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The c-MET receptor tyrosine kinase contributes to neutrophil-driven pathology in cutaneous leishmaniasis

['Katiuska Passelli', 'Department Of Biochemistry', 'Faculty Of Biology', 'Medicine', 'University Of Lausanne', 'Epalinges', 'World Health Organization Collaborating Centre For Immunology Research', 'Training', 'Borja Prat-Luri', 'Margot Merlot']

Date: 2022-02 

Neutrophils are the first line of defence against invading pathogens. Although neutrophils are well-known professional killers, some pathogens including Leishmania (L.) parasites survive in neutrophils, using these cells to establish infection. Manipulation of neutrophil recruitment to the infection site is therefore of interest in this cutaneous disease. The c-MET tyrosine kinase receptor was shown to promote neutrophil migration to inflamed sites. Here, we investigated the importance of c-MET expression on neutrophils in their recruitment to the infection site and the role of c-Met expression in the pathology of leishmaniasis. Following infection with L. mexicana, mice with conditional deletion of c-MET in neutrophils controlled significantly better their lesion development and parasite burden compared to similarly infected wild type mice. Our data reveal a specific role for c-MET activation in Leishmania-induced neutrophil infiltration, a process correlating with their negative role in the pathology of the diseases. We further show that c-MET phosphorylation is observed in established cutaneous lesions. Exposure to L. mexicana upregulated c-Met expression predominantly in infected neutrophils and c-Met expression influenced ROS release by neutrophils. In addition, pharmacological inhibition of c-MET, administrated once the lesion is established, induced a significant decrease in lesion size associated with diminished infiltration of neutrophils. Both genetic ablation of c-MET in neutrophils and systemic inhibition of c-MET locally resulted in higher levels of CD4 + T cells producing IFNγ, suggesting a crosstalk between neutrophils and these cells. Collectively, our data show that c-MET activation in neutrophils contributes to their recruitment following infection, and that L. mexicana induction of c-MET on neutrophils impacts the local pathology associated with this disease. Our results suggest a potential use for this inhibitor in the control of the cutaneous lesion during this parasitic infection.

The leishmaniases are a complex of diseases caused by Leishmania protozoan parasites. There exist different forms of the disease, ranging from cutaneous lesions to visceral disease, the latter being fatal if not treated. Cutaneous leishmaniasis is the most common form with one million new cases occurring annually. Increasing evidence points to an important role for neutrophils in disease outcome and these cells were shown to play a deleterious role following infection with several Leishmania spp., including L. mexicana. Here, we investigated the role of the c-MET receptor tyrosine kinase in the outcome of cutaneous leishmaniasis. c-MET has been shown to be involved in neutrophil recruitment to inflamed sites. We show here that L. mexicana induce c-MET expression and phosphorylation predominantly in neutrophils. Using mice genetically deficient for c-MET in neutrophils, a better control of lesion size and parasite load was observed. Furthermore, injection of a specific inhibitor of c-MET, once the lesion is established, significantly decreased the cutaneous pathology. The results presented here reveal that c-MET is contributing to the pathology induced by L. mexicana and suggest that the use of c-MET inhibitors could ameliorate the cutaneous lesion pathology.

Leishmania (L.) are obligate intracellular protozoan parasites that cause the leishmaniases, a spectrum of vector-borne infectious diseases ranging from cutaneous to visceral forms. During their blood meals, female sandfly vectors deposit metacyclic promastigotes in the host skin. At the site of infection, Leishmania are first internalized within hours by recruited neutrophils and then transferred to macrophages where they differentiate into the replicative non-flagellated amastigote form [ 10 , 11 ]. L. mexicana is a New World Leishmania species, causing chronic unhealing lesions in humans and mice. The severity of the disease correlates with the limited recruitment of monocytes and poor development of a Th1 immune response [ 12 – 14 ]. We previously showed that the early neutrophil recruitment to the infection site plays a detrimental role during L. mexicana infection, as in contrast to wild type mice, neutropenic mice showed an early increase in monocytes and dendritic cells recruitment inducing the subsequent development of a protective Th1 immune response and mice were able to heal their lesion and better control their parasite burden [ 8 ].

