TIM Polymorphisms – Genetics and Function

Abstract

The transmembrane immunoglobulin and mucin domain (TIM) family was identified more than a decade ago. Although the founding member of the family was first described in a rat model of ischemia reperfusion injury (IRI), much of the recent interest in the TIM family members has focused on their potential roles in immunity. There are now a large number of genetic studies that have investigated the possible association of various TIM1 and TIM3 polymorphisms with different diseases. Here we review this body of literature, and highlight some of the most interesting studies.

TIM Family Proteins

The transmembrane immunoglobulin and mucin domain (TIM) family has more often been referred to as the T cell immunoglobulin and mucin domain family, although we will employ the former term because it more accurately reflects the nature of these proteins and their expression patterns. TIM proteins are characterized by an N-terminal Ig domain of the V subset, followed by a mucin-like domain, single transmembrane domain and a cytoplasmic tail of variable length. The human genome contains three TIM genes, which encode the proteins TIM-1, TIM-3 and TIM-4. This is rather different from the situation with mice, for example, which also contain an expressed gene designated Tim2, as well as several apparent pseudogenes^1–4.

The founding member of the family, which we will refer to as TIM-1, was discovered by Kaplan and colleagues, as a protein expressed on monkey and human cells that they called HAVcr1, based on its ability to act as a cellular receptor for hepatitis A virus (HAV)⁵. Subsequently, this protein was found to be upregulated in a rat model of kidney ischemia and thus designated kidney injury molecule 1 (Kim-1)⁶. During ischemic kidney injury, a soluble form of Kim1/TIM1 is produced, and the detection of Kim1/TIM1 in urine is currently a clinical diagnostic marker for acute kidney injury^7–9. Immunological interest in the TIMs was piqued in part by a report that mapped the susceptibility of particular mouse strains to develop atopic asthma to a genomic region containing the Tim locus¹⁰. Importantly, in this genetic linkage analysis, the Tim locus segregated away from the relatively nearby Th2 cytokine locus, which had previously been implicated in susceptibility to atopic asthma. This report also identified the human homolog of Tim-1 as the hepatitis A virus receptor (hHAVcr1). Meanwhile, Kuchroo and colleagues were studying a novel transmembrane protein (Tim-3) that they identified in mice as a relatively specific marker for Th1, but not naïve or Th2, T cells¹¹. Subsequent studies have defined a role for TIM-3 in negative regulation of T cell-mediated immune responses, although it is also expressed by cells of the innate arm of the immune systems of mice and humans³. The initial report of the TIM family by McIntire et al. noted several polymorphisms each in Tim1 and Tim3, spurring efforts by many groups to search for polymorphisms in human TIM1 and TIM3, as well as their possible connection to various diseases.

TIM-1 and atopic diseases

Atopic diseases, including asthma, atopic dermatitis, and allergic rhinitis, are caused by a complex interplay between environmental and genetic factors. Since the Tim gene locus was discovered as a major candidate locus for allergy and asthma susceptibility in mice, many epidemiological studies have been conducted to assess the link between atopy and human TIM1 polymorphisms. Given the discovery of the Tim locus in a mouse model of asthma and the emerging role of TIM-1 in the regulation of Th2 cell activation and function^{10; 12–14}, we will first discuss relevant studies that tested TIM1 polymorphisms in specific human populations for possible associations between specific TIM1 variants and atopic diseases. Some of the notable TIM-1 variants in this regard are summarized in Table I. These and other variants discussed below are also shown in the context of the TIM-1 gene and protein, in Figure 1.

Table I.

Notable Studies on the role of TIM-1 Variants in Atopic Disease

Study	Findings	Study Design/Strengths	Limitations
McIntire et al. (2003; Nature)	6 aa insertion in mucin domain (157insMTTTVP) confers protection from atopy, but only in HAV+ individuals	Case-control study; HAV status determined serologically	No mechanistic/molecular correlate data (see Kim et al., below)
Gao et al. (2005; J. Allergy Clin Immunol.)	157insMTTTVP and intronic polymorphism each associated with increased risk of asthma, but independently of HAV	Case-control and family-based study; HAV status determined	No mechanistic/molecular correlate data (see Kim et al., below)
Graves et al. (2005; J. Allergy Clin Immunol.)	Several polymorphisms associated with atopy and eczema, but not asthma (some TIM-3 variants also yielded same pattern of association)	Case-control, longitudinal study; adjustment for multiple comparisons; atopy assessed by skin prick	No stratified analysis by ethnicity; asthma and eczema assessed only by questionnaire
Kim et al. (2011; J. Clin. Invest.)	157insMTTTVP polymorphism associated with more severe HAV-induced liver disease	Data presented on HAV binding and NKT cell activation	Relatively small no. of patients

Figure 1.

Overview of TIM1 polymorphism disease associations in both genomic and protein contexts. Major polymorphisms are shown above the genomic scheme, while coding changes are also shown below the protein scheme. See text for complete description of the individual polymorphisms.

