated the reciprocal pattern of their infiltration into the lung. The nitric oxide-producing cells were Ly-6C(+)Ly-6G(-) and they downmodulated T-cell activation, recruited T(reg) cells, and dramatically downregulated antigen-induced airway hyperresponsiveness. The superoxide-producing cells were Ly-6C(-)Ly-6G(+) and they expressed proinflammatory activities, exacerbating airway hyperresponsiveness in a superoxide-dependent fashion. A smaller population of Ly-6C(+)Ly-6G(+) cells also suppressed T-cell responses, but in an iNOS- and arginase-independent fashion. These regulatory myeloid cells represent important targets for asthma therapy.

Early upregulation of T cell IL-10 production plays an important role in oral tolerance induction.

Early in oral tolerance induction, IL-10-producing CD4(+) T cells were increased, and adoptive transfer of IL-10-deficient CD4(+) T cells failed induction of oral tolerance, suggesting a key role of IL-10 production in such a process.

Unexpected characteristics of the IFN-gamma reporters in nontransformed T cells.

Analysis of the IFN-gamma promoter has primarily been conducted by transient expression of reporter constructs in transformed cells. However, the activity of cis elements may differ when expressed transiently compared with their activity within native chromatin. Furthermore, the transcription factors and signaling mechanisms in transformed cells may differ from those in normal T cells. To analyze IFN-gamma promoter regulation in normal T cells, we developed a novel retroviral bottom-strand reporter system to allow the chromatin integration of promoter regions in primary developing T cells. As controls, both the IL-2 and IL-4 promoters were inducible in this system, with the IL-4 reporter having Th2-specific activity. Strikingly, the IFN-gamma promoter exhibited constitutive activity in both Th1 and Th2 subsets, in contrast to the behavior of the endogenous IFN-gamma gene, which is inducible only in Th1 cells. In mapping this activity, we found that the AP-1/GM-CSF site in the distal promoter element is the most critical element for the constitutive activity. Transgenic reporter lines for the IFN-gamma promoter confirmed the constitutive behavior of the isolated IFN-gamma promoter. This constitutive activity was resistant to inhibition by cyclosporin A and was independent of Stat4 and p38 mitogen-activated protein kinase. These results suggest that IFN-gamma promoter regulation may require cis elements residing either downstream or >3.4 kb upstream of the transcriptional start site, involving repression of constitutive activity.

Immuno-bacterial homeostasis in the gut: new insights into an old enigma.

The intestinal mucosa is the interface between the immune system and the massive antigenic load represented by the commensal enteric bacteria. These commensal bacteria drive the development of the mucosal immune system, and in turn most of the lymphocytes in the intestinal mucosa appear to be specific for enteric bacteria antigens. Proper regulation of the responses of these anti-bacterial lymphocytes are extremely important because T cell effectors reactive to enteric bacterial antigens have been shown to cause chronic intestinal inflammation in an adoptive transfer system. The cells and molecules important in regulating mucosal immune response are now being identified. Insights into the mechanisms of mucosal regulation have come from a number of genetically manipulated mouse strains which develop inflammatory bowel disease in response to the enteric bacterial flora. CD4(+)T cells with regulatory function in the mucosa are being identified; other cell types such as CD8(+)T cells. NK cells, and B cells may also have a role in mucosal immune regulation. A model for T cell-immune homeostasis in the intestinal mucosa is presented.

Differential expression of Fas ligand in Th1 and Th2 cells is regulated by early growth response gene and NF-AT family members.

Inducible expression of Fas ligand (CD95 ligand) by activated T cells and the resulting apoptosis of CD95-bearing cells is a critical component of peripheral T cell homeostasis and cytotoxic effector mechanisms. Transcriptional control of the expression of Fas ligand has been attributed to a number of factors, including early growth response gene 2 (Egr2), Egr3, Sp1, and NF-AT, although a direct contribution of NF-AT is controversial. The present study confirms a role for Egr factors and indicates that NF-AT is essential for optimal expression of murine Fas ligand through a direct interaction with an NF-AT consensus element. The role of these factors was further defined by studying the differential expression of Fas ligand in Th1 and Th2 lines derived from DO11.10 TCR transgenic mice. EMSA analyses of a composite Egr/NF-AT site showed recruitment of Sp1 to this site in Th2 cells, but not in Th1 cells. Furthermore, gel-shift analyses demonstrated the binding of Egr1, 2, and 3 in Th2 cells and Egr1 and 2, but not Egr3 in Th1 cells at a known Egr site. Northern analysis corroborated the lack of Egr3 in Th1 cells. Differential usage of these transcription factors by Th1 and Th2 cells suggests a potential mechanism underlying the differential expression of Fas ligand by distinct T cell lineages.