c-MET is a tyrosine kinase encoded by the c-MET proto-oncogene, it is the only known receptor for the hepatocyte growth factor (HGF). HGF/c-MET signalling regulates numerous biological pathways such as proliferation, survival, migration and tissue regeneration [ 1 ]. Aberrant activation of c-MET signalling has been shown to participate in tumour progression [ 2 – 5 ], prompting the development of c-MET inhibitors. However, c-MET inhibitors showed only limited success in clinical trials [ 6 ]. Notably, this restricted efficacy of systemic c-MET inhibition in cancer is in part related to the inhibition of c-MET in neutrophils. Indeed, c-MET was shown to be crucial for the recruitment of anti-tumoral neutrophils, that kill cancer cells following nitric oxide production [ 7 ].

Results

Leishmania parasites induce c-MET mRNA expression in neutrophils In order to investigate the role of c-MET in neutrophils during L. mexicana infection, we first assessed by qPCR whether the parasite could induce c-MET mRNA expression in vitro. To this end, bone marrow neutrophils (BMNs) were isolated from the femur of C57BL/6 mice and the cells were exposed for 16h to L. mexicana metacyclic promastigotes, the infective form of the parasite, at a multiplicity of infection (MOI) of 2 and 5. As positive control, neutrophils were treated for 16h with LPS. Induction of c-MET mRNA in neutrophils was increased ten to twenty-fold at MOI of 2 and 5, respectively, compared to the mRNA levels observed in non-exposed neutrophils (Fig 1A). To assess if infection impacted survival of neutrophils in vitro, BMNs were exposed to L. mexicana at the indicated MOIs and 16 hours post infection, neutrophils were stained with AnnexinV and DAPI and their apoptotic status analysed by flow cytometry. Exposure to L. mexicana did not impact the survival (AnnexinV-DAPI-) nor the apoptotic status (AnnexinV+DAPI+/-) of neutrophils in vitro (S1A Fig). These data demonstrate that the increase in c-MET mRNA observed in infected neutrophils is not due to differences in neutrophil survival between uninfected and infected neutrophils. We further analysed whether L. mexicana promotes c-MET expression in other myeloid cells participating in the innate immune response against the parasite, such as macrophages and dendritic cells. Bone marrow-derived macrophages (BMMs) were incubated in vitro for 16h with L. mexicana promastigotes. Compared to non-exposed macrophages, no difference (MOI of 2) or only a slight increase (MOI of 5) in c-MET mRNA expression was observed following incubation with Leishmania promastigotes (Fig 1B). Similarly, L. mexicana promastigotes induced only a slight increase in c-MET expression in bone marrow-derived dendritic cells (BMDCs) (Fig 1C). We then investigated whether amastigotes, the replicative form of the parasite which is present during the chronic phase of the disease, was also able to induce c-MET mRNA expression in neutrophils. BMNs, BMMs and BMDCs were co-cultured with amastigotes for 16 hours, and c-MET mRNA was measured. Amastigotes induced c-MET mRNA, with levels up to ten-fold at MOI2 and fifteen-fold at MOI5 higher than those observed in uninfected (control) neutrophils (Fig 1D). Very low mRNA induction was observed in BMMs and BMDCs exposed to L. mexicana amastigotes (Fig 1E and 1F, respectively). These findings indicate that L. mexicana promotes a strong dose-dependent upregulation of c-MET mRNA expression specifically in neutrophils. c-MET mRNA levels were also selectively upregulated in neutrophils following exposure to L. major LV39 (S2 Fig), showing that the upregulation of c-MET on neutrophils by Leishmania mexicana extends to other Leishmania spp. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Leishmania parasites upregulate c-MET mRNA expression in neutrophils. A) BM isolated neutrophils (BMNs) were co-cultured at the indicated multiplicity of infection (MOI) with the metacyclic (infecting) form of the L. mexicana parasites as indicated. 16h later mRNA was extracted, and c-MET mRNA levels analysed by RT-qPCR. Uninfected cells were used as negative control and cells treated with 50 ng/mL of LPS were used as positive control. B) BM-derived macrophages (BMMs) and C) BM-derived dendritic cells (BMDCs) were processed similarly. D-F) The indicated bone marrow-derived myeloid cells were similarly infected with amastigotes, the replicating form of the parasites. The data are normalized to endogenous levels of HPRT mRNA and expressed as fold increase relative to expression levels detected in control cells. The data are representative of ≥ 3 independent experiments, n = 3/group. https://doi.org/10.1371/journal.ppat.1010247.g001

