Marika C. Kullberg

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.

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

Although interferon γ (IFN-γ) 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-γ effector functions. We analyzed the source of regulatory IL-10 in mice infected with the protozoan parasite Toxoplasma gondii. Unexpectedly, IFN-γ–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-γγ 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-γ, IL-10 production could be stimulated in IL-10−IFN-γ+ cells by further activation in vitro. In addition, experiments with T. gondii–specific IL-10+IFN-γ+ CD4 clones revealed that although IFN-γ 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.

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 γ and IL-17 responses that together synergize to trigger severe intestinal inflammation.

Role of T‐Cell Derived Cytokines in the Downregulation of Immune Responses in Parasitic and Retroviral Infection

Parasitic infection is frequently accompanied by a downregulation in host cell-mediated immunity. Recent studies suggest that this modulation of helper T cells and effector cell function can at least in part be attributed to the action of a set of inhibitory cytokines produced by T lymphocytes as well as by a number of other cell types. The best characterized of these inhibitory lymphokines are IL-4, IL-10 and TGF-beta. Interestingly, both IL-4 and IL-10 are produced by the Th2 but not the Th1 subset of CD4+ helper cells. The former subset dominates in many situations of chronic or exacerbated parasitic infection and is thought to suppress Th1 function as a consequence of the cross-regulatory activity of these two cytokines. The latter hypothesis is supported by recent experiments demonstrating that mAb-mediated neutralization of IL-10 reverses suppressed IFN-gamma responses and/or disease susceptibility in mice with parasitic infections. In vivo neutralization of TGF-beta has also been reported to increase host resistance to parasite challenge. In addition to suppressing T-cell differentiation, function or proliferation, IL-4, IL-10 and TGF-beta each inhibit the ability of IFN-gamma to activate macrophages for killing of both intracellular and extracellular parasites. Moreover, the three cytokines are able to synergize with each other in downregulating these parasiticidal effects. Interestingly, each of the cytokines inhibits the production of reactive nitrogen oxides, an effector mechanism previously demonstrated to play a major role in parasite killing by activated macrophages. In the case of IL-10, this suppression of nitrogen oxide production appears to result from an inhibition of TNF-alpha synthesis leading to defective macrophage stimulation. While distant from parasites in their biology and phylogeny, some retroviruses also appear to induce an over-production in downregulatory cytokines which is closely associated with the onset of immunodeficiency. Thus, in an animal model involving infection of mice with LP-BM5 MuLV and in human HIV infection, Th2 (IL-10 and/or IL-4) cytokine synthesis is increased while Th1 (IFN-gamma and/or IL-2) cytokine production is suppressed. These observations suggest that cytokine-mediated cross-regulation may play a role in the pathogenesis of acquired immune deficiency disease, contributing both to the progression of retroviral infection and the increase in susceptibility to opportunistic infections and malignancy. Observations of similar cytokine cross-regulatory activities in organisms as diverse as helminths, protozoa and retroviruses predict that comparable mechanisms may operate in a wide variety of infectious diseases.

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 CD45RBlow 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− CD45RBlow CD4+ cells block colitis involves IL-10 and not transforming growth factor (TGF)-β, as treatment with anti–IL-10R but not anti–TGF-β monoclonal antibody abrogated their protective effect. In vitro, CD45RBlow CD4+ cells from infected WT mice were shown to produce IL-10 and suppress interferon-γ 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.

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.

Single Cell Analysis Reveals That IL-4 Receptor/Stat6 Signaling Is Not Required for the In Vivo or In Vitro Development of CD4+ Lymphocytes with a Th2 Cytokine Profile

The concept that IL-4 is the primary signal for Th2 lymphocyte differentiation has recently been put in doubt by studies in which the production of Th2-associated cytokines was detected in mice deficient in IL-4 synthesis or IL-4R triggering. In this study, we formally demonstrate by single cell analysis that CD4+ lymphocytes with a classical Th2 phenotype (IL-4+, IL-5+, IFN-γ−, IL-2−) develop in significant numbers in helminth-infected mice deficient in either IL-4R α-chain or Stat6. While an expanded population of Th1 (IL-4−, IL-5−, IFN-γ+, IL-2+) lymphocytes was observed in the same animals, surprisingly, cells with a mixed Th0 cytokine pattern were rare. The cytokine production phenotypes of the Th1 and Th2 subpopulations generated in infected Stat6-deficient mice were unaffected by in vitro neutralization of endogenous IL-4 or IFN-γ. Nevertheless, while addition of exogenous rIL-12 resulted in transitory IFN-γ production by Th2 lymphocytes from both wild-type and Stat6-deficient mice, IL-4 synthesis was preserved in the former, but temporarily ablated in the latter cells. Importantly, IL-4+ IFN-γ− and IL-4− IFN-γ+ populations similar to those arising in helminth-infected Stat6-deficient mice could also be generated in vitro by repetitive polyclonal stimulation of CD4+CD62Lhigh lymphocytes from uninfected mice of the same strain. Together, the results of these single cell analysis experiments demonstrate that IL-4R/Stat6 signaling, while influencing the final frequency of Th2 lymphocytes, is not essential for Th2 cell development, and suggest that this pathway has a previously unrecognized function in stabilizing Th2 populations once they have emerged.

