The role of IL-10 in immune regulation during M. tuberculosis infection.

During gaseous exchange the lungs are exposed to a vast variety of pathogens, allergens, and innocuous particles. A feature of the lung immune response to lung-tropic aerosol-transmitted bacteria such as Mycobacterium tuberculosis (Mtb) is a balanced immune response that serves to restrict pathogen growth while not leading to host-mediated collateral damage of the delicate lung tissues. One immune-limiting mechanism is the inhibitory and anti-inflammatory cytokine interleukin (IL)-10. IL-10 is made by many hematopoietic cells and a major role is to suppress macrophage and dendritic cell (DC) functions, which are required for the capture, control, and initiation of immune responses to pathogens such as Mtb. Here, we review the role of IL-10 on bacterial control during the course of Mtb infection, from early innate to adaptive immune responses. We propose that IL-10 is linked with the ability of Mtb to evade immune responses and mediate long-term infections in the lung.

Potential role of interleukin-10-secreting regulatory T cells in allergy and asthma.

Allergic diseases are caused by aberrant T-helper-2 immune responses in susceptible individuals. Both naturally occurring CD4(+)CD25(+) regulatory T cells and inducible populations of antigen-specific interleukin-10-secreting regulatory T cells inhibit these inappropriate immune responses in experimental models. This article discusses the evidence that regulatory T-cell function might be impaired in allergic and asthmatic disease and that certain therapeutic regimens might function, at least in part, to promote regulatory T-cell generation. Current research strategies seek to exploit these observations to improve the generation of allergen-specific regulatory T-cell populations with the potential to provide the safe and long-term alleviation of disease symptoms.

Mouse type I IFN-producing cells are immature APCs with plasmacytoid morphology.

We show here that mouse interferon-alpha (IFN-alpha)-producing cells (mIPCs) are a unique subset of immature antigen-presenting cells (APCs) that secrete IFN-alpha upon stimulation with viruses. mIPCs have a plasmacytoid morphology, can be stained with an antibody to Ly6G and Ly6C (anti-Ly6G/C) and are Ly6C+B220+CD11cloCD4+; unlike other dendritic cell subsets, however, they do not express CD8alpha or CD11b. Although mIPCs undergo apoptosis in vitro, stimulation with viruses, IFN-alpha or CpG oligonucleotides enhanced their survival and T cell stimulatory activity. In vivo, mIPCs were the main producers of IFN-alpha in cytomegalovirus-infected mice, as depletion of Ly6G+/C+ cells abrogated IFN-alpha production. mIPCs produced interleukin 12 (IL-12) in response to viruses and CpG oligodeoxynucleotides, but not bacterial products. Although different pathogens can selectively engage various APC subsets for IL-12 production, IFN-alpha production is restricted to mIPCs' response to viral infection.

1alpha,25-Dihydroxyvitamin d3 has a direct effect on naive CD4(+) T cells to enhance the development of Th2 cells.

1alpha,25-Dihydroxyvitamin D3 (vitD3) is an immunoregulatory hormone with beneficial effects on Th1 mediated autoimmune diseases. Although the inhibitory effects of vitD3 on macrophages and dendritic cells are well documented, any direct effects of vitD3 on Th cell development are not clearly defined. Using CD4(+)Mel14(+) T cells derived from mice on a BALB/c and a C57BL/6 genetic background we examined the effect of vitD3 on Th cell development. We demonstrated that vitD3 affects Th cell polarization by inhibiting Th1 (IFN-gamma production) and augmenting Th2 cell development (IL-4, IL-5, and IL-10 production). These effects were observed in cultures driven with splenic APC and Ag, as well as with anti-CD3 and anti-CD28 alone, indicating that CD4(+) cells can also be direct targets for vitD3. The enhanced Th2 development by vitD3 was found in both BALB/c and C57BL/6 mice. An increased expression of the Th2-specific transcription factors GATA-3 and c-maf correlated with the increased production of Th2 cytokines after vitD3 treatment. The vitD3-induced effects were largely mediated via IL-4, because neutralization of IL-4 almost completely abrogated the augmented Th2 cell development after vitD3 treatment. These findings suggest that vitD3 acts directly on Th cells and can, in the absence of APC, enhance the development of a Th2 phenotype and augment the expression of the transcription factors c-maf and GATA-3. Our findings suggest that the beneficial effects of vitD3 in autoimmune diseases and transplantation operate through prevention of strong Th1 responses via the action on the APC, while simultaneously directly acting on the T cell to enhance Th2 cell development.