The first human study to provide evidence for functional consequences of TIM1 polymorphisms in human disease was reported in 2003¹⁵. The authors identified several TIM1 variants, including two single-amino acid changes, 195delT and A206T, and a six amino acid insertion at position 157 (157insMTTTVP) that resulted in lengthening of the TIM-1 mucin domain by 12–14%. More importantly, a cross-sectional study of 375 racially diverse individuals revealed that the 157 insMTTTVP polymorphism, which was present in 63%, 46% and 64% of Caucasians, Asians and African Americans in the study, respectively, conferred protection against atopy, but only in HAV sero-positive individuals (OR=0.257; 95% CI=0.116–0.570). This result was confirmed by subgroup analysis with the Asian and Caucasian participants. It is noteworthy that this is one of the few studies that assessed the role of TIM1 variants in atopy in the context of HAV infection. As a human cellular receptor for Hepatitis A virus, TIM-1 was proposed to interact with HAV for efficient viral binding and uncoating¹⁶. Since the 157insMTTTVP variant of TIM1 is a common allele, this study provided a possible explanation for the inverse relationship between the declining incidence of HAV infection and increased atopy in the industrialized world. Specifically, McIntire et al. suggested that individuals with certain TIM1 alleles, such as the 157insMTTTVP variant, are protected against atopy¹⁵. However, this protective effect may only occur in individuals with prior HAV infection. Subsequent studies have focused on identifying additional TIM1 polymorphisms, with particular emphasis on the mucin domain.

In a series of case-control studies, Chae et al. tested novel polymorphisms in the promoter and coding regions of TIM1 in Korean populations of unrelated asthma and atopic dermatitis patients for association with asthma or atopic dermatitis^{17; 18}. Analysis revealed ten novel TIM1 variants, five of which were SNPs in the promoter and coding regions (−1166C>G, −416G>C, −232A>G, 5365C>T, and 5529A>G), one novel duplication in the promoter (−2278_−2276dupATT), and three variations in exon 4 (5383_5397del, 5387_5389insACC, and 5509_5511delCAA). Genotype and allele frequency tests of association suggested that the homozygous sequence of 5509_5511delCAA was associated with asthma (p=0.011) but not with atopic dermatitis. However, the 5383_5397del variant, also known as 157insMTTTVP, was not significantly associated with either asthma or atopy (p=0.439; p=0.063 respectively). Due to the small sample size, this study was mainly a screening for TIM1 polymorphisms in a Korean population and a preliminary asthma/atopy association survey. The authors subsequently examined the alleles 5383_5397del and 5509_5511delCAA in exon 4 in a larger population involving 200 asthma patients 112 atopic dermatitis patients and 201 control subjects¹⁸. Genotype frequency comparisons confirmed an association between 5509_5511delCAA and asthma (p=0.037). In this larger study, 5383_5397del was found to be strongly associated with atopic dermatitis (p=0.005). Molecular analyses presented in this study found neither serum IgE levels nor eosinophil counts to be associated with TIM1 haplotype. The interpretation of these findings was somewhat difficult because it was unclear whether the subjects also included patients assessed in a previous study. In addition, there were significant differences in case-control study populations, specifically the relative representation of men and women.

Natural selection tends to act at genetic regions containing functionally important variants. Thus, studies of variations in TIM1/HAVCR1 revealed a large degree of genetic diversity in exon 4 (which encodes the mucin domain), as indicated by the high frequency of non-synonymous amino acid substitution and insertion/deletion variants in this region¹⁹. Despite the relatively similar patterns of haplotype frequencies in exon 4 of Korean and Japanese populations, the 5383_5397del variant, which was associated with atopy in a Korean population, was not significantly associated with atopic asthma in a Japanese pediatric patient family study²⁰. Thus, Noguchi et al. identified seven polymorphisms in TIM1 from 144 families: −1634A>G; −1606A>C; −1547G>C; −1453G>A; two insertion/deletion polymorphisms in exon 4, c469-470insCAATGACAACGACTGTTC; c589-590delGTG and c469-470insTGACGACTGTTCCA termed hHAVcr-1* allele 2 and 3, respectively, and an intronic variant. With adequate power to detect an odds ratio (OR) greater than 2, the transmission disequilibrium test (TDT) indicated that none of the hHAVcr1/TIM1 variant alleles was preferentially transmitted to asthmatic children. In addition, serum IgE levels did not differ by TIM1 haplotype for the variants analyzed. It is worth noting that although HAV infection status was unknown in this subject population, the prevalence of HAV sero-positivity in Japan has been reported to be low^{21; 22}. Therefore, while polymorphisms in the region encoding the mucin domain of TIM-1 are not linked to atopic diseases in a Japanese population, it is possible that these variations might alter susceptibility to asthma and atopy in other populations where the prevalence of HAV exposure is higher or in which other genetic factors are at work.

