Major Neutrophilia Observed in Acute Phase of Human Leptospirosis Is Not Associated with Increased Expression of Granulocyte Cell Activation Markers

Abstract

It has long been known that pathogenic Leptospira can mobilize the immune system but the specific contribution of neutrophils to control the infectious challenge remains to be clarified. We herein analyzed the phenotype of circulating neutrophils of patients with leptospirosis and healthy controls for the expression of toll-like receptor (TLR) type 2 (TLR2, to sense the leptospiral LPS) and several activation markers: interleukin 8 chemokine receptor CD182 (CXCR2), CD11b of the integrin/opsonin complement receptor type 3 (CR3) and CD15 (ligand of the selectin). The plasmatic level of the main CD182 ligand, interleukin 8 (CXCL8), was measured by ELISA. Hospitalized leptospirosis cases showed marked neutrophilia, particularly in the most severe cases. Interestingly, TLR2 was significantly increased in leptospirosis but identical levels of CD182 and CD11b were detected when compared to controls. CD15 was significantly decreased on neutrophils in leptospirosis but returned to normal within 1 month. Basal levels of IL-8 were measured in control subjects and were not increased in leptospirosis cases at the initial stage of the disease. In conclusion, we observed that neutrophils failed to regulate the expression of several of the receptors involved in cell activation and recruitment. This study further emphasizes the paradigm that neutrophils may be impaired in their overall capacity to thwart bacterial infection in leptospirosis patients.

Fig 1. Bacterial burden is higher in severe leptospirosis.

Plasma bacterial load is established from the plasma PCR values (n = 12) and using the log-transformed standard curve as described [27]. Horizontal bars indicate the median. Comparison with non-parametric Mann-Whitney test. * indicates P-value inferior to 0.05.

Fig 2. Neutrophil levels at admittance and during the disease course of leptospirosis patients.

(A) Counts of immune cells for healthy controls (circles) and leptospirosis patients (squares) at admittance (M0). (B) The maximal value of PMN count reached during hospital stay was compared between mild and severe forms. For A and B, the largest horizontal bars indicate the median value, upper and lower bars the interquartile ranges. Comparisons with non-parametric Mann-Whitney test. *, *** indicate P-value inferior to 0.05 and 0.0001 respectively. (C) Among the patients with leptospirosis, the maximal value of PMN was correlated to the maximal value of creatinine with Spearman test.

Fig 3. Expression of cell surface molecules implicated in PMN recruitment and activation during leptospirosis.

(A) Expression levels of receptors and ligands on circulating PMN at admittance, assessed by flow cytometry. The mean fluorescence intensity (MFI) values were evaluated while isotypic negative controls were set with an MFI of 10⁻¹. One value is missing for CD15. (B) Evolution of CD15 expression among 7 leptospirosis patients and comparison between admittance (M0) and at 1-month (M1) post infection. Largest horizontal bars indicate the median value, upper and lower bars the interquartile ranges. Comparison of the patient group and healthy donor group with non-parametric Mann-Whitney test, and comparison within patients between M0 and M1 with non-parametric Wilcoxon paired test. *, **, *** indicate P-value inferior to 0.05, 0.001 and 0.0001 respectively.

Fig 4. Circulating CXCL8 (interleukin 8) levels are not significantly elevated in leptospirosis cases.

CXCL8 levels were assessed by ELISA of plasma from healthy control individuals (circles, n = 13) and leptospirosis cases (squares, n = 15). For leptospirosis cases, assessment was performed with sampling at hospital admittance corresponding to acute phase (M0), with PCR positive patients. Comparison between two groups with non-parametric Mann-Whitney U-test showed no significant difference.