Proinflammatory Role of Monocyte-Derived CX3CR1int Macrophages in Helicobacter hepaticus-Induced Colitis.

Cells of the monocyte/macrophage lineage play important roles in the pathogenesis of inflammatory bowel diseases, but they are also present in the normal healthy intestine, where they are critical for maintaining homeostasis. It has been unclear whether the proinflammatory roles of intestinal macrophages reflect altered behavior of the existing resident cells, or whether they involve recruitment of a distinct cell type. Here, we have explored these ideas using the model of colitis induced by Helicobacter hepaticus in the context of neutralization or deletion of interleukin-10 (IL-10). Granulocytes and monocytes made up most of the inflammatory myeloid infiltrates found in the colon of H. hepaticus-infected colitic mice, rising to a peak within 2 weeks of H. hepaticus inoculation but taking several months to resolve completely. The inflammatory response was dependent on the combined presence of H. hepaticus and absence of IL-10 and was accompanied by increased production of inflammatory mediators such as IL-1ß, tumor necrosis factor alpha (TNF-α), IL-6, and IL-23p19 by infiltrating myeloid cells, mostly relatively immature cells of the macrophage lineage that express intermediate levels of CX3CR1. In contrast, the population of mature CX3CR1hi macrophages did not expand as markedly during colitis, and these cells made little contribution to inflammatory mediator production. Taking into account their numerical dominance in the myeloid compartment, we conclude that newly recruited monocytes are the main source of proinflammatory mediators in colitis induced in the absence of IL-10 signaling and that altered behavior of mature macrophages is not a major component of this pathology.

ASPASIA: A toolkit for evaluating the effects of biological interventions on SBML model behaviour.

A calibrated computational model reflects behaviours that are expected or observed in a complex system, providing a baseline upon which sensitivity analysis techniques can be used to analyse pathways that may impact model responses. However, calibration of a model where a behaviour depends on an intervention introduced after a defined time point is difficult, as model responses may be dependent on the conditions at the time the intervention is applied. We present ASPASIA (Automated Simulation Parameter Alteration and SensItivity Analysis), a cross-platform, open-source Java toolkit that addresses a key deficiency in software tools for understanding the impact an intervention has on system behaviour for models specified in Systems Biology Markup Language (SBML). ASPASIA can generate and modify models using SBML solver output as an initial parameter set, allowing interventions to be applied once a steady state has been reached. Additionally, multiple SBML models can be generated where a subset of parameter values are perturbed using local and global sensitivity analysis techniques, revealing the model's sensitivity to the intervention. To illustrate the capabilities of ASPASIA, we demonstrate how this tool has generated novel hypotheses regarding the mechanisms by which Th17-cell plasticity may be controlled in vivo. By using ASPASIA in conjunction with an SBML model of Th17-cell polarisation, we predict that promotion of the Th1-associated transcription factor T-bet, rather than inhibition of the Th17-associated transcription factor RORγt, is sufficient to drive switching of Th17 cells towards an IFN-γ-producing phenotype. Our approach can be applied to all SBML-encoded models to predict the effect that intervention strategies have on system behaviour. ASPASIA, released under the Artistic License (2.0), can be downloaded from http://www.york.ac.uk/ycil/software.

Tbet or Continued RORγt Expression Is Not Required for Th17-Associated Immunopathology.

The discovery of Th17 cell plasticity, in which CD4(+) IL-17-producing Th17 cells give rise to IL-17/IFN-γ double-producing cells and Th1-like IFNγ(+) ex-Th17 lymphocytes, has raised questions regarding which of these cell types contribute to immunopathology during inflammatory diseases. In this study, we show using Helicobacter hepaticus-induced intestinal inflammation that IL-17A(Cre)- or Rag1(Cre)-mediated deletion of Tbx21 has no effect on the generation of IL-17/IFN-γ double-producing cells, but leads to a marked absence of Th1-like IFNγ(+) ex-Th17 cells. Despite the lack of Th1-like ex-Th17 cells, the degree of H. hepaticus-triggered intestinal inflammation in mice in which Tbx21 was excised in IL-17-producing or Rag1-expressing cells is indistinguishable from that observed in control mice. In stark contrast, using experimental autoimmune encephalomyelitis, we show that IL-17A(Cre)-mediated deletion of Tbx21 prevents the conversion of Th17 cells to IL-17A/IFN-γ double-producing cells as well as Th1-like IFN-γ(+) ex-Th17 cells. However, IL-17A(Cre)-mediated deletion of Tbx21 has only limited effects on disease course in this model and is not compensated by Ag-specific Th1 cells. IL-17A(Cre)-mediated deletion of Rorc reveals that RORγt is essential for the maintenance of the Th17 cell lineage, but not immunopathology during experimental autoimmune encephalomyelitis. These results show that neither the single Th17 subset, nor its progeny, is solely responsible for immunopathology or autoimmunity.