Epidemiologic data so far indicate that a connection between TIM1 polymorphisms and atopy was present in some, but not all, populations examined. Atopy and asthma are complex diseases whose phenotypes may vary during child development and throughout adulthood. As a result, it is important to assess the link between TIM1 polymorphisms and risk factors for asthma and atopy that occur during childhood. In a large cohort study²³, newborns in Tucson, AZ were recruited for a longitudinal study to monitor the development of atopy, asthma, and eczema at ages 6, 11, and 16. Seven TIM1 polymorphisms were identified in 564 subjects. Of these, 5383_5397del was found to have an association with atopy that was strong enough to remain statistically significant after adjustment for multiple comparisons (relative risk (RR)=1.24; 95% CI=1.07–1.45, p=0.005). 5383_5397del was associated with eczema (RR=1.43; 95% CI=1.01–2.01, p=0.004), but the association was not statistically significant after adjustments for multiple comparisons. 5383_5397del not associated with asthma in this study. This lack of an apparent association may be partly due to the method of obtaining data, since patients were assessed for eczema and asthma through a series of questionnaires, rather than by physician diagnosis, while atopy was assessed by skin prick tests to common allergens. Phenotypic manifestations of asthma and allergic diseases can vary throughout life, so it is difficult to accurately assess them based solely on questionnaires. In addition, HAV infection status was not obtained from these children, so the authors could not determine whether this factor affected the relationship between 5383_5397ins/del and allergic disease. Nevertheless, this study aimed to remove potential bias through adjustment for multiple comparisons and stratified analysis of polymorphism frequency by ethnicity. Therefore, the increased risk of atopy attributed to the presence of the 5383_5397del variant in this study is likely to be physiologically relevant and independent of ethnicity. In another study of TIM1 polymorphisms and childhood asthma, Wu et al. examined the promoter SNP −232G>A and the 5383_5397ins/del variation in Chinese children with atopic and non-atopic asthma, and found no association between these two variants and the development of asthma²⁴. Intriguingly, patients with the heterozygous variant 5383_5397ins/del had significantly higher total serum IgE levels even after adjustment for sex and age (p=0.0112). Since increased serum IgE is associated with an increased risk of asthma, it is possible that this particular TIM1 variant contributes to higher IgE production, thereby enhancing susceptibility to developing childhood asthma in a subset of patients.

Gao et al. conducted a family-based study involving 40 case-parent trios, with a nested case-control study design including 89 asthmatic patients and 94 controls, to assess the association of TIM1 sequence variation, HAV sero-positivity and asthma in an African American population²⁵. This study found that 157delMTTTVP was present at a higher frequency in asthmatic individuals than control subjects (OR=3.09; 95% CI=1.05–9.82), but this association was also found in HAV sero-negative individuals (OR=3.00; 95% CI=0.89–11.6). The association of 157delMTTTVP with asthma was further substantiated by a case-parent trios study. In addition, heterozygous and homozygous sequences carrying the minor T allele in the intronic variant rs2277025 correlated with asthma risk in this African American population (OR=2.027; 95% CI= 1.013–4.058 and OR=2.779; 95%CI=1.176–6.567, respectively). Haplotype analysis across the TIM1 gene revealed that the T-G-del-G allele was more frequent in asthmatics, while the allele C-G-ins-G appeared at a higher frequency in healthy individuals. Because 157insMTTTVP has been implicated in protection against atopy in HAV sero-positive individuals¹⁵, Gao et al. determined whether 157ins/delMTTTVP also played a role in HAV infection-related protection in this population. Surprisingly, 157delMTTTVP was linked to asthma in both the family-based and the nested case-control analysis in HAV sero-negative individuals. Because only two patients with asthma were HAV sero-positive, the authors were unable to examine this association in HAV-infected individuals. Hence, this study suggested that 157delMTTTVP was linked to asthma susceptibility independent of HAV status, at least in this population.

Since most of the above studies were conducted in relatively developed countries where HAV infection has become quite rare, it is of interest to assess the relationship between TIM1 variants and atopic diseases in places where HAV exposure is still relatively high. According to epidemiological data, as of 1990 China was reported to have a high incidence of HAV infection, with 73.6% of the rural Chinese population having circulating antibodies to HAV²⁶. China also experiences occasional outbreaks of Hepatitis A, with the most current occurrence in 2006²⁷. Based on polymorphisms found in linkage studies of TIM1 and allergic diseases, Li et al. examined the insertion/deletion coding polymorphisms, rs45623443 (a.k.a. 5383_5397ins/del) and 5509_5511delCAA in TIM1 exon 4, as well as the SNP IVS 8+9G/A in intron 8, in a case-control study of 352 asthma patients and 309 controls who were unrelated Han Chinese²⁸. Genotype and allele frequency tests indicated that these three variants were not significantly associated with asthma, despite the fact that 5383_5397del was linked to atopic dermatitis in a Korean population¹⁸ and to asthma in an HAV sero-negative population of African Americans²⁵. In addition, 5509_5511delCAA has also been linked to asthma in a Korean population¹⁷. While the study of Li et al. suggested that the above polymorphisms were not associated with asthma in a Han Chinese population, the authors did not investigate other polymorphisms in either the promoter or coding regions of TIM1, where there might exist significant genetic variations unique to this particular Chinese population. In addition, subjects in this study were not serologically tested for HAV exposure, so the authors could not properly determine whether there is a physiological connection between HAV exposure and asthma susceptibility in individuals with various TIM1 polymorphisms.

