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Purpose: Toll-like receptors play a pivotal role in an innate immunity system, which controls inflammation responses and further instructs development of adaptive immunity. The aim was to study the expression profile and polymorphism of TLR-2 and TLR-4 in the peripheral blood lymphocytes of Fulminant Hepatic Failure (FHF).
Methods: Our study included 45 cases with FHF, 45 cases with Acute Hepatitis (AH) and 25 healthy controls. The mean age in years (range) of subjects in different groups were FHF, 29.84 (14 - 65yrs); FHF-E, 27.65 (17 - 55yrs); FHF-S, 33.00 (14 - 65yrs); AVH, 29.47 (16 - 68yrs). Peripheral blood lymphocytes were isolated for studying the polymorphism, total RNA was extracted and reverse transcribed into cDNA. The mRNA expression level of TLR-2 and TLR-4 were quantitatively determined by semi-quantitative RT-PCR.
Results: : Expression of TLR 2 and TLR 4 was significantly more in patients with FHF 31/45 (68.9%) and AVH 20/45 (44.4%) than normal controls (p<.001). Patients with FHF also showed a significant greater expression than those with AVH (p<0.019). Greater expression was seen in patients with FHF-E 20/26 (76.9%) vs. FHF-S 11/19 (57.9%) but however results here were not significant (p=0.17). Polymorphism for four types of SNPs -TLR2 Arg677, TLR2 Arg753, TLR4 Asp299 and TLR4 Thr399 were studied which failed to show a significant difference.
Conclusion: In conclusion, our study reveals a possible interaction between Fulminant Hepatic Failure, TLR signaling and the innate immune response, which may partially explain the mechanism of infection induced immuno-tolerance.
Acute Viral Hepatitis (AVH) is a global public health problem and which is highly prominent in India. It is an acute self-limited disease with a serum aspartate amino transferase (AST) elevation of at least five folds or clinical jaundice or both.11 However, in about 1% of patients this can be complicated by development of Fulminant Hepatic Failure (FHF). While FHF has numerous causes, most cases are due to viral hepatitis and drug toxicity or idiosyncratic reactions. In India, more than 90% cases are due to acute viral hepatitis, the most common viruses being hepatitis E followed by hepatitis B.1 Not much is known about the pathophysiology of this mortal condition, which even in the west has 50-80% mortality rate. Recently, interest has been created to study the possible pathophysiological mechanisms of hepatic damage. Inciting agents including viruses and toxins, leading to a critical degree of liver cell damage and death due to apoptosis or necrosis which is not adequately compensated by hepatocellular regeneration, there by resulting in massive hepatic necrosis in FHF.2 Although the inciting agents may directly damage hepatocytes, recent evidences have suggested that inflammatory cells and their products (viz: TNF-ïŸ and IL-1) can contribute to the hepatic necrosis.3 Furthermore, in patients with liver failure endotoxemia occurs which contributes to the pathogenesis of this disorder. Recent studies have focused on the intermediate pathways activated during the initial injury that leads to progressive and rapid hepatocyte destruction. Different types of stimuli such as endotoxins, lipopolysacchride (LPS), tumor necrosis factor (TNF-ïŸ), Interleukin 1(IL-1), IL-2 etc. can trigger rapid activation of a cellular proinflammatory transcription factor, NF-ïŸB and subsequent modification of its downstream target genes and proteins.4
Toll-like receptors (TLRs) are a family of pattern-recognition membrane receptors of innate immunity that recognize unique molecular signatures of microbes, called pathogen-associated molecular patterns (PAMPs).5 TLRs are the first line of host defense and TLR activation by their respective PAMPs results in pro inflammatory cytokine cascades and induction of both innate and adaptive immune responses. TLR 4 recognizes LPS of Gram-negative bacteria, whereas TLR 2 responds to various lipoproteins, including components of Gram-positive bacteria, such as peptidoglycan.5
Members of the TLR family also detect viruses that enter the endosome through endocytosis. This pathway induces production of interferons through several signaling proteins that ultimately lead to the activation of the transcription factors NF-B, IRF3 and IRF7. 6-8
While role of TLR expression and presence of single nucleotide polymorphisms of Toll-like receptors has been found to be positively correlated with susceptibility to several diseases9; studies investigating their possible role in Acute Viral Hepatitis (AVH) and Fulminant Hepatic Failure (FHF) are lacking. This is the first human study concerning the expression of TLR in AVH and FHF.