Differential Requirements for IL-17A and IL-22 in Cecal versus Colonic Inflammation Induced by Helicobacter hepaticus.

Type 17 helper T-cell cytokines have been implicated in the pathogenesis of inflammatory bowel disease, a chronic condition affecting the gastrointestinal tract, but information regarding their contribution to pathology in different regions of the gut is lacking. By using a murine model of bacteria-induced typhlocolitis, we investigated the role of IL-17A, IL-17F, and IL-22 in cecal versus colonic inflammation. Cecal, but not colonic, pathology in C57BL/6 mice inoculated with Helicobacter hepaticus plus anti-IL-10 receptor (IL-10R) monoclonal antibody was exacerbated by co-administration of anti-IL-17A monoclonal antibody, suggesting a disease-protective role for IL-17A in the cecum. In contrast, anti-IL-17F had no effect on H. hepaticus-induced intestinal pathology. Neutralization of IL-22 prevented the development of colonic, but not cecal, inflammation in H. hepaticus-infected anti-IL-10R-treated mice, demonstrating a pathogenic role for IL-22 in the colon. Analysis of transcript levels revealed differential expression of IL-22R, IL-22 binding protein, and IL-23R between cecum and colon, a finding that may help explain why these tissues respond differently after anti-IL-22 treatment. Analysis of microarray data from healthy human intestine further revealed significant differences in cytokine receptor transcript levels (including IL-22RA1 and IL-23R) in distinct parts of the human gut. Together, our findings demonstrate that individual type 17 helper T-cell cytokines can have proinflammatory or anti-inflammatory effects in different regions of the intestine, an observation that may have implications for interventions against human inflammatory bowel disease.

Conventional T-bet(+)Foxp3(-) Th1 cells are the major source of host-protective regulatory IL-10 during intracellular protozoan infection.

Although interferon gamma (IFN-gamma) secretion is essential for control of most intracellular pathogens, host survival often also depends on the expression of interleukin 10 (IL-10), a cytokine known to counteract IFN-gamma effector functions. We analyzed the source of regulatory IL-10 in mice infected with the protozoan parasite Toxoplasma gondii. Unexpectedly, IFN-gamma-secreting T-bet(+)Foxp3(-) T helper type 1 (Th1) cells were found to be the major producers of IL-10 in these animals. Further analysis revealed that the same IL-10(+)IFN-gamma(gamma) population displayed potent effector function against the parasite while, paradoxically, also inducing profound suppression of IL-12 production by antigen-presenting cells. Although at any given time point only a fraction of the cells appeared to simultaneously produce IL-10 and IFN-gamma, IL-10 production could be stimulated in IL-10(-)IFN-gamma(+) cells by further activation in vitro. In addition, experiments with T. gondii-specific IL-10(+)IFN-gamma(+) CD4 clones revealed that although IFN-gamma expression is imprinted and triggered with similar kinetics regardless of the state of Th1 cell activation, IL-10 secretion is induced more rapidly from recently activated than from resting cells. These findings indicate that IL-10 production by CD4(+) T lymphocytes need not involve a distinct regulatory Th cell subset but can be generated in Th1 cells as part of the effector response to intracellular pathogens.

Interleukin-23 drives innate and T cell-mediated intestinal inflammation.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract involving aberrant activation of innate and adaptive immune responses. We have used two complementary models of IBD to examine the roles of interleukin (IL)-12 family cytokines in bacterially induced intestinal inflammation. Our results clearly show that IL-23, but not IL-12, is essential for the induction of chronic intestinal inflammation mediated by innate or adaptive immune mechanisms. Depletion of IL-23 was associated with decreased proinflammatory responses in the intestine but had little impact on systemic T cell inflammatory responses. These results newly identify IL-23 as a driver of innate immune pathology in the intestine and suggest that selective targeting of IL-23 represents an attractive therapeutic approach in human IBD.

