IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans.

The intracellular protozoan parasite Leishmania donovani causes debilitating human diseases that involve visceral and dermal manifestations. Type 3 interferons (IFNs), also referred to as lambda IFNs (IFNL, IFN-L, or IFN-λ), are known to play protective roles against intracellular pathogens at the epithelial surfaces. Herein, we show that L. donovani induces IFN-λ3 in human as well as mouse cell line-derived macrophages. Interestingly, IFN-λ3 treatment significantly decreased parasite load in infected cells, mainly by increasing reactive oxygen species production. Microscopic examination showed that IFN-λ3 inhibited uptake but not replication, while the phagocytic ability of the cells was not affected. This was confirmed by experiments that showed that IFN-λ3 could decrease parasite load only when added to the medium at earlier time points, either during or soon after parasite uptake, but had no effect on parasite load when added at 24 h post-infection, suggesting that an early event during parasite uptake was targeted. Furthermore, the parasites could overcome the inhibitory effect of IFN-λ3, which was added at earlier time points, within 2-3 days post-infection. BALB/c mice treated with IFN-λ3 before infection led to a significant increase in expression of IL-4 and ARG1 post-infection in the spleen and liver, respectively, and to different pathological changes, especially in the liver, but not to changes in parasite load. Treatment with IFN-λ3 during infection did not decrease the parasite load in the spleen either. However, IFN-λ3 was significantly increased in the sera of visceral leishmaniasis patients, and the IFNL genetic variant rs12979860 was significantly associated with susceptibility to leishmaniasis.

A Novel Inactive Isoform with a Restored Reading Frame Is Expressed from the Human Interferon Lambda 4 TT Allele at rs368234815.

The TT allele of the dinucleotide variant rs368234815 (TT/ΔG) abolishes the open reading frame (ORF) created by the ancestral ΔG allele of the human interferon lambda 4 (IFNL4) gene, thus preventing the expression of a functional IFN-λ4 protein. While probing the expression of IFN-λ4 in human peripheral blood mononuclear cells (PBMCs), using a monoclonal antibody that binds to the C-terminus of IFN-λ4, surprisingly, we observed that PBMCs obtained from TT/TT genotype individuals could also express proteins that reacted with the IFN-λ4-specific antibody. We confirmed that these products did not emanate from the IFNL4 paralog, IF1IC2 gene. Using cell lines and overexpressing human IFNL4 gene constructs, we obtained evidence from Western blots to show that the TT allele could express a protein that reacted with the IFN-λ4 C-terminal-specific antibody. It had a molecular weight similar if not identical to IFN-λ4 expressed from the ΔG allele. Furthermore, the same start and stop codons used by the ΔG allele were used to express the novel isoform from the TT allele suggesting that a restoration of the ORF had occurred in the body of the mRNA. However, this TT allele isoform did not induce any IFN-stimulated gene expression. Our data do not support a ribosomal frameshift that leads to the expression of this new isoform, implying that an alternate splicing event may be responsible. An N-terminal-specific monoclonal antibody did not react with the novel protein isoform suggesting that the alternate splicing event likely occurs beyond exon 2. The new isoform is glycosylated similar to the functional IFN-λ4 and is also secreted. Furthermore, we show that the ΔG allele can also potentially express a similarly frameshifted isoform. The splicing event that leads to the generation of these novel isoforms and their functional significance remains to be elucidated.

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.