Colitis induced by enteric bacterial antigen-specific CD4+ T cells requires CD40-CD40 ligand interactions for a sustained increase in mucosal IL-12.

C3H/HeJBir is a mouse substrain that is highly susceptible to colitis. Their CD4+ T cells react to Ags of the commensal enteric bacteria, and the latter can mediate colitis when activated by these Ags and transferred to histocompatible scid recipients. In this study, multiple long-term C3H/HeJBir CD4+ T cell (Bir) lines reactive to commensal enteric bacterial Ags have been generated. All these were Ag specific, pauciclonal, and Th1 predominant; most induced colitis uniformly after transfer to scid recipients. Lesions were focal and marked by increased expression of IL-12p40 and IFN-gamma mRNA and protein. Pathogenic Bir T cell lines expressed CD40 ligand (CD40L) when cultured with Ag-pulsed APCs in vitro. Production of IL-12 was also increased in such cultures, an effect that was Ag- and T cell-dependent and required costimulation by CD40, but not by B7. The two Bir T cell lines that did not induce lesions after transfer failed to significantly express CD40L or increase IL-12 when cultured with Ag-pulsed APCs. Administration of anti-CD40L blocked disease expression induced by pathogenic T cells. We conclude that interactions in the colon mucosa between CD40L-expressing Bir Th1 cells with APCs endogenously loaded with commensal bacterial Ags are critical for sustained increases in local IL-12 production and progression to colitis.

Interleukin-2 expression by a subpopulation of primary T cells is linked to enhanced memory/effector function.

Single cell studies have identified intraclonal heterogeneity of cytokine production by activated T cells. To investigate implications of cytokine heterogeneity for cell fate, an interleukin (IL)-2 promoter-green fluorescent protein (GFP) reporter transgenic model was developed to track IL-2+ and IL-2- T cells during differentiation from naive precursors. Antigen-activated IL-2+ and IL-2- cells had comparable proliferative capacities in primary responses. However, T cells that expressed IL-2 in primary responses demonstrated enhanced antigenic sensitivity and increased expression of effector cytokines in secondary responses in vitro and in vivo. Thus, heterogeneity of activation during a primary response translates into heterogeneous secondary responses, in which enhanced memory/effector function is linked to cells that previously exceeded an activation threshold associated with IL-2 gene transcription.

Memory/effector T cells in TCR transgenic mice develop via recognition of enteric antigens by a second, endogenous TCR.

The majority of clonotypic CD4(+) T cells in the intestinal lamina propria of DO11.10 TCR transgenic mice have an activated/memory phenotype and produce effector cytokines despite the absence of prior exposure to ovalbumin (OVA), the transgene-specific antigen. A small number of splenic T cells have a similar phenotype. Clonotypic T cells from Peyer's patch are intermediate in both phenotype and effector cytokine production. Flow cytometric analysis of cells isolated from thymectomized, OVA-naive DO11.10 mice treated with continuous administration of BrdU indicated that a significant fraction of clonotype-positive T cells in the lamina propria and Peyer's patch were in the cell cycle, with significantly fewer cycling cells in the spleen. Most of the cycling cells from each anatomic site expressed low levels of CD45RB. Effector cytokine expression was enriched in the CD45RB(low) populations. These memory/effector cell populations were eliminated in DO11.10/SCID and DO11.10/RAG-2(-/-) mice, suggesting that recognition of non-OVA antigens through a second, non-clonotypic TCR was driving differentiation of memory/effector cells in naive BALB/c DO11.10 mice. Clonotypic CD4(+) T cells isolated from DO11.10, but not from DO11.10/SCID or DO11.10/RAG-2(-/-) mice, were stimulated to enter the cell cycle by antigen-presenting cells pulsed with an intestinal bacterial antigen extract. These data provide direct evidence that enteric bacterial antigens can activate transgenic T cells through a second, non-clonotypic TCR, and support the notion that the development and turnover of memory/effector cells in vivo is driven by the intestinal flora.

Heterogeneity in the clonal T cell response. Implications for models of T cell activation and cytokine phenotype development.

The T cell can be defined in the context of two properties--the recognition specificity of the T cell receptor (TCR) heterodimer and the functional response of the T cell after TCR stimulation. Once a particular TCR heterodimer is expressed and successfully selected during thymic development, the antigen specificity is fixed for all the clonal progeny of that cell. In contrast, the potential functional responses that may be generated in response to specific antigen in the postthymic environment are quite extensive. These range from programmed cell death to initiation of alternate programs of phenotype development that generate effector populations with distinct cytokine expression patterns and regulatory properties. Recent advances in analytical methods that have permitted multiparametric characterizations of the T cell response at the single cell, rather than population level, have necessitated a modified view of T cell activation and the clonal T cell response, and have generated new insights into the regulation of immunity. In this brief review, we highlight studies that have characterized heterogeneity of the CD4+ T cell clonal response based on single-cell analyses, and discuss implications for models of T cell activation and cytokine phenotype development.

