The Genetic Association of IFN-λs with Human Inflammatory Disorders Remains a Conundrum

Abstract

Type III Interferons (IFNs) or lambda IFNs (IFN-λs or IFNLs) although are primarily antiviral cytokines, may have roles to play in shaping immune responses, including those during inflammation. Genetic variants within the IFNL locus have been shown to be associated with various inflammatory conditions in humans ranging from metabolic to autoimmune and allergic diseases. The mechanism behind these genetic associations is not clear. Appropriate data analysis methods and functional evidence should be complimentarily used to identify the causal variants and mechanisms.

Inflammation is a bodily response to an insult arising from different origins, but invariably involves the immune cells in initiating and perpetuating it. Type III interferons (IFNs) or the lambda IFNs (IFNLs or IFN-λs) are newly discovered cytokines, active at epithelial surfaces; they have immunomodulatory functions besides their primary roles as antiviral molecules. Several genetic variants have been identified at the IFNL locus on chromosome 19 that associate with many human diseases of both infectious and noninfectious nature (reviewed in Bhushan and Chinnaswamy 2018). No clear explanation about the molecular mechanism behind these associations is available, except in the case of chronic hepatitis C virus (HCV) infections (Bhushan and Chinnaswamy 2018).

Fine-mapping and in vitro studies have identified 3 functional genetic variants at the IFNL locus: the first 2 are single nucleotide polymorphisms, (SNPs) rs28416813 and rs4803217, present in the 5′UTR and 3′UTR, respectively, of IFNL3 and they potentially regulate IFNL3 expression by disparate mechanisms (reviewed in Bhushan and Chinnaswamy 2018); and a third variant, a dinucleotide polymorphism rs368234815 generates a new type III IFN, IFN-λ4 (Fig. 1). Some nonsynonymous SNPs, including rs117648444 within IFNL4 affect in vitro activity and function of IFN-λ4. Even though, many other SNPs like rs12979860 and rs8099917 have been genotyped and tested in different studies, it is believed that they are all correlated to the above 3 functional variants by linkage disequilibrium (LD).

Fig. 1.

Three functional variants at the IFNL locus. IFN-λ3 functional variants can influence IFNL3 transcription (rs28416813; C allele increases and G allele decreases) or translation (rs4803217; G allele increases and T allele decreases) while IFN-λ4 functional variant gives rise to a new ORF for IFNL4 to be translated. The task of determining which of the 3 functional variants are likely causal to a given phenotype is challenging in the absence of alternate methods and unequivocal functional evidence. IFN-λ, interferon lambda; ORF open reading frame. Color images are available online.

In the absence of other convincing evidence to implicate any other SNP/variant in the IFNL region to have functional roles, it is sufficient to conclude at this point that the above-mentioned variants are the most likely ones to have causal roles in the development of phenotypes described in different studies. However, strong LD in the IFNL region in many world populations, except the African and its lineage, prevent us from conclusively assigning one or the other functional variant as being causal to a phenotype.

Recent studies have shown that the IFNL locus SNPs are associated with a variety of inflammatory disorders (Table 1) ranging from metabolic and autoimmune to respiratory diseases. As Table 1 shows, one underlying pattern to these associations is that the IFN-λ4-generating ΔG allele (or alleles at other SNP positions that correlate to it by LD) shows a protective phenotype in many of these disorders. The alleles of the 2 functional variants that potentially increase IFNL3 expression (C allele at rs28416813 and G allele at rs4803217) are correlated by LD to the TT allele at rs368234815 that abolishes the expression of a functional IFN-λ4. Therefore, due to prevalent LD, it will be a challenge to genetically ascertain whether the protection is offered by decreased expression of IFN-λ3 or that the presence of an extra type III IFN (IFN-λ4) is somehow subverting the excessive inflammation.

Table 1. Summary of Genetic Association of Interferon Lambda Polymorphisms with Various Inflammatory Diseases in Humans.