Patients and Clinical Samples
Patients with Acute Viral Hepatitis and Fulminant Hepatic Failure presenting to the Department of Medicine, Lok Nayak Hospital, Delhi were prospectively recruited.
A patient was diagnosed to have Fulminant hepatic failure when after a typical acute onset, patient became deeply jaundiced, developed coagulopathy (Prothrombin activity < 40% of normal) and went into hepatic encephalopathy within eight weeks of onset of disease without any past history of chronic liver disease.10 Acute viral hepatitis was defined as those cases, which had an acute self-limiting disease and a serum aspartate transaminase elevation of at least five fold or jaundice or both.11
Written informed consent was obtained from all subjects. Medical history was obtained from all participants, including details of previous episodes of jaundice; any associated systemic diseases. Biochemical parameters including detailed liver function tests (AST, ALT, and alkaline phosphatase, total serum bilirubin and its fractions, total proteins and fractions and prothrombin time) were conducted in all. Age and sex matched healthy control subjects without any history of any liver disease were recruited. Blood samples were collected into sterile, anticoagulant (EDTA)-coated tubes and immediately transported to the laboratory for DNA and RNA extraction.
PCR RFLP of the Toll-like receptor-2 and -4 gene
Determination of the TLR 2 and 4 gene mutations was accomplished with polymerase chain reaction (PCR) and restriction fragment length polymorphism. The total volume of the PCR was 25 Âµl (TLR2: 2 Âµl) containing 100 ng of genomic DNA, 1xPCRbuffer(Qiagen, Hilden, Germany), 0ïƒ-2 mM of each dNTP (Sigma,Taufkirchen, Germany), 0ïƒ-75 units (TLR2: 0ïƒ-5 units) of HotStar-Taqïƒ”DNA polymerase (Qiagen) and 7ïƒ-5 pmol (TLR2: 5 pmol)of each primer (TIB MOLBIOL, Berlin, Germany). The final concentration of MgCl2 was 4 mM for Asp299Gly, 1ïƒ-5 mM for Thr399Ile, and 3 mM for Arg753Gln and Arg677Trp. The PCR was performed in a Thermocycler MJ Research with an initial denaturation step (95oC for 15 min), 35 cycles (94oC for 30 sec, 62oC (Asp299Gly), 60oC (Thr399Ile), or 65oC (Arg677Trp and Arg753Gln) for 30 s, 72oC for 30 s), and a final extension step (72oC for 10 min). The restriction assay contained 1 restriction buffer, 20 units (TLR2 Arg677Trp: 12ïƒ-5 units) of the respective restriction enzyme (New England Biolabs, Beverly, MD, USA) and 15 ml (TLR2: 20 ml) of the PCR product. It was incubated overnight at 37OC and analyzed by electrophoresis on a 2ïƒ-5% agarose gel. The primer sequences are presented in Table 2. Full length PCR products were digested into the restriction fragments as listed in Table 3. The PCR was performed in a total volume of 100 ml and the final concentration of MgCl2 was 1ïƒ-5 mM, the other components and concentrations were as described above. The PCR comprised an initial denaturation step of 15 min at 95oC, 35 cycles of denaturation for 30 s at 94oC, annealing for 30 s at 65oC and extension for 60 s at 72oC, and a final extension step for 10 min at 72oC.
Table 1: The Primer sequences for TLR 2 and 4 genotyping by PCR- RFLP method:
F: 5 - AGCATACTTAGACTACTACCTCCATG-3
R: 5 - GAGAGATTTGAGTTTCAATGTGGG-3
F:5 - GTTGCTGTTCTCAAAGTGATTTTGGGAGAA-3
R:5 - GAAATCCAGATGTTCTAGTTGTTCTAAGCC-3
F: 5 -CCCCTTCAAGTTGTGGCTTCATAAG-3
R: 5 -AGTCCAGTTCATACTTGCACCAC-3
F: 5 -CATTCCCCAGCGCTTCTGCAAGCTCC-3
R: 5 -GGAACCTAGGACTTTATCGCAGCTC-3
Table 2: Restriction enzymes and length of the restriction fragments
Restriction temp ï‚°C
Length of the Restriction fragments:
Wild type (allele A):ï½ 188 bp
Asp299Gly (allele G):ï½ 168 bp + 20 bp
Wild type (allele C):ï½ 124 bp
Thr399Ile (allele T):ï½ 98 bp + 26 bp
Wild type (allele C):ï½ï½ 130 bp + 22 bp
Arg677Trp (allele T):ï½ 152 bp
Wild type (allele G):ï½ 104 bp + 25 bp
Arg753Trp (allele A):ï½ 129 bp
DETECTION OF TLR 2 and 4 EXPRESSION
RNA isolation and TLR2/TLR4 reverse transcription polymerase chain reaction (RT-PCR) analysis.