Experimental models to study molecular mechanisms underlying intestinal inflammation.

Experimental animal models, particularly the newer mouse models, have convincingly demonstrated that CD+ T cells play a central role in chronic intestinal inflammation. Such CD4+ effector T cells are induced by the bacterial flora. In at least one model, it is conventional protein antigens that are stimulating these pathogenic T cells. The antigens driving disease seem to be a selective subset of immunodominant proteins, likely derived from a subset of organisms. Multiple genes contribute to colitis susceptibility and a number of these genes are being localized.

Single-cell analyses of CD4+ T cells from alpha beta T cell receptor-transgenic mice: a distinct mucosal cytokine phenotype in the absence of transgene-specific antigen.

Development of distinct CD4+ T cell cytokine phenotypes may be conditioned by the anatomic site in which activation occurs. A double-label in situ hybridization technique was used to characterize co-expression of cytokine mRNA in antigen-specific responses of Peyer's patch (PP), lamina propria (LP), and splenic (SP) CD4+ T cells isolated from alpha beta T cell receptor-transgenic mice. Interleukin (IL)-2 was the dominant cytokine expressed by antigen-stimulated PP and SP populations, though it was expressed by a minority of the activated T cells. Cells that expressed interferon (IFN)-gamma were less frequent, and IL-4, IL-5, and IL-10 were infrequent. In contrast, cells that expressed IFN-gamma or IL-10 were most frequent in the LP population, with lower frequencies of IL-2, and few IL-4- and IL-5-positive cells. Co-expression of two cytokines by the same cell was the exception, regardless of the anatomic site from which the T cells were isolated. The surface phenotype of transgene-positive T cells isolated from each anatomic site was distinct, despite the absence of in vivo exposure to antigen for which the transgenic T cell receptor is specific. These data suggest that the cytokine responses of CD4+ T cells may be conditioned by the microenvironment, independently of specific antigen, and that the LP CD4+ T population has a distinct cytokine expression pattern with counter-regulatory properties that may be important for homeostasis in mucosal immune tissues.

Visualization of antigen-specific T cell activation and cytokine expression in vivo.

A transgenic TCR adoptive transfer system was used to visualize Ag-specific T cell activation and cytokine expression in vivo. After s.c. injection of peptide in adjuvant the entire Ag-specific population up-regulated IL-2 receptor alpha-chain expression, underwent blast transformation, and developed a memory-surface phenotype. A minority of the Ag-specific T cells produced predominantly IL-2 mRNA and localized at the T cell/B cell junction in draining lymph nodes. In the secondary response, a mixed cytokine pattern of both Th1 and Th2 cytokines was demonstrated. When peptide was administered i.v. without adjuvant, 50% of the Ag-specific cells expressed IL-2, but the peak of expression occurred before IL-2 receptor alpha-chain up-regulation, and only a minority of the Ag-specific T cells underwent blast transformation.

The mucosal adjuvanticity of cholera toxin involves enhancement of costimulatory activity by selective up-regulation of B7.2 expression.

Cholera toxin (CT) is a potent mucosal immunogen and adjuvant that can strongly prime mucosal T cells. The present study was undertaken to investigate the effects of CT on the expression and functional activity of the costimulatory molecules B7.1 and B7.2 on macrophages and the relationship of these effects to the mucosal adjuvanticity of CT. Bone marrow macrophages (BMM) were generated by culturing bone marrow with macrophage CSF or granulocyte-macrophage CSF. After treatment with either CT alone or IFN-gamma alone, B7.2 expression on BMM was moderately up-regulated and was further increased when BMM were treated with both CT and IFN-gamma together. Interestingly, CT had no effect on B7.1 expression despite the close relationship between these two molecules. Up-regulation of B7.2 expression by CT was mediated by intracellular cAMP production, in that CT-B subunit had no effect and dibutyryl cAMP could mimic the effect. CT increased functional costimulatory activity of macrophages for both anti-CD3-stimulated and allostimulated T cells, an increase that was blocked by anti-B7.2, but not anti-B7.1, Ab. B7.2 expression by Mac1+ Peyer's patch cells was increased after intraluminal exposure to CT in vivo. Treatment of mice with anti-B7.2 Ab in vivo inhibited both the mucosal adjuvanticity and the immunogenicity of CT. We conclude that CT enhances the costimulatory activity of mucosal APC by differentially up-regulating B7.2 expression, an effect that appears to be important for its mucosal adjuvanticity and immunogenicity.

Single cell analysis of cytokine gene coexpression during CD4+ T-cell phenotype development.