A critical role for interleukin 18 in primary and memory effector responses to Listeria monocytogenes that extends beyond its effects on Interferon gamma production.

The stimulation of interferon (IFN)-gamma by interleukin (IL)-12 has been shown to provide protection from intracellular pathogens such as Listeria monocytogenes. Tumor necrosis factor (TNF) is also a major player in the resolution of Listeria infections and is suggested to have more global effects than can be explained by the induction of IFN-gamma alone. Since IL-18 synergizes with IL-12 to induce IFN-gamma production by natural killer and T helper (Th)1 cells, we determined its role in responses to Listeria. IL-18 appeared to be even more potent than either IL-12 or IFN-gamma for protection against this pathogen and IL-18 enhanced bacterial clearance in the complete absence of IFN-gamma. Indeed IL-18 was comparable to TNF in its ability to resolve the infection and showed a lowered protective capacity in the absence of TNF. Moreover, IL-18 induced macrophages to secrete both TNF and nitric oxide after a Listeria infection. IL-18 was also essential for optimal IFN-gamma production by antigen-specific T cells. Therefore, IL-18 operates via its effects on both the innate immune response, including macrophages, as well as on Th1 cells, to protect against Listeria.

Aberrant in vivo T helper type 2 cell response and impaired eosinophil recruitment in CC chemokine receptor 8 knockout mice.

Chemokine receptors transduce signals important for the function and trafficking of leukocytes. Recently, it has been shown that CC chemokine receptor (CCR)8 is selectively expressed by Th2 subsets, but its functional relevance is unclear. To address the biological role of CCR8, we generated CCR8 deficient (-/-) mice. Here we report defective T helper type 2 (Th2) immune responses in vivo in CCR8(-/)- mice in models of Schistosoma mansoni soluble egg antigen (SEA)-induced granuloma formation as well as ovalbumin (OVA)- and cockroach antigen (CRA)-induced allergic airway inflammation. In these mice, the response to SEA, OVA, and CRA showed impaired Th2 cytokine production that was associated with aberrant type 2 inflammation displaying a 50 to 80% reduction in eosinophils. In contrast, a prototypical Th1 immune response, elicited by Mycobacteria bovis purified protein derivative (PPD) was unaffected by CCR8 deficiency. Mechanistic analyses indicated that Th2 cells developed normally and that the reduction in eosinophil recruitment was likely due to systemic reduction in interleukin 5. These results indicate an important role for CCR8 in Th2 functional responses in vivo.

Interleukin-10 and the interleukin-10 receptor.

Interleukin-10 (IL-10), first recognized for its ability to inhibit activation and effector function of T cells, monocytes, and macrophages, is a multifunctional cytokine with diverse effects on most hemopoietic cell types. The principal routine function of IL-10 appears to be to limit and ultimately terminate inflammatory responses. In addition to these activities, IL-10 regulates growth and/or differentiation of B cells, NK cells, cytotoxic and helper T cells, mast cells, granulocytes, dendritic cells, keratinocytes, and endothelial cells. IL-10 plays a key role in differentiation and function of a newly appreciated type of T cell, the T regulatory cell, which may figure prominently in control of immune responses and tolerance in vivo. Uniquely among hemopoietic cytokines, IL-10 has closely related homologs in several virus genomes, which testify to its crucial role in regulating immune and inflammatory responses. This review highlights findings that have advanced our understanding of IL-10 and its receptor, as well as its in vivo function in health and disease.

Cytokine networking in lungs of immunocompetent mice in response to inhaled Aspergillus fumigatus.