IL-23 plays a key role in Helicobacter hepaticus-induced T cell-dependent colitis.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract that is caused in part by a dysregulated immune response to the intestinal flora. The common interleukin (IL)-12/IL-23p40 subunit is thought to be critical for the pathogenesis of IBD. We have analyzed the role of IL-12 versus IL-23 in two models of Helicobacter hepaticus-triggered T cell-dependent colitis, one involving anti-IL-10R monoclonal antibody treatment of infected T cell-sufficient hosts, and the other involving CD4+ T cell transfer into infected Rag-/- recipients. Our data demonstrate that IL-23 and not IL-12 is essential for the development of maximal intestinal disease. Although IL-23 has been implicated in the differentiation of IL-17-producing CD4+ T cells that alone are sufficient to induce autoimmune tissue reactivity, our results instead support a model in which IL-23 drives both interferon gamma and IL-17 responses that together synergize to trigger severe intestinal inflammation.

Interleukin-23 and T helper 17-type responses in intestinal inflammation: from cytokines to T-cell plasticity.

Interleukin-23 (IL-23) plays an essential role in driving intestinal pathology in experimental models of both T-cell-dependent and innate colitis. Furthermore, genome-wide association studies have identified several single-nucleotide polymorphisms in the IL-23 receptor (IL-23R) gene that are associated with either susceptibility or resistance to inflammatory bowel disease in humans. Although initially found to support the expansion and maintenance of CD4(+) T helper 17 (Th17) cells, IL-23 is now recognized as having multiple effects on the immune response, including restraining Foxp3(+) regulatory T-cell activity and inducing the expression of Th17-type cytokines from non-T-cell sources. Here we focus on Th17 cells and their associated cytokines IL-17A, IL-17F, IL-21 and IL-22. We review studies performed in mouse models of colitis where these effector cytokines have been shown to have either a pathogenic or a tissue-protective function. We also discuss the heterogeneity found within the Th17 population and the phenomenon of plasticity of Th17 cells, in particular the ability of these lymphocytes to extinguish IL-17 expression and turn on interferon-γ production to become Th1-like 'ex-Th17' cells. Interleukin-23 has been identified as a key driver in this process, and this may be an additional mechanism by which IL-23 promotes pathology in the intestinal tract. These 'ex-Th17' cells may contribute to disease pathogenesis through their secretion of pro-inflammatory mediators.

Bacteria-triggered CD4(+) T regulatory cells suppress Helicobacter hepaticus-induced colitis.

We have previously demonstrated that interleukin (IL)-10-deficient (IL-10 knockout [KO]) but not wild-type (WT) mice develop colitis after infection with Helicobacter hepaticus. Here, we show that infected recombination activating gene (RAG) KO mice develop intestinal inflammation after reconstitution with CD4(+) T cells from IL-10 KO animals and that the cotransfer of CD4(+) T cells from H. hepaticus-infected but not uninfected WT mice prevents this colitis. The disease-protective WT CD4(+) cells are contained within the CD45RB(low) fraction and unexpectedly were found in both the CD25(+) and the CD25(-) subpopulations of these cells, their frequency being higher in the latter. The mechanism by which CD25(+) and CD25(-) CD45RB(low) CD4(+) cells block colitis involves IL-10 and not transforming growth factor (TGF)-beta, as treatment with anti-IL-10R but not anti-TGF-beta monoclonal antibody abrogated their protective effect. In vitro, CD45RB(low) CD4(+) cells from infected WT mice were shown to produce IL-10 and suppress interferon-gamma production by IL-10 KO CD4(+) cells in an H. hepaticus antigen-specific manner. Together, our data support the concept that H. hepaticus infection results in the induction in WT mice of regulatory T cells that prevent bacteria-induced colitis. The induction of such cells in response to gut flora may be a mechanism protecting normal individuals against inflammatory bowel disease.

Sweet! Helicobacter Sugar Calms Intestinal Macrophages.

In the absence of IL-10, Helicobacter hepaticus (Hh) induces colitis. In this issue of Cell Host & Microbe, Danne et al. (2017) report that Hh produces a polysaccharide that induces an anti-inflammatory response in macrophages, providing a potential clue as to why this bacterium is normally tolerated by the immune system.

Mechanisms underlying helminth- induced Th2 polarization: default, negative or positive pathways?

Since the initial description of Th1 and Th2 subsets in the 1980s, there has been enormous progress in identifying the molecular events and the transcriptional factors that regulate Th differentiation in response to a specific stimulus (e.g. antigen dose, co-receptors, cytokines). Although TCR cross-linking and engagement of co-stimulatory molecules are necessary for activation of CD4+ lymphocytes, these two events do not appear in themselves to explain Th1/Th2 commitment. Among pathogens, helminths are the main examples of Th2-cell inducers in both humans and experimental models. This review will focus on our recent findings on the requirements for Th2 polarization by the parasitic trematode Schistosoma mansoni. In particular, we will address the ongoing controversy as to whether Th2 development depends on positive vs. negative vs. the absence of signals from antigen-presenting cells. In addition, we will discuss the similarities between the pathways involved in parasite- and allergen-induced Th2 differentiation.