c-MET expression has a minor impact on the effector functions of L. mexicana infected neutrophils c-MET expression was previously shown to impact neutrophil function in the tumor environment [7]. To assess if c-MET expression and activation had an impact on L. mexicana-induced neutrophil effector functions, BMNs derived from mice genetically deficient for c-MET in neutrophils (Mrp8;Metfl/fl) and control littermate mice (Mrp8;Metwt/wt) were isolated and exposed to metacyclic L. mexicana-DsRed parasites for 2 and 20 hours at an MOI of 2 and 5. Flow cytometry analysis showed a comparable frequency of infected DsRed+ neutrophils between both groups 2 hours post infection (Fig 4A) suggesting similar parasite internalisation. The frequency of intracellular parasites present in neutrophils 20h post-L. mexicana-DsRed infection was analyzed by flow cytometry to determine if the parasite presence was impacted in absence of c-MET. A 16% and 26% frequency of infected neutrophils was observed at MOI2 and MOI5, respectively. However, no difference in the frequency of infected neutrophils was observed between c-MET deficient and control neutrophils (Fig 4B). No differences in neutrophil survival and apoptosis were observed between Mrp8;Metfl/fl and Mrp8;Metwt/wt neutrophils at 2 and 20 hours post infection (S1B Fig). To assess if the absence of c-MET would have an impact on the parasite number per neutrophils, BMNs from WT (Mrp8;Metwt/wt) and c-MET deficient (Mrp8;Metfl/fl) neutrophils were infected and 16 hours later analysed by imaging flow cytometry. No difference in parasite number was observed between c-MET deficient and control neutrophils (Fig 4C and 4D), confirming our results obtained by conventional flow cytometry. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Impact of c-MET on neutrophil functions. BMNs were isolated from mice genetically deficient for c-MET in PMNs (Mrp8;Metfl/fl) and control littermate (Mrp8;Metwt/wt). PMNs were co-cultured in vitro with L. mexicana-DsRed at the indicated MOI. A) 2h post infection free parasites were extensively washed and internalization was assessed by flow cytometry. A representative flow cytometry plot and the frequency of infected PMNs are shown. B) A similar analysis was performed 20 hours after infection. C) Representative images taken by imaging flow cytometry representing L. mexicana parasites in Mrp8;Metfl/fl and control Mrp8;Metwt/wt BMNs 24 hours after L. mexicana infection. D) Quantitative assessment of the number of parasites in Mrp8;Metfl/fl and control Mrp8;Metwt/wt neutrophils, 24 hours after infection. E) To analyse ROS production, PMNs were exposed for 2h to L. mexicana parasites at MOI of 5. As control, PMNs similarly grown in medium were analysed. Luminol was added to the co-culture and the chemiluminescence was measured for 2h to evaluate ROS production. Data are shown as relative light units (RLU). F) The corresponding area under the curve (AUC) is shown. G) Limiting dilution analysis of Mrp8;Metfl/fl and Mrp8;Metwt/wt neutrophils infected with L. mexicana (MOI 2). H) Mrp8;Metfl/fl and Mrp8;Metwt/wt mice were infected with 106 L. mexicana metacyclic promastigotes in the ear dermis. 24 hours later, the infected ears were collected, and ROS production was assessed by flow cytometry in CD45+CD11b+Ly6G+ PMNs using the DHR123 probe. A representative flow cytometry plot, and the corresponding MFI (right) of a representative experiment are shown. Shaded histograms are the FMO control for DHR123. I) NO levels were analyzed by flow cytometry in Mrp8;Metfl/fl and Mrp8;Metwt/wt neutrophils 24 hours after infection. Data are representative of ≥ 2 experiments, n≥3/group. https://doi.org/10.1371/journal.ppat.1010247.g004 ROS production is one of the major defence mechanisms of neutrophils, we thus analysed a potential impact of c-MET expression on ROS production. BMNs isolated from the femurs of Mrp8;Metfl/fl or of Mrp8;Metwt/wt mice, were exposed to L. mexicana metacyclic promastigotes for 2h at MOI of 5. ROS production was measured by the chemiluminescence induced by the reaction of luminol with ROS and analyzed over 120 min. Unexposed neutrophils were used as control. Higher levels of ROS were produced by neutrophils deficient for c-MET compared to WT neutrophils (Fig 4E and 4F). As ROS may impact parasite survival, neutrophils were similarly exposed to L. mexicana and limiting dilution analysis performed. A small but statistically significant decrease in parasite survival was observed in c-MET-deficient neutrophils (Fig 4G), suggesting that even though a similar number of DsRed-L. mexicana is observed in c-MET deficient and WT neutrophils, there appears to be a reduced fitness of parasites present in c-MET deficient neutrophils. These data correlate with higher ROS production observed in c-MET-deficient neutrophils. We then measured in vivo the production of ROS in c-MET deficient and control neutrophils at the site of infection. Mrp8;Metfl/fl and Mrp8;Metwt/wt mice were infected in the ear dermis with 106 L. mexicana metacyclic promastigotes, and ROS production was assessed in neutrophils 24 hours later by flow cytometry, at a time when neutrophil presence peaks following infection [8]. ROS production in CD45+CD11b+Ly6G+ neutrophils was analyzed using the DHR123 probe. No difference was observed between c-MET deficient and WT neutrophils (Fig 4H). The level of nitric oxide (NO) was also assessed by flow cytometry using the DAF-FM probe. No difference between Mrp8;Metfl/fl and Mrp8;Metwt/wt mice was observed (Fig 4I). These findings indicate that c-MET expression on neutrophils has no impact on L. mexicana internalisation and neutrophil effector function except a small effect observed in ROS production in vitro, a phenomenon that is compensated in vivo.

