Roelof A. de Paus

IL-12Rβ1 deficiency: mutation update and description of the IL12RB1 variation database

IL‐12Rβ1 deficiency is an autosomal recessive disorder characterized by predisposition to recurrent and/or severe infections caused by otherwise poorly pathogenic mycobacteria and salmonella. IL‐12Rβ1 is a receptor chain of both the IL‐12 and the IL‐23 receptor and deficiency of IL‐12Rβ1 thus abolishes both IL‐12 and IL‐23 signaling. IL‐12Rβ1 deficiency is caused by bi‐allelic mutations in the IL12RB1 gene. Mutations resulting in premature stop codons, such as nonsense, frame shift, and splice site mutations, represent the majority of IL‐12Rβ1 deficiency causing mutations (66%; 46/70). Also every other morbid mutation completely inactivates the IL‐12Rβ1 protein. In addition to disease‐causing mutations, rare and common variations with unknown functional effect have been reported in IL12RB1. All these variants have been deposited in the online IL12RB1 variation database (www.LOVD.nl/IL12RB1). In this article, we review the function of IL‐12Rβ1 and molecular genetics of human IL12RB1.

IL-23 modulates CD56+/CD3− NK Cell and CD56+/CD3+ NK-like T Cell function differentially from IL-12

NK and NK-like T cells play an essential role in linking innate and adaptive immunity through their ability to secrete IFN-gamma. The exact trigger initiating production of IFN-gamma is uncertain. Antigen-presenting cell (APC)-derived IL-12 is thought to be the classical IFN-gamma-inducing cytokine but requires an additional stimulus such as IFN-gamma itself. IL-23 and IL-18 are among the first cytokines secreted by APC in response to binding of pathogen-associated molecular patterns such as LPS. Thus, early APC-derived IL-23 may be an initial trigger of IFN-gamma production in NK and NK-like T cells. Herein, we characterized the effect of IL-23 on IFN-gamma secretion by NK and NK-like T cells. Our findings show that IL-23 and IL-18 synergistically elicit IFN-gamma production in NK-like T cells but not in NK cells. In contrast, IL-12 together with IL-18-induced secretion of IFN-gamma in both populations. The observed synergy between IL-23 and IL-18 in NK-like T cells coincided with IL-23-mediated up-regulation of IL-18Ralpha. Furthermore, IL-23 up-regulated CD56 expression in NK-like T cells and, together with IL-18, induced proliferation of NK and NK-like T cells. We postulate a role for APC-derived IL-23 in the activation of NK and NK-like T cells early in infection and in shaping T(h)1 differentiation, via induction of IFN-gamma, which provides the additional stimulus needed for APC to subsequently produce IL-12.

Salmonella Induced IL-23 and IL-1β Allow for IL-12 Production by Monocytes and Mφ1 through Induction of IFN-γ in CD56+ NK/NK-Like T Cells

Background The type-1 cytokine pathway plays a pivotal role in immunity against intracellular bacterial pathogens such as Salmonellae and Mycobacteria. Bacterial stimulation of pattern recognition receptors on monocytes, macrophages and dendritic cells initiates this pathway, and results in the production of cytokines that activate lymphocytes to produce interferon (IFN)-γ. Interleukin (IL)-12 and IL-23 are thought to be the key cytokines required for initiating a type-1 cytokine immune response to Mycobacteria and Salmonellae. The relative contribution of IL-23 and IL-12 to this process is uncertain. Methodology/Principal Findings We show that various TLR agonists induce the production of IL-23 but not IL-12 in freshly isolated human monocytes and cultured human macrophages. In addition, type 1 pro-inflammatory macrophages (Mϕ1) differentiated in the presence of GM-CSF and infected with live Salmonella produce IL-23, IL-1β and IL-18, but not IL-12. Supernatants of Salmonella-infected Mϕ1 contained more IL-18 and IL-1β as compared with supernatants of Mϕ1 stimulated with isolated TLR agonists, and induced IFN-γ production in human CD56+ cells in an IL-23 and IL-1β-dependent but IL-12-independent manner. In addition, IL-23 together with IL-18 or IL-1β led to the production of GM-CSF in CD56+ cells. Both IFN-γ and GM-CSF enhanced IL-23 production by monocytes in response to TLR agonists, as well as induced IL-12 production. Conclusions/Significance The findings implicate a positive feedback loop in which IL-23 can enhance its release via induction of IFN-γ and GM-CSF. The IL-23 induced cytokines allow for the subsequent production of IL-12 and amplify the IFN-γ production in the type-1 cytokine pathway.

Inhibition of the type I immune responses of human monocytes by IFN-α and IFN-β.

