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

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

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

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

Demographic and epidemiologic features of patients with cutaneous leishmaniasis and household contacts with and without evidence of immune response to leishmania antigen.

<p>Values with identical superscripts are significantly different. Statistical tests and p-values are below.</p><p>P-values for pair-wise Bonferroni post-hoc tests:</p>a<p> = 0.002,</p>b<p> = 0.004,</p>c<p> = 0.005,</p>d<p> = 0.003,</p>e<p> = 0.009,</p>f<p><0.001.</p>*<p>one-way ANOVA test.</p>**<p>Pearson's chi-square test.</p>***<p>before 4:00 pm.</p

Gene categories modulated by SGS pre-immunization.

<p>BALB/c mice were inoculated 3 times in the right ear pinna every 2 wks with SGS from 1 pair of <i>Lu. intermedia</i> salivary glands and then challenged 2 wks later with <i>Lu. intermedia</i> SGS plus 1×10⁶<i>L. braziliensis</i> parasites. (A) Lesion development was monitored weekly. Each point is the mean ±SEM of 5 animals per group. (B) Lesion images of ear pinna at 8 wks p.i. and these data are representative of two independent experiments. (C) BALB/c mice were pre-immunized (Imm) 3 times in the right ear every 2 wks with <i>Lu. intermedia</i> SGS or PBS and then challenged (Chl) in the left ear 2 wks later with <i>Lu. intermedia</i> SGS. The challenged left ears were collected after 2 wks, homogenized and gene expression was determined by microarray analysis. (D) Global significant differences in gene expression shown in log₂ were determined comparing mice pre-immunized with SGS to those given PBS (n = 3 mice per group) and hierarchical clustering revealed genes differentially expressed >1.5× with a p value of <0.05 are presented in a heat map (E) and separated based on functional categories in a pie chart (F).</p

IFN-inducible genes are upregulated in response to SGS pre-immunization.

<p>BALB/c mice were inoculated 3 times in the right ear every 2 wks with <i>Lu. intermedia</i> SGS or PBS and then challenged in the left ear 2 wks later with <i>Lu. intermedia</i> SGS. The left ears were collected 2 wks after SGS challenge, homogenized and the expression of IFN-inducible genes such as (A) Ifit1, Irgm1 and Irgm2, and the expression of IFN-related genes like (B) Stat1 and CXCL9 was determined by real-time quantitative PCR normalized relative to HPRT mRNA levels. Similar analysis of ear pinna of naïve mice that did not receive any injections and were not challenged with SGS is also shown. Data are an independent biological replicate to the microarray analysis; relative mRNA expression normalized to the housekeeping gene HPRT is presented as the mean +SEM with 3–5 mice per group; ** p<0.005, * p<0.05 by Student's <i>t</i>-test.</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