Highlights d Visceral leishmaniasis patients co-infected with HIV suffer from frequent VL relapse d VL relapse is associated with low CD4 + T cell counts and low production of IFN-g d VL relapse is associated with increased PD1 expression on CD4 + and CD8 + T cells d These three markers can predict whether and when these patients relapse

One of the key immunological characteristics of active visceral leishmaniasis (VL) is a profound immunosuppression and impaired production of Interferon-γ (IFN-γ). However, recent studies from Bihar in India showed using a whole blood assay, that whole blood cells have maintained the capacity to produce IFN-γ. Here we tested the hypothesis that a population of low-density granulocytes (LDG) might contribute to T cell responses hyporesponsiveness via the release of arginase. Our results show that this population is affected by the anticoagulant used to collect blood: the frequency of LDGs is significantly lower when the blood is collected with heparin as compared to EDTA; however, the anticoagulant does not impact on the levels of arginase released. Next, we assessed the capacity of whole blood cells from patients with active VL to produce IFN-γ and IL-10 in response to antigen-specific and polyclonal activation. Our results show that whole blood cells produce low or levels below detection limit of IFN-γ and IL-10, however, after successful treatment of VL patients, these cells gradually regain their capacity to produce IFN-γ, but not IL-10, in response to activation. These results suggest that in contrast to VL patients from Bihar, India, whole blood cells from VL patients from Gondar, Ethiopia, have lost their ability to produce IFN-γ during active VL and that active disease is not associated with sustained levels of IL-10 production following stimulation.

Visceral leishmaniasis (VL) has emerged as a clinically important opportunistic infection in HIV patients, as VL/HIV co-infected patients suffer from frequent VL relapse. Here, we followed cohorts of VL patients with or without HIV co-infections in Ethiopia and collected detailed clinical and immunological data during 12 months of follow-up. By the end of the study 78.1% of VL/HIV patients, but none of the VL only patients, had relapsed. Despite clinically defined cure, VL/HIV patients maintained high parasite loads, low BMI, hepatosplenomegaly and pancytopenia throughout follow-up. During detailed immunological study throughout the follow-up period, we identified three markers associated with VL relapse: i) failure to restore antigen-specific production of IFNγ, ii) persistently low CD4+ T cell counts, and iii) high expression of PD1 on CD4+ T cells. We show that these three markers combine well in predicting VL relapse, and that all three measurements are needed for optimal predictive power. These three immunological markers can be measured in primary hospital settings in Ethiopia and can predict VL relapse after anti-leishmanial therapy. The use of our prediction model has the potential to improve disease management and patient care.

Immunologically, active visceral leishmaniasis (VL) is characterized by profound immunosuppression, severe systemic inflammatory responses, and an impaired capacity to control parasite replication. Neutrophils are highly versatile cells, which play a crucial role in the induction as well as the resolution of inflammation, the control of pathogen replication, and the regulation of immune responses. Neutrophil functions have been investigated in human cutaneous leishmaniasis; however, their role in human VL is poorly understood. In the present study we evaluated the activation status and effector functions of neutrophils in patients with active VL and after successful anti-leishmanial treatment. Our results show that neutrophils are highly activated and have degranulated; high levels of arginase, myeloperoxidase, and elastase, all contained in neutrophils’ granules, were found in the plasma of VL patients. In addition, we show that a large proportion of these cells are immature. We also analyzed effector functions of neutrophils that are essential for pathogen clearance and show that neutrophils have an impaired capacity to release neutrophil extracellular traps, produce reactive oxygen species, and phagocytose bacterial particles, but not Leishmania parasites. Our results suggest that impaired effector functions, increased activation, and immaturity of neutrophils play a key role in the pathogenesis of VL.

Malnutrition is commonly associated with increased infectious disease susceptibility and severity. Whereas malnutrition might enhance the incidence of disease as well as its severity, active infection can in turn exacerbate malnutrition. Therefore, in a malnourished individual suffering from a severe infection, it is not possible to determine the contribution of the pre-existing malnutrition and/or the infection itself to increased disease severity. In the current study we focussed on two groups of malnourished, but otherwise healthy individuals: moderately malnourished (BMI: 18.4–16.5) and severely malnourished (BMI <16.5) and compared several immune parameters with those of individuals with a normal BMI (≥18.5). Our results show a similar haematological profile in all three groups, as well as a similar ratio of CD4+ and CD8+ T cells. We found significant correlations between low BMI and increased levels of T helper (Th) 1 (Interferon (IFN)-γ, (interleukin (IL)-2, IL-12), Th2 (IL-4, IL-5, IL-13), as well as IL-10, IL-33 and tumor necrosis factor-α, but not IL-8 or C reactive protein. The activities of arginase, an enzyme associated with immunosuppression, were similar in plasma, peripheral blood mononuclear cells (PBMC) and neutrophils from all groups and no differences in the expression levels of CD3ζ, a marker of T cell activation, were observed in CD4+ and CD8+T cells. Furthermore, whereas the capacity of neutrophils from the malnourished groups to phagocytose particles was not impaired, their capacity to produce reactive oxygen species was impaired. Finally we evaluated the frequency of a subpopulation of low-density neutrophils and show that they are significantly increased in the malnourished individuals. These differences were more pronounced in the severely malnourished group. In summary, our results show that even in the absence of apparent infections, healthy malnourished individuals display dysfunctional immune responses that might contribute to increased susceptibility and severity to infectious diseases.