Interleukin-12 (IL-12), IL-23 and interferon-γ (IFN-γ) are pivotal cytokines acting in concert with tumor necrosis factor (TNF) and IL-1β to shape type I immune responses against bacterial pathogens. Recently, several groups reported that type I immunity can be inhibited by IFN-α/β. Here we show the extent of the inhibitory effects of IFN-α and IFN-β on the responsiveness of human monocytes to Toll like receptor-ligands and IFN-γ. Both IFN-α and IFN-β strongly reduced the production of IL-12p40, IL-1β and TNF and the IFN-γ induced CD54 and CD64 expression. High IFN-γ concentrations could not counterbalance the inhibitions and IFN-α still inhibited monocytes 24h after stimulation in vitro as well as in vivo in patients undergoing IFN-α treatment. Next, we explored the mechanism of inhibition. We confirm that IFN-α/β interferes with the IFN-γR1 expression, by studying the kinetics of IFN-γR1 downregulation. However, IFN-γR1 downregulation occurred only after two hours of IFN-α/β stimulation and was transient, which cannot explain the IFN-γ unresponsiveness observed directly and late after IFN-α/β stimulation. Additional experiments indeed indicate that other mechanisms are involved. IFN-α may interfere with IFN-γ-elicited phosphorylation of signal transducer and activator of transcription 1 (STAT1). IFN-α may also activate methyltransferases which in turn reduce, at least partly, the TNF and IL-1β production and CD54 expression. IFN-α also induces the protein inhibitor of activated STAT1 (PIAS1). In conclusion, IFN-α and IFN-β strongly inhibit the IFN-γ responsiveness and the production of type I cytokines of monocytes, probably via various mechanisms. Our findings indicate that IFN-α/β play a significant role in the immunopathogenesis of bacterial infections, for example Mycobacterium tuberculosis infection.

IL-23 and IL-12 responses in activated human T cells retrovirally transduced with IL-23 receptor variants.

Interleukin-23 (IL-23) is a regulator of cellular immune responses involved in controlling infections and autoimmune diseases. Effects of IL-23 on T cells are mediated via a receptor complex consisting of an IL-12Rbeta1 and a specific IL-23R chain. The R381Q and P310L variants of the IL-23R were recently reported to be associated with autoimmune diseases, suggesting they have an effect on IL-23R function. To investigate this matter, these variants and a newly identified variant, Y173H, were retrovirally transduced into human T cell blasts and functionally characterized by measuring the IL-23-induced signal transduction pathway (i.e., STAT1, STAT3 and STAT4 phosphorylation), and IFN-gamma and IL-10 production. No differences were detected between the genetic variants and wild-type in the function of the IL-23R-chain. Furthermore, while comparing IFN-gamma and IL-10 production in response to IL-23 and IL-12, we found IL-23 to be a more potent IL-10 inducer, and IL-12 a more potent IFN-gamma inducer. In addition, IL-23 also exerted a minor IL-12-like effect by inducing IL-23R-independent, IL-12Rbeta1-dependent STAT4 phosphorylation and IFN-gamma production. In conclusion, the reported association between R381Q and P310L variants of the IL-23R and autoimmune diseases does not depend on differences in functional activity between wild-type and R381Q and P310L variants of the IL-23R.

Differential expression and function of human IL-12Rβ2 polymorphic variants

The receptor for interleukin-12, formed by IL-12Rβ1 and IL-12Rβ2, mediates the type I immune responses of various types of lymphocytes. Polymorphisms in IL12RB2, the gene encoding IL-12Rβ2, were reported to be associated with several immune related diseases, such as Crohns disease. Because the IL23R and IL12RB2 genes are located in close proximity on the genome, the reported associations might also be attributable to linked polymorphisms in IL23R, which were found to be associated with immune related diseases as well. To clarify the role of IL-12Rβ2 in immune diseases, we investigated the functional consequences of thirteen amino acid substitutions in IL-12Rβ2. We developed a model with retroviral expression of IL-12Rβ2 in B cell lines. With the use of this model the expression and function of the variants was compared within the same genetic background. Four of the IL-12Rβ2 variants, N271Y, R313G, A604V and L808R showed reduced IL-12 responses compared to the wild type variant. Two of these are relatively common in some populations and may be used in future association studies to reveal a role for IL-12 in infectious and/or immune related diseases.

Disseminated Mycobacterium genavense infection in a patient with a novel partial interleukin-12/23 receptor β1 deficiency

A patient presented with late onset disseminated nontuberculous mycobacterium (NTM) infection due to a novel interleukin-12/interleukin-23 receptor β1 (IL-12/IL-23Rβ1) mutation, r.1561C>G, leading to the amino acid substitution R521G. This is the second patient reported with a partial IL-12/IL-23Rβ1 defect.