In a separate family-based association study, Wu et al. analyzed 118 case-parent trios and selected five polymorphisms in the TIM1 gene for analysis of childhood asthma risk²⁹. A pairwise linkage disequilibrium test showed that these polymorphisms were in moderate LD with each other, but individual polymorphisms, including the 5383_5397ins/del, did not yield significant association with asthma as determined by TDT and haplotype relative risk. Nevertheless, when a combination of polymorphisms was assessed, G-A-ins-C-G was over-transmitted to asthmatic children (TDT p=0.03; haplotype RR (HRR)=1.91; 95% CI=1.06–3.41) suggesting that this haplotype, with the insertion variant of 5383_5397 ins/del, may confer susceptibility to asthma. Overall, this study indicated that a single polymorphism in this population was not sufficient to confer genetic susceptibility to (or protection from) asthma, but rather a combination of these polymorphisms was needed to observe functional effects. As this study was limited to a small number of genotypes, a more comprehensive assessment of TIM1 polymorphisms in a larger population might provide more insight regarding functionally significant SNPs.

In addition to asthma and atopic dermatitis, allergic rhinitis has also been analyzed in several studies of TIM1 polymorphisms. Allergic rhinitis (AR) is characterized by airway hyper-responsiveness, overproduction of Th2 cytokines, and influx of eosinophils in the nasal mucosa. Upon comparing two TIM1 SNPs in the promoter region, −416G>C and −1454G>A, in healthy controls and AR patients, Mou et al. found that the CC genotype of −416G>C and the AA genotype of −1454G>A were significantly associated with development of AR (OR=2.567, 95%CI=1.151–3.012; OR=5.234; 95%CI=2.339–6.571, respectively); these associations were statistically significant even after Bonferroni correction and adjustment for sex and age³⁰. Also, both SNPs were associated with significantly higher allergen-specific serum IgE but not total serum IgE. The authors concluded that −416G>C and −1454G>A in the TIM1 promoter region may be associated with allergic rhinitis in a Han Chinese population, a phenomenon that was not observed in either Korean or Japanese patients with asthma and atopic disease. However, this study did not assess whether these SNPs affect mRNA expression level of TIM1, which could influence Th2 responses observed in allergic rhinitis. The 5383_5397ins/del and 5509_5511delCAA variations in the mucin domain of TIM-1 failed to exhibit any association with allergic rhinitis, while these variations were implicated in atopic dermatitis and asthma, respectively, in a Korean population¹⁸. Overall, this study agrees with previous reports examining Han Chinese populations in both case-control and family-based studies, suggesting that atopic and asthma susceptibility are not linked to individual variations in the mucin domain of TIM-1²⁹. This in part can be explained by the relatively low heterozygosity of exon 4 in Chinese populations¹⁹. Thus, results from various populations, despite limitations in study design, suggest that the role of TIM-1 in establishing Th2-biased responses may be influenced at least in part by factors that differ by ethnicity.

Despite the intensive search for TIM1 haplotype association with allergy and asthma, few studies have offered insights into the potential mechanism of this association. Xu et al. reported that TIM1 mRNA expression was upregulated along with the Th2 transcription factor GATA-3, thereby suggesting that TIM-1 is involved in differentiation of Th2 T cells³¹. McIntire et al. proposed that the longer TIM-1 mucin domain encoded by 157insMTTTVP allows for more efficient viral uncoating and entry, leading to either inhibition of or diversion from the Th2 responses seen in atopic diseases¹⁶. On the other hand, by constructing major TIM1 haplotypes with various SNPs and exon 4 variations identified in a Spanish Caucasian population, Garcia Lozano et al. found that 157insMTTTVP belonged to a low mRNA expression haplotype³². Thus, lower expression of TIM-1 may also lead to the reduced Th2 responses, as suggested by McIntire et al. Recent studies of acute liver failure in an Argentinean population of children infected with HAV also provided supporting evidence to the above proposed model by McIntire et al.³³. Patients with severe liver failure had an increased tendency to possess the 157insMTTTVP variant or the “long form” of TIM-1. Using fusion proteins consisting of various forms of TIM-1, it was found that the “long form” bound HAV most efficiently. When expressed on NKT cells, this TIM-1 variant was associated with greater cytotoxicity for HAV-infected cells, which was abolished by an anti-TIM1 antibody³³.