Cytokine networking in the lung in response to inhaled Aspergillus fumigatus was assessed using a murine model of primary pulmonary aspergillosis in immunocompetent Crl:CF-1 mice. Inhalation of virulent A. fumigatus (6 x 10(6) CFU) resulted in the induction of interleukin 18 (IL-18), tumor necrosis factor alpha (TNF-alpha), IL-12, and gamma interferon (IFN-gamma) protein in bronchoalveolar lavage fluid and/or lung tissue. Induction of immunoreactive IL-18 preceded induction of TNF-alpha protein, which preceded induction of immunoreactive IL-12 and IFN-gamma. Real-time reverse transcriptase (RT) PCR analysis of infected lung tissue demonstrated that induction of IL-18 protein also preceded induction of pulmonary TNF-alpha, IL-12, and IFN-gamma mRNAs. Mice were subsequently treated with cytokine-specific neutralizing monoclonal antibodies (MAbs) to the IL-18 receptor (anti-IL-18R MAb), TNF-alpha (anti-TNF-alpha MAb), IL-12 (anti-IL-12 MAb), and/or IFN-gamma (anti-IFN-gamma MAb), and effects on intrapulmonary cytokine activity and growth of A. fumigatus were assessed in infected lung homogenates. Simultaneous neutralization of IL-12 and IL-18 resulted in decreased levels of immunoreactive TNF-alpha, while neutralization of IL-18, TNF-alpha, or IL-12 alone or of IL-18 and IL-12 together resulted in decreased levels of immunoreactive IFN-gamma. Simultaneous neutralization of IL-12 and IL-18 or neutralization of TNF-alpha alone or in combination with IL-12, IL-18, or IFN-gamma also resulted in a significant increase in A. fumigatus CFU in lung tissue. Taken together, these results demonstrate that endogenous IL-18, IL-12, and TNF-alpha, through their modulatory effects on both intrapulmonary cytokine activity and growth of A. fumigatus, play key roles in host defense against primary pulmonary aspergillosis.

The molecular basis of T helper 1 and T helper 2 cell differentiation.

Cytokines such as interleukin 12 (IL-12) and IL-4 are dominant factors in driving the development of T helper 1 (Th1) and Th2 cells, respectively, through specific signalling pathways. In addition, it has been demonstrated more recently that T helper-cell-specific transcription factors exist that determine the commitment of Th1 and Th2 cells for the production of distinct profiles of cytokines. In addition to the expression of distinct cytokine genes and transcription factors, the molecular basis for commitment to a Th1 or Th2 phenotype can probably be explained by multiple mechanisms, including differential cytokine signalling, exclusive cytokine receptor expression, differential expression of transcription factors and/or differential chromatin remodelling of Th1- and Th2-specific genes.

Cutting edge: chromatin remodeling at the IL-4/IL-13 intergenic regulatory region for Th2-specific cytokine gene cluster.

During the differentiation of naive Th cells into Th2 effector cells, the entire IL-4/IL-13 locus is remodeled into an accessible chromatin conformation. Here we show that ectopic expression and activation of Stat6 or GATA-3 in Th cells developing under Th1-polarizing conditions lead to the induction of chromatin remodeling not only at the flanking regions of the IL-4 and IL-13 genes but also at the IL-4/IL-13 intergenic regulatory region for the IL-4/IL-13/IL-5 gene cluster. Furthermore, we demonstrate that GATA-3 and another Th2-specific, inducible protein complex interact with the IL-4/IL-13 intergenic DNase I hypersensitive region specifically in Th2 cells.

Anti-interleukin 10 receptor monoclonal antibody is an adjuvant for T helper cell type 1 responses to soluble antigen only in the presence of lipopolysaccharide.

Soluble foreign antigen usually leads to a transient clonal expansion of antigen-specific T cells followed by the deletion and/or functional inactivation of the cells. As interleukin (IL)-10 is a key immunoregulatory cytokine, we questioned whether neutralization of IL-10 during priming with soluble antigen could prime for a subsequent T helper cell type 1 (Th1) effector recall response. By using an adoptive transfer model to track the fate of antigen-specific T cell receptor (TCR)-transgenic CD4(+) T cells, we show that administration of soluble ovalbumin (OVA) protein, but not OVA(323-339) peptide antigen, together with an anti-IL-10 receptor (R) mAb led to the enhancement of a Th1 response upon rechallenge. Lipopolysaccharide (LPS) present in the protein was necessary for priming for Th1 recall responses in the presence of anti-IL-10R mAb, as removal of LPS abrogated this effect. Moreover, addition of LPS to the peptide did not itself allow priming for recall Th1 effector responses unless endogenous levels of IL-10 were neutralized with an anti-IL-10R mAb. A significant increase in OVA-specific IgG1 and IgG2a isotypes was observed when the protein antigen was administered with anti-IL-10R mAb; however, this was not the case with peptide antigen administered together with anti-IL-10R and LPS. Our data, showing that LPS receptor signaling and neutralization of endogenous immunosuppressive cytokines is essential for Th1 priming, has important implications for the design of relevant vaccines for effective in vivo immunotherapy.

