Dusan Bogunovic

Aberrant miR-182 expression promotes melanoma metastasis by repressing FOXO3 and microphthalmia-associated transcription factor.

The highly aggressive character of melanoma makes it an excellent model for probing the mechanisms underlying metastasis, which remains one of the most difficult challenges in treating cancer. We find that miR-182, member of a miRNA cluster in a chromosomal locus (7q31–34) frequently amplified in melanoma, is commonly up-regulated in human melanoma cell lines and tissue samples; this up-regulation correlates with gene copy number in a subset of melanoma cell lines. Moreover, miR-182 ectopic expression stimulates migration of melanoma cells in vitro and their metastatic potential in vivo, whereas miR-182 down-regulation impedes invasion and triggers apoptosis. We further show that miR-182 over-expression promotes migration and survival by directly repressing microphthalmia-associated transcription factor-M and FOXO3, whereas enhanced expression of either microphthalmia-associated transcription factor-M or FOXO3 blocks miR-182s proinvasive effects. In human tissues, expression of miR-182 increases with progression from primary to metastatic melanoma and inversely correlates with FOXO3 and microphthalmia-associated transcription factor levels. Our data provide a mechanism for invasion and survival in melanoma that could prove applicable to metastasis of other cancers and suggest that miRNA silencing may be a worthwhile therapeutic strategy.

Mycobacterial disease and impaired IFN-γ immunity in humans with inherited ISG15 deficiency.

Tuberculosis Vaccine Conundrum Some children experience severe clinical disease when they are vaccinated against tuberculosis, an attenuated live vaccine that is normally innocuous in humans. Several germline mutations have been identified that account for this susceptibility, and now Bogunovic et al. (p. 1684, published online 2 August) add another to the list—ISG15. Uncovering this mutation, which is inherited in an autosomal recessive manner, was a surprise because studies with mice deficient in ISG15 showed enhanced susceptibility to some viral, but not bacterial, infections. Nevertheless, patients lacking ISG15 were not able to produce adequate amounts of interferon-γ, a cytokine critical for clearance of the bacteria. A mutation that accounts for adverse reactions to the Bacille Calmette-Guérin vaccine against tuberculosis is identified. ISG15 is an interferon (IFN)-α/β–inducible, ubiquitin-like intracellular protein. Its conjugation to various proteins (ISGylation) contributes to antiviral immunity in mice. Here, we describe human patients with inherited ISG15 deficiency and mycobacterial, but not viral, diseases. The lack of intracellular ISG15 production and protein ISGylation was not associated with cellular susceptibility to any viruses that we tested, consistent with the lack of viral diseases in these patients. By contrast, the lack of mycobacterium-induced ISG15 secretion by leukocytes—granulocyte, in particular—reduced the production of IFN-γ by lymphocytes, including natural killer cells, probably accounting for the enhanced susceptibility to mycobacterial disease. This experiment of nature shows that human ISGylation is largely redundant for antiviral immunity, but that ISG15 plays an essential role as an IFN-γ–inducing secreted molecule for optimal antimycobacterial immunity.

Human intracellular ISG15 prevents interferon-α/β over-amplification and auto-inflammation

Intracellular ISG15 is an interferon (IFN)-α/β-inducible ubiquitin-like modifier which can covalently bind other proteins in a process called ISGylation; it is an effector of IFN-α/β-dependent antiviral immunity in mice. We previously published a study describing humans with inherited ISG15 deficiency but without unusually severe viral diseases. We showed that these patients were prone to mycobacterial disease and that human ISG15 was non-redundant as an extracellular IFN-γ-inducing molecule. We show here that ISG15-deficient patients also display unanticipated cellular, immunological and clinical signs of enhanced IFN-α/β immunity, reminiscent of the Mendelian autoinflammatory interferonopathies Aicardi–Goutières syndrome and spondyloenchondrodysplasia. We further show that an absence of intracellular ISG15 in the patients’ cells prevents the accumulation of USP18, a potent negative regulator of IFN-α/β signalling, resulting in the enhancement and amplification of IFN-α/β responses. Human ISG15, therefore, is not only redundant for antiviral immunity, but is a key negative regulator of IFN-α/β immunity. In humans, intracellular ISG15 is IFN-α/β-inducible not to serve as a substrate for ISGylation-dependent antiviral immunity, but to ensure USP18-dependent regulation of IFN-α/β and prevention of IFN-α/β-dependent autoinflammation.

Immune profile and mitotic index of metastatic melanoma lesions enhance clinical staging in predicting patient survival

Although remission rates for metastatic melanoma are generally very poor, some patients can survive for prolonged periods following metastasis. We used gene expression profiling, mitotic index (MI), and quantification of tumor infiltrating leukocytes (TILs) and CD3+ cells in metastatic lesions to search for a molecular basis for this observation and to develop improved methods for predicting patient survival. We identified a group of 266 genes associated with postrecurrence survival. Genes positively associated with survival were predominantly immune response related (e.g., ICOS, CD3d, ZAP70, TRAT1, TARP, GZMK, LCK, CD2, CXCL13, CCL19, CCR7, VCAM1) while genes negatively associated with survival were cell proliferation related (e.g., PDE4D, CDK2, GREF1, NUSAP1, SPC24). Furthermore, any of the 4 parameters (prevalidated gene expression signature, TILs, CD3, and in particular MI) improved the ability of Tumor, Node, Metastasis (TNM) staging to predict postrecurrence survival; MI was the most significant contributor (HR = 2.13, P = 0.0008). An immune response gene expression signature and presence of TILs and CD3+ cells signify immune surveillance as a mechanism for prolonged survival in these patients and indicate improved patient subcategorization beyond current TNM staging.