Helicobacter hepaticus-Induced Colitis in Interleukin-10-Deficient Mice: Cytokine Requirements for the Induction and Maintenance of Intestinal Inflammation

ABSTRACT We have previously shown that specific-pathogen-free interleukin-10 (IL-10)-deficient (IL-10 KO) mice reconstituted withHelicobacter hepaticus develop severe colitis associated with a Th1-type cytokine response. In the present study, we formally demonstrate that IL-12 is crucial for disease induction, because mice deficient for both IL-10 and IL-12 p40 show no intestinal pathology following H. hepaticus infection. By using monoclonal antibodies (MAbs) to IL-12, gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α), we have further analyzed the role of these cytokines in the maintenance of the Th1 response and inflammation in IL-10 KO mice with established H. hepaticus-induced colitis. Treatment of infected colitic IL-10 KO mice with anti-IL-12 p40 resulted in markedly reduced intestinal inflammation, colonic IFN-γ, TNF-α, and inducible nitric oxide synthase (iNOS) mRNA levels, and H. hepaticus-specific IFN-γ secretion by mesenteric lymph node (MLN) cells compared to the findings in control MAb-treated mice. Moreover, the diminished pathology was associated with decreased numbers of colonic CD3+ T cells and significantly reduced frequencies ofHelicobacter-reactive CD4+ Th1 cells in MLN. In contrast, anti-IFN-γ and/or anti-TNF-α had no effect on intestinal inflammation in IL-10 KO mice with established colitis. Using IL-10/IFN-γ double-deficient mice, we further show that IFN-γ is not required for the development of colitis follwing H. hepaticus infection. MLN cells from infected IL-10/IFN-γ KO animals secreted elevated amounts of IL-12 and TNF-α following bacterial antigen stimulation, indicating alternative pathways of disease induction. Taken together, our results demonstrate a crucial role for IL-12 in both inducing and sustaining intestinal inflammation through recruitment and maintenance of a pool of pathogenic Th1 cells.

Maintenance of Pulmonary Th1 Effector Function in Chronic Tuberculosis Requires Persistent IL-12 Production

The mechanisms that prevent reactivation of latent Mycobacterium tuberculosis infection in asymptomatic individuals are poorly understood. Although IL-12 is critical for the induction of IFN-γ-dependent host control of M. tuberculosis, the requirement for the cytokine in the maintenance of host resistance and pulmonary Th1 effector function has not yet been formally examined. In this study, we reconstituted IL-12p40-deficient mice with IL-12 during the first 4 wk of infection and then assessed the effects of cytokine withdrawal. Although IL-12 administration initially resulted in restricted mycobacterial growth and prolonged survival, the reconstituted animals eventually succumbed to infection. This breakdown in bacterial control was accompanied by a marked reduction in the numbers of IFN-γ-producing CD4+ T cells in lungs. Moreover, whereas CD4+ T cells isolated from chronically infected wild-type mice expanded and transferred long-term protection to M. tuberculosis-challenged RAG−/− mice, they failed to do so in IL-12p40-deficient RAG−/− recipients and were clearly reduced in frequency within pulmonary granulomas in the latter animals. These studies establish that continuous IL-12 production is necessary for maintenance of the pulmonary Th1 cells required for host control of persistent M. tuberculosis infection and suggest that breakdown of this mechanism could be a contributing factor in reactivated disease.

TGF-β1 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‐β1 and effector T cell responsiveness to TGF‐β in CD4+CD25+ T cell‐mediated suppression of inflammatory bowel disease (IBD). We show that CD4+CD25+ Treg cells from either TGF‐β1+/+ or neonatal TGF‐β1–/– mice can suppress the incidence and severity of IBD as well as colonic IFN‐γ mRNA expression induced by WT CD4+CD25– effector T cells. Furthermore, TGF‐β‐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‐β 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‐β1 or effector T cell/Treg cell responsiveness to TGF‐β via Smad3.