Variants of CARD8 in Leishmania guyanensis-cutaneous leishmaniasis and influence of the variants genotypes on circulating plasma cytokines IL-1ß, TNFα and IL-8.

Nucleotide-binding oligomerization domain, leucine-rich repeat-containing protein family (NLR) are intracellular pathogen recognition receptors mediating innate immunity, releasing proinflammatory cytokines IL-1ß and IL-18, and promoting pyroptotic cell death, upon sensing pathogenic or endogenous danger signals. In animal models, NLRP3 inflammasome has a dual role, pathogenic or protective in Leishmania-infection, depending on the Leishmania species and mice strain. Caspase recruitment containing domain 8 (CARD8) is a negative regulator of NLRP3 inflammasome and also an inhibitor of transcription factor NFĸB, a major transcription factor of proinflammatory cytokines. We investigated whether single nucleotide variants in CARD8 may partially explain why only a proportion of individuals coming from the same area of endemicity of leishmaniasis develop cutaneous leishmaniasis caused by Leishmania guyanensis. We genotyped four single nucleotide variants of the CARD8 gene by direct nucleotide sequencing in 1741 individuals from an endemic area of leishmaniasis, constituting 850 patients with CL and 891 healthy controls. The frequencies of the genotypes of the variants rs2288877 T>C, rs73944113 C>T, and rs2043211 A>T are similar among the patients with CL and HC, while the variant rs2288876 A>G) reveals an excess of the genotype AA among the patients with CL (44%) compared to 37% in the HC group. Allele A of the variant rs2288876 A>G) is associated with susceptibility to CL (OR = 1.2 [95%CI 1.03-1.4]; P = 0.01). Haplotype analysis showed that individuals harboring the haplotype CCAA have 280% odds of developing CL caused by L. guyanensis (OR = 3.8 [95% CI 2.0-7.7]; p = 0.00004). The variants rs2288877 T>C and rs2288876 A>G correlate with the plasma level of IL-8. Spearman correlation showed a significant positive correlation between the rs2288876 A>G allele A and the level of IL-8 (ρ = 0.22; p = 0.0002). CARD8 may partially contribute to the development of CL caused by L. guyanensis.

Distinct plasma chemokines and cytokines signatures in Leishmania guyanensis-infected patients with cutaneous leishmaniasis.

The immunopathology associated with Leishmaniasis is a consequence of inflammation. Upon infection with Leishmania, the type of host-immune response is determinant for the clinical manifestations that can lead to either self-healing or chronic disease. Multiple pathways may determine disease severity. A comparison of systemic immune profiles in patients with cutaneous leishmaniasis caused by L. guyanensis and healthy individuals with the same socio-epidemiological characteristics coming from the same endemic areas as the patients is performed to identify particular immune profile and pathways associated with the progression of disease development. Twenty-seven plasma soluble circulating factors were evaluated between the groups by univariate and multivariate analysis. The following biomarkers pairs IL-17/IL-9 (ρ=0,829), IL-17/IL-12 (ρ=0,786), IL-6/IL-1ra (ρ=0,785), IL-6/IL-12 (ρ=0,780), IL-1ß/G-CSF (ρ=0,758) and IL-17/MIP-1ß (ρ=0,754) showed the highest correlation mean among the patient while only INF-γ/IL-4 (ρ=0.740), 17/MIP-1ß (ρ=0,712) and IL-17/IL-9 (ρ=0,707) exhibited positive correlation among the control group. The cytokine IL-17 and IL1ß presented the greater number of positive pair correlation among the patients. The linear combinations of biomarkers displayed IP-10, IL-2 and RANTES as the variables with the higher discriminatory activity in the patient group compared to PDGF, IL-1ra and eotaxin among the control subjects. IP-10, IL-2, IL-1ß, RANTES and IL-17 seem to be predictive value of progression to the development of disease among the Lg-infected individuals.

Use of Recombinant Antigens for Sensitive Serodiagnosis of American Tegumentary Leishmaniasis Caused by Different Leishmania Species.

American tegumentary leishmaniasis (ATL) (also known as cutaneous leishmaniasis [CL]) is caused by various species of protozoa of the genus Leishmania The diagnosis is achieved on a clinical, epidemiological, and pathological basis, supported by positive parasitological exams and demonstration of leishmanin delayed-type hypersensitivity. Serological assays are not routinely used in the diagnosis because many are considered to have low sensitivity and the particular Leishmania species causing the disease can lead to variable performance. In the present study, we generated recombinant versions of two highly conserved Leishmania proteins, Leishmania (Viannia) braziliensis-derived Lb8E and Lb6H, and evaluated both in enzyme-linked immunosorbent assays (ELISA). Recombinant Lb6H (rLb6H) had better performance and reacted with 100.0% of the ATL and 89.4% of the VL samples. These reactions with rLb6H were highly specific (98.5%) when compared against those for samples from healthy control individuals. We then assessed rLb6H against sera from ATL patients infected with different species of Leishmania prevalent in Brazil [Leishmania (Leishmania) amazonensis, L (Viannia) braziliensis, and L (V) guyanensis] and samples from patients with other infectious diseases. In analyses of 500 sera, ELISA using rLb6H detected all 219 ATL samples (sensitivity of 100.0%) with an overall specificity of 93.9% (considering healthy individuals and other infectious diseases patients). Only a minority of samples from Chagas disease patients possessed antibodies against rLb6H, and all of these responses were low (with a highest reactivity index of 2.2). Taken together, our data support further evaluation of rLb6H and the potential for its routine use in the serological diagnosis of ATL.

Polymorphisms in the TOLLIP Gene Influence Susceptibility to Cutaneous Leishmaniasis Caused by Leishmania guyanensis in the Amazonas State of Brazil.

INTRODUCTION: The clinical outcome to Leishmania-infection is determined by the individual adaptive immune T helper cell responses and their interactions with parasitized host cells. An early development of a proinflammatory immune response (Th1 response) is necessary for Leishmania-infection resolution. The Toll-interacting protein (TOLLIP) regulates human Toll-like receptors signaling pathways by down regulating the proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) and inducing the ant-inflammatory cytokine interleukin-10 (IL-10). Polymorphisms in the TOLLIP gene are associated with infectious diseases. MATERIAL AND METHODS: The polymorphisms rs5743899 and rs3750920 in the TOLLIP gene were genotyped by polymerase chain reaction and restriction fragment length polymorphism (RFLP) analysis in 631 patients with cutaneous leishmaniasis (CL) caused by L. guyanensis and 530 individuals with no history of leishmaniasis. RESULTS: The G and T alleles of the rs5743899 and rs3750920 were more common in patients with CL than in healthy individuals (P = 2.6 x10(-8) ; odds ratio [OR], 1.7 [ 95% confidence interval (CI) 1.4-2.0] and P = 1.9 x10(-8) ; OR, 1.6 [95% CI 1.4-1.9] respectively). The r2 and D' linkage disequilibrium between the two polymorphisms are 0.05 and 0.473 with a confidence bounds of 0.37 to 0.57 respectively. CONCLUSION: The two polymorphisms are independently associated with an increased risk of developing CL.