Missile embolisation to the right common femoral artery following thoracic injury, without any conspicuous source of entry.

Embolisation of a projectile metallic body in the arterial system is not an unusual entity and has been reported off and on, in literature. We report the successful treatment and survival of a patient with thoracic injury, with a metallic projectile embolisation to the right common femoral artery and acute limb ischemia. Curiously, there was no identifiable entry point, on multiple imaging. The patient remained functionally independent, and without any complication on discharge. Supplementary Information: The online version contains supplementary material available at 10.1007/s12055-021-01291-1.

Distinct molecular phenotypes involving several human diseases are induced by IFN-λ3 and IFN-λ4 in monocyte-derived macrophages.

Human Interferon (IFN) lambda 3 (IFN-λ3) and IFN-λ4 are closely linked at the IFNL locus and show association with several diseases in genetic studies. Since they are only ~30% identical to each other, to better understand their roles in disease phenotypes, comparative studies are needed. Monocytes are precursors to macrophages (monocyte-derived macrophages; MDMs) that get differentiated under the influence of various immune factors, including IFNs. In a recent study, we characterized lipopolysaccharide-activated M1 and M2-MDMs that were differentiated in presence of IFN-λ3 or IFN-λ4. In this study, we performed transcriptomics on these M1 and M2-MDMs to further understand their molecular phenotypes. We identified over 760 genes that were reciprocally regulated by IFN-λ3 and IFN-λ4, additionally we identified over 240 genes that are significantly affected by IFN-λ4 but not IFN-λ3. We observed that IFN-λ3 was more active in M2-MDMs while IFN-λ4 showed superior response in M1-MDMs. Providing a structural explanation for these functional differences, molecular modeling showed differences in expected interactions of IFN-λ3 and IFN-λ4 with the extracellular domain of IFN-λR1. Further, pathway analysis showed several human infectious diseases and even cancer-related pathways being significantly affected by IFN-λ3 and/or IFN-λ4 in both M1 and M2-MDMs.

Functional genetic variants of the IFN-λ3 (IL28B) gene and transcription factor interactions on its promoter.

Interferon lambda 3 (IFN-λ3 or IFNL3, formerly IL28B), a type III interferon, modulates immune responses during infection/inflammation. Several human studies have reported an association of single nucleotide polymorphisms (SNP) in the IFNL3 locus with expression level of IFNL3. Previous genetic studies, in the context of hepatitis C virus infections, had predicted three regulatory SNPs: rs4803219, rs28416813 and rs4803217 that could have functional/causal roles. Subsequent studies confirmed this prediction for rs28416813 and rs4803217. A dinucleotide TA-repeat variant (rs72258881) has also been reported to be regulating the IFN-λ3 promoter. In this study, we tested all these genetic variants using a sensitive reporter assay. We show that the minor/ancestral alleles of both rs28416813 and rs4803217, together have a strong inhibitory effect on reporter gene expression. We also show an interaction between the two principal transcription factors regulating IFNL3 promoter: IRF7 and NF-kB RelA/p65. We show that IRF7 and p65 physically interact with each other. By using a transient ChIP assay, we show that presence of p65 increases the promoter occupancy of IRF7, thereby leading to synergistic activation of the IFNL3 promoter. We reason that, in contrast to p65, a unique nature of IRF7 binding to its specific DNA sequence makes it more sensitive to changes in DNA phasing. As a result, we see that IRF7, but not p65-mediated transcriptional activity is affected by the phase changes introduced by the TA-repeat polymorphism. Overall, we see that three genetic variants: rs28416813, rs4803217 and rs72258881 could have functional roles in controlling IFNL3 gene expression.

Monocytes differentiated into macrophages and dendritic cells in the presence of human IFN-λ3 or IFN-λ4 show distinct phenotypes.

Human IFN-λ4 is expressed by only a subset of individuals who possess the ΔG variant allele at the dinucleotide polymorphism rs368234815. Recent genetic studies have shown an association between rs368234815 and different infectious and inflammatory disorders. It is not known if IFN-λ4 has immunomodulatory activity. The expression of another type III IFN, IFN-λ3, is also controlled by genetic polymorphisms that are strongly linked to rs368234815. Therefore, it is of interest to compare these two IFNs for their effects on immune cells. Herein, using THP-1 cells, it was confirmed that IFN-λ4 could affect the differentiation status of macrophage-like cells and dendritic cells (DCs). The global gene expression changes induced by IFN-λ4 were also characterized in in vitro generated primary macrophages. Next, human PBMC-derived CD14+ monocytes were used to obtain M1 and M2 macrophages and DCs in the presence of IFN-λ3 or IFN-λ4. These DCs were cocultured with CD4+ Th cells derived from allogenic donors and their in vitro cytokine responses were measured. The specific activity of recombinant IFN-λ4 was much lower than that of IFN-λ3, as shown by induction of IFN-stimulated genes. M1 macrophages differentiated in the presence of IFN-λ4 showed higher IL-10 secretion than those differentiated in IFN-λ3. Coculture experiments suggested that IFN-λ4 could confer a Th2-biased phenotype to allogenic Th cells, wherein IFN-λ3, under similar circumstances, did not induce a significant bias toward either a Th1 or Th2 phenotype. This study shows for the first time that IFN-λ4 may influence immune responses by immunomodulation.

