Josiane Ragimbeau

Evolutionary dynamics of human Toll-like receptors and their different contributions to host defense.

Infectious diseases have been paramount among the threats to health and survival throughout human evolutionary history. Natural selection is therefore expected to act strongly on host defense genes, particularly on innate immunity genes whose products mediate the direct interaction between the host and the microbial environment. In insects and mammals, the Toll-like receptors (TLRs) appear to play a major role in initiating innate immune responses against microbes. In humans, however, it has been speculated that the set of TLRs could be redundant for protective immunity. We investigated how natural selection has acted upon human TLRs, as an approach to assess their level of biological redundancy. We sequenced the ten human TLRs in a panel of 158 individuals from various populations worldwide and found that the intracellular TLRs—activated by nucleic acids and particularly specialized in viral recognition—have evolved under strong purifying selection, indicating their essential non-redundant role in host survival. Conversely, the selective constraints on the TLRs expressed on the cell surface—activated by compounds other than nucleic acids—have been much more relaxed, with higher rates of damaging nonsynonymous and stop mutations tolerated, suggesting their higher redundancy. Finally, we tested whether TLRs have experienced spatially-varying selection in human populations and found that the region encompassing TLR10-TLR1-TLR6 has been the target of recent positive selection among non-Africans. Our findings indicate that the different TLRs differ in their immunological redundancy, reflecting their distinct contributions to host defense. The insights gained in this study foster new hypotheses to be tested in clinical and epidemiological genetics of infectious disease.

The tyrosine kinase Tyk2 controls IFNAR1 cell surface expression.

The four mammalian Jak tyrosine kinases are non‐covalently associated with cell surface receptors binding helical bundled cytokines. In the type I interferon receptor, Tyk2 associates with the IFNAR1 receptor subunit and positively influences ligand binding to the receptor complex. Here, we report that Tyk2 is essential for stable cell surface expression of IFNAR1. In the absence of Tyk2, mature IFNAR1 is weakly expressed on the cell surface. Rather, it is localized into a perinuclear endosomal compartment which overlaps with that of recycling transferrin receptors and with early endosomal antigen‐1 (EEA1) positive vesicles. Conversely, co‐expressed Tyk2 greatly enhances surface IFNAR1 expression. Importantly, we demonstrate that Tyk2 slows down IFNAR1 degradation and that this is due, at least in part, to inhibition of IFNAR1 endocytosis. In addition, Tyk2 induces plasma membrane relocalization of the R2 subunit of the interleukin‐10 receptor. These results reveal a novel function of a Jak protein on internalization of a correctly processed cytokine receptor. This function is distinct from the previously reported effect of other Jak proteins on receptor exit from the endoplasmic reticulum.

A natural mutation in the Tyk2 pseudokinase domain underlies altered susceptibility of B10.Q/J mice to infection and autoimmunity

The B10.Q/J strain of mice was serendipitously discovered to be highly susceptible to infection by the intracellular protozoan parasite, Toxoplasma gondii but markedly resistant to induction of autoimmune arthritis. We have previously shown that the B10.Q/J phenotype is controlled by a single recessive locus and is associated with lymphocyte hyporesponsiveness to IL-12. Using genetic approaches, we have now localized the B10.Q/J locus to chromosome 9 and established its identity as Tyk2, a Janus kinase essential for IL-12 and IFN-α/β cytokine signaling. The B10.Q/J Tyk2 gene contained a single missense mutation resulting in a nonconservative amino acid substitution (E775K) in an invariant motif of the pseudokinase (Janus kinase homology 2) domain. This mutation appeared to result in the absence of the B10.Q/J-encoded Tyk2 protein, despite presence of Tyk2-specific transcripts. Phenotypically, B10.Q/J cells were indistinguishable from Tyk2-deficient cells, showing impaired signaling and biologic responses to IL-12, IL-23, and type I IFNs. The analogous E782K mutant of human Tyk2 failed to restore IFN-α responsiveness in Tyk2 null 11,1 cells. Our results indicate a crucial role for Tyk2 in T helper 1-mediated protective and pathogenic immune responses. An additional implication of our findings is that naturally occurring mutations in the Tyk2 gene may underlie altered susceptibilities to infectious or autoimmune diseases in human and animal populations.

