Characterization of Neutrophil Function in Human Cutaneous Leishmaniasis Caused by <i>Leishmania braziliensis</i>

<div><p>Infection with different <i>Leishmania</i> spp. protozoa can lead to a variety of clinical syndromes associated in many cases with inflammatory responses in the skin. Although macrophages harbor the majority of parasites throughout chronic infection, neutrophils are the first inflammatory cells to migrate to the site of infection. Whether neutrophils promote parasite clearance or exacerbate disease in murine models varies depending on the susceptible or resistant status of the host. Based on the hypothesis that neutrophils contribute to a systemic inflammatory state in humans with symptomatic <i>L</i>. <i>braziliensis</i> infection, we evaluated the phenotype of neutrophils from patients with cutaneous leishmaniasis (CL) during the course of <i>L</i>. <i>braziliensis</i> infection. After <i>in vitro</i> infection with <i>L</i>. <i>braziliensis</i>, CL patient neutrophils produced more reactive oxygen species (ROS) and higher levels of CXCL8 and CXCL9, chemokines associated with recruitment of neutrophils and Th1-type cells, than neutrophils from control healthy subjects (HS). Despite this, CL patient and HS neutrophils were equally capable of phagocytosis of <i>L</i>. <i>braziliensis</i>. There was no difference between the degree of activation of neutrophils from CL versus healthy subjects, assessed by CD66b and CD62L expression using flow cytometry. Of interest, these studies revealed that both parasite-infected and bystander neutrophils became activated during incubation with <i>L</i>. <i>braziliensis</i>. The enhanced ROS and chemokine production in neutrophils from CL patients reverted to baseline after treatment of disease. These data suggest that the circulating neutrophils during CL are not necessarily more microbicidal, but they have a more pro-inflammatory profile after parasite restimulation than neutrophils from healthy subjects.</p></div

Chemokine production by neutrophils from patients with CL before or after treatment.

<p>Neutrophils from CL patients (n = 11) were obtained before or after therapeutic cure, and incubated with or without 5:1 <i>L</i>. braziliensis for 180 minutes. The release of of CXCL9 (A) or CXCL8 (B) into supernatants was evaluated by ELISA. Data points show the median of triplicate samples for each subject/condition. Statistical analyses were performed using Wilcoxon test (*p<0.05).</p

Uptake of <i>L</i>. <i>braziliensis</i> by neutrophils (PMN) from CL patients or healthy subjects (HS).

<p>Neutrophils from CL patients (n = 13) or healthy subjects (n = 7) were incubated with stationary phase <i>L</i>. <i>braziliensis</i> at a 5:1 parasite:neutrophil ratio, under conditions that allow phagocytosis. After 10, 90 or 180 minutes of incubation at 37°C, 5% CO₂, cytocentrifuge slides were prepared and stained with Giemsa. The percentage of infected cells (A) and the number of intracellular <i>L</i>. <i>braziliensis</i> (B) per 100 neutrophils were determined microscopically. Each symbol represents the mean value of neutrophils from a different patient and lines represent the median of the group. Statistical analyses were performed using the Mann-Whitney test (*p<0.05, **p<0.01).</p

Effect of <i>L</i>. <i>braziliensis</i> infection on expression of neutrophil activation markers CD62L and CD66b.

<p>Panel (A) shows a histogram demonstrating staining in neutrophils infected with CFSE-stained <i>L</i>. <i>braziliensis</i> versus uninfected neutrophils from a CL patient. Panel (B) shows a representative scatter plot indicating the purity of PMN population based CD16+ expression. Panel (C) shows collated results of surface staining for activation markers CD62L and CD66b by flow cytometry. Each value represents the MFI of one subjects’ neutrophils. “Inf” and “Bystand” represent the staining on neutrophils that were either infected (CFSE+) with CFSE- labeled <i>L</i>. <i>braziliensis</i>, or uninfected bystander neutrophils (CFSE-). The two left graphs represent data from subjects with CL; the two graphs on the right show data from healthy control subjects. Statistical analysis was performed using the Wilcoxon test, comparing stimulated to unstimulated cells (*p<0.05, **p<0.01, ***p<0.001).</p

Expression of neutrophil surface markers CD62L and CD66b from CL patients before or after therapeutic cure.

<p>Neutrophils from CL patients (n = 11) were isolated before or after CL treatment, and incubated with CFSE stained <i>L</i>. <i>braziliensis</i> at a 5:1 ratio. After 90 minutes cells were stained for flow cytometry and the expression of CD62L (A) and CD66b (B) was assessed by flow cytometry. Points on graphs represent the median MFI for neutrophils from each subject, incubated in medium alone or with CFSE labeled <i>L</i>. <i>braziliensis</i>. Statistical analyses utilized Wilcoxon test (**p<0.01, ***p<0.001).</p

Release of reactive oxidants by neutrophils from subjects with CL or healthy control subjects (HS) induced by phagocytosis of <i>L</i>. <i>braziliensis</i>.

<p>(A) Representative histograms showing DHR-123 fluorescence due to reactive oxidants in unexposed neutrophils from a healthy subject, or the same neutrophils exposed to either <i>L</i>. <i>braziliensis</i> or PMA. (B) Graphical presentation of the MFI of DHR-123 staining in neutrophils from subjects with CL (n = 13) or healthy control subjects (n = 9), incubated with medium, <i>L</i>. <i>braziliensis</i> or PMA. Horizontal lines indicate the mean MFI of all subjects. Statistical analyses were performed using the Mann-Whitney test to evaluate differences between the groups, and the Wilcoxon test to compare results of different conditions in the same subject. (**p<0.01, ***p<0.001).</p

Neutrophil reactive oxidant production from subjects with CL before or after treatment.

<p>Neutrophils from CL patients (n = 11) were obtained before and after successful treatment of the disease, in which there was local resolution of the lesion. Cells were incubated in dihydrorhodamine 123 (DHR-123), and incubated with medium alone, 5:1 <i>L</i>. <i>braziliensis</i>, or PMA for ten minutes. The concentration of reactive oxidant species in neutrophils was assessed by flow cytometry. Data represent the median MFI samples from each patient. Statistical analyses were performed using the Wilcoxon test. (**p<0.01, ***p<0.001).</p

Chemokines produced by neutrophils from CL patients or healthy controls.

<p>Neutrophils from CL patients (n = 13) or healthy controls (n = 9) were incubated with <i>L</i>. <i>braziliensis</i> at a 5:1 ratio for 180 minutes. The concentrations of the indicated chemokines in the supernatants were evaluated by ELISA. Each symbol represents mean values from a different patient, and lines represent the median of each group. The Mann-Whitney test was used to compare differences between CL <i>versus</i> Healthy Control groups (*p<0.05, ***p<0.001).</p

Correlation between IFN-γ and CXCL10 production to soluble <i>Leishmania</i> antigen.

<p>The data was obtained from 54 household contacts (HC) with evidence of immune response and 51 household contacts without evidence of immune response which were selected among individuals living in the same households as HC with evidence of immune response. Values of IFN-γ (pg/ml) and CXCL10 (pg/ml) from 105 HC were plotted in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001947#pntd-0001947-g003" target="_blank">Figure 3</a> and these data were analyzed by the Spearman correlation test. P<0.0001, r = 0,74.</p

Establishment of a cohort of HC of CL patients in <i>L. braziliensis</i> endemic area.

<p>Establishment of a cohort of HC of CL patients in <i>L. braziliensis</i> endemic area.</p