It has become increasingly evident that atopic disease like asthma are quite heterogeneous, with contributions from both the innate and adaptive arms of the immune system³³. The variants of TIM-1 and their differential effects in various populations reflects the involvement of a viral receptor that has been shaped by selective pressure of natural selection to become a relevant modulator of allergic responses in the absence of viral infection. While the significance of prior HAV infection in the susceptibility to atopy conferred by TIM1 polymorphisms is still debatable, recent studies raise the questions of whether HAV infection needs to occur during childhood and whether HAV vaccination can produce the same effects as natural infection.

TIM-1 and Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease that primarily affects the synovial membrane, cartilage and bone, leading to joint destruction. RA has been thought of as a Th1-driven autoimmune disorder, but the importance of Th17 cells has been emerging. In established disease, the synovial fluid of RA patients contains relatively high levels of pro-inflammatory cytokines such as TNF-α, IL-6, IL-12, IL-23, but relatively low amounts of IL-4 and IL-13. As discussed above, Chae et al. studied the association of multiple SNPs and variations in the promoter and coding regions of the TIM1 gene with allergic disease in a Korean population^{17; 18}. In a separate study, Chae et al. assessed the association of these variants with rheumatoid arthritis (RA)³⁴. By comparing the genotype and allele frequency between RA patients and controls, these investigators found that the homozygous sequence of 5383_5397del was significantly associated with reduced susceptibility to RA (OR=0.25; 95%CI=0.09–0.68). In addition, homozygosity of 5509_5511delCAA was significantly linked with susceptibility to RA (OR= 5.15; 95%CI=.70–15.65). However, these two variants did not correlate with levels of C-reactive protein (CRP) and rheumatoid factor (RF), which are characteristic of RA. It is worth noting that the homozygous variation sequence of 5383_5397del was significantly associated with susceptibility to atopy in their previous study¹⁸. Furthermore, the homozygous variation sequence of 5509_5511delCAA is under-represented in asthmatic patients, compared with healthy controls. Results from these studies indicated that variations in exon 4 may be linked to a tendency to develop imbalanced Th1 or Th2 responses. Nevertheless, it remains to be determined whether variations in exon 4 of the TIM1 gene are associated with differences in TIM-1 expression or expression and, in turn, Th1/Th2 differentiation.

Chae et al. subsequently assessed the relationship between polymorphisms in the TIM1 promoter, exon 1, intron 1 and susceptibility to RA³⁵. By combining data from newly recruited RA and control individuals with those in the previous RA study³⁴, the authors showed that only the −1637A>G polymorphism in the promoter region was associated with development of RA. Specifically, the G allele was present at a higher frequency in RA patients (OR=1.85, 95%CI=1.29–2.66). According to data not shown, this association was not affected by gender, likely because the RA patients in this study were predominantly female. Haplotypes generated using the −1637A>G, −416G>C and −232A>G promoter polymorphisms showed that the A-C-G and A-C-A haplotypes were present at a significantly higher frequency in control patients compared to RA patients (p=0.001 and p=0.023, respectively). However, a more robust connection of −1637A>G with RA could not be made since this SNP was not associated with CRP and RF levels. Overall, this study provided evidence that promoter polymorphisms may influence susceptibility to developing RA, but did not directly address whether levels of TIM-1 protein expression are affected by these polymorphisms.

TIM-1 and Multiple Sclerosis (MS)

Multiple sclerosis is a T-cell-mediated autoimmune disease of the central nervous system. Khademi et al. reported that TIM1 mRNA expression was significantly upregulated in cerebrospinal fluid mononuclear cells of patients with MS, compared to control patients³⁶. Curiously, the upregulation of TIM1 message was also linked to lower IFNγ levels and the inactive phase of MS, suggesting that TIM-1 expression may be a regulator or marker of remission. To determine if the TIM1 polymorphisms were indeed associated with MS in a Western Austrian population, Grabmer et al. developed a PCR with sequence-specific priming (PCR-SSP) approach to map the SNPs in the TIM1 promoter and coding regions³⁷. Fine mapping revealed five SNPs in the promoter, two SNPs in exon 4, two SNPs in intron 4, and one SNP in intron 7. However, there was no significant association between disease state and any of the above SNPs after the Bonferroni correction for multiple comparisons. The authors proposed that failure to detect any significant difference might be due to the relatively low statistical power of 30% to detect an OR of 2.51. Hence, a larger sample of MS patients would likely be necessary to accurately pinpoint small differences between MS and control.

