In-vitro effect of human cathelicidin antimicrobial peptide LL-37 on dengue virus type 2.

Human Cathelicidin antimicrobial peptide LL-37 is known to have antiviral activity against many viruses. In the present study, we investigated the in-vitro effect of LL-37 on dengue virus type 2 (DENV-2) infection and replication in Vero E6 cells. To study the effect of pretreatment of virus or cells with LL-37, the virus was pretreated with different concentrations of LL-37 (2.5µM-15µM) or scrambled (Scr) LL-37(5µM-15µM) and used for infection or the cells were first treated with LL-37 and infected. To study the effect of LL-37 post infection (PI), the cells were infected first followed by addition of LL-37 to the culture medium 24h after infection. In all conditions, after the incubation, the culture supernatant was assessed for viral RNA copy number by real time RT-PCR, infectious virus particles by focus forming unit assay (FFU) and non structural protein 1 (NS1) antigen levels by ELISA. Percentage of infection was assessed using immunoflourescence assay (IFA). The results revealed that pretreatment of virus with 10-15µM LL-37 significantly reduced its infectivity as compared to virus control (P<0.0001). Moreover, pretreatment of virus with 10-15µM LL-37 significantly reduced the levels of viral genomic RNA and NS1 antigen (P<0.0001). Treatment of virus with 10-15µM LL-37 resulted in two to three log reduction of mean log10 FFU/ml as compared to virus control (P<0.0001). Treatment of the virus with scrambled LL-37 had no effect on percentage of infection and viral load as compared to virus control cultures (P>0.05). Pretreatment of cells before infection or addition of LL-37 to the culture 24h PI had no effect on viral load. Molecular docking studies revealed possible binding of LL-37 to both the units of DENV envelope (E) protein dimer. Together, the in-vitro experiments and in-silico analyses suggest that LL-37 inhibits DENV-2 at the stage of entry into the cells by binding to the E protein. The results might have implications for prophylaxis against DENV infections and need further in-vivo studies.

Altered profile of regulatory T cells and associated cytokines in mild and moderate dengue.

The pathogenesis of dengue is immune-mediated. Regulatory T cells suppress immune response and may contribute to better prognosis. The present study evaluates Tregs and cytokines in dengue patients in the context of disease severity, time of sampling and immune status. The cohort included 90 patients (51 mild, 39 moderate) and 27 healthy controls. Frequencies of Tregs, CD4(+)CD25(-)Foxp3(+) T cells and CD3(+), CD3(+)CD4(+) and CD3(+)CD8(+) T cells were enumerated by flow cytometry. Circulating levels of 15 cytokines/chemokines were measured using Luminex technology and mRNA levels of Foxp3, IL-10 and TGF-ß were assessed by real-time polymerase chain reaction (PCR). Significantly higher frequencies of Tregs were observed in mild cases, especially during post-defervescence. The difference between mild and moderate cases was more evident in secondary infections. Frequencies of T cells were higher in mild cases but during pre-defervescence. On the other hand, the levels of IL-6, IL-7, IL-8, TNF-α and IL-10 were significantly higher in moderate cases. IL-6 and IL-8 levels correlated negatively with Treg frequencies during post-defervescence and in secondary infections. Higher levels of IL-10 and TGF-ß in moderate cases were not reflected by their corresponding mRNA levels. Platelet counts correlated positively with Treg frequencies and TGF-ß levels, and negatively with IL-10 levels. Higher Treg frequencies may favour a beneficial outcome in dengue. Higher cytokine levels may indirectly contribute to disease severity by exerting an inhibitory influence on Tregs. The dichotomy between mRNA and proteins levels for IL-10 and TGF-ß is suggestive of increased translational efficiency.

Association of combinations of interleukin-10 and pro-inflammatory cytokine gene polymorphisms with dengue hemorrhagic fever.

Pro-inflammatory and anti-inflammatory cytokines have been shown to play an important role in dengue disease pathogenesis. In the present study, to find out whether single nucleotide polymorphisms (SNPs) in the pro-inflammatory and anti-inflammatory cytokine genes are associated with dengue disease severity, SNPs in TNF, IFNG, IL1B, IL8, IL0, IL17A and IL17F genes were investigated using polymerase chain reaction based methods in 132 dengue (DEN) cases [87 dengue fever (DF), 45 dengue hemorrhagic fever (DHF) cases] and 108 apparently healthy controls (HC) from Pune, Maharashtra, western India. Under recessive genetic model (C/C vs. T/T+T/C), the TNF rs1799964 C/C genotype was significantly associated with DEN [P=0.014, OR with 95% CI 3.07 (1.18-7.98)]. Frequency of T/C genotype of IL17F rs763780 was significantly lower in DEN group as compared to HC [P=0.033, OR with 95% CI 0.43 (0.19-0.95)]. Under overdominant genetic model (A/T vs. A/A+T/T), IL8 rs4973 A/T genotype was negatively associated with DHF compared to HCs [p=0.029, OR with 95% CI 0.43 (0.20-0.93)]. Under overdominant genetic model, A/G genotype of IL10 rs1800871 was significantly negatively associated with DHF compared to DF cases [p=0.014, OR with 95% CI 0.35 (0.15-0.84)]. Significantly higher frequency of the combined genotype IL10 A/A-IFNG A/T and lower frequency of the combined genotypes IL10 A/G-IL1B A/A, IL10 A/G-IL8 A/T and IL10 A/G-IL17F T/T were observed in DHF cases compared to DF. The results suggest that heterozygous genotypes of IL8 rs4973 and IL10 rs1800871 are associated with reduced risk of DHF. Combinations of IL10 rs1800871 and pro-inflammatory cytokine genotypes influence the risk of DHF.