Effect of titanium implants along with silver ions and tetracycline on type I interferon-beta expression during implant-related infections in co-culture and mouse model.

Type I interferon-beta (IFN-ß) is a crucial component of innate and adaptive immune systems inside the host. The formation of bacterial biofilms on medical implants can lead to inflammatory diseases and implant failure. Biofilms elicit IFN-ß production inside the host that, in turn, restrict bacterial growth. Biofilms pose strong antibiotic resistance, whereas surface modification of medical implants with antibacterial agents may demonstrate strong antimicrobial effects. Most of the previous investigations were focused on determining the antibacterial activities of implant surfaces modified with antibacterial agents. The present study, for the first time, measured antibacterial activities and IFN-ß expression of titanium surfaces along with silver or tetracycline inside co-culture and mouse models. A periodontal pathogen: Aggregatibacter actinomycetemcomitans reported to induce strong inflammation, was used for infection. Silver and tetracycline were added to the titanium surface using the heat evaporation method. Macrophages showed reduced compatibility on titanium surfaces with silver, and IFN-ß expression inside cultured cells significantly decreased. Macrophages showed compatibility on implant surfaces with tetracycline, but IFN-ß production significantly decreased inside seeded cells. The decrease in IFN-ß production inside macrophages cultured on implant surfaces with silver and tetracycline was not related to the downregulation of Ifn-ß gene. Bacterial infection significantly upregulated mRNA expression levels of Isg15, Mx1, Mx2, Irf-3, Irf-7, Tlr-2, Tnf-α, Cxcl-1, and Il-6 genes. Notably, mRNA expression levels of Mx1, Irf7, Tlr2, Tnf-α, Cxcl1, and Il-6 genes inside macrophages significantly downregulated on implant surfaces with silver or tetracycline. Titanium with tetracycline showed higher antibacterial activities than silver. The in vivo evaluation of IFN-ß expression around implants was measured inside transgenic mice constitutive for IFN-ß expression. Of note, the non-invasive in vivo imaging revealed a significant decrease in IFN-ß expression around subcutaneous implants with silver compared to titanium and titanium with tetracycline in sterile or infected situations. The histology of peri-implant tissue interfaces around infected implants with silver showed a thick interface with a significantly higher accumulation of inflammatory cells. Titanium implants with silver and tetracycline remained antibacterial in mice. Findings from this study unequivocally indicate that implant surfaces with silver decrease IFN-ß expression, a crucial component of host immunity.

ISG15 deficiency features a complex cellular phenotype that responds to treatment with itaconate and derivatives.

BACKGROUND: Congenital ISG15 deficiency is a rare autoinflammatory disorder that is driven by chronically elevated systemic interferon levels and predominantly affects central nervous system and skin. METHODS AND RESULTS: We have developed induced pluripotent stem cell-derived macrophages and endothelial cells as a model to study the cellular phenotype of ISG15 deficiency and identify novel treatments. ISG15-/- macrophages exhibited the expected hyperinflammatory responses, but normal phagocytic function. In addition, they displayed a multifaceted pathological phenotype featuring increased apoptosis/pyroptosis, oxidative stress, glycolysis, and acylcarnitine levels, but decreased glutamine uptake, BCAT1 expression, branched chain amino acid catabolism, oxidative phosphorylation, ß-oxidation, and NAD(P)H-dependent oxidoreductase activity. Furthermore, expression of genes involved in mitochondrial biogenesis and respiratory chain complexes II-V was diminished in ISG15-/- cells. Defective mitochondrial respiration was restored by transduction with wild-type ISG15, but only partially by a conjugation-deficient variant, suggesting that some ISG15 functions in mitochondrial respiration require ISGylation to cellular targets. Treatment with itaconate, dimethyl-itaconate, 4-octyl-itaconate, and the JAK1/2 inhibitor ruxolitinib ameliorated increased inflammation, propensity for cell death, and oxidative stress. Furthermore, the treatments greatly improved mitochondria-related gene expression, BCAT1 levels, redox balance, and intracellular and extracellular ATP levels. However, efficacy differed among the compounds according to read-out and cell type, suggesting that their effects on cellular targets are not identical. Indeed, only itaconates increased expression of anti-oxidant genes NFE2L2, HMOX1, and GPX7, and dimethyl-itaconate improved redox balance the most. Even though itaconate treatments normalized the elevated expression of interferon-stimulated genes, ISG15-/- macrophages maintained their reduced susceptibility to influenza virus infection. CONCLUSIONS: These findings expand the cellular phenotype of human ISG15 deficiency and reveal the importance of ISG15 for regulating oxidative stress, branched chain amino acid metabolism, and mitochondrial function in humans. The results validate ruxolitinib as treatment for ISG15 deficiency and suggest itaconate-based medications as additional therapeutics for this rare disorder.

Congenital deficiency reveals critical role of ISG15 in skin homeostasis.

Ulcerating skin lesions are manifestations of human ISG15 deficiency, a type I interferonopathy. However, chronic inflammation may not be their exclusive cause. We describe two siblings with recurrent skin ulcers that healed with scar formation upon corticosteroid treatment. Both had a homozygous nonsense mutation in the ISG15 gene, leading to unstable ISG15 protein lacking the functional domain. We characterized ISG15-/- dermal fibroblasts, HaCaT keratinocytes, and human induced pluripotent stem cell-derived vascular endothelial cells. ISG15-deficient cells exhibited the expected hyperinflammatory phenotype, but also dysregulated expression of molecules critical for connective tissue and epidermis integrity, including reduced collagens and adhesion molecules, but increased matrix metalloproteinases. ISG15-/- fibroblasts exhibited elevated ROS levels and reduced ROS scavenger expression. As opposed to hyperinflammation, defective collagen and integrin synthesis was not rescued by conjugation-deficient ISG15. Cell migration was retarded in ISG15-/- fibroblasts and HaCaT keratinocytes, but normalized under ruxolitinib treatment. Desmosome density was reduced in an ISG15-/- 3D epidermis model. Additionally, there were loose architecture and reduced collagen and desmoglein expression, which could be reversed by treatment with ruxolitinib/doxycycline/TGF-ß1. These results reveal critical roles of ISG15 in maintaining cell migration and epidermis and connective tissue homeostasis, whereby the latter likely requires its conjugation to yet unidentified targets.