Transcription of toll-like receptors 2, 3, 4 and 9, FoxP3 and Th17 cytokines in a susceptible experimental model of canine Leishmania infantum infection

Canine leishmaniosis (CanL) due to Leishmania infantum is a chronic zoonotic systemic disease resulting from complex interactions between protozoa and the canine immune system. Toll-like receptors (TLRs) are essential components of the innate immune system and facilitate the early detection of many infections. However, the role of TLRs in CanL remains unknown and information describing TLR transcription during infection is extremely scarce. The aim of this research project was to investigate the impact of L. infantum infection on canine TLR transcription using a susceptible model. The objectives of this study were to evaluate transcription of TLRs 2, 3, 4 and 9 by means of quantitative reverse transcription polymerase chain reaction (qRT-PCR) in skin, spleen, lymph node and liver in the presence or absence of experimental L. infantum infection in Beagle dogs. These findings were compared with clinical and serological data, parasite densities in infected tissues and transcription of IL-17, IL-22 and FoxP3 in different tissues in non-infected dogs (n = 10), and at six months (n = 24) and 15 months (n = 7) post infection. Results revealed significant down regulation of transcription with disease progression in lymph node samples for TLR3, TLR4, TLR9, IL-17, IL-22 and FoxP3. In spleen samples, significant down regulation of transcription was seen in TLR4 and IL-22 when both infected groups were compared with controls. In liver samples, down regulation of transcription was evident with disease progression for IL-22. In the skin, upregulation was seen only for TLR9 and FoxP3 in the early stages of infection. Subtle changes or down regulation in TLR transcription, Th17 cytokines and FoxP3 are indicative of the silent establishment of infection that Leishmania is renowned for. These observations provide new insights about TLR transcription, Th17 cytokines and Foxp3 in the liver, spleen, lymph node and skin in CanL and highlight possible markers of disease susceptibility in this model

Balance of IL-10 and Interferon-γ plasma levels in human visceral leishmaniasis: Implications in the pathogenesis

BACKGROUND: Leishmaniasis remains a serious public health problem in several parts of the developing world. Effective prophylactic measurements are hampered by imprecise comprehension of different aspects of the disease, including its immunoregulation. A better comprehension of immunoregulation in human VL may be useful both for designing and evaluating immunoprophylaxis. METHODS: To explore immunoregulatory mechanisms, 20 visceral leishmaniasis (VL) patients were evaluated during active disease and at different periods up to one year after treatment determining their plasma cytokine levels, clinical parameters (palpable spleen and liver) and antibody levels. RESULTS: Elevated plasma levels of IFN-γ and of IL-12 p40 were observed during active disease, significantly decreasing after treatment whereas in vitro Leishmania antigen-stimulated IFN-γ production by PBMC exhibited an inverse pattern being low during disease and increasing steadily thereafter. Absence of IFN-γ activity is a hallmark of VL. The main candidate for blunting IFN-γ activity is IL-10, a cytokine highly elevated in plasma with sharp decrease after treatment. Activity of IL-10 is inferred by high levels of anti-Leishmania specific IgG1 and IgG3. TGF-β had elevated total, but not of active, levels lessening the likelihood of being the IFN-γ counterpart. Spleen or liver size presented a steady decrease but return to normal values at only 120 days after treatment. Anti-Leishmania IgG (total and subclasses) levels and DTH or Leishmania-stimulated lymphocyte proliferation conversion to positive also present a slow decrease after treatment. IL-6 plasma levels were elevated in only a few patients. CONCLUSION: Taken together our results suggest that IFN-γ and IL-10 are the molecules most likely involved in determining fate of disease. After treatment, there is a long delay before the immune profile returns to normal what precludes using plasma cytokine levels as criteria of cure as simpler clinical evaluations, as a palpable spleen or liver, can be used

IgG1 as a potential biomarker of post-chemotherapeutic relapse in visceral leishmaniasis, and adaptation to a rapid diagnostic test.

BACKGROUND: Visceral leishmaniasis (VL), caused by protozoa of the Leishmania donovani complex, is a widespread parasitic disease of great public health importance; without effective chemotherapy symptomatic VL is usually fatal. Distinction of asymptomatic carriage from progressive disease and the prediction of relapse following treatment are hampered by the lack of prognostic biomarkers for use at point of care. METHODOLOGY/PRINCIPAL FINDINGS: All IgG subclass and IgG isotype antibody levels were determined using unpaired serum samples from Indian and Sudanese patients with differing clinical status of VL, which included pre-treatment active VL, post-treatment cured, post-treatment relapsed, and post kala-azar dermal leishmaniasis (PKDL), as well as seropositive (DAT and/or rK39) endemic healthy controls (EHCs) and seronegative EHCs. L. donovani antigen-specific IgG1 levels were significantly elevated in relapsed versus cured VL patients (p<0.0001). Using paired Indian VL sera, consistent with the known IgG1 half-life, IgG1 levels had not decreased significantly at day 30 after the start of treatment (p = 0.8304), but were dramatically decreased by 6 months compared to day 0 (p = 0.0032) or day 15 (p<0.0001) after start of treatment. Similarly, Sudanese sera taken soon after treatment did not show a significant change in the IgG1 levels (p = 0.3939). Two prototype lateral flow immunochromatographic rapid diagnostic tests (RDTs) were developed to detect IgG1 levels following VL treatment: more than 80% of the relapsed VL patients were IgG1 positive; at least 80% of the cured VL patients were IgG1 negative (p<0.0001). CONCLUSIONS/SIGNIFICANCE: Six months after treatment of active VL, elevated levels of specific IgG1 were associated with treatment failure and relapse, whereas no IgG1 or low levels were detected in cured VL patients. A lateral flow RDT was successfully developed to detect anti-Leishmania IgG1 as a potential biomarker of post-chemotherapeutic relapse