CD4+ T cells from alpha beta-T-cell receptor transgenic mice were analyzed for coexpression of cytokine mRNAs during phenotype development using a double-label in situ hybridization technique. T cells that produced cytokines in the primary response were a fraction of the activated population, and only a minority of the cytokine-positive cells coexpressed two cytokines. In secondary responses, frequencies of double-positive cells increased, although they remained a minority of the total. Of the cytokine pairs examined, interleukin (IL)-4 and IL-5 were the most frequently coexpressed. IL-4 and interferon gamma showed the greatest tendency toward segregation of expression, being rarely coexpressed after the primary stimulation. These data indicate that there is significant heterogeneity of cytokine gene expression by individual CD4+ T cells during early antigenic responses. Coexpression of any pairs of cytokines, much less Th1 and Th2 cytokines, is generally the exception. The Th0 phenotype is a population phenotype rather than an individual cell phenotype.

In situ hybridization for cytokine mRNA with digoxigenin-labeled riboprobes. Sensitivity of detection and double label applications.

A sensitive in situ hybridization procedure using both digoxigenin and 35S-labeled riboprobes is described that allows detection of single T cells expressing cytokine mRNA species in both single and double label formats. Modifications to existing procedures have been developed that allow in situ hybridization to be performed in either fresh frozen tissue sections or cytocentrifuge preparations of cultured cells. For single label studies, the digoxigenin labeling technique is equivalent to 35S labeling for sensitivity of detection and is superior with respect to precise localization and ease of use. A procedure to detect two cytokine mRNA species in individual cells can be performed using one digoxigenin-labeled riboprobe and one 35S-riboprobe, with equivalent sensitivity between the two labels and no non-specific mixing of the two signals. Since production of many T cell cytokines are controlled by transcriptional mechanisms, the use of in situ hybridization will be useful to investigate the biology of T cell activation, patterns of cytokine phenotype development, and histological localization of cytokine expressing cells in inflammatory lesions. Initial studies using this method to examine cytokine expression by a panel of T cell clones reveals that individual cytokine genes are not necessarily expressed in coordination in individual cells and relatively few individual cells in a Th0 clone express Th1-like and Th2-like cytokines simultaneously.

Heterogeneity of single cell cytokine gene expression in clonal T cell populations.

T helper type 0 (Th0), Th1, and Th2 CD4+ T cell clones derived from a T cell receptor alpha/beta (TCR-alpha/beta) transgenic mouse were activated by antigen presented on "artificial" antigen-presenting cells that expressed or lacked the costimulatory molecule B7-1, and were analyzed for single cell cytokine mRNA expression by in situ hybridization. There was significant heterogeneity in the frequency of T cells that expressed individual cytokine mRNAs within each clonal population, suggesting that transcriptional control of each of the cytokine genes was not coordinate within an individual cell. The majority of antigen-stimulated Th1 cells expressed mRNA for interferon gamma (IFN-gamma), but far fewer cells in the same population expressed interleukin 2 (IL-2). Similarly, the frequency of IL-4-expressing cells was greater than that of IL-5- or IL-10-expressing cells in the same Th2 population, but the difference in expression frequencies was more variable between clones. The expression frequencies of each of the cytokines was quite heterogeneous in the antigen-activated Th0 population. The principal effect of increased antigen on the activation of individual cytokine genes in each of the clonal populations was to increase recruitment of mRNA-positive cells, with little or no effect on the level of cytokine mRNA expression in individual positive cells. The effects of B7 costimulation were variable depending on the cytokine gene analyzed. B7 costimulation markedly increased the frequency and the level of IL-2 mRNA expression in individual positive cells in the Th1 and Th0 populations, with less effect on the recruitment and single cell expression level of IFN-gamma. IL-4 frequencies were modestly increased by B7 costimulation of the Th2 clones, but there was no detectable increase in single cell IL-4 expression level. The observed patterns of cytokine mRNA expression favor a model of T cell activation in which all-or-none, rather than graded, responses of cytokine genes are dominant.

CD8+ T-cell clones deficient in the expression of the CD45 protein tyrosine phosphatase have impaired responses to T-cell receptor stimuli.

CD45 is a high-molecular-weight transmembrane protein tyrosine phosphatase expressed only by nucleated cells of hematopoietic origin. To examine function, mouse CD8+ cytolytic T-cell clones were derived that had a specific defect in the expression of CD45. Northern (RNA) blot analysis indicates that the CD45 deficiency is due to either a transcriptional defect or mRNA instability. The CD45-deficient cells were greatly diminished in their ability to respond to antigen. All functional parameters of T-cell receptor signalling analyzed (cytolysis of targets, proliferation, and cytokine production) were markedly diminished. A CD45+ revertant was isolated, and the ability to respond to antigen was restored. These results support a central and immediate role for this transmembrane protein tyrosine phosphatase in T-cell receptor signalling.