IFN-α cannot substitute lack of IFN-γ responsiveness in cells of an IFN-γR1 deficient patient.

Patients with complete IFN-γR deficiency are unable to respond to IFN-γ and have impaired Th1-immunity and recurrent, severe infections with weakly virulent Mycobacteria. Since IFN-α and IFN-γ share signalling pathways, treatment with IFN-α has been proposed in complete IFN-γR deficiency. We stimulated cells from healthy controls and from a patient lacking IFN-γR1 with IFN-α and IFN-γ, to establish whether IFN-α would substitute for IFN-γ effects. IFN-α induced STAT1 phosphorylation in monocytes of the IFN-γR1(-/-) patient, but did not prime for LPS-induced IL-12p70, IL-12p40, IL-23 or TNF production. In control cells, IFN-α inhibited the priming effect of IFN-γ on LPS-induced pro-inflammatory cytokine release. Finally, IFN-γ but not IFN-α induced killing of M. smegmatis in cultured macrophages. In conclusion, no evidence was found to support the use of IFN-α in IFN-γR-deficient patients as intervention against mycobacterial infection; on the contrary, treatment of individuals with IFN-α may even adversely affect host defence against Mycobacteria.

Functional analysis of naturally occurring amino acid substitutions in human IFN-gammaR1.

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.

Salmonella induced IL-23 and IL-1beta allow for IL-12 production by monocytes and Mphi1 through induction of IFN-gamma 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)-gamma. 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 (Mphi1) differentiated in the presence of GM-CSF and infected with live Salmonella produce IL-23, IL-1beta and IL-18, but not IL-12. Supernatants of Salmonella-infected Mphi1 contained more IL-18 and IL-1beta as compared with supernatants of Mphi1 stimulated with isolated TLR agonists, and induced IFN-gamma production in human CD56(+) cells in an IL-23 and IL-1beta-dependent but IL-12-independent manner. In addition, IL-23 together with IL-18 or IL-1beta led to the production of GM-CSF in CD56(+) cells. Both IFN-gamma 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-gamma and GM-CSF. The IL-23 induced cytokines allow for the subsequent production of IL-12 and amplify the IFN-gamma production in the type-1 cytokine pathway.

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.

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.