Total cellular RNA was extracted from the isolated PBMC using an acid phenol-chloroform extraction method, as described previously. The RNA concentration was quantitated spectrophotometrically, and cDNA of PBMC was obtained by reverse transcriptase using 1 Âµg of RNA and oligo-dT primers. PCR for TLR2, TLR4 and Î²-actin was performed with primer pairs spanning exon-exon conjunctions. The following oligonucleotide primer sequences were used with Î²-actin by densitometric analysis of the amplified cDNA product. The primer used for Î²-actin (226 bp) (F: GGC GAC GAG GCC CAGA & R: CGA TTT CCC GCT CGGC) as internal control. Cycling conditions were 94oC for 1 min, 55 oC for 1 min, and 72oC for 1 min at 30 cycles. Human TLR2 (394 bp) (F): 5'-GCC AAA GTC TTG ATT GAT TGG, (R): 5'-TTG AAG TTC TCC AGC TCC TG; humanTLR4 (506 bp) (F): 5'-TGG ATA CGT TTC CTTATA AG, (R): 5'-GAA ATG GAG GCA CCC CTTC. Cycling conditions were 95 oC for 40 s, 54 oC for 40 s, and 72 oC for 1 min for both TLR2 and TLR4.Optimal results for TLR2 were obtained at 28 cycles in PBMC, and for TLR4 at 32 cycles. The amplified products were separated by electrophoresis on a 2% agarose gel and visualized by ultraviolet light illumination using ethidium bromide staining. As performed by other authors, semiquantitative PCR results were generated by grading a ratio between the densitometry results of the target gene (TLR2, TLR4) and the housekeeping enzyme Î²-actin.
Immunohistochemistry and analysis of NF-B expression
Post-mortem liver biopsy was performed on FHF patients who expired using tru-cut biopsy needle (n=10). Postmortem liver biopsy done in patients, who died of a cause unrelated to liver disease and whose biopsy revealed a normal histology (n=10), done for diagnostic or therapeutic reasons served as biopsy controls. Informed consent was obtained from the nearest relative or the patient in all cases. Briefly, after deparafinization and rehydration, the tissue sections were blocked in 3% bovine serum albumin for 30 min and incubated overnight at 37ï‚°C with the primary antibody. Immunoreactivity was visualized using an ABC Staining System Kit from Santa Cruz Biotechnology following the manufacturer's protocol. The slides were then counterstained regressively in Mayer's hematoxylin, dipped in methanol for a few seconds, cleared in xylene, and mounted in Permount. To assess the specificity of staining, sections were processed without primary or without secondary antibodies as controls. The same antibodies were used for the immunohistochemical study.
The data obtained from the study was tabulated and transferred to a personal computer. Quantitative data was expressed as mean Â± standard deviation and/or as medians. The information thus collected was analyzed using standard statistical tests (Chi square test of significance or Student's t-test or Fisher test as applicable). P < 0.05 was considered to be statistically significant in all analysis.
Forty five patients each with acute viral hepatitis and fulminant hepatic failure satisfying the study inclusion and exclusion criteria were studied. The mean age in years (range) of subjects in different groups were FHF, 29.84 (14 - 65yrs); FHF-E, 27.65 (17 - 55yrs); FHF-S, 33.00 (14 - 65yrs); AVH, 29.47 (16 - 68yrs). Equal number of age and sex matched normal people served as controls.
Patients with FHF and AVH showed mild to moderate expression of TLR 2 and moderate to high expression of TLR 4. Controls showed mild expression of TLR 2 and medium expression of TLR 4 (Fig. 1, Table 3).
A significant higher level of TLR-2 as well as TLR-4 expression was observed in patients with FHF than AVH (p=.019) and also in AVH than healthy subjects (p< .001). Similarly, amongst FHF group; greater expression was seen in patients who expired than those who survived but this could not attain statically significance (p=.17).
Table 3: Expression of TLR 4 & TLR 2
Fig. 1Semiquantification of TLR 2 & 4 Expression in reference to Î² actin
Lane 1 2 3 4 5 6 7
Lane 2 & 5 - FHF cases; Lane 3 & 6 - Controls; Lane 4 & 7 - AVH
Also, no difference was observed in the levels of TLR 2 or 4 expressions depending on whether etiology of FHF was viral hepatitis A to E or non A-E.