Functional analysis of naturally occurring amino acid substitutions in human IFN-γR1

IFN-gamma plays an essential role in the IL-12/IL-23/IFN-gamma pathway that is required for the defense against intracellular pathogens. In the IFN-gammaR1 several amino acid substitutions have been reported that abrogate IFN-gamma signaling. These substitutions can lead to a null phenotype and enhanced susceptibility to infection by poorly pathogenic mycobacteria, a disorder known as Mendelian Susceptibility to Mycobacterial Disease (MSMD). More common amino acid variations in the IFN-gammaR1 may also influence IFN-gammaR function, albeit more subtle. To determine the effect of various amino acid substitutions on IFN-gammaR1 expression and function we cloned two newly identified amino acid substitutions (S149L, I352M), four common variations (V14M, V61I, H335P, L467P), seven reported missense mutations (V61Q, V63G, Y66C, C77Y, C77F, C85Y, I87T) and the 818delTTAA mutation in a retroviral expression vector. IFN-gammaR1 expression was determined as well as responsiveness to IFN-gamma stimulation. The two newly discovered variants, and the four common polymorphisms could be detected on the cell surface, however, the V14M, H335P and I352M variants were significantly lower expressed at the cell membrane, compared to the wild type receptor. Despite the variance in cell surface expression, these IFN-gammaR1 variants did not affect function. In contrast to literature, in our model the expression of the V63G variant was severely reduced and its function was severely impaired but not completely abrogated. In addition, we confirmed the severely reduced function of the I87T mutant receptor, the completely abrogated expression and function of the V61E, V61Q, C77F, C77Y and the C85Y mutations, as well as the overexpression pattern of the 818delTTAA mutant receptor. The Y66C mutation was expressed at the cell surface, it was however, not functional. We conclude that the V14M, V61I, S149L, H335P, I352M and L467P are functional polymorphisms. The other variants are deleterious mutations with V61E, V61Q, Y66C, C77F, C77Y and C85Y leading to complete IFN-gammaR1 deficiency, while V63G and I87T lead to partial IFN-gammaR1 deficiency.

The influence of influenza virus infections on the development of tuberculosis

Recently, it was shown that interferon-γ mediated immune responses, which play a major role in the control of infection with Mycobacterium tuberculosis (Mtb), can be inhibited by type I interferons. Since type I interferons are abundantly induced during viral infections, we hypothesized that infections with influenza viruses might play a role in the development of active TB disease either directly after exposure to Mtb or through reactivation of latent Mtb infection. To explore this hypothesis we investigated in a retrospective study whether newly diagnosed adult tuberculosis patients from Indonesia had had recent influenza infection. Plasma samples from TB patients and controls were assayed for antibodies against two subtypes of at that time relevant, seasonal influenza A viruses. Overall, no correlation was observed with the presence of antibodies and manifest tuberculosis. Still, antibody titers against circulating A/H3N2 influenza virus were slightly enhanced in tuberculosis patients as compared to controls, and highest in cases of advanced tuberculosis. This suggests that tuberculosis patients were recently infected with influenza, before clinical manifestation of the disease. Alternatively, the production of antibodies and susceptibility to tuberculosis may be influenced by a common confounding factor, for example the ability of patients to induce interferon-α. We conclude that in an endemic country like Indonesia, an influenza virus infection is not a major determinant for developing clinically manifest tuberculosis.

Immunological characterization of latent tuberculosis infection in a low endemic country

The diagnosis of a latent tuberculosis infection (LTBI) is based on detection of immunity against Mycobacterium tuberculosis (Mtb). The tuberculin skin test (TST), the Quantiferon (QFT) and a prolonged lymphocyte stimulation test using either ESAT-6/CFP-10 (LST-EC) or PPD (LST-PPD) were evaluated in a cohort of 495 individuals, suspected to have LTBI, in a low endemic country. While the TST and LST-PPD were both positive in the majority (75%) of individuals, only one third responded in the LST-EC and in the QFT. The choice for LTBI treatment was significantly associated with ESAT6/CFP10 recognition, however the LST-EC detected considerably more individuals (21%) with immunity against Mtb, who might also be at risk for development of active TB, although none of them did during follow up. Follow-up for 2 years showed 7% conversions and 32% reversions for the QFT. The LST-EC showed higher conversion rates (∼45%), although the percentage of individuals positive in the LST-EC did not change significantly within the follow-up period. LTBI treatment did not alter immune recognition of Mtb antigens. In conclusion, the sensitivity of tests for detection of cellular immunity to Mtb specific antigens depends on test methodology and may vary considerably over time in a low endemic region.