Immunomodulatory role of endogenous interleukin-18 in gamma interferon-mediated resolution of replicative Legionella pneumophila lung infection.

The in vivo role of endogenous interleukin-18 (IL-18) in modulating gamma interferon (IFN-gamma)-mediated resolution of replicative Legionella pneumophila lung infection was assessed using a murine model of Legionnaires' disease. Intratracheal inoculation of A/J mice with virulent bacteria (10(6) L. pneumophila organisms per mouse) resulted in induction of IL-18 protein in bronchoalveolar lavage fluid (BALF) and intrapulmonary expression of IL-18 mRNA. Real-time quantitative RT-PCR analysis of infected lung tissue demonstrated that induction of IL-18 in BALF preceded induction of IL-12 and IFN-gamma mRNAs in the lung. Blocking intrapulmonary IL-18 activity by administration of a monoclonal antibody (MAb) to the IL-18 receptor (anti-IL-18R MAb) prior to L. pneumophila infection inhibited induction of intrapulmonary IFN-gamma production but did not significantly alter resolution of replicative L. pneumophila lung infection. In contrast, blocking endogenous IL-12 activity by administration of anti-IL-12 MAb) alone or in combination with anti-IL-18R MAb inhibited induction of intrapulmonary IFN-gamma and resulted in enhanced intrapulmonary growth of the bacteria within 5 days postinfection. Taken together, these results demonstrate that IL-18 plays a key role in modulating induction of IFN-gamma in the lung in response to L. pneumophila and that together with IL-12, IL-18 regulates intrapulmonary growth of the bacteria.

IL-18 receptors, their role in ligand binding and function: anti-IL-1RAcPL antibody, a potent antagonist of IL-18.

IL-18 is critical in eliciting IFN-gamma production from Th1 cells both in vitro and in vivo. Th1 cells have been implicated in the pathogenesis of autoimmune disorders, making antagonists of IL-18 promising therapeutics. However, specificity and binding characteristics of IL-18R components have only been superficially explored. In this study, we show that IL-1R related protein 1 (IL-1Rrp1) and IL-1R accessory protein-like (IL-1RAcPL) confer responsiveness to IL-18 in a highly specific (no response to other IL-1 ligands) and unique manner (no functional pairing with other IL-1Rs and IL-1R-like molecules). Cotransfection with both receptor components resulted in expression of both low and high affinity binding sites for IL-18 (K:(d) of 11 and 0.4 nM, respectively). We prepared anti-IL-1RAcPL mAb TC30-28E3, which, in contrast to soluble R proteins, effectively inhibited the IL-18-induced activation of NF-kappaB. Quantitative PCR showed that Th1 but not Th2 cells are unique in that they coexpress IL-1Rrp1 and IL-1RAcPL. mAb TC30-28E3 inhibited IL-18-induced production of IFN-gamma by Th1 cells, being at least 10-fold more potent than anti-IL-18 ligand mAb. This study shows that IL-1RAcPL is highly specific to IL-18, is required for high affinity binding of IL-18, and that the anti-IL-1RAcPL mAb TC30-28E3 potently antagonizes IL-18 responses in vitro, providing a rationale for the use of anti-IL-1RAcPL Abs to inhibit Th1-mediated inflammatory pathologies.

TGF-beta1 down-regulates Th2 development and results in decreased IL-4-induced STAT6 activation and GATA-3 expression.

TGF-beta plays an important role in immune regulation in vivo and affects T cell differentiation in vitro. Here we describe how TGF-beta modulates Th2 development in vitro and investigate its mechanisms of action. TGF-beta down-regulated Th2 development of naive CD4+ Mel-14high T cells derived from the DO11.10 ovalbumin-specific TCR-transgenic mouse, and this was observed both in cultures driven with anti-CD3 and anti-CD28 and with splenic APC and antigen. TGF-beta down-regulated GATA-3 expression in developing Th2 and these cells showed a diminished IL-4-induced STAT6 activation. We found, however, that naive cells driven in Th2 conditions with TGF-beta did not show a significantly decreased STAT6 activation, suggesting that TGF-beta inhibits Th2 development via a STAT6-independent mechanism.

Commitment factors for T helper cells.

Cytokines are dominant factors in driving the development of T helper cell subsets via specific signaling pathways. Transcription factors specific to the T helper cell lineages have also been shown to determine the commitment of Th1 and Th2 cells to the production of distinct profiles of cytokines.