Postmortem liver biopsies were collected from 10 patients. Ten persons in whom liver biopsy was performed for some other diagnostic purpose and biopsy specimen showed normal histology on H & E constituted the control group.
Table 4: Expression of p50 & p65
Figure 2: Immunohistochemical analysis of expression and localization of p50 and p65 in normal as well as post-mortem liver biopsies of FHF patients
Paraffin-embedded sections (4-5 Î¼m thick) were immunolocalized with the Santa-Cruz antibodies specific for p50 and p65 of NF-kB. (A) IHC using p65 antibody in normal liver tissue; (B) IHC using p50 antibody in normal liver tissue; (C) IHC using p65 antibody in postmortem liver biopsy; (D) IHC using p50 antibody in postmortem liver biopsy.
When we analyzed the expression profile of p50 and p65 proteins that heterodimerizes to form functional NF-ïŸB complex by immunohistochemistry we observed that most patients with FHF also showed a moderate to high level of p50 expression while p65 had a mild expression (Table 4, Figure 2). A decrease of p65 expression and increased p50 expression was found to be both nuclear as well as cytoplasmic (Figure 2 C, D). In contrast, nuclear localization of both p50 and p65 was observed in normal liver biopsies (Figure 2 A, B).
TLR2 and -4 Genotypes of Patients
In this study, we have analyzed TLR genotype polymorphisms of TLR2 Arg677, TLR2 Arg753, TLR4 Asp299 and TLR4 Thr399. TLR2 Arg677 polymorphism was detected in none of the subjects as well as the control group. TLR2 Arg753 polymorphism was detected in 8.8% of patients with FHF and 2.2% and 4% in AVH and control, respectively.
TLR4 Asp299 polymorphism was seen in 8.8% and TLR4 Thr399 polymorphism in 11.1% of patients of FHF. Both of these polymorphisms were detected in about 4% of patients of AVH and normal controls.
Thus prevalence of these SNPs failed to show any significant difference between the AVH, FHF and controls.
TLRs belong to a class of cell surface molecules known as pattern recognition receptors. The ligands for these receptors are components of pathogenic microbes called pathogen-associated molecular patterns (PAMPs).5 TLRs are part of the innate immune system, although they also modulate mechanisms that impact the development of adaptive immune responses.
Stimulation of TLRs triggers the association of MyD88 (myeloid differentiation primary-response protein 88), which in turn recruits IRAK4 (IL-1R-associated kinase 4), thereby allowing the association of IRAK1. IRAK4 then induces the phosphorylation of IRAK1. TRAF6 (tumour-necrosis-factorreceptor- associated factor 6) is also recruited to the receptor complex, by associating with phosphorylated IRAK1. Phosphorylated IRAK1 and TRAF6 then dissociate from the receptor and form a complex with TAK1 (transforming-growthfactor-Î²-activated kinase), TAB1 (TAK1-binding protein 1) and TAB2 at the plasma membrane , which induces the phosphorylation of TAB2 and TAK1. IRAK1 is degraded at the plasma membrane, and the remaining complex (consisting of TRAF6, TAK1, TAB1 and TAB2) translocates to the cytosol, where it associates with the ubiquitin ligases UBC13 (ubiquitinconjugating enzyme 13) and UEV1A (ubiquitin-conjugating enzyme E2 variant 1). This leads to the ubiquitylation of TRAF6, which induces the activation of TAK1. TAK1, in turn, phosphorylates both mitogen-activated protein (MAP) kinases and the IKK complex (inhibitor of nuclear factor-ÎºB (IÎºB)-kinase complex), which consists of IKK-Î±, IKK-Î² and IKK-Î³. The IKK complex then phosphorylates IÎºB, which leads to its ubiquitylation and subsequent degradation. This allows NF-ÎºB to translocate to the nucleus and induce the expression of its target genes.
Several lines of evidence indicate that TLRs are implicated in inflammatory and immune disorders. Earlier studies have shown pivotal role of TLRs in pathogenesis of chronic enterocolitis,12 allograft rejection,13 development of atherosclerosis,14,15 SLE and rheumatoid arthritis. In addition to these immune-related disorders, TLR recognition of commensal bacteria has been shown to play a crucial role in the maintenance of intestinal epithelial homeostasis.16 Thus, TLR-mediated pathways are probably involved in many aspects of immune responses, even in the absence of infection.