TIM-1 and Systemic Lupus Erythematosus (SLE)

SLE is a complex systemic autoimmune disease, characterized by tissue damage, due to deposition of immune complexes in various organs, including the kidney. Lupus nephritis (LN) is a major contributor to the mortality of lupus patients, and has been associated with dysregulation of either Th1 or Th2 responses^{34; 38}. Based on its potential influence on Th1/Th2 regulation, the TIM1 gene was investigated for its association with development of SLE. Wang et al. examined TIM-1 expression on PBMC of nineteen SLE patients and found a significant increase in TIM-1 expression in these patients, compared to sex-matched healthy controls (p=0.021)³⁹. In addition, production of IL-10 was upregulated in these patients and was also positively correlated with TIM-1 expression (r_s=0.4188, p=0.0372). However, there was only a marginal negative correlation between expression of TIM-1 and active SLE (r_s=0.4544, p=0.0498). In another study linking TIM1 polymorphisms to the progression of SLE, Li et al. examined the genotype and allele frequency of the SNPs rs12522248 (A>G) in exon 4 and rs1501909 (C>A) in intron 4 from SLE patients and healthy controls⁴⁰. This study also assessed the frequency of these polymorphisms in SLE patients with and without lupus nephritis (LN), according to clinical disease activity. However, there was no association between these two SNPs and SLE after adjustment for sex and age, possibly due to low statistical power to detect a true-positive association (< 15%). Therefore, it is possible that other TIM1 variants in this region as well as in the promoter may be associated with SLE when larger samples from different populations are examined.

TIM-1 and Cerebral Malaria

Cerebral malaria is a major cause of death after infection by the malaria parasite Plasmodium falciparum. Inflammatory cytokines such as IFNγ produced by Th1 cells are essential for elimination of the parasites, but the same cytokines are also involved in the development of cerebral malaria⁴¹. Therefore, Nuchnoi et al. assessed whether TIM1 polymorphisms could potentially regulate the balance between Th1 and Th2 cytokine production⁴². In this study, 478 Thai malaria patients were subdivided into mild, non-cerebral, and cerebral malaria. The three promoter SNPs −1637G>A, −1549G>C, and −1454G>A maintained significant difference in allele frequency between mild and cerebral malaria even after the Bonferroni correction (all p=0.0009). In addition, a TIM1 haplotype comprised of −1637A, −1549C, and −1454A was significantly associated with protection against cerebral malaria (OR=0.41; 95% CI=0.24–0.71). Since the above promoter SNPs were in complete linkage disequilibrium (r²=1), promoter haplotypes were constructed to determine the functional effect of these polymorphisms on TIM1 transcription. The haplotype H2 (−1637A, −1549C, −1454A) was found to confer a TIM1 mRNA transcription level that was 3.9 times higher than that of haplotype H1 (−1637G, −1549G, −1454G). However, due to the complete LD between these SNPs, this study was not able to identify the primary polymorphism responsible for protection. These results demonstrated that TIM1 promoter haplotype consisting of −1637A, −1549C, −1454A was associated with protection against cerebral malaria, potentially due to higher TIM-1 expression and consequently higher Th2 cytokine (and corresponding lower Th1 cytokine) production. Therefore, it would be interesting to compare TIM-1 expression or the levels of Th1 and Th2 cytokines in mild versus cerebral malaria patients.

TIM-1 and HIV/AIDS

Th1 cells are essential for immune responses against intracellular infection caused by viruses and parasites. Wichukchinda et al. investigated the association of six TIM1 haplotypes with progression of HIV/AIDS in a population of HIV-1-infected Thai females⁴³. The D3-A haplotype was marginally associated with higher CD4+ T cell count (p=0.0615) compared to other haplotypes. D3-A carriers also had a lower rate of HIV-1 related symptoms, which was not significant after correction for multiple comparisons. On the other hand, haplotype D3-C was associated with the opposite phenotypes to D3-A, such as lower CD4+ T cell count (p=0.07) and a higher rate of HIV-1 related symptoms (p=0.05). Given the potential positive effect of D3-A on HIV-1 progression, the authors further examined the relationship between D3-A and mortality. Indeed, D3-A carriers had a significantly higher survival (p=0.044) than non-D3-A patients, even after adjustment for the antiviral treatment that was started by some patients during the course of this study (p=0.019). Consistent with a higher survival rate, D3-A patients had lower risk of death in the untreated period compared to others (HR=0.51; 95% CI=0.29–0.90). Importantly, the association was maintained after adjustment for age and plasma viral load, but not after adjustment for CD4 cell count baseline, suggesting that the protective effects of D3-A haplotype may be connected to CD4 cell count. This group also analyzed the association of the promoter SNPs −1637G>A and −1549G>C with TIM-1 expression, but could not detect any difference in TIM-1 expression⁴³. This absence of association is contrary to a previous study that observed higher TIM-1 mRNA transcription with the promoter haplotype −1637A/−1549C⁴². However, a B cell line carrying the D3-A haplotype had significantly higher TIM-1 transcript levels than non-D3-A cell lines (p=0.0421). Thus, it would be interesting to compare TIM-1 expression on CD4 T cells of HIV-1 infected patients and healthy controls.