Identifying causal variants at the interferon lambda locus in case-control studies: Utilizing non-synonymous variant rs117648444 to probe the role of IFN-λ4.

Genetic variants at the interferon lambda (IFNL) locus have been associated with several human phenotypes in both disease and health. In chronic hepatitis C virus (HCV) infections, where the IFNL variants were first identified to be associated with response to interferon-α-ribavirin therapy, the available data clearly suggests that the causal variant could be the dinucleotide polymorphism rs368234815 that causes an open reading frame-shift in the IFNL4 gene resulting in expression of a functional IFN-λ4, a new type III IFN. In other human diseases/phenotypes where IFNL variants have been recently associated with, the causal mechanism remains unclear. In vitro evidence has shown that other IFNL variants (rs28416813, rs4803217) may regulate expression of another type III IFN, IFN-λ3. Therefore, expression of a functional IFN-λ4 and quantitative differences in IFN-λ3 expression are two potential causal mechanisms behind the observed phenotypes. Since these two potential causal mechanisms involve features of mutual exclusivity and overlapping functions, it is difficult to differentiate one from the other, in vivo, in absence of other implicating evidences. In addition, the strong linkage disequilibrium (LD) observed in many populations at the IFNL locus makes it difficult to tease out the actual functional/causal variants responsible for the phenotypes. The non-synonymous single nucleotide polymorphism rs117648444 that alters the activity of IFN-λ4 and the LD structure in the IFNL region which leads to a confounding effect of rs117648444 on other IFNL variants, provide us with additional tools in case-control studies to probe the role of IFN-λ4.

Confounding by Single Nucleotide Polymorphism rs117648444 (P70S) Affects the Association of Interferon Lambda Locus Variants with Response to Interferon-α-Ribavirin Therapy in Patients with Chronic Genotype 3 Hepatitis C Virus Infection.

Genome-wide association studies discovered interferon lambda (IFNL or IFN-λ) locus on chromosome 19 to be involved in clearance of chronic hepatitis C virus (HCV) infection in patients following interferon-α-ribavirin (IFN-RBV) therapy. Subsequent studies established a dinucleotide polymorphism rs368234815, as the prime causal variant behind this association. The ΔG allele of this variant gives rise to a new IFNL gene, IFNL4, coding for IFN-λ4 whose activity paradoxically associates with lesser viral clearance rates. A low-frequency, nonsynonymous single nucleotide polymorphism (SNP) rs117648444 within the 2nd exon of IFNL4 changes the 70th amino acid from proline to serine resulting in lower activity of the functional IFN-λ4 protein, thereby increasing HCV clearance rates. In the present study, we used a cohort of genotype 3 HCV-infected patients, drawn from different geographical regions of India who underwent IFN-RBV therapy, to examine the association of several important IFNL locus SNPs/variants with sustained virological response (SVR). Intriguingly, the causal variant rs368234815 did not show the best strength and significance of association with SVR, while further analysis revealed that a negative confounding effect of rs117648444 was responsible for this phenomenon. Our results indicate that IFNL locus SNPs are subject to either a positive or a negative confounding effect by rs117648444; the nature of confounding depends on the linkage of the IFNL SNPs with the low-activity IFN-λ4-generating minor allele of rs117648444. Thus, our work demonstrates that the linkage disequilibrium structure of the IFNL region may confound the results of association studies. These results have implications for the design and understanding of future case-control studies involving IFNL locus SNPs/variants.

Roles for Transcription Factors Sp1, NF-κB, IRF3, and IRF7 in Expression of the Human IFNL4 Gene.

The expression of a biologically active human IFNλ4 depends on the presence of a frameshift deletion polymorphism within the first exon of the interferon lambda 4 (IFNL4) gene. In this report, we use the lung carcinoma-derived cell line, A549, which is genetically viable to express a functional IFNλ4, to address transcriptional requirements of the IFNL4 gene. We show that the GC-rich DNA-binding transcription factor (TF) specificity protein 1 (Sp1) is recruited to the IFNL4 promoter and has a role in induction of gene expression upon stimulation with viral RNA mimic poly(I:C). By using RNAi and overexpression strategies, we also show key roles in IFNL4 gene expression for the virus-inducible TFs, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), IFN regulatory factor 3 (IRF3), and IRF7. Interestingly, we also observe that overexpression of IFNλ4 influences IFNL4 promoter activity, which may further be dependent on the retinoic acid-inducible gene-I (RIG-I)-like receptor pathway. Together, our work for the first time reports on the functional characterization of the human IFNL4 promoter.