GATA-3 induces T helper cell type 2 (Th2) cytokine expression and chromatin remodeling in committed Th1 cells.

Committed T helper type 1 (Th1) and Th2 effector cells, resulting from chronic antigenic stimulation in interleukin (IL)-12 and IL-4, are implicated in the pathology of autoimmune and allergic diseases. Committed Th1 cells cannot be induced to change their cytokine profiles in response to antigenic stimulation and Th2 cytokine-inducing conditions. Here, we report that ectopic expression of GATA-3 induced Th2-specific cytokine expression not only in developing Th1 cells but also in otherwise irreversibly committed Th1 cells and a Th1 clone, HDK1. Moreover, cAMP, an inhibitor of cytokine production by Th1 cells, markedly augmented Th2 cytokine production in GATA-3-expressing Th1 cells. Ectopic expression of GATA-3 in developing Th1 cells, but not in Th1 clone HDK1, induced endogenous GATA-3, suggesting an autoregulatory mechanism for maintenance of GATA-3 expression in Th2 cells. Structure-function analyses of GATA-3 revealed that the NH(2)-terminal transactivation domain and the COOH-terminal zinc finger domain of GATA-3 were critical, whereas the NH(2)-terminal zinc finger domain was dispensable for the induction of IL-4. Both zinc fingers, however, were required for IL-5 induction. A Th2-specific DNaseI-hypersensitive site of the IL-4 locus was detected in GATA-3-expressing Th1 cells. Thus, GATA-3 can change the phenotype of committed Th1 cells, previously considered to be irreversible.

Cutting edge: ectopic expression of the IL-12 receptor-beta 2 in developing and committed Th2 cells does not affect the production of IL-4 or induce the production of IFN-gamma.

The IL-12 receptor-beta 2 (IL-12R beta 2) chain is expressed on Th1 cells and lost upon differentiation to the Th2 phenotype. This has been suggested as the basis for commitment of Th1 cells, because early differentiated Th2 cells do not reverse their phenotype and do not produce IFN-gamma on restimulation in the presence of IL-12. In this study, we ectopically expressed the IL-12 receptor-beta 2 (IL-12R beta 2) bicistronically with enhanced green fluorescent protein by retroviral infection in developing and committed Th2 cells. Restimulation of Th2 cells expressing this ectopic IL-12R beta 2 in the presence of IL-12 led to levels of IL-4 production similar to those in control Th2 cells. The expression of IL-12R beta 2 in Th2 cells did not lead to significant levels of IFN-gamma production, although IL-12-mediated STAT signaling and proliferation were restored. Thus, although the IL-12R beta 2 and IL-12-dependent STAT4 activation are required for Th1 responses, activation of this pathway is not sufficient to restore a Th1 phenotype in developing or committed Th2 cells.

Differential representations of memory T cell subsets are characteristic of polarized immunity in leprosy and atopic diseases.

We identified functionally polarized subsets of CD4 memory T cells on the basis of the expression of CD11a, CD45RA and CD62L. Within the several phenotypically distinct subsets of CD4 memory cells are two that, upon stimulation, produce primarily IL-4 (MT(2), CD45RA(-)CD62L(+)CD11a(dim)) or primarily IFN-gamma (MT(1), CD45RA(-)CD62L(-)CD11a(bright)). In addition, four other phenotypically distinct subsets of CD4 cells have unique cytokine profiles. To determine the clinical relevance of the representation of these cell types, we analyzed blood from patients with the chronic diseases leprosy and atopy. These diseases are characterized as immunologically polarized, since T cell responses in affected individuals are often strongly biased towards T(h)1 (dominated by IFN-gamma production) or T(h)2 (IL-4 production). We show here that this polarization reflects homeostatic or differentiation mechanisms affecting the representation of the functionally distinct subsets of memory CD4 T cells, MT(1) and MT(2). Significantly, the representation of the MT(1) and MT(2) subsets differs dramatically between subjects with tuberculoid leprosy (a T(h)1 disease), or lepromatous leprosy or atopic disease (T(h)2 diseases). However, there was no difference in the cytokine profiles of these or any of the other finely resolved CD4 subsets, when compared between individuals across all disease states. Thus, it is the representation of these subsets in peripheral blood that is diagnostic of the polarized state of the immune system.