TIM-3 and Allergic Diseases

TIM-3 is preferentially expressed on Th1 cells versus Th2 cells and known to negatively regulate Th1 T cell responses. However, TIM-3 may also be able to affect Th2-driven allergic diseases by indirectly modulating the balance between Th1 and Th2 type responses. Administration of anti-Tim-3 antibody decreased the production of Th2 cytokines and infiltration of eosinophils and Th2 T cells, preventing allergen-induced airway inflammation in a mouse model of asthma⁴⁴. Tim-3 is constitutively expressed by mast cells and Tim-3 polyclonal antibody treatment enhanced production of Th2 cytokines such as IL-4, IL-6 and IL-13 from mast cells, without inducing degranulation⁴⁵. Several genetic studies on TIM-3 polymorphisms suggest that TIM-3 is involved in susceptibility to Th2-driven allergic diseases. Some of the notable TIM-3 variants in this regard are summarized in Table II. These and other variants discussed below are also shown in the context of the TIM-3 gene and protein, in Figure 2. Chae et al. investigated frequencies of TIM3 polymorphisms in asthma and allergic rhinitis patients from a Korean population (n=254, 133 males and 121 females). The −574T allele was found only in asthma and rhinitis patients at the frequency of 1% and 1.5% respectively, while all healthy controls carried the G allele only. The minor T allele of 4259T>G was found more frequently in rhinitis patients, but not in asthma patients⁴⁶. However, while these polymorphisms correlated with symptoms of allergic asthma or rhinitis, a significant correlation with serum IgE level or blood eosinophil counts was not seen. Graves et al. investigated a possible correlation between atopic diseases in children from white or Hispanic parents (n=1088, DNA sample from 568 subjects) and three polymorphisms −882C>T, 4259T>G and 22713A>G²³. None of the TIM-3 polymorphisms tested appeared to be associated with asthma in children. 22713A>G and −882C>T were shown to be associated with atopic dermatitis, but 4259T>G (which was associated with asthma in a Korean population) did not appear to be associated with either atopic dermatitis or asthma in the study of Graves et al. This study has a limitation in that asthma and eczema were assessed by questionnaire, rather than by physician’s diagnosis and, in spite of adjustment, there is the possibility of false positive results from multiple comparisons. On the contrary, two other studies on children-parent trios did not find evidence of an association of polymorphisms of TIM-3 with allergic diseases. Gao et al. studied the two intronic polymorphisms rs11134551A>G and rs11742259 C>T, and a novel polymorphism in the 3′ UTR of TIM3 in an African American population. In this case-controlled study, neither genotypic, haplotypic nor family based transmission analysis found an increased risk of asthma with these TIM-3 variants²⁵. Another study conducted on Chinese children-parents trios, using transmission disequilibrium test and haplotype relative risk analysis for single TIM-3 polymorphisms, could not find evidence of an effect of TIM-3 polymorphisms on asthma susceptibility. However, haplotype analysis for the combination of three TIM-3 polymorphisms revealed that the G-G-G haplotype for rs10053538G>T, rs13170556A>G and rs9313441G>A was under-transmitted to asthmatic children²⁹. Since each study investigated the effect of different polymorphisms on allergic diseases, it may be too early to generalize the consequences of TIM3 polymorphisms for allergic asthma and rhinitis. Thus, TIM-3 is expressed on - and negatively regulates -Th1 cells, and treatment with an agonistic antibody to TIM-3 induces Th2 cytokine production in mast cells. The polymorphisms of TIM-3 associated with Th2-mediated diseases may be due to indirect effects of skewing the Th1/Th2 balance towards Th2. However, it has not been investigated whether variations within the coding or non-coding regions of Tim-3 lead to increased Tim-3 protein expression, stability, or function.

Table II.

Notable Studies on the role of TIM-3 Variants in Atopic Disease

Study	Findings	Study Design/Strengths	Limitations
Chae et al. (2004; Human Immunology)	Several SNPs associated with asthma and/or allergic rhinitis (but not with serum IgE or peripheral blood eosinophils)	Case-control; Large population; selection based on physician diagnosis	No adjustment for multiple comparisons
Wu et al. (2009; Int. Arch. Allergy Immunol.)	No single polymorphism was associated with childhood asthma, but a combination of 3 polymorphisms was	Family trios study; Selection based on physician diagnosis; family trio study; TDT and haplotype relative risk assessed	No mechanistic/molecular correlate data
Graves et al. (2005; J. Allergy Clin. Immunol.)	Several polymorphisms associated with atopy and eczema, but not asthma	Case-control, longitudinal, study; adjustment for multiple comparisons; atopy assessed by skin prick	No stratified analysis by ethnicity asthma and eczema assessed only by questionnaire
Gao et al. (2005; J. Allergy Clin Immunol.)	Several TIM-3 variants found not to be associated with asthma	Case-control and family- based study	No mechanistic/molecular correlate data

Figure 2.

Overview of TIM3 polymorphism disease associations in both genomic and protein contexts. Major polymorphisms are shown above the genomic scheme, while coding changes are also shown below the protein scheme. See text for complete description of the individual polymorphisms.