Amongst liver diseases, TLR-mediated signals have been associated with infectious,17-23 granulomatous19,20, alcoholic liver disease24-26, ischemia-reperfusion injury27-31 and liver regeneration32-38. Recent studies also provide evidence for a role of TLRs in the pathophysiology of nonalcoholic steatohepatitis (NASH)39,40, autoimmune hepatitis41, primary biliary cirrhosis (PBC)42-46, hepatic fibrosis47-50, and hepatocarcinogenesis51, and in the function of RNA-helicases, particularly in chronic hepatitis C virus (HCV) infection52-58. Experimental studies in animals have even co-related increased expression of TLR 2 and 4 in acute liver failure.59-61
Both acute viral hepatitis and fulminant viral hepatitis have same etiological factors but a markedly different clinical course. While former is a self-limited disease, the later is a highly mortal condition often requiring liver transplant. This difference is likely due to host immune factors. This is due to the fact that in fulminant hepatitis B virus (HVB) infection, humoral antibody response is enhanced and a significantly faster rate of clearance of HBsAg is seen compared to patients with severe uncomplicated hepatitis. On admission anti-HBs Ab is found in about 40% of patients with fulminant hepatitis whereas it is very rarely detected in uncomplicated hepatitis at this phase.
The key elements that determine this difference in host response are unclear. In our study, we observed that significantly higher levels of TLR2 and 4 expression are associated with fulminant hepatitis than in patients with acute viral hepatitis. Patients with acute viral hepatitis also had significant increased expression as compared to normal controls. Among fulminant hepatic failure group, patients who died also showed greater expression than was seen in patients who survived however results here were not significant. Thus, increased immune response involving TLR 2 and 4 has an association with differential expression of AVH and FHF.
NF-B is the master regulator of all TLR-induced responses and its activation of is the pivotal event in TLR-mediated activation of cytokines that mediate the development of innate immunity. To assess functional significance of the increased TLR expression, immunohistochemistry for p50 and p65 proteins that heterodimerizes to form functional NF-B was done in liver tissue. We observed that patients with FHF mostly showed a moderate to higher level of p50 expression while p65 was expressed very weakly while a reverse pattern of expression of NF-kB components was also observed in control group which predominately showed weak p50 expression but moderate to high expression of p65.
Thus, proposed etiopathogenesis of differential regulation of NF-kB signaling pathway at the transcriptional level through upregulation and homodimerization of p50 subunits but selective suppression of its canonical dimerization partner p65 and thereby leading to complete breakdown of the immune system with severe liver damage is further supported. Similar results have been shown in a larger study on HEV infected pregnant women with FHF.62
Genetic polymorphisms-for the most part, single nucleotide polymorphisms (SNPs)-are common variants within a population that are found at a frequency of over 1%. SNPs may alter the aminoacid sequence (non-synonymous SNPs), affect promoter characteristics, or may be completely "silent". Mutations do not occur randomly within the genome, but rather depend on the particular genomic region, as well as on selective pressure.63 Thus, one might expect that genes encoding for proteins involved in immunity or disease resistance should exhibit a high degree of polymorphisms, a hypothesis that was strengthened by data obtained from genome-sequencing approaches.64,65 Within the genes involved in immune recognition, several non-synonymous SNPs have been described, including pattern recognition molecules and cytokines. However in our study presence of SNPs failed to show a significant difference between the three groups.
Studies have suggested an association of these SNPs (particularly: TLR2 Arg677Try; Arg753Gln; TLR4 Asp299Gly and Thr399Ile Asp299Gly ) with incidence of premature birth, 66 crohn's 67, lepromatous leprosy68, severity of asthma69, decreased susceptibility to rheumatoid arthritis70 , lower incidence of diabetic neuropathy in type 2 diabetics71 and lower rate of acute allograft rejection72. However in our study presence of SNPs failed to show a significant difference between the three groups.
In conclusion, it is suggested that immune response mediated by TLR 2 and TLR 4 in parallel with activation of NF-B plays a major role in etiopathogenesis of both AVH and FHF. The study also indicates that increased TLR 2 and 4 levels are related to hepatic injury in hepatitis and the activity correlates with severity of the disease, but such a correlation may not be extended directly to mortality. The question remains as to whether increased TLR 4 is an example of dysregulation or normal immune response.
However, functionally effective polymorphisms of the Toll-like receptor-2 and -4 genes appear to have apparently no influence on the individual susceptibility for AVH and FHF.
TLRmediated signals have been associated with numerous diseases including liver diseases such as ischemiareperfusion injury and liver regeneration.
What is new:
TLR 2 and 4 expression is also increased in Fulminant Hepatic Failure and it is significantly more than that in acute viral hepatitis.