LXR promotes the maximal egress of monocyte-derived cells from mouse aortic plaques during atherosclerosis regression

We have previously shown that mouse atherosclerosis regression involves monocyte-derived (CD68+) cell emigration from plaques and is dependent on the chemokine receptor CCR7. Concurrent with regression, mRNA levels of the gene encoding LXRalpha are increased in plaque CD68+ cells, suggestive of a functional relationship between LXR and CCR7. To extend these results, atherosclerotic Apoe-/- mice sufficient or deficient in CCR7 were treated with an LXR agonist, resulting in a CCR7-dependent decrease in plaque CD68+ cells. To test the requirement for LXR for CCR7-dependent regression, we transplanted aortic arches from atherosclerotic Apoe-/- mice, or from Apoe-/- mice with BM deficiency of LXRalpha or LXRbeta, into WT recipients. Plaques from both LXRalpha and LXRbeta-deficient Apoe-/- mice exhibited impaired regression. In addition, the CD68+ cells displayed reduced emigration and CCR7 expression. Using an immature DC line, we found that LXR agonist treatment increased Ccr7 mRNA levels. This increase was blunted when LXRalpha and LXRbeta levels were reduced by siRNAs. Moreover, LXR agonist treatment of primary human immature DCs resulted in functionally significant upregulation of CCR7. We conclude that LXR is required for maximal effects on plaque CD68+ cell expression of CCR7 and monocyte-derived cell egress during atherosclerosis regression in mice.

Human TYK2 deficiency: Mycobacterial and viral infections without hyper-IgE syndrome

Kreins et al. report the identification and immunological characterization of a group of TYK2-deficient patients.

Human USP18 deficiency underlies type 1 interferonopathy leading to severe pseudo-TORCH syndrome

Meuwissen and collaborators define a novel genetic cause of pseudo-TORCH syndrome, which resembles the sequelae of congenital infection and represents a novel type I interferonopathy.

A novel form of human STAT1 deficiency impairing early but not late responses to interferons

Autosomal recessive STAT1 deficiency is associated with impaired cellular responses to interferons and susceptibility to intracellular bacterial and viral infections. We report here a new form of partial STAT1 deficiency in 2 siblings presenting mycobacterial and viral diseases. Both carried a homozygous missense mutation replacing a lysine with an asparagine residue at position 201 (K201N) of STAT1. This mutation causes the abnormal splicing out of exon 8 from most STAT1 mRNAs, thereby decreasing (by ~ 70%) STAT1 protein levels. The mutant STAT1 proteins are not intrinsically deleterious, in terms of tyrosine phosphorylation, dephosphorylation, homodimerization into γ-activating factor and heterotrimerization into ISGF-3, binding to specific DNA elements, and activation of the transcription. Interestingly, the activation of γ-activating factor and ISGF3 was impaired only at early time points in the various cells from patient (within 1 hour of stimulation), whereas sustained impairment occurs in other known forms of complete and partial recessive STAT1 deficiency. Consequently, delayed responses were normal; however, the early induction of interferon-stimulated genes was selectively and severely impaired. Thus, the early cellular responses to human interferons are critically dependent on the amount of STAT1 and are essential for the appropriate control of mycobacterial and viral infections.

TLR4 Engagement during TLR3-Induced Proinflammatory Signaling in Dendritic Cells Promotes IL-10–Mediated Suppression of Antitumor Immunity

Toll-like receptor (TLR) agonists are promising adjuvants for immune therapy of cancer, but their potential efficacy as single or combinatorial agents has yet to be fully evaluated. Here, we report that among all TLR agonists tested, dendritic cells (DC) stimulated with the TLR3 agonist polyI:C displayed the strongest activity in stimulating proinflammatory responses and the production of melanoma antigen-specific CD8(+) T cells. Simultaneous treatment with TLR7/8 agonists further improved these responses, but the inclusion of bacterial lipopolysaccharide (LPS), a TLR4 agonist, suppressed proinflammatory cytokine production. This inhibition was contingent upon rapid induction of the suppressive cytokine interleukin (IL)-10 by LPS, leading to dysregulated immune responses and it could be reversed by signal transducers and activators of transcription 3 knockdown, p38 blockade or antibodies to IL-10 and its receptor. Our findings show how certain TLR agonist combinations can enhance or limit DC responses associated with antitumor immunity, through their relative ability to induce IL-10 pathways that are immune suppressive.

ISG15: leading a double life as a secreted molecule

ISG15 is a well-known intracellular ubiquitin-like molecule involved in ISGylation. However, a recent study has revived the notion first put forward two decades ago that ISG15 is also a secreted molecule. Human neutrophils, monocytes and lymphocytes can release ISG15, even though this protein has no detectable signal peptide sequence. ISG15 has also been found in the secretory granules of granulocytes. The mechanism underlying ISG15 secretion is unknown. Secreted ISG15 acts on at least T and natural killer (NK) lymphocytes, in which it induces interferon (IFN)-γ production. However, the mechanism by which ISG15 stimulates these cells also remains unclear. ISG15 and IFN-γ seem to define an innate circuit that operates preferentially, but not exclusively, between granulocytes and NK cells. Inherited ISG15 deficiency is associated with severe mycobacterial disease in both mice and humans. This infectious phenotype probably results from the lack of secreted ISG15, because patients and mice with other inborn errors of IFN-γ immunity also display mycobacterial diseases. In addition to raising mechanistic issues, the studies described here pave the way for clinical studies of various aspects, ranging from the use of recombinant ISG15 in patients with infectious diseases to the use of ISG15-blocking agents in patients with inflammatory diseases.