TYK2 activity promotes ligand-induced IFNAR1 proteolysis

The type I IFNR (interferon receptor) is a heterodimer composed of two transmembrane chains, IFNAR1 (interferon-alpha receptor 1 subunit) and IFNAR2, which are associated with the tyrosine kinases Tyk2 and Jak1 (Janus kinase 1) respectively. Ligand-induced down-regulation of the type I IFNR is a major mechanism of negative regulation of cellular signalling and involves the internalization and lysosomal degradation of IFNAR1. IFNalpha promotes the phosphorylation of IFNAR1 on Ser535, followed by recruitment of the E3 ubiquitin ligase, beta-TrCP2 (beta-transducin repeats-containing protein 2), ubiquitination of IFNAR1 and proteolysis. The non-catalytic role of Tyk2 in sustaining the steady-state IFNAR1 level at the plasma membrane is well documented; however, little is known about the function of Tyk2 in the steps that precede and succeed serine phosphorylation and ubiquitination of IFNAR1 in response to ligand binding. In the present study, we show that catalytic activation of Tyk2 is not essential for IFNAR1 internalization, but is required for ligand-induced IFNAR1 serine phosphorylation, ubiquitination and efficient lysosomal proteolysis.

Differential responsiveness to IFN-α and IFN-β of human mature DC through modulation of IFNAR expression

In human monocyte‐derived dendritic cells (DC), infection with Mycobacterium tuberculosis and viruses or stimulation with Toll‐like receptor type 3 and 4 agonists causes the release of type I interferon (IFN). Here, we describe that the IFN‐β released upon stimulation with lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (poly I:C) is responsible for a rapid and sustained signal transducer and activator of transcription 1 and 2 activation and expression of IFN‐stimulated genes, such as the transcription factor IFN regulatory factor 7 and the chemokine CXC chemokine ligand 10. The autocrine production of IFN‐β from LPS and poly I:C‐matured DC (mDC) induced a temporary saturation of the response to type I IFN and a marked decline in the level of the two IFN receptor (IFNAR) subunits. It is interesting that we found that upon clearing of the released cytokines, LPS‐stimulated DC reacquired full responsiveness to IFN‐β but only partial responsiveness to IFN‐α, and their maturation process was unaffected. Monitoring of surface and total levels of the receptor subunits showed that maximal expression of IFNAR2 resumed within 24 h of clearing, and IFNAR1 expression remained low. Thus, mDC can modulate their sensitivity to two IFN subtypes through a differential regulation of the IFNAR subunits.

Comparable potency of IFNα2 and IFNβ on immediate JAK/STAT activation but differential down-regulation of IFNAR2

Type I IFNs (interferons) (IFNα/β) form a family of related cytokines that control a variety of cellular functions through binding to a receptor composed of IFNAR (IFNα receptor subunit) 1 and 2. Among typexa0I IFNs, the α2 and β subtypes exhibit a large difference in their binding affinities to IFNAR1, and it was suggested that high concentrations of IFNAR1 may compensate for its low intrinsic binding affinity for IFNα2. We tested whether receptor-proximal signalling events are sensitive to IFNAR1 surface concentration by investigating the relationship between relative IFNAR1/IFNAR2 surface levels and IFNα2 and IFNβ signalling potencies in several cell lines. For this, we monitored the activation profile of JAK (Janus kinase)/STAT (signal transducer and activator of transcription) proteins, measured basal and ligand-induced surface decay of each receptor subunit and tested the effect of variable IFNAR1 levels on IFNα2 signalling potency. Our data show that the cell-surface IFNAR1 level is indeed a limiting factor for assembly of the functional complex, but an increased concentration of it does not translate into an IFNα/β differential JAK/STAT signalling nor does it change the dynamics of the engaged receptor. Importantly, however, our data highlight a differential effect upon routing of IFNAR2. Following binding of IFNα2, IFNAR2 is internalized, but, instead of being routed towards degradation as it is when complexed to IFNβ, it recycles back to the cell surface. These observations suggest strongly that the stability and the intracellular lifetime of the ternary complex account for the differential control of IFNAR2. Moreover, the present study opens up the attractive possibility that endosomal-initiated signalling may contribute to IFNα/β differential bioactivities.

Ligand-independent pathway that controls stability of interferon alpha receptor.

Ligand-specific negative regulation of cytokine-induced signaling relies on down regulation of the cytokine receptors. Down regulation of the IFNAR1 sub-unit of the Type I interferon (IFN) receptor proceeds via lysosomal receptor proteolysis, which is triggered by ubiquitination that depends on IFNAR1 serine phosphorylation. While IFN-inducible phosphorylation, ubiquitination, and degradation requires the catalytic activity of the Tyk2 Janus kinase, here we found the ligand- and Tyk2-independent pathway that promotes IFNAR1 phosphorylation, ubiquitination, and degradation when IFNAR1 is expressed at high levels. A major cellular kinase activity that is responsible for IFNAR1 phosphorylation in vitro does not depend on either ligand or Tyk2 activity. Inhibition of ligand-independent IFNAR1 degradation suppresses cell proliferation. We discuss the signaling events that might lead to ubiquitination and degradation of IFNAR1 via ligand-dependent and independent pathways and their potential physiologic significance.