				Possible correlating allele at:
				IFN-λ3 SNPs		IFN-λ4 variant
Disease (study population)	Phenotype tested	SNP/variant tested (alleles)	Effect allele	rs28416813 (C/G)	rs4803217 (G/T)	rs368234815 (TT/ΔG)	Association p-value	Effect allele is protective (P)/risk (R)	Reference
Asthma (Australian)	Allergy	rs12979860 (C/T)	T	G	T	ΔG	0.004	R	Gaudieri and others 2012
Allergic Asthma (Polish)	Atopy	rs368234815(TT/ΔG)	ΔG	G	T	—	0.018	P	Chinnaswamy and others 2017
COPD (European)	Time to severe exacerbation	rs8099917 (T/G)	G	G	T	ΔG	0.037	R	Egli and others 2018
	Severe exacerbation rate						0.032	R
	BODE index prognosis	rs12979860 (C/T)	C	C	G	TT	0.031	R
Fibrosis in chronic HCV infection (European)	Hepatic inflammation	rs12979860 (C/T)	C	C	G	TT	1.3 × 10−16	R	Eslam and others 2017
		rs368234815 (TT/ΔG)	TT	C	G	—	1.1X 10−16	R
		rs4803217 (G/T)	G	C	—	TT	5.5 × 10 − 12	R
	Fibrosis	rs12979860 (C/T)	C	C	G	TT	1.1X 10−6	R
		rs368234815 (TT/ΔG)	TT	C	G	—	5.0X 10−6	R
		rs4803217 (G/T)	G	C	—	TT	5.2X 10−5	R
	Fast FPR	rs12979860 (C/T)	C	C	G	TT	7.0X 10−5	R
		rs368234815 (TT/ΔG)	TT	C	G	—	3.3X 10−4	R
		rs4803217 (G/T)	G	C	—	TT	6.4X 10−4	R
NAFLD (European)	Fibrosis (F3-F4)	rs368234815 (TT/ΔG)	TT	C	G	—	0.005	R	Petta and others 2017
	Lobular necroinflammation (G2-G3)	rs368234815 (TT/ΔG)	TT	C	G	—	0.002	R
HT (Turkish)	HT	rs8099917 (T/G)	G	G	T	ΔG	0.001	P	Arpaci and others 2016
SLE (Taiwanese)	Nephritis	rs8099917 (T/G)	T	C	G	TT	0.002	R	Chen and others 2018
		rs12979860 (C/T)	C	C	G	TT	0.003	R
		rs368234815 (TT/ΔG)	TT	C	G	—	0.002	R
		rs4803217 (G/T)	G	C	—	TT	0.002	R
	SLE in nephritis-negative patients	rs8099917 (T/G)	G	G	T	ΔG	0.001	R
		rs12979860 (C/T)	T	G	T	ΔG	0.002	R
		rs368234815 (TT/ΔG)	ΔG	G	T	—	0.001	R
		rs4803217 (G/T)	T	G	—	ΔG	0.001	R
CP (Iranian)	CP	rs8099917 (T/G)	G	G	T	ΔG	<0.0001	R	Heidari and others 2017
		rs12979860 (C/T)	T	G	T	ΔG	<0.0001	R

BODE, body-mass index, airflow obstruction, dyspnea, and exercise; COPD, chronic obstructive pulmonary disease; CP, chronic periodontitis; FPR, fibrosis progression rate; HCV, hepatitis C virus; HT, Hashimoto’s thyroiditis; IFN-λ, interferon lambda; NAFLD, nonalcoholic fatty liver disease; SLE, systemic lupus erythematosus; SNP, single nucleotide polymorphism.

On the contrary, since the alleles that increase IFN-λ3 expression and the one that abolishes expression of a functional IFN-λ4 are more likely to be present on opposite chromosomes, it may be advantageous in that a test for the best-fitting genetic model (dominant, recessive, additive, multiplicative etc.), which can explain the observed data, may tell us which of the functional variants could be behind the phenotypes. This test may not discriminate between rs28416813 and rs4803217, but certainly can distinguish between these 2 SNPs and rs368234815, thereby implicating either IFN-λ3 or IFN-λ4 as being responsible for the observed phenotype.

A classic example to support the notion that a test of the best-fitting genetic model could provide insights into the likely causal mechanism comes from the well-studied HCV-IFNL association. Shebl and others (2011) published a study, much before IFN-λ4 was discovered, which dealt with spontaneous clearance of HCV and association of the IFNL SNP rs12979860 in which they compared different genetic models to explain their data. They showed that a supra-additive model for the favorable C allele at rs12979860 was the closest fit to their observed data and concluded that the unfavorable T allele was contributing more than the favorable C allele to the observed phenotype, thus putting the onus on the minor T allele to be the more likely causal allele. This was later corroborated by the discovery of IFN-λ4 that comes from the minor allele background, which is now accepted as the causal mechanism behind HCV persistence (Bhushan and Chinnaswamy 2018).

In addition, IFN-λ4 activity-modifying nonsynonymous variant, rs117648444, could be an advantage as it can be used as a tool to probe the role of IFN-λ4 in a given phenotype in case–control studies (Bhushan and Chinnaswamy 2018).

One study has shown that increased expression of IFN-λ3 may induce excessive inflammation in HCV-associated fibrosis (Eslam and others 2017) and further concluded that IFN-λ4 may not have an active role in the expression of this phenotype. The authors claim this because their data on inflammatory scores could not be stratified based on the perceived differences in the IFN-stimulated gene (ISG) activity of IFN-λ4 as reported by the variation at rs117648444. One presumption, in this study, is that the ISG activity is a proxy for IFN-λ4 functions; however, it remains to be seen if this is true. In fact, a recent report in mice shows that IFN-λs can modify specific leukocyte functions without following the ISG stimulation pathway (Broggi and others 2017). While the study by Eslam and others (2017) shows a relatively large amount of genetic and functional data to justify the claim that IFN-λ3 may be causal, a test of different genetic models that best explains their observed data could have been more conclusive. Moreover, the study does not show any direct evidence that IFN-λ3 is indeed behind the excessive inflammation other than the fact that it was noted to be expressed at higher levels in those patients with more severe fibrosis. In other words, the observation could be a mere correlation and not necessarily causation.