Specific deletion of c-MET in neutrophils has a major impact on L. mexicana lesion development Early neutrophils infiltration in L. mexicana lesion contributes to the cutaneous pathology [8]. Thus, we hypothesised that the deletion of c-MET in neutrophils might decrease their presence in infected tissues and thereby be beneficial to the disease. To assess the impact of c-MET in neutrophils on the disease outcome, Mrp8;Metfl/fl and Mrp8;Metwt/wt mice were infected i.d. with L. mexicana-DsRed metacyclic promastigotes. Following inoculation of a high dose (106) of L. mexicana, elevated HGF levels were observed in ear lysates 24 hours p.i. with similar levels observed between PBS or L. mexicana injected ears, suggesting that the needle injection itself is inducing HGF release. Accordingly, no difference in neutrophil recruitment was observed at that time point between mice injected either with PBS or a high dose of L. mexicana (S5 Fig). Thus, to analyse the impact of the parasite infection on c-MET activation on neutrophils and its effect on the disease, and to be closer to the physiological parasite dose inoculated during a sand fly blood meal, we infected mice with a low dose (104) of parasites. Parasite-specific neutrophil recruitment to the infected ear takes place between 3–4 weeks p.i. Following infection, Mrp8;Metfl/fl and Mrp8;Metwt/wt control mice developed a small lesion that did not differ until 3–4 weeks post infection. From there on, Mrp8;Metfl/fl mice controlled better the infection and developed a significantly smaller lesion than similarly infected Mrp8;Metwt/wt control mice (Fig 5A and 5B). Ten weeks post-infection, the number of CD11b+ cells recruited in the infected ear was comparable between the two groups of mice (Fig 5C) but the frequency of infected (DsRed+) cells in the CD11b+ population was significantly reduced in Mrp8;Metfl/fl infected ears (Fig 5D and 5E). Accordingly, neutrophils, monocytes and dendritic cells were less infected in Mrp8;Metfl/fl compared to control Mrp8;Metwt/wt mice (Fig 5E). In addition, the parasite load in the infected ear, as determined by limiting dilution analysis (LDA), was significantly lower in Mrp8;Metfl/fl than in Mrp8;Metwt/wt infected ears (Fig 5F). PPT PowerPoint slide