Jamip1 (Marlin-1) defines a family of proteins interacting with Janus kinases and microtubules

Jamip1 (Jak and microtubule interacting protein), an alias of Marlin-1, was identified for its ability to bind to the FERM (band 4.1 ezrin/radixin/moesin) homology domain of Tyk2, a member of the Janus kinase (Jak) family of non-receptor tyrosine kinases that are central elements of cytokine signaling cascades. Jamip1 belongs to a family of three genes conserved in vertebrates and is predominantly expressed in neural tissues and lymphoid organs. Jamip proteins lack known domains and are extremely rich in predicted coiled coils that mediate dimerization. In our initial characterization of Jamip1 (73 kDa), we found that it comprises an N-terminal region that targets the protein to microtubule polymers and, when overexpressed in fibroblasts, profoundly perturbs the microtubule network, inducing the formation of tight and stable bundles. Jamip1 was shown to associate with two Jak family members, Tyk2 and Jak1, in Jurkat T cells via its C-terminal region. The restricted expression of Jamip1 and its ability to associate to and modify microtubule polymers suggest a specialized function of these proteins in dynamic processes, e.g. cell polarization, segregation of signaling complexes, and vesicle traffic, some of which may involve Jak tyrosine kinases.

Two Rare Disease-Associated Tyk2 Variants Are Catalytically Impaired but Signaling Competent

Tyk2 belongs to the Janus protein tyrosine kinase family and is involved in signaling of immunoregulatory cytokines (type I and III IFNs, IL-6, IL-10, and IL-12 families) via its interaction with shared receptor subunits. Depending on the receptor complex, Tyk2 is coactivated with either Jak1 or Jak2, but a detailed molecular characterization of the interplay between the two enzymes is missing. In human populations, the Tyk2 gene presents high levels of genetic diversity with >100 nonsynonymous variants being detected. In this study, we characterized two rare Tyk2 variants, I684S and P1104A, which have been associated with susceptibility to autoimmune disease. Specifically, we measured their in vitro catalytic activity and their ability to mediate Stat activation in fibroblasts and genotyped B cell lines. Both variants were found to be catalytically impaired but rescued signaling in response to IFN-α/β, IL-6, and IL-10. These data, coupled with functional study of an engineered Jak1 P1084A, support a model of nonhierarchical activation of Janus kinases in which one catalytically competent Jak is sufficient for signaling provided that its partner behaves as proper scaffold, even if inactive. Through the analysis of IFN-α and IFN-γ signaling in cells with different Jak1 P1084A levels, we also illustrate a context in which a hypomorphic Jak can hamper signaling in a cytokine-specific manner. Given the multitude of Tyk2-activating cytokines, the cell context–dependent requirement for Tyk2 and the catalytic defect of the two disease-associated variants studied in this paper, we predict that these alleles are functionally significant in complex immune disorders.

NF-κB is required for STAT-4 expression during dendritic cell maturation

The transcription factor STAT‐4 plays a pivotal role in the IL‐12‐mediated development of naive CD4+ T cells into the Th1 phenotype. Initially thought to be restricted to the lymphoid lineage, STAT‐4 was subsequently shown to be expressed in the myeloid compartment, mainly in activated monocytes, macrophages, and dendritic cells (DC). Here, we have studied STAT‐4 in human monocyte‐derived DC, and we demonstrated that its expression can be induced by multiple stimuli, such as the ligands for TLR‐4, TLR‐2, and TLR‐3, different pathogens, CD40 ligand, and the proinflammatory cytokines TNF‐α and IL‐1β. It is interesting that we found that STAT‐4 is tyrosine‐phosphorylated in response to type I IFN but not IL‐12 in human mature DC. Cloning and functional analysis of the STAT‐4 promoter showed that a NF‐κB binding site, localized at –969/–959 bp upstream of the transcriptional start site, is involved in the regulation of this gene in primary human DC. EMSAs using a probe containing this NF‐κB binding sequence and chromatin immunoprecipitation indicated that p65/p50 and p50/p50 dimers were the main NF‐κB/Rel proteins involved in STAT‐4 gene expression in maturing DC. The mutation of this κB site or the overexpression of the repressor IκBα exerted an inhibitory effect on a STAT‐4 promoter‐driven reporter as well as on STAT‐4 expression. Altogether, these results indicate that STAT‐4 can be finely tuned along with DC maturation through NF‐κB activation and that its induction may be involved in preparing the DC to be receptive to the cytokine environment present in lymphoid organs.