Evidence That Lipopolisaccharide May Contribute to the Cytokine Storm and Cellular Activation in Patients with Visceral Leishmaniasis

Abstract

Background

Visceral leishmaniasis (VL) is characterized by parasite-specific immunosuppression besides an intense pro-inflammatory response. Lipopolisaccharide (LPS) has been implicated in the immune activation of T-cell deficient diseases such as HIV/AIDS and idiopathic lymphocytopenia. The source of LPS is gram-negative bacteria that enter the circulation because of immunological mucosal barrier breakdown. As gut parasitization also occurs in VL, it was hypothesized that LPS may be elevated in leishmaniasis, contributing to cell activation.

Methodology/Principal Findings

Flow cytometry analysis and immunoassays (ELISA and luminex micro-beads system) were used to quantify T-cells and soluble factors. Higher LPS and soluble CD14 levels were observed in active VL in comparison to healthy subjects, indicating that LPS was bioactive; there was a positive correlation between these molecules (r = 0.61; p<0.05). Interestingly, LPS was negatively correlated with CD4 ⁺ (r = −0.71; p<0.01) and CD8 ⁺ T-cells (r = −0.65; p<0.05). Moreover, higher levels of activation-associated molecules (HLA-DR, CD38, CD25) were seen on T lymphocytes, which were positively associated with LPS levels. Pro-inflammatory cytokines and macrophage migration inhibitory factor (MIF) were also augmented in VL patients. Consistent with the higher immune activation status, LPS levels were positively correlated with the inflammatory cytokines IL-6 (r = 0.63; p<0.05), IL-8 (r = 0.89; p<0.05), and MIF (r = 0.64; p<0.05). Also, higher plasma intestinal fatty acid binding protein (IFABP) levels were observed in VL patients, which correlated with LPS levels (r = 0.57; p<0.05).

Conclusions/Significance

Elevated levels of LPS in VL, in correlation with T-cell activation and elevated pro-inflammatory cytokines and MIF indicate that this bacterial product may contribute to the impairment in immune effector function. The cytokine storm and chronic immune hyperactivation status may contribute to the observed T-cell depletion. LPS probably originates from microbial translocation as suggested by IFABP levels and, along with Leishmania antigen-mediated immune suppression, may play a role in the immunopathogenesis of VL. These findings point to possible benefits of antimicrobial prophylaxis in conjunction with anti- Leishmania therapy.

Author Summary

Visceral leishmaniasis (VL) affects organs rich in lymphocytes, being characterized by intense Leishmania-induced T-cell depletion and reduction in other hematopoietic cells. In other infectious and non-infectious diseases in which the immune system is affected, such as HIV-AIDS and inflammatory bowel disease, damage to gut-associated lymphocyte tissues occurs, enabling luminal bacteria to enter into the circulation. Lipopolisaccharide (LPS) is a bacterial product that stimulates macrophages, leading to the production of pro-inflammatory cytokines and other soluble factors such as MIF, which in turn activate lymphocytes. Continuous and exaggerated stimulation causes exhaustion of the T-cell compartment, contributing to immunosuppression.

Herein, we show that an increment in LPS plasma levels also occurs in VL; the higher the LPS levels, the lower the TCD4 ⁺ and TCD8 ⁺ cell count in the blood. This T-cell depletion may affect the mucosal immune system, which, along with intestinal parasitization by amastigotes, may contribute to gut barrier damage and consequent microbial translocation. LPS levels were correlated with T-cell activation, pro-inflammatory cytokine plasma levels, MIF, and IFABP, showing that a bacterial molecule, probably from luminal origin, not associated with Leishmania infection can negatively affect the immune system. These findings points to possible benefits of antimicrobial prophylaxis in conjunction with anti- Leishmania therapy.