Tyk2 and JAK1, members of the Janus kinase (JAK) family of protein tyrosine kinases, are required for interferon-alpha/beta binding and signaling. Both enzymes are associated with the interferon-alpha/beta receptor, and upon ligand binding, they undergo tyrosine phosphorylation and catalytic activation in an interdependent manner. To identify residues involved in Tyk2 regulation and to understand the basis of the interdependence of Tyk2 and JAK1, six mutated versions of Tyk2 bearing single or multiple point mutations in the tyrosine kinase domain were studied in a cell line lacking endogenous Tyk2. The Y1054F/Y1055F substitutions in the putative activation loop prevented ligand-dependent activation of Tyk2, without abolishing its catalytic potential. The K930R mutation in the ATP binding site generated a kinase-negative protein, which however, still became phosphorylated upon interferon-alpha treatment. The Y1054F/Y1055F substitutions in this kinase-negative Tyk2 abolished the induced phosphorylation. These results indicate that Tyk2 is activated by phosphorylation on Tyr-1054 and/or Tyr-1055 and that this phosphorylation requires another kinase, most likely JAK1. While the Tyk2 forms mutated on Tyr-1054 and Tyr-1055 or on Lys-930 allowed some inducible gene expression, the combination of the three point mutations totally abolished signaling.

The chemokine system mediates acute inflammation by driving leukocyte migration to damaged or infected tissues. However, elevated expression of chemokines and their receptors can contribute to chronic inflammation and malignancy. Thus, great effort has been taken to target these molecules. The first hint of the druggability of the chemokine system was derived from the role of chemokine receptors in HIV infection. CCR5 and CXCR4 function as essential co-receptors for HIV entry, with the former accounting for most new HIV infections worldwide. Not by chance, an anti-CCR5 compound, maraviroc, was the first FDA-approved chemokine receptor-targeting drug. CCR5, by directing leukocytes to sites of inflammation and regulating their activation, also represents an important player in the inflammatory response. This function is shared with CCR2 and its selective ligand CCL2, which constitute the primary chemokine axis driving the recruitment of monocytes/macrophages to inflammatory sites. Both receptors are indeed involved in the pathogenesis of several immune-mediated diseases, and dual CCR5/CCR2 targeting is emerging as a more efficacious strategy than targeting either receptor alone in the treatment of complex human disorders. In this review, we focus on the distinctive and complementary contributions of CCR5 and CCR2/CCL2 in HIV infection, multiple sclerosis, liver fibrosis and associated hepatocellular carcinoma. The emerging therapeutic approaches based on the inhibition of these chemokine axes are highlighted.

Dendritic cells (DCs) generated by a single-step exposure of human monocytes to type I IFN and GM-CSF (IFN-DCs) are endowed with potent immunostimulatory activities and a distinctive migratory response to specific chemokines. In this study, we evaluated the effects of 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), the biologically active metabolite of vitamin D₃, on the DC differentiation/activation induced by type I IFN. We found that 1,25(OH)₂D₃ prevented the generation of IFN-DCs when added to freshly isolated monocytes, and was capable of redirecting already differentiated IFN-DCs toward a more immature stage, as revealed by their immunophenotype, reduced allostimulatory activity, and impaired LPS-induced production of Th1-polarizing cytokines. Control and 1,25(OH)₂D₃-treated IFN-DCs exhibited a similar expression of vitamin D receptor, as well as comparable cell death rates. Furthermore, the chemotactic response of IFN-DCs to CCL4 and CCL19 was markedly reduced or completely abrogated by 1,25(OH)₂D₃. Despite these changes in the IFN-DC migratory behavior, the expression of CCR5 and CCR7 and the calcium fluxes triggered by CCL4 and CCL19 were not affected. These findings indicate that, in this innovative single-step DC generation model from monocytes, the suppressive effect of 1,25(OH)₂D₃ is associated with a potent impairment of DC migration in response to inflammatory and lymph node-homing chemokines, thus unraveling a novel mechanism involved in 1,25(OH)₂D₃-mediated immunomodulation.

Tyk2 belongs to the Janus kinase (JAK) family of receptor associated tyrosine kinases, characterized by a large N-terminal region, a kinase-like domain and a tyrosine kinase domain. It was previously shown that Tyk2 contributes to interferon-α (IFN-α) signaling not only catalytically, but also as an essential intracellular component of the receptor complex, being required for high affinity binding of IFN-α. For this function the tyrosine kinase domain was found to be dispensable. Here, it is shown that mutant cells lacking Tyk2 have significantly reduced IFN-α receptor 1 (IFNAR1) protein level, whereas the mRNA level is unaltered. Expression of the N-terminal region of Tyk2 in these cells reconstituted wild-type IFNAR1 level, but did not restore the binding activity of the receptor. Studies of mutant Tyk2 forms deleted at the N terminus indicated that the integrity of the N-terminal region is required to sustain IFNAR1. These studies also showed that the N-terminal region does not directly modulate the basal autophosphorylation activity of Tyk2, but it is required for efficientin vitroIFNAR1 phosphorylation and for rendering the enzyme activatable by IFN-α. Overall, these results indicate that distinct Tyk2 domains provide different functions to the receptor complex: the N-terminal region sustains IFNAR1 level, whereas the kinase-like domain provides a function toward high affinity ligand binding.

We investigated the effect of vpr, physiologically expressed during the course of an acute HIV-1 infection, on the response of infected cells to apoptotic stimuli as well as on the HIV-induced apoptosis. At 48 h after infection, Jurkat cells exhibited a lower susceptibility to undergo apoptosis with respect to uninfected cells. This effect was not observed following infection with either a vpr-mutated virus or a wild-type strain in the presence of antisense oligodeoxynucleotides targeted at vpr mRNA. Single-cell analysis, aimed at simultaneously identifying apoptotic and infected cells, revealed that resistance to apoptosis correlated with productive infection. Notably, vpr-dependent protection from induced apoptosis was also observed in HIV-1-infected PBMC. In contrast, at later stages of infection, a marked increase in the number of cells spontaneously undergoing apoptosis was detected in infected cultures. This virus-induced apoptosis involved vpr expression and predominantly occurred in productively infected cells. These results indicate that HIV-1 vpr can exert opposite roles in the regulation of apoptosis, which may depend on the level of its intracellular expression at different stages of HIV-1 infection. The dual function of vpr represents a novel mechanism in the complex strategy evolved by HIV to influence the turnover of T lymphocytes leading to either viral persistence or virus release and spreading.