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TIFF original image Download: Fig 5. Specific deletion of c-MET in neutrophils has a major impact on L. mexicana lesion development. Mrp8;Metfl/fl mice selectively deficient for c-MET in PMNs and Mrp8;Metwt/wt control littermate were infected i.d. with 104 L. mexicana-DsRed metacyclic promastigotes. A) Lesion development was measured on a weekly basis using a caliper and lesion score was determined. Briefly, the score is based on the inflammation of the ear, the lesion size (mm) and appearance of necrosis. B) Representative pictures of ear lesions ten weeks post infection are shown. Ten weeks post infection, at the end of the experiment, ears and draining lymph nodes (dLNs) were excised and digested. C) The number of CD11b+ myeloid cells was assessed by flow cytometry at the site of infection. D) Representative flow cytometry plot showing the gating strategy for CD45+CD11b+DsRed+ cells. E) The frequency of infected myeloid CD45+CD11b+DsRed+ cells and the frequency of infected CD45+CD11b+Ly6G+DsRed+ PMNs, CD45+CD11b+Ly6G-Ly6C+DsRed+ monocytes and CD45+CD11c+DsRed+ DCs in the ear was assessed by flow cytometry. F) The parasite load in the infected ears was determined by limiting dilution assay (LDA). G) Representative flow cytometry plot of ear derived dermal cells. H) Frequency of total CD45+CD11b+Ly6G+ PMNs, CD45+CD11b+Ly6G-Ly6C+ monocytes, CD45+CD11c+ DCs and CD45+CD11c+Ly6C+CD11b+ monocyte derived DCs (MoDCs). I) The frequency of CD45+CD11b+Ly6G+ PMNs in the ears of Mrp8;Metfl/fl mice and Mrp8;Metwt/wt control littermates was analysed by flow cytometry 0, 3, 4 and 10 weeks post i.d. infection with 104 metacyclic L. mexicana. The data in I are pooled from two independent experiments involving ≥6 mice/ group J) The frequency of CD45+CD4+IFN-γ+ and CD45+CD4+IL-4+ T cells in dLNs and K) in the ears is presented. Data are representative of ≥3 experiments, n≥6/group, *p <0.05; **p <0.01. https://doi.org/10.1371/journal.ppat.1010247.g005 The ear cellularity at the infected site was analysed by flow cytometry (Fig 5G). The frequency of neutrophils recruited to the ear dermis was significantly lower in Mrp8;Metfl/fl than Mrp8;Metwt/wt mice at ten weeks post-infection, whereas no differences were observed in the frequency of monocytes, DCs and monocytes derived DCs (MoDC) (Fig 5H). To further demonstrate the importance of c-MET in neutrophil recruitment to the site of infection, we also analyzed the frequency of ear-recruited neutrophils early after infection, when no difference in lesion size was yet observed between Mrp8;Metfl/fl and Mrp8;Metwt/wt mice. Ears were isolated, digested and neutrophil presence was analyzed by flow cytometry. Only a very low neutrophil frequency was observed in Mrp8;Metfl/fl and Mrp8;Metwt/wt ears at basal levels (time 0) which was similar between both groups. Three weeks post infection, a few neutrophils started to be recruited to the site of infection, and already a tendency to a lower frequency of recruited neutrophils was observed in Mrp8;Metfl/fl compared to Mrp8;Metwt/wt ears. Four weeks post infection, higher levels of neutrophils were recruited to the infected site and a significant decrease in neutrophil recruitment was observed in Mrp8;Metfl/fl compared to Mrp8;Metwt/wt ears. Ten weeks post infection, the difference in neutrophil frequency observed between Mrp8;Metfl/fl and Mrp8;Metwt/wt infected ears was further increased (Fig 5I). To analyse the type of adaptive immune response developing in these mice, we measured the frequency of CD4+IFN-γ+ and CD4+IL-4+ cells present in the draining lymph node (dLN) and infected ear. In the dLN, the frequency of CD4+ T cells expressing IFN-γ and IL-4 was similar between the two mouse strains (Fig 5J). At the site of infection, an increase in the frequency of CD4+IFN-γ+ T cells was consistently observed in the ears of Mrp8;Metfl/fl compared to control mice, although it was not statistically significant in all experiments (Fig 5K). Correspondingly, the frequency of CD4+IL-4+ T cells was lower in the ears of Mrp8;Metfl/fl compared to control group (Fig 5K). Collectively, our results indicate that c-MET signalling in neutrophils is contributing to the development of the pathology associated with L. mexicana infection.

[END]

[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010247

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