A secondary wave of neutrophil infiltration causes necrosis and ulceration in lesions of experimental American cutaneous leishmaniasis

<div><p>We evaluated the importance of neutrophils in the development of chronic lesions caused by <i>L</i>. <i>Viannia spp</i>. using the hamster as experimental model of American Cutaneous Leishmaniasis (ACL). Neutrophils infiltrated the lesion within the first six hours post-infection. Inhibition of this early infiltration using a polyclonal antibody or cyclophosphamide was associated with transient parasite control but the protective effect vanished when lesions became clinically apparent. At lesion onset (approximately 10 days p.i.), there was an increased proportion of both uninfected and infected macrophages, and subsequently a second wave of neutrophils infiltrated the lesion (after 19 days p.i.) This second neutrophil infiltration was associated with lesion necrosis and ulceration (R² = 0.75) and maximum parasite burden. Intradermal delivery of N-formylmethionyl-leucyl-phenylalanine (fMLP), aimed to increase neutrophil infiltration, resulted in larger lesions with marked necrosis and higher parasite burden than in mock treated groups (p<0.001 each). In contrast, reduced neutrophil infiltration via cyclophosphamide-mediated depletion led to more benign lesions and lower parasite loads compared to controls (p<0.001 each). Neutrophils of the second wave expressed significantly lower GM-CSF, reactive oxygen species and nitric oxide than those of the first wave, suggesting that they had less efficient anti-leishmania activity. However, there was increased inflammatory cytokines and expression of neutrophil proteases (myeloperoxidase, cathepsin G and elastase) in lesions during the second wave of neutrophil infiltration compared with the levels reached during the first wave (6h p.i.). This suggests that augmented neutrophil proteases and inflammatory cytokines during the secondary wave of neutrophils could contribute to skin inflammation, ulceration and necrosis in ACL. The overall results indicate that neutrophils were unable to clear the infection in this model, and that the second wave of neutrophils played an important role in the severity of ACL.</p></div

Impact of early neutrophil infiltration in parasite killing and lesion development in hamsters infected with <i>L</i>. <i>V</i>. <i>panamensis</i>.

<p>(A), Parasite burden in the cutaneous lesion of hamsters treated to inhibit neutrophil recruitment with either a polyclonal anti-serum (Ab) (6h before infection) or 200mg/kg cyclophosphamide (cycloph.) (24h before infection) or treated to increase neutrophil recruitment with 25 μg fMLP (6h before infection), evaluated at 6h post-infection; (B), Parasite burden in lesion of hamsters treated to inhibit neutrophil with cyclophosphamide or treated to recruit neutrophils with fMLP, evaluated at 19 days p.i. Determined in skin sections stained with H&E at 100X magnification. Number of cells = proportion of cells x total number of cells / 100 (determined in a standard area of 0.21mm² at 100X magnification). (C-D), Lesion size and severity of the skin ulcer and necrosis followed during 19 days of infection. *p<0.05; **p<0.01, with reference to control group, n = 8 hamsters per group.</p

Protease expression was enhanced with the infection and during the second wave of neutrophil infiltration.

<p>(A-C), Expression of myeloperoxidase, cathepsin G, matrix metallopeptidase 8 (MMP8), proteinase-3, neutrophil elastase and matrix metallopeptidase 9 (MMP9) determined in skin from uninfected hamsters (Un), or in the infection site of hamsters infected with <i>L</i>. <i>V</i>. <i>panamensis</i>. Lesions obtained during the first wave of neutrophil infiltration (1^st wave, 6h p.i.) or during the second wave of neutrophil infiltration (2^nd wave, 28d p.i.). Determined by qPCR.*p<0.01; **p<0.001; ***p<0.001, n = 5–7 lesions per group. One-way Anova, Kruskal-Wallis statistic.</p

Impact of late neutrophil infiltration on lesion severity and parasite burden in hamsters infected with <i>L</i>. <i>V</i>. <i>panamensis</i>.

<p>(A-C), Modulation of the second wave of neutrophils from 16–22 days of infection: showing lesion size (A), severity of skin necrosis and ulcer (B), and parasite burden at 22d p.i. (C) in lesions of hamsters treated to recruit neutrophils with fMLP or treated to inhibit neutrophils with cyclophosphamide; D-F, Modulation of the second wave of neutrophils from 16–45 days of infection: showing lesion size (D), severity of skin necrosis and ulceration (E), and parasite burden at 45 days of infection (F); G-I, Modulation of first and second wave of neutrophil infiltration up to 24 days post-infection showing lesion size (G), severity of skin necrosis and ulcer (H), and parasite burden at 24 days of infection (I). Parasite burden was evaluated in skin sections stained with H&E at 100X magnification. Number of cells = proportion of cells x total number of cells / 100 (determined in a standard area of 0.21mm² at 100X magnification). * p<0.05, **p<0.001, comparison against control group, n = 8 hamsters per group; (J), Representative snout lesions at 22 days p.i.: (i) Control group (PBS), (ii) Increased neutrophil recruitment (fMLP), (iii) Decreased neutrophil recruitment (Cyclophosphamide).</p

Neutrophils are associated with severity of cutaneous lesions in hamsters infected with <i>L</i>. <i>V</i>. <i>panamensis</i>.

<p>(A), Number of neutrophils or macrophages infiltrating the lesion (primary Y-axis) and lesion size (secondary Y-axis) post-infection; (B), Neutrophils during the development of cutaneous lesions (first wave, 2–6 hours after infection; second wave, after 19 days of infection); (C), Neutrophil infiltration and clinical severity of ulcer and necrosis from 0 to 45 days post-infection; (D), Intensity of neutrophil infiltrate and severity of ulcer and necrosis; Determined in a standard area of 0.21mm² in two different samples per time point; (E), Representative histopathology of tissue sections (100X) stained with H&E obtained from snout of hamsters: (i) Naïve tissue without inflammatory infiltrate, (ii) Skin tissue at 6 h p.i. showing presence of neutrophils in reticular dermis (ret) and hypodermis (hyp) (black arrows) and typical tri-lobulated nuclei of neutrophils (inset, 1000X magnification), (iii) Cutaneous lesions at 21 days p.i. showing dense neutrophil infiltrate involving the hypodermis and papillary dermis (pap) (black arrows), (iv) Cutaneous ulcer at 26 days p.i., showing disruption of epidermis (white arrows), disseminated necrosis across the dermis and hypodermis (black circles), with abundant exudative material (grey arrows).</p