Recent studies on IFN-λs, although from mice, show evidence contrary to the speculation that IFN-λs could be mediating excessive inflammation (Wack and others 2015; reviewed in Andreakos and others 2019). Several reports on mice models of virus infection and other inflammatory disorders have shown that IFN-λs display a relative protective effect in dealing with the infection/inflammation stimulus compared with their type I IFN counterparts.

The mechanism behind this phenomenon could be: IFN-λs exert an immediate and effective antiviral response against viral infections of the epithelium and prevent a subsequent type I response that is more systemic and inflammatory in nature (Mahlakõiv and others 2015); IFN-λs effectively get rid of the stimulus when presented at low doses without causing excess harm to the host (Galani and others 2017); IFN-λs act directly on neutrophils by restricting their recruitment to sites of insult and also by altering their functions such that they produce less inflammatory mediators (Blazek and others 2015; Broggi and others 2017). If these observations could be extrapolated to humans (with a caveat that several discrepancies exist in the biology of IFN-λs of mice and humans) then it goes on to suggest that there could be alternate sources of the excessive inflammation we observe in human diseases, despite the fact that these disorders show strong genetic association with the IFN-λ locus (Table 1).

This needs a clearer understanding of the different players in the inflammatory cascade that a particular stimulus/insult would provoke; the type I IFNs are an obvious candidate given their pathogenic roles in several inflammatory diseases, such as systemic lupus erythematosus, psoriasis etc. (Crow and Rönnblom 2018). A likely explanation could be that, in humans IFN-λs respond to and effectively deal with the initial inflammatory insult (could be viral or metabolic insults that may be mediated by NF-kB, which is a transcription factor for driving both type I and III IFN gene expression) by their antimicrobial actions directly on the cells and/or indirectly by modulating immune cell responses without invoking a strong inflammatory response, similar to their roles in mice. Only when this preliminary defense fails, the other players, such as type I IFNs, come to play that mediate a strong Th1 immune response to get rid of the inflammatory insult, which will also bring in a compensating Th2 response that eventually repairs the “wound” by replacing the functional tissue mass with fibrotic tissue.

Alternately, unlike in mice, both type I and III IFNs can participate in the inflammatory cascade at different levels and grades and could function in synergy to get rid of the initial insult. In either case, the outcome would be that a defective IFN-λ response would result in an excess of inflammation; the defect in IFN-λ will manifest as a relative defect by way of a genetic association between one of the IFN-λ locus variants and the disease phenotype.

In this scenario, the following models can be proposed to explain the genetic association of human inflammatory disorders with the IFN-λ locus, keeping in view our current knowledge on the possible causal variants in the region (Fig. 1). The TT allele carriers at rs368234815 would be at an advantage in that they can express higher amounts of IFN-λ3, which is a typical IFN in that it is secreted out of the cells and thereby amplifies the signal optimally to perform its protective functions. The ΔG allele carriers on the other hand may express IFN-λ4, which is poorly secreted (Obajemu and others 2017) and additionally its expression may be transcriptionally or translationally silenced (Hong and others 2016). This could explain why the TT allele carriers show a protective phenotype in some disorders (Table 1).

In other disorders (the majority in Table 1), where the ΔG allele carriers show a protective phenotype, they would express IFN-λ4, which is known to be more potent than IFN-λ3 (Obajemu and others 2017) and also IFN-λ4 is known to induce the expression of certain anti-inflammatory genes (Obajemu and others 2017). Functional evidence like mRNA or protein level differences of the key molecules in the target tissue/organ could explain which of the above possibilities are true in each case. The recent availability of commercial IFN-λ4 antibodies would help researchers enormously to trace the presence of IFN-λ4 in affected tissue/organs. This, in combination with best-fitting genetic model evidence, would go a long way in gaining a better understanding of the disease mechanism.

Interestingly, IFN-λs also are known to have dual functions both as tumor suppressors and promoters (Lasfar and others 2019). Recent studies show that IFN-λ4 associates with a transcriptional signature that associates with an inflammatory phenotype in prostate tumors of African American men; this inflammation, however, proves to be a risk for disease progression, including poorer response to radiation and chemotherapy (Tang and other 2018). Furthermore, the risk of prostate cancer due to IFN-λ4 is higher in men who have relatively more number of sexual partners (Minas and others 2018). In another gender-specific cancer, mucinous ovarian carcinoma, the minor alleles of IFNL locus SNPs (which may correlate with the IFN-λ4-generating ΔG allele at rs368234815) were associated with lesser risk of cancer in a genome-wide association study (Kelemen and others 2015).

Clearly, how IFN-λs influence inflammation and immunity is an area that needs more efforts by the research community, particularly in humans, to explain the association of IFNL locus variants with the inflammatory disorders.