TIM-3 and Systemic Lupus Erythematosus

Wang et al. demonstrated the association of TIM genotype with SLE³⁹. While significantly increased TIM-1 expression was found in PMBC isolated from SLE patients, TIM-3 expression levels were not statistically different. An increase in IL-10 production was observed with PMBC from SLE patients, accompanied by a corresponding decrease in IFN-γ. These findings suggest that skewing of the Th1/Th2 balance toward Th2 by the TIM-3-galectin-9 interaction contributes to SLE pathogenesis. Recently, the relationship of two TIM3 polymorphisms rs9313439 (22813 C>G, 3′ UTR) and rs10515746 (−574G>T) to SLE was explored. Li et al. compared frequencies of TIM3 polymorphisms in Chinese SLE patients without a history of atopic diseases (172 female and 30 male) and sex-matched healthy controls⁴⁰. Neither genotype nor allele frequency analysis found differences in the frequency of TIM-3 variants between patients with SLE and healthy controls. These investigators also compared genotype and allele frequencies between patients with active and inactive disease (n=94 and 105 respectively), sub-grouped according to SLE disease activity index (SLEDAI), and again no significant difference was observed⁴⁰.

TIM-3 and Rheumatoid Arthritis

It has been suggested that TIM-3 plays a protective role in RA. Thus, galectin-9 administration reduced pro-inflammatory Th1 and Th17 cytokine levels in joints and Th1 cytokine production in splenocytes, resulting in suppressed CIA symptoms in mice⁴⁷. TIM-3 expression was upregulated in peripheral monocytes and T cells, and synovial tissue in RA patients. In addition, TIM-3 expression on PBMC, T cells, and NKT cells was inversely correlated with plasma TNF-α levels and disease severity^48,49. Chae et al. investigated genotype and allele frequencies of three TIM-3 polymorphisms in RA patients⁵⁰. With 295 Korean RA patients (43 males, 253 females, mean age=38.7 years), these investigators showed that −574G>T and 4259T>G were associated with rheumatoid arthritis. The minor allele of −574 T>G was found only in RA patients (p=0.001) and the minor allele of 4259 G>T was present at significantly higher frequency in RA patients, compared to healthy controls (p=0.003). A possible correlation between C-reactive protein (CRP) and rheumatoid factor (RF) levels and the three TIM3 polymorphisms in RA patients was examined. However, no significant differences by genotype were observed. The positive correlation found between rheumatoid arthritis and TIM-3 polymorphisms −574 G>T and 4259 T>G may seem contradictory to the study done by the same group showing that the same variants are associated with Th2-type allergic diseases⁴⁶. Also, Tim-3 ligation and blocking were protective in mouse models of autoimmune arthritis and allergen-induced airway inflammation, respectively^{47; 51}. A cohort study using a large population reported that the presence of the Th1 type disease RA does not lower the susceptibility to the Th2-dominant disease asthma, but the incidence of asthma was higher in children with RA than children without RA⁵². Recently, another study conducted on Korean RA patients (41 males, 325 females, mean age 51 years, 389 healthy controls) investigated five polymorphisms, including −575 G>T (rs10515746) and 4259 T>G (rs1036199) ⁵³. The minor allele frequency of −574 G>T was higher in RA patients (p=0.023), but 4259 T>G was not. This study also examined the frequency of rs35960726, located in exon2, whose minor allele frequency was significantly elevated in RA patients (p<0.001). After stratification of HLA-DRB1 epitope checked patients (with SE=107 and without SE=58) by shared epitope status frequency of −574 G>T rs35960726 was significantly associated with in both RA patients with SE (p=0.006 and <0.001) and without SE (p=0.015 and 0.001). Significant association of 4259T>G frequency with the disease was found in RA patients with SE, but not those without SE.

Summary

As detailed here, a large number of studies have now examined the possible association of TIM variants with a variety of diseases. The greatest body of literature thus far has focused on a possible role for TIM1 or TIM3 polymorphisms in susceptibility to atopic disease, particularly asthma. However, most of these studies fail to take into account HAV infection status, which certainly seems to be relevant in the case of studies on TIM-1^{5; 15}, and perhaps even for TIM-3 as well⁵⁴. Furthermore, it is important to keep in mind that in the initial study that identified the entire Tim locus in mice, as a possible controller of asthma and atopy susceptibility, polymorphisms in the coding regions of both Tim-1 and Tim-3 were described¹⁰. In addition, the TIM genes are linked, something, which is not taken into account in all studies. One exception is the work of Graves et al.²³, discussed above. It will also be important in the future to obtain more objective data on the disease status of subjects, since many of the previous studies relied on questionnaires, rather than laboratory tests. Finally, there is a need for more thorough mechanistic study of the various TIM polymorphisms that have been implicated in disease susceptibility. Thus, how do the TIM protein variants differ in not only HAV binding but also interactions with soluble or cell-bound ligands, and how do these differences affect intracellular signaling pathways that control leukocyte function?