TGF-beta1 production by CD4+ CD25+ regulatory T cells is not essential for suppression of intestinal inflammation.

Naturally occurring CD4+ CD25+ regulatory T cells (Treg) are potent suppressors of CD4+ and CD8+ T cell responses in vitro and inhibit several organ-specific autoimmune diseases. While most in vitro studies suggest that CD4+ CD25+ Treg cells adopt a cytokine-independent but cell contact-dependent mode of T cell regulation, their precise mechanism of suppression in vivo remains largely unknown. Here we examine the functional contribution of Treg cell-derived TGF-beta1 and effector T cell responsiveness to TGF-beta in CD4+ CD25+ T cell-mediated suppression of inflammatory bowel disease (IBD). We show that CD4+ CD25+ Treg cells from either TGF-beta1+/+ or neonatal TGF-beta1-/- mice can suppress the incidence and severity of IBD as well as colonic IFN-gamma mRNA expression induced by WT CD4+ CD25- effector T cells. Furthermore, TGF-beta-resistant Smad3-/- CD4+ CD25+ Treg cells are equivalent to WT Treg cells in their capacity to suppress disease induced by either WT or Smad3-/- CD4+ CD25- effector T cells. Finally, anti-TGF-beta treatment exacerbates the colitogenic potential of CD4+ CD25- effector T cells in the absence of CD4+ CD25+ Treg cells. Together, these data demonstrate that in certain situations CD4+ CD25+ T cells are able to suppress intestinal inflammation by a mechanism not requiring Treg cell-derived TGF-beta1 or effector T cell/Treg cell responsiveness to TGF-beta via Smad3.

Parasite-induced Th2 polarization is associated with down-regulated dendritic cell responsiveness to Th1 stimuli and a transient delay in T lymphocyte cycling.

The nature of the signals that bias Th effector choice is still not completely understood. Using parasite extracts from pathogens known to induce polarized Th1 or Th2 responses and an in vitro experimental model for priming murine CD4(+) cells, we demonstrated that splenic dendritic cells (DC), but not B cells, promote Th1/Th2 differentiation of naive CD4(+) lymphocytes. Th polarization in this system was found not to depend on DC secretion of the polarizing cytokines IL-12/IL-4, but instead correlated with distinct states of DC activation induced by the different parasite preparations. As expected, conditioning of DC for Th1 development was associated with up-regulation of costimulatory molecules and enhanced chemokine production and required intact MyD88 signaling. In contrast, conditioning of DC for Th2 differentiation correlated with down-regulation of many of the same functions and was MyD88 independent. This dampened DC activation was accompanied in the cocultures by a reduction in the frequency of CD4(+) lymphocytes exiting the first division of the cell cycle. When the latter was mimicked by drug-induced arrest of peptide-primed CD4(+) cells after the S phase of the first cycle, a marked Th2 polarization was also observed. Together, these findings suggest that the emergence of IL-4-producing CD4(+) lymphocytes results from a suppression in DC function leading to a temporary delay in initial T cell cycling.

In the absence of IL-12, CD4(+) T cell responses to intracellular pathogens fail to default to a Th2 pattern and are host protective in an IL-10(-/-) setting.

IL-12-deficient mice exposed to nonlethal infections with intracellular pathogens or repeatedly immunized with a pathogen extract developed lowered but nevertheless substantial numbers of IFN-gamma(+) CD4(+) T cells compared to those observed in wild-type animals. Moreover, the CD4(+) responses in these knockout animals failed to default to a Th2 pattern. The protective efficacy of the Th1 cells developing in an IL-12-deficient setting was found to be limited by IL-10 since mice doubly deficient in IL-10 and IL-12 survived, while animals deficient in IL-12 alone succumbed to pathogen challenge. In contrast to IL-12 knockout mice, MyD88-deficient animals exposed to a Th1 microbial stimulus developed a pure Th2 response, arguing that this signaling element plays a more critical function than IL-12 in determining pathogen-induced CD4 polarization.

Transgenic mice expressing human interleukin-10 in the antigen-presenting cell compartment show increased susceptibility to infection with Mycobacterium avium associated with decreased macrophage effector function and apoptosis.

Interleukin-10 (IL-10) is thought to play an important role in the regulation of microbial immunity. While T-cell-derived IL-10 has been shown to suppress cell-mediated immunity, there has been debate as to whether antigen presenting cell (APC)-derived cytokine can perform the same function in vivo. To assess the influence of APC-produced IL-10 on host resistance to mycobacterial infection, transgenic mice expressing human IL-10 under the control of the major histocompatibility complex class II promoter (hu10Tg) were infected with Mycobacterium avium, and bacterial burdens and immune responses were compared with those observed in wild-type (wt) animals. Hu10Tg mice harbored substantially higher numbers of M. avium and succumbed 16 to 18 weeks postinfection. The granulomas in infected hu10Tg mice showed marked increases in both acid-fast bacilli and host macrophages. In addition, these animals displayed a dramatic increase in hepatic fibrosis. The increased susceptibility of the hu10Tg mice to M. avium infection is independent of T-cell-produced endogenous murine IL-10, since bacterial burdens in mice derived by crossing hu10Tg mice with murine IL-10-deficient mice were not significantly different from those in hu10Tg mice. Importantly, gamma interferon (IFN-gamma) responses were not decreased in the infected transgenic animals from those in wt animals, suggesting the normal development of Th1 effector cells. In contrast, mycobacterium-induced macrophage apoptosis as well as production of TNF, nitric oxide, and IL-12p40 were strongly inhibited in hu10Tg mice. Together, these data indicate that APC-derived IL-10 can exert a major inhibitory effect on control of mycobacterial infection by a mechanism involving the suppression of macrophage effector function and apoptosis.

Induction of colitis by a CD4+ T cell clone specific for a bacterial epitope.

It is now well established that the intestinal flora plays an important role in the pathogenesis of inflammatory bowel disease (IBD). However, whether bacteria serve as the sole target of the immune response in this process or whether they act indirectly by triggering an anti-self response is still unclear. We have previously shown that specific pathogen-free IL-10-deficient (IL-10 KO) mice develop a T helper (Th1)-cytokine associated colitis after experimental infection with Helicobacter hepaticus. We here show that H. hepaticus Ag (SHelAg)-specific CD4+ Th1 clones transfer disease to H. hepaticus-infected T cell-deficient RAG KO hosts. Importantly, uninfected recipients of the SHelAg-specific clones did not develop intestinal inflammation, and a control Schistosoma mansoni-specific Th1 clone did not induce colitis upon transfer to infected RAG KO mice. The disease-inducing T cell clones recognized antigen(s) (Ag) specifically expressed by certain Helicobacter species as they responded when stimulated in vitro with H. hepaticus and Helicobacter typhlonius Ag, but not when cultured with Ag preparations from Helicobacter pylori, various non-helicobacter bacteria, or with cecal bacterial lysate from uninfected mice. Characterization of the Ag specificity of one of the clones showed that it reacts uniquely with a 15-mer peptide epitope on the flagellar hook protein (FlgE) of H. hepaticus presented by I-Ab. Together, our results demonstrate that colitis can be induced by clonal T cell populations that are highly specific for target Ag on intestinal bacteria, suggesting that an aberrant T cell response directed against gut flora is sufficient to trigger IBD.

Role of cytokines in the formation and downregulation of hepatic circumodal granulomas and hepatic fibrosis in Schistosoma mansoni-infected mice

Schistosoma mansoni infections are associated with a strong Th2 cytokine response. Treatment of mice with IL-12 or anti-IL-2 or anti-IL-4 before i.v. injection of eggs increased IFN-gamma production and downregulated Th2 responses and pulmonary granuloma size. Conversely, anti-IFN-gamma antibody treatment increased Th2 responses and granoloma size. Similar manipulation produced less dramatic results in infected mice. However, sensitization of mice with eggs + IL-12 before infection augmented the Th1 response and decreased Th2 cytokines, granoloma size and fibrosis. Antisera to IFN-gamma, TNF-alpha or IL-12 during IL-12-egg immunization partly restored granuloma size and fibrosis following infection. Variations in the size of granulomas in acute (8 weeks) infections may be influenced primarily by the number and state of activation of T cells. In chronic (12-16 week) infections immunologic downmodulation proceed normally in mice without functional CD8 + cells and in IFN-gamma KO mice but not in B cell KO (µMT) mice or in mice deficient in FcR expression in spite of the fact that these mice downregulated their T cell and cytokine responses. It is evident that the participation of cytokines in granuloma formation and regulation is complicated and that the mechanisms controlling both these phenomena are likely to involve both T